A Natural System of Chromosome Transfer in Yersinia pseudotuberculosis

The High Pathogenicity Island of Yersinia pseudotuberculosis IP32637 was previously shown to be horizontally transferable as part of a large chromosomal segment. We demonstrate here that at low temperature other chromosomal loci, as well as a non-mobilizable plasmid (pUC4K), are also transferable. This transfer, designated GDT4 (Generalized DNA Transfer at 4°C), required the presence of an IP32637 endogenous plasmid (pGDT4) that carries several mobile genetic elements and a conjugation machinery. We established that cure of this plasmid or inactivation of its sex pilus fully abrogates this process. Analysis of the mobilized pUC4K recovered from transconjugants revealed the insertion of one of the pGDT4–borne ISs, designated ISYps1, at different sites on the transferred plasmid molecules. This IS belongs to the IS6 family, which moves by replicative transposition, and thus could drive the formation of cointegrates between pGDT4 and the host chromosome and could mediate the transfer of chromosomal regions in an Hfr-like manner. In support of this model, we show that a suicide plasmid carrying ISYps1 is able to integrate itself, flanked by ISYps1 copies, at multiple locations into the Escherichia coli chromosome. Furthermore, we demonstrate the formation of RecA-independent cointegrates between the ISYps1-harboring plasmid and an ISYps1-free replicon, leading to the passive transfer of the non-conjugative plasmid. We thus demonstrate here a natural mechanism of horizontal gene exchange, which is less constrained and more powerful than the classical Hfr mechanism, as it only requires the presence of an IS6-type element on a conjugative replicon to drive the horizontal transfer of any large block of plasmid or chromosomal DNA. This natural mechanism of chromosome transfer, which occurs under conditions mimicking those found in the environment, may thus play a significant role in bacterial evolution, pathogenesis, and adaptation to new ecological niches

Homeostatic Interplay between Bacterial Cell-Cell Signaling and Iron in Virulence

Pathogenic bacteria use interconnected multi-layered regulatory networks, such as quorum sensing (QS) networks to sense and respond to environmental cues and external and internal bacterial cell signals, and thereby adapt to and exploit target hosts. Despite the many advances that have been made in understanding QS regulation, little is known regarding how these inputs are integrated and processed in the context of multi-layered QS regulatory networks. Here we report the examination of the Pseudomonas aeruginosa QS 4-hydroxy-2-alkylquinolines (HAQs) MvfR regulatory network and determination of its interaction with the QS acyl-homoserine-lactone (AHL) RhlR network. The aim of this work was to elucidate paradigmatically the complex relationships between multi-layered regulatory QS circuitries, their signaling molecules, and the environmental cues to which they respond. Our findings revealed positive and negative homeostatic regulatory loops that fine-tune the MvfR regulon via a multi-layered dependent homeostatic regulation of the cell-cell signaling molecules PQS and HHQ, and interplay between these molecules and iron. We discovered that the MvfR regulon component PqsE is a key mediator in orchestrating this homeostatic regulation, and in establishing a connection to the QS rhlR system in cooperation with RhlR. Our results show that P. aeruginosa modulates the intensity of its virulence response, at least in part, through this multi-layered interplay. Our findings underscore the importance of the homeostatic interplay that balances competition within and between QS systems via cell-cell signaling molecules and environmental cues in the control of virulence gene expression. Elucidation of the fine-tuning of this complex relationship offers novel insights into the regulation of these systems and may inform strategies designed to limit infections caused by P. aeruginosa and related human pathogens

Inhibitors of Pathogen Intercellular Signals as Selective Anti-Infective Compounds

Long-term antibiotic use generates pan-resistant super pathogens. Anti-infective compounds that selectively disrupt virulence pathways without affecting cell viability may be used to efficiently combat infections caused by these pathogens. A candidate target pathway is quorum sensing (QS), which many bacterial pathogens use to coordinately regulate virulence determinants. The Pseudomonas aeruginosa MvfR-dependent QS regulatory pathway controls the expression of key virulence genes; and is activated via the extracellular signals 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), whose syntheses depend on anthranilic acid (AA), the primary precursor of 4-hydroxy-2-alkylquinolines (HAQs). Here, we identified halogenated AA analogs that specifically inhibited HAQ biosynthesis and disrupted MvfR-dependent gene expression. These compounds restricted P. aeruginosa systemic dissemination and mortality in mice, without perturbing bacterial viability, and inhibited osmoprotection, a widespread bacterial function. These compounds provide a starting point for the design and development of selective anti-infectives that restrict human P. aeruginosa pathogenesis, and possibly other clinically significant pathogens

Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa

<p>Abstract</p> <p>Background</p> <p>The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in various bacteria and fungi. The procedure consists of electroporating a polymerase chain reaction (PCR) fragment that was obtained with a 1- or 3-step PCR protocol and that carries an antibiotic cassette flanked by a region homologous to the target locus into a strain that expresses the lambda Red recombination system.</p> <p>Results</p> <p>This system has been modified for use in <it>Pseudomonas aeruginosa</it>. Chromosomal DNA deletions of single genes were generated using 3-step PCR products containing flanking regions 400–600 nucleotides (nt) in length that are homologous to the target sequence. A 1-step PCR product with a homologous extension flanking region of only 100 nt was in some cases sufficient to obtain the desired mutant. We further showed that the <it>P. aeruginosa </it>strain PA14 non-redundant transposon library can be used in conjunction with the lambda Red technique to rapidly generate large chromosomal deletions or transfer mutated genes into various PA14 isogenic mutants to create multi-locus knockout mutants.</p> <p>Conclusion</p> <p>The lambda Red-based technique can be used efficiently to generate mutants in <it>P. aeruginosa</it>. The main advantage of this procedure is its rapidity as mutants can be easily obtained in less than a week if the 3-step PCR procedure is used, or in less than three days if the mutation needs to be transferred from one strain to another.</p

A Quorum Sensing Regulated Small Volatile Molecule Reduces Acute Virulence and Promotes Chronic Infection Phenotypes

A significant number of environmental microorganisms can cause serious, even fatal, acute and chronic infections in humans. The severity and outcome of each type of infection depends on the expression of specific bacterial phenotypes controlled by complex regulatory networks that sense and respond to the host environment. Although bacterial signals that contribute to a successful acute infection have been identified in a number of pathogens, the signals that mediate the onset and establishment of chronic infections have yet to be discovered. We identified a volatile, low molecular weight molecule, 2-amino acetophenone (2-AA), produced by the opportunistic human pathogen Pseudomonas aeruginosa that reduces bacterial virulence in vivo in flies and in an acute mouse infection model. 2-AA modulates the activity of the virulence regulator MvfR (multiple virulence factor regulator) via a negative feedback loop and it promotes the emergence of P. aeruginosa phenotypes that likely promote chronic lung infections, including accumulation of lasR mutants, long-term survival at stationary phase, and persistence in a Drosophila infection model. We report for the first time the existence of a quorum sensing (QS) regulated volatile molecule that induces bistability phenotype by stochastically silencing acute virulence functions in P. aeruginosa. We propose that 2-AA mediates changes in a subpopulation of cells that facilitate the exploitation of dynamic host environments and promote gene expression changes that favor chronic infections

Horizontal Transfer of the High-Pathogenicity Island of Yersinia pseudotuberculosis

The horizontal transfer of genetic elements plays a major role in bacterial evolution. The high-pathogenicity island (HPI), which codes for an iron uptake system, is present and highly conserved in various Enterobacteriaceae, suggesting its recent acquisition by lateral gene transfer. The aim of this work was to determine whether the HPI has kept its ability to be transmitted horizontally. We demonstrate here that the HPI is indeed transferable from a donor to a recipient Yersinia pseudotuberculosis strain. This transfer was observable only when the donor and recipient bacteria were cocultured at low temperatures in a liquid medium. When optimized conditions were used (bacteria actively growing in an iron-deprived medium at 4°C), the frequency of HPI transfer reached ∼10(−8). The island was transferable to various serotype I strains of Y. pseudotuberculosis and to Yersinia pestis, but not to Y. pseudotuberculosis strains of serotypes II and IV or to Yersinia enterocolitica. Upon transfer, the HPI was inserted almost systematically into the asn3 tRNA locus. Acquisition of the HPI resulted in the loss of the resident island, suggesting an incompatibility between two copies of the HPI within the same strain. Transfer of the island did not require a functional HPI-borne insertion-excision machinery and was RecA dependent in the recipient but not the donor strain, suggesting that integration of the island into the recipient chromosome occurs via a mechanism of homologous recombination. This lateral transfer also involved the HPI-adjacent sequences, leading to the mobilization of a chromosomal region at least 46 kb in size

Use of the lambda Red recombinase system to rapidly generate mutants in -5

He mutants generated in this study after overnight growth in LB at 37°C.<p><b>Copyright information:</b></p><p>Taken from "Use of the lambda Red recombinase system to rapidly generate mutants in "</p><p>http://www.biomedcentral.com/1471-2199/9/20</p><p>BMC Molecular Biology 2008;9():20-20.</p><p>Published online 4 Feb 2008</p><p>PMCID:PMC2287187.</p><p></p

Use of the lambda Red recombinase system to rapidly generate mutants in -7

and Fout/Rout were used. The Δmutant was verified with primer pairs Fout-lasR/R-kan and Fout-lasR/Rout-lasR, Δand Δwith Fout-kynBU/R-kan and Fout-kynBU/Rout-kynBU. . To confirm that the MAR2xT7 transposon replaced the 24 kb HSI-II locus, primer pairs F-MAR2xT7/R-PA14_42880 and F-PA14_43100/RPA14_42880 were used. No amplification is achieved when wild type genomic DNA is used.<p><b>Copyright information:</b></p><p>Taken from "Use of the lambda Red recombinase system to rapidly generate mutants in "</p><p>http://www.biomedcentral.com/1471-2199/9/20</p><p>BMC Molecular Biology 2008;9():20-20.</p><p>Published online 4 Feb 2008</p><p>PMCID:PMC2287187.</p><p></p

Use of the lambda Red recombinase system to rapidly generate mutants in -1

He mutants generated in this study after overnight growth in LB at 37°C.<p><b>Copyright information:</b></p><p>Taken from "Use of the lambda Red recombinase system to rapidly generate mutants in "</p><p>http://www.biomedcentral.com/1471-2199/9/20</p><p>BMC Molecular Biology 2008;9():20-20.</p><p>Published online 4 Feb 2008</p><p>PMCID:PMC2287187.</p><p></p