Recipient UvrD helicase is involved in single- to double-stranded DNA conversion during conjugative plasmid transfer.

Dissemination of antibiotic resistance, a current societal challenge, is often driven by horizontal gene transfer through bacterial conjugation. During conjugative plasmid transfer, single-stranded (ss) DNA is transferred from the donor to the recipient cell. Subsequently, a complete double-stranded (ds) plasmid molecule is generated and plasmid-encoded genes are expressed, allowing successful establishment of the transconjugant cell. Such dynamics of transmission can be modulated by host- or plasmid-encoded factors, either in the donor or in the recipient cell. We applied transposon insertion sequencing to identify host-encoded factors that affect conjugative transfer frequency in Escherichia coli. Disruption of the recipient uvrD gene decreased the acquisition frequency of conjugative plasmids belonging to different incompatibility groups. Results from various UvrD mutants suggested that dsDNA binding activity and interaction with RNA polymerase are dispensable, but ATPase activity is required for successful plasmid establishment of transconjugant cells. Live-cell microscopic imaging showed that the newly transferred ssDNA within a uvrD- recipient often failed to be converted to dsDNA. Our work suggested that in addition to its role in maintaining genome integrity, UvrD is also key for the establishment of horizontally acquired plasmid DNA that drives genome diversity and evolution.

Analysis of HubP-dependent cell pole protein targeting in Vibrio cholerae uncovers novel motility regulators.

In rod-shaped bacteria, the emergence and maintenance of long-axis cell polarity is involved in key cellular processes such as cell cycle, division, environmental sensing and flagellar motility among others. Many bacteria achieve cell pole differentiation through the use of polar landmark proteins acting as scaffolds for the recruitment of functional macromolecular assemblies. In Vibrio cholerae a large membrane-tethered protein, HubP, specifically interacts with proteins involved in chromosome segregation, chemotaxis and flagellar biosynthesis. Here we used comparative proteomics, genetic and imaging approaches to identify additional HubP partners and demonstrate that at least six more proteins are subject to HubP-dependent polar localization. These include a cell-wall remodeling enzyme (DacB), a likely chemotaxis sensory protein (HlyB), two presumably cytosolic proteins of unknown function (VC1210 and VC1380) and two membrane-bound proteins, named here MotV and MotW, that exhibit distinct effects on chemotactic motility. We show that while both ΔmotW and ΔmotV mutants retain monotrichous flagellation, they present significant to severe motility defects when grown in soft agar. Video-tracking experiments further reveal that ΔmotV cells can swim in liquid environments but are unable to tumble or penetrate a semisolid matrix, whereas a motW deletion affects both tumbling frequency and swimming speed. Motility suppressors and gene co-occurrence analyses reveal co-evolutionary linkages between MotV, a subset of non-canonical CheV proteins and flagellar C-ring components FliG and FliM, whereas MotW regulatory inputs appear to intersect with specific c-di-GMP signaling pathways. Together, these results reveal an ever more versatile role for the landmark cell pole organizer HubP and identify novel mechanisms of motility regulation.

Plasmid replication-associated single-strand-specific methyltransferases.

Analysis of genomic DNA from pathogenic strains of Burkholderia cenocepacia J2315 and Escherichia coli O104:H4 revealed the presence of two unusual MTase genes. Both are plasmid-borne ORFs, carried by pBCA072 for B. cenocepacia J2315 and pESBL for E. coli O104:H4. Pacific Biosciences SMRT sequencing was used to investigate DNA methyltransferases M.BceJIII and M.EcoGIX, using artificial constructs. Mating properties of engineered pESBL derivatives were also investigated. Both MTases yield promiscuous m6A modification of single strands, in the context SAY (where S = C or G and Y = C or T). Strikingly, this methylation is asymmetric in vivo, detected almost exclusively on one DNA strand, and is incomplete: typically, around 40% of susceptible motifs are modified. Genetic and biochemical studies suggest that enzyme action depends on replication mode: DNA Polymerase I (PolI)-dependent ColE1 and p15A origins support asymmetric modification, while the PolI-independent pSC101 origin does not. An MTase-PolI complex may enable discrimination of PolI-dependent and independent plasmid origins. M.EcoGIX helps to establish pESBL in new hosts by blocking the action of restriction enzymes, in an orientation-dependent fashion. Expression and action appear to occur on the entering single strand in the recipient, early in conjugal transfer, until lagging-strand replication creates the double-stranded form.

A multidomain hub anchors the chromosome segregation and chemotactic machinery to the bacterial pole.

The cell poles constitute key subcellular domains that are often critical for motility, chemotaxis, and chromosome segregation in rod-shaped bacteria. However, in nearly all rods, the processes that underlie the formation, recognition, and perpetuation of the polar domains are largely unknown. Here, in Vibrio cholerae, we identified HubP (hub of the pole), a polar transmembrane protein conserved in all vibrios, that anchors three ParA-like ATPases to the cell poles and, through them, controls polar localization of the chromosome origin, the chemotactic machinery, and the flagellum. In the absence of HubP, oriCI is not targeted to the cell poles, chemotaxis is impaired, and a small but increased fraction of cells produces multiple, rather than single, flagella. Distinct cytoplasmic domains within HubP are required for polar targeting of the three ATPases, while a periplasmic portion of HubP is required for its localization. HubP partially relocalizes from the poles to the mid-cell prior to cell division, thereby enabling perpetuation of the polar domain in future daughter cells. Thus, a single polar hub is instrumental for establishing polar identity and organization.

High-resolution genetic analysis of the requirements for horizontal transmission of the ESBL plasmid from Escherichia coli O104:H4.

Horizontal dissemination of the genes encoding extended spectrum beta-lactamases (ESBLs) via conjugative plasmids is facilitating the increasingly widespread resistance of pathogens to beta-lactam antibiotics. However, there is relatively little known about the regulatory factors and mechanisms that govern the spread of these plasmids. Here, we carried out a high-throughput, transposon insertion site sequencing analysis (TnSeq) to identify genes that enable the maintenance and transmission of pESBL, an R64 (IncI1)-related resistance plasmid that was isolated from Escherichia coli O104:H4 linked to a recent large outbreak of gastroenteritis. With a few exceptions, the majority of the genes identified as required for maintenance and transmission of pESBL matched those of their previously defined R64 counterparts. However, our analyses of the high-density transposon insertion library in pESBL also revealed two very short and linked regions that constitute a previously unrecognized regulatory system controlling spread of IncI1 plasmids. In addition, we investigated the function of the pESBL-encoded M.EcoGIX methyltransferase, which is also encoded by many other IncI1 and IncF plasmids. This enzyme proved to protect pESBL from restriction in new hosts, suggesting it aids in expanding the plasmid's host range. Collectively, our work illustrates the power of the TnSeq approach to enable rapid and comprehensive analyses of plasmid genes and sequences that facilitate the dissemination of determinants of antibiotic resistance.

The origin of the Haitian cholera outbreak strain.

BACKGROUND: Although cholera has been present in Latin America since 1991, it had not been epidemic in Haiti for at least 100 years. Recently, however, there has been a severe outbreak of cholera in Haiti. METHODS: We used third-generation single-molecule real-time DNA sequencing to determine the genome sequences of 2 clinical Vibrio cholerae isolates from the current outbreak in Haiti, 1 strain that caused cholera in Latin America in 1991, and 2 strains isolated in South Asia in 2002 and 2008. Using primary sequence data, we compared the genomes of these 5 strains and a set of previously obtained partial genomic sequences of 23 diverse strains of V. cholerae to assess the likely origin of the cholera outbreak in Haiti. RESULTS: Both single-nucleotide variations and the presence and structure of hypervariable chromosomal elements indicate that there is a close relationship between the Haitian isolates and variant V. cholerae El Tor O1 strains isolated in Bangladesh in 2002 and 2008. In contrast, analysis of genomic variation of the Haitian isolates reveals a more distant relationship with circulating South American isolates. CONCLUSIONS: The Haitian epidemic is probably the result of the introduction, through human activity, of a V. cholerae strain from a distant geographic source. (Funded by the National Institute of Allergy and Infectious Diseases and the Howard Hughes Medical Institute.).

Regulatory cross-talk links Vibrio cholerae chromosome II replication and segregation.

There is little knowledge of factors and mechanisms for coordinating bacterial chromosome replication and segregation. Previous studies have revealed that genes (and their products) that surround the origin of replication (oriCII) of Vibrio cholerae chromosome II (chrII) are critical for controlling the replication and segregation of this chromosome. rctB, which flanks one side of oriCII, encodes a protein that initiates chrII replication; rctA, which flanks the other side of oriCII, inhibits rctB activity. The chrII parAB2 operon, which is essential for chrII partitioning, is located immediately downstream of rctA. Here, we explored how rctA exerts negative control over chrII replication. Our observations suggest that RctB has at least two DNA binding domains--one for binding to oriCII and initiating replication and the other for binding to rctA and thereby inhibiting RctB's ability to initiate replication. Notably, the inhibitory effect of rctA could be alleviated by binding of ParB2 to a centromere-like parS site within rctA. Furthermore, by binding to rctA, ParB2 and RctB inversely regulate expression of the parAB2 genes. Together, our findings suggest that fluctuations in binding of the partitioning protein ParB2 and the chrII initiator RctB to rctA underlie a regulatory network controlling both oriCII firing and the production of the essential chrII partitioning proteins. Thus, by binding both RctB and ParB2, rctA serves as a nexus for regulatory cross-talk coordinating chrII replication and segregation.

Real-time visualisation of the intracellular dynamics of conjugative plasmid transfer.

Conjugation is a contact-dependent mechanism for the transfer of plasmid DNA between bacterial cells, which contributes to the dissemination of antibiotic resistance. Here, we use live-cell microscopy to visualise the intracellular dynamics of conjugative transfer of F-plasmid in E. coli, in real time. We show that the transfer of plasmid in single-stranded form (ssDNA) and its subsequent conversion into double-stranded DNA (dsDNA) are fast and efficient processes that occur with specific timing and subcellular localisation. Notably, the ssDNA-to-dsDNA conversion determines the timing of plasmid-encoded protein production. The leading region that first enters the recipient cell carries single-stranded promoters that allow the early and transient synthesis of leading proteins immediately upon entry of the ssDNA plasmid. The subsequent conversion into dsDNA turns off leading gene expression, and activates the expression of other plasmid genes under the control of conventional double-stranded promoters. This molecular strategy allows for the timely production of factors sequentially involved in establishing, maintaining and disseminating the plasmid.

Vibrio cholerae Chromosome Partitioning without Polar Anchoring by HubP.

Partition systems are widespread among bacterial chromosomes. They are composed of two effectors, ParA and ParB, and cis acting sites, parS, located close to the replication origin of the chromosome (oriC). ParABS participate in chromosome segregation, at least in part because they serve to properly position sister copies of oriC. A fourth element, located at cell poles, is also involved in some cases, such as HubP for the ParABS1 system of Vibrio cholerae chromosome 1 (ch1). The polar anchoring of oriC of ch1 (oriC1) is lost when HubP or ParABS1 are inactivated. Here, we report that in the absence of HubP, ParABS1 actively maintains oriC1 at mid-cell, leading to the subcellular separation of the two ch1 replication arms. We further show that parS1 sites ectopically inserted in chromosome 2 (ch2) stabilize the inheritance of this replicon in the absence of its endogenous partition system, even without HubP. We also observe the positioning interference between oriC1 and oriC of ch2 regions when their positionings are both driven by ParABS1. Altogether, these data indicate that ParABS1 remains functional in the absence of HubP, which raises questions about the role of the polar anchoring of oriC1 in the cell cycle.

The three vibrio cholerae chromosome II-encoded ParE toxins degrade chromosome I following loss of chromosome II.

Three homologues of the plasmid RK2 ParDE toxin-antitoxin system are present in the Vibrio cholerae genome within the superintegron on chromosome II. Here we found that these three loci-two of which have identical open reading frames and regulatory sequences-encode functional toxin-antitoxin systems. The ParE toxins inhibit bacterial division and reduce viability, presumably due to their capacity to damage DNA. The in vivo effects of ParE1/3 mimic those of ParE2, which we have previously demonstrated to be a DNA gyrase inhibitor in vitro, suggesting that ParE1/3 is likewise a gyrase inhibitor, despite its relatively low degree of sequence identity. ParE-mediated DNA damage activates the V. cholerae SOS response, which in turn likely accounts for ParE's inhibition of cell division. Each toxin's effects can be prevented by the expression of its cognate ParD antitoxin, which acts in a toxin-specific fashion both to block toxicity and to repress the expression of its parDE operon. Derepression of ParE activity in ΔparAB2 mutant V. cholerae cells that have lost chromosome II contributes to the prominent DNA degradation that accompanies the death of these cells. Overall, our findings suggest that the ParE toxins lead to the postsegregational killing of cells missing chromosome II in a manner that closely mimics postsegregational killing mediated by plasmid-encoded homologs. Thus, the parDE loci aid in the maintenance of the integrity of the V. cholerae superintegron and in ensuring the inheritance of chromosome II.

Targeting the replication initiator of the second Vibrio chromosome: towards generation of vibrionaceae-specific antimicrobial agents.

The Vibrionaceae is comprised of numerous aquatic species and includes several human pathogens, such as Vibrio cholerae, the cause of cholera. All organisms in this family have two chromosomes, and replication of the smaller one depends on rctB, a gene that is restricted to the Vibrionaceae. Given the increasing prevalence of multi-drug resistance in pathogenic vibrios, there is a need for new targets and drugs to combat these pathogens. Here, we carried out a high throughput cell-based screen to find small molecule inhibitors of RctB. We identified a compound that blocked growth of an E. coli strain bearing an rctB-dependent plasmid but did not influence growth of E. coli lacking this plasmid. This compound, designated vibrepin, had potent cidal activity against V. cholerae and inhibited the growth of all vibrio species tested. Vibrepin blocked RctB oriCII unwinding, apparently by promoting formation of large non-functional RctB complexes. Although vibrepin also appears to have targets other than RctB, our findings suggest that RctB is an attractive target for generation of novel antibiotics that only block growth of vibrios. Vibrio-specific agents, unlike antibiotics currently used in clinical practice, will not engender resistance in the normal human flora or in non-vibrio environmental microorganisms.

ATP negatively regulates the initiator protein of Vibrio cholerae chromosome II replication.

Vibrio cholerae, the agent of cholera, has two circular chromosomes. In bacteria that contain a single chromosome, initiation of chromosome DNA replication is mediated by DnaA, a AAA+ ATPase that unwinds the origin of replication. There is little knowledge regarding initiation of chromosome replication in bacteria with more than one chromosome. Here, we purified V. cholerae DnaA and RctB, which have been implicated in the replication of V. cholerae chromosome II, and characterized their activities in vitro. We found that RctB has origin-specific unwinding activity and can melt the origin of chromosome II (oriCIIvc) but not the origin of chromosome I (oriCIvc); conversely, DnaA promoted the unwinding of oriCIvc and not oriCIIvc. The activity of DnaA and several plasmid initiator proteins is stimulated by ATP binding. We found that RctB bound and hydrolyzed ATP even though RctB lacks any apparent ATP-binding motifs. However, we unexpectedly found that ATP inhibited the oriCIIvc binding activity of RctB, suggesting that the ATP-bound form of RctB cannot initiate replication of chromosome II. Supporting this idea, we identified an RctB mutant that does not bind ATP and found that expression of this ATP-blind RctB mutant in V. cholerae leads to significant overinitiation of chromosome II and marked inhibition of V. cholerae growth. These observations suggest that the rules that license the replication of the two V. cholerae chromosomes differ.

Protocol for High-Throughput Analysis of Sister-Chromatids Contacts.

Sister chromatid interactions are a key step to ensure the successful segregation of sister chromatids after replication. Our knowledge about this phenomenon is mostly based on microscopy approaches, which have some constraints such as resolution limit and the impossibility of studying several genomic positions at the same time. Here, we present a protocol for Hi-SC2, a high-throughput sequencing-based method, to monitor sister chromatid contacts after replication at high resolution throughout the genome, which we applied to study cohesion in Vibrio cholerae. For complete details on the use and execution of this protocol, please refer to Espinosa et al. (2020).

Additive effects of metal excess and superoxide, a highly toxic mixture in bacteria.

Heavy metal contamination is a serious environmental problem. Understanding the toxicity mechanisms may allow to lower concentration of metals in the metal-based antimicrobial treatments of crops, and reduce metal content in soil and groundwater. Here, we investigate the interplay between metal efflux systems and the superoxide dismutase (SOD) in the purple bacterium Rubrivivax gelatinosus and other bacteria through analysis of the impact of metal accumulation. Exposure of the Cd2+ -efflux mutant ΔcadA to Cd2+ caused an increase in the amount and activity of the cytosolic Fe-Sod SodB, thereby suggesting a role of SodB in the protection against Cd2+ . In support of this conclusion, inactivation of sodB gene in the ΔcadA cells alleviated detoxification of superoxide and enhanced Cd2+ toxicity. Similar findings were described in the Cu+ -efflux mutant with Cu+ . Induction of the Mn-Sod or Fe-Sod in response to metals in other bacteria, including Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Vibrio cholera and Bacillus subtilis, was also shown. Both excess Cd2+ or Cu+ and superoxide can damage [4Fe-4S] clusters. The additive effect of metal and superoxide on the [4Fe-4S] could therefore explain the hypersensitive phenotype in mutants lacking SOD and the efflux ATPase. These findings underscore that ROS defence system becomes decisive for bacterial survival under metal excess.

Increasing the copper sensitivity of microorganisms by restricting iron supply, a strategy for bio-management practices.

Pollution by copper (Cu2+ ) extensively used as antimicrobial in agriculture and farming represents a threat to the environment and human health. Finding ways to make microorganisms sensitive to lower metal concentrations could help decreasing the use of Cu2 + in agriculture. In this respect, we showed that limiting iron (Fe) uptake makes bacteria much more susceptible to Cu2 + or Cd2+ poisoning. Using efflux mutants of the purple bacterium Rubrivivax gelatinosus, we showed that Cu+ and Cd2+ resistance relies on the expression of the Fur-regulated FbpABC and Ftr iron transporters. To support this conclusion, inactivation of these Fe-importers in the Cu+ or Cd2+ -ATPase efflux mutants gave rise to hypersensitivity towards these ions. Moreover, in metal overloaded cells the expression of FbpA, the periplasmic iron-binding component of the ferric ion transport FbpABC system was induced, suggesting that cells perceived an 'iron-starvation' situation and responded to it by inducing Fe-importers. In this context, the Fe-Sod activity increased in response to Fe homoeostasis dysregulation. Similar results were obtained for Vibrio cholerae and Escherichia coli, suggesting that perturbation of Fe-homoeostasis by metal excess appeared as an adaptive response commonly used by a variety of bacteria. The presented data support a model in which metal excess induces Fe-uptake to support [4Fe-4S] synthesis and thereby induce ROS detoxification system.

Vertical and Horizontal Transmission of ESBL Plasmid from Escherichia coli O104:H4.

Multidrug resistance (MDR) often results from the acquisition of mobile genetic elements (MGEs) that encode MDR gene(s), such as conjugative plasmids. The spread of MDR plasmids is founded on their ability of horizontal transference, as well as their faithful inheritance in progeny cells. Here, we investigated the genetic factors involved in the prevalence of the IncI conjugative plasmid pESBL, which was isolated from the Escherichia coli O104:H4 outbreak strain in Germany in 2011. Using transposon-insertion sequencing, we identified the pESBL partitioning locus (par). Genetic, biochemical and microscopic approaches allowed pESBL to be characterized as a new member of the Type Ib partitioning system. Inactivation of par caused mis-segregation of pESBL followed by post-segregational killing (PSK), resulting in a great fitness disadvantage but apparent plasmid stability in the population of viable cells. We constructed a variety of pESBL derivatives with different combinations of mutations in par, conjugational transfer (oriT) and pnd toxin-antitoxin (TA) genes. Only the triple mutant exhibited plasmid-free cells in viable cell populations. Time-lapse tracking of plasmid dynamics in microfluidics indicated that inactivation of pnd improved the survival of plasmid-free cells and allowed oriT-dependent re-acquisition of the plasmid. Altogether, the three factors-active partitioning, toxin-antitoxin and conjugational transfer-are all involved in the prevalence of pESBL in the E. coli population.

Single molecule super-resolution imaging of bacterial cell pole proteins with high-throughput quantitative analysis pipeline.

Bacteria show sophisticated control of their cellular organization, and many bacteria deploy different polar landmark proteins to organize the cell pole. Super-resolution microscopy, such as Photo-Activated Localization Microscopy (PALM), provides the nanoscale localization of molecules and is crucial for better understanding of organization and dynamics in single-molecule. However, analytical tools are not fully available yet, in particular for bacterial cell biology. For example, quantitative and statistical analyses of subcellular localization with multiple cells from multiple fields of view are lacking. Furthermore, brightfield images are not sufficient to get accurate contours of small and low contrast bacterial cells, compared to subpixel presentation of target molecules. Here we describe a novel analytic tool for PALM which integrates precisely drawn cell outlines, of either inner membrane or periplasm, labelled by PALM-compatible fluorescent protein fusions, with molecule data for >10,000 molecules from >100 cells by fitting each cell into an oval arc. In the vibrioid bacterium Vibrio cholerae, the polar anchor HubP constitutes a big polar complex which includes multiple proteins involved in chemotaxis and the flagellum. With this pipeline, HubP is shown to be slightly skewed towards the inner curvature side of the cell, while its interaction partners showed rather loose polar localization.

Mutation in ESBL Plasmid from Escherichia coli O104:H4 Leads Autoagglutination and Enhanced Plasmid Dissemination.

Conjugative plasmids are one of the main driving force of wide-spreading of multidrug resistance (MDR) bacteria. They are self-transmittable via conjugation as carrying the required set of genes and cis-acting DNA locus for direct cell-to-cell transfer. IncI incompatibility plasmids are nowadays often associated with extended-spectrum beta-lactamases producing Enterobacteria in clinic and environment. pESBL-EA11 was isolated from Escherichia coli O104:H4 outbreak strain in Germany in 2011. During the previous study identifying transfer genes of pESBL-EA11, it was shown that transposon insertion at certain DNA region of the plasmid, referred to as Hft, resulted in great enhancement of transfer ability. This suggested that genetic modifications can enhance dissemination of MDR plasmids. Such 'superspreader' mutations have attracted little attention so far despite their high potential to worsen MDR spreading. Present study aimed to gain our understanding on regulatory elements that involved pESBL transfer. While previous studies of IncI plasmids indicated that immediate downstream gene of Hft, traA, is not essential for conjugative transfer, here we showed that overexpression of TraA in host cell elevated transfer rate of pESBL-EA11. Transposon insertion or certain nucleotide substitutions in Hft led strong TraA overexpression which resulted in activation of essential regulator TraB and likely overexpression of conjugative pili. Atmospheric Scanning Electron Microscopy observation suggested that IncI pili are distinct from other types of conjugative pili (such as long filamentous F-type pili) and rather expressed throughout the cell surface. High transfer efficiency in the mutant pESBL-EA11 was involved with hyperpiliation which facilitates cell-to-cell adhesion, including autoagglutination. The capability of plasmids to evolve to highly transmissible mutant is alarming, particularly it might also have adverse effect on host pathogenicity.

Distribution of centromere-like parS sites in bacteria: insights from comparative genomics.

Partitioning of low-copy-number plasmids to daughter cells often depends on ParA and ParB proteins acting on centromere-like parS sites. Similar chromosome-encoded par loci likely also contribute to chromosome segregation. Here, we used bioinformatic approaches to search for chromosomal parS sites in 400 prokaryotic genomes. Although the consensus sequence matrix used to search for parS sites was derived from two gram-positive species, putative parS sites were identified on the chromosomes of 69% of strains from all branches of bacteria. Strains that were not found to contain parS sites clustered among relatively few branches of the prokaryotic evolutionary tree. In the vast majority of cases, parS sites were identified in origin-proximal regions of chromosomes. The widespread conservation of parS sites across diverse bacteria suggests that par loci evolved very early in the evolution of bacterial chromosomes and that the absence of parS, parA, and/or parB in certain strains likely reflects the loss of one of more of these loci much later in evolution. Moreover, the highly conserved origin-proximal position of parS suggests par loci are primarily devoted to regulating processes that involve the origin region of bacterial chromosomes. In species containing multiple chromosomes, the parS sites found on secondary chromosomes diverge significantly from those found on their primary chromosomes, suggesting that chromosome segregation of multipartite genomes requires distinct replicon-specific par loci. Furthermore, parS sites on secondary chromosomes are not well conserved among different species, suggesting that the evolutionary histories of secondary chromosomes are more diverse than those of primary chromosomes.