Picture of Marcin S. Filutowicz

Marcin S. Filutowicz

Professor of Bacteriology

Address: 1550 Linden Drive, Room 6550
Phone: (608) 262-6947
Lab Phone: (608) 262-8553
Email: msfiluto@wisc.edu
Overview · Lab · Publications · Lab Website · The Raper Strain Archive
J. Bacteriol., Nucleic Acids Res., Gene, J. Mol. Biol., J. Biol. Chem., Plasmid, J Gen Microbiol., Mol Gen Genet., Proc Natl Acad Sci U S A., Mutat Res. , J Biol Chem. , J Mol Biol., In: Plasmids in Bacteria, ed. D.R. Helinski et al. Plenum Press. New York., J. Cell Sci. Suppl., Encyclopedia of Microbiology (Third Edition), J Ocul Biol, Acta Biochim. Polon., Arch Microbiol., J Burn Care Res, FEMS Microbiol. Lett., J. Microbiol. Methods, Biochem. Biophys. Res. Commun., Nat. Struct. Biol., Acta Biochim. Pol., Proc. Natl. Acad. Sci. U.S.A., Prog. Nucleic Acid Res. Mol. Biol., Mol. Gen. Genet., New Biol., J Ocular Biol,

    Research Papers

  • Broekema NM, Larsen IV, Naruzawa ES, Filutowicz M, Kolb AW, Teixeira LB, Brandt CR (2016) A Mouse Model of Multi-Drug Resistant Staphylococcus aureus-induced Ocular Disease. J Ocul Biol 4(2): (PMC5123590) View Abstract · Pubmed Record
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    Staphylococcus aureus infection of the cornea is a significant threat to vision. The percentage of bacterial isolates resistant to antibiotics is increasing as is the percentage of infections caused by methicillin resistant isolates. There is a critical need for additional therapeutic approaches and their development will require the use of animal models to test efficacy. Two mouse models of S. aureus keratitis have been described but only quantified stromal keratitis (corneal clouding and perforation). We have extended these models using the methicillin resistant S. aureus USA300 LAC strain and show that eyelid inflammation and swelling (blepharitis) and corneal neovascularization can be quantified. This expanded model should prove useful in assessing additional effects of antibacterial therapies and additional pathological mechanisms involved in bacterial ocular infection.

  • Broekema NM, Larsen IV, Naruzawa ES, Filutowicz MS, Kolb AW, Teixeira LBC, Brandt CR (2016) A Mouse Model of Multi-Drug Resistant Staphylococcus aureus-induced Ocular Disease J Ocular Biol 4(2):1-5 View Abstract
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    Staphylococcus aureus infection of the cornea is a significant threat to vision. The percentage of bacterial isolates resistant to antibiotics is increasing as is the percentage of infections caused by methicillin resistant isolates. There is a critical need for additional therapeutic approaches and their development will require the use of animal models to test efficacy. Two mouse models of S. aureus keratitis have been described but only quantified stromal keratitis (corneal clouding and perforation). We have extended these models using the methicillin resistant S. aureus USA300 LAC strain and show that eyelid inflammation and swelling (blepharitis) and corneal neovascularization can be quantified. This expanded model should prove useful in assessing additional effects of antibacterial therapies and additional pathological mechanisms involved in bacterial ocular infection.

  • Rakowski SA, Filutowicz M (2013) Plasmid R6K replication control. Plasmid 69(3):231-42 (PMC3691012) View Abstract · Pubmed Record
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    The focus of this minireview is the replication control of the 39.9-kb plasmid R6K and its derivatives. Historically, this plasmid was thought to have a narrow host range but more recent findings indicate that its derivatives can replicate in a variety of enteric and non-enteric bacterial species (Wild et al., 2004). In the four-plus decades since it was first described, R6K has proven to be an excellent model for studies of plasmid DNA replication. In part this is because of its similarities to other systems in which replication is activated and regulated by Rep protein and iteron-containing DNA. However its apparent idiosynchracies have also added to its significance (e.g., independent and co-dependent replication origins, and Rep dimers that stably bind iterons). Here, we survey the current state of knowledge regarding R6K replication and place individual regulatory elements into a proposed homeostatic model with implications for the biological significance of R6K and its multiple origins of replication.

  • Filutowicz, M (2009) Plasmids, Bacterial. Encyclopedia of Microbiology (Third Edition) :644-55 10.1016/B978-012373944-5.00014-6 View Abstract · DOI Record
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    No abstract available.

  • Filutowicz M, Burgess R, Gamelli RL, Heinemann JA, Kurenbach B, Rakowski SA, Shankar R (2008) Bacterial conjugation-based antimicrobial agents. Plasmid 60(1):38-44 View Abstract · Pubmed Record
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    A clear imperative exists to generate radically different antibacterial technologies that will reduce the usage of conventional chemical antibiotics. Here we trace one route into this new frontier of drug discovery, a concept that we call the bacterial conjugation-based technologies (BCBT). One of the objectives of the BCBT is to exploit plasmid biology for combating the rising tide of antibiotic-resistant bacteria. Specifically, the concept utilizes conjugationally delivered plasmids as antimicrobial agents, and it builds on the accumulated work of many scientists dating back to the discoveries of conjugation and plasmids themselves. Each of the individual components that comprise the approach has been demonstrated to be feasible. We discuss the properties of bacterial plasmids to be employed in BCBT.

  • Bowers LM, Filutowicz M (2008) Cooperative binding mode of the inhibitors of R6K replication, pi dimers. J. Mol. Biol. 377(3):609-15 (PMC2518315) View Abstract · Pubmed Record
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    The replication initiator protein, pi, plays an essential role in the initiation of plasmid R6K replication. Both monomers and dimers of pi bind to iterons in the gamma origin of plasmid R6K, yet monomers facilitate open complex formation, while dimers, the predominant form in the cell, do not. Consequently, pi monomers activate replication, while pi dimers inhibit replication. Recently, it was shown that the monomeric form of pi binds multiple tandem iterons in a strongly cooperative fashion, which might explain how monomers outcompete dimers for replication initiation when plasmid copy number and pi supply are low. Here, we examine cooperative binding of pi dimers and explore the role that these interactions may have in the inactivation of gamma origin. To examine pi dimer/iteron interactions in the absence of competing pi monomer/iteron interactions using wild-type pi, constructs were made with key base changes to each iteron that eliminate pi monomer binding yet have no impact on pi dimer binding. Our results indicate that, in the absence of pi monomers, pi dimers bind with greater cooperativity to alternate iterons than to adjacent iterons, thus preferentially leaving intervening iterons unbound and the origin unsaturated. We discuss new insights into plasmid replication control by pi dimers.

  • Kunnimalaiyaan S, Rakowski SA, Filutowicz M (2007) Structure-based functional analysis of the replication protein of plasmid R6K: key amino acids at the pi/DNA interface. J. Bacteriol. 189(13):4953-6 (PMC1913429) View Abstract · Pubmed Record
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    In previous work, we characterized the bases in an iteron of plasmid R6K that are important for the binding of pi protein monomers and dimers. Here we investigate the following six amino acids of pi, encoded by pir, hypothesized to be important for DNA contact: Ser71, Try74, Gly131, Gly211, Arg225, and Arg254.

  • Bowers LM, Krüger R, Filutowicz M (2007) Mechanism of origin activation by monomers of R6K-encoded pi protein. J. Mol. Biol. 368(4):928-38 (PMC2001305) View Abstract · Pubmed Record
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    One recurring theme in plasmid duplication is the recognition of the origin of replication (ori) by specific Rep proteins that bind to DNA sequences called iterons. For plasmid R6K, this process involves a complex interplay between monomers and dimers of the Rep protein, pi, with seven tandem iterons of gamma ori. Remarkably, both pi monomers and pi dimers can bind to iterons, a new paradigm in replication control. Dimers, the predominant form in the cell, inhibit replication, while monomers facilitate open complex formation and activate the ori. Here, we investigate a mechanism by which pi monomers out-compete pi dimers for iteron binding, and in so doing activate the ori. With an in vivo plasmid incompatibility assay, we find that pi monomers bind cooperatively to two adjacent iterons. Cooperative binding is eliminated by insertion of a half-helical turn between two iterons but is diminished only slightly by insertion of a full helical turn between two iterons. These studies show also that pi bound to a consensus site promotes occupancy of an adjacent mutated site, another hallmark of cooperative interactions. pi monomer/iteron interactions were quantified using a monomer-biased pi variant in vitro with the same collection of two-iteron constructs. The cooperativity coefficients mirror the plasmid incompatibility results for each construct tested. pi dimer/iteron interactions were quantified with a dimer-biased mutant in vitro and it was found that pi dimers bind with negligible cooperativity to two tandem iterons.

  • Shankar R, He LK, Szilagyi A, Muthu K, Gamelli RL, Filutowicz M, Wendt JL, Suzuki H, Dominguez M (2007) A novel antibacterial gene transfer treatment for multidrug-resistant Acinetobacter baumannii-induced burn sepsis. J Burn Care Res 28(1):6-12 View Abstract · Pubmed Record
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    Sepsis caused by multidrug-resistant bacterial infections in critically injured patients has become a major clinical problem. Recently, Acinetobacter baumannii (AB) wound infections, especially in our critically injured soldiers fighting in Iraq and Afghanistan, is posing a major clinical problem and an economic burden. ConjuGon, Inc., has developed a novel antibacterial therapeutic technology using bacterial conjugation. The donor cells are attenuated Escherichia coli carrying a conjugative plasmid. The expression of bactericidal genes cloned on the plasmid is tightly repressed in the donor cells but becomes de-repressed once mobilized into a pathogen and disrupts protein synthesis. Here, we tested the efficacy of this novel conjugation technology to control and eradicate a drug-resistant clinical isolate of AB wound infection both in vitro and in a murine burn sepsis model. C57Blk/6J mice were divided into burn (B) and burn sepsis (BS) groups. All animals received a 12% TBSA dorsal scald full-thickness burn. The BS group was inoculated with multidrug-resistant AB (1 x 10(5) colony-forming units [CFU]) at the burn wound site. BS animals were either untreated or treated with increasing concentrations (10(3) - 19(10) CFU) of attenuated donor E. coli encoding bactericidal proteins. The survival rate was monitored for 10 days. The ability of donor cells to significantly diminish AB levels in the burn wound 24 hours after injury was determined by quantitative cultures. Donor cells were highly effective in killing AB in vitro. In the burn sepsis model, 90% B group animals survived, and 40% to 50% BS animals survived with no treatment in 5 to 6 days. Treatment with donor cells at 10(10) to 10(6) provided significant survival advantage (P < .05). Quantitative cultures of burn wounds revealed that AB numbers increased from 3 x 10(4) CFU to 7.8 +/- 4.4 x 10(9) CFU in 24 hours in the untreated group. Single treatment with donor cells (10(10) CFU) significantly reduced AB in the burn wound to less than the levels seeded into the wound (1.23 +/- 0.5 x 10(4) CFU; P < .05). Taken together, these results indicate that this novel technology is an efficient method to control drug-resistant AB burn wound infections and prevent their systemic spread.

  • Peng Y, Rakowski SA, Filutowicz M (2006) Small deletion variants of the replication protein, pi, and their potential for over-replication-based antimicrobial activity. FEMS Microbiol. Lett. 261(2):245-52 View Abstract · Pubmed Record
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    The emergence of multiply antibiotic-resistant microorganisms in the environment has become a serious public health threat. To address this, our lab has devised a methodology in which antimicrobial agents are transferred into unwanted cells using the process of bacterial conjugation. In the work described here, we pursued proteins that cause plasmid over-replication as potential antimicrobial agents. Our focus was on the pir-encoded pi protein of plasmid R6K that possesses both positive and negative functions in controlling gamma origin-based replication. We observed that three of four pir mutations examined, including two in-frame deletions, severely impaired negative plasmid-replication control. The resulting over-replication phenotype was particularly strong when a pir mutant was placed in cis to gamma origin. In conjugative mating experiments with several representatives of the family Enterobacteriaceae, the plasmids expressed postconjugational antimicrobial activity. The potential utility of a conjugation-based antimicrobial approach is discussed. Additionally, we describe the replication inhibitory function of a novel and useful Rep protein variant, pi*M36A;M38A, which binds iteron DNA exclusively as dimers.

  • Kunnimalaiyaan S, Inman RB, Rakowski SA, Filutowicz M (2005) Role of pi dimers in coupling ("handcuffing") of plasmid R6K's gamma ori iterons. J. Bacteriol. 187(11):3779-85 (PMC1112066) View Abstract · Pubmed Record
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    One proposed mechanism of replication inhibition in iteron-containing plasmids (ICPs) is "handcuffing," in which the coupling of origins via iteron-bound replication initiator (Rep) protein turns off origin function. In minimal R6K replicons, copy number control requires the interaction of plasmid-encoded pi protein with the seven 22-bp iterons of the gamma origin of replication. Like other related Rep proteins, pi exists as both monomers and dimers. However, the ability of pi dimers to bind iterons distinguishes R6K from most other ICPs, where only monomers have been observed to bind iterons. Here, we describe experiments to determine if monomers or dimers of pi protein are involved in the formation of handcuffed complexes. Standard ligation enhancement assays were done using pi variants with different propensities to bind iterons as monomers or dimers. Consistent with observations from several ICPs, a hyperreplicative variant (pi.P106L(wedge)F107S) exhibits deficiencies in handcuffing. Additionally, a novel dimer-biased variant of pi protein (pi.M36A(wedge)M38A), which lacks initiator function, handcuffs iteron-containing DNA more efficiently than does wild-type pi. The data suggest that pi dimers mediate handcuffing, supporting our previously proposed model of handcuffing in the gamma ori system. Thus, dimers of pi appear to possess three distinct inhibitory functions with respect to R6K replication: transcriptional autorepression of pi expression, in cis competition (for origin binding) with monomeric activator pi, and handcuffing-mediated inhibition of replication in trans.

  • Bowers LM, Lapoint K, Anthony L, Pluciennik A, Filutowicz M (2004) Bacterial expression system with tightly regulated gene expression and plasmid copy number. Gene 340(1):11-8 View Abstract · Pubmed Record
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    A new Escherichia coli host/vector system has been engineered to allow tight and uniform modulation of gene expression and gamma origin (ori) plasmid copy number. Regulation of gamma ori plasmid copy number is achieved through arabinose-inducible expression of the necessary Rep protein, pi, whose gene was integrated into the chromosome of the host strain under control of the P(BAD) promoter. gamma ori replication can be uniformly modulated over 100-fold by changing the concentration of l-arabinose in the growth medium. This strain avoids the problem of all-or-nothing induction of P(BAD) because it is deficient in both arabinose uptake and degradation genes. Arabinose enters the cell by a mutant LacY transporter, LacYA177C, which is expressed from the host chromosome. Although this strain could be compatible with any gamma ori plasmid, we describe the utility of a gamma ori expression vector that allows especially tight regulation of gene expression. With this host/vector system, it is possible to independently modulate gene expression and gene dosage, facilitating the cloning and overproduction of toxic gene products. We describe the successful use of this system for cloning a highly potent toxin, Colicin E3, in the absence of its cognate immunity protein. This system could be useful for cloning genes encoding other potent toxins, screening libraries for potential toxins, and maintaining any gamma ori vector at precise copy levels in a cell.

  • Kunnimalaiyaan S, Krüger R, Ross W, Rakowski SA, Filutowicz M (2004) Binding modes of the initiator and inhibitor forms of the replication protein pi to the gamma ori iteron of plasmid R6K. J. Biol. Chem. 279(39):41058-66 View Abstract · Pubmed Record
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    Discerning the interactions between initiator protein and the origin of replication should provide insights into the mechanism of DNA replication initiation. In the gamma origin of plasmid R6K, the Rep protein, pi, is distinctive in that it can bind the seven 22-bp iterons in two forms; pi monomers activate replication, whereas pi dimers act as inhibitors. In this work, we used wild type and variants of the pi protein with altered monomer/dimer ratios to study iteron/pi interactions. High resolution contact mapping was conducted using multiple techniques (missing base contact probing, methylation protection, base modification, and hydroxyl radical footprinting), and the electrophoretic separation of nucleoprotein complexes allowed us to discriminate between contact patterns produced by pi monomers and dimers. We also isolated iteron mutants that affected the binding of pi monomers (only) or both monomers and dimers. The mutational studies and footprinting analyses revealed that, when binding DNA, pi monomers interact with nucleotides spanning the entire length of the iteron. In contrast, pi dimers interact with only the left half of the iteron; however, the retained interactions are strikingly similar to those seen with monomers. These results support a model in which Rep protein dimerization disturbs one of two DNA binding domains important for monomer/iteron interaction; the dimer/iteron interaction utilizes only one DNA binding domain.

  • Anthony LC, Suzuki H, Filutowicz M (2004) Tightly regulated vectors for the cloning and expression of toxic genes. J. Microbiol. Methods 58(2):243-50 View Abstract · Pubmed Record
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    A series of low-copy expression vectors that permits the stable maintenance and regulated expression of highly toxic gene products has been developed. These vectors utilize the lactose promoter/operator system, and protect against read-through transcription from other promoters on the plasmid by placement of the rrnB T1T2 terminators upstream of the lactose promoter. For additional regulatory control, the vectors utilize low-copy origins of replication. Either the pMPP6 origin (pSC101-derived) is used for cloning into Escherichia coli or related species, or the broad-host-range RK2 origin of replication is utilized for cloning into the majority of Gram-negative bacteria. The resulting plasmids have no detectable leaky expression. To test these vectors, the genes for the bacteriocidal colicins D, E3, and E7 were cloned and stably maintained in the absence of their immunity genes. Upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), cell death was observed, indicating expression of each colicin. These low-copy expression vectors will be useful for the cloning and expression of toxic genes in bacterial systems.

  • Krüger R, Rakowski SA, Filutowicz M (2004) Isomerization and apparent DNA bending by pi, the replication protein of plasmid R6K. Biochem. Biophys. Res. Commun. 313(4):834-40 View Abstract · Pubmed Record
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    Plasmid R6K-encoded pi protein has multiple regulatory functions in replication and transcription. These functions rely, in part, on a complex set of interactions between monomers and dimers of the protein and distinct DNA targets, the direct and inverted repeats (DRs, IRs). In the work described here, we examine the isomerization and DNA bending properties of pi using electrophoretic mobility shift assays and circular permutation assays. Our data suggest that pi dimers can bend IRs, and dimer subunits seem to readily associate in head-to-head and head-to-tail fashion. The ability of pi to bend DRs is also reexamined using techniques that allow us to discriminate between bending induced by its different isomeric forms. We find that both monomers and dimers bend a single DR to similar degrees while results with 2DRs are more complex. The significance of the bending data in regard to a possible mechanism for replication initiation by pi protein is discussed.

  • Krüger R, Filutowicz M (2003) pi protein- and ATP-dependent transitions from 'closed' to 'open' complexes at the gamma ori of plasmid R6K. Nucleic Acids Res. 31(20):5993-6003 (PMC219486) View Abstract · Pubmed Record
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    R6K-encoded pi protein can bind to the seven, 22 bp tandem iterons of the gamma origin. In this work, we use a variant of pi, His-pi.F107S, that is hyperactive in replication. In vitro, His-pi.F107S-dependent local DNA melting (open complex formation) occurs in the absence of host proteins (IHF/HU or DnaA) and it is positioned in the A + T-rich region adjacent to iterons. Experiments described here examine the effects of ATP, Mg2+ and temperature on the opening reaction. We show that the opening of the gamma origin can occur in the presence of ATP as well as AMP-PCP (a non-hydrolyzable ATP analog). This suggests that, for gamma origin, ATP hydrolysis may be unnecessary for open complex formation facilitated by His-pi.F107S. In the absence of ATP or Mg2+, His-pi.F107S yielded data suggestive of distortions in the iteron attributable to DNA bending rather than DNA melting. Our findings also demonstrate that ATP and pi stimulate open complex formation over a wide range of temperatures, but not at 0 degrees C. These and other results indicate that ATP and/or Mg2+ are not needed for His-pi.F107S binding to iterons and that ATP effects an allosteric change in the protein bound to gamma origin.

  • Krüger R, Filutowicz M (2003) Characterization of His-tagged, R6K-encoded pi protein variants. Plasmid 50(1):80-5 View Abstract · Pubmed Record
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    The pi protein of plasmid R6K is a multifunctional replication (Rep) protein, its different activities attributable, in part, to different oligomeric states: monomers and dimers. We have previously shown that His-tagged variants of the protein can exhibit alterations in dimer stability. Herein, we examined the functional properties of selected His-tagged derivatives of pi (His-pi x wt and three hyperactive replication variants) to determine if the functionality of these proteins in replication, DNA binding, and oligomerization is altered. Our results indicate that these tagged proteins retain the characteristics previously demonstrated for their non-tagged counterparts making them suitable for ongoing studies of pi protein structure and functions in replication and transcription.

  • Forest KT, Filutowicz MS (2003) Remodeling of replication initiator proteins. Nat. Struct. Biol. 10(7):496-8 View Abstract · Pubmed Record
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    No abstract available.

  • Pluciennik A, Iyer RR, Napierala M, Larson JE, Filutowicz M, Wells RD (2002) Long CTG.CAG repeats from myotonic dystrophy are preferred sites for intermolecular recombination. J. Biol. Chem. 277(37):34074-86 View Abstract · Pubmed Record
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    Homologous recombination was shown to enable the expansion of CTG.CAG repeat sequences. Other prior investigations revealed the involvement of replication and DNA repair in these genetic instabilities. Here we used a genetic assay to measure the frequency of homologous intermolecular recombination between two CTG.CAG tracts. When compared with non-repeating sequences of similar lengths, long (CTG.CAG)(n) repeats apparently recombine with an approximately 60-fold higher frequency. Sequence polymorphisms that interrupt the homogeneity of the CTG.CAG repeat tracts reduce the apparent recombination frequency as compared with the pure uninterrupted repeats. The orientation of the repeats relative to the origin of replication strongly influenced the apparent frequency of recombination. This suggests the involvement of DNA replication in the recombination process of triplet repeats. We propose that DNA polymerases stall within the CTG.CAG repeat tracts causing nicks or double-strand breaks that stimulate homologous recombination. The recombination process is RecA-dependent.

  • Krüger R, Konieczny I, Filutowicz M (2001) Monomer/dimer ratios of replication protein modulate the DNA strand-opening in a replication origin. J. Mol. Biol. 306(5):945-55 View Abstract · Pubmed Record
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    DNA opening is an essential step in the initiation of replication via the Cairns mode of replication. The opening reaction was investigated in a gamma ori system by using hyperactive variants of plasmid R6K-encoded initiator protein, pi. Reactivity to KMnO4 (indicative of opening) within gamma ori DNA occurred in both strands of a superhelical template upon the combined addition of wt pi, DnaA and integration host factor (IHF), each protein known to specifically bind gamma ori. IHF, examined singly, enhanced reactivity to KMnO4. The IHF-dependent reactive residues, however, are distinct from those dependent on pi (wt and hyperactive variants). Remarkably, the DNA helix opening does not require IHF and/or DnaA when hyperactive variants of pi were used instead of wt protein. We present three lines of evidence consistent with the hypothesis that DNA strand separation is facilitated by pi monomers despite the fact that both monomers and dimers of the protein can bind to iterons (pi binding sites). Taken together, our data suggest that pi elicits its ability to modulate plasmid copy number at the DNA helix-opening step.

  • Krüger R, Filutowicz M (2000) Dimers of pi protein bind the A+T-rich region of the R6K gamma origin near the leading-strand synthesis start sites: regulatory implications. J. Bacteriol. 182(9):2461-7 (PMC111308) View Abstract · Pubmed Record
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    The replication of gamma origin, a minimal replicon derived from plasmid R6K, is controlled by the Rep protein pi. At low intracellular concentrations, pi activates the gamma origin, while it inhibits replication at elevated concentrations. Additionally, pi acts as a transcription factor (auto)repressing its own synthesis. These varied regulatory functions depend on pi binding to reiterated DNA sequences bearing a TGAGNG motif. However, pi also binds to a "non-iteron" site (i.e., not TGAGNG) that resides in the A+T-rich region adjacent to the iterons. This positioning places the non-iteron site near the start sites for leading-strand synthesis that also occur in the A+T-rich region of gamma origin. We have hypothesized that origin activation (at low pi levels) would require the binding of pi monomers to iterons, while the binding of pi dimers to the non-iteron site (at high pi levels) would be required to inhibit priming. Although monomers as well as dimers can bind to an iteron, we demonstrate that only dimers bind to the non-iteron site. Two additional pieces of data support the hypothesis of negative replication control by pi binding to the non-iteron site. First, pi binds to the non-iteron site about eight times less well than it binds to a single iteron. Second, hyperactive variants of pi protein (called copy-up) either do not bind to the non-iteron site or bind to it less well than wild-type pi. We propose a replication control mechanism whereby pi would directly inhibit primer formation.

  • Wu J, Filutowicz M (2000) Hexahistidine (His6)-tag dependent protein dimerization: a cautionary tale. Acta Biochim. Pol. 46(3):591-9 View Abstract · Pubmed Record
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    Nickel nitrilotriacetic acid (Ni2+-NTA) immobilization of hexahistidine (His6) tagged proteins has become one of the most commonly used methods of affinity chromatography. Perhaps the greatest utility of this protein purification method stems from the general belief that His-tagged proteins (comprised of His6) are little affected in their activities or efficiencies, while alterations in specificity are unexpected. Although this is certainly true in many instances, we present a case in which the biochemical properties of proteins being studied were fundamentally altered due to the presence of His-tags. We carried out these studies using variants of the pi(30.5) protein of plasmid R6K, a DNA binding protein which negatively regulates plasmid replication. Pi(30.5) can bind DNA containing a target sequence (TGAGR) arranged either asymmetrically (direct repeats) in the gamma origin, or symmetrically in inverted half-repeats (IR's) in the operator of its own gene, pir. Importantly, dimers of pi protein bind to an IR; this property allows researchers to quickly assess whether different regulatory variants of pi proteins exhibit altered dimerization properties. For example, pi(30.5) containing a single amino-acid substitution, F107S (pi200(30.5)), has been shown to be monomeric in solution and dimers were not observed bound to IR's. Here we demonstrate that the presence of a His-tag partially restores the ability of pi200(30.5) to dimerize in solution and bind to an IR in dimeric form. This report sends an important message that (other) proteins containing His-tags may differ from their wild type counterparts in dimerization/oligomerization properties.

  • Filutowicz M, Rakowski SA (1998) Regulatory implications of protein assemblies at the gamma origin of plasmid R6K - a review. Gene 223(1-2):195-204 View Abstract · Pubmed Record
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    Recognition of the replication origin (ori) by initiator protein is a recurring theme for the regulated initiation of DNA replication in diverse biological systems. The objective of the work reviewed here is to understand the initiation process focusing specifically on the gamma-ori of the antibiotic-resistance plasmid R6K. The control of gamma-ori copy number is determined by both plasmid-encoded and host-encoded factors. The two central regulatory elements of the plasmid are a multifunctional initiator protein pi, and sequence-related DNA target sites, the inverted half-repeats (IRs) and the direct repeats (DRs). The replication activator and inhibitor activities of pi seem to be at least partially distributed between two naturally occurring pi polypeptides (designated by their molecular weights pi35.0 and pi30.5). Regulatory variants of pi with altered states of oligomerization in nucleoprotein complexes with DRs and IRs have been isolated. The properties of these mutants laid the foundation for our model of pi protein activity which proposes that different protein surfaces are required for the formation of functionally distinct complexes of pi with DRs and IRs. These mutants also suggest that pi polypeptides have a modular structure; the C-terminus contains the DNA-binding domain while the N-terminus controls protein oligomerization. Additionally, pi35.0 binds to a novel DNA sequence in the A+T-rich segment of gamma-ori. This binding site is at or near the site from which synthesis of the leading strand begins.

  • Urh M, Wu J, Wu J, Forest K, Inman RB, Filutowicz M (1998) Assemblies of replication initiator protein on symmetric and asymmetric DNA sequences depend on multiple protein oligomerization surfaces. J. Mol. Biol. 283(3):619-31 View Abstract · Pubmed Record
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    The pi35.0 protein of plasmid R6K regulates transcription and replication by binding a DNA sequence motif (TGAGR) arranged either asymmetrically into 22 bp direct repeats (DRs) in the gamma origin, or symmetrically into inverted half-repeats (IRs) in the operator of its own gene, pir. The binding patterns of the two natural forms of the pi protein and their heterodimers revealed that the predominant species, pi35.0 (35.0 kDa), can bind to a single copy of the DR as either a monomer or a dimer while pi30.5 (30.5 kDa) binds only as a dimer. We demonstrate that only one subunit of a pi35.0 dimer makes specific contact with DNA. Electron microscopic (EM) analysis of the nucleoprotein complexes formed by pi35.0 and DNA fragments containing all seven DRs revealed coupled ("hand-cuffed") DNA molecules that are aligned in a parallel orientation. Antiparallel orientations of the DNA were not observed. Thus, hand-cuffing depends on a highly ordered oligomerization of pi35.0 in such structures. The pi protein (pi35.0, pi30.5) binds to an IR as a dimer or heterodimer but not as a monomer. Moreover, a single amino acid residue substitution, F200S (pir200), introduced into pi30.5 severely destabilizes dimers of this protein in solution and concomitantly prevents binding of this protein to the IR. This mutation also changes the stability of pi35.0 dimers but it does not change the ability of pi35.0 to bind IRs. To explain these observations we propose that the diverse interactions of pi variants with DNA are controlled by multiple surfaces for protein oligomerization.

  • Chen D, Feng J, Krüger R, Urh M, Inman RB, Filutowicz M (1998) Replication of R6K gamma origin in vitro: discrete start sites for DNA synthesis dependent on pi and its copy-up variants. J. Mol. Biol. 282(4):775-87 View Abstract · Pubmed Record
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    The regulation of the plasmid R6K gamma origin (gamma ori) is accomplished through the ability of the pi protein to act as an initiator and inhibitor of replication. Hyperactive variants of this protein, called copy-up pi, allow four to tenfold increases of gamma ori plasmid DNA in vivo. The higher activity of copy-up pi variants could be explained by an increase in the initiator function, a decrease in the inhibitor activity, or a derepression of a more efficient mechanism of replication that can be used by wt pi (pi35. 0) only under certain conditions. We have compared the replication activities of wt pi35.0 and copy-up pi mutants in vitro, and analyzed the replication products. It is shown that copy-up variants are several-fold more active than wt pi35.0 in replication. This appears to be due to enhanced specific replication activity of copy-up mutants rather than elevated fractions of protein proficient in DNA binding. Furthermore, biochemical complementation revealed that pi200 (copy-up) is dominant over wt pi35.0. The elevated activity of copy-up pi is not caused by an increased rate of replisome assembly as inferred from in vitro replication assays in which the lag periods observed were similar to that of wt pi35.0. Moreover, only one round of semiconservative, unidirectional replication occurred in all the samples analyzed indicating that copy-up pi proteins do not initiate multiple rounds of DNA synthesis. Rather, a larger fraction of DNA template replicates in the presence of copy-up pi as determined by electron microscopy. Two clusters of discrete DNA synthesis start sites are mapped by primer extension near the stability (stb) locus of the gamma ori. We show that the start sites are the same in the presence of wt pi35.0 or copy-up proteins. This comparative analysis suggests that wt pi35.0 and copy-up variants utilize fundamentally similar mechanism(s) of replication priming.

  • Wu J, Sektas M, Chen D, Filutowicz M (1997) Two forms of replication initiator protein: positive and negative controls. Proc. Natl. Acad. Sci. U.S.A. 94(25):13967-72 (PMC28416) View Abstract · Pubmed Record
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    The pir gene of plasmid R6K encodes the protein, pi, a replication and transcription factor. Two translational options for the pir gene give rise to two forms of pi protein: a 35.0-kDa form (pi35.0) and a shortened 30.5-kDa form (pi30.5). Although both proteins bind to a series of 22-bp direct repeats essential for plasmid R6K replication, only pi35.0 can bind to a site in the (A.T)-rich segment of its gamma ori and activate the gamma ori in vivo and in vitro. However, unlike pi35.0, pi30.5can inhibit in vivo and in vitro replication (activated by pi35.0). We propose that the two forms of pi might have distinct functions in replication. We show that although both forms of pi produce dimers, the nature of these dimers is not identical. The N-terminal 37 amino acid residues appear to control the formation of the more stable pi35.0 dimers, whereas another, apparently weaker interface holds together dimers of pi30.5. We speculate that the leucine zipper-like motif, absent in pi30.5, controls very specific functions of pi protein.

  • Levchenko I, Inman RB, Filutowicz M (1997) Replication of the R6K gamma origin in vitro: dependence on wt pi and hyperactive piS87N protein variant. Gene 193(1):97-103 View Abstract · Pubmed Record
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    The pi protein of plasmid R6K is involved in control of replication. The aim of this study was to use an in vitro replication system dependent on an R6K-derived gamma origin of replication (gamma ori) to compare replication characteristics of wt pi and a hyperactive variant of pi protein (piS87N; Filutowicz et al., 1994b. Cooperative binding of initiator protein to replication origin conferred by single amino acid substitution. Nucleic Acids Res. 22, 4211-4215). The characteristics of in vitro replication from gamma ori reported in this investigation are as follows: (i) piS87N is considerably more active in comparison to wt pi. (ii) Replication proceeds through Cairns-type intermediates and the initiation site and directionality of the fork movement are similar in the presence of both proteins. (iii) Replication forks emanate unidirectionally in the vicinity of the cluster of seven 22-bp direct repeats within gamma ori. (iv) Replication dependent on wt pi, but not piS87N, is stimulated up to 1.5-fold by rifampicin.

  • Wu F, Wu J, Ehley J, Filutowicz M (1996) Preponderance of Fis-binding sites in the R6K gamma origin and the curious effect of the penicillin resistance marker on replication of this origin in the absence of Fis. J. Bacteriol. 178(16):4965-74 (PMC178281) View Abstract · Pubmed Record
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    Fis protein is shown here to bind to 10 sites in the gamma origin of plasmid R6K. The Fis-binding sites overlap all the previously identified binding sites in the gamma origin for the plasmid-encoded pi initiator protein and three host-encoded proteins, DnaA, integration host factor, and RNA polymerase. However, the requirement of Fis for R6K replication depends on the use of copy-up pi-protein variants and, oddly, the antibiotic resistance marker on the plasmid. In Fis-deficient cells, copy-up pi variants cannot drive replication of R6K gamma-origin plasmids carrying the bla gene encoding resistance to penicillin (Penr) but can drive replication of plasmids with the same origin but carrying the chloramphenicol acetyltransferase gene encoding chloramphenicol resistance (Cmr). In contrast, R6K replication driven by wild-type pi is unaffected by the antibiotic resistance marker in the absence of Fis protein. Individually, none of these elements (copy-up pi, Fis deficiency, or drug markers) prevents R6K replication. The replication defect is not caused by penicillin in the medium or runaway replication and is unaffected by the orientation of the bla gene relative to the origin. Replication remains inhibited when part of the bla coding segment is deleted but the bla promoter is left intact. However, replication is restored by insertion of transcriptional terminators on either side of the gamma origin, suggesting that excess transcription from the bla gene may inactivate replication driven by pi copy-up mutants in the absence of Fis. This study suggests that vector sequences such as drug markers may not be inconsequential in replication studies, as is generally assumed.

  • Levchenko I, Filutowicz M (1996) Initiator protein pi can bind independently to two domains of the gamma origin core of plasmid R6K: the direct repeats and the A+T-rich segment. Nucleic Acids Res. 24(10):1936-42 (PMC145866) View Abstract · Pubmed Record
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    The pi protein of plasmid R6K functions in both replication and transcription. pi autoregulates its own synthesis and is required for replication of the RISK gamma origin. pi performs these functions by binding to specific DNA sites arranged as pairs of 6-10 bp inverted repeats (IRs) or as a cluster of seven tandem 22 bp direct repeats (DRs) which lack symmetry. The sites share the TGAGRG nucleotide motif (where R is A or G). The DRs and IRs flank the central A+T-rich segment of the gamma origin. In this work we carried out DNase I and hydroxyl radical protection experiments on various deletion derivatives of the gamma origin complexed with pi protein. These experiments revealed binding of pi to a novel site embedded within the A+T-rich segment. This interaction manifests primarily by the appearance of the enhanced scissions of DNA by DNase I and hydroxyl radicals. pi interaction with the A+T-rich site is independent of pi binding to the DRs and IRs. We propose that pi protein can recognize distinct families of DNA sequences in the gamma origin.

  • Dellis S, Feng J, Filutowicz M (1996) Replication of plasmid R6K gamma origin in vivo and in vitro: dependence on IHF binding to the ihf1 site. J. Mol. Biol. 257(3):550-60 View Abstract · Pubmed Record
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    The gamma origin of plasmid R6K requires the specific initiator protein pi for initiation of replication. However, increased pi concentrations inhibit replication. The host-encoded integration host factor (IHF) protein permits gamma origin replication at otherwise inhibitory pi levels. IHF is thought to mediate this positive effect by directly binding to the gamma origin. In this study we demonstrate that IHF binding to one IHF site in the gama origin, ihf1, but not to the other side, ihf2, is necessary for the gamma origin to replicate at high pi protein levels. We also show that in vitro replication of the gamma origin plasmid requires IHF binding to the ihf1 site. Finally, we demonstrate both in vivo and in vitro that, when mutant pi proteins (hyperactive) are provided instead of wild-type pi, gamma origin plasmids can replicate in the absence of IHF. This supports a previously proposed hypothesis that the pi mutants can bypass the IHF requirement for gamma origin replication.

  • Urh M, Flashner Y, Shafferman A, Filutowicz M (1995) Altered (copy-up) forms of initiator protein pi suppress the point mutations inactivating the gamma origin of plasmid R6K. J. Bacteriol. 177(23):6732-9 (PMC177536) View Abstract · Pubmed Record
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    The R6K gamma origin core contains the P2 promoter, whose -10 and -35 hexamers overlap two of the seven binding sites for the R6K-encoded pi protein. Two mutations, P2-201 and P2-203, which lie within the -35 region of P2, are shown to confer a promoter-down phenotype. We demonstrate here that these mutations prevent replication of a gamma origin core plasmid. To determine whether or not the reduced promoter activity caused by these mutations is responsible for their effect on replication, we generated two new mutations (P2-245-6-7 and P2-246) in the -10 hexamer of the P2 promoter. Although these new mutations inhibit P2 activity as much as the P2-201 and P2-203 mutations, they do not prevent replication of the gamma origin core. Therefore, activity of the P2 promoter does not appear to be required for replication. We also show that the inability of the gamma origin to function in the presence of the P2-201 and P2-203 mutations is reversed by the hyperactive variants of pi protein called copy-up pi. This suppression occurs despite the fact that in vivo dimethyl sulfate methylation protection patterns of the gamma origin iterons are identical in cells producing wild-type pi and those producing copy-up pi variants. We discuss how the P2-201 and P2-203 mutations could inhibit replication of the gamma origin core and what mechanisms might allow the copy-up pi mutants to suppress this deficiency.

  • Wu F, Levchenko I, Filutowicz M (1995) A DNA segment conferring stable maintenance on R6K gamma-origin core replicons. J. Bacteriol. 177(22):6338-45 (PMC177482) View Abstract · Pubmed Record
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    The plasmid R6K gamma origin consists of two adjacent modules, the enhancer and the core, and requires R6K initiator protein pi for replication. While the core alone can replicate at a low level of wild-type pi protein, we show here that host cells do not stably maintain core plasmids. The presence of the enhancer segment confers stable inheritance on core plasmids without a significant change in average plasmid copy number. Deletions and site-directed mutagenesis indicated that the stability of core plasmids is not mediated by binding sites or consensus sequences in the enhancer for DnaA, pi protein, gyrase, Fis, or Dcm methylase. Proper segregation of core plasmids requires only the R6K stb or stability-related region, which includes the 20-bp segment of the 100-bp enhancer adjacent to the core. The use of the pi 116 mutant protein, which increases plasmid copy number fourfold, does not stabilize core plasmids lacking the enhancer. We also show that at an elevated level of wild-type pi, the gamma-origin plasmid is unstable, even in the presence of the enhancer. We discuss the differences and similarities between the R6K stability system and those found in other plasmids.

  • Urh M, York D, Filutowicz M (1995) Buffer composition mediates a switch between cooperative and independent binding of an initiator protein to DNA. Gene 164(1):1-7 View Abstract · Pubmed Record
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    The regulation of many biological processes, including DNA replication, is frequently achieved by protein-protein interactions, as well as protein-DNA interactions. Multiple protein-binding sites are often involved. For example, the replication of plasmid R6K involves binding of the initiator protein pi to seven 22-bp direct repeats (DR) in the gamma origin of replication (gamma ori). A mutant protein pi S87N has been isolated, that in Tris.borate buffer (TB) binds cooperatively to seven DR, whereas wild-type (wt) pi binds independently [Filutowicz et al., Nucleic Acids Res. 22 (1994) 4211-4215]. Surprisingly, we found that wt pi can also bind cooperatively when Tris.acetate (TA), Tris.succinate or Tris.glutamate buffers are used instead of TB. The cooperative binding of the wt pi protein was also observed in the TB buffer at high concentrations of Na2EDTA. These results suggest that pi may be able to assume two functionally distinct conformations as a result of either mutation or buffer composition. Moreover, we found that the mode of pi binding is determined not by the composition of the buffer in which the reaction was assembled, but by the composition of the electrophoresis buffer. We discuss the general implications of these findings.

  • Wu F, Levchenko I, Filutowicz M (1994) Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein. J. Bacteriol. 176(22):6795-801 (PMC197046) View Abstract · Pubmed Record
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    Replication of the gamma origin of Escherichia coli plasmid R6K requires pi protein, encoded by the R6K pir gene, and many host factors, including DnaA protein. Pi has dual roles, activating replication at low levels and inhibiting replication at high levels. The inhibitory function of pi is counteracted by integration host factor and a specific sequence of the origin called the enhancer. This 106-bp DNA segment contains a binding site for DnaA protein (DnaA box 1). In this study, we mutated this site to determine if it was required for the enhancer's function. Using gamma origin derivative plasmids with the DnaA box 1 altered or deleted, we show that this site is necessary to protect the origin against levels of wild-type pi protein that would otherwise inhibit replication. To show that the base substitutions in DnaA box 1 weakened the binding of DnaA, we developed a new application of the agarose gel retardation assay. This quick and easy assay has broad applicability, as shown in binding studies with DNA fragments carrying a different segment of the R6K origin, the chromosomal origin (oriC), or the pUC origin. The gel retardation assay suggests a stoichiometry of DnaA binding different from that deduced from other assays.

  • Filutowicz M, Dellis S, Levchenko I, Urh M, Wu F, York D (1994) Regulation of replication of an iteron-containing DNA molecule. Prog. Nucleic Acid Res. Mol. Biol. 48:239-73 View Abstract · Pubmed Record
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    No abstract available.

  • Filutowicz M, York D, Levchenko I (1994) Cooperative binding of initiator protein to replication origin conferred by single amino acid substitution. Nucleic Acids Res. 22(20):4211-5 (PMC331923) View Abstract · Pubmed Record
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    The replication initiator protein pi of plasmid R6K binds seven 22 bp direct repeats (DR) in the gamma origin. The pi protein also binds to an inverted repeat (IR) in the operator of its own gene, pir, which lies outside the gamma origin sequences. A genetic system was devised to select for pi protein mutants which discriminate between IR and DR (York et al., Gene (Amst.) 116, 7-12, 1992; York and Filutowicz, J. Biol. Chem. 268, 21854-21861, 1993). From this selection the mutant pi S87N protein was isolated which is deficient in repressing the pir gene's expression because it cannot bind to IR at the pir gene operator. Remarkably, we discovered that pi S87N binds to DR cooperatively under conditions where wt pi binds independently. Moreover, the pi S87N is more active as a replication initiator in vivo when supplied at the same level as wt pi. Quantitative binding assays showed that both wt pi and pi S87N bind a DNA fragment containing a single DR unit with a similar affinity (Kd = 0.3 x 10(-12) M). Thus, cooperativity of pi S87N is most likely achieved through altered interactions between promoters bound at adjacent DR units.

  • Filutowicz M, Grimek H, Appelt K (1994) Purification of the Escherichia coli integration host factor (IHF) in one chromatographic step. Gene 147(1):149-50 View Abstract · Pubmed Record
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    The integration host factor (IHF) participates in a diverse array of DNA transactions such as replication, recombination and gene expression. We describe a fast and very efficient isolation procedure which yields highly purified IHF in one chromatographic step.

  • Levchenko I, York D, Filutowicz M (1994) The dimerization domain of R6K plasmid replication initiator protein pi revealed by analysis of a truncated protein. Gene 145(1):65-8 View Abstract · Pubmed Record
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    Replication of plasmid R6K is controlled by the homodimeric initiator protein pi, which binds to seven 22-bp direct repeats (iterons) in the gamma-origin. One of the genetically engineered pi variants (delta C164 pi) contains only the 164 N-terminal amino acids (aa) of the 305-aa pi molecule. This truncated pi polypeptide retains the ability to function as a specific inhibitor of R6K replication in vivo, though it neither drives replication, nor binds to iterons [Greener et al., Mol. Gen. Genet. 224 (1990) 24-32]. In order to define the region of pi responsible for dimerization, we have performed chemical crosslinking experiments with purified delta C164 pi and shown that this polypeptide is dimeric. We did not observe an exchange between protein monomers upon mixing of wild-type pi and delta C164 pi homodimers. However, heterodimers, as well as each type of homodimers, were formed when these polypeptides refolded after guanidine hydrochloride treatment. Thus, both dimerization and dimer stability are determined by the N-terminal domain of pi. We speculate that these properties might depend on the leucine zipper and RGD motifs that have been identified in the two regions of the N-terminal domain of pi.

  • Pósfai G, Koob M, Hradecná Z, Hasan N, Filutowicz M, Szybalski W (1994) In vivo excision and amplification of large segments of the Escherichia coli genome. Nucleic Acids Res. 22(12):2392-8 (PMC523700) View Abstract · Pubmed Record
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    In vivo excision and amplification of large segments of a genome offer an alternative to heterologous DNA cloning. By obtaining predetermined fragments of the chromosome directly from the original organism, the problems of clone stability and clone identification are alleviated. This approach involves the insertion of two recognition sequences for a site-specific recombinase into the genome at predetermined sites, 50-100 kb apart. The integration of these sequences, together with a conditional replication origin (ori), is targeted by homologous recombination. The strain carrying the insertions is stably maintained until, upon induction of specifically engineered genes, the host cell expresses the site-specific recombinase and an ori-specific replication protein. The recombinase then excises and circularizes the genomic segment flanked by the two insertions. This excised DNA, which contains ori, is amplified with the aid of the replication protein and can be isolated as a large plasmid. The feasibility of such an approach is demonstrated here for E. coli. Using the yeast FLP/FRT site-specific recombination system and the pi/gamma-ori replication initiation of plasmid R6K, we have devised a procedure that should allow the isolation of virtually any segment of the E. coli genome. This was shown by excising, amplifying and isolating the 51-kb lacZ--phoB and the 110-kb dapX--dsdC region of the E. coli MG1655 genome.

  • York D, Filutowicz M (1993) Autoregulation-deficient mutant of the plasmid R6K-encoded pi protein distinguishes between palindromic and nonpalindromic binding sites. J. Biol. Chem. 268(29):21854-61 View Abstract · Pubmed Record
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    The autogenously regulated gene pir of Escherichia coli plasmid R6K encodes the replication protein pi. This protein binds to two sites in the operator region of the pir gene: a 22-base pair nonpalindromic sequence and a pair of palindromic 9-base pair sequences. These pi-binding sites are similar, suggesting that pi uses a single DNA-binding domain in recognizing them. We devised a plasmid system permitting isolation of mutants of the pi protein which are altered in autoregulation. A Ser87 to Asn substitution in one such mutant, designated pi 87, reduces the protein's ability to repress the pir gene promoter in vivo. DNase I protection and gel retardation assays were carried out with highly purified pi 87 protein. In these studies pi 87 exhibited altered binding to the palindromic but not to the nonpalindromic part of the operator of the pir gene. Chemical cross-linking and gel filtration analyses have shown that the dimerization properties of wild type pi and pi 87 proteins are similar in solution. We propose that the interaction of pi protein with the palindromic part of the pir operator is essential for autoregulation; we also propose that there is a fundamental difference in the mechanisms of pi protein recognition of palindromic and nonpalindromic sequences.

  • Dellis S, Schatz T, Rutlin K, Inman RB, Filutowicz M (1992) Two alternative structures can be formed by IHF protein binding to the plasmid R6K gamma origin. J. Biol. Chem. 267(34):24426-32 View Abstract · Pubmed Record
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    Escherichia coli integration host factor (IHF) contributes to the regulation of R6K plasmid copy number by counteracting the inhibitory activity of the plasmid-encoded replication protein pi. Two IHF-binding sites (ihf1 and ihf2) flank seven iterons in the origin which bind pi protein. As previously shown by electron microscopy, IHF can compact a large segment of the R6K gamma origin DNA, encompassing site ihf1, an AT-rich domain containing ihf1, and some of the seven iterons located downstream of ihf1. We termed this phenomenon IHF-mediated DNA folding. This folding requires a high IHF concentration, and the region of the origin (replication enhancer) located to the left of the AT-rich domain. However, site ihf2 is not necessary in forming the folded structure. As reported here, IHF binding to ihf2 can be detected in gel mobility shift assays only if the leftmost enhancer region is absent. Sites ihf1 and ihf2 each contain two consensus IHF sequences. Site-directed mutagenesis was performed to determine which sequences are recognized by IHF protein and which sites are involved in forming the various gamma origin-IHF complexes. Finally, we define the boundaries of protection from DNaseI digestion when IHF is bound to ihf2. We propose a model in which IHF protein bound to ihf1, in the absence of the enhancer region, facilitates IHF binding to ihf2.

  • York D, Ivanov V, Gan J, Filutowicz M (1992) Translational options for the pir gene of plasmid R6K: multiple forms of the replication initiator protein pi. Gene 116(1):7-12 View Abstract · Pubmed Record
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    The autogenously controlled pir gene of plasmid R6K was believed to encode a single polypeptide that plays multiple roles in the plasmid's biology. We have isolated an opal (op) mutant at the 18th codon of the pir coding frame which does not totally abolish translation of pir mRNA. In extracts of cells containing this mutation two translational products (35 kDa and 30.2 kDa) have been detected. We propose that the 35-kDa polypeptide produced by the pir18 op mutation contains Trp substituted for Arg18 as the result of an opal readthrough. Translation, which results in the 30.2-kDa polypeptide, originates downstream from the UGA stop signal created by the mutation. Moreover, we realize now that the 30.2-kDa polypeptide is also produced in cells containing a wild-type (wt) pir gene. The shorter variant of the pi protein lacks replication initiation and inhibition functions, as well as autorepressor activity in vivo. We also show that an in-frame fusion of seven N-terminal codons of the trpE gene with a pir gene lacking the first two codons produces two polypeptides which replace the 35-kDa pi protein and are of similar molecular weight. Thus, at least three options exist in the translation of the wt pir mRNA. Start codons are most likely at codon positions 1, 6 or 7, and 36 or 38. Each of these five AUG codons is preceded by a consensus ribosome-binding site (RBS).

  • Mukerji P, Greener A, Filutowicz M (1992) Identification of a novel promoter in the replication control region of plasmid R6K. J. Bacteriol. 174(14):4777-82 (PMC206275) View Abstract · Pubmed Record
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    A novel source of transcription has been detected in the replication region of plasmid R6K by using fusions involving the galK reporter gene. The -35 and -10 consensus RNA polymerase binding sites were identified in the region overlapping the binding sites for the R6K-encoded replication protein pi. Transcription from this promoter, designated P2, is repressed in vivo by pi-protein levels that are inhibitory for replication. Promoter-down mutations in P2 induced in vitro by bisulfite mutagenesis result in a reduced copy number of a beta-replicon but not of a gamma-replicon. Implications of the role of P2 in R6K replication are discussed.

  • Wu F, Goldberg I, Filutowicz M (1992) Roles of a 106-bp origin enhancer and Escherichia coli DnaA protein in replication of plasmid R6K. Nucleic Acids Res. 20(4):811-7 (PMC312022) View Abstract · Pubmed Record
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    A dnaA 'null' strain could not support replication of intact plasmid R6K or derivatives containing combinations of its three replication origins (alpha, gamma, beta). DnaA binds in vitro to sites in two functionally distinct segments of the central gamma origin. The 277-bp core segment is common to all three origins and contains DnaA box 2, which cannot be deleted without preventing replication. Immediately to the left of the core lies the 106-bp origin enhancer, which contains DnaA box 1. When the origin enhancer is deleted, the core alone can still initiate replication if levels of wt pi protein are decreased or if copy-up pi mutant proteins are provided in trans. DnaA does not effect expression of R6K replication initiator protein pi, although several DnaA boxes were identified in the coding segment of the pir gene, which encodes pi. Together these data suggest that a single DnaA box, 2, is sufficient for initiation from the gamma origin and might be sufficient for initiation from the gamma origin and might be sufficient and required for the activity of the alpha and beta origins as well. Implications of the DnaA protein binding to two domains of the gamma origin and the role of the 106-bp origin enhancer in replication are discussed.

  • Filutowicz M, Ross W, Wild J, Gourse RL (1992) Involvement of Fis protein in replication of the Escherichia coli chromosome. J. Bacteriol. 174(2):398-407 (PMC205730) View Abstract · Pubmed Record
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    We report evidence indicating that Fis protein plays a role in initiation of replication at oriC in vivo. At high temperatures, fis null mutants form filamentous cells, show aberrant nucleoid segregation, and are unable to form single colonies. DNA synthesis is inhibited in these fis mutant strains following upshift to 44 degrees C. The pattern of DNA synthesis inhibition upon temperature upshift and the requirement for RNA synthesis, but not protein synthesis, for resumed DNA synthesis upon downshift to 32 degrees C indicate that synthesis is affected in the initiation phase. fis mutations act synergistically with gyrB alleles known to affect initiation. oriC-dependent plasmids are poorly established and maintained in fis mutant strains. Finally, purified Fis protein interacts in vitro with sites in oriC. These interactions could be involved in mediating the effect of Fis on DNA synthesis in vivo.

  • Filutowicz M, Inman R (1991) A compact nucleoprotein structure is produced by binding of Escherichia coli integration host factor (IHF) to the replication origin of plasmid R6K. J. Biol. Chem. 266(35):24077-83 View Abstract · Pubmed Record
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    Understanding the role of Escherichia coli histone-like protein integration host factor (IHF) in replication of R6K plasmid (Dellis, S., and Filutowicz, M. (1991) J. Bacteriol. 173, 1279-1286) requires detailed analyses of the interaction of IHF protein with the plasmid's replication origin (gamma ori). We describe an electron microscopic analysis which shows that a compact structure can be formed in the presence of IHF, in which, on average, a 102-base pair (bp) ori segment is involved. IHF.gamma ori complexes also undergo a two-step conformational change in an IHF concentration-dependent manner when analysed by band shift assay. We believe that the DNA is bent at low IHF concentrations, but folded at high IHF concentrations. This idea is supported by the fact that electrophoretic mobility of the IHF.gamma ori complexes is faster at higher concentrations of IHF. Furthermore, it is shown that the formation of a compact nucleoprotein structure depends on the two regions flanking the AT-rich segment; the iterons to the right and the 106-bp ori domain to the left. Finally we show that IHF protects the entire AT-rich segment of the ori against nuclease cleavage. In addition to the protection, an altered cleavage pattern by DNase I, in the presence of high levels of IHF, was observed within the iterons but not within the 106-bp domain of the ori. Implications of the IHF-mediated gamma ori folding as a possible mechanism protecting the ori from replication inhibition by R6K initiator protein tau are discussed.

  • Dellis S, Filutowicz M (1991) Integration host factor of Escherichia coli reverses the inhibition of R6K plasmid replication by pi initiator protein. J. Bacteriol. 173(3):1279-86 (PMC207252) View Abstract · Pubmed Record
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    Integration host factor (IHF) protein is the only host-encoded protein known to bind and to affect replication of the gamma origin of Escherichia coli plasmid R6K. We examined the ability of R6K origins to replicate in cells lacking either of the two subunits of IHF. As shown previously, the gamma origin cannot replicate in IHF-deficient cells. However, this inability to replicate was relieved under the following conditions: underproduction of the wild-type pi replication protein of R6K or production of normal levels of mutant pi proteins which exhibit relaxed replication control. The copy number of plasmids containing the primary R6K origins (alpha and beta) is substantially reduced in IHF-deficient bacteria. Furthermore, replication of these plasmids is completely inhibited if the IHF-deficient strains contain a helper plasmid producing additional wild-type pi protein. IHF protein has previously been shown to bind to two sites within the gamma origin. These sites flank a central repeat segment which binds pi protein. We propose a model in which IHF binding to its sites reduces the replication inhibitor activity of pi protein at all three R6K origins.

  • Greener A, Filutowicz MS, McEachern MJ, Helinski DR (1990) N-terminal truncated forms of the bifunctional pi initiation protein express negative activity on plasmid R6K replication. Mol. Gen. Genet. 224(1):24-32 View Abstract · Pubmed Record
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    The replication initiation protein pi of the Escherichia coli plasmid R6K is a dual regulator in the control of plasmid copy number, functioning both as a specific initiator and inhibitor of replication. While the biochemical basis of these activities is not known, initiator activity requires binding of the protein to the seven 22 bp direct repeats within the gamma-origin region. By deleting C-terminal segments of the pi coding region, we have found that the N-terminal polypeptides of pi that are produced, corresponding to the first 117 and 164 amino acids, respectively, retain the negative activity of the bifunctional protein, i.e. these truncated pi proteins specifically inhibit R6K replication in vivo. These negatively acting polypeptides, however, are incapable of initiating replication in vivo and fail to bind to the gamma-origin of the R6K DNA in vitro. A correspondence between the observed negative activity of the N-terminal peptide and the negative regulatory activity of the intact pi protein is supported by the finding that point mutations introduced into the 164 amino acid N-terminal peptide that result in a decrease in its inhibitory activity also produce a plasmid high-copy phenotype when these mutations are incorporated into the full-length pi protein. These findings demonstrate that the negative domain of pi resides in the N-terminal segment of the protein. Furthermore, the data obtained suggest that inhibition of R6K replication by pi does not require direct binding to DNA.

  • Filutowicz M, Roll J (1990) The requirement of IHF protein for extrachromosomal replication of the Escherichia coli oriC in a mutant deficient in DNA polymerase I activity. New Biol. 2(9):818-27 View Abstract · Pubmed Record
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    It is shown here that plasmids containing the replication origin of Escherichia coli (oriC) cannot replicate in an extrachromosomal state in E. coli cells with the polA1hip3 double mutation. This E. coli mutant is deficient in the polymerizing function of DNA polymerase I (Pol I) and is unable to produce functional IHF protein. The inability of the oriC minichromosomes to replicate in the absence of IHF is dependent on the absence of Pol I; cells with the polA+himA- or polA+hip- mutation, which are deficient in the alpha and beta subunits of the IHF heterodimer, respectively, can support replication of the oriC replicons. We propose that IHF-deficient cells utilize an alternative pathway of the DNA replication in which Pol I is required. In vitro DNA binding assays revealed that the IHF binding site resides between the oriC coordinates 110 and 122 and is adjacent to the DnaA "box" 1. Within the area protected by IHF we found at least 1 out of 11 GATC methylation sites present in oriC. The consequences of lack of IHF protein binding to the oriC and the indirect effects of the IHF deficiency on the oriC replication are discussed.

  • Filutowicz M, McEachern MJ, Mukhopadhyay P, Greener A, Yang SL, Helinski DR (1988) DNA and protein interactions in the regulation of plasmid replication. J. Cell Sci. Suppl. 7:15-31 View Abstract · Pubmed Record
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    As for bacterial and animal viruses that employ different mechanisms for their duplication in a host cell, plasmids have evolved different strategies to assure their hereditary stability or maintenance at a specific copy number during cell growth and division. A characteristic feature of plasmid replication control, however, is an involvement of one or more negatively controlling elements. Furthermore, a majority of the bacterial plasmids examined to date contain direct nucleotide sequence repeats at their origin of replication and encode a replication protein that binds to these repeat sequences. The binding of the replication protein (pi protein) specified by the antibiotic resistance plasmid R6K to a set of 22 base pair direct nucleotide sequence repeats is required for the initiation of replication at each of three origins of replication (alpha, beta and gamma) within a 4 Kb segment of R6K. The pi initiation protein is multifunctional in that it has both positive and negative activities in both controlling the initiation of replication and autoregulating its own synthesis. Similarly, the direct repeats of plasmid R6K and several other plasmid systems play more than one role in plasmid replication. These repeats, termed iterons, are not only required for origin activity but also exert a negative effect on plasmid copy number possibly as a result of their 'titration' of a plasmid encoded replication protein. The properties of plasmid replication proteins and direct nucleotide sequence repeats that are important for their opposing positive and negative roles in the regulation of the initiation of replication are described with particular emphasis on plasmid R6K of Escherichia coli.

  • Filutowicz M, Appelt K (1988) The integration host factor of Escherichia coli binds to multiple sites at plasmid R6K gamma origin and is essential for replication. Nucleic Acids Res. 16(9):3829-43 (PMC336559) View Abstract · Pubmed Record
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    Examination of the effect of the himA and himD mutants of E. coli on the maintenance of plasmid R6K has revealed that the gamma origin-containing replicons cannot be established in any of the mutants deficient in the production of E. coli Integration Host Factor (IHF). Contrary, the R6K derivatives containing other origins of the plasmid (alpha and/or beta) replicate in a host lacking functional IHF protein. We show that IHF protein binds specifically to a segment of the replication region which is essential for the activity of all three R6K origins. Mapping the IHF binding sequence with neocarzinostatin showed that the protein protects three segments of the origin: two strong binding sites reside within an AT-rich block, while the third, considerably weaker site is separated from the other two by a cluster of the seven 22 bp direct repeats. These seven repeats have been shown previously to bind the R6K-encoded initiator protein pi. We also demonstrate that the establishment of pi-origin complexes prior to IHF addition prevents the binding of the IHF protein to the gamma origin. The binding sequences of IHF and pi proteins do not overlap, therefore, we propose that the binding of pi protein alters the structure of the DNA and thereby prevents the subsequent binding of IHF protein.

  • Filutowicz M, McEachern MJ, Helinski DR (1986) Positive and negative roles of an initiator protein at an origin of replication. Proc Natl Acad Sci U S A. 83(24):9645-9 View Abstract · Pubmed Record
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    No abstract available.

  • Saraswat LD, Filutowicz M, Taylor SS (1986) Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli. J Biol Chem. 261(24):11091-6 View Abstract · Pubmed Record
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    No abstract available.

  • Mukhopadhyay P, Filutowicz M, Helinski DR (1986) Replication from one of the three origins of the plasmid R6K requires coupled expression of two plasmid-encoded proteins. J Biol Chem. 261(20):9534-9 View Abstract · Pubmed Record
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    No abstract available.

  • Filutowicz M, Uhlenhopp E, Helinski DR (1986) Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K. J Mol Biol. 187(2):225-39 View Abstract · Pubmed Record
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    No abstract available.

  • McEachern MJ, Filutowicz M, Helinski DR (1985) Mutations in direct repeat sequences and in a conserved sequence adjacent to the repeats result in a defective replication origin in plasmid R6K. Proc Natl Acad Sci U S A. 82(5):1480-4 View Abstract · Pubmed Record
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    No abstract available.

  • Filutowicz M, Davis G, Greener A, Helinski DR (1985) Autorepressor properties of the pi-initiation protein encoded by plasmid R6K. Nucleic Acids Res. 13(1):103-14 View Abstract · Pubmed Record
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    No abstract available.

  • Filutowicz M, McEachern M, Greener A, Mukhopadhyay P, Uhlenhopp E, Durland R, Helinski D (1985) Role of the pi initiation protein and direct nucleotide sequence repeats in the regulation of plasmid R6K replication. In: Plasmids in Bacteria, ed. D.R. Helinski et al. Plenum Press. New York. 30:125-40 View Abstract · Pubmed Record
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    No abstract available.

  • Stalker DM, Filutowicz M, Helinski DR (1983) Release of initiation control by a mutational alteration in the R6K pi protein required for plasmid DNA replication. Proc Natl Acad Sci U S A. 80(18):5500-4 View Abstract · Pubmed Record
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    No abstract available.

  • Schmidhauser TJ, Filutowicz M, Helinski DR (1983) Replication of derivatives of the broad host range plasmid RK2 in two distantly related bacteria. Plasmid 9(3):325-30 View Abstract · Pubmed Record
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    No abstract available.

  • Filutowicz M, Jonczyk P (1983) The gyrB gene product functions in both initiation and chain polymerization of Escherichia coli chromosome replication: suppression of the initiation deficiency in gyrB-ts mutants by a class of rpoB mutations. Mol Gen Genet. 191(2):282-7 View Abstract · Pubmed Record
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    No abstract available.

  • Wiater A, Filutowicz M, Hulanicka D (1982) A new class of mutants of the cysB regulatory gene for cysteine biosynthesis in Salmonella typhimurium. J Gen Microbiol. 128(8):1785-90 View Abstract · Pubmed Record
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    No abstract available.

  • Filutowicz M, Wiater A, Hulanicka D (1982) Delayed inducibility of sulphite reductase in cysM mutants of Salmonella typhimurium under anaerobic conditions. J Gen Microbiol. 128(8):1791-4 View Abstract · Pubmed Record
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    No abstract available.

  • Filutowicz M, Jonczyk P (1981) Essential role of the gyrB gene product in the transcriptional event coupled to dnaA-dependent initiation of Escherichia coli chromosome replication. Mol Gen Genet. 183(1):134-8 View Abstract · Pubmed Record
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    No abstract available.

  • Ciesla Z, Filutowicz M, Klopotowski T (1980) Involvement of the L-cysteine biosynthetic pathway in azide-induced mutagenesis in Salmonella typhimurium. Mutat Res. 70(3):261-8 View Abstract · Pubmed Record
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    No abstract available.

  • Filutowicz M (1980) Requirement of DNA gyrase for the initiation of chromosome replication in Escherichia coli K-12. Mol Gen Genet. 177(2):301-9 View Abstract · Pubmed Record
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    No abstract available.

  • Filutowicz M, Ciesla Z, Klopotowski T (1979) Interference of azide with cysteine biosynthesis in Salmonella typhimurium. J Gen Microbiol. 113(1):45-55 View Abstract · Pubmed Record
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    No abstract available.

  • Ciesla Z, Filutowicz M (1979) Azide mutagenesis in gram-negative bacteria: reversion of the mutagenic effect by L-cystine. Mutat Res. 66(3):301-5 View Abstract · Pubmed Record
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    No abstract available.

  • Stepien E, Filutowicz M, Fikus M (1979) Effect of temperature and 4,6'-diamidine-2-phenylindole on restriction of supercoiled Col E1 DNA by Eco RI endonuclease. Acta Biochim. Polon. 26(1-2):29-38 View Abstract · Pubmed Record
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    No abstract available.

  • Wild J, Filutowicz M, Klopotowski T (1978) Utilization of D-amino acids by dadR mutants of Salmonella typhimurium Arch Microbiol. 118(1):71-7 View Abstract · Pubmed Record
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    No abstract available.