Two Independent Pathways for Self-Recognition in Proteus Mirabilis Are Linked by Type VI-Dependent Export

Swarming colonies of the bacterium Proteus mirabilis are capable of self-recognition and territorial behavior. Swarms of independent P. mirabilis isolates can recognize each other as foreign and establish a visible boundary where they meet; in contrast, genetically identical swarms merge. The ids genes, which encode self-identity proteins, are necessary but not sufficient for this territorial behavior. Here we have identified two new gene clusters: one (idr) encodes rhs-related products, and another (tss) encodes a putative type VI secretion (T6S) apparatus. The Ids and Idr proteins function independently of each other in extracellular transport and in territorial behaviors; however, these self-recognition systems are linked via this type VI secretion system. The T6S system is required for export of select Ids and Idr proteins. Our results provide a mechanistic and physiological basis for the fundamental behaviors of self-recognition and territoriality in a bacterial model system.Molecular and Cellular Biolog

Genome-wide analysis of the PreA/PreB (QseB/QseC) regulon of Salmonella enterica serovar Typhimurium

<p>Abstract</p> <p>Background</p> <p>The <it>Salmonella </it>PreA/PreB two-component system (TCS) is an ortholog of the QseBC TCS of <it>Escherichia coli</it>. In both <it>Salmonella </it>and <it>E. coli</it>, this system has been shown to affect motility and virulence in response to quorum-sensing and hormonal signals, and to affect the transcription of the <it>Salmonella enterica </it>serovar Typhimurium (<it>S</it>. Typhimurium) <it>pmrAB </it>operon, which encodes an important virulence-associated TCS.</p> <p>Results</p> <p>To determine the PreA/PreB regulon in <it>S</it>. Typhimurium, we performed DNA microarrays comparing the wild type strain and various <it>preA </it>and/or <it>preB </it>mutants in the presence of ectopically expressed <it>preA </it>(<it>qseB</it>). These data confirmed our previous findings of the negative effect of PreB on PreA gene regulation and identified candidate PreA-regulated genes. A proportion of the activated loci were previously identified as PmrA-activated genes (<it>yibD</it>, <it>pmrAB</it>, <it>cptA</it>, etc.) or were genes located in the local region around <it>preA</it>, including the <it>preAB </it>operon. The transcriptional units were defined in this local region by RT-PCR, suggesting three PreA activated operons composed of <it>preA-preB</it>, <it>mdaB-ygiN</it>, and <it>ygiW</it>-STM3175. Several putative virulence-related phenotypes were examined for <it>preAB </it>mutants, resulting in the observation of a host cell invasion and slight virulence defect of a <it>preAB </it>mutant. Contrary to previous reports on this TCS, we were unable to show a PreA/PreB-dependent effect of the quorum-sensing signal AI-2 or of epinephrine on <it>S</it>. Typhimurium with regard to bacterial motility.</p> <p>Conclusion</p> <p>This work further characterizes this unorthadox OmpR/EnvZ class TCS and provides novel candidate regulated genes for further study. This first in-depth study of the PreA/PreB regulatory system phenotypes and regulation suggests significant comparative differences to the reported function of the orthologous QseB/QseC in <it>E. coli</it>.</p

Prevalence and diversity of type VI secretion systems in a model beneficial symbiosis

The type VI secretion system (T6SS) is widely distributed in diverse bacterial species and habitats where it is required for interbacterial competition and interactions with eukaryotic cells. Previous work described the role of a T6SS in the beneficial symbiont, Vibrio fischeri, during colonization of the light organ of Euprymna scolopes squid. However, the prevalence and diversity of T6SSs found within the distinct symbiotic structures of this model host have not yet been determined. Here, we analyzed 73 genomes of isolates from squid light organs and accessory nidamental glands (ANGs) and 178 reference genomes. We found that the majority of these bacterial symbionts encode diverse T6SSs from four distinct classes, and most share homology with T6SSs from more distantly related species, including pathogens of animals and humans. These findings indicate that T6SSs with shared evolutionary histories can be integrated into the cellular systems of host-associated bacteria with different effects on host health. Furthermore, we found that one T6SS in V. fischeri is located within a genomic island with high genomic plasticity. Five distinct genomic island genotypes were identified, suggesting this region encodes diverse functional potential that natural selection can act on. Finally, analysis of newly described T6SSs in roseobacter clade ANG isolates revealed a novel predicted protein that appears to be a fusion of the TssB-TssC sheath components. This work underscores the importance of studying T6SSs in diverse organisms and natural habitats to better understand how T6SSs promote the propagation of bacterial populations and impact host health

Coordination of the arc regulatory system and pheromone-mediated positive feedback in controlling the Vibrio fischeri lux operon.

Bacterial pheromone signaling is often governed both by environmentally responsive regulators and by positive feedback. This regulatory combination has the potential to coordinate a group response among distinct subpopulations that perceive key environmental stimuli differently. We have explored the interplay between an environmentally responsive regulator and pheromone-mediated positive feedback in intercellular signaling by Vibrio fischeri ES114, a bioluminescent bacterium that colonizes the squid Euprymna scolopes. Bioluminescence in ES114 is controlled in part by N-(3-oxohexanoyl)-L-homoserine lactone (3OC6), a pheromone produced by LuxI that together with LuxR activates transcription of the luxICDABEG operon, initiating a positive feedback loop and inducing luminescence. The lux operon is also regulated by environmentally responsive regulators, including the redox-responsive ArcA/ArcB system, which directly represses lux in culture. Here we show that inactivating arcA leads to increased 3OC6 accumulation to initiate positive feedback. In the absence of positive feedback, arcA-mediated control of luminescence was only ∼2-fold, but luxI-dependent positive feedback contributed more than 100 fold to the net induction of luminescence in the arcA mutant. Consistent with this overriding importance of positive feedback, 3OC6 produced by the arcA mutant induced luminescence in nearby wild-type cells, overcoming their ArcA repression of lux. Similarly, we found that artificially inducing ArcA could effectively repress luminescence before, but not after, positive feedback was initiated. Finally, we show that 3OC6 produced by a subpopulation of symbiotic cells can induce luminescence in other cells co-colonizing the host. Our results suggest that even transient loss of ArcA-mediated regulation in a sub-population of cells can induce luminescence in a wider community. Moreover, they indicate that 3OC6 can communicate information about both cell density and the state of ArcA/ArcB

Models of how subpopulations with heterogeneous Arc status could coordinate population-wide responses.

<p>Triangles indicate 3OC6 pheromone, white cells indicate no luminescence, and blue cells indicate luminescent cells. Panel A illustrates an interpretation of the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049590#pone-0049590-g004" target="_blank">Figure 4C</a>, and panel B illustrates how similar signaling might occur in the host environment. In panel B we propose that even if only a subset of cells in the light organ experience physiological changes that silence ArcB kinase activity, this could result in loss of ArcA-dependent repression of <i>lux</i> followed by increased production of 3OC6 pheromone signals. This 3OC6 could then diffuse into neighboring ArcA-repressed cells to initiate positive feedback regulation of <i>lux,</i> thereby inducing luminescence in a population-wide response to conditions sensed by ArcB in a sub-population of symbionts.</p

Effect of controlled ArcA expression on bioluminescence.

<p>Cultures of either the Δ<i>arcA</i> mutant with the inducible-<i>arcA</i> vector pAS104 (circles and squares) or wild type (diamonds) were grown in SWTO medium in duplicate aerobic shake flasks. Specific luminescence (luminescence per OD₅₉₅) was observed for cultures grown with no addition (black) or with 2 mM IPTG added at the time of inoculation (T₀, gray squares) or when cultures reached an OD₅₉₅ of ∼1.0 (T₁, gray circles).</p

ArcA/ArcB and LuxR-LuxI-mediated regulation of bioluminescence in <i>V. fischeri</i>.

<p>LuxI synthesizes 3OC6, a diffusible pheromone that upon reaching a sufficient concentration combines with LuxR. 3OC6-LuxR binds to the “<i>lux</i> box” and stimulates transcription of the <i>luxICDABEG</i> operon, which produces more 3OC6 and bioluminescence. The ArcA/ArcB two-component regulatory system responds to reducing conditions, and ArcA-P binds near the <i>lux</i> box, effectively inhibiting bioluminescence. Two other autoinducer pheromones AI-2 and C8-HSL are not shown, although the latter can also function with LuxR.</p

Effect of <i>luxI</i> and 3OC6 on derepression of the <i>lux</i> operon in an <i>arcA</i> mutant.

<p>Luminescence or <i>lux</i> reporter expression was measured in strains ES114 (WT), AMJ2 (Δ<i>arcA</i>), VCW2G7 (<i>luxI</i> mutant), or ANS7 (Δ<i>arcA luxI</i>). (A) Peak specific luminescence (luminescence per OD₅₉₅) of strains grown in SWTO in aerobic shake flasks. Error bars indicate standard deviation (n = 3). (B) Plasmid based P<i>_luxI</i>-<i>lacZ</i> transcriptional reporter assays. Strains containing pJLB171 or the promoterless vector pAKD702 were grown in duplicate aerobic shake flasks in SWTO medium. Cells were harvested at peak luminescence for ß–galactosidase assays. Error bars indicate standard deviation (n = 2). Asterisk indicates p-value of <0.05 with a Student’s t-test comparing the <i>arcA</i> mutant to its respective parent strain. (C) Peak specific luminescence values for aerobic cultures grown in SWTO medium with 50 nM 3OC6 added where indicated. Error bars (some too small to see) indicate standard deviation (n = 2). Each panel is representative of at least three independent experiments.</p

Inter-strain bioluminescence induction in mixed and spatially separated <i>arcA</i>⁺ and <i>arcA</i> mutant cells.

<p>Wild-type cells were mixed with either the dark <i>arcA</i> Δ<i>luxCDABEG</i> strain (JB33) able to produce 3OC6 (A) or the dark <i>arcA</i> Δ<i>luxICDABEG</i> strain (ANS6) lacking the <i>luxI</i> 3OC6 synthase gene (B). Wild-type and <i>arcA</i> mutant cultures and co-cultures were grown in SWTO broth in duplicate aerobic shake flasks to peak luminescence. A sample was removed and dilution plated to determine the CFU ml⁻¹ and the percent of wild-type cells in each co-culture (52% for A, 68% for B). Luminescence values are presented as luminescence per 10⁸ CFU. Luminescence values presented for co-cultures are luminescence per 10⁸ wild-type CFU. (C) Cultures of wild type or the <i>luxI</i> mutant were streaked onto SWTO agar plates next to spotted culture of the dark <i>arcA</i> Δ<i>luxCDABEG</i> strain (JB33) able to produce 3OC6. Inoculated plates were incubated at room temperature overnight and negative images of bioluminescence were captured with a BioRad Fluor-S MultiImager. Scale bar indicates approximately 5 mm.</p