Microbial communities associated with herbivores are fundamental to ecosystem functioning in almost all environments on Earth. Specifically, these communities facilitate the conversion of plant biomass into nutrients usable by their host, thereby bridging primary producers and secondary consumers. My research is focused on understanding the evolution and ecology of herbivore-associated microbial communities on three different scales. At the broadest scale, I am interested in how these communities evolve across host type, host diet, and geographical distribution. At an intermediate level, I am interested in how members of these communities interact with each other to coordinate the breakdown of plant biomass and produce nutrients for their host. At the finest scale, I am interested in understanding how microbes fundamentally degrade polysaccharides (cellulose and hemicelluloses) in plant cell walls. My work can be applied to two specific fields as described below.
Biofuels. A major global challenge is to reduce our reliance on fossil fuels. Biofuels have been proposed as an alternative fuel source because of their cleanliness and sustainability. One proposed biofuel is cellulosic ethanol, which can supplement gasoline and integrate with our current transportation infrastructure. The generation of cellulosic ethanol requires the conversion of cellulose into simple sugars followed by fermentation into ethanol. My work directly impacts the first bottleneck of cellulose degradation. I use as a model system, ruminants, which are arguably one of the most efficient natural cellulose-degrading systems. Ruminants such as domesticated cattle harbor a specialized community of plant-degrading bacteria that ferment cellulose and other polysaccharides into small chain fatty acids. We are attempting to understand this process at a base level by characterizing the mechanisms through which ruminal bacteria like Fibrobacter succinogenes S85 and Ruminococcus albus 7 degrade cellulose. In particular, Ruminococcus albus 7 is capable of fermenting ethanol using cellulose in vitro. This work incorporates whole-genome sequencing, transcript sequencing using RNA-seq, prediction of protein-protein interactions using functional genomics, and in vitro cell-free expression of cellulolytic enzymes. Finally, these bacteria are known to work with other hemicellulolytic ruminal bacteria to synergistically enhance their overall cellulolytic and fermentative abilities. We are also investigating these interactions to gain an understanding of this process.
Animal Health and Production. Ruminants are major agricultural resources, particularly for the production of products like beef and milk. Milk production is linked to the ruminal microbial community, as the small chain fatty acids produced by these microbes directly impacts the quality of milk produced by cows. We are interested in understanding the ecology and evolution of these ruminal communities and their impact on milk production. Specifically, we are working to understand the confluence of diet, host genotype, and ruminal microbiota on milk quality. We use a combination of metagenomics, metatranscriptomics, milk production metrics, and cow health to assess these factors. We are also interested in understanding how rumen microbiota become established in developing calves, and in particular, how diet influences microbial composition. Finally, these studies have direct impact on human health and disease, particularly in development, lactation, and the influence of diet on host microbiota. Ruminants are excellent models for understanding these factors in humans as they share a large number of genes, can be directly manipulated at the ruminal level, and can be reared on controlled diets.
Microbiology 526: Physiology of Microorganisms
Trainer, Molecular Biosciences Training Grant Program
Trainer, Microbes in Health and Disease Training Program
Trainer, Computational and Informatics in Biology and Medicine
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
Tall fescue, the predominant Southeastern United States cool season forage grass, frequently becomes infected with an ergot alkaloid-producing toxic endophyte, Consumption of endophyte-infected fescue results in fescue toxicosis (FT), a condition that lowers beef cow productivity. Limited data on the influence of ergot alkaloids on rumen fermentation profiles or ruminal bacteria that could degrade the ergot alkaloids are available, but how FT influences the grazing bovine fecal microbiota, or what role fecal microbiota might play in FT etiology, and associated production losses, has yet to be investigated. Here, we used 16S rRNA gene sequencing of fecal samples from weaned Angus steers grazing toxic (n = 6; E+) or non-toxic (n = 6; Max-Q) tall fescue before, 1, 2, 14, and 28 days post-pasture assignment. Bacteria in the and phyla comprised 90% of the Max-Q and E+ steer fecal microbiota throughout the trial. Early decreases in the family and delayed increases of the and families were among the major effects of E+ grazing. E+ also increased the abundances within the , , and phyla and the family. Multiple operational taxonomic units classified to the and were correlated negatively with weight gains (lower in E+) and positively with respiration rates (increased by E+). These data provide insights into how E+ grazing alters the Angus steer microbiota and the relationship of fecal microbiota dynamics with FT. Consumption of toxic endophyte-infected (E+) tall fescue has an estimated annual $1 billion negative impact on the U.S. beef industry, with one driver of these costs being lowered weight gains. As global agricultural demand continues to grow, mitigating production losses resultant from grazing the predominant Southeastern United States forage grass is of great value. Our investigation of the effects of E+ grazing on the fecal microbiota furthers our understanding of bovine fescue toxicosis in a real-world grazing production setting and provides a starting point for identifying easy-to-access fecal bacteria that could serve as potential biomarkers of animal productivity and/or FT severity for tall fescue grazing livestock.
Laryngotracheal stenosis is an obstructive respiratory disease that leads to voicing difficulties and dyspnea with potential life-threatening consequences. The majority of incidences are due to iatrogenic etiology from endotracheal tube intubation; however, airway scarring also has idiopathic causes. While recent evidence suggests a microbial contribution to mucosal inflammation, the microbiota associated with different types of stenosis has not been characterized. High-throughput sequencing of the V4 region of the16S rRNA gene was performed to characterize the microbial communities of 61 swab samples from 17 iatrogenic and 10 adult idiopathic stenosis patients. Nonscar swabs from stenosis patients were internal controls, and eight swabs from four patients without stenosis represented external controls. Significant differences in diversity were observed between scar and nonscar samples and among sample sites, with decreased diversity detected in scar samples and the glottis region. Permutational analysis of variance (PERMANOVA) results revealed significant differences in community composition for scar versus nonscar samples, etiology type, sample site, groups (iatrogenic, idiopathic, and internal and external controls), and individual patients. Pairwise Spearman's correlation revealed a strong inverse correlation between and among all samples. Finally, bacteria in the family were found to be distinctly associated with idiopathic stenosis samples in comparison with external controls. Our findings suggest that specific microbiota and community shifts are present with laryngotracheal stenosis in adults, with members of the family , including the known pathogens and , identified in idiopathic scar. Further work is warranted to elucidate the contributing role of bacteria on the pathogenesis of laryngotracheal stenosis. The laryngotracheal region resides at the intersection between the heavily studied nasal cavity and lungs; however, examination of the microbiome in chronic inflammatory conditions of the subglottis and trachea remains scarce. To date, studies have focused on the microbiota of the vocal folds, or the glottis, for laryngeal carcinoma, as well as healthy larynges, benign vocal fold lesions, and larynges exposed to smoking and refluxate. In this study, we seek to examine the structure and composition of the microbial community in adult laryngotracheal stenosis of various etiologies. Due to the heterogeneity among the underlying pathogenesis mechanisms and clinical outcomes seen in laryngotracheal stenosis disease, we hypothesized that different microbial profiles will be detected among various stenosis etiology types. Understanding differences in the microbiota for subglottic stenosis subtypes may shed light upon etiology-specific biomarker identification and offer novel insights into management approaches for this debilitating disease.
Early gut microbial colonization is important for postnatal metabolic and immune development. However, little is known about the effects of different feeding modes (suckling versus bottle-feeding) or microbial sources on this process in farm animals. We found that suckled and bottle-fed newborn lambs had their own distinct gut microbiota. Results from 16S rRNA gene sequencing and qPCR showed that, compared with suckling, bottle feeding significantly increased the abundances of Escherichia/Shigella, Butyricicoccus, and Clostridium XlVa, while significantly decreased the abundance of Clostridium XI. The higher levels of Escherichia/Shigella in bottle-fed lambs suggest that artificial feeding may increase the number of potential pathogens and delay the establishment of the anaerobic environment and anaerobic microbes. Feeding modes also affected the direct transmission of bacteria from the mother and the environment to newborns. The SourceTracker analysis estimated that the early gut microbes of suckled lambs were mainly derived from the mother's teats (43%) and ambient air (28%); whereas those of bottle-fed lambs were dominated by bacteria from the mother's vagina (46%), ambient air (31%), and the sheep pen floor (12%). These findings advance our understanding of gut microbiota in early life and may help design techniques to improve gut microbiota and health.
The objective of this research was to evaluate the effects of corn silage inclusion in starter feed provided to calves after birth through weaning at 7 wk of age. Thirty-six heifer calves and 9 bull calves were individually housed in hutches. Calves in treatment groups received pasteurized milk with all calf starter, 25% calf starter and 75% corn silage, or all corn silage. Values were recorded daily for feed intake and health, which included fecal, respiratory, and attitude scores; and at wk 2, 4, and 8 for concentrations of serum protein, hematocrit, and serum β-hydroxybutyrate. Body weight, withers height, and hip height were measured at wk 2, 4, 8, and 52. Nine bull calves (3/treatment) were killed at 8 wk of age for assessment of rumen and intestinal tissue morphology. Feed intake and average daily gain were not different among treatments. Least squares means of rumen papillae lengths were significantly different and decreased as corn silage inclusion increased. Crypt depths and total thickness of epithelium were reduced for the corn silage group. Least squares means of body weight, heart girth, hip height, withers height, serum protein, hematocrit, and β-hydroxybutyrate concentrations did not differ among treatments. These data indicated that the mixture of corn silage and starter did not affect growth, feed intake, or intestinal morphology but did affect rumen wall morphology. Feeding solely corn silage as starter feed stunted the growth of rumen papillae and tended to impair intestinal morphology, indicating that only calf starter or a mixture of calf starter and corn silage is more appropriate.
Gut -associated microbes ('gut microbiota') impact the nutrition of their hosts, especially in ruminants and pseudoruminants that consume high-cellulose diets. Examples include the pseudoruminant alpaca. To better understand how body site and diet influence the alpaca microbiota, we performed three 16S rRNA gene surveys. First, we surveyed the compartment 1 (C1), duodenum, jejunum, ileum, cecum, and large intestine (LI) of alpacas fed a grass hay (GH; tall fescue) or alfalfa hay (AH) diet for 30 days. Second, we performed a C1 survey of alpacas fed a series of 2-week mixed grass hay (MGH) diets supplemented with ∼25% dry weight barley, quinoa, amaranth, or soybean meal. Third, we examined the microbial differences of alpacas with normal versus poor body condition. Samples from GH- and AH-fed alpacas grouped by diet and body site but none of the four supplements significantly altered C1 microbiota composition, relative to each other, and none of the OTUs were differentially abundant between alpacas with normal versus poor body conditions. Taken together, the findings of a diet- and body-site specific alpaca microbiota are consistent with previous findings in ruminants and other mammals, but we provide no evidence to link changes in alpaca body condition with variation in microbiota relative abundance or identity.
In mammals, microbial colonization of the digestive tract (GIT) occurs right after birth by several bacterial phyla. Numerous human and mouse studies have reported the importance of early gut microbial inhabitants on host health. However, few attempts have been undertaken to directly interrogate the role of early gut/rumen microbial colonization on GIT development or host health in neonatal ruminants through artificial manipulation of the rumen microbiome. Thus, the molecular changes associated with bacterial colonization are largely unknown in cattle. In this study, we dosed young calves with exogenous rumen fluid obtained from an adult donor cow, starting at birth, and repeated every other week until six weeks of age. Eight Holstein bull calves were included in this study and were separated into two groups of four: the first group was treated with rumen content freshly extracted from an adult cow, and the second group was treated with sterilized rumen content. Using whole-transcriptome RNA-sequencing, we investigated the transcriptional changes in the host liver, which is a major metabolic organ and vital to the calf's growth performance. Additionally, the comparison of rumen epimural microbial communities between the treatment groups was performed using the rRNA reads generated by sequencing. Liver transcriptome changes were enriched with genes involved in cell signaling and protein phosphorylation. Specifically, up-regulation of SGPL1 suggests a potential increase in the metabolism of sphingolipids, an essential molecular signal for bacterial survival in digestive tracts. Notably, eight genera, belonging to four phyla, had significant increases in abundance in treated calves. Our study provides insight into host liver transcriptome changes associated with early colonization of the microbial communities in neonatal calves. Such knowledge provides a foundation for future probiotics-based research in microbial organism mediated rumen development and nutrition in ruminants.
Members of the genus are cellulose-degrading bacteria and common constituents of the gastrointestinal microbiota of herbivores. Although considerable phylogenetic diversity is observed among members of this group, few functional differences explaining the distinct ecological distributions of specific phylotypes have been described. In this study, we sequenced and performed a comparative analysis of whole genomes from 38 novel strains against the type strains for the two formally described species strain S85 and strain NR9. Significant differences in the number of genes encoding carbohydrate-active enzyme families involved in plant cell wall polysaccharide degradation were observed among phylotypes. genomes were consistently enriched in genes encoding carbohydrate-active enzymes compared to those of strains. Moreover, genomes of phylotypes that are dominant in the rumen had significantly more genes annotated to major families involved in hemicellulose degradation (e.g., CE6, GH10, and GH43) than did the genomes of phylotypes typically observed in the lower gut of large hindgut-fermenting herbivores such as horses. Genes encoding a putative urease were also identified in 12 of the genomes, which were primarily isolated from hindgut-fermenting hosts. Screening for growth on urea as the sole source of nitrogen provided strong evidence that the urease was active in these strains. These results represent the strongest evidence reported to date for specific functional differences contributing to the ecology of spp. in the herbivore gut. The herbivore gut microbiome is incredibly diverse, and a functional understanding of this diversity is needed to more reliably manipulate this community for specific gain, such as increased production in ruminant livestock. Microbial degraders of plant cell wall polysaccharides in the herbivore gut, particularly spp., are of fundamental importance to their hosts for digestion of a diet consisting primarily of recalcitrant plant fibers. Considerable phylogenetic diversity exists among members of the genus , but much of this diversity remains cryptic. Here, we used comparative genomics, applied to a diverse collection of recently isolated strains, to identify a robust association between carbohydrate-active enzyme gene content and the phylogeny. Our results provide the strongest evidence reported to date for functional differences among phylotypes associated with either the rumen or the hindgut and emphasize the general significance of carbohydrate-active enzymes in the evolution of fiber-degrading bacteria.
Cellulose is the most abundant biological polymer on earth, making it an attractive substrate for the production of next-generation biofuels and commodity chemicals. However, the economics of cellulose utilization are currently unfavorable due to a lack of efficient methods for its hydrolysis. strain S85, originally isolated from the bovine rumen, is among the most actively cellulolytic mesophilic bacteria known, producing succinate as its major fermentation product. In this study, we examined the transcriptome of S85 grown in continuous culture at several dilution rates on cellulose, cellobiose, or glucose to gain a system-level understanding of cellulose degradation by this bacterium. Several patterns of gene expression were observed for the major cellulases produced by S85. A large proportion of cellulase genes were constitutively expressed, including the gene encoding for Cel51A, the major cellulose-binding endoglucanase produced by this bacterium. Moreover, other cellulase genes displayed elevated expression during growth on cellulose relative to growth on soluble sugars. Growth rate had a strong effect on global gene expression, particularly with regard to genes predicted to encode carbohydrate-binding modules and glycoside hydrolases implicated in hemicellulose degradation. Expression of hemicellulase genes was tightly regulated, with these genes displaying elevated expression only during slow growth on soluble sugars. Clear differences in gene expression were also observed between adherent and planktonic populations within continuous cultures growing on cellulose. This work emphasizes the complexity of the fiber-degrading system utilized by S85, and reinforces the complementary role of hemicellulases for accessing cellulose by these bacteria. We report for the first time evidence of global differences in gene expression between adherent and planktonic populations of an anaerobic bacterium growing on cellulose at steady state during continuous cultivation. Finally, our results also highlight the importance of controlling for growth rate in investigations of gene expression.
Gastrointestinal tract (GIT) microorganisms play important roles in the health of ruminant livestock and affect the production of agriculturally relevant products, including milk and meat. Despite this link, interventions to alter the adult microbiota to improve production have proven ineffective, as established microbial communities are resilient to change. In contrast, developing communities in young animals may be more easily altered but are less well studied. Here, we measured the GIT-associated microbiota of 45 Holstein dairy cows from 2 weeks to the first lactation cycle, using Illumina amplicon sequencing of bacterial (16S rRNA V4), archaeal (16S rRNA V6 to V8), and fungal (internal transcribed region 1 [ITS1]) communities. Fecal and ruminal microbiota of cows raised on calf starter grains and/or corn silage were correlated to lifetime growth as well as milk production during the first lactation cycle, in order to determine whether early-life diets have long-term impacts. Significant diet-associated differences in total microbial communities and specific taxa were observed by weaning (8 weeks), but all animals reached an adult-like composition between weaning and 1 year. While some calf-diet-driven differences were apparent in the microbiota of adult cows, these dissimilarities did not correlate with animal growth or milk production. This finding suggests that initial microbial community establishment is affected by early-life diet but postweaning factors have a greater influence on adult communities and production outcomes. The gut microbiota is essential for the survival of many organisms, including ruminants that rely on microorganisms for nutrient acquisition from dietary inputs for the production of products such as milk and meat. While alteration of the adult ruminant microbiota to improve production is possible, changes are often unstable and fail to persist. In contrast, the early-life microbiota may be more amenable to sustained modification. However, few studies have determined the impact of early-life interventions on downstream production. Here, we investigated the impact of agriculturally relevant calf diets, including calf starter and corn silage, on gut microbial communities, growth, and production through the first lactation cycle. Thus, this work serves to further our understanding of early-life microbiota acquisition, as well as informing future practices in livestock management.
The objective of this longitudinal cohort study was to describe the milk microbiota of dairy cow mammary glands based on inflammation status before and after the dry period. Individual mammary quarters were assigned to cohorts based on culture results and somatic cell count () at dryoff and twice in the first 2 weeks post-calving. Mammary glands that were microbiologically negative and had low SCC (< 100,000 cells/mL) at all 3 sampling periods were classified as Healthy ( = 80). Microbiologically negative mammary glands that had SCC ≥150,000 cells/mL at dryoff and the first post-calving sample were classified as Chronic Culture-Negative Inflammation (CHRON; = 17). Quarters that did not have both culture-negative milk and SCC ≥ 150,000 cells/mL at dryoff but were culture-negative with SCC ≥ 150,000 at both post-calving sampling periods were classified as Culture-Negative New Inflammation (NEWINF; = 6). Mammary glands with bacterial growth and SCC ≥ 150,000 cells/mL at all 3 periods were classified as Positive (POS; = 3). Milk samples were collected from all enrolled quarters until 150 days in milk and subjected to microbiota analysis. Milk samples underwent total DNA extraction, a 40-cycle PCR to amplify the V4 region of the bacterial 16S rRNA gene, and next-generation sequencing. Healthy quarters had the lowest rate of PCR and sequencing success (53, 67, 83, and 67% for Healthy, CHRON, NEWINF, and POS, respectively). Chao richness was greatest in milk collected from Healthy quarters and Shannon diversity was greater in milk from Healthy and CHRON quarters than in milk collected from glands in the NEWINF or POS cohorts. Regardless of cohort, season was associated with both richness and diversity, but stage of lactation was not. The most prevalent OTUs included typical gut- and skin-associated bacteria such as those in the phylum Bacteroidetes and the genera and . The increased sequencing success in quarters with worse health outcomes, combined with the lack of bacterial growth in most samples and the high PCR cycle number required for amplification of bacterial DNA, suggests that the milk microbiota of culture-negative, healthy mammary glands is less abundant than that of culture-negative glands with a history of inflammation.
The rumen is a complex ecosystem composed of anaerobic bacteria, protozoa, fungi, methanogenic archaea and phages. These microbes interact closely to breakdown plant material that cannot be digested by humans, whilst providing metabolic energy to the host and, in the case of archaea, producing methane. Consequently, ruminants produce meat and milk, which are rich in high-quality protein, vitamins and minerals, and therefore contribute to food security. As the world population is predicted to reach approximately 9.7 billion by 2050, an increase in ruminant production to satisfy global protein demand is necessary, despite limited land availability, and whilst ensuring environmental impact is minimized. Although challenging, these goals can be met, but depend on our understanding of the rumen microbiome. Attempts to manipulate the rumen microbiome to benefit global agricultural challenges have been ongoing for decades with limited success, mostly due to the lack of a detailed understanding of this microbiome and our limited ability to culture most of these microbes outside the rumen. The potential to manipulate the rumen microbiome and meet global livestock challenges through animal breeding and introduction of dietary interventions during early life have recently emerged as promising new technologies. Our inability to phenotype ruminants in a high-throughput manner has also hampered progress, although the recent increase in "omic" data may allow further development of mathematical models and rumen microbial gene biomarkers as proxies. Advances in computational tools, high-throughput sequencing technologies and cultivation-independent "omics" approaches continue to revolutionize our understanding of the rumen microbiome. This will ultimately provide the knowledge framework needed to solve current and future ruminant livestock challenges.
The milk microbiota is an intriguing area of research because milk with no bacterial growth in culture was long thought to be sterile. Recent DNA sequencing techniques have been developed that do not require bacteria to be culturable, and DNA from new bacteria have been reported in milk from dairy cow mammary glands with or without mastitis. Methodologies and results vary among research groups, and not enough is known about the milk microbiota for the results to be used for diagnosis or prognosis of mastitis.
Perturbations in the gastrointestinal microbiome caused by antibiotics are a major risk factor for Clostridium difficile infection (CDI). Probiotics are often recommended to mitigate CDI symptoms; however, there exists only limited evidence showing probiotic efficacy for CDI. Here, we examined changes to the GI microbiota in a study population where probiotic treatment was associated with significantly reduced duration of CDI diarrhea. Subjects being treated with standard of care antibiotics for a primary episode of CDI were randomized to probiotic treatment or placebo for 4 weeks. Probiotic treatment consisted of a daily multi-strain capsule (Lactobacillus acidophilus NCFM, ATCC 700396; Lactobacillus paracasei Lpc-37, ATCC SD5275; Bifidobacterium lactis Bi-07, ATCC SC5220; Bifidobacterium lactis B1-04, ATCC SD5219) containing 1.7 x 1010 CFUs. Stool was collected and analyzed using 16S rRNA sequencing. Microbiome analysis revealed apparent taxonomic differences between treatments and timepoints. Subjects administered probiotics had reduced Verrucomicrobiaceae at week 8 compared to controls. Bacteroides were significantly reduced between weeks 0 to 4 in probiotic treated subjects. Ruminococcus (family Lachnospiraceae), tended to be more abundant at week 8 than week 4 within the placebo group and at week 8 than week 0 within the probiotic group. Similar to these results, previous studies have associated these taxa with probiotic use and with mitigation of CDI symptoms. Compositional prediction of microbial community function revealed that subjects in the placebo group had microbiomes enriched with the iron complex transport system, while probiotic treated subjects had microbiomes enriched with the antibiotic transport system. Results indicate that probiotic use may impact the microbiome function in the face of a CDI; yet, more sensitive methods with higher resolution are warranted to better elucidate the roles associated with these changes. Continuing studies are needed to better understand probiotic effects on microbiome structure and function and the resulting impacts on CDI.
Heifers emit more enteric methane (CH ) than adult cows and these emissions tend to decrease per unit feed intake as they age. However, common mitigation strategies like expensive high-quality feeds are not economically feasible for these pre-production animals. Given its direct role in CH production, altering the rumen microbiota is another potential avenue for reducing CH production by ruminants. However, to identify effective microbial targets, a better understanding of the rumen microbiota and its relationship to CH production across heifer development is needed. Here, we investigate the relationship between rumen bacterial, archaeal, and fungal communities as well as CH emissions and a number of production traits in prepubertal (PP), pubertal (PB), and pregnant heifers (PG). Overall, PG heifers emitted the most CH , followed by PB and PP heifers. The bacterial genus Acetobacter and the archaeal genus Methanobrevibacter were positively associated, while Eubacterium and Methanosphaera were negatively associated with raw CH production by heifers. When corrected for dietary intake, both Eubacterium and Methanosphaera remained negatively associated with CH production. We suggest that Eubacterium and Methanosphaera represent likely targets for CH mitigation efforts in heifers as they were negatively associated with CH production and not significantly associated with production traits. © 2018 Society of Chemical Industry.
The objective of this pilot study was to evaluate the influence of sampling technique and exposure to different bedding types on the milk microbiome of healthy primiparous cows. Primiparous Holstein cows (n = 20) with no history of clinical mastitis or monthly somatic cell counts >150,000 cells/mL were selected for this study. From each enrolled cow, a composite milk sample was aseptically collected from all 4 mammary quarters (individual quarter somatic cell counts <100,000 cells/mL), 1 individual quarter milk sample was collected using conventional aseptic technique, and 2 individual quarter milk samples were collected directly from the gland cistern using a needle and vacuum tube. All milk samples were cultured using standard milk microbiological techniques and DNA was extracted. Extracted DNA was subjected to PCR and next-generation sequencing for microbiota determination. All samples yielded relatively little total DNA. Amplification of PCR was successful in 45, 40, and 83% of composite, conventional, and cisternal samples, respectively. Bacteria were successfully cultured from 35% of composite milk samples but from none of the quarter milk samples collected using conventional or cisternal sampling techniques. Bacterial DNA sequences were assigned to operational taxonomic units (OTU) based on 97% sequence similarity, and bacterial richness and diversity were determined. Most samples were dominated by low-prevalence OTU and of the 4,051 identified OTU, only 14 were prevalent at more than 1% each. These included bacteria typically recovered from environmental sources. Chao richness was greatest in composite samples and was 636, 347, and 356 for composite, conventional quarter, and cisternal milk samples, respectively. Shannon diversity was similar among sample types and ranged from 3.88 (quarter) to 4.17 (composite). Richness and diversity did not differ by bedding type among cisternal samples, but the power of this pilot study was limited due to small sample size. Despite the small sample size, for milk samples collected from the gland cistern, overall bacterial community composition differed among bedding types. These results demonstrate that sampling technique and bedding type may be associated with the microbiota detected in bovine milk, and we suggest that these variables should be considered in designing and reporting studies about the milk microbiota.
Prevention of multidrug-resistant organism (MDRO) infections, such as those caused by methicillin-resistant , vancomycin-resistant enterococci, fluoroquinolone-resistant Gram-negative bacteria and is crucial. Evidence suggests that dietary fibre increases gut microbial diversity, which may help prevent colonisation and subsequent infection by MDROs. The aim of the Winning the War on Antibiotic Resistance (WARRIOR) project is to examine associations of dietary fibre consumption with the composition of the gut microbiota and gut colonisation by MDROs. The secondary purpose of the study is to create a biorepository of multiple body site specimens for future microbiota research. The WARRIOR project collects biological specimens, including nasal, oral and skin swabs and saliva and stool samples, along with extensive data on diet and MDRO risk factors, as an ancillary study of the Survey of the Health of Wisconsin (SHOW). The SHOW is a population-based health survey collecting data on several different health determinants and outcomes, as well as objective body measurements and biological specimens. WARRIOR participants include 600 randomly selected Wisconsin residents age 18 and over. Specimens are screened for MDRO colonisation and DNA is extracted for 16S ribosomal RNA-based microbiota sequencing. Data will be analysed to assess the relationship between dietary fibre, the gut microbiota composition and gut MDRO colonisation. The WARRIOR project is approved by the University of Wisconsin Institutional Review Board. The main results of this study will be published in a peer-reviewed scientific journal.
Mammalian gut microbial communities form intricate mutualisms with their hosts, which have profound implications on overall health. One group of important gut microbial mutualists are bacteria in the genus Ruminococcus, which serve to degrade and convert complex polysaccharides into a variety of nutrients for their hosts. Isolated decades ago from the bovine rumen, ruminococci have since been cultured from other ruminant and non-ruminant sources, and next-generation sequencing has further shown their distribution to be widespread in a diversity of animal hosts. While most ruminococci that have been studied are those capable of degrading cellulose, much less is known about non-cellulolytic, nonruminant-associated species, such as those found in humans. Furthermore, a mechanistic understanding of the role of Ruminococcus spp. in their respective hosts is still a work in progress. This review highlights the broad work done on species within the genus Ruminococcus with respect to their physiology, phylogenetic relatedness, and their potential impact on host health.
Microbial communities play critical roles in the gastrointestinal tracts (GIT) of preruminant calves by influencing performance and health. However, little is known about the establishment of microbial communities in the calf GIT or their dynamics during development. In this study, next-generation sequencing was used to assess changes in the bacterial communities of the rumen, jejunum, cecum, and colon in 26 crossbred calves at four developmental stages (7, 28, 49, and 63 days old). Alpha diversity differed among GIT regions with the lowest diversity and evenness in the jejunum, whereas no changes in alpha diversity were observed across developmental stage. Beta diversity analysis showed both region and age effects, with low numbers of operational taxonomic units (OTUs) shared between regions within a given age group or between ages in a given region. Taxonomic analysis revealed that several taxa coexisted in the rumen, jejunum, cecum, and colon but that their abundances differed considerably by GIT region and age. As calves aged, we observed lower abundances of taxa such as , , and with higher abundances of and in the rumen. The jejunum also displayed taxonomic changes with increases in and taxa in older calves. In the lower gut, taxa such as , , and decreased and S24-7, , and increased as calves aged. These data support a model whereby early and successive colonization by bacteria occurs across the GIT of calves and provides insights into the temporal dynamics of the GIT microbiota of dairy calves during preweaning development. The gastrointestinal tracts (GIT) of ruminants, such as dairy cows, house complex microbial communities that contribute to their overall health and support their ability to produce milk. For example, the rumen microbiota converts feed into usable nutrients, while the jejunal microbiota provides access to protein. Thus, establishing a properly functioning GIT microbiota in dairy calves is critical to their productivity as adult cows. However, little is known about the establishment, maintenance, and dynamics of the calf GIT microbiota in early life. In this study, we evaluated the bacterial communities in the rumen, jejunum, cecum, and colon in dairy calves across preweaning development and show that they are highly variable early on in life before transitioning to a stable community. Understanding the dairy calf GIT microbiota has implications for ensuring proper health during early life and will aid in efforts to develop strategies for improving downstream production.
Mammalian herbivores have developed numerous adaptations to utilize their plant-based diets including a modified gastrointestinal tract (GIT) and symbiosis with a GIT microbiota that plays a major role in digestion and the maintenance of host health. The red panda () is a herbivorous carnivore that lacks the specialized GIT common to other herbivores but still relies on microorganisms for survival on its almost entirely bamboo diet. The GIT microbiota is of further importance in young red pandas, as high cub mortality is problematic and has been attributed to failure to meet nutritional requirements. To gain insight into the establishment of the GIT microbiota of red pandas, we examined microbial communities in two individuals following dietary changes associated with weaning using next-generation 16S rRNA Illumina MiSeq paired-end sequencing of faecal samples. Across all four stages (pre-weaning, during weaning, post-weaning and adult), the GIT microbial community displayed low diversity and was dominated by bacteria in the phylum Firmicutes with lesser contributions from the Proteobacteria. A core community was found consistently across all weaning stages and included species within the taxa , and an unclassified Clostridiaceae. Analysis of the overall community composition and structure showed that although the GIT microbiota is established early in red pandas, dietary changes during weaning further shape the community and are correlated with the presence of new bacterial species. This work is the first analysis of the GIT microbiota for red panda cubs during weaning and provides a framework for understanding how diet and host microbiota impact the development of these threatened animals.
This experiment aimed to determine the effects of camelina seed (CS) supplementation at different dietary fat levels on ruminal bacterial community composition and how it relates to changes in ruminal fermentation in a dual-flow continuous culture system. Diets were randomly assigned to 8 fermenters (1,200-1,250 mL) in a 2 × 2 factorial arrangement of treatments in a replicated 4 × 4 Latin square with four 10-day experimental periods that consisted of 7 days for diet adaptation and 3 days for sample collection. Treatments were: (1) no CS at 5% ether extract (EE, NCS5); (2) no CS at 8% EE (NCS8); (3) 7.7% CS at 5% EE (CS5); and (4) 17.7% CS at 8% EE (CS8). Megalac was used as a control to adjust EE levels. Diets contained 55% orchardgrass hay and 45% concentrate, and fermenters were equally fed a total of 72 g/day (DM basis) twice daily. The bacterial community was determined by sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform. Sequencing data were analyzed using mothur and statistical analyses were performed in R and SAS. The most abundant phyla across treatments were the and , accounting for 49 and 39% of the total sequences, respectively. The bacterial community composition in both liquid and solid fractions of the effluent digesta changed with CS supplementation but not by dietary EE. Including CS in the diets decreased the relative abundances of spp., spp., and spp. The most abundant genus across treatments, , was reduced by high dietary EE levels, while and were increased by CS supplementation in the liquid fraction. Correlatively, the concentration of acetate was decreased while propionate increased; C18:0 was decreased and polyunsaturated fatty acids, especially C18:2 n-6 and C18:3 n-3, were increased by CS supplementation. Based on the correlation analysis between genera and fermentation end products, this study revealed that CS supplementation could be energetically beneficial to dairy cows by increasing propionate-producing bacteria and suppressing ruminal bacteria associated with biohydrogenation. However, attention should be given to avoid the effects of CS supplementation on suppressing cellulolytic bacteria.
The evaluation of how the gut microbiota affects both methane emissions and animal production is necessary in order to achieve methane mitigation without production losses. Toward this goal, the aim of this study was to correlate the rumen microbial communities (bacteria, archaea, and fungi) of high (HP), medium (MP), and low milk producing (LP), as well as dry (DC), Holstein dairy cows in an actual tropical production system with methane emissions and animal production traits. Overall, DC cows emitted more methane, followed by MP, HP and LP cows, although HP and LP cow emissions were similar. Using next-generation sequencing, it was found that bacteria affiliated with Christensenellaceae, Mogibacteriaceae, S24-7, Butyrivibrio, Schwartzia, and Treponema were negatively correlated with methane emissions and showed positive correlations with digestible dry matter intake (dDMI) and digestible organic matter intake (dOMI). Similar findings were observed for archaea in the genus Methanosphaera. The bacterial groups Coriobacteriaceae, RFP12, and Clostridium were negatively correlated with methane, but did not correlate with dDMI and dOMI. For anaerobic fungal communities, no significant correlations with methane or animal production traits were found. Based on these findings, it is suggested that manipulation of the abundances of these microbial taxa may be useful for modulating methane emissions without negatively affecting animal production.
At birth, calves display an underdeveloped rumen that eventually matures into a fully functional rumen as a result of solid food intake and microbial activity. However, little is known regarding the gradual impact of pre-weaning diet on the establishment of the rumen microbiota. Here, we employed next-generation sequencing to investigate the effects of the inclusion of starter concentrate (M: milk-fed vs. MC: milk plus starter concentrate fed) on archaeal, bacterial and anaerobic fungal communities in the rumens of 45 crossbred dairy calves across pre-weaning development (7, 28, 49, and 63 days). Our results show that archaeal, bacterial, and fungal taxa commonly found in the mature rumen were already established in the rumens of calves at 7 days old, regardless of diet. This confirms that microbiota colonization occurs in the absence of solid substrate. However, diet did significantly impact some microbial taxa. In the bacterial community, feeding starter concentrate promoted greater diversity of bacterial taxa known to degrade readily fermentable carbohydrates in the rumen (e.g., , and ). Shifts in the ruminal bacterial community also correlated to changes in fermentation patterns that favored the colonization of sp. A4 in the rumen of MC calves. In contrast, M calves displayed a bacterial community dominated by taxa able to utilize milk nutrients (e.g., , and ). In both diet groups, the dominance of these milk-associated taxa decreased with age, suggesting that diet and age simultaneously drive changes in the structure and abundance of bacterial communities in the developing rumen. Changes in the composition and abundance of archaeal communities were attributed exclusively to diet, with more highly abundant and less abundant in MC calves. Finally, the fungal community was dominated by members of the genus SK3 and . Relative anaerobic fungal abundances did not change significantly in response to diet or age, likely due to high inter-animal variation and the low fiber content of starter concentrate. This study provides new insights into the colonization of archaea, bacteria, and anaerobic fungi communities in pre-ruminant calves that may be useful in designing strategies to promote colonization of target communities to improve functional development.
The genus Fibrobacter contains cellulolytic bacteria originally isolated from the rumen. Culture-independent investigations have since identified Fibrobacter populations in the gastrointestinal tracts of numerous hindgut-fermenting herbivores, but their physiology is poorly characterized due to few representative axenic cultures. To test the hypothesis that novel Fibrobacter diversity exists in hindgut fermenters, we performed culturing and 16S rRNA gene amplicon sequencing on samples collected from phylogenetically diverse herbivorous hosts. Using a unique approach for recovering axenic Fibrobacter cultures, we isolated 45 novel strains from 11 different hosts. Full-length 16S rRNA gene sequencing of these isolates identified nine discrete phylotypes (cutoff = 0.03%) among them, including several that were only isolated from hindgut-fermenting hosts, and four previously unrepresented by axenic cultures. Our phylogenetic analysis indicated that six of the phylotypes are more closely related to previously described subspecies of Fibrobacter succinogenes, while the remaining three were more closely related to F. intestinalis. Culture-independent bacterial community profiling confirmed that most isolates were representative of numerically dominant phylotypes in their respective samples and strengthened the association of certain phylotypes with either ruminants or hindgut-fermenters. Despite considerable phylogenetic diversity observed among the Fibrobacter strains isolated here, phenotypic characterization suggests a conserved specialization for growth on cellulose.
The objectives of this study were to determine if milk production efficiency (MPE) is altered by near-total exchange of ruminal contents between high- (HE) and low-MPE (LE) cows and to characterize ruminal bacterial community composition (BCC) before exchange and over time postexchange. Three pairs of ruminally cannulated, third-lactation cows were selected whose MPE (energy-corrected milk per unit of dry matter intake) differed over their first 2 lactations. Approximately 95% of ruminal contents were exchanged between cows within each pair. Ruminal pH and volatile fatty acid (VFA) profiles, along with BCC (characterized by sequencing of the variable 4 region of 16S rRNA genes), were assessed just before feeding on d -8, -7, -5, -4, -1, 1, 2, 3, 7, 10, 14, 21, 28, 35, 42, and 56, relative to the exchange date. High-MPE cows had higher total ruminal VFA concentrations, higher molar percentages of propionate and valerate, and lower molar percentages of acetate and butyrate than did LE cows, and re-established these differences 1 d after contents exchange. Across all LE cows, MPE increased during 7 d postexchange but declined thereafter. Two of the 3 HE cows displayed decreases in MPE following introduction of the ruminal contents from the corresponding LE cow, but MPE increased in the third HE cow, which was determined to be an outlier. For all 6 cows, both liquid- and solids-associated BCC differed between individuals within a pair before contents exchange. Upon exchange, BCC of both phases in all 3 pairs was more similar to that of the donor inoculum than to preexchange host BCC. For 5 of 6 cows, the solids-associated community returned within 10 d to more resemble the preexchange community of that host than that of the donor community. Individual variability before the exchange was greater in liquids than in solids, as was the variability in response of bacterial communities to the exchange. Individual cows varied in their response, but generally moved toward re-establishment of their preexchange communities by 10 d after contents exchange. By contrast, ruminal pH and VFA profiles returned to preexchange levels within 1 d. Despite the small number of cows studied, the data suggest an apparent role for the ruminal bacterial community as a determinant of MPE.
It has become increasingly clear that the composition of mammalian gut microbial communities is substantially diet driven. These microbiota form intricate mutualisms with their hosts, which have profound implications on overall health. For example, many gut microbes are involved in the conversion of host-ingested dietary polysaccharides into host-usable nutrients. One group of important gut microbial symbionts are bacteria in the genus Ruminococcus. Originally isolated from the bovine rumen, ruminococci have been found in numerous mammalian hosts, including other ruminants, and non-ruminants such as horses, pigs and humans. All ruminococci require fermentable carbohydrates for growth, and their substrate preferences appear to be based on the diet of their particular host. Most ruminococci that have been studied are those capable of degrading cellulose, much less is known about non-cellulolytic non-ruminant-associated species, and even less is known about the environmental distribution of ruminococci as a whole. Here, we capitalized on the wealth of publicly available 16S rRNA gene sequences, genomes and large-scale microbiota studies to both resolve the phylogenetic placement of described species in the genus Ruminococcus, and further demonstrate that this genus has largely unexplored diversity and a staggering host distribution. We present evidence that ruminococci are predominantly associated with herbivores and omnivores, and our data supports the hypothesis that very few ruminococci are found consistently in non-host-associated environments. This study not only helps to resolve the phylogeny of this important genus, but also provides a framework for understanding its distribution in natural systems.
Development of the dairy calf gastrointestinal tract (GIT) and its associated microbiota are essential for survival and milk production, as this community is responsible for converting plant-based feeds into accessible nutrients. However, little is known regarding the establishment of microbes in the calf GIT. Here, we measured fecal-associated bacterial, archaeal, and fungal communities of dairy cows from 2 weeks to the middle of first lactation (>2 years) as well as rumen-associated communities from weaning (8 weeks) to first lactation. These communities were then correlated to animal growth and health. Although succession of specific operational taxonomic units (OTUs) was unique to each animal, beta-diversity decreased while alpha-diversity increased as animals aged. Calves exhibited similar microbial families and genera but different OTUs than adults, with a transition to an adult-like microbiota between weaning and 1 year of age. This suggests that alterations of the microbiota for improving downstream milk production may be most effective during, or immediately following, the weaning transition.
Prolonged diffuse laryngeal inflammation from smoking and/or reflux is commonly diagnosed as chronic laryngitis and treated empirically with expensive drugs that have not proven effective. Shifts in microbiota have been associated with many inflammatory diseases, though little is known about how resident microbes may contribute to chronic laryngitis. We sought to characterize the core microbiota of disease-free human laryngeal tissue and to investigate shifts in microbial community membership associated with exposure to cigarette smoke and reflux. Using 454 pyrosequencing of the 16S rRNA gene, we compared bacterial communities of laryngeal tissue biopsies collected from 97 non-treatment-seeking volunteers based on reflux and smoking status. The core community was characterized by a highly abundant OTU within the family Comamonadaceae found in all laryngeal tissues. Smokers demonstrated less microbial diversity than nonsmokers, with differences in relative abundances of OTUs classified as Streptococcus, unclassified Comamonadaceae, Cloacibacterium, and Helicobacter. Reflux status did not affect microbial diversity nor community structure nor composition. Comparison of healthy laryngeal microbial communities to benign vocal fold disease samples revealed greater abundance of Streptococcus in benign vocal fold disease suggesting that mucosal dominance by Streptococcus may be a factor in disease etiology.
Dietary shifts can result in changes to the gastrointestinal tract (GIT) microbiota, leading to negative outcomes for the host, including inflammation. Giant pandas (Ailuropoda melanoleuca) are physiologically classified as carnivores; however, they consume an herbivorous diet with dramatic seasonal dietary shifts and episodes of chronic GIT distress with symptoms including abdominal pain, loss of appetite and the excretion of mucous stools (mucoids). These episodes adversely affect the overall nutritional and health status of giant pandas. Here, we examined the fecal microbiota of two giant pandas' non-mucoid and mucoid stools and compared these to samples from a previous winter season that had historically few mucoid episodes. To identify the microbiota present, we isolated and sequenced the 16S rRNA using next-generation sequencing. Mucoids occurred following a seasonal feeding switch from predominately bamboo culm (stalk) to leaves. All fecal samples displayed low diversity and were dominated by bacteria in the phyla Firmicutes and to a lesser extent, Proteobacteria. Fecal samples immediately prior to mucoid episodes had lower microbial diversity as compared to mucoids. Mucoids were mostly comprised of common mucosal-associated taxa including Streptococcus and Leuconostoc species, and exhibited increased abundance for bacteria in the family Pasteurellaceae. Taken together, these findings indicate that mucoids may represent an expulsion of the mucosal lining that is driven by changes in diet. We suggest that these occurrences serve to reset their GIT microbiota following changes in bamboo part preference, as giant pandas have retained a carnivorous GIT anatomy while shifting to an herbivorous diet.
Many microorganisms with specialized lifestyles have reduced genomes. This is best understood in beneficial bacterial symbioses, where partner fidelity facilitates loss of genes necessary for living independently. Specialized microbial pathogens may also exhibit gene loss relative to generalists. Here, we demonstrate that Escovopsis weberi, a fungal parasite of the crops of fungus-growing ants, has a reduced genome in terms of both size and gene content relative to closely related but less specialized fungi. Although primary metabolism genes have been retained, the E. weberi genome is depleted in carbohydrate active enzymes, which is consistent with reliance on a host with these functions. E. weberi has also lost genes considered necessary for sexual reproduction. Contrasting these losses, the genome encodes unique secondary metabolite biosynthesis clusters, some of which include genes that exhibit up-regulated expression during host attack. Thus, the specialized nature of the interaction between Escovopsis and ant agriculture is reflected in the parasite's genome.
The first report of the effect of hibernation on the gut microbiota of bears reveals trends both similar and distinct from those found in small hibernators. A model mouse system also suggested possible roles of the microbiota for healthy weight gain and insulin tolerance in bears during their active season.
The purpose of this study was to compare the effects of feeding virginiamycin or bacitracin methylene disalicylate (BMD), two in-feed antibiotics typically used by commercial poultry producers in the United States, on the chicken gastrointestinal microbiota. 454 pyrosequencing of the V6-V8 region of the 16S rRNA gene and quantitative PCR were employed to examine the bacterial microbiota and Clostridium perfringens, respectively, in the jejunum and caecum of market-age broiler chickens over four replicate grow-outs. Our results suggest that virginiamycin has a more pronounced impact on broiler gastrointestinal tract bacterial communities, relative to BMD, manifested primarily through significant enrichments in the genus Faecalibacterium in the caecum and a distinct population of Lactobacillus, OTU_02, in both the jejunum and caecum. No evidence for a difference among the diets in Cl. perfringens levels in the jejunum or caecum was observed. This work represents the highest resolution comparison to date of the jejunum and caecum microbiota in broilers fed either virginiamycin or BMD, and provides evidence for specific bacterial OTUs potentially involved in the health and performance benefits typically attributed to these in-feed antibiotics.
Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further clarify the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.
Symbiotic microbial communities are critical to the function and survival of animals. This relationship is obligatory for herbivores that engage gut microorganisms for the conversion of dietary plant materials into nutrients such as short-chain organic acids (SCOAs). The constraint on body size imposed by their arboreal lifestyle is thought to make this symbiosis especially important for sloths. Here, we use next-generation sequencing to identify the bacteria present in the fore and distal guts of wild two- and three-toed sloths, and correlate these communities with both diet and SCOAs. We show that, unlike other mammalian herbivores, sloth gut communities are dominated by the bacterial phyla Proteobacteria and Firmicutes. Specifically, three-toed sloths possess a highly conserved, low-diversity foregut community with a highly abundant Neisseria species associated with foregut lactate. In contrast, two-toed sloths have a more variable and diverse foregut microbiota correlated with a variety of SCOAs. These differences support the hypothesis that feeding behaviour selects for specific gut bacterial communities, as three-toed sloths subsist primarily on Cecropia tree leaves while two-toed sloths have a more generalist diet. The less diverse diet and gut microbiota of three-toed sloths may render them more susceptible to habitat loss and other diet-altering conditions.
All organisms must synthesize the enzymatic cofactor coenzyme A (CoA) from the precursor pantothenate. Most bacteria can synthesize pantothenate de novo by the condensation of pantoate and β-alanine. The synthesis of β-alanine is catalyzed by L-aspartate-α-decarboxylase (PanD), a pyruvoyl enzyme that is initially synthesized as a zymogen (pro-PanD). Active PanD is generated by self-cleavage of pro-PanD at Gly24-Ser25 creating the active-site pyruvoyl moiety. In Salmonella enterica, this cleavage requires PanM, an acetyl-CoA sensor related to the Gcn5-like N-acetyltransferases. PanM does not acetylate pro-PanD, but the recent publication of the three-dimensional crystal structure of the PanM homologue PanZ in complex with the PanD zymogen of Escherichia coli provides validation to our predictions and provides a framework in which to further examine the cleavage mechanism. In contrast, PanD from bacteria lacking PanM efficiently cleaved in the absence of PanM in vivo. Using phylogenetic analyses combined with in vivo phenotypic investigations, we showed that two classes of bacterial L-aspartate-α-decarboxylases exist. This classification is based on their posttranslational activation by self-cleavage of its zymogen. Class I L-aspartate-α-decarboxylase zymogens require the acetyl-CoA sensor PanM to be cleaved into active PanD. This class is found exclusively in the Gammaproteobacteria. Class II L-aspartate-α-decarboxylase zymogens self cleave efficiently in the absence of PanM, and are found in a wide number of bacterial phyla. Several members of the Euryarchaeota and Crenarchaeota also contain Class II L-aspartate-α-decarboxylases. Phylogenetic and amino acid conservation analyses of PanM revealed a conserved region of PanM distinct from conserved regions found in related Gcn5-related acetyltransferase enzymes (Pfam00583). This conserved region represents a putative domain for interactions with L-aspartate-α-decarboxylase zymogens. This work may inform future biochemical and structural studies of pro-PanD-PanM interactions. Experimental results indicate that S. enterica and C. glutamicum L-aspartate-α-decarboxylases represent two different classes of homologues of these enzymes. Class I homologues require PanM for activation, while Class II self cleave in the absence of PanM. Computer modeling of conserved amino acids using structure coordinates of PanM and L-aspartate-α-decarboxylase available in the protein data bank (RCSB PDB) revealed a putative site of interactions, which may help generate models to help understand the molecular details of the self-cleavage mechanism of L-aspartate-α-decarboxylases.
Fourteen Holstein cows of similar ages were monitored through their first two lactation cycles, during which ruminal solids and liquids, milk samples, production data, and feed consumption data were collected for each cow during early (76 to 82 days in milk [DIM]), middle (151 to 157 DIM), and late (251 to 257 DIM) lactation periods. The bacterial community of each ruminal sample was determined by sequencing the region from V6 to V8 of the 16S rRNA gene using 454 pyrosequencing. Gross feed efficiency (GFE) for each cow was calculated by dividing her energy-corrected milk by dry matter intake (ECM/DMI) for each period of both lactation cycles. Four pairs of cows were identified that differed in milk production efficiency, as defined by residual feed intake (RFI), at the same level of ECM production. The most abundant phyla detected for all cows were Bacteroidetes (49.42%), Firmicutes (39.32%), Proteobacteria (5.67%), and Tenericutes (2.17%), and the most abundant genera included Prevotella (40.15%), Butyrivibrio (2.38%), Ruminococcus (2.35%), Coprococcus (2.29%), and Succiniclasticum (2.28%). The bacterial microbiota between the first and second lactation cycles were highly similar, but with a significant correlation between total community composition by ruminal phase and specific bacteria whose relative sequence abundances displayed significant positive or negative correlation with GFE or RFI. These data suggest that the ruminal bacterial community is dynamic in terms of membership and diversity and that specific members are associated with high and low milk production efficiency over two lactation cycles.
Bacteria in the genus Ruminococcus are ubiquitous members of the mammalian gastrointestinal tract. In particular, they are important in ruminants where they digest a wide range of plant cell wall polysaccharides. For example, Ruminococcus albus 7 is a primary cellulose degrader that produces acetate usable by its bovine host. Moreover, it is one of the few organisms that ferments cellulose to form ethanol at mesophilic temperatures in vitro. The mechanism of cellulose degradation by R. albus 7 is not well-defined and is thought to involve pilin-like proteins, unique carbohydrate-binding domains, a glycocalyx, and cellulosomes. Here, we used a combination of comparative genomics, fermentation analyses, and transcriptomics to further clarify the cellulolytic and fermentative potential of R. albus 7. A comparison of the R. albus 7 genome sequence against the genome sequences of related bacteria that either encode or do not encode cellulosomes revealed that R. albus 7 does not encode for most canonical cellulosomal components. Fermentation analysis of R. albus 7 revealed the ability to produce ethanol and acetate on a wide range of fibrous substrates in vitro. Global transcriptomic analysis of R. albus 7 grown at identical dilution rates on cellulose and cellobiose in a chemostat showed that this bacterium, when growing on cellulose, utilizes a carbohydrate-degrading strategy that involves increased transcription of the rare carbohydrate-binding module (CBM) family 37 domain and the tryptophan biosynthetic operon. Our data suggest that R. albus 7 does not use canonical cellulosomal components to degrade cellulose, but rather up-regulates the expression of CBM37-containing enzymes and tryptophan biosynthesis. This study contributes to a revised model of carbohydrate degradation by this key member of the rumen ecosystem.
The rich and diverse microbiota of the rumen provides ruminant animals the capacity to utilize highly fibrous feedstuffs as their energy source, but there is surprisingly little information on the composition of the microbiome of ruminants fed all-forage diets, despite the importance of such agricultural production systems worldwide. In three 28-day periods, three ruminally-cannulated Holstein heifers sequentially grazed orchardgrass pasture (OP), then were fed orchardgrass hay (OH), then returned to OP. These heifers displayed greater shifts in ruminal bacterial community composition (determined by automated ribosomal intergenic spacer analysis and by pyrotag sequencing of 16S rRNA genes) than did two other heifers maintained 84 d on the same OP. Phyla Firmicutes and Bacteroidetes dominated all ruminal samples, and quantitative PCR indicated that members of the genus Prevotella averaged 23% of the 16S rRNA gene copies, well below levels previously reported with cows fed total mixed rations. Differences in bacterial community composition and ruminal volatile fatty acid (VFA) profiles were observed between the OP and OH despite similarities in gross chemical composition. Compared to OP, feeding OH increased the molar proportion of ruminal acetate (P = 0.02) and decreased the proportion of ruminal butyrate (P < 0.01), branched-chain VFA (P < 0.01) and the relative population size of the abundant genus Butyrivibrio (P < 0.001), as determined by pyrotag sequencing. Despite the low numbers of animals examined, the observed changes in VFA profile in the rumens of heifers on OP vs. OH are consistent with the shifts in Butyrivibrio abundance and its known physiology as a butyrate producer that ferments both carbohydrates and proteins.
The ability to cultivate food is an innovation that has produced some of the most successful ecological strategies on the planet. Although most well recognized in humans, where agriculture represents a defining feature of civilization, species of ants, beetles, and termites have also independently evolved symbioses with fungi that they cultivate for food. Despite occurring across divergent insect and fungal lineages, the fungivorous niches of these insects are remarkably similar, indicating convergent evolution toward this successful ecological strategy. Here, we characterize the microbiota of ants, beetles, and termites engaged in nutritional symbioses with fungi to define the bacterial groups associated with these prominent herbivores and forest pests. Using culture-independent techniques and the in silico reconstruction of 37 composite genomes of dominant community members, we demonstrate that different insect-fungal symbioses that collectively shape ecosystems worldwide have highly similar bacterial microbiotas comprised primarily of the genera Enterobacter, Rahnella, and Pseudomonas. Although these symbioses span three orders of insects and two phyla of fungi, we show that they are associated with bacteria sharing high whole-genome nucleotide identity. Due to the fine-scale correspondence of the bacterial microbiotas of insects engaged in fungal symbioses, our findings indicate that this represents an example of convergence of entire host-microbe complexes. The cultivation of fungi for food is a behavior that has evolved independently in ants, beetles, and termites and has enabled many species of these insects to become ecologically important and widely distributed herbivores and forest pests. Although the primary fungal cultivars of these insects have been studied for decades, comparatively little is known of their bacterial microbiota. In this study, we show that diverse fungus-growing insects are associated with a common bacterial community composed of the same dominant members. Furthermore, by demonstrating that many of these bacteria have high whole-genome similarity across distantly related insect hosts that reside thousands of miles apart, we show that these bacteria are an important and underappreciated feature of diverse fungus-growing insects. Because of the similarities in the agricultural lifestyles of these insects, this is an example of convergence between both the life histories of the host insects and their symbiotic microbiota.
The gut microbiota plays important roles in animal nutrition and health. This relationship is particularly dynamic in hibernating mammals where fasting drives the gut community to rely on host-derived nutrients instead of exogenous substrates. We used 16S rRNA pyrosequencing and caecal tissue protein analysis to investigate the effects of hibernation on the mucosa-associated bacterial microbiota and host responses in 13-lined ground squirrels. The mucosal microbiota was less diverse in winter hibernators than in actively feeding spring and summer squirrels. UniFrac analysis revealed distinct summer and late winter microbiota clusters, while spring and early winter clusters overlapped slightly, consistent with their transitional structures. Communities in all seasons were dominated by Firmicutes and Bacteroidetes, with lesser contributions from Proteobacteria, Verrucomicrobia, Tenericutes and Actinobacteria. Hibernators had lower relative abundances of Firmicutes, which include genera that prefer plant polysaccharides, and higher abundances of Bacteroidetes and Verrucomicrobia, some of which can survive solely on host-derived mucins. A core mucosal assemblage of nine operational taxonomic units shared among all individuals was identified with an average total sequence abundance of 60.2%. This core community, together with moderate shifts in specific taxa, indicates that the mucosal microbiota remains relatively stable over the annual cycle yet responds to substrate changes while potentially serving as a pool for 'seeding' the microbiota once exogenous substrates return in spring. Relative to summer, hibernation reduced caecal crypt length and increased MUC2 expression in early winter and spring. Hibernation also decreased caecal TLR4 and increased TLR5 expression, suggesting a protective response that minimizes inflammation.
To measure the impact of supplementing a forage diet with tree-based browse on the ruminal bacterial communities of Nigerian West African Dwarf (WAD) sheep. Fifteen WAD sheep were fed a control diet of forage (Panicum maximum), with 12 animals shifted in groups of three to one of four browse-supplemented diets (Albizia saman, Bridelia micrantha, Ficus sur, or Gmelina arborea). These browse plants were shown in a concurrent but separate study to be reasonably nutritious (based on chemical composition and fibre constituents) and nontoxic (based on tannin, phytate, saponin, alkaloid and oxalate levels). Rumen liquids and solids for DNA extraction were collected via intubation from two animals in each group before and after dietary shift. Bacterial 16S rRNA gene regions V6-V8 were sequenced by 454 pyrosequencing. All communities were highly diverse and dominated by the phyla Firmicutes, Bacteroidetes, Tenericutes, Actinobacteria and Proteobacteria. All communities shared members of the genera Butryivibrio, Prevotella and Ruminococcus. Our analysis defined a core sets of bacteria shared by all animals, forage-fed animals and browse-fed animals. Community structure shifted dramatically in animals fed A. saman or G. arborea. The impact of tree-based browse on the ruminal bacterial community of Nigerian WAD sheep varies by browse species, likely due to differences in browse composition. Our study describes the first neotropical small ruminant bacterial microbiome and supports diet supplementation with specific tree-based browse for WAD sheep.
Members of the phylum Fibrobacteres are highly efficient cellulolytic bacteria, best known for their role in rumen function and as potential sources of novel enzymes for bioenergy applications. Despite being key members of ruminants and other digestive microbial communities, our knowledge of this phylum remains incomplete, as much of our understanding is focused on two recognized species, Fibrobacter succinogenes and F. intestinalis. As a result, we lack insights regarding the environmental niche, host range, and phylogenetic organization of this phylum. Here, we analyzed over 1000 16S rRNA Fibrobacteres sequences available from public databases to establish a phylogenetic framework for this phylum. We identify both species- and genus-level clades that are suggestive of previously unknown taxonomic relationships between Fibrobacteres in addition to their putative lifestyles as host-associated or free-living. Our results shed light on this poorly understood phylum and will be useful for elucidating the function, distribution, and diversity of these bacteria in their niches.
Genomes of eusocial insects code for dramatic examples of phenotypic plasticity and social organization. We compared the genomes of seven ants, the honeybee, and various solitary insects to examine whether eusocial lineages share distinct features of genomic organization. Each ant lineage contains ∼4000 novel genes, but only 64 of these genes are conserved among all seven ants. Many gene families have been expanded in ants, notably those involved in chemical communication (e.g., desaturases and odorant receptors). Alignment of the ant genomes revealed reduced purifying selection compared with Drosophila without significantly reduced synteny. Correspondingly, ant genomes exhibit dramatic divergence of noncoding regulatory elements; however, extant conserved regions are enriched for novel noncoding RNAs and transcription factor-binding sites. Comparison of orthologous gene promoters between eusocial and solitary species revealed significant regulatory evolution in both cis (e.g., Creb) and trans (e.g., fork head) for nearly 2000 genes, many of which exhibit phenotypic plasticity. Our results emphasize that genomic changes can occur remarkably fast in ants, because two recently diverged leaf-cutter ant species exhibit faster accumulation of species-specific genes and greater divergence in regulatory elements compared with other ants or Drosophila. Thus, while the "socio-genomes" of ants and the honeybee are broadly characterized by a pervasive pattern of divergence in gene composition and regulation, they preserve lineage-specific regulatory features linked to eusociality. We propose that changes in gene regulation played a key role in the origins of insect eusociality, whereas changes in gene composition were more relevant for lineage-specific eusocial adaptations.
The gastrointestinal tracts (GIT) of herbivores harbor dense and diverse microbiota that has beneficial interactions with the host, particularly for agriculturally relevant animals like ruminants such as cattle. When assessing ruminant health, microbiological indicators are often derived from the rumen or feces. However, it is probable that ruminal and fecal microbiota do not reflect the microbial communities within the GIT of ruminants. To test this, we investigated the compartments of the GIT from a Brazilian Nelore steer and performed a 16S rRNA pyrosequencing analysis on the collected samples. Our results showed high intra-individual variation, with samples clustering according to their location in the GIT including the forestomach, small intestine, and large intestine. Although sequences related to the phyla Firmicutes and Bacteroidetes predominated all samples, there was a remarkable variation at the family level. Comparisons between the microbiota in the rumen, feces, and other GIT components showed distinct differences in microbial community. This work is the first intensive non-culture based GIT microbiota analysis for any ruminant and provides a framework for understanding how host microbiota impact the health of bovines.
The mountain pine beetle, Dendroctonus ponderosae, is a subcortical herbivore native to western North America that can kill healthy conifers by overcoming host tree defenses, which consist largely of high terpene concentrations. The mechanisms by which these beetles contend with toxic compounds are not well understood. Here, we explore a component of the hypothesis that beetle-associated bacterial symbionts contribute to the ability of D. ponderosae to overcome tree defenses by assisting with terpene detoxification. Such symbionts may facilitate host tree transitions during range expansions currently being driven by climate change. For example, this insect has recently breached the historical geophysical barrier of the Canadian Rocky Mountains, providing access to näive tree hosts and unprecedented connectivity to eastern forests. We use culture-independent techniques to describe the bacterial community associated with D. ponderosae beetles and their galleries from their historical host, Pinus contorta, and their more recent host, hybrid P. contorta-Pinus banksiana. We show that these communities are enriched with genes involved in terpene degradation compared with other plant biomass-processing microbial communities. These pine beetle microbial communities are dominated by members of the genera Pseudomonas, Rahnella, Serratia, and Burkholderia, and the majority of genes involved in terpene degradation belong to these genera. Our work provides the first metagenome of bacterial communities associated with a bark beetle and is consistent with a potential microbial contribution to detoxification of tree defenses needed to survive the subcortical environment.
Sphingomonads comprise a physiologically versatile group within the Alphaproteobacteria that includes strains of interest for biotechnology, human health, and environmental nutrient cycling. In this study, we compared 26 sphingomonad genome sequences to gain insight into their ecology, metabolic versatility, and environmental adaptations. Our multilocus phylogenetic and average amino acid identity (AAI) analyses confirm that Sphingomonas, Sphingobium, Sphingopyxis, and Novosphingobium are well-resolved monophyletic groups with the exception of Sphingomonas sp. strain SKA58, which we propose belongs to the genus Sphingobium. Our pan-genomic analysis of sphingomonads reveals numerous species-specific open reading frames (ORFs) but few signatures of genus-specific cores. The organization and coding potential of the sphingomonad genomes appear to be highly variable, and plasmid-mediated gene transfer and chromosome-plasmid recombination, together with prophage- and transposon-mediated rearrangements, appear to play prominent roles in the genome evolution of this group. We find that many of the sphingomonad genomes encode numerous oxygenases and glycoside hydrolases, which are likely responsible for their ability to degrade various recalcitrant aromatic compounds and polysaccharides, respectively. Many of these enzymes are encoded on megaplasmids, suggesting that they may be readily transferred between species. We also identified enzymes putatively used for the catabolism of sulfonate and nitroaromatic compounds in many of the genomes, suggesting that plant-based compounds or chemical contaminants may be sources of nitrogen and sulfur. Many of these sphingomonads appear to be adapted to oligotrophic environments, but several contain genomic features indicative of host associations. Our work provides a basis for understanding the ecological strategies employed by sphingomonads and their role in environmental nutrient cycling.
Plants represent a large reservoir of organic carbon comprised primarily of recalcitrant polymers that most metazoans are unable to deconstruct. Many herbivores gain access to nutrients in this material indirectly by associating with microbial symbionts, and leaf-cutter ants are a paradigmatic example. These ants use fresh foliar biomass as manure to cultivate gardens composed primarily of Leucoagaricus gongylophorus, a basidiomycetous fungus that produces specialized hyphal swellings that serve as a food source for the host ant colony. Although leaf-cutter ants are conspicuous herbivores that contribute substantially to carbon turnover in Neotropical ecosystems, the process through which plant biomass is degraded in their fungus gardens is not well understood. Here we present the first draft genome of L. gongylophorus, and, using genomic and metaproteomic tools, we investigate its role in lignocellulose degradation in the gardens of both Atta cephalotes and Acromyrmex echinatior leaf-cutter ants. We show that L. gongylophorus produces a diversity of lignocellulases in ant gardens and is likely the primary driver of plant biomass degradation in these ecosystems. We also show that this fungus produces distinct sets of lignocellulases throughout the different stages of biomass degradation, including numerous cellulases and laccases that likely play an important role in lignocellulose degradation. Our study provides a detailed analysis of plant biomass degradation in leaf-cutter ant fungus gardens and insight into the enzymes underlying the symbiosis between these dominant herbivores and their obligate fungal cultivar.
Actinobacteria in the genus Cellulomonas are the only known and reported cellulolytic facultative anaerobes. To better understand the cellulolytic strategy employed by these bacteria, we sequenced the genome of the Cellulomonas fimi ATCC 484(T). For comparative purposes, we also sequenced the genome of the aerobic cellulolytic "Cellvibrio gilvus" ATCC 13127(T). An initial analysis of these genomes using phylogenetic and whole-genome comparison revealed that "Cellvibrio gilvus" belongs to the genus Cellulomonas. We thus propose to assign "Cellvibrio gilvus" to the genus Cellulomonas. A comparative genomics analysis between these two Cellulomonas genome sequences and the recently completed genome for Cellulomonas flavigena ATCC 482(T) showed that these cellulomonads do not encode cellulosomes but appear to degrade cellulose by secreting multi-domain glycoside hydrolases. Despite the minimal number of carbohydrate-active enzymes encoded by these genomes, as compared to other known cellulolytic organisms, these bacteria were found to be proficient at degrading and utilizing a diverse set of carbohydrates, including crystalline cellulose. Moreover, they also encode for proteins required for the fermentation of hexose and xylose sugars into products such as ethanol. Finally, we found relatively few significant differences between the predicted carbohydrate-active enzymes encoded by these Cellulomonas genomes, in contrast to previous studies reporting differences in physiological approaches for carbohydrate degradation. Our sequencing and analysis of these genomes sheds light onto the mechanism through which these facultative anaerobes degrade cellulose, suggesting that the sequenced cellulomonads use secreted, multidomain enzymes to degrade cellulose in a way that is distinct from known anaerobic cellulolytic strategies.
Herbivores gain access to nutrients stored in plant biomass largely by harnessing the metabolic activities of microbes. Leaf-cutter ants of the genus Atta are a hallmark example; these dominant neotropical herbivores cultivate symbiotic fungus gardens on large quantities of fresh plant forage. As the external digestive system of the ants, fungus gardens facilitate the production and sustenance of millions of workers. Using metagenomic and metaproteomic techniques, we characterize the bacterial diversity and physiological potential of fungus gardens from two species of Atta. Our analysis of over 1.2 Gbp of community metagenomic sequence and three 16S pyrotag libraries reveals that in addition to harboring the dominant fungal crop, these ecosystems contain abundant populations of Enterobacteriaceae, including the genera Enterobacter, Pantoea, Klebsiella, Citrobacter and Escherichia. We show that these bacterial communities possess genes associated with lignocellulose degradation and diverse biosynthetic pathways, suggesting that they play a role in nutrient cycling by converting the nitrogen-poor forage of the ants into B-vitamins, amino acids and other cellular components. Our metaproteomic analysis confirms that bacterial glycosyl hydrolases and proteins with putative biosynthetic functions are produced in both field-collected and laboratory-reared colonies. These results are consistent with the hypothesis that fungus gardens are specialized fungus-bacteria communities that convert plant material into energy for their ant hosts. Together with recent investigations into the microbial symbionts of vertebrates, our work underscores the importance of microbial communities in the ecology and evolution of herbivorous metazoans.
Epulopiscium sp. type B, a large intestinal bacterial symbiont of the surgeonfish Naso tonganus, does not reproduce by binary fission. Instead, it forms multiple intracellular offspring using a process with morphological features similar to the survival strategy of endospore formation in other Firmicutes. We hypothesize that intracellular offspring formation in Epulopiscium evolved from endospore formation and these two developmental programs share molecular mechanisms that are responsible for the observed morphological similarities. To test this, we sequenced the genome of Epulopiscium sp. type B to draft quality. Comparative analysis with the complete genome of its close, endospore-forming relative, Cellulosilyticum lentocellum, identified homologs of well-known sporulation genes characterized in Bacillus subtilis. Of the 147 highly conserved B. subtilis sporulation genes used in this analysis, we found 57 homologs in the Epulopiscium genome and 87 homologs in the C. lentocellum genome. Genes coding for components of the central regulatory network which govern the expression of forespore and mother-cell-specific sporulation genes and the machinery used for engulfment appear best conserved. Low conservation of genes expressed late in endospore formation, particularly those that confer resistance properties and encode germinant receptors, suggest that Epulopiscium has lost the ability to form a mature spore. Our findings provide a framework for understanding the evolution of a novel form of cellular reproduction.
Ants (Hymenoptera, Formicidae) represent one of the most successful eusocial taxa in terms of both their geographic distribution and species number. The publication of seven ant genomes within the past year was a quantum leap for socio- and ant genomics. The diversity of social organization in ants makes them excellent model organisms to study the evolution of social systems. Comparing the ant genomes with those of the honeybee, a lineage that evolved eusociality independently from ants, and solitary insects suggests that there are significant differences in key aspects of genome organization between social and solitary insects, as well as among ant species. Altogether, these seven ant genomes open exciting new research avenues and opportunities for understanding the genetic basis and regulation of social species, and adaptive complex systems in general.
Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.
Ruminococcus albus 7 is a highly cellulolytic ruminal bacterium that is a member of the phylum Firmicutes. Here, we describe the complete genome of this microbe. This genome will be useful for rumen microbiology and cellulosome biology and in biofuel production, as one of its major fermentation products is ethanol.
Sirex noctilio is an invasive wood-feeding wasp that threatens the world's commercial and natural pine forests. Successful tree colonization by this insect is contingent on the decline of host defenses and the ability to utilize the woody substrate as a source of energy. We explored its potential association with bacterial symbionts that may assist in nutrient acquisition via plant biomass deconstruction using growth assays, culture-dependent and -independent analysis of bacterial frequency of association and whole-genome analysis. We identified Streptomyces and γ-Proteobacteria that were each associated with 94% and 88% of wasps, respectively. Streptomyces isolates grew on all three cellulose substrates tested and across a range of pH 5.6 to 9. On the basis of whole-genome sequencing, three Streptomyces isolates have some of the highest proportions of genes predicted to encode for carbohydrate-active enzymes (CAZyme) of sequenced Actinobacteria. γ-Proteobacteria isolates grew on a cellulose derivative and a structurally diverse substrate, ammonia fiber explosion-treated corn stover, but not on microcrystalline cellulose. Analysis of the genome of a Pantoea isolate detected genes putatively encoding for CAZymes, the majority predicted to be active on hemicellulose and more simple sugars. We propose that a consortium of microorganisms, including the described bacteria and the fungal symbiont Amylostereum areolatum, has complementary functions for degrading woody substrates and that such degradation may assist in nutrient acquisition by S. noctilio, thus contributing to its ability to be established in forested habitats worldwide.
The signal transduction networks that initiate multicellular development in bacteria remain largely undefined. Here, we report that Myxococcus xanthus regulates entry into its multicellular developmental program using a novel strategy: a cascade of transcriptional activators known as enhancer binding proteins (EBPs). The EBPs in the cascade function in sequential stages of early development, and several lines of evidence indicate that the cascade is propagated when EBPs that function at one stage of development directly regulate transcription of an EBP gene important for the next developmental stage. We also show that the regulatory cascade is designed in a novel way that extensively expands on the typical use of EBPs: Instead of using only one EBP to regulate a particular gene or group of genes, which is the norm in other bacterial systems, the cascade uses multiple EBPs to regulate EBP genes that are positioned at key transition points in early development. Based on the locations of the putative EBP promoter binding sites, several different mechanisms of EBP coregulation are possible, including the formation of coregulating EBP transcriptional complexes. We propose that M. xanthus uses an EBP coregulation strategy to make expression of EBP genes that modulate stage-stage transitions responsive to multiple signal transduction pathways, which provide information that is important for a coordinated decision to advance the developmental process.
Streptomyces griseus strain XylebKG-1 is an insect-associated strain of the well-studied actinobacterial species S. griseus. Here, we present the genome of XylebKG-1 and discuss its similarity to the genome of S. griseus subsp. griseus NBRC13350. XylebKG-1 was isolated from the fungus-cultivating Xyleborinus saxesenii system. Given its similarity to free-living S. griseus subsp. griseus NBRC13350, comparative genomics will elucidate critical components of bacterial interactions with insects.
Cellulosilyticum lentocellum DSM 5427 is an anaerobic, endospore-forming member of the Firmicutes. We describe the complete genome sequence of this cellulose-degrading bacterium, which was originally isolated from estuarine sediment of a river that received both domestic and paper mill waste. Comparative genomics of cellulolytic clostridia will provide insight into factors that influence degradation rates.
We report the draft genome sequence of the red harvester ant, Pogonomyrmex barbatus. The genome was sequenced using 454 pyrosequencing, and the current assembly and annotation were completed in less than 1 y. Analyses of conserved gene groups (more than 1,200 manually annotated genes to date) suggest a high-quality assembly and annotation comparable to recently sequenced insect genomes using Sanger sequencing. The red harvester ant is a model for studying reproductive division of labor, phenotypic plasticity, and sociogenomics. Although the genome of P. barbatus is similar to other sequenced hymenopterans (Apis mellifera and Nasonia vitripennis) in GC content and compositional organization, and possesses a complete CpG methylation toolkit, its predicted genomic CpG content differs markedly from the other hymenopterans. Gene networks involved in generating key differences between the queen and worker castes (e.g., wings and ovaries) show signatures of increased methylation and suggest that ants and bees may have independently co-opted the same gene regulatory mechanisms for reproductive division of labor. Gene family expansions (e.g., 344 functional odorant receptors) and pseudogene accumulation in chemoreception and P450 genes compared with A. mellifera and N. vitripennis are consistent with major life-history changes during the adaptive radiation of Pogonomyrmex spp., perhaps in parallel with the development of the North American deserts.
Ants are some of the most abundant and familiar animals on Earth, and they play vital roles in most terrestrial ecosystems. Although all ants are eusocial, and display a variety of complex and fascinating behaviors, few genomic resources exist for them. Here, we report the draft genome sequence of a particularly widespread and well-studied species, the invasive Argentine ant (Linepithema humile), which was accomplished using a combination of 454 (Roche) and Illumina sequencing and community-based funding rather than federal grant support. Manual annotation of >1,000 genes from a variety of different gene families and functional classes reveals unique features of the Argentine ant's biology, as well as similarities to Apis mellifera and Nasonia vitripennis. Distinctive features of the Argentine ant genome include remarkable expansions of gustatory (116 genes) and odorant receptors (367 genes), an abundance of cytochrome P450 genes (>110), lineage-specific expansions of yellow/major royal jelly proteins and desaturases, and complete CpG DNA methylation and RNAi toolkits. The Argentine ant genome contains fewer immune genes than Drosophila and Tribolium, which may reflect the prominent role played by behavioral and chemical suppression of pathogens. Analysis of the ratio of observed to expected CpG nucleotides for genes in the reproductive development and apoptosis pathways suggests higher levels of methylation than in the genome overall. The resources provided by this genome sequence will offer an abundance of tools for researchers seeking to illuminate the fascinating biology of this emerging model organism.
Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs), carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation.
Leaf-cutter ants are one of the most important herbivorous insects in the Neotropics, harvesting vast quantities of fresh leaf material. The ants use leaves to cultivate a fungus that serves as the colony's primary food source. This obligate ant-fungus mutualism is one of the few occurrences of farming by non-humans and likely facilitated the formation of their massive colonies. Mature leaf-cutter ant colonies contain millions of workers ranging in size from small garden tenders to large soldiers, resulting in one of the most complex polymorphic caste systems within ants. To begin uncovering the genomic underpinnings of this system, we sequenced the genome of Atta cephalotes using 454 pyrosequencing. One prediction from this ant's lifestyle is that it has undergone genetic modifications that reflect its obligate dependence on the fungus for nutrients. Analysis of this genome sequence is consistent with this hypothesis, as we find evidence for reductions in genes related to nutrient acquisition. These include extensive reductions in serine proteases (which are likely unnecessary because proteolysis is not a primary mechanism used to process nutrients obtained from the fungus), a loss of genes involved in arginine biosynthesis (suggesting that this amino acid is obtained from the fungus), and the absence of a hexamerin (which sequesters amino acids during larval development in other insects). Following recent reports of genome sequences from other insects that engage in symbioses with beneficial microbes, the A. cephalotes genome provides new insights into the symbiotic lifestyle of this ant and advances our understanding of host-microbe symbioses.
Flexible genomes facilitate bacterial evolution and are classically organized into polymorphic strain-specific segments called regions of genomic plasticity (RGPs). Using a new web tool, RGPFinder, we investigated plasticity units in bacterial genomes, by exhaustive description of the RGPs in two Photorhabdus and two Xenorhabdus strains, belonging to the Enterobacteriaceae and interacting with invertebrates (insects and nematodes). RGPs account for about 60% of the genome in each of the four genomes studied. We classified RGPs into genomic islands (GIs), prophages and two new classes of RGP without the features of classical mobile genetic elements (MGEs) but harboring genes encoding enzymes catalyzing DNA recombination (RGPmob), or with no remarkable feature (RGPnone). These new classes accounted for most of the RGPs and are probably hypervariable regions, ancient MGEs with degraded mobilization machinery or non canonical MGEs for which the mobility mechanism has yet to be described. We provide evidence that not only the GIs and the prophages, but also RGPmob and RGPnone, have a mosaic structure consisting of modules. A module is a block of genes, 0.5 to 60 kb in length, displaying a conserved genomic organization among the different Enterobacteriaceae. Modules are functional units involved in host/environment interactions (22-31%), metabolism (22-27%), intracellular or intercellular DNA mobility (13-30%), drug resistance (4-5%) and antibiotic synthesis (3-6%). Finally, in silico comparisons and PCR multiplex analysis indicated that these modules served as plasticity units within the bacterial genome during genome speciation and as deletion units in clonal variants of Photorhabdus. This led us to consider the modules, rather than the entire RGP, as the true unit of plasticity in bacterial genomes, during both short-term and long-term genome evolution.
Herbivores can gain indirect access to recalcitrant carbon present in plant cell walls through symbiotic associations with lignocellulolytic microbes. A paradigmatic example is the leaf-cutter ant (Tribe: Attini), which uses fresh leaves to cultivate a fungus for food in specialized gardens. Using a combination of sugar composition analyses, metagenomics, and whole-genome sequencing, we reveal that the fungus garden microbiome of leaf-cutter ants is composed of a diverse community of bacteria with high plant biomass-degrading capacity. Comparison of this microbiome's predicted carbohydrate-degrading enzyme profile with other metagenomes shows closest similarity to the bovine rumen, indicating evolutionary convergence of plant biomass degrading potential between two important herbivorous animals. Genomic and physiological characterization of two dominant bacteria in the fungus garden microbiome provides evidence of their capacity to degrade cellulose. Given the recent interest in cellulosic biofuels, understanding how large-scale and rapid plant biomass degradation occurs in a highly evolved insect herbivore is of particular relevance for bioenergy.
Blastomyces dermatitidis belongs to a group of human pathogenic fungi that exhibit thermal dimorphism. At 22 degrees C, these fungi grow as mold that produce conidia or infectious particles, whereas at 37 degrees C they convert to budding yeast. The ability to switch between these forms is essential for virulence in mammals and may enable these organisms to survive in the soil. To identify genes that regulate this phase transition, we used Agrobacterium tumefaciens to mutagenize B. dermatitidis conidia and screened transformants for defects in morphogenesis. We found that the GATA transcription factor SREB governs multiple fates in B. dermatitidis: phase transition from yeast to mold, cell growth at 22 degrees C, and biosynthesis of siderophores under iron-replete conditions. Insertional and null mutants fail to convert to mold, do not accumulate significant biomass at 22 degrees C, and are unable to suppress siderophore biosynthesis under iron-replete conditions. The defect in morphogenesis in the SREB mutant was independent of exogenous iron concentration, suggesting that SREB promotes the phase transition by altering the expression of genes that are unrelated to siderophore biosynthesis. Using bioinformatic and gene expression analyses, we identified candidate genes with upstream GATA sites whose expression is altered in the null mutant that may be direct or indirect targets of SREB and promote the phase transition. We conclude that SREB functions as a transcription factor that promotes morphogenesis and regulates siderophore biosynthesis. To our knowledge, this is the first gene identified that promotes the conversion from yeast to mold in the dimorphic fungi, and may shed light on environmental persistence of these pathogens.
Leaf-cutter ants use fresh plant material to grow a mutualistic fungus that serves as the ants' primary food source. Within fungus gardens, various plant compounds are metabolized and transformed into nutrients suitable for ant consumption. This symbiotic association produces a large amount of refuse consisting primarily of partly degraded plant material. A leaf-cutter ant colony is thus divided into two spatially and chemically distinct environments that together represent a plant biomass degradation gradient. Little is known about the microbial community structure in gardens and dumps or variation between lab and field colonies. Using microbial membrane lipid analysis and a variety of community metrics, we assessed and compared the microbiota of fungus gardens and refuse dumps from both laboratory-maintained and field-collected colonies. We found that gardens contained a diverse and consistent community of microbes, dominated by Gram-negative bacteria, particularly gamma-Proteobacteria and Bacteroidetes. These findings were consistent across lab and field gardens, as well as host ant taxa. In contrast, dumps were enriched for Gram-positive and anaerobic bacteria. Broad-scale clustering analyses revealed that community relatedness between samples reflected system component (gardens/dumps) rather than colony source (lab/field). At finer scales samples clustered according to colony source. Here we report the first comparative analysis of the microbiota from leaf-cutter ant colonies. Our work reveals the presence of two distinct communities: one in the fungus garden and the other in the refuse dump. Though we find some effect of colony source on community structure, our data indicate the presence of consistently associated microbes within gardens and dumps. Substrate composition and system component appear to be the most important factor in structuring the microbial communities. These results thus suggest that resident communities are shaped by the plant degradation gradient created by ant behavior, specifically their fungiculture and waste management.
Bacteria-mediated acquisition of atmospheric N2 serves as a critical source of nitrogen in terrestrial ecosystems. Here we reveal that symbiotic nitrogen fixation facilitates the cultivation of specialized fungal crops by leaf-cutter ants. By using acetylene reduction and stable isotope experiments, we demonstrated that N2 fixation occurred in the fungus gardens of eight leaf-cutter ant species and, further, that this fixed nitrogen was incorporated into ant biomass. Symbiotic N2-fixing bacteria were consistently isolated from the fungus gardens of 80 leaf-cutter ant colonies collected in Argentina, Costa Rica, and Panama. The discovery of N2 fixation within the leaf-cutter ant-microbe symbiosis reveals a previously unrecognized nitrogen source in neotropical ecosystems.
We identified Xenorhabdus nematophila transposon mutants with defects in lipase activity. One of the mutations, in yigL, a conserved gene of unknown function, resulted in attenuated virulence against Manduca sexta insects. We discuss possible connections between lipase production, YigL, and specific metabolic pathways.
The family Rhizobiaceae contains plant-associated bacteria with critical roles in ecology and agriculture. Within this family, many Rhizobium and Sinorhizobium strains are nitrogen-fixing plant mutualists, while many strains designated as Agrobacterium are plant pathogens. These contrasting lifestyles are primarily dependent on the transmissible plasmids each strain harbors. Members of the Rhizobiaceae also have diverse genome architectures that include single chromosomes, multiple chromosomes, and plasmids of various sizes. Agrobacterium strains have been divided into three biovars, based on physiological and biochemical properties. The genome of a biovar I strain, A. tumefaciens C58, has been previously sequenced. In this study, the genomes of the biovar II strain A. radiobacter K84, a commercially available biological control strain that inhibits certain pathogenic agrobacteria, and the biovar III strain A. vitis S4, a narrow-host-range strain that infects grapes and invokes a hypersensitive response on nonhost plants, were fully sequenced and annotated. Comparison with other sequenced members of the Alphaproteobacteria provides new data on the evolution of multipartite bacterial genomes. Primary chromosomes show extensive conservation of both gene content and order. In contrast, secondary chromosomes share smaller percentages of genes, and conserved gene order is restricted to short blocks. We propose that secondary chromosomes originated from an ancestral plasmid to which genes have been transferred from a progenitor primary chromosome. Similar patterns are observed in select Beta- and Gammaproteobacteria species. Together, these results define the evolution of chromosome architecture and gene content among the Rhizobiaceae and support a generalized mechanism for second-chromosome formation among bacteria.
Fungus-growing ants (Attini: Formicidae) engage in an obligate mutualism with fungi they cultivate for food. Although biologists have been fascinated with fungus-growing ants since the resurgence of natural history in the modern era, the early stages of research focused mainly on the foraging behavior of the leaf-cutters (the most derived attine lineage). Indeed, the discovery that the ants actually use leaf fragments to manure a fungus did not come until the 1800s. More recently, three additional microbial symbionts have been described, including specialized microfungal parasites of the ant's fungus garden, antibiotic-producing actinobacteria that help protect the fungus garden from the parasite, and a black yeast that parasitizes the ant-actinobacteria mutualism. The fungus-growing ant symbiosis serves as a particularly useful model system for studying insect-microbe symbioses, because, to date, it contains four well-characterized microbial symbionts, including mutualists and parasites that encompass micro-fungi, macro-fungi, yeasts, and bacteria. Here, we discuss approaches for studying insect-microbe symbioses, using the attine ant-microbial symbiosis as our framework. We draw attention to particular challenges in the field of symbiosis, including the establishment of symbiotic associations and symbiont function. Finally, we discuss future directions in insect-microbe research, with particular focus on applying recent advances in DNA sequencing technologies.
No abstract available.
The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strain's complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase-like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.
Automated DNA sequencing technology is so rapid that analysis has become the rate-limiting step. Hundreds of prokaryotic genome sequences are publicly available, with new genomes uploaded at the rate of approximately 20 per month. As a result, this growing body of genome sequences will include microorganisms not previously identified, isolated, or observed. We hypothesize that evolutionary pressure exerted by an ecological niche selects for a similar genetic repertoire in those prokaryotes that occupy the same niche, and that this is due to both vertical and horizontal transmission. To test this, we have developed a novel method to classify prokaryotes, by calculating their Pfam protein domain distributions and clustering them with all other sequenced prokaryotic species. Clusters of organisms are visualized in two dimensions as 'mountains' on a topological map. When compared to a phylogenetic map constructed using 16S rRNA, this map more accurately clusters prokaryotes according to functional and environmental attributes. We demonstrate the ability of this map, which we term a "niche map", to cluster according to ecological niche both quantitatively and qualitatively, and propose that this method be used to associate uncharacterized prokaryotes with their ecological niche as a means of predicting their functional role directly from their genome sequence.
xanthusBase (http://www.xanthusbase.org) is the official model organism database (MOD) for the social bacterium Myxococcus xanthus. In many respects, M.xanthus represents the pioneer model organism (MO) for studying the genetic, biochemical, and mechanistic basis of prokaryotic multicellularity, a topic that has garnered considerable attention due to the significance of biofilms in both basic and applied microbiology research. To facilitate its utility, the design of xanthusBase incorporates open-source software, leveraging the cumulative experience made available through the Generic Model Organism Database (GMOD) project, MediaWiki (http://www.mediawiki.org), and dictyBase (http://www.dictybase.org), to create a MOD that is both highly useful and easily navigable. In addition, we have incorporated a unique Wikipedia-style curation model which exploits the internet's inherent interactivity, thus enabling M.xanthus and other myxobacterial researchers to contribute directly toward the ongoing genome annotation.
No abstract available.
Accurate determination of functional interactions among proteins at the genome level remains a challenge for genomic research. Here we introduce a genome-scale approach to functional protein annotation--phylogenomic mapping--that requires only sequence data, can be applied equally well to both finished and unfinished genomes, and can be extended beyond single genomes to annotate multiple genomes simultaneously. We have developed and applied it to more than 200 sequenced bacterial genomes. Proteins with similar evolutionary histories were grouped together, placed on a three dimensional map and visualized as a topographical landscape. The resulting phylogenomic maps display thousands of proteins clustered in mountains on the basis of coinheritance, a strong indicator of shared function. In addition to systematic computational validation, we have experimentally confirmed the ability of phylogenomic maps to predict both mutant phenotype and gene function in the delta proteobacterium Myxococcus xanthus.
Long-term results of 122 patients with advanced rectal cancer who were randomly treated with three different methods from July 1984 to July 1986. Of 122 patients, 44 were treated with endocavitary 915 MHz microwave applicators combined with 10 MeV X-ray or 60CO followed by surgery (group A), 38 with preoperative radiation (group B) and 40 with surgery (group C) as a control group. The temperature on the surface of the applicator touching the middle of the caudad to cephaladic extent of disease was 45-50 degrees C for 45 min twice a week for 6-8 sessions. Radiation dose was 30 Gy or 40 Gy/4 weeks. Of cases with stages 0 and A, 45.5% (20/44) were in group A, 23.7% (9/38) in group B and 12.5% (5/40) in group C (chi 2 test p < 0.05 and p < 0.01, respectively). Five-year survival rate was 66.7% (24/36) in group A, 50% (14/28) in group B and 40.5% (15/37) in group C. Percentage of survival at 5 years was 73.7% (14/19) for 40 Gy plus heat, 57.1% (8/14) for 40 Gy alone, 58.8% (10/17) for 30 Gy plus heat, and 42.9% (6/14) for 30 Gy alone. Data suggest a survival advantage for patients treated with preoperative radiation combined with endocavitary hyperthermia.
Two 12-wk experiments were conducted to determine the adequate dietary niacin levels for juvenile hybrid tilapia, Oreochromis niloticus x O. aureus, when diets containing either 38% D(+)-glucose or 38% dextrin (type III, from corn) as the carbohydrate source were fed. In Experiment 1, we used 0, 40, 80, 120, 160 and 200 mg/kg of supplemental niacin in both the glucose- and dextrin-containing diets. In Experiment 2, 0, 10, 25, 40 and 55 mg/kg or 0, 10, 25, 40, 80, 120 and 160 mg/kg of supplemental niacin was incorporated in diets containing glucose or dextrin, respectively. In both experiments, fish fed niacin-deficient diets grew poorly. They developed skin, fin and mouth lesions and hemorrhages; the snout was deformed and there was gill edema. These pathologies began 6 wk after the experiments started. Plasma glucose concentrations were higher in fish fed glucose diets than those fed dextrin diets. Weight gain analyzed by broken-line regression indicated that the adequate dietary niacin level for maximal growth in rapidly growing tilapia fingerlings is 26 mg/kg in fish fed the glucose diet and 121 mg/kg in fish fed the dextrin diet.