The Interaction of the F-Like Plasmid-Encoded TraN Isoforms with Their Cognate Outer Membrane Receptors.

Horizontal gene transfer via conjugation plays a major role in bacterial evolution. In F-like plasmids, efficient DNA transfer is mediated by close association between donor and recipient bacteria. This process, known as mating pair stabilization (MPS), is mediated by interactions between the plasmid-encoded outer membrane (OM) protein TraN in the donor and chromosomally-encoded OM proteins in the recipient. We have recently reported the existence of 7 TraN sequence types, which are grouped into 4 structural types, that we named TraNα, TraNß, TraNγ, and TraNδ. Moreover, we have shown specific pairing between TraNα and OmpW, TraNß and OmpK36 of Klebsiella pneumoniae, TraNγ and OmpA, and TraNδ and OmpF. In this study, we found that, although structurally similar, TraNα encoded by the Salmonella enterica pSLT plasmid (TraNα2) binds OmpW in both Escherichia coli and Citrobacter rodentium, while TraNα encoded by the R100-1 plasmid (TraNα1) only binds OmpW in E. coli. AlphaFold2 predictions suggested that this specificity is mediated by a single amino acid difference in loop 3 of OmpW, which we confirmed experimentally. Moreover, we show that single amino acids insertions into loop 3 of OmpK36 affect TraNß-mediated conjugation efficiency of the K. pneumoniae resistance plasmid pKpQIL. Lastly, we report that TraNß can also mediate MPS by binding OmpK35, making it the first TraN variant that can bind more than one OM protein in the recipient. Together, these data show that subtle sequence differences in the OM receptors can impact TraN-mediated conjugation efficiency. IMPORTANCE Conjugation plays a central role in the spread of antimicrobial resistance genes among bacterial pathogens. Efficient conjugation is mediated by formation of mating pairs via a pilus, followed by mating pair stabilization (MPS), mediated by tight interactions between the plasmid-encoded outer membrane protein (OMP) TraN in the donor (of which there are 7 sequence types grouped into the 4 structural isoforms α, ß, γ, and δ), and an OMP receptor in the recipient (OmpW, OmpK36, OmpA, and OmpF, respectively). In this study, we found that subtle differences in OmpW and OmpK36 have significant consequences on conjugation efficiency and specificity, highlighting the existence of selective pressure affecting plasmid-host compatibility and the flow of horizontal gene transfer in bacteria.

IncFV plasmid pED208: Sequence analysis and evidence for translocation of maintenance/leading region proteins through diverse type IV secretion systems.

Two phylogenetically distantly-related IncF plasmids, F and pED208, serve as important models for mechanistic and structural studies of F-like type IV secretion systems (T4SSFs) and F pili. Here, we present the pED208 sequence and compare it to F and pUMNF18, the closest match to pED208 in the NCBI database. As expected, gene content of the three cargo regions varies extensively, although the maintenance/leading regions (MLRs) and transfer (Tra) regions also carry novel genes or motifs with predicted modulatory effects on plasmid stability, dissemination and host range. By use of a Cre recombinase assay for translocation (CRAfT), we recently reported that pED208-carrying donors translocate several products of the MLR (ParA, ParB1, ParB2, SSB, PsiB, PsiA) intercellularly through the T4SSF. Here, we extend these findings by reporting that pED208-carrying donors translocate 10 additional MLR proteins during conjugation. In contrast, two F plasmid-encoded toxin components of toxin-antitoxin (TA) modules, CcdB and SrnB, were not translocated at detectable levels through the T4SSF. Remarkably, most or all of the pED208-encoded MLR proteins and CcdB and SrnB were translocated through heterologous T4SSs encoded by IncN and IncP plasmids pKM101 and RP4, respectively. Together, our sequence analyses underscore the genomic diversity of the F plasmid superfamily, and our experimental data demonstrate the promiscuous nature of conjugation machines for protein translocation. Our findings raise intriguing questions about the nature of T4SS translocation signals and of the biological and evolutionary consequences of conjugative protein transfer.

CpxR negatively regulates IncFII-replicon plasmid pEC011 conjugation by directly binding to multi-promoter regions.

CpxAR is a global regulatory protein and has important roles in plasmid mating. However, except for traJ, the regulatory effect of CpxAR on other tra genes is unclear. The aim of this study was to explore the effects of CpxAR on conjugative transfer of the epidemic plasmid pEC011 (IncFII replicon) in Escherichia coli. The plasmid mating frequencies were significantly higher for the single deletion mutant strain FΔcpxR than for the parental strain F25922. Additionally, expression levels of traM, traJ and traY in FΔcpxR were significantly higher than those in F25922. Further investigations revealed that His6-CpxR protein could directly bind to the traM, traJ and traY promoter regions with the binding sites of 5'-TTTACATT-3' (PM), 5'-ATAAGAAT-3' (PJ), and 5'-AATTTTAT-3' (PY), respectively. Taken together, our results demonstrate that CpxAR can downregulate the expression of traM, traJ and traY by directly binding to the CpxR box-like sites of promoters, thus significantly reducing the mating rates of IncFII replicon plasmid pEC011.

Mating pair stabilization mediates bacterial conjugation species specificity.

Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating the spread of virulence and resistance plasmids. Here we describe how variants of the plasmid-encoded donor outer membrane (OM) protein TraN cooperate with distinct OM receptors in recipients to mediate mating pair stabilization and efficient DNA transfer. We show that TraN from the plasmid pKpQIL (Klebsiella pneumoniae) interacts with OmpK36, plasmids from R100-1 (Shigella flexneri) and pSLT (Salmonella Typhimurium) interact with OmpW, and the prototypical F plasmid (Escherichia coli) interacts with OmpA. Cryo-EM analysis revealed that TraNpKpQIL interacts with OmpK36 through the insertion of a ß-hairpin in the tip of TraN into a monomer of the OmpK36 porin trimer. Combining bioinformatic analysis with AlphaFold structural predictions, we identified a fourth TraN structural variant that mediates mating pair stabilization by binding OmpF. Accordingly, we devised a classification scheme for TraN homologues on the basis of structural similarity and their associated receptors: TraNα (OmpW), TraNß (OmpK36), TraNγ (OmpA), TraNδ (OmpF). These TraN-OM receptor pairings have real-world implications as they reflect the distribution of resistance plasmids within clinical Enterobacteriaceae isolates, demonstrating the importance of mating pair stabilization in mediating conjugation species specificity. These findings will allow us to predict the distribution of emerging resistance plasmids in high-risk bacterial pathogens.

Contributions of F-specific subunits to the F plasmid-encoded type IV secretion system and F pilus.

F plasmids circulate widely among the Enterobacteriaceae through encoded type IV secretion systems (T4SSF s). Assembly of T4SSF s and associated F pili requires 10 VirB/VirD4-like Tra subunits and eight or more F-specific subunits. Recently, we presented evidence using in situ cryoelectron tomography (cryoET) that T4SSF s undergo structural transitions when activated for pilus production, and that assembled pili are deposited onto alternative basal platforms at the cell surface. Here, we deleted eight conserved F-specific genes from the MOBF12C plasmid pED208 and quantitated effects on plasmid transfer, pilus production by fluorescence microscopy, and elaboration of T4SSF structures by in situ cryoET. Mutant phenotypes supported the assignment of F-specific subunits into three functional Classes: (i) TraF, TraH, and TraW are required for all T4SSF -associated activities, (ii) TraU, TraN, and TrbC are nonessential but contribute significantly to distinct T4SSF functions, and (iii) TrbB is essential for F pilus production but not for plasmid transfer. Equivalent mutations in a phylogenetically distantly related MOB12A F plasmid conferred similar phenotypes and generally supported these Class assignments. We present a new structure-driven model in which F-specific subunits contribute to distinct steps of T4SSF assembly or activation to regulate DNA transfer and F pilus dynamics and deposition onto alternative platforms.

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.

A role for the last C-terminal helix of the F plasmid segregating protein SopA in nucleoid binding and plasmid maintenance.

The rapid emergence and spread of antibiotic resistance is a growing global burden. Antibiotic resistance is often associated with large single or low copy number plasmids, which rely upon cytoskeletal proteins for their stable maintenance. While the mechanism of plasmid partitioning has been well established for the R plasmids, the molecular details by which the F plasmid is maintained is only beginning to emerge. The partitioning function of the F plasmid depends upon a ParA/ MinD family of proteins known as SopA. SopA, by virtue of its ATP-dependent non-specific DNA binding activity and association with the bacterial nucleoid, drives the segregation of the F plasmid into the daughter cells. This function further depends upon the stimulation of the ATPase activity of SopA by the SopBC complex. Here, we report that several residues in the last C-terminal helix in SopA play a crucial but distinct role in SopA function and plasmid maintenance. While the deletion of the last five residues in SopA does not affect its ability to bind the nucleoid or SopB, they severely affect the plasmid partitioning function. Further, we show that while mutations in certain polar residues in the C-terminal helix only mildly affect its localisation to the nucleoid, others cause defects in nsDNA binding and disrupt plasmid maintenance functions.

Structure of a type IV secretion system core complex encoded by multi-drug resistance F plasmids.

Bacterial type IV secretion systems (T4SSs) are largely responsible for the proliferation of multi-drug resistance. We solved the structure of the outer-membrane core complex (OMCCF) of a T4SS encoded by a conjugative F plasmid at <3.0 Å resolution by cryoelectron microscopy. The OMCCF consists of a 13-fold symmetrical outer ring complex (ORC) built from 26 copies of TraK and TraV C-terminal domains, and a 17-fold symmetrical central cone (CC) composed of 17 copies of TraB ß-barrels. Domains of TraV and TraB also bind the CC and ORC substructures, establishing that these proteins undergo an intraprotein symmetry alteration to accommodate the C13:C17 symmetry mismatch. We present evidence that other pED208-encoded factors stabilize the C13:C17 architecture and define the importance of TraK, TraV and TraB domains to T4SSF function. This work identifies OMCCF structural motifs of proposed importance for structural transitions associated with F plasmid dissemination and F pilus biogenesis.

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

Bacterial type IV secretion systems (T4SSs) mediate the conjugative transfer of mobile genetic elements (MGEs) and their cargoes of antibiotic resistance and virulence genes. Here, we report that the pED208-encoded T4SS (TrapED208) translocates not only this F plasmid but several plasmid-encoded proteins, including ParA, ParB1, single-stranded DNA-binding protein SSB, ParB2, PsiB, and PsiA, to recipient cells. Conjugative protein translocation through the TrapED208 T4SS required engagement of the pED208 relaxosome with the TraD substrate receptor or coupling protein. T4SSs translocate MGEs as single-stranded DNA intermediates (T-strands), which triggers the SOS response in recipient cells. Transfer of pED208 deleted of psiB or ssb, which, respectively, encode the SOS inhibitor protein PsiB and single-stranded DNA-binding protein SSB, elicited a significantly stronger SOS response than pED208 or mutant plasmids deleted of psiA, parA, parB1, or parB2. Conversely, translocation of PsiB or SSB, but not PsiA, through the TrapED208 T4SS suppressed the mating-induced SOS response. Our findings expand the repertoire of known substrates of conjugation systems to include proteins with functions associated with plasmid maintenance. Furthermore, for this and other F-encoded Tra systems, docking of the DNA substrate with the TraD receptor appears to serve as a critical activating signal for protein translocation. Finally, the observed effects of PsiB and SSB on suppression of the mating-induced SOS response establishes a novel biological function for conjugative protein translocation and suggests the potential for interbacterial protein translocation to manifest in diverse outcomes influencing bacterial communication, physiology, and evolution. IMPORTANCE 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. The repertoire of translocated proteins includes the plasmidic SOS inhibitor protein PsiB, single-stranded DNA-binding protein SSB, and several partitioning proteins. We demonstrate that intercellular transmission of PsiB and SSB suppresses the SOS response, which is triggered in recipient cells upon acquisition of the single-stranded DNA transfer intermediate during mating. Our findings identify a new biological function for conjugative protein translocation in mitigating potentially deleterious consequences to plasmid and genome integrity resulting from SOS-induced recombination and mutation events.

Grand scale genome manipulation via chromosome swapping in Escherichia coli programmed by three one megabase chromosomes.

In bacterial synthetic biology, whole genome transplantation has been achieved only in mycoplasmas that contain a small genome and are competent for foreign genome uptake. In this study, we developed Escherichia coli strains programmed by three 1-megabase (Mb) chromosomes by splitting the 3-Mb chromosome of a genome-reduced strain. The first split-chromosome retains the original replication origin (oriC) and partitioning (par) system. The second one has an oriC and the par locus from the F plasmid, while the third one has the ori and par locus of the Vibrio tubiashii secondary chromosome. The tripartite-genome cells maintained the rod-shaped form and grew only twice as slowly as their parent, allowing their further genetic engineering. A proportion of these 1-Mb chromosomes were purified as covalently closed supercoiled molecules with a conventional alkaline lysis method and anion exchange columns. Furthermore, the second and third chromosomes could be individually electroporated into competent cells. In contrast, the first split-chromosome was not able to coexist with another chromosome carrying the same origin region. However, it was exchangeable via conjugation between tripartite-genome strains by using different selection markers. We believe that this E. coli-based technology has the potential to greatly accelerate synthetic biology and synthetic genomics.

Identification of a Potential Membrane-Targeting Sequence in the C-Terminus of the F Plasmid Segregation Protein SopA.

Stable maintenance and partitioning of the 'Fertility' plasmid or the F plasmid in its host Escherichia coli require the function of a ParA superfamily of proteins known as SopA. The mechanism by which SopA mediates plasmid segregation is well studied. SopA is a nucleoid-binding protein and binds DNA in an ATP-dependent but sequence non-specific manner. ATP hydrolysis stimulated by the binding of the SopBC complex mediates the release of SopA from the nucleoid. Cycles of ATP-binding and hydrolysis generate an ATPase gradient that moves the plasmid through a chemophoresis force. Nucleoid binding of SopA thus assumes a central role in its plasmid-partitioning function. However, earlier work also suggests that the F plasmid can be partitioned into anucleate cells, thus implicating nucleoid independent partitioning. Interestingly, SopA is also reported to be associated with the inner membrane of the bacteria. Here, we report the identification of a possible membrane-targeting sequence, a predicted amphipathic helix, at the C-terminus of SopA. Molecular dynamics simulations indicate that the predicted amphipathic helical motif of SopA has weak affinity for membranes. Moreover, we experimentally show that SopA can associate with bacterial membranes, is detectable in the membrane fractions of bacterial lysates, and is sensitive to the membrane potential. Further, unlike the wild-type SopA, a deletion of the C-terminal 29 amino acids results in the loss of F plasmids from bacterial cells.

Plasmid Transfer by Conjugation in Gram-Negative Bacteria: From the Cellular to the Community Level.

Bacterial conjugation, also referred to as bacterial sex, is a major horizontal gene transfer mechanism through which DNA is transferred from a donor to a recipient bacterium by direct contact. Conjugation is universally conserved among bacteria and occurs in a wide range of environments (soil, plant surfaces, water, sewage, biofilms, and host-associated bacterial communities). Within these habitats, conjugation drives the rapid evolution and adaptation of bacterial strains by mediating the propagation of various metabolic properties, including symbiotic lifestyle, virulence, biofilm formation, resistance to heavy metals, and, most importantly, resistance to antibiotics. These properties make conjugation a fundamentally important process, and it is thus the focus of extensive study. Here, we review the key steps of plasmid transfer by conjugation in Gram-negative bacteria, by following the life cycle of the F factor during its transfer from the donor to the recipient cell. We also discuss our current knowledge of the extent and impact of conjugation within an environmentally and clinically relevant bacterial habitat, bacterial biofilms.

Interaction of the maturation protein of the bacteriophage MS2 and the sex pilus of the Escherichia coli F plasmid.

One promising strategy to combat antimicrobial resistance is to use bacteriophages that attach to the sex pili produced by transmissible antimicrobial resistance (AMR) plasmids, infect AMR bacteria and select for loss of the AMR plasmids, prolonging the life of existing antimicrobials. The maturation protein of the bacteriophage MS2 attaches to the pili produced by Incompatibility group F plasmid-containing bacteria. This interaction initiates delivery of the viral genetic material into the bacteria. Using protein-protein docking we constructed a model of the F pilus comprising a trimer of subunits binding to the maturation protein. Interactions between the maturation protein and the F pilus were investigated using molecular dynamics simulations. In silico alanine scanning and in silico single-point mutations were explored, with the longer term aim of increasing the affinity of the maturation protein to other Incompatibility group pili, without reducing the strength of binding to F pilin. We report our computational findings on which residues are required for the maturation protein and F pilin to interact, those which had no effect on the interaction and the mutations which led to a stronger interaction.

F Plasmids Are the Major Carriers of Antibiotic Resistance Genes in Human-Associated Commensal Escherichia coli.

The evolution and propagation of antibiotic resistance by bacterial pathogens are significant threats to global public health. Contemporary DNA sequencing tools were applied here to gain insight into carriage of antibiotic resistance genes in Escherichia coli, a ubiquitous commensal bacterium in the gut microbiome in humans and many animals, and a common pathogen. Draft genome sequences generated for a collection of 101 E. coli strains isolated from healthy undergraduate students showed that horizontally acquired antibiotic resistance genes accounted for most resistance phenotypes, the primary exception being resistance to quinolones due to chromosomal mutations. A subset of 29 diverse isolates carrying acquired resistance genes and 21 control isolates lacking such genes were further subjected to long-read DNA sequencing to enable complete or nearly complete genome assembly. Acquired resistance genes primarily resided on F plasmids (101/153 [67%]), with smaller numbers on chromosomes (30/153 [20%]), IncI complex plasmids (15/153 [10%]), and small mobilizable plasmids (5/153 [3%]). Nearly all resistance genes were found in the context of known transposable elements. Very few structurally conserved plasmids with antibiotic resistance genes were identified, with the exception of an ∼90-kb F plasmid in sequence type 1193 (ST1193) isolates that appears to serve as a platform for resistance genes and may have virulence-related functions as well. Carriage of antibiotic resistance genes on transposable elements and mobile plasmids in commensal E. coli renders the resistome highly dynamic.IMPORTANCE Rising antibiotic resistance in human-associated bacterial pathogens is a serious threat to our ability to treat many infectious diseases. It is critical to understand how acquired resistance genes move in and through bacteria associated with humans, particularly for species such as Escherichia coli that are very common in the human gut but can also be dangerous pathogens. This work combined two distinct DNA sequencing approaches to allow us to explore the genomes of E. coli from college students to show that the antibiotic resistance genes these bacteria have acquired are usually carried on a specific type of plasmid that is naturally transferrable to other E. coli, and likely to other related bacteria.

Antimicrobial antisense RNA delivery to F-pili producing multidrug-resistant bacteria via a genetically engineered bacteriophage.

Multidrug-resistant bacteria are a growing issue worldwide. This study developed a convenient and effective method to downregulate the expression of a specific gene to produce a novel antimicrobial tool using a small (140 nucleotide) RNA with a 24-nucleotide antisense (as) region from an arabinose-inducible expression phagemid vector in Escherichia coli. Knockdown effects of rpoS encoding RNA polymerase sigma factor were observed using this inducible artificial asRNA approach. asRNAs targeting several essential E. coli genes produced significant growth defects, especially when targeted to acpP and ribosomal protein coding genes rplN, rplL, and rpsM. Growth inhibited phenotypes were facilitated in hfq- conditions. Phage lysates were prepared from cells harboring phagemids as a lethal-agent delivery tool. Targeting the rpsM gene by phagemid-derived M13 phage infection of E. coli containing a carbapenem-producing F-plasmid and multidrug-resistant Klebsiella pneumoniae containing an F-plasmid resulted in the death of over 99.99% of infected bacteria. This study provides a possible strategy for treating bacterial infection and can be applied to any F-pilus producing bacterial species.

Depression and Abuse During Medical Internships in Peruvian Hospitals.

OBJECTIVE: To estimate the prevalence of depression and abuse and identify the associated factors in medical interns in Peruvian hospitals. METHODS: We conducted a multicentre, cross-sectional and analytical study during the months of March to May 2016. We included medical interns from 18 Peruvian hospitals, in Lima and provinces. We used a survey that included sociodemographic data and a scale to measure the perception of abuse (psychological, physical and sexual). In addition, we used the Spanish version of the PHQ-9 to evaluate depression. We used Poisson regression with robust variances to calculate prevalence ratios (PRs). RESULTS: A total of 402 medical interns participated in the study, the median age was 25 [IQR: 21-33], and 52.7% were male. Moreover, 25.4% of them suffered from depression. In the adjusted model, the variables associated with depression were age (PRa=1.15), the number of hours they sleep per day (PRa=1.23), being a woman (PRa=3.33), performing the internship in a province (PRa=0.25), studying at a public university (PRa=0.64), living with parents (PRa=0.65) or alone (PRa=0.33), and having perceived some type of abuse (PRa=1.07). CONCLUSIONS: A quarter of surveyed medical interns had depression. Early screening is necessary to identify perceived abuse and other triggering depression factors that may adversely affect work performance and overall mental health during medical internships.

Structural bases for F plasmid conjugation and F pilus biogenesis in Escherichia coli.

Bacterial conjugation systems are members of the large type IV secretion system (T4SS) superfamily. Conjugative transfer of F plasmids residing in the Enterobacteriaceae was first reported in the 1940s, yet the architecture of F plasmid-encoded transfer channel and its physical relationship with the F pilus remain unknown. We visualized F-encoded structures in the native bacterial cell envelope by in situ cryoelectron tomography (CryoET). Remarkably, F plasmids encode four distinct structures, not just the translocation channel or channel-pilus complex predicted by prevailing models. The F1 structure is composed of distinct outer and inner membrane complexes and a connecting cylinder that together house the envelope-spanning translocation channel. The F2 structure is essentially the F1 complex with the F pilus attached at the outer membrane (OM). Remarkably, the F3 structure consists of the F pilus attached to a thin, cell envelope-spanning stalk, whereas the F4 structure consists of the pilus docked to the OM without an associated periplasmic density. The traffic ATPase TraC is configured as a hexamer of dimers at the cytoplasmic faces of the F1 and F2 structures, where it respectively regulates substrate transfer and F pilus biogenesis. Together, our findings present architectural renderings of the DNA conjugation or "mating" channel, the channel-pilus connection, and unprecedented pilus basal structures. These structural snapshots support a model for biogenesis of the F transfer system and allow for detailed comparisons with other structurally characterized T4SSs.

Role of AcrAB-TolC multidrug efflux pump in drug-resistance acquisition by plasmid transfer.

Drug-resistance dissemination by horizontal gene transfer remains poorly understood at the cellular scale. Using live-cell microscopy, we reveal the dynamics of resistance acquisition by transfer of the Escherichia coli fertility factor-conjugation plasmid encoding the tetracycline-efflux pump TetA. The entry of the single-stranded DNA plasmid into the recipient cell is rapidly followed by complementary-strand synthesis, plasmid-gene expression, and production of TetA. In the presence of translation-inhibiting antibiotics, resistance acquisition depends on the AcrAB-TolC multidrug efflux pump, because it reduces tetracycline concentrations in the cell. Protein synthesis can thus persist and TetA expression can be initiated immediately after plasmid acquisition. AcrAB-TolC efflux activity can also preserve resistance acquisition by plasmid transfer in the presence of antibiotics with other modes of action.

Cooperative Function of TraJ and ArcA in Regulating the F Plasmid tra Operon.

The F plasmid tra operon encodes most of the proteins required for bacterial conjugation. TraJ and ArcA are known activators of the tra operon promoter PY, which is subject to H-NS-mediated silencing. Donor ability and promoter activity assays indicated that PY is inactivated by silencers and requires both TraJ and ArcA for activation to support efficient F conjugation. The observed low-level, ArcA-independent F conjugation is caused by tra expression from upstream alternative promoters. Electrophoretic mobility shift assays showed that TraJ alone weakly binds to PY regulatory DNA; however, TraJ binding is significantly enhanced by ArcA binding to the same DNA, indicating cooperativity of the two proteins. Analysis of binding affinities between ArcA and various DNA fragments in the PY regulatory region defined a 22-bp tandem repeat sequence (from -76 to -55 of PY) sufficient for optimal ArcA binding, which is immediately upstream of the predicted TraJ-binding site (from -54 to -34). Deletion analysis of the PY promoter in strains deficient in TraJ, ArcA, and/or H-NS determined that sequences upstream of -103 are required by silencers including H-NS for PY silencing, whereas sequences downstream of -77 are targeted by TraJ and ArcA for activation. TraJ and ArcA appear not only to counteract PY silencers but also to directly activate PY in a cooperative manner. Our data reveal the cooperativity of TraJ and ArcA during PY activation and provide insights into the regulatory circuit controlling F-family plasmid-mediated bacterial conjugation.IMPORTANCE Conjugation is a major mechanism for dissemination of antibiotic resistance and virulence among bacterial populations. The tra operon in the F family of conjugative plasmids encodes most of the proteins involved in bacterial conjugation. This work reveals that activation of tra operon transcription requires two proteins, TraJ and ArcA, to bind cooperatively to adjacent sites immediately upstream of the major tra promoter PY The interaction of TraJ and ArcA with the tra operon not only relieves PY from silencers but also directly activates it. These findings provide insights into the regulatory circuit of the F-family plasmid-mediated bacterial conjugation.

New approach for the simultaneous detection of somatic coliphages and F-specific RNA coliphages as indicators of fecal pollution.

Two groups of coliphages have been recently included in different water management policies as indicators of viral fecal pollution in water and food: somatic coliphages, which infect E. coli through cell wall receptors, and F-specific RNA coliphages, which infect through the F-pili. Somatic coliphages are more abundant in fecally contaminated waters, except reclaimed waters, those disinfected by UV irradiation, and some groundwater samples that show a higher level of F-specific coliphages. Somatic coliphages are morphologically similar to DNA enteric viruses while F-specific coliphages are similar to RNA viruses such as norovirus and hepatitis A viruses, which are the viral pathogens of concern in sewage. The use of strains sensitive to both types of phages has been proposed for total coliphage enumeration, thereby avoiding double analysis. The standardized methods available for coliphage detection are robust and cost-effective, but the introduction of ready-to-use methods would facilitate routine implementation in laboratories. The fastest available tool for somatic coliphage enumeration is the recently developed Bluephage, which uses a modified ß-glucuronide-overexpressing E. coli strain unable to take up the glucuronide substrate. The overexpressed enzyme accumulates inside the bacterial cells until released by phage-induced cell lysis, whereupon it encounters its substrate and the medium changes from yellow to blue. The present method uses E. coli strain CB12, sensitive to somatic coliphages and F-specific coliphages due to the expression of the F-pili. The Bluephage approach incorporating CB12 detects both types of coliphages in a time range of 1:30 to 4:00 h, as assayed with coliphages from raw sewage, river water, sludge and mussels. This strategy can be applied to obtain qualitative and quantitative results and is applicable to microplates as well as to large sample volumes (100 ml). Moreover it can provide monitoring of water bodies at real time, as for example for ambient recreational beach monitoring.