The Secreted Protease PrtA Controls Cell Growth, Biofilm Formation and Pathogenicity in Xylella fastidiosa.

Pierce's disease (PD) is a deadly disease of grapevines caused by the Gram-negative bacterium Xylella fastidiosa. Though disease symptoms were formerly attributed to bacteria blocking the plant xylem, this hypothesis is at best overly simplistic. Recently, we used a proteomic approach to characterize the secretome of X. fastidiosa, both in vitro and in planta, and identified LesA as one of the pathogenicity factors of X. fastidiosa in grapevines that leads to leaf scorching and chlorosis. Herein, we characterize another such factor encoded by PD0956, designated as an antivirulence secreted protease "PrtA" that displays a central role in controlling in vitro cell proliferation, length, motility, biofilm formation, and in planta virulence. The mutant in X. fastidiosa exhibited reduced cell length, hypermotility (and subsequent lack of biofilm formation) and hypervirulence in grapevines. These findings are supported by transcriptomic and proteomic analyses with corresponding plant infection data. Of particular interest, is the hypervirulent response in grapevines observed when X. fastidiosa is disrupted for production of PrtA, and that PD-model tobacco plants transformed to express PrtA exhibited decreased symptoms after infection by X. fastidiosa.

Genetics of Natural Competence in Vibrio cholerae and other Vibrios.

Many Gram-positive and Gram-negative bacteria can become naturally competent to take up extracellular DNA from the environment via a dedicated uptake apparatus. The genetic material that is acquired can (i) be used for nutrients, (ii) aid in genome repair, and (iii) promote horizontal gene transfer when incorporated onto the genome by homologous recombination, the process of "transformation." Recent studies have identified multiple environmental cues sufficient to induce natural transformation in Vibrio cholerae and several other Vibrio species. In V. cholerae, nutrient limitation activates the cAMP receptor protein regulator, quorum-sensing signals promote synthesis of HapR-controlled QstR, chitin stimulates production of TfoX, and low extracellular nucleosides allow CytR to serve as an additional positive regulator. The network of signaling systems that trigger expression of each of these required regulators is well described, but the mechanisms by which each in turn controls competence apparatus genes is poorly understood. Recent work has defined a minimal set of genes that encode apparatus components and begun to characterize the architecture of the machinery by fluorescence microscopy. While studies with a small set of V. cholerae reference isolates have identified regulatory and competence genes required for DNA uptake, future studies may identify additional genes and regulatory connections, as well as revealing how common natural competence is among diverse V. cholerae isolates and other Vibrio species.

Evolutionary dynamics of Vibrio cholerae O1 following a single-source introduction to Haiti.

UNLABELLED: Prior to the epidemic that emerged in Haiti in October of 2010, cholera had not been documented in this country. After its introduction, a strain of Vibrio cholerae O1 spread rapidly throughout Haiti, where it caused over 600,000 cases of disease and >7,500 deaths in the first two years of the epidemic. We applied whole-genome sequencing to a temporal series of V. cholerae isolates from Haiti to gain insight into the mode and tempo of evolution in this isolated population of V. cholerae O1. Phylogenetic and Bayesian analyses supported the hypothesis that all isolates in the sample set diverged from a common ancestor within a time frame that is consistent with epidemiological observations. A pangenome analysis showed nearly homogeneous genomic content, with no evidence of gene acquisition among Haiti isolates. Nine nearly closed genomes assembled from continuous-long-read data showed evidence of genome rearrangements and supported the observation of no gene acquisition among isolates. Thus, intrinsic mutational processes can account for virtually all of the observed genetic polymorphism, with no demonstrable contribution from horizontal gene transfer (HGT). Consistent with this, the 12 Haiti isolates tested by laboratory HGT assays were severely impaired for transformation, although unlike previously characterized noncompetent V. cholerae isolates, each expressed hapR and possessed a functional quorum-sensing system. Continued monitoring of V. cholerae in Haiti will illuminate the processes influencing the origin and fate of genome variants, which will facilitate interpretation of genetic variation in future epidemics. IMPORTANCE: Vibrio cholerae is the cause of substantial morbidity and mortality worldwide, with over three million cases of disease each year. An understanding of the mode and rate of evolutionary change is critical for proper interpretation of genome sequence data and attribution of outbreak sources. The Haiti epidemic provides an unprecedented opportunity to study an isolated, single-source outbreak of Vibrio cholerae O1 over an established time frame. By using multiple approaches to assay genetic variation, we found no evidence that the Haiti strain has acquired any genes by horizontal gene transfer, an observation that led us to discover that it is also poorly transformable. We have found no evidence that environmental strains have played a role in the evolution of the outbreak strain.

Natural competence in Vibrio cholerae is controlled by a nucleoside scavenging response that requires CytR-dependent anti-activation.

Competence for genetic transformation in Vibrio cholerae is triggered by chitin-induced transcription factor TfoX and quorum sensing (QS) regulator HapR. Transformation requires expression of ComEA, described as a DNA receptor in other competent bacteria. A screen for mutants that poorly expressed a comEA-luciferase fusion identified cytR, encoding the nucleoside scavenging cytidine repressor, previously shown in V. cholerae to be a biofilm repressor and positively regulated by TfoX, but not linked to transformation. A ΔcytR mutant was non-transformable and defective in expression of comEA and additional TfoX-induced genes, including pilA (transformation pseudopilus) and chiA-1 (chitinase). In Escherichia coli, 'anti-activation' of nucleoside metabolism genes, via protein-protein interactions between critical residues in CytR and CRP (cAMP receptor protein), is disrupted by exogenous cytidine. Amino acid substitutions of the corresponding V. cholerae CytR residues impaired expression of comEA, pilA and chiA-1, and halted DNA uptake; while exogenous cytidine hampered comEA expression levels and prevented transformation. Our results support a speculative model that when V. cholerae reaches high density on chitin, CytR-CRP interactions 'anti-activate' multiple genes, including a possible factor that negatively controls DNA uptake. Thus, a nucleoside scavenging mechanism couples nutrient stress and cell-cell signalling to natural transformation in V. cholerae as described in other bacterial pathogens.

Quorum-sensing autoinducer molecules produced by members of a multispecies biofilm promote horizontal gene transfer to Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera and a natural inhabitant of aquatic environments, regulates numerous behaviors using a quorum-sensing (QS) system conserved among many members of the marine genus Vibrio. The Vibrio QS response is mediated by two extracellular autoinducer (AI) molecules: CAI-I, which is produced only by Vibrios, and AI-2, which is produced by many bacteria. In marine biofilms on chitinous surfaces, QS-proficient V. cholerae become naturally competent to take up extracellular DNA. Because the direct role of AIs in this environmental behavior had not been determined, we sought to define the contribution of CAI-1 and AI-2 in controlling transcription of the competence gene, comEA, and in DNA uptake. In this study we demonstrated that comEA transcription and the horizontal acquisition of DNA by V. cholerae are induced in response to purified CAI-1 and AI-2, and also by autoinducers derived from other Vibrios co-cultured with V. cholerae within a mixed-species biofilm. These results suggest that autoinducer communication within a consortium may promote DNA exchange among Vibrios, perhaps contributing to the evolution of these bacterial pathogens.