scholarly journals TcpA, an FtsK/SpoIIIE Homolog, Is Essential for Transfer of the Conjugative Plasmid pCW3 in Clostridium perfringens

2007 ◽  
Vol 189 (21) ◽  
pp. 7782-7790 ◽  
Author(s):  
Jennifer A. Parsons ◽  
Trudi L. Bannam ◽  
Rodney J. Devenish ◽  
Julian I. Rood
Keyword(s):  
Clostridium Perfringens ◽  
Essential Role ◽  
Tetracycline Resistance ◽  
Conjugative Plasmid ◽  
Conjugative Transfer ◽  
Binding Motifs ◽  
Coupling Protein ◽  
Conjugation Process ◽  
Dna Translocase

ABSTRACT The conjugative tetracycline resistance plasmid pCW3 is the paradigm conjugative plasmid in the anaerobic gram-positive pathogen Clostridium perfringens. Two closely related FtsK/SpoIIIE homologs, TcpA and TcpB, are encoded on pCW3, which is significant since FtsK domains are found in coupling proteins of gram-negative conjugation systems. To develop an understanding of the mechanism of conjugative transfer in C. perfringens, we determined the role of these proteins in the conjugation process. Mutation and complementation analysis was used to show that the tcpA gene was essential for the conjugative transfer of pCW3 and that the tcpB gene was not required for transfer. Furthermore, complementation of a pCW3ΔtcpA mutant with divergent tcpA homologs provided experimental evidence that all of the known conjugative plasmids from C. perfringens use a similar transfer mechanism. Functional genetic analysis of the TcpA protein established the essential role in conjugative transfer of its Walker A and Walker B ATP-binding motifs and its FtsK-like RAAG motif. It is postulated that TcpA is the essential DNA translocase or coupling protein encoded by pCW3 and as such represents a key component of the unique conjugation process in C. perfringens.

scholarly journals The Putative Coupling Protein TcpA Interacts with Other pCW3-Encoded Proteins To Form an Essential Part of the Conjugation Complex

2009 ◽  
Vol 191 (9) ◽  
pp. 2926-2933 ◽  
Author(s):  
Jennifer A. Steen ◽  
Trudi L. Bannam ◽  
Wee Lin Teng ◽  
Rodney J. Devenish ◽  
Julian I. Rood
Keyword(s):  
Protein Interactions ◽  
Tetracycline Resistance ◽  
Conjugative Plasmid ◽  
Conjugative Transfer ◽  
Protein Protein Interactions ◽  
Coupling Protein ◽  
Derivatives Of ◽  
Two Hybrid ◽  
Chemical Cross Linking ◽  
Transfer Apparatus

ABSTRACT Conjugative plasmids encode antibiotic resistance determinants or toxin genes in the anaerobic pathogen Clostridium perfringens. The paradigm conjugative plasmid in this bacterium is pCW3, a 47-kb tetracycline resistance plasmid that encodes the unique tcp transfer locus. The tcp locus consists of 11 genes, intP and tcpA-tcpJ, at least three of which, tcpA, tcpF, and tcpH, are essential for the conjugative transfer of pCW3. In this study we examined protein-protein interactions involving TcpA, the putative coupling protein. Use of a bacterial two-hybrid system identified interactions between TcpA and TcpC, TcpG, and TcpH. This analysis also demonstrated TcpA, TcpC, and TcpG self-interactions, which were confirmed by chemical cross-linking studies. Examination of a series of deletion and site-directed derivatives of TcpA identified the domains and motifs required for these interactions. Based on these results, we have constructed a model for this unique conjugative transfer apparatus.

scholarly journals Two Novel Membrane Proteins, TcpD and TcpE, Are Essential for Conjugative Transfer of pCW3 in Clostridium perfringens

2014 ◽  
Vol 197 (4) ◽  
pp. 774-781 ◽  
Author(s):  
Jessica A. Wisniewski ◽  
Wee L. Teng ◽  
Trudi L. Bannam ◽  
Julian I. Rood
Keyword(s):  
Clostridium Perfringens ◽  
Cell Envelope ◽  
Tetracycline Resistance ◽  
Conjugative Plasmid ◽  
Conjugative Transfer ◽  
Western Blots ◽  
Content Type ◽  
Essential Components ◽  
Encoded Proteins ◽  
Transfer Apparatus

The anaerobic pathogenClostridium perfringensencodes either toxin genes or antibiotic resistance determinants on a unique family of conjugative plasmids that have a novel conjugation region, thetcplocus. Studies of the paradigm conjugative plasmid fromC. perfringens, the 47-kb tetracycline resistance plasmid pCW3, have identified severaltcp-encoded proteins that are involved in conjugative transfer and form part of the transfer apparatus. In this study, the role of the conserved hypothetical proteins TcpD, TcpE, and TcpJ was examined. Mutation and complementation analyses showed that TcpD and TcpE were essential for the conjugative transfer of pCW3, whereas TcpJ was not required. To analyze the TcpD and TcpE proteins inC. perfringens, functional hemagglutinin (HA)-tagged derivatives were constructed. Western blots showed that TcpD and TcpE localized to the cell envelope fraction independently of the presence of other pCW3-encoded proteins. Finally, examination of the subcellular localization of TcpD and TcpE by immunofluorescence showed that these proteins were concentrated at both poles ofC. perfringensdonor cells, where they are postulated to form essential components of the multiprotein complex that comprises the transfer apparatus.

Studies on the role of IS1216E in the formation and dissemination of poxtA-carrying plasmids in an Enterococcus faecium clade A1 isolate

2020 ◽  
Vol 75 (11) ◽  
pp. 3126-3130
Author(s):  
Xinxin Shan ◽  
Xin-Sheng Li ◽  
Nannan Wang ◽  
Stefan Schwarz ◽  
Su-Mei Zhang ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Blot Hybridization ◽  
Tetracycline Resistance ◽  
Southern Blot Hybridization ◽  
Conjugative Plasmid ◽  
Fusion Event ◽  
Mobilizable Plasmid ◽  
Antimicrobial Resistance Genes ◽  
Positive Isolate

Abstract Objectives To analyse the role of IS1216E in the dissemination of the phenicol-oxazolidinone-tetracycline resistance gene poxtA in an Enterococcus faecium clade A1 isolate. Methods MICs were determined by broth microdilution. The poxtA-positive isolate was typed by MLST. The two plasmids were characterized by PCR, conjugation, S1-PFGE, Southern blot hybridization and WGS analysis. The presence of translocatable units (TUs) was examined by PCR and sequencing. Results Isolate E1077 contains the 217661 bp conjugative plasmid pE1077-217 and the 23710 bp mobilizable plasmid pE1077-23. pE1077-217 harbours erm(B), aac(A)-aph(D), aadE, spw, lsa(E), lnu(B), aphA3 and dfrG, whereas pE1077-23 carries a Tn6657-like transposon containing poxtA and fexB. pE1077-23 was apparently formed by an IS1216E-mediated composite transposon–plasmid fusion event, involving a replicative transposition process. Conjugation experiments showed that pE1077-23 is mobilizable by pE1077-217. Moreover, a novel 31742 bp plasmid, pT-E1077-31, was found in a transconjugant. WGS analysis indicated that pT-E1077-31 was formed by the integration of a Tn6657-derived, IS1216E-based translocatable unit, which carried fexB and poxtA, into a copy of pE1077-23. Conclusions This study showed the presence of two cointegrate formation events in the formation and spread of a poxtA/fexB-carrying plasmid in E. faecium. One was the integration of a transposon into a plasmid while the other was the integration of a TU into a different site of the same type of plasmid-borne transposon from which it originated. In both events, IS1216E played a major role, suggesting that IS1216E-mediated transposition and translocation processes aid the dissemination and persistence of important antimicrobial resistance genes, such as poxtA, among enterococci.

scholarly journals Functional Characterization and Localization of the TcpH Conjugation Protein from Clostridium perfringens

2008 ◽  
Vol 190 (14) ◽  
pp. 5075-5086 ◽  
Author(s):  
Wee Lin Teng ◽  
Trudi L. Bannam ◽  
Jennifer A. Parsons ◽  
Julian I. Rood
Keyword(s):  
Clostridium Perfringens ◽  
Protein Interactions ◽  
Functional Characterization ◽  
Pair Formation ◽  
Conjugative Plasmid ◽  
Mating Pair ◽  
Protein Protein Interactions ◽  
Form Part ◽  
Coupling Protein ◽  
Two Hybrid

ABSTRACT In Clostridium perfringens, conjugative plasmids encode important virulence factors, such as toxins and resistance determinants. All of these plasmids carry a conjugation locus that consists of 11 genes: intP and tcpA to tcpJ. Three proteins, TcpA, a potential coupling protein, TcpF, a putative ATPase that is similar to ORF15 from Tn916, and TcpH, which contains VirB6-like domains, are essential for conjugation in the prototype conjugative plasmid pCW3. To analyze the functional domains of TcpH, a putative structural component of the mating-pair formation complex and deletion and site-directed mutants were constructed and analyzed. The results showed that the N-terminal 581 residues and the conserved 242VQQPW246 motif were required for conjugative transfer. Bacterial two-hybrid and biochemical studies showed that TcpH interacted with itself and with TcpC. An analysis of the tcpH mutants demonstrated that the region required for these interactions also was localized to the N-terminal 581 residues and that the function of the C-terminal region of TcpH was independent of protein-protein interactions. Finally, immunofluorescence studies showed that TcpH and TcpF were located at both cell poles of donor C. perfringens cells. The results provide evidence that TcpH is located in the cell membrane, where it oligomerizes and interacts with TcpC to form part of the mating-pair formation complex, which is located at the cell poles and is closely associated with TcpF.

scholarly journals Conjugation-Mediated Horizontal Gene Transfer of Clostridium perfringens Plasmids in the Chicken Gastrointestinal Tract Results in the Formation of New Virulent Strains

2017 ◽  
Vol 83 (24) ◽  
Author(s):  
Jake A. Lacey ◽  
Anthony L. Keyburn ◽  
Mark E. Ford ◽  
Ricardo W. Portela ◽  
Priscilla A. Johanesen ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Clostridium Perfringens ◽  
Plasmid Transfer ◽  
Conjugative Plasmid ◽  
Conjugative Transfer ◽  
Necrotic Enteritis ◽  
Content Type ◽  
Gastrointestinal Pathogen

ABSTRACT Clostridium perfringens is a gastrointestinal pathogen capable of causing disease in a variety of hosts. Necrotic enteritis in chickens is caused by C. perfringens strains that produce the pore-forming toxin NetB, the major virulence factor for this disease. Like many other C. perfringens toxins and antibiotic resistance genes, NetB is encoded on a conjugative plasmid. Conjugative transfer of the netB-containing plasmid pJIR3535 has been demonstrated in vitro with a netB-null mutant. This study has investigated the effect of plasmid transfer on disease pathogenesis, with two genetically distinct transconjugants constructed under in vitro conditions, within the intestinal tract of chickens. This study also demonstrates that plasmid transfer can occur naturally in the host gut environment without the need for antibiotic selective pressure to be applied. The demonstration of plasmid transfer within the chicken host may have implications for the progression and pathogenesis of C. perfringens-mediated disease. Such horizontal gene transfer events are likely to be common in the clostridia and may be a key factor in strain evolution, both within animals and in the wider environment. IMPORTANCE Clostridium perfringens is a major gastrointestinal pathogen of poultry. C. perfringens strains that express the NetB pore-forming toxin, which is encoded on a conjugative plasmid, cause necrotic enteritis. This study demonstrated that the conjugative transfer of the netB-containing plasmid to two different nonpathogenic strains converted them into disease-causing strains with disease-causing capability similar to that of the donor strain. Plasmid transfer of netB and antibiotic resistance was also demonstrated to occur within the gastrointestinal tract of chickens, with approximately 14% of the isolates recovered comprising three distinct, in vivo-derived, transconjugant types. The demonstration of in vivo plasmid transfer indicates the potential importance of strain plasticity and the contribution of plasmids to strain virulence.

scholarly journals NetF-producing Clostridium perfringens and its associated diseases in dogs and foals

2020 ◽  
Vol 32 (2) ◽  
pp. 230-238 ◽  
Author(s):  
Iman Mehdizadeh Gohari ◽  
Stefan Unterer ◽  
Ashley E. Whitehead ◽  
John F. Prescott
Keyword(s):  
Clostridium Perfringens ◽  
Conjugative Plasmid ◽  
Current Evidence ◽  
Toxin Gene ◽  
Enteric Diseases ◽  
Form Part ◽  
Putative Toxin ◽  
Toxigenic Strains ◽  
Diarrhea Syndrome

The role of type A Clostridium perfringens in canine acute hemorrhagic diarrhea syndrome and foal necrotizing enteritis is poorly characterized. However, a highly significant association between the presence of novel toxigenic C. perfringens and these specific enteric diseases has been described. These novel toxigenic strains produce 3 novel putative toxins, which have been designated NetE, NetF, and NetG. Although not conclusively demonstrated, current evidence suggests that NetF is likely the major virulence factor in strains responsible for canine acute hemorrhagic diarrhea syndrome and foal necrotizing enteritis. NetF is a beta–pore-forming toxin that belongs to the same toxin superfamily as CPB and NetB toxins produced by C. perfringens. The netF gene is encoded on a conjugative plasmid that, in the case of netF, also carries another putative toxin gene, netE. In addition, these strains consistently also carry a cpe tcp-conjugative plasmid, and a proportion also carry a separate netG tcp-conjugative plasmid. The netF and netG genes form part of a locus with all the features of the pathogenicity loci of tcp-conjugative plasmids. The netF-positive isolates are clonal in origin and fall into 2 clades. Disease in dogs or foals can be associated with either clade. Thus, these are strains with unique virulence-associated characteristics associated with serious and sometimes fatal cases of important enteric diseases in 2 animal species.

scholarly journals Genetic and Functional Analyses of the mob Operon on Conjugative Transposon CTn341 from Bacteroides spp.

2010 ◽  
Vol 192 (18) ◽  
pp. 4643-4650 ◽  
Author(s):  
Lindsay Peed ◽  
Anita C. Parker ◽  
C. Jeffrey Smith
Keyword(s):  
Protein A ◽  
Regulatory System ◽  
Tetracycline Resistance ◽  
Conjugative Transposons ◽  
Coupling Protein ◽  
Nick Site ◽  
Transfer Origin ◽  
Functional Analyses ◽  
Tetracycline Induction

ABSTRACT Bacteroides are Gram-negative anaerobes indigenous to the intestinal tract of humans, and they are important opportunistic pathogens. Mobile genetic elements, such as conjugative transposons (CTns), have contributed to an increase in antibiotic resistance in these organisms. CTns are self-transmissible elements that belong to the superfamily of integrative and conjugative elements (ICEs). CTn341 is 52 kb; it encodes tetracycline resistance and its transfer is induced by tetracycline. The mobilization region of CTn341 was shown to be comprised of a three-gene operon, mobABC, and the transfer origin, oriT. The three genes code for a nicking accessory protein, a relaxase, and a VirD4-like coupling protein, respectively. The Mob proteins were predicted to mediate the formation of the relaxosome complex, nick DNA at the oriT, and shuttle the DNA/protein complex to the mating-pore apparatus. The results of mutational studies indicated that the three genes are required for maximal transfer of CTn341. Mob gene transcription was induced by tetracycline, and this regulation was mediated through the two-component regulatory system, RteAB. The oriT region of CTn341 was located within 100 bp of mobA, and a putative Bacteroides consensus nicking site was observed within this region. Mutation of the putative nick site resulted in a loss of transfer. This study demonstrated a role of the mobilization region for transfer of Bacteroides CTns and that tetracycline induction occurs for the mob gene operon, as for the tra gene operon(s), as shown previously.

scholarly journals tISCpe8, an IS1595-Family Lincomycin Resistance Element Located on a Conjugative Plasmid in Clostridium perfringens

2009 ◽  
Vol 191 (20) ◽  
pp. 6345-6351 ◽  
Author(s):  
Dena Lyras ◽  
Vicki Adams ◽  
Susan A. Ballard ◽  
Wee L. Teng ◽  
Pauline M. Howarth ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Antibiotic Resistance Genes ◽  
Tetracycline Resistance ◽  
Conjugative Plasmid ◽  
Insertion Sequences ◽  
Genetic Element ◽  
Significant Similarity ◽  
Conjugative Transposon ◽  
Resistance Determinants ◽  
Lincomycin Resistance

ABSTRACT Clostridium perfringens is a normal gastrointestinal organism that is a reservoir for antibiotic resistance genes and can potentially act as a source from which mobile elements and their associated resistance determinants can be transferred to other bacterial pathogens. Lincomycin resistance in C. perfringens is common and is usually encoded by erm genes that confer macrolide-lincosamide-streptogramin B resistance. In this study we identified strains that are lincomycin resistant but erythromycin sensitive and showed that the lincomycin resistance determinant was plasmid borne and could be transferred to other C. perfringens isolates by conjugation. The plasmid, pJIR2774, is the first conjugative C. perfringens R-plasmid to be identified that does not confer tetracycline resistance. Further analysis showed that resistance was encoded by the lnuP gene, which encoded a putative lincosamide nucleotidyltransferase and was located on tISCpe8, a functional transposable genetic element that was a member of the IS1595 family of transposon-like insertion sequences. This element had significant similarity to the mobilizable lincomycin resistance element tISSag10 from Streptococcus agalactiae. Like tISSag10, tISCpe8 carries a functional origin of transfer within the resistance gene, allowing the element to be mobilized by the conjugative transposon Tn916. The similarity of these elements and the finding that they both contain an oriT-like region support the hypothesis that conjugation may result in the movement of DNA modules that are not obviously mobile since they are not linked to conjugation or mobilization functions. This process likely plays a significant role in bacterial adaptation and evolution.

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