Functional and Mutational Analysis of Conjugative Transfer Region 2 (Tra2) from the IncHI1 Plasmid R27

ABSTRACT The transfer 2 region (Tra2) of the conjugative plasmid drR27 (derepressed R27) was analyzed by PSI-BLAST, insertional mutagenesis, genetic complementation, and an H-pilus assay. Tra2 contains 11 mating-pair formation (Mpf) genes that are essential for conjugative transfer, 9 of which are essential for H-pilus production (trhA, -L, -E, -K, -B, -V, -C, -P, and -W). TrhK has similarity to secretin proteins, suggesting a mechanism by which DNA could traverse the outer membrane of donors. The remaining two Mpf genes, trhU and trhN, play an auxiliary role in H-pilus synthesis and are proposed to be involved in DNA transfer and mating-pair stabilization, respectively. Conjugative transfer abilities were restored for each mutant when complemented with the corresponding transfer gene. In addition to the essential Mpf genes, three genes, trhO, trhZ, and htdA, modulate R27 transfer frequency. Disruption of trhO and trhZ severely reduced the transfer frequencies of drR27, whereas disruption of htdA greatly increased the transfer frequency of wild-type R27 to drR27 levels. A comparison of the essential transfer genes encoded by the Tra2 and Tra1 (T. D. Lawley, M. W. Gilmour, J. E. Gunton, L. J. Standeven, and D. E. Taylor, J. Bacteriol. 184:2173-2183, 2002) of R27 to other transfer systems illustrates that the R27 conjugative transfer system is a chimera composed of IncF-like and IncP-like transfer systems. Furthermore, the Mpf/type IV secretion systems encoded by IncH and IncF transfer systems are distinct from that of the IncP transfer system. The phenotypic and ecological significance of these observations is discussed.

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Functional and Mutational Analysis of Conjugative Transfer Region 1 (Tra1) from the IncHI1 Plasmid R27

Journal of Bacteriology ◽

10.1128/jb.184.8.2173-2180.2002 ◽

2002 ◽

Vol 184 (8) ◽

pp. 2173-2180 ◽

Cited By ~ 42

Author(s):

Trevor D. Lawley ◽

Matthew W. Gilmour ◽

James E. Gunton ◽

Leah J. Standeven ◽

Diane E. Taylor

Keyword(s):

Leucine Zipper ◽

Mutational Analysis ◽

Dna Transfer ◽

Genetic Complementation ◽

Conjugative Transfer ◽

Transfer System ◽

Sequence Motifs ◽

Specific Phage ◽

Transfer Region ◽

Coupling Protein

ABSTRACT The conjugative transfer region 1 (Tra1) of the IncHI1 plasmid R27 was subjected to DNA sequence analysis, mutagenesis, genetic complementation, and an H-pilus-specific phage assay. Analysis of the nucleotide sequence indicated that the Tra1 region contains genes coding for mating pair formation (Mpf) and DNA transfer replication (Dtr) and a coupling protein. Insertional disruptions of 9 of the 14 open reading frames (ORFs) in the Tra1 region resulted in a transfer-deficient phenotype. Conjugative transfer was restored for each transfer mutant by genetic complementation. An intergenic region between traH and trhR was cloned and mobilized by R27, indicating the presence of an origin of transfer (oriT). The five ORFs immediately downstream of the oriT region are involved in H-pilus production, as determined by an H-pilus-specific phage assay. Three of these ORFs encode proteins homologous to Mpf proteins from IncF plasmids. Upstream of the oriT region are four ORFs required for plasmid transfer but not H-pilus production. TraI contains sequence motifs that are characteristic of relaxases from the IncP lineage but share no overall homology to known relaxases. TraJ contains both an Arc repressor motif and a leucine zipper motif. A putative coupling protein, TraG, shares a low level of homology to the TraG family of coupling proteins and contains motifs that are important for DNA transfer. This analysis indicates that the Mpf components of R27 share a common lineage with those of the IncF transfer system, whereas the relaxase of R27 is ancestrally related to that of the IncP transfer system.

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Functional and Mutational Analysis of P19, a DNA Transfer Protein with Muramidase Activity

Journal of Bacteriology ◽

10.1128/jb.183.10.3176-3183.2001 ◽

2001 ◽

Vol 183 (10) ◽

pp. 3176-3183 ◽

Cited By ~ 33

Author(s):

Michaela Bayer ◽

Robert Iberer ◽

Karin Bischof ◽

Edith Rassi ◽

Edith Stabentheiner ◽

...

Keyword(s):

Mutational Analysis ◽

Cell Envelope ◽

Large Family ◽

Conjugative Plasmid ◽

Secretion Systems ◽

Type Iv ◽

Lytic Transglycosylase ◽

Plasmid R1 ◽

Muramidase Activity

ABSTRACT Protein P19 encoded by the conjugative resistance plasmid R1 has been identified as being one member of a large family of muramidases encoded by bacteriophages and by type III and type IV secretion systems. We carried out a mutational analysis to investigate the function of protein P19 and used in vivo complementation assays to test those of several P19 mutants. The results indicated that conserved residues present in the presumed catalytic center of P19 are absolutely essential for its function in conjugation of plasmid R1 and infection by the RNA phage R17. Overexpression of protein P19 in an early growth phase resulted in a massive lysis of Escherichia coli cells in liquid culture, as indicated by a rapid and distinct decrease in cell culture densities after induction. Change of the proposed catalytic glutamate at position 44 to glutamine completely abolished this effect. P19-induced cell lysis was directly shown by transmission and scanning electron microscopy. Typically, P19-overexpressing cells showed bulges protruding from the cell surfaces. Our interpretation is that these protrusions arose from a localized and spatially confined disruption of the bacterial cell wall. To our knowledge such an effect has not previously been documented for any member of the lytic transglycosylase family. From the data presented here, we conclude that protein P19 possesses the proposed localized peptidoglycan-hydrolyzing activity. This activity would be a prerequisite for efficient penetration of the cell envelope by the DNA translocation complex encoded by the conjugative plasmid.

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Agrobacterium VirD2-Binding Protein Is Involved in Tumorigenesis and Redundantly Encoded in Conjugative Transfer Gene Clusters

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-10-1201 ◽

2007 ◽

Vol 20 (10) ◽

pp. 1201-1212 ◽

Cited By ~ 17

Author(s):

Minliang Guo ◽

Qingming Hou ◽

Choy L. Hew ◽

Shen Q. Pan

Keyword(s):

Virulence Factor ◽

Mutational Analysis ◽

Gene Clusters ◽

Conjugative Transfer ◽

Fingerprint Matching ◽

Secretion Systems ◽

T Complex ◽

Affinity Ligand ◽

Type Iv ◽

Peptide Mass

Agrobacterium tumefaciens can transfer oncogenic T-DNA into plant cells; T-DNA transfer is mechanistically similar to a conjugation process. VirD2 is the pilot protein that guides the transfer, because it is covalently associated with single-stranded T-DNA to form the transfer substrate T-complex. We used the VirD2 protein as an affinity ligand to isolate VirD2-binding proteins (VBPs). By pull-down assays and peptide-mass-fingerprint matching, we identified an A. tumefaciens protein designated VBP1 that could bind VirD2 directly. Genome-wide sequence analysis showed that A. tumefaciens has two additional genes encoding proteins highly similar to VBP1, designated vbp2 and vbp3. Like VBP1, both VBP2 and VBP3 also could bind VirD2; all three VBPs contain a putative nucleotidyltransferase motif. Mutational analysis of vbp demonstrated that the three vbp genes could functionally complement each other. Consequently, only inactivation of all three vbp genes highly attenuated the bacterial ability to cause tumors on plants. Although vbp1 is harbored on the megaplasmid pAtC58, vbp2 and vbp3 reside on the linear chromosome. The vbp genes are clustered with conjugative transfer genes, suggesting linkage between the conjugation and virulence factor. The three VBPs appear to contain C-terminal positively charged residues, often present in the transfer substrate proteins of type IV secretion systems. Inactivation of the three vbp genes did not affect the T-strand production. Our data indicate that VBP is a newly identified virulence factor that may affect the transfer process subsequent to T-DNA production.

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A cryptic plasmid of Yersinia enterocolitica encodes a conjugative transfer system related to the regions of CloDF13 Mob and IncX Pil

Microbiology ◽

10.1099/mic.0.26418-0 ◽

2003 ◽

Vol 149 (10) ◽

pp. 2829-2845 ◽

Cited By ~ 20

Author(s):

Eckhard Strauch ◽

Greta Goelz ◽

Dorothea Knabner ◽

Antje Konietzny ◽

Erich Lanka ◽

...

Keyword(s):

Yersinia Enterocolitica ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Kanamycin Resistance ◽

Cryptic Plasmid ◽

Cosmid Library ◽

Conjugative Transfer ◽

Transfer System ◽

Type Iv ◽

K 12

Yersinia enterocolitica 29930 (biotype 1A; O : 7,8), the producing strain of the phage-tail-like bacteriocin enterocoliticin, possesses a plasmid-encoded conjugative type IV transfer system. The genes of the conjugative system were found by screening of a cosmid library constructed from total DNA of strain 29930. The cosmid Cos100 consists of the vector SuperCos1 and an insert DNA of 40 303 bp derived from a cryptic plasmid of strain 29930. The conjugative transfer system consists of genes encoding a DNA transfer and replication system (Dtr) with close relationship to the mob region of the mobilizable plasmid CloDF13 and a gene cluster encoding a mating pair formation system (Mpf) closely related to the Mpf system of the IncX plasmid R6K. However, a gene encoding a homologue of TaxB, the coupling protein of the IncX system, is missing. The whole transfer region has a size of approximately 17 kb. The recombinant plasmid Cos100 was shown to be transferable between Escherichia coli and Yersinia with transfer frequencies up to 0·1 transconjugants per donor. Mutations generated by inserting a tetracycline cassette into putative tri genes yielded a transfer-deficient phenotype. Conjugative transfer of the cryptic plasmid could not be demonstrated in the original host Y. enterocolitica 29930. However, a kanamycin-resistance-conferring derivative of the plasmid was successfully introduced into E. coli K-12 by transformation and was shown to be self-transmissible. Furthermore, Southern blot hybridization and PCR experiments were carried out to elucidate the distribution of the conjugative transfer system in Yersinia. In total, six Y. enterocolitica biotype 1A strains harbouring closely related systems on endogenous plasmids were identified.

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Characteristics of the Conjugative Transfer System of the IncM Plasmid pCTX-M3 and Identification of Its Putative Regulators

Journal of Bacteriology ◽

10.1128/jb.00234-18 ◽

2018 ◽

Vol 200 (18) ◽

Cited By ~ 9

Author(s):

Michał Dmowski ◽

Marcin Gołębiewski ◽

Izabela Kern-Zdanowicz

Keyword(s):

Host Range ◽

Deletion Analysis ◽

Transcriptional Analysis ◽

Conjugative Transfer ◽

Transfer System ◽

Secretion Systems ◽

Conjugation System ◽

Content Type ◽

Transfer Genes ◽

Plasmid Mobilization

ABSTRACTPlasmid conjugative transfer systems comprise type IV secretion systems (T4SS) coupled to DNA processing and replication. The T4SSs are divided into two phylogenetic subfamilies, namely, IVA and IVB, or on the basis of the phylogeny of the VirB4 ATPase, into eight groups. The conjugation system of the IncM group plasmid pCTX-M3, fromCitrobacter freundii, is classified in the IVB subfamily and in the MPFIgroup, as are the conjugation systems of IncI1 group plasmids. Although the majority of the conjugative genes of the IncM and IncI1 plasmids display conserved synteny, there are several differences. Here, we present a deletion analysis of 27 genes in the conjugative transfer regions of pCTX-M3. Notably, the deletion of either of two genes dispensable for conjugative transfer, namely,orf35andorf36, resulted in an increased plasmid mobilization efficiency. Transcriptional analysis of theorf35andorf36deletion mutants suggested an involvement of these genes in regulating the expression of conjugative transfer genes. We also revised the host range of the pCTX-M3 replicon by finding that this replicon is unable to support replication inAgrobacterium tumefaciens,Ralstonia eutropha, andPseudomonas putida, though its conjugation system is capable of introducing plasmids bearingoriTpCTX-M3into these bacteria, which are representatives ofAlpha-,Beta-, andGammaproteobacteria, respectively. Thus, the conjugative transfer system of pCTX-M3 has a much broader host range than its replicon.IMPORTANCEHorizontal gene transfer is responsible for rapid changes in bacterial genomes, and the conjugative transfer of plasmids has a great impact on the plasticity of bacteria. Here, we present a deletion analysis of the conjugative transfer system genes of the pCTX-M3 plasmid of the IncM group, which is responsible for the dissemination of antibiotic resistance genes inEnterobacteriaceae. We found that the deletion of either of theorf35andorf36genes, which are dispensable for conjugative transfer, increased the plasmid mobilization efficiency. Real-time quantitative PCR (RT-qPCR) analysis suggested the involvement oforf35andorf36in regulating the expression of transfer genes. We also revised the host range of pCTX-M3 by showing that its conjugative transfer system has a much broader host range than its replicon.

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Protein Transfer through an F Plasmid-Encoded Type IV Secretion System Suppresses the Mating-Induced SOS Response

mBio ◽

10.1128/mbio.01629-21 ◽

2021 ◽

Author(s):

Abu Amar M. Al Mamun ◽

Kouhei Kishida ◽

Peter J. Christie

Keyword(s):

Heavy Metals ◽

Sos Response ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Conjugative Transfer ◽

Secretion Systems ◽

Protein Transfer ◽

Type Iv ◽

Genes Encoding

Many bacteria carry plasmids and other mobile genetic elements (MGEs) whose conjugative transfer through encoded type IV secretion systems (T4SSs), or “mating channels,” can lead to a rapid intra- and interspecies proliferation of genes encoding resistance to antibiotics or heavy metals or virulence traits. Here, we show that a model IncF plasmid-encoded T4SS translocates not only DNA but also several proteins intercellularly.

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Structural and Biochemical Analysis of OrfG: The VirB8-like Component of the Conjugative Type IV Secretion System of ICESt3 From Streptococcus thermophilus

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.642606 ◽

2021 ◽

Vol 8 ◽

Author(s):

Julien Cappele ◽

Abbas Mohamad Ali ◽

Nathalie Leblond-Bourget ◽

Sandrine Mathiot ◽

Tiphaine Dhalleine ◽

...

Keyword(s):

Antibiotic Resistance ◽

Structural Analysis ◽

Pathogenic Bacteria ◽

Quaternary Structure ◽

Streptococcus Thermophilus ◽

Type Iv Secretion ◽

Conjugative Transfer ◽

Structural Level ◽

Secretion Systems ◽

Type Iv

Conjugative transfer is a major threat to global health since it contributes to the spread of antibiotic resistance genes and virulence factors among commensal and pathogenic bacteria. To allow their transfer, mobile genetic elements including Integrative and Conjugative Elements (ICEs) use a specialized conjugative apparatus related to Type IV secretion systems (Conj-T4SS). Therefore, Conj-T4SSs are excellent targets for strategies that aim to limit the spread of antibiotic resistance. In this study, we combined structural, biochemical and biophysical approaches to study OrfG, a protein that belongs to Conj-T4SS of ICESt3 from Streptococcus thermophilus. Structural analysis of OrfG by X-ray crystallography revealed that OrfG central domain is similar to VirB8-like proteins but displays a different quaternary structure in the crystal. To understand, at a structural level, the common and the diverse features between VirB8-like proteins from both Gram-negative and -positive bacteria, we used an in silico structural alignment method that allowed us to identify different structural classes of VirB8-like proteins. Biochemical and biophysical characterizations of purified OrfG soluble domain and its central and C-terminal subdomains indicated that they are mainly monomeric in solution but able to form an unprecedented 6-mer oligomers. Our study provides new insights into the structural analysis of VirB8-like proteins and discusses the interplay between tertiary and quaternary structures of these proteins as an essential component of the conjugative transfer.

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Genetic and Sequence Analysis of the pTiC58 trb Locus, Encoding a Mating-Pair Formation System Related to Members of the Type IV Secretion Family

Journal of Bacteriology ◽

10.1128/jb.180.23.6164-6172.1998 ◽

1998 ◽

Vol 180 (23) ◽

pp. 6164-6172 ◽

Cited By ~ 40

Author(s):

Pei-Li Li ◽

Dawn M. Everhart ◽

Stephen K. Farrand

Keyword(s):

Sequence Analysis ◽

Pair Formation ◽

Type Iv Secretion ◽

Ti Plasmid ◽

Open Reading Frames ◽

Conjugal Transfer ◽

Mating Pair ◽

Secretion Systems ◽

Type Iv ◽

And Function

ABSTRACT Conjugal transfer of pTiC58 requires two regions, trawhich contains the oriT and several genes involved in DNA processing and a region of undefined size and function that is located at the 2-o’clock position of the plasmid. Using transposon mutagenesis with Tn3HoHo1 and a binary transfer system, we delimited this second region, called trb, to an 11-kb interval between the loci for vegetative replication and nopaline catabolism. DNA sequence analysis of this region identified 13 significant open reading frames (ORFs) spanning 11,003 bp. The first, encodingtraI, already has been described and is responsible for the synthesis of Agrobacterium autoinducer (AAI) (I. Hwang, P.-L. Li, L. Zhang, K. R. Piper, D. M. Cook, M. E. Tate, and S. K. Farrand, Proc. Natl. Acad. Sci. USA 91:4639–4643, 1994). Translation products of the next 11 ORFs showed similarities to those of trbB, -C, -D,-E, -J, -K, -L,-F, -G, -H, and -I of the trb region of the octopine-type Ti plasmid pTi15955 and of the tra2 core region of RP4. In RP4, these genes encode mating-pair formation functions and are essential for the conjugal transfer of the IncP plasmid. Each of the trb gene homologues is oriented counterclockwise on the Ti plasmid. Expression of these genes, as measured by using the lacZ fusions formed by Tn3HoHo1, required the traI promoter and the transcriptional activator TraR along with its coinducer, AAI. While related to that of RP4, the trb system of pTiC58 did not allow propagation of the trb-specific bacteriophages PRD1, PRR1, and Pf3. The products of several trb genes of the Ti plasmid are similar to those of other loci that encode DNA transfer or protein secretion systems, all of which are members of the type IV secretion family.

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Subcellular localization and functional domains of the coupling protein, TraG, from IncHI1 plasmid R27

Microbiology ◽

10.1099/mic.0.28255-0 ◽

2005 ◽

Vol 151 (11) ◽

pp. 3549-3561 ◽

Cited By ~ 14

Author(s):

James E. Gunton ◽

Matthew W. Gilmour ◽

Guillermina Alonso ◽

Diane E. Taylor

Keyword(s):

Mutational Analysis ◽

Transcriptional Analysis ◽

Nucleoside Triphosphate ◽

Cell Periphery ◽

Conjugative Transfer ◽

Dependent Manner ◽

Transfer Event ◽

Binding Domains ◽

Type Iv ◽

Coupling Protein

Bacterial conjugation is a horizontal gene transfer event mediated by the type IV secretion system (T4SS) encoded by bacterial plasmids. Within the T4SS, the coupling protein plays an essential role in linking the membrane-associated pore-forming proteins to the cytoplasmic, DNA-processing proteins. TraG is the coupling protein encoded by the incompatibility group HI plasmids. A hallmark feature of the IncHI plasmids is optimal conjugative transfer at 30 °C and an inability to transfer at 37 °C. Transcriptional analysis of the transfer region 1 (Tra1) of R27 has revealed that traG is transcribed in a temperature-dependent manner, with significantly reduced levels of expression at 37 °C as compared to expression at 30 °C. The R27 coupling protein contains nucleoside triphosphate (NTP)-binding domains, the Walker A and Walker B boxes, which are well conserved among this family of proteins. Site-specific mutagenesis within these motifs abrogated the conjugative transfer of R27 into recipient cells. Mutational analysis of the TraG periplasmic-spanning residues, in conjunction with bacterial two-hybrid and immunoprecipitation analysis, determined that this region is essential for a successful interaction with the T4SS protein TrhB. Further characterization of TraG by immunofluorescence studies revealed that the R27 coupling protein forms membrane-associated fluorescent foci independent of R27 conjugative proteins. These foci were found at discrete positions within the cell periphery. These results allow the definition of domains within TraG that are involved in conjugative transfer, and determination of the cellular location of the R27 coupling protein.

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Transfer of DNA from Bacteria to Eukaryotes

mBio ◽

10.1128/mbio.00863-16 ◽

2016 ◽

Vol 7 (4) ◽

Cited By ~ 64

Author(s):

Benoît Lacroix ◽

Vitaly Citovsky

Keyword(s):

Bartonella Henselae ◽

Bacterial Species ◽

Symbiotic Bacterium ◽

Host Cells ◽

Conjugative Plasmid ◽

Secretion Systems ◽

Type Iv ◽

Conjugative Plasmid Transfer ◽

Eukaryotic Species ◽

Potential Applications

ABSTRACTHistorically, the members of theAgrobacteriumgenus have been considered the only bacterial species naturally able to transfer and integrate DNA into the genomes of their eukaryotic hosts. Yet, increasing evidence suggests that this ability to genetically transform eukaryotic host cells might be more widespread in the bacterial world. Indeed, analyses of accumulating genomic data reveal cases of horizontal gene transfer from bacteria to eukaryotes and suggest that it represents a significant force in adaptive evolution of eukaryotic species. Specifically, recent reports indicate that bacteria other thanAgrobacterium, such asBartonella henselae(a zoonotic pathogen),Rhizobium etli(a plant-symbiotic bacterium related toAgrobacterium), or evenEscherichia coli, have the ability to genetically transform their host cells under laboratory conditions. This DNA transfer relies on type IV secretion systems (T4SSs), the molecular machines that transport macromolecules during conjugative plasmid transfer and also during transport of proteins and/or DNA to the eukaryotic recipient cells. In this review article, we explore the extent of possible transfer of genetic information from bacteria to eukaryotic cells as well as the evolutionary implications and potential applications of this transfer.

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