Presence of Functionally Active Cytolethal Distending Toxin Genes on a Conjugative Plasmid in a Clinical Isolate of Providencia rustigianii.

Providencia rustigianii is potentially enteropathogenic in humans. Recently, we identified a P. rustigianii strain carrying a part of the cdtB gene homologous to that of Providencia alcalifacines that produces an exotoxin called cytolethal distending toxin (CDT), encoded by three subunit genes (cdtA, cdtB, and cdtC). In this study, we analyzed the P. rustigianii strain for possible presence of the entire cdt gene cluster and its organization, location, and mobility, as well as expression of the toxin as a putative virulence factor of P. rustigianii. Nucleotide sequence analysis revealed the presence of the three cdt subunit genes in tandem, and over 94% homology to the corresponding genes carried by P. alcalifaciens both at nucleotide and amino acid sequence levels. The P. rustigianii strain produced biologically active CDT, which caused distension of eukaryotic cell lines with characteristic tropism of CHO and Caco-2 cells but not of Vero cells. S1-nuclease digested pulsed-field gel electrophoresis followed by Southern hybridization analysis demonstrated that the cdt genes in both P. rustigianii and P. alcalifaciens strains are located on large plasmids (140 to 170 kb). Subsequently, conjugation assays using a genetically marked derivative of the P. rustigianii strain showed that the plasmid carrying cdt genes in the P. rustigianii was transferable to cdt gene-negative recipient strains of P. rustigianii, Providencia rettgeri, and Escherichia coli. Our results demonstrated the presence of cdt genes in P. rustigianii for the first time, and further showed that the genes are located on a transferable plasmid, which can potentially spread to other bacterial species.

Baicalein Inhibits Plasmid-Mediated Horizontal Transmission of the blaKPC Multidrug Resistance Gene from Klebsiella pneumoniae to Escherichia coli.

Carbapenem-resistant bacterial infections pose an urgent threat to public health worldwide. Horizontal transmission of the ß-lacatamase Klebsiella pneumoniae carbapenemase (blaKPC) multidrug resistance gene is a major mechanism for global dissemination of carbapenem resistance. Here, we investigated the effects of baicalein, an active ingredient of a Chinese herbal medicine, on plasmid-mediated horizontal transmission of blaKPC from a meropenem-resistant K. pneumoniae strain (JZ2157) to a meropenem-sensitive Escherichia coli strain (E600). Baicalein showed no direct effects on the growth of JZ2157 or E600. Co-cultivation of JZ2157 and E600 caused the spread of meropenem resistance from JZ2157 to E600. Baicalein at 40 and 400 µg/mL significantly inhibited the spread of meropenem resistance. Co-cultivation also resulted in plasmid-mediated transmission of blaKPC from JZ2157 to E600, which was inhibited by baicalein. Therefore, baicalein may be used in clinical practice to prevent or contain outbreaks of carbapenem-resistant infections by inhibiting the horizontal transfer of resistance genes across bacteria species.

Analysis of the complete genome sequences of Clostridium perfringens strains harbouring the binary enterotoxin BEC gene and comparative genomics of pCP13-like family plasmids.

BACKGROUND: BEC-producing Clostridium perfringens is a causative agent of foodborne gastroenteritis. It was first reported in 2014, and since then, several isolates have been identified in Japan and the United Kingdom. The novel binary ADP-ribosylating toxin BEC, which consists of two components (BECa and BECb), is encoded on a plasmid that is similar to pCP13 and harbours a conjugation locus, called Pcp, encoding homologous proteins of the type 4 secretion system. Despite the high in vitro conjugation frequency of pCP13, its dissemination and that of related plasmids, including bec-harbouring plasmids, in the natural environment have not been characterised. This lack of knowledge has limited our understanding of the genomic epidemiology of bec-harbouring C. perfringens strains. RESULTS: In this study, we determined the complete genome sequences of five bec-harbouring C. perfringens strains isolated from 2009 to 2019. Each isolate contains a ~ 3.36 Mbp chromosome and 1-3 plasmids of either the pCW3-like family, pCP13-like family, or an unknown family, and the bec-encoding region in all five isolates was located on a ~ 54 kbp pCP13-like plasmid. Phylogenetic and SNP analyses of these complete genome sequences and the 211 assembled C. perfringens genomes in GenBank showed that although these bec-harbouring strains were split into two phylogenetic clades, the sequences of the bec-encoding plasmids were nearly identical (>99.81%), with a significantly smaller SNP accumulation rate than that of their chromosomes. Given that the Pcp locus is conserved in these pCP13-like plasmids, we propose a mechanism in which the plasmids were disseminated by horizontal gene transfer. Data mining showed that strains carrying pCP13-like family plasmids were unexpectedly common (58/216 strains) and widely disseminated among the various C. perfringens clades. Although these plasmids possess a conserved Pcp locus, their 'accessory regions' can accommodate a wide variety of genes, including virulence-associated genes, such as becA/becB and cbp2. These results suggest that this family of plasmids can integrate various foreign genes and is transmissible among C. perfringens strains. CONCLUSION: This study demonstrates the potential significance of pCP13-like plasmids, including bec-encoding plasmids, for the characterisation and monitoring of the dissemination of pathogenic C. perfringens strains.

An easily modifiable conjugative plasmid for studying horizontal gene transfer.

Horizontal gene transfer is an important mechanism in bacterial evolution and can occur at striking frequencies when mediated by mobile genetic elements. Conjugative plasmids are mobile genetic elements that are main drivers of horizontal transfer and a major facilitator in the spread of antibiotic resistance genes. However, conjugative plasmid models that readily can be genetically modified with the aim to study horizontal transfer are not currently available. The aim of this study was to develop a conjugative plasmid model where the insertion of gene cassettes such as reporter genes (e.g., fluorescent proteins) or antibiotic resistance genes would be efficient and convenient. Here, we introduced a single attTn7 site into the conjugative broad-host-range IncP-1 plasmid pKJK5 in a non-disruptive manner. Furthermore, a version with lower transfer rate and a non-conjugative version of pKJK5-attTn7 were also constructed. The advantage of having the attTn7 sites is that genes of interest can be introduced in a single step with very high success rate using the Tn7 transposition system. In addition, larger genetic fragments can be inserted. To illustrate the efficacy of the constructed pKJK5 plasmids, they were complemented with sfGFP (a gene encoding superfolder green fluorescent protein) in addition to seven different ß-lactamase genes representing the four known classes of ß-lactamases.

Complete sequence of classic F-type plasmid pRK100 shows unique conservation over time and geographic location.

Plasmids exhibit great diversity of gene content and host ranges and are famous for quick adaptation to the genetic background of the bacterial host cell. In addition to observing ever evolving plasmids, some plasmids have conserved backbones: a stable core composition and arrangement of genes in addition to variable regions. There are a few reports of extremely conserved plasmids. Here we report the complete sequence of pRK100 plasmid - a large, well-characterized conjugative F-like plasmid found in an Escherichia coli strain isolated from a urinary tract infection patient in 1990. The sequence shows that the 142 kb-long pRK100 plasmid is nearly identical to plasmids circulating in distant geographical locations and found in different host E. coli strains between 2007 and 2017. We also performed additional functional characterization of pRK100. Our results showed that pRK100 does not have a strong pathogenicity phenotype in porcine primary bladder epithelial cell culture. Moreover, the conjugation of pRK100 seems to strongly depend on recipient characteristics. These observations and identification of the pRK100 plasmid in different strain genotypes leave the extreme sequence conservation and broad distribution of this plasmid unexplained.

Hyper-stimulation of Pyrococcus furiosus CRISPR DNA uptake by a self-transmissible plasmid.

Pyrococcus furiosus is a hyperthermophilic archaeon with three effector CRISPR complexes (types I-A, I-B, and III-B) that each employ crRNAs derived from seven CRISPR arrays. Here, we investigate the CRISPR adaptation response to a newly discovered and self-transmissible plasmid, pT33.3. Transconjugant strains of Pyrococcus furiosus exhibited dramatically elevated levels of new spacer integration at CRISPR loci relative to the strain harboring a commonly employed, laboratory-constructed plasmid. High-throughput sequence analysis demonstrated that the vast majority of the newly acquired spacers were preferentially selected from DNA surrounding a particular region of the pT33.3 plasmid and exhibited a bi-directional pattern of strand bias that is a hallmark of primed adaptation by type I systems. We observed that one of the CRISPR arrays of our Pyrococcus furiosus laboratory strain encodes a spacer that closely matches the region of the conjugative plasmid that is targeted for adaptation. The hyper-adaptation phenotype was found to strictly depend both on the presence of this single matching spacer as well as the I-B effector complex, known to mediate primed adaptation. Our results indicate that Pyrococcus furiosus naturally encountered this conjugative plasmid or a related mobile genetic element in the past and responds to reinfection with robust primed adaptation.

Unraveling the impact and mechanism of antipyretic paracetamol on intergenera conjugative plasmid transfer.

Antimicrobial resistance has been considered as a great threat to biosecurity and human health. And the transmission of antibiotic resistance genes (ARGs) by conjugated plasmid is a key factor in the prevalence of antimicrobial resistance. Paracetamol (PRC), one of nonopioid analgesics, is an extensively used antipyretic and mild analgesic worldwide available for numerous prescriptions. It was unclear whether PRC could promote the spread of ARGs. Here, it was demonstrated that PRC promoted intergenera conjugative plasmid transfer in an established conjugation model. Both donor and recipient strains treated by PRC emerged the variations of reactive oxygen species (ROS), SOS response and cell membrane permeability. Correspondingly, transcriptome analysis revealed that the gene expression involved in cell membrane permeability and SOS response was up-regulated significantly after PRC exposure. More directly, PRC also increased the expressions of conjugation related genes of trbG and trbP in donor. This study proved for the first time that PRC could enhance the intergenera conjugative plasmid transfer. Collectively, these findings manifested the potential threat associated with the existence of non-antibiotic substance PRC, which could provide an important insight into antimicrobial resistance spread.

Linezolid Resistance Genes in Enterococci Isolated from Sediment and Zooplankton in Two Italian Coastal Areas.

Linezolid is a last-resort antibiotic for the treatment of severe infections caused by multidrug-resistant Gram-positive organisms; although linezolid resistance remains uncommon, the number of linezolid-resistant enterococci has increased in recent years due to worldwide spread of acquired resistance genes (cfr, optrA, and poxtA) in clinical, animal, and environmental settings. In this study, we investigated the occurrence of linezolid-resistant enterococci in marine samples from two coastal areas in Italy. Isolates grown on florfenicol-supplemented Slanetz-Bartley agar plates were investigated for their carriage of optrA, poxtA, and cfr genes; optrA was found in one Enterococcus faecalis isolate, poxtA was found in three Enterococcus faecium isolates and two Enterococcus hirae isolates, and cfr was not found. Two of the three poxtA-carrying E. faecium isolates and the two E. hirae isolates showed related pulsed-field gel electrophoresis (PFGE) profiles. Two E. faecium isolates belonged to the new sequence type 1710, which clustered in clonal complex 94, encompassing nosocomial strains. S1 PFGE/hybridization assays showed a double (chromosome and plasmid) location of poxtA and a plasmid location of optrA Whole-genome sequencing revealed that poxtA was contained in a Tn6657-like element carried by two plasmids (pEfm-EF3 and pEh-GE2) of similar size, found in different species, and that poxtA was flanked by two copies of IS1216 in both plasmids. In mating experiments, all but one strain (E. faecalis EN3) were able to transfer the poxtA gene to E. faecium 64/3. The occurrence of linezolid resistance genes in enterococci from marine samples is of great concern and highlights the need to improve practices aimed at limiting the transmission of linezolid-resistant strains to humans from environmental reservoirs.IMPORTANCE Linezolid is one of the few antimicrobials available to treat severe infections due to drug-resistant Gram-positive bacteria; therefore, the emergence of linezolid-resistant enterococci carrying transferable resistance determinants is of great concern for public health. Linezolid resistance genes (cfr, optrA, and poxtA), often plasmid located, can be transmitted via horizontal gene transfer and have the potential to spread globally. This study highlights the detection of enterococci carrying linezolid resistance genes from sediment and zooplankton samples from two coastal urban areas in Italy. The presence of clinically relevant resistant bacteria, such as linezolid-resistant enterococci, in marine environments could reflect their spillover from human and/or animal reservoirs and could indicate that coastal seawaters also might represent a source of these resistance genes.

Probiotic Bacillus Affects Enterococcus faecalis Antibiotic Resistance Transfer by Interfering with Pheromone Signaling Cascades.

Enterococcus faecalis, a member of the commensal flora in the human gastrointestinal tract, has become a threatening nosocomial pathogen because it has developed resistance to many known antibiotics. More concerningly, resistance gene-carrying E. faecalis cells may transfer antibiotic resistance to resistance-free E. faecalis cells through their unique quorum sensing-mediated plasmid transfer system. Therefore, we investigated the role of probiotic bacteria in the transfer frequency of the antibiotic resistance plasmid pCF10 in E. faecalis populations to mitigate the spread of antibiotic resistance. Bacillus subtilis subsp. natto is a probiotic strain isolated from Japanese fermented soybean foods, and its culture fluid potently inhibited pCF10 transfer by suppressing peptide pheromone activity from chromosomally encoded CF10 (cCF10) without inhibiting E. faecalis growth. The inhibitory effect was attributed to at least one 30- to 50-kDa extracellular protease present in B. subtilis subsp. natto. Nattokinase of B. subtilis subsp. natto was involved in the inhibition of pCF10 transfer and cleaved cCF10 (LVTLVFV) into LVTL plus VFV fragments. Moreover, the cleavage product LVTL (L peptide) interfered with the conjugative transfer of pCF10. In addition to cCF10, faecalis-cAM373 and gordonii-cAM373, which are mating inducers of vancomycin-resistant E. faecalis, were also cleaved by nattokinase, indicating that B. subtilis subsp. natto can likely interfere with vancomycin resistance transfer in E. faecalis. Our work shows the feasibility of applying fermentation products of B. subtilis subsp. natto and L peptide to mitigate E. faecalis antibiotic resistance transfer. IMPORTANCE Enterococcus faecalis is considered a leading cause of hospital-acquired infections. Treatment of these infections has become a major challenge for clinicians because some E. faecalis strains are resistant to multiple clinically used antibiotics. Moreover, antibiotic resistance genes can undergo efficient intra- and interspecies transfer via E. faecalis peptide pheromone-mediated plasmid transfer systems. Therefore, this study provided the first experimental demonstration that probiotics are a feasible approach for interfering with conjugative plasmid transfer between E. faecalis strains to stop the transfer of antibiotic resistance. We found that the extracellular protease(s) of Bacillus subtilis subsp. natto cleaved peptide pheromones without affecting the growth of E. faecalis, thereby reducing the frequency of conjugative plasmid transfer. In addition, a specific cleaved pheromone fragment interfered with conjugative plasmid transfer. These findings provide a potential probiotic-based method for interfering with the transfer of antibiotic resistance between E. faecalis strains.

Molecular Characterization of Cephalosporin-Resistant Salmonella Enteritidis ST11 Isolates Carrying blaCTX-M from Children with Diarrhea.

Salmonella Enteritidis is an important foodborne pathogen with high prevalence of resistance to cephalosporins, imposing a serious threat to public health. Therefore, a total of 162 Salmonella Enteritidis isolates collected from child patients in China from 2007 to 2017 were characterized for their resistance to cephalosporins and investigated the transmission characteristics of cephalosporin resistance gene. We found that 15 (9.26%) isolates were all resistant to cefalotin (minimum inhibitory concentration [MIC] ≥512 µg/mL), ceftazidime (MIC 16-128 µg/mL), ceftriaxone (MIC 64 to ≥512 µg/mL), ceftiofur (MIC 64-256 µg/mL), and cefotaxime (MIC 64 to ≥512 µg/mL) with the possession of cephalosporin resistance genes blaCTX-M-55 (n = 13), blaCTX-M-101 (n = 1), and blaCTX-M-153 (n = 1). Molecular typing further revealed that these 15 isolates belonged to sequence type ST11 and shared close pulsed-field gel electrophoresis patterns, suggesting the possibility of clonal spread in Salmonella Enteritidis interspecies. Furthermore, conjugation experiments were successfully performed in 13 of 15 isolates, and blaCTX-M-55 was present on conjugative plasmids with sizes ranging from 54.7 to 173.4 kb. Compared with recipient Escherichia coli C600, transconjugants conferred elevated MICs for cephalosporins ranging from 2- to 2048-fold. The genetic structure surrounding of blaCTX-M-55 gene in transconjugants were ΔISEcp1-blaCTX-M-55-orf477 (n = 8) and ISEcp1-blaCTX-M-55-orf477 (n = 3), respectively. Taken together, blaCTX-M on the plasmids might contribute to cephalosporin resistance in Salmonella Enteritidis, and conjugative transfer of blaCTX-M-55 might facilitate the spread of cephalosporin resistance in Salmonella Enteritidis. Hence, effective mitigation measurements are needed to reduce the threat caused by cephalosporin-resistant Salmonella Enteritidis to public health.

Entry Exclusion of Conjugative Plasmids of the IncA, IncC, and Related Untyped Incompatibility Groups.

Conjugative plasmids of incompatibility group C (IncC), formerly known as A/C2, disseminate antibiotic resistance genes globally in diverse pathogenic species of Gammaproteobacteria. Salmonella genomic island 1 (SGI1) can be mobilized by IncC plasmids and was recently shown to reshape the conjugative type IV secretion system (T4SS) encoded by these plasmids to evade entry exclusion. Entry exclusion blocks DNA translocation between cells containing identical or highly similar plasmids. Here, we report that the protein encoded by the entry exclusion gene of IncC plasmids (eexC) mediates entry exclusion in recipient cells through recognition of the IncC-encoded TraGC protein in donor cells. Phylogenetic analyses based on EexC and TraGC homologs predicted the existence of at least three different exclusion groups among IncC-related conjugative plasmids. Mating assays using Eex proteins encoded by representative IncC and IncA (former A/C1) and related untyped plasmids confirmed these predictions and showed that the IncC and IncA plasmids belong to the C exclusion group, thereby explaining their apparent incompatibility despite their compatible replicons. Representatives of the two other exclusion groups (D and E) are untyped conjugative plasmids found in Aeromonas sp. Finally, we determined through domain swapping that the carboxyl terminus of the EexC and EexE proteins controls the specificity of these exclusion groups. Together, these results unravel the role of entry exclusion in the apparent incompatibility between IncA and IncC plasmids while shedding light on the importance of the TraG subunit substitution used by SGI1 to evade entry exclusion.IMPORTANCE IncA and IncC conjugative plasmids drive antibiotic resistance dissemination among several pathogenic species of Gammaproteobacteria due to the diversity of drug resistance genes that they carry and their ability to mobilize antibiotic resistance-conferring genomic islands such as SGI1 of Salmonella enterica While historically grouped as "IncA/C," IncA and IncC replicons were recently confirmed to be compatible and to abolish each other's entry into the cell in which they reside during conjugative transfer. The significance of our study is in identifying an entry exclusion system that is shared by IncA and IncC plasmids. It impedes DNA transfer to recipient cells bearing a plasmid of either incompatibility group. The entry exclusion protein of this system is unrelated to any other known entry exclusion proteins.

Detection of plasmid-mediated tigecycline-resistant gene tet(X4) in Escherichia coli from pork, Sichuan and Shandong Provinces, China, February 2019.

The plasmid-mediated high-level tigecycline resistance gene, tet(X4), was detected in seven Escherichia coli isolates from pork in two Chinese provinces. Two isolates belonged to the epidemic spreading sequence type ST101. Tet(X4) was adjacent to ISVsa3 and concurrent with floR in all seven isolates. In addition to IncFIB, the replicon IncFII was found to be linked to tet(X4). This report follows a recent detection of tet(X3)/(X4) in E. coli from animals and humans in China.

Identification and Characterization of Conjugative Plasmids That Encode Ciprofloxacin Resistance in Salmonella.

This study aimed to characterize novel conjugative plasmids that encode transferable ciprofloxacin resistance in Salmonella In this study, 157 nonduplicated Salmonella isolates were recovered from food products, of which 55 were found to be resistant to ciprofloxacin. Interestingly, 37 of the 55 CiprSalmonella isolates (67%) did not harbor any mutations in the quinolone resistance-determining regions (QRDR). Six Salmonella isolates were shown to carry two novel types of conjugative plasmids that could transfer the ciprofloxacin resistance phenotype to Escherichia coli J53 (azithromycin resistant [Azir]). The first type of conjugative plasmid belonged to the ∼110-kb IncFIB-type conjugative plasmids carrying qnrB-bearing and aac(6')-Ib-cr-bearing mobile elements. Transfer of the plasmid between E. coli and Salmonella could confer a ciprofloxacin MIC of 1 to 2 µg/ml. The second type of conjugative plasmid belonged to ∼240-kb IncH1/IncF plasmids carrying a single PMQR gene, qnrS Importantly, this type of conjugative ciprofloxacin resistance plasmid could be detected in clinical Salmonella isolates. The dissemination of these conjugative plasmids that confer ciprofloxacin resistance poses serious challenges to public health and Salmonella infection control.

Deciphering conjugative plasmid permissiveness in wastewater microbiomes.

Wastewater treatment plants (WWTPs) are designed to robustly treat polluted water. They are characterized by ceaseless flows of organic, chemical and microbial matter, followed by treatment steps before environmental release. WWTPs are hotspots of horizontal gene transfer between bacteria via conjugative plasmids, leading to dissemination of potentially hazardous genetic material such as antimicrobial resistance genes (AMRGs). While current focus is on the threat of AMRGs spreading and their environmental maintenance, conjugative plasmid transfer dynamics within and between bacterial communities still remains largely uncharted. Furthermore, current in vitro methods used to assess conjugation in complex microbiomes do not include in situ behaviours of recipient cells, resulting in partial understanding of transfers. We investigated the in vitro conjugation capacities of WWTP microbiomes from inlet sewage and outlet treated water using the broad-host range IncP-1 conjugative plasmid, pKJK5. A thorough molecular approach coupling metagenomes to 16S rRNA DNA/cDNA amplicon sequencing was established to characterize microbiomes using the ecological concept of functional response groups. A broad diversity of recipient bacterial phyla for the plasmid was observed, especially in WWTP outlets. We also identified permissive bacteria potentially able to cross WWTPs and engage in conjugation before and after water treatment. Bacterial activity and lifestyle seem to influence conjugation extent, as treated water copiotrophs were the most represented strategist amongst transconjugants. Correlation analysis highlighted possible plasmid transmission routes into communities between the sewage to the environment, with identification of keystone members (e.g., Arcobacter) potentially involved in cross-border exchanges between distant Gram-positive and Gram-negative phyla.

Sequence of the R1 plasmid and comparison to F and R100.

The R1 antibiotic resistance plasmid, originally discovered in a clinical Salmonella isolate in London, 1963, has served for decades as a key model for understanding conjugative plasmids. Despite its scientific importance, a complete sequence of this plasmid has never been reported. We present the complete genome sequence of R1 along with a brief review of the current knowledge concerning its various genetic systems and a comparison to the F and R100 plasmids. R1 is 97,566 nucleotides long and contains 120 genes. The plasmid consists of a backbone largely similar to that of F and R100, a Tn21-like transposon that is nearly identical to that of R100, and a unique 9-kb sequence that bears some resemblance to sequences found in certain Klebsiella oxytoca strains. These three regions of R1 are separated by copies of the insertion sequence IS1. Overall, the structure of R1 and comparison to F and R100 suggest a fairly stable shared conjugative plasmid backbone into which a variety of mobile elements have inserted to form an "accessory" genome, containing multiple antibiotic resistance genes, transposons, remnants of phage genes, and genes whose functions remain unknown.

A large conjugative Acinetobacter baumannii plasmid carrying the sul2 sulphonamide and strAB streptomycin resistance genes.

Acinetobacter baumannii is an important nosocomial pathogen that often complicates treatment because of its high level of resistance to antibiotics. Though plasmids can potentially introduce various genes into bacterial strains, compared to other Gram-negative bacteria, information about the unique A. baumannii plasmid repertoire is limited. Here, whole genome sequence data was used to determine the plasmid content of strain A297 (RUH875), the reference strain for the globally disseminated multiply resistant A. baumannii clone, global clone 1(GC1). A297 contains three plasmids. Two known plasmids were present; one, pA297-1 (pRAY*), carries the aadB gentamicin, kanamycin and tobramycin resistance gene and another is an 8.7kb cryptic plasmid often found in GC1 isolates. The third plasmid, pA297-3, is 200kb and carries the sul2 sulphonamide resistance gene and strAB streptomycin resistance gene within Tn6172 and a mer mercuric ion resistance module elsewhere. pA297-3 transferred sulphonamide, streptomycin and mercuric ion resistance at high frequency to a susceptible A. baumannii recipient, and contains several genes potentially involved in conjugative transfer. However, a relaxase gene was not found. It also includes several genes encoding proteins involved in DNA metabolism such as partitioning. However, a gene encoding a replication initiation protein could not be found. pA297-3 includes two copies of a Miniature Inverted-Repeat Transposable Element (MITE), named MITE-297, bracketing a 77.5kb fragment, which contains several IS and the mer module. Several plasmids related to but smaller than pA297-3 were found in the GenBank nucleotide database. They were found in different A. baumannii clones and are wide spread. They all contain either Tn6172 or a variant in the same position in the backbone as Tn6172 in pA297-3. Some related plasmids have lost the segment between the MITE-297 copies and retain only one MITE-297. Others have segments of various lengths between two MITE-297 copies, and these can be derived from the region in pA297-3 via a deletion adjacent to IS related to IS26 such as IS1007 or IS1007-like. pA297-3 and its relatives represent a third type of conjugative Acinetobacter plasmid that contributes to the dissemination of antibiotic resistance in this species.

NetB and necrotic enteritis: the hole movable story.

Clostridium perfringens is the primary causative agent of avian necrotic enteritis. Our understanding of the pathogenesis of this economically important disease has been enhanced by the discovery of C. perfringens NetB toxin, which belongs to the α-haemolysin family of ß-pore-forming toxins. In a chicken disease model, the analysis of an isogenic set of strains comprising the wild type, a netB mutant, and its complemented derivative, fulfilled molecular Koch's postulates and revealed that NetB was essential for disease. These results were consistent with epidemiological surveys, which generally found that there was a higher prevalence of netB carriage in C. perfringens isolates from diseased poultry compared to healthy birds. The netB gene has been shown to be located on large conjugative plasmids that are closely related to other toxin plasmids from C. perfringens, which has potential implications for the epidemiology of necrotic enteritis infections. The crystal structures of both monomeric NetB and the heptameric NetB pore have been determined, the latter revealed a central pore diameter of approximately 26 Å. Finally, it has been shown that vaccine preparations that include NetB can protect chickens against disease and a series of single amino acid substitution derivatives of NetB that have potential value for vaccine formulations have been isolated and analysed. It is likely that NetB will be an important antigen to include in an effective, commercially viable, necrotic enteritis vaccine.

Plasmid carriage can limit bacteria-phage coevolution.

Coevolution with bacteriophages is a major selective force shaping bacterial populations and communities. A variety of both environmental and genetic factors has been shown to influence the mode and tempo of bacteria-phage coevolution. Here, we test the effects that carriage of a large conjugative plasmid, pQBR103, had on antagonistic coevolution between the bacterium Pseudomonas fluorescens and its phage, SBW25ϕ2. Plasmid carriage limited bacteria-phage coevolution; bacteria evolved lower phage-resistance and phages evolved lower infectivity in plasmid-carrying compared with plasmid-free populations. These differences were not explained by effects of plasmid carriage on the costs of phage resistance mutations. Surprisingly, in the presence of phages, plasmid carriage resulted in the evolution of high frequencies of mucoid bacterial colonies. Mucoidy can provide weak partial resistance against SBW25ϕ2, which may have limited selection for qualitative resistance mutations in our experiments. Taken together, our results suggest that plasmids can have evolutionary consequences for bacteria that go beyond the direct phenotypic effects of their accessory gene cargo.

The transmembrane domain of the T4SS coupling protein TrwB and its role in protein-protein interactions.

Bacteria use type IV secretion systems to transfer genetic material and proteins from donor to recipient cells, using proteins encoded by conjugative plasmids. Among those proteins the so-called Type IV Coupling Protein plays a central role in the process. One of the best studied members of this family is TrwB, the conjugative coupling protein of R388 plasmid. Previous studies indicated that the transmembrane domain of TrwB plays a role beyond the mere anchoring of the protein to the membrane. TrwB has also been shown to interact with other conjugative proteins, such as the VirB10-like protein of R388 TrwE. The goal of this study is to elucidate the role of the different domains of TrwB and TrwE in their biological function, and in both self- and TrwB-TrwE interactions. To this aim, a series of TrwB and TrwE deletion mutant proteins were constructed. Conjugation and interaction studies revealed that the transmembrane domain of TrwB, and particularly its second transmembrane helix, is needed for TrwB self-interaction and for R388 conjugative transfer and that there are contacts between TrwB and TrwE in the membrane. On the contrary, the lack of the TMD of TrwE does not completely abolish R388 conjugation although the interaction between TrwE-TrwB is lost. These results identify protein-protein interactions inside the membrane needed for T4SS function.