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Faculty & Staff

  • Image of Michael G. Thomas

    Michael G. Thomas

    Professor of Bacteriology
    Alfred Toepfer Faculty Fellow

    6159 Microbial Sciences Building
    Office: (608) 263-9075
    Lab: (608) 265-7767

Start and Promotion Dates

  • Assistant Professor: 2002
  • Associate Professor: 2009
  • Full Professor: 2014


B.S. Rutgers University 1992 
M.S. Washington State University 1994 
Ph.D. University of Wisconsin-Madison 1999 
Postdoctoral Research: Harvard Medical School

Areas of Study

Enzyme Evolution
Natural Product Discovery
Bioenergy Research

Research Overview

Bacterial secondary metabolism has proven to be a rich source of natural products with medically and agriculturally relevant biological activities. Additionally, the enzymes employed by the producing organisms to generate these metabolites have proven to be a fascinating fusion of reactions that are typically seen in primary metabolism, with slight modifications to generate unusual enzymatic reactions. Thus, analysis of bacterial secondary metabolism offers the opportunity to study processes that have both basic and applied scientific interests. The overall goals of my research program are to discover, decipher, and direct bacterial secondary metabolism with these two scientific interests in mind.

Directed Evolution of Natural Product Biosynthesis Enzymes

Two of the largest classes of natural products are the nonribosomal peptides and the polyketides. The nonribosomal peptides are assembled by large protein complexes called nonribosomal peptide synthetases (NRPSs). As the name implies, NRPSs synthesize peptides independent of the ribosome. This enzymology involves a set of repeating catalytic domains that are grouped into modules. Each module contains all the catalytic domains for the incorporation and modification of one precursor (typically an amino acid) into the growing peptide chain. Nature has generated the enormous structural diversity of nonribosomal peptides by changing the number of modules, the precursors recognized and incorporated by these modules, and the modifications to the incorporated precursors. Polyketide biosynthesis works in a similar manner with repeating, modular catalytic domains controlling the incorporation of thioesterified carboxylic acids. These enzyme complexes are called polyketide synthases (PKSs) and the changing of the number of modules, precursors incorporated, and modifying domains generates enormous structural diversity in these natural products as well. While Nature has been able to generate structural diversity of nonribosomal peptides and polyketides by shuffling the NRPS and PKS domains/modules, we have not been able to fully understand what controls substrate recognition and proper protein-protein interactions in these large protein complexes to do the equivalent in a directed manner to make designer molecules. To address these issues, we have developed a set of NRPS and PKS tools that can be harnessed for directed evolution approaches to dissect NRPS and PKS enzymology. Our ultimate goal is to enable us to redirect this enzymology to generate desired molecules for drug developement.

Bioenergy Research

It is essential that we move from a petroleum-based economy to a renewable-energy based economy. The Great Lakes Bioenergy Research Center (GLBRC) is a leader in moving science towards this goal. Of particular interest to my group is adding value to the biological material remaining after a desired biofuel has been produced and extracted from the starting plant hydrolysate. This material is called conversion residue and has up to 2/3 of the carbon from the starting material. Currently this material is dried and burned to generate energy for the biorefinery. Our goal is to capture some of this carbon to produce value-added bioproducts. To do this, we are using Streptomyces species to metabolize the carbon in the conversion residue and convert it to desired bioproducts. Streptomyces species are phylogenetically and metabolically diverse and are well-known for producing secondary metabolites that are chemically similar to economically valuable bioproducts. We are using a select number of Streptomyces species as biological chassis to efficiently transform conversion residue carbon into terpene- and fatty acid-based value-added bioproducts.

Discovery of Metallophores Produced by the Human Microbiota

The acquisition of essential micronutrient transition metals (Fe, Co, Ni, Cu, Zn, Mg, and Mn) is a largely unexplored space in the commensal bacteria or microorganisms that make up the human microbiota. This area of research has been overlooked mainly due to a heavy emphasis on Fe acquisition by pathogens using metallophores called siderophores and the prevailing belief that the iron acquisition driven by the endogenous production of siderophores is not needed by the vast majority of commensals, specifically those associated with the gut. Recently, there have been reports that siderophores have biological functions beyond Fe acquisition, and many may actually have roles in acquiring other transition metals protecting the producing strain from oxidative stress, or providing a mechanism for dealing with metal toxicity. In collaboration with Profs. Federico Rey and Tim Bugni we are mining the human microbiota for metallophores to provide insights into how the producing organism accesses or blocks the intake of these micronutrients to survive in the host, while at the same time providing insights into the coevolution of the host and commensal for metal acquisition, the evolution of metallophore structural diversity, and the development of new therapeutics.


Microbiology 526: Physiology of Microorganisms


Trainer, Microbiology Doctoral Training Program
Trainer, Biotechnology Training Program
Trainer, Chemical Biology Interface Program
Editorial Board, Applied and Environmental Microbiology
Editor, Microbiology and Molecular Biology Reviews


  • 2015-2020, E. B. Fred Professor of Bacteriology
  • 2010, CALS Pound Research Award
  • 2006-2008, Alfred Toepfer Faculty Fellow

Lab Personnel

Picture of Conley
Erin Conley
Grad Student
Picture of Cook
Taylor Cook
Picture of Miller
Neil Miller
Picture of Spencer
Mallory Spencer
Grad Student
Picture of Wadler
Caryn Wadler

Research Papers

  • Cook TB, Jacobson TB, Venkataraman MV, Hofstetter H, Amador-Noguez D, Thomas MG, Pfleger BF (2021) Stepwise genetic engineering of Pseudomonas putida enables robust heterologous production of prodigiosin and glidobactin A. Metabolic engineering 67:112-124 PMC8434984 · Pubmed · DOI

    No abstract available.

  • Vinnik V, Zhang F, Park H, Cook TB, Throckmorton K, Pfleger BF, Bugni TS, Thomas MG (2020) Structural and Biosynthetic Analysis of the Fabrubactins, Unusual Siderophores from Agrobacterium fabrum Strain C58. ACS chemical biology 16((1)):125-135 PMC7882191 · Pubmed · DOI

    No abstract available.

  • Wu Q, Throckmorton K, Maity M, Chevrette MG, Braun DR, Rajski SR, Currie CR, Thomas MG, Bugni TS (2020) Bacillibactins E and F from a Marine Sponge-Associated Bacillus sp. Journal of natural products 84((1)):136-141 PMC7856188 · Pubmed · DOI

    No abstract available.

  • Zhang F, Wyche TP, Zhu Y, Braun DR, Yan JX, Chanana S, Ge Y, Guzei IA, Chevrette MG, Currie CR, Thomas MG, Rajski SR, Bugni TS (2020) MS-Derived Isotopic Fine Structure Reveals Forazoline A as a Thioketone-Containing Marine-Derived Natural Product. Organic letters 22((4)):1275-1279 PMC7494057 · Pubmed · DOI

    No abstract available.

  • Throckmorton K, Vinnik V, Chowdhury R, Cook T, Chevrette M, Maranas C, Pfleger B, Thomas MG (2019) Directed Evolution Reveals the Functional Sequence Space of an Adenylation Domain Specificity Code ACS Chem. Biol. 14(9):2044-2054 · DOI

    Nonribosomal peptides are important natural products biosynthesized by nonribosomal peptide synthetases (NRPSs). Adenylation (A) domains of NRPSs are highly specific for the substrate they recognize. This recognition is determined by 10 residues in the substrate-binding pocket, termed the specificity code. This finding led to the proposal that nonribosomal peptides could be altered by specificity code swapping. Unfortunately, this approach has proven, with few exceptions, to be unproductive; changing the specificity code typically results in broadened specificity or poor function. To enhance our understanding of A domain substrate selectivity, we carried out a detailed analysis of the specificity code from the A domain of EntF, an NRPS involved in enterobactin biosynthesis in Escherichia coli. Using directed evolution and a genetic selection, we determined which sites in the code have strict residue requirements and which are tolerant of variation. We showed that the EntF A domain, and other l-Ser-specific A domains, have a functional sequence space for l-Ser recognition, rather than a single code. This functional space is more expansive than the aggregate of all characterized l-Ser-specific A domains: we identified 152 new l-Ser specificity codes. Together, our data provide essential insights into how to overcome the barriers that prevent rational changes to A domain specificity.

  • Esquilín-Lebrón KJ, Boynton TO, Shimkets LJ, Thomas MG (2018) An Orphan MbtH-Like Protein Interacts with Multiple Nonribosomal Peptide Synthetases in Myxococcus xanthus DK1622. J. Bacteriol. 200(21): (PMC6182236) · Pubmed · DOI

    One mechanism by which bacteria and fungi produce bioactive natural products is the use of nonribosomal peptide synthetases (NRPSs). Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility or function. Although MLPs are known to interact with the adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within the biosynthetic gene clusters (BGCs) that also code for the NRPSs that interact with the MLP. Here, we identify 50 orphan MLPs from diverse bacteria. An orphan MLP is one that is encoded by a gene that is not directly adjacent to genes predicted to be involved in nonribosomal peptide biosynthesis. We targeted the orphan MLP MXAN_3118 from DK1622 for characterization. The DK1622 genome contains 15 NRPS-encoding BGCs but only one MLP-encoding gene (MXAN_3118). We tested the hypothesis that MXAN_3118 interacts with one or more NRPS using a combination of and assays. We determined that MXAN_3118 interacts with at least seven NRPSs from distinct BGCs. We show that one of these BGCs codes for NRPS enzymology that likely produces a valine-rich natural product that inhibits the clumping of DK1622 in liquid culture. MXAN_3118 is the first MLP to be identified that naturally interacts with multiple NRPS systems in a single organism. The finding of an MLP that naturally interacts with multiple NRPS systems suggests it may be harnessed as a "universal" MLP for generating functional hybrid NRPSs. MbtH-like proteins (MLPs) are essential accessory proteins for the function of many nonribosomal peptide synthetases (NRPSs). We identified 50 MLPs from diverse bacteria that are coded by genes that are not located near any NRPS-encoding biosynthetic gene clusters (BGCs). We define these as orphan MLPs because their NRPS partner(s) is unknown. Investigations into the orphan MLP from DK1622 determined that it interacts with NRPSs from at least seven distinct BGCs. Support for these MLP-NRPS interactions came from the use of a bacterial two-hybrid assay and copurification of the MLP with various NRPSs. The flexibility of this MLP to naturally interact with multiple NRPSs led us to hypothesize that this MLP may be used as a "universal" MLP during the construction of functional hybrid NRPSs.

  • Schomer RA, Park H, Barkei JJ, Thomas MG (2018) Alanine Scanning of YbdZ, an MbtH-like Protein, Reveals Essential Residues for Functional Interactions with Its Nonribosomal Peptide Synthetase Partner EntF. Biochemistry 57(28):4125-4134 (PMC6050124) · Pubmed · DOI

    Nonribosomal peptide synthetases (NRPSs) are megasynthetases that require complex and specific interactions between multiple domains and proteins to functionally produce a metabolite. MbtH-like proteins (MLPs) are integral components of many NRPSs and interact directly with the adenylation domain of the megasynthetases to stimulate functional enzymology. All of the MLP residues that are essential for functional interactions between the MLP and NRPS have yet to be defined. Here we probe the interactions between YbdZ, an MLP, and EntF, an NRPS, from Escherichia coli by performing a complete alanine scan of YbdZ. A phenotypic screen identified 11 YbdZ variants that are unable to replace the wild-type MLP, and these YbdZ variants were characterized using a series of in vivo and in vitro assays in an effort to explain why functional interactions with EntF were disrupted. All of the YbdZ variants enhanced the solubility of overproduced EntF, suggesting they were still capable of direct interactions with the megasynthase. Conversely, we show that EntF also influences the solubility of YbdZ and its variants. In vitro biochemical analyses of EntF function with each of the YbdZ variants found the impact that an amino acid substitution will have on NRPS function is difficult to predict, highlighting the complex interaction between these proteins.

  • Schomer RA, Thomas MG (2017) Characterization of the Functional Variance in MbtH-like Protein Interactions with a Nonribosomal Peptide Synthetase. Biochemistry 56(40):5380-5390 (PMC5902190) · Pubmed · DOI

    Many nonribosomal peptide synthetases (NRPSs) require MbtH-like proteins (MLPs) for solubility or for activation of amino acid substrate by the adenylation domain. MLPs are capable of functional crosstalk with noncognate NRPSs at varying levels. Using enterobactin biosynthesis in Escherichia coli as a model MLP-dependent NRPS system, we use in vivo and in vitro techniques to characterize how seven noncognate MLPs influence the function of the enterobactin NRPS EntF when the cognate MLP, YbdZ, is absent. Using a series of in vitro assays to analyze EntF solubility, adenylation, aminoacylation, and in vitro enterobactin production, we show that interactions between MLPs and NRPSs are multifaceted and more complex than previously appreciated. We separate MLP influence on solubility and function in a manner that shows altered solubility is not indicative of a functional MLP/NRPS pair. Although much of the functional variation among these noncognates can be explained by differences in EntF affinity for an MLP or the extent an MLP alters EntF l-Ser affinity, we demonstrate that MLPs can have a broader impact beyond solubility and adenylation. First, we show that a noncognate MLP can affect formation of l-Ser-S-EntF. Second, under in vitro conditions saturating for substrate and MLP, enterobactin production remains compromised in the absence of an appropriate MLP partner. These data suggest that we expand our investigations into how the MLPs influence NRPS enzymology. A more detailed understanding of these influences will be essential for downstream engineering of hybrid NRPS systems.

  • Smanski MJ, Mead D, Gustafsson C, Thomas MG (2017) Meeting Report for Synthetic Biology for Natural Products 2017: The Interface of (Meta)Genomics, Machine Learning, and Natural Product Discovery. ACS Synth Biol 6(5):737-743 · Pubmed · DOI

    No abstract available.

  • Lozano GL, Holt J, Ravel J, Rasko DA, Thomas MG, Handelsman J (2016) Draft Genome Sequence of Biocontrol Agent Bacillus cereus UW85. Genome Announc 4(5): (PMC5009980) · Pubmed

    Bacillus cereus UW85 was isolated from a root of a field-grown alfalfa plant from Arlington, WI, and identified for its ability to suppress damping off, a disease caused by Phytophthora megasperma f. sp. medicaginis on alfalfa. Here, we report the draft genome sequence of B. cereus UW85, obtained by a combination of Sanger and Illumina sequencing.

  • Stulberg ER, Lozano GL, Morin JB, Park H, Baraban EG, Mlot C, Heffelfinger C, Phillips GM, Rush JS, Phillips AJ, Broderick NA, Thomas MG, Stabb EV, Handelsman J (2016) Genomic and Secondary Metabolite Analyses of Streptomyces sp. 2AW Provide Insight into the Evolution of the Cycloheximide Pathway. Front Microbiol 7:573 (PMC4853412) · Pubmed

    The dearth of new antibiotics in the face of widespread antimicrobial resistance makes developing innovative strategies for discovering new antibiotics critical for the future management of infectious disease. Understanding the genetics and evolution of antibiotic producers will help guide the discovery and bioengineering of novel antibiotics. We discovered an isolate in Alaskan boreal forest soil that had broad antimicrobial activity. We elucidated the corresponding antimicrobial natural products and sequenced the genome of this isolate, designated Streptomyces sp. 2AW. This strain illustrates the chemical virtuosity typical of the Streptomyces genus, producing cycloheximide as well as two other biosynthetically unrelated antibiotics, neutramycin, and hygromycin A. Combining bioinformatic and chemical analyses, we identified the gene clusters responsible for antibiotic production. Interestingly, 2AW appears dissimilar from other cycloheximide producers in that the gene encoding the polyketide synthase resides on a separate part of the chromosome from the genes responsible for tailoring cycloheximide-specific modifications. This gene arrangement and our phylogenetic analyses of the gene products suggest that 2AW holds an evolutionarily ancestral lineage of the cycloheximide pathway. Our analyses support the hypothesis that the 2AW glutaramide gene cluster is basal to the lineage wherein cycloheximide production diverged from other glutarimide antibiotics. This study illustrates the power of combining modern biochemical and genomic analyses to gain insight into the evolution of antibiotic-producing microorganisms.

  • Weerth RS, Michalska K, Bingman CA, Yennamalli RM, Li H, Jedrzejczak R, Wang F, Babnigg G, Joachimiak A, Thomas MG, Phillips GN (2015) Structure of a cupin protein Plu4264 from Photorhabdus luminescens subsp. laumondii TTO1 at 1.35 Å resolution. Proteins 83(2):383-8 (PMC4300268) · Pubmed

    Proteins belonging to the cupin superfamily have a wide range of catalytic and noncatalytic functions. Cupin proteins commonly have the capacity to bind a metal ion with the metal frequently determining the function of the protein. We have been investigating the function of homologous cupin proteins that are conserved in more than 40 species of bacteria. To gain insights into the potential function of these proteins we have solved the structure of Plu4264 from Photorhabdus luminescens TTO1 at a resolution of 1.35 Å and identified manganese as the likely natural metal ligand of the protein.

  • Park H, Kevany BM, Dyer DH, Thomas MG, Forest KT (2014) A polyketide synthase acyltransferase domain structure suggests a recognition mechanism for its hydroxymalonyl-acyl carrier protein substrate. PLoS ONE 9(10):e110965 (PMC4207774) · Pubmed

    We have previously shown that the acyl transferase domain of ZmaA (ZmaA-AT) is involved in the biosynthesis of the aminopolyol polyketide/nonribosomal peptide hybrid molecule zwittermicin A from cereus UW85, and that it specifically recognizes the precursor hydroxymalonyl-acyl carrier protein (ACP) and transfers the hydroxymalonyl extender unit to a downstream second ACP via a transacylated AT domain intermediate. We now present the X-ray crystal structure of ZmaA-AT at a resolution of 1.7 Å. The structure shows a patch of solvent-exposed hydrophobic residues in the area where the AT is proposed to interact with the precursor ACP. We addressed the significance of the AT/ACP interaction in precursor specificity of the AT by testing whether malonyl- or methylmalonyl-ACP can be recognized by ZmaA-AT. We found that the ACP itself biases extender unit selection. Until now, structural information for ATs has been limited to ATs specific for the CoA-linked precursors malonyl-CoA and (2S)-methylmalonyl-CoA. This work contributes to polyketide synthase engineering efforts by expanding our knowledge of AT/substrate interactions with the structure of an AT domain that recognizes an ACP-linked substrate, the rare hydroxymalonate. Our structure suggests a model in which ACP interaction with a hydrophobic motif promotes secondary structure formation at the binding site, and opening of the adjacent substrate pocket lid to allow extender unit binding in the AT active site.

  • McMahon MD, Rush JS, Thomas MG (2012) Analyses of MbtB, MbtE, and MbtF suggest revisions to the mycobactin biosynthesis pathway in Mycobacterium tuberculosis. J. Bacteriol. 194(11):2809-18 (PMC3370630) · Pubmed

    The production of mycobactin (MBT) by Mycobacterium tuberculosis is essential for this bacterium to access iron when it is in an infected host. Due to this essential function, there is considerable interest in deciphering the mechanism of MBT assembly, with the goal of targeting select biosynthetic steps for antituberculosis drug development. The proposed scheme for MBT biosynthesis involves assembly of the MBT backbone by a hybrid nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) megasynthase followed by the tailoring of this backbone by N(6) acylation of the central l-Lys residue and subsequent N(6)-hydroxylation of the central N(6)-acyl-l-Lys and the terminal caprolactam. A complete testing of this hypothesis has been hindered by the inability to heterologously produce soluble megasynthase components. Here we show that soluble forms of the NRPS components MbtB, MbtE, and MbtF are obtained when these enzymes are coproduced with MbtH. Using these soluble enzymes we determined the amino acid specificity of each adenylation (A) domain. These results suggest that the proposed tailoring enzymes are actually involved in precursor biosynthesis since the A domains of MbtE and MbtF are specific for N(6)-acyl-N(6)-hydroxy-l-Lys and N(6)-hydroxy-l-Lys, respectively. Furthermore, the preference of the A domain of MbtB for l-Thr over l-Ser suggests that the megasynthase produces MBT derivatives with β-methyl oxazoline rings. Since the most prominent form of MBT produced by M. tuberculosis lacks this β-methyl group, a mechanism for demethylation remains to be discovered. These results suggest revisions to the MBT biosynthesis pathway while also identifying new targets for antituberculosis drug development.

  • McMahon MD, Guan C, Handelsman J, Thomas MG (2012) Metagenomic analysis of Streptomyces lividans reveals host-dependent functional expression. Appl. Environ. Microbiol. 78(10):3622-9 (PMC3346366) · Pubmed

    Most functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA in Escherichia coli, which has been the predominant surrogate host to date. To expand the range of expressed genes, we developed tools for construction and functional screening of metagenomic libraries in Streptomyces lividans. We expanded on previously published protocols by constructing a system that enables retrieval and characterization of the metagenomic DNA from biologically active clones. To test the functionality of these methods, we constructed and screened two metagenomic libraries in S. lividans. One was constructed with pooled DNA from 14 bacterial isolates cultured from Alaskan soil and the second with DNA directly extracted from the same soil. Functional screening of these libraries identified numerous clones with hemolytic activity, one clone that produces melanin by a previously unknown mechanism, and one that induces the overproduction of a secondary metabolite native to S. lividans. All bioactive clones were functional in S. lividans but not in E. coli, demonstrating the advantages of screening metagenomic libraries in more than one host.

  • Felnagle EA, Podevels AM, Barkei JJ, Thomas MG (2011) Mechanistically distinct nonribosomal peptide synthetases assemble the structurally related antibiotics viomycin and capreomycin. Chembiochem 12(12):1859-67 (PMC3389393) · Pubmed

    No abstract available.

  • Felnagle EA, Barkei JJ, Park H, Podevels AM, McMahon MD, Drott DW, Thomas MG (2010) MbtH-like proteins as integral components of bacterial nonribosomal peptide synthetases. Biochemistry 49(41):8815-7 (PMC2974439) · Pubmed

    The biosynthesis of many natural products of clinical interest involves large, multidomain enzymes called nonribosomal peptide synthetases (NRPSs). In bacteria, many of the gene clusters coding for NRPSs also code for a member of the MbtH-like protein superfamily, which are small proteins of unknown function. Using MbtH-like proteins from three separate NRPS systems, we show that these proteins copurify with the NRPSs and influence amino acid activation. As a consequence, MbtH-like proteins are integral components of NRPSs.

  • Park H, Thomas MG (2010) Using surrogates to bypass missing catalytic components. Chem. Biol. 17(10):1045-6 (PMC2987683) · Pubmed

    Bryostatin is a natural product that has many medically promising biological activities. Understanding how bryostatin is assembled by the producting symbiotic bacterium has been hampered by the limited availability of genetic information. In the new report, Buchholz et al. (2010) circumvented this issue by using surrogates to replace missing catalytic components.

  • Chan YA, Thomas MG (2010) Recognition of (2S)-aminomalonyl-acyl carrier protein (ACP) and (2R)-hydroxymalonyl-ACP by acyltransferases in zwittermicin A biosynthesis. Biochemistry 49(17):3667-77 (PMC2860681) · Pubmed

    Polyketide synthases elongate a polyketide backbone by condensing carboxylic acid precursors that are thioesterified to either coenzyme A or an acyl carrier protein (ACP). Two of the three known ACP-linked extender units, (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP, are found in the biosynthesis of the agriculturally important antibiotic zwittermicin A. We previously reconstituted the formation of (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP from the primary metabolites l-serine and 1,3-bisphospho-d-glycerate. In this report, we characterize the two acyltransferases involved in the specific transfer of the (2S)-aminomalonyl and (2R)-hydroxymalonyl moieties from the ACPs associated with extender unit formation to the ACPs integrated into the polyketide synthase. This work establishes which acyltransferase recognizes each extender unit and also provides insight into the substrate selectivity of these enzymes. These are important step toward harnessing these rare polyketide synthase extender units for combinatorial biosynthesis.

  • Helmetag V, Samel SA, Thomas MG, Marahiel MA, Essen LO (2009) Structural basis for the erythro-stereospecificity of the L-arginine oxygenase VioC in viomycin biosynthesis. FEBS J. 276(13):3669-82 (PMC2771579) · Pubmed

    The nonheme iron oxygenase VioC from Streptomyces vinaceus catalyzes Fe(II)-dependent and alpha-ketoglutarate-dependent Cbeta-hydroxylation of L-arginine during the biosynthesis of the tuberactinomycin antibiotic viomycin. Crystal structures of VioC were determined in complexes with the cofactor Fe(II), the substrate L-arginine, the product (2S,3S)-hydroxyarginine and the coproduct succinate at 1.1-1.3 A resolution. The overall structure reveals a beta-helix core fold with two additional helical subdomains that are common to nonheme iron oxygenases of the clavaminic acid synthase-like superfamily. In contrast to other clavaminic acid synthase-like oxygenases, which catalyze the formation of threo diastereomers, VioC produces the erythro diastereomer of Cbeta-hydroxylated L-arginine. This unexpected stereospecificity is caused by conformational control of the bound substrate, which enforces a gauche(-) conformer for chi(1) instead of the trans conformers observed for the asparagine oxygenase AsnO and other members of the clavaminic acid synthase-like superfamily. Additionally, the substrate specificity of VioC was investigated. The side chain of the L-arginine substrate projects outwards from the active site by undergoing interactions mainly with the C-terminal helical subdomain. Accordingly, VioC exerts broadened substrate specificity by accepting the analogs L-homoarginine and L-canavanine for Cbeta-hydroxylation.

  • Berti AD, Thomas MG (2009) Analysis of achromobactin biosynthesis by Pseudomonas syringae pv. syringae B728a. J. Bacteriol. 191(14):4594-604 (PMC2704727) · Pubmed

    Pseudomonas syringae pv. syringae B728a is known to produce the siderophore pyoverdine under iron-limited conditions. It has also been proposed that this pathovar has the ability to produce a second siderophore, achromobactin. Here we present genetic and biochemical evidence supporting the hypothesis that P. syringae pv. syringae B728a produces both of these siderophores. We show that strains unable to synthesize either pyoverdine or achromobactin are unable to grow under iron-limiting conditions, which is consistent with these two molecules being the only siderophores synthesized by P. syringae pv. syringae B728a. Enzymes associated with achromobactin biosynthesis were purified and analyzed for substrate recognition. We showed that AcsD, AcsA, and AcsC together are able to condense citrate, ethanolamine, 2,4-diaminobutyrate, and alpha-ketoglutarate into achromobactin. Replacement of ethanolamine with ethylene diamine or 1,3-diaminopropane in these reactions resulted in the formation of achromobactin analogs that were biologically active. This work provides insights into the biosynthetic steps in the formation of achromobactin and is the first in vitro reconstitution of achromobactin biosynthesis.

  • Chan YA, Thomas MG (2009) Formation and characterization of acyl carrier protein-linked polyketide synthase extender units. Meth. Enzymol. 459:143-63 (PMC2765370) · Pubmed

    Polyketide natural products are assembled by the condensation of an initiating precursor, or starter unit, with a series of additional precursors referred to as extender units. While there are a number of polyketide synthase starter units, there are currently only seven known polyketide synthase extender units. Polyketide synthase extender units thioesterified to coenzyme A have been known for some time; however, polyketide synthase extender units thioesterified to acyl carrier proteins (ACPs) have been identified only recently. Two of them, (2R)-hydroxymalonyl-ACP and (2S)-aminomalonyl-ACP, are found in the biosynthetic pathway of the antibiotic zwittermicin A in Bacillus cereus UW85. The focus of this chapter is the in vitro formation of (2R)-hydroxymalonyl-ACP and (2S)-aminomalonyl-ACP and the characterization of these extender units using high performance liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

  • Kevany BM, Rasko DA, Thomas MG (2009) Characterization of the complete zwittermicin A biosynthesis gene cluster from Bacillus cereus. Appl. Environ. Microbiol. 75(4):1144-55 (PMC2643575) · Pubmed

    Bacillus cereus UW85 produces the linear aminopolyol antibiotic zwittermicin A (ZmA). This antibiotic has diverse biological activities, such as suppression of disease in plants caused by protists, inhibition of fungal and bacterial growth, and amplification of the insecticidal activity of the toxin protein from Bacillus thuringiensis. ZmA has an unusual chemical structure that includes a d amino acid and ethanolamine and glycolyl moieties, as well as having an unusual terminal amide that is generated from the modification of the nonproteinogenic amino acid beta-ureidoalanine. The diverse biological activities and unusual structure of ZmA have stimulated our efforts to understand how this antibiotic is biosynthesized. Here, we present the identification of the complete ZmA biosynthesis gene cluster from B. cereus UW85. A nearly identical gene cluster is identified on a plasmid from B. cereus AH1134, and we show that this strain is also capable of producing ZmA. Bioinformatics and biochemical analyses of the ZmA biosynthesis enzymes strongly suggest that ZmA is initially biosynthesized as part of a larger metabolite that is processed twice, resulting in the formation of ZmA and two additional metabolites. Additionally, we propose that the biosynthesis gene cluster for the production of the amino sugar kanosamine is contained within the ZmA biosynthesis gene cluster in B. cereus UW85.

  • Chan YA, Podevels AM, Kevany BM, Thomas MG (2009) Biosynthesis of polyketide synthase extender units. Nat Prod Rep 26(1):90-114 (PMC2766543) · Pubmed

    This review covers the biosynthesis of extender units that are utilized for the assembly of polyketides by polyketide synthases. The metabolic origins of each of the currently known polyketide synthase extender units are covered.

  • Barkei JJ, Kevany BM, Felnagle EA, Thomas MG (2009) Investigations into viomycin biosynthesis by using heterologous production in Streptomyces lividans. Chembiochem 10(2):366-76 (PMC2765823) · Pubmed

    Viomycin and capreomycin are members of the tuberactinomycin family of antituberculosis drugs. As with many antibacterial drugs, resistance to the tuberactinomycins is problematic in treating tuberculosis; this makes the development of new derivatives of these antibiotics to combat this resistance of utmost importance. To take steps towards developing new derivatives of this family of antibiotics, we have focused our efforts on understanding how these antibiotics are biosynthesized by the producing bacteria so that metabolic engineering of these pathways can be used to generate desired derivatives. Here we present the heterologous production of viomycin in Streptomyces lividans 1326 and the use of targeted-gene deletion as a mechanism for investigating viomycin biosynthesis as well as the generation of viomycin derivatives. Deletion of vioQ resulted in nonhydroxylated derivatives of viomycin, while strains lacking vioP failed to acylate the cyclic pentapeptide core of viomycin with beta-lysine. Surprisingly, strains lacking vioL produced derivatives that had the carbamoyl group of viomycin replaced by an acetyl group. Additionally, the acetylated viomycin derivatives were produced at very low levels. These two observations suggested that the carbamoyl group of the cyclic pentapeptide core of viomycin was introduced at an earlier step in the biosynthetic pathway than previously proposed. We present biochemical evidence that the carbamoyl group is added to the beta-amino group of L-2,3-diaminopropionate prior to incorporation of this amino acid by the nonribosomal peptide synthetases that form the cyclic pentapeptide cores of both viomycin and capreomycin.

  • Felnagle EA, Jackson EE, Chan YA, Podevels AM, Berti AD, McMahon MD, Thomas MG (2008) Nonribosomal peptide synthetases involved in the production of medically relevant natural products. Mol. Pharm. 5(2):191-211 (PMC3131160) · Pubmed

    Natural products biosynthesized wholly or in part by nonribosomal peptide synthetases (NRPSs) are some of the most important drugs currently used clinically for the treatment of a variety of diseases. Since the initial research into NRPSs in the early 1960s, we have gained considerable insights into the mechanism by which these enzymes assemble these natural products. This review will present a brief history of how the basic mechanistic steps of NRPSs were initially deciphered and how this information has led us to understand how nature modified these systems to generate the enormous structural diversity seen in nonribosomal peptides. This review will also briefly discuss how drug development and discovery are being influenced by what we have learned from nature about nonribosomal peptide biosynthesis.

  • Berti AD, Greve NJ, Christensen QH, Thomas MG (2007) Identification of a biosynthetic gene cluster and the six associated lipopeptides involved in swarming motility of Pseudomonas syringae pv. tomato DC3000. J. Bacteriol. 189(17):6312-23 (PMC1951903) · Pubmed

    Pseudomonas species are known to be prolific producers of secondary metabolites that are synthesized wholly or in part by nonribosomal peptide synthetases. In an effort to identify additional nonribosomal peptides produced by these bacteria, a bioinformatics approach was used to "mine" the genome of Pseudomonas syringae pv. tomato DC3000 for the metabolic potential to biosynthesize previously unknown nonribosomal peptides. Herein we describe the identification of a nonribosomal peptide biosynthetic gene cluster that codes for proteins involved in the production of six structurally related linear lipopeptides. Structures for each of these lipopeptides were proposed based on amino acid analysis and mass spectrometry analyses. Mutations in this cluster resulted in the loss of swarming motility of P. syringae pv. tomato DC3000 on medium containing a low percentage of agar. This phenotype is consistent with the loss of the ability to produce a lipopeptide that functions as a biosurfactant. This work gives additional evidence that mining the genomes of microorganisms followed by metabolite and phenotypic analyses leads to the identification of previously unknown secondary metabolites.

  • Felnagle EA, Rondon MR, Berti AD, Crosby HA, Thomas MG (2007) Identification of the biosynthetic gene cluster and an additional gene for resistance to the antituberculosis drug capreomycin. Appl. Environ. Microbiol. 73(13):4162-70 (PMC1932801) · Pubmed

    Capreomycin (CMN) belongs to the tuberactinomycin family of nonribosomal peptide antibiotics that are essential components of the drug arsenal for the treatment of multidrug-resistant tuberculosis. Members of this antibiotic family target the ribosomes of sensitive bacteria and disrupt the function of both subunits of the ribosome. Resistance to these antibiotics in Mycobacterium species arises due to mutations in the genes coding for the 16S or 23S rRNA but can also arise due to mutations in a gene coding for an rRNA-modifying enzyme, TlyA. While Mycobacterium species develop resistance due to alterations in the drug target, it has been proposed that the CMN-producing bacterium, Saccharothrix mutabilis subsp. capreolus, uses CMN modification as a mechanism for resistance rather than ribosome modification. To better understand CMN biosynthesis and resistance in S. mutabilis subsp. capreolus, we focused on the identification of the CMN biosynthetic gene cluster in this bacterium. Here, we describe the cloning and sequence analysis of the CMN biosynthetic gene cluster from S. mutabilis subsp. capreolus ATCC 23892. We provide evidence for the heterologous production of CMN in the genetically tractable bacterium Streptomyces lividans 1326. Finally, we present data supporting the existence of an additional CMN resistance gene. Initial work suggests that this resistance gene codes for an rRNA-modifying enzyme that results in the formation of CMN-resistant ribosomes that are also resistant to the aminoglycoside antibiotic kanamycin. Thus, S. mutabilis subsp. capreolus may also use ribosome modification as a mechanism for CMN resistance.

  • Pacholec M, Sello JK, Walsh CT, Thomas MG (2007) Formation of an aminoacyl-S-enzyme intermediate is a key step in the biosynthesis of chloramphenicol. Org. Biomol. Chem. 5(11):1692-4 · Pubmed

    Herein we report the first biochemical characterization of an enzyme involved in the biosynthesis of chloramphenicol that provides new insights into the origins of the antibiotic.

  • Chan YA, Boyne MT, Podevels AM, Klimowicz AK, Handelsman J, Kelleher NL, Thomas MG (2006) Hydroxymalonyl-acyl carrier protein (ACP) and aminomalonyl-ACP are two additional type I polyketide synthase extender units. Proc. Natl. Acad. Sci. U.S.A. 103(39):14349-54 (PMC1599966) · Pubmed

    Combinatorial biosynthesis of type I polyketide synthases is a promising approach for the generation of new structural derivatives of polyketide-containing natural products. A target of this approach has been to change the extender units incorporated into a polyketide backbone to alter the structure and activity of the natural product. One limitation to these efforts is that only four extender units were known: malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, and methoxymalonyl-acyl carrier protein (ACP). The chemical attributes of these extender units are quite similar, with the exception of the potential hydrogen bonding interactions by the oxygen of the methoxy moiety. Furthermore, the incorporated extender units are not easily modified by using simple chemical approaches when combinatorial biosynthesis is coupled to semisynthetic chemistry. We recently proposed the existence of two additional extender units, hydroxymalonyl-ACP and aminomalonyl-ACP, involved in the biosynthesis of zwittermicin A. These extender units offer unique possibilities for combinatorial biosynthesis and semisynthetic chemistry because of the introduction of free hydroxyl and amino moieties into a polyketide structure. Here, we present the biochemical and mass spectral evidence for the formation of these extender units. This evidence shows the formation of ACP-linked extender units for polyketide synthesis. Interestingly, aminomalonyl-ACP formation involves enzymology typically found in nonribosomal peptide synthesis.

  • Rondon MR, Ballering KS, Thomas MG (2004) Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58. Microbiology (Reading, Engl.) 150(Pt 11):3857-66 · Pubmed

    Using the complete genome sequence from Agrobacterium tumefaciens C58, the authors identified a secondary metabolite gene cluster that encodes the biosynthesis of a metabolite with siderophore activity. Support for this conclusion came from genetic and regulatory analysis of the gene cluster, along with the purification of a metabolite from A. tumefaciens C58 with iron-chelating activity. Genetic analysis of mutant strains disrupted in this gene cluster showed that these strains grew more slowly than the wild-type strain in medium lacking iron. Additionally, the mutant strains failed to produce a chrome-azurol-S-reactive material in liquid or solid medium, and failed to produce the metabolite with iron-chelating characteristics that was identified in the wild-type strain. Addition of this purified metabolite to the growth medium of a mutant strain restored its ability to grow in iron-deficient medium. Furthermore, expression of this gene cluster was induced by growth under iron-limiting conditions, suggesting that expression of this gene cluster occurs when iron is scarce. These data are all consistent with the proposal that the proteins encoded by this gene cluster are involved in the production of a siderophore. Interestingly, these proteins show the highest level of amino acid similarity to proteins from a gene cluster found in the filamentous cyanobacterium Nostoc sp. PCC7120, rather than to known siderophore biosynthetic enzymes. Given these properties, it is proposed that the siderophore produced by A. tumefaciens C58 will have a unique chemical structure. Production of the siderophore was not required for virulence of A. tumefaciens when tested with a standard stem inoculation assay.

  • Ju J, Ozanick SG, Shen B, Thomas MG (2004) Conversion of (2S)-arginine to (2S,3R)-capreomycidine by VioC and VioD from the viomycin biosynthetic pathway of Streptomyces sp. strain ATCC11861. Chembiochem 5(9):1281-5 · Pubmed

    No abstract available.

  • Emmert EA, Klimowicz AK, Thomas MG, Handelsman J (2004) Genetics of zwittermicin a production by Bacillus cereus. Appl. Environ. Microbiol. 70(1):104-13 (PMC321298) · Pubmed

    Zwittermicin A represents a new chemical class of antibiotic and has diverse biological activities, including suppression of oomycete diseases of plants and potentiation of the insecticidal activity of Bacillus thuringiensis. To identify genes involved in zwittermicin A production, we generated 4,800 transposon mutants of B. cereus UW101C and screened them for zwittermicin A accumulation. Nine mutants did not produce detectable zwittermicin A, and one mutant produced eightfold more than the parent strain. The DNA flanking the transposon insertions in six of the nine nonproducing mutants contains significant sequence similarity to genes involved in peptide and polyketide antibiotic biosynthesis. The mutant that overproduced zwittermicin A contained a transposon insertion immediately upstream from a gene that encodes a deduced protein that is a member of the MarR family of transcriptional regulators. Three genes identified by the mutant analysis mapped to a region that was previously shown to carry the zwittermicin A self-resistance gene, zmaR, and a biosynthetic gene (E. A. Stohl, J. L. Milner, and J. Handelsman, Gene 237:403-411, 1999). Further sequencing of this region revealed genes proposed to encode zwittermicin A precursor biosynthetic enzymes, in particular, those involved in the formation of the aminomalonyl- and hydroxymalonyl-acyl carrier protein intermediates. Additionally, nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) homologs are present, suggesting that zwittermicin A is synthesized by a mixed NRPS/PKS pathway.

  • Thomas MG, Chan YA, Ozanick SG (2003) Deciphering tuberactinomycin biosynthesis: isolation, sequencing, and annotation of the viomycin biosynthetic gene cluster. Antimicrob. Agents Chemother. 47(9):2823-30 (PMC182626) · Pubmed

    The tuberactinomycin antibiotics are essential components in the drug arsenal against Mycobacterium tuberculosis infections and are specifically used for the treatment of multidrug-resistant tuberculosis. These antibiotics are also being investigated for their targeting of the catalytic RNAs involved in viral replication and for the treatment of bacterial infections caused by methicillin-resistant Staphylococcus aureus strains and vancomycin-resistant enterococci. We report on the isolation, sequencing, and annotation of the biosynthetic gene cluster for one member of this antibiotic family, viomycin, from Streptomyces sp. strain ATCC 11861. This is the first gene cluster for a member of the tuberactinomycin family of antibiotics sequenced, and the information gained can be extrapolated to all members of this family. The gene cluster covers 36.3 kb of DNA and encodes 20 open reading frames that we propose are involved in the biosynthesis, regulation, export, and activation of viomycin, in addition to self-resistance to the antibiotic. These results enable us to predict the metabolic logic of tuberactinomycin production and begin steps toward the combinatorial biosynthesis of these antibiotics to complement existing chemical modification techniques to produce novel tuberactinomycin derivatives.

  • Pootoolal J, Thomas MG, Marshall CG, Neu JM, Hubbard BK, Walsh CT, Wright GD (2002) Assembling the glycopeptide antibiotic scaffold: The biosynthesis of A47934 from Streptomyces toyocaensis NRRL15009. Proc. Natl. Acad. Sci. U.S.A. 99(13):8962-7 (PMC124406) · Pubmed

    The glycopeptide antibiotics vancomycin and teicoplanin are vital components of modern anti-infective chemotherapy exhibiting outstanding activity against Gram-positive pathogens including members of the genera Streptococcus, Staphylococcus, and Enterococcus. These antibiotics also provide fascinating examples of the chemical and associated biosynthetic complexity exploitable in the synthesis of natural products by actinomycetes group of bacteria. We report the sequencing and annotation of the biosynthetic gene cluster for the glycopeptide antibiotic from Streptomyces toyocaensis NRRL15009, the first complete sequence for a teicoplanin class glycopeptide. The cluster includes 34 ORFs encompassing 68 kb and includes all of the genes predicted to be required to synthesize and regulate its biosynthesis. The gene cluster also contains ORFs encoding enzymes responsible for glycopeptide resistance. This role was confirmed by insertional inactivation of the d-Ala-d-lactate ligase, vanAst, which resulted in the predicted -sensitive phenotype and impaired antibiotic biosynthesis. These results provide increased understanding of the biosynthesis of these complex natural products.

  • Thomas MG, Burkart MD, Walsh CT (2002) Conversion of L-proline to pyrrolyl-2-carboxyl-S-PCP during undecylprodigiosin and pyoluteorin biosynthesis. Chem. Biol. 9(2):171-84 · Pubmed

    Several medically and agriculturally important natural products contain pyrrole moieties. Precursor labeling studies of some of these natural products have shown that L-proline can serve as the biosynthetic precursor for these moieties, including those found in coumermycin A(1), pyoluteorin, and one of the pyrroles of undecylprodigiosin. This suggests a novel mechanism for pyrrole biosynthesis. The biosynthetic gene clusters for these three natural products each encode proteins homologous to adenylation (A) and peptidyl carrier protein (PCP) domains of nonribosomal peptide synthetases in addition to novel acyl-CoA dehydrogenases. Here we show that the three proteins from the undecylprodigiosin and pyoluteorin biosynthetic pathways are sufficient for the conversion of L-proline to pyrrolyl-2-carboxyl-S-PCP. This establishes a novel mechanism for pyrrole biosynthesis and extends the hypothesis that organisms use A/PCP pairs to partition an amino acid into secondary metabolism.