Structure and expression of gene <Emphasis Type="Italic">vfr</Emphasis> in <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

Gene vfr of Pseudomonas chlororaphis 449 previously described only in Pseudomonas aeruginosa was identified, cloned, and sequenced; its localization in the chromosome was determined. Amino acid sequence of the protein encoded by gene vfr in P. chlororaphis 449 was shown to have a 83% identity with the Vfr protein of P. aeruginosa PAO1 and a 63% identity with the CRP protein of Escherichia coli. Amino acid residues that ensure the most important structural properties of the CRP protein, i.e., its binding to cAMP, RNA polymerase, and DNA, were identical or highly conserved in Vfr proteins of P. aeruginosa and P. chlororaphis 449. The cloned vfr gene of P. chlororaphis 449 was complemented partially the mutation at gene crp in cells of E. coli AM306 enhancing ten times synthesis of β-galactosidase dependent on the CRP protein. Unlike P. aeruginosa, the Vfr protein in cells of P. chlororaphis 449 does not participate in the regulation of synthesis of N-acyl-homoserine lactones.     

Exogenous N‐acyl‐homoserine lactones enhance the expression of flagella of Pseudomonas syringae and activate defence responses in plants

In order to cope with pathogens, plants have evolved sophisticated mechanisms to sense pathogenic attacks and to induce defence responses. The N‐acyl‐homoserine lactone (AHL)‐mediated quorum sensing in bacteria regulates diverse physiological processes, including those involved in pathogenicity. In this work, we study the interactions between AHL‐producing transgenic tobacco plants and Pseudomonas syringae pv. tabaci 11528 (P. syringae 11528). Both a reduced incidence of disease and decrease in the growth of P. syringae 11528 were observed in AHL‐producing plants compared with wild‐type plants. The present data indicate that plant‐produced AHLs enhance disease resistance against this pathogen. Subsequent RNA‐sequencing analysis showed that the exogenous addition of AHLs up‐regulated the expression of P. syringae 11528 genes for flagella production. Expression levels of plant defence genes in AHL‐producing and wild‐type plants were determined by quantitative real‐time polymerase chain reaction. These data showed that plant‐produced AHLs activated a wide spectrum of defence responses in plants following inoculation, including the oxidative burst, hypersensitive response, cell wall strengthening, and the production of certain metabolites. These results demonstrate that exogenous AHLs alter the gene expression patterns of pathogens, and plant‐produced AHLs either directly or indirectly enhance plant local immunity during the early stage of plant infection.     

A quorum sensing‐defective mutant of Pectobacterium carotovorum ssp. brasiliense 1692 is attenuated in virulence and unable to occlude xylem tissue of susceptible potato plant stems

Pectobacterium carotovorum ssp. brasiliense 1692 (Pcb1692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during post‐harvest storage. Blackleg diseases involve the bacterial colonization of vascular tissue and the formation of aggregates, also known as biofilms. To understand the role of quorum sensing in vascular colonization by Pcb1692, we generated a Pcb1692ΔexpI mutant strain. Inactivation of expI led to the reduced production of plant cell wall‐degrading enzymes (PCWDEs), the inability to produce acyl homoserine lactone (AHL) and reduced virulence in potato tubers and stems. Complementation of the mutant strain with the wild‐type expI gene in trans successfully restored AHL and PCWDE production as well as virulence. Transmission electron microscopy and in vitro motility assays demonstrated hyperpiliation and loss of flagella and swimming motility in the mutant strain compared with the wild‐type Pcb1692. Furthermore, we noted that, in the early stages of infection, Pcb1692 wild‐type cells had intact flagella which were shed at the later stages of infection. Confocal laser microscopy of PcbΔexpI‐inoculated plants showed that the mutant strain tended to aggregate in intercellular spaces, but was unable to transit to xylem tissue. On the contrary, the wild‐type strain was often observed forming aggregates within xylem tissue of potato stems. Gene expression analyses confirmed that flagella are part of the quorum sensing regulon, whereas fimbriae and pili appear to be negatively regulated by quorum sensing. The relative expression levels of other important putative virulence genes, such as those encoding different groups of PCWDEs, were down‐regulated in the mutant compared with the wild‐type strain.     

Pseudomonas corrugata contains a conserved N-acyl homoserine lactone quorum sensing system; its role in tomato pathogenicity and tobacco hypersensitivity response

Pseudomonas corrugata is a phytopathogenic bacterium, causal agent of tomato pith necrosis, yet it is an ubiquitous bacterium that is part of the microbial community in the soil and in the rhizosphere of different plant species. Although it is a very heterogeneous species, all the strains tested were able to produce short chain acyl homoserine lactone (AHL) quorum sensing signal molecules. The main AHL produced was N-hexanoyl-L-homoserine lactone (C(6)-AHL). An AHL quorum sensing system, designated PcoI/PcoR, was identified and characterized. The role of the quorum sensing system in the expression of a variety of traits was evaluated. Inactivation of pcoI abolished the production of AHLs. The pcoR mutant, but not the pcoI mutant, was impaired in swarming, unable to cause a hypersensitivity response on tobacco and resulted in a reduced tomato pith necrosis phenotype. The pcoI mutant showed a reduced antimicrobial activity against various fungi and bacteria when assayed on King's B medium. These results demonstrate that the AHL quorum sensing in Ps. corrugata regulates traits that contribute to virulence, antimicrobial activity and fitness. This is the first report of genes of Ps. corrugata involved in the disease development and biological control activity.     

sarA‐mediated repression of protease production plays a key role in the pathogenesis of Staphylococcus aureus USA300 isolates

Mutation of staphylococcal accessory regulator (sarA) results in increased production of extracellular proteases in Staphylococcus aureus, which has been correlated with decreased biofilm formation and decreased accumulation of extracellular toxins. We used murine models of implant‐associated biofilm infection and S. aureus bacteraemia (SAB) to compare virulence of USA300 strain LAC, its isogenic sarA mutant, and derivatives of each of these strains with mutations in all 10 of the genes encoding recognized extracellular proteases. The sarA mutant was attenuated in both models, and this was reversed by eliminating production of extracellular proteases. To examine the mechanistic basis, we identified proteins impacted by sarA in a protease‐dependent manner. We identified 253 proteins where accumulation was reduced in the sarA mutant compared with the parent strain, and was restored in the sarA/protease mutant. Additionally, in SAB, the LAC protease mutant exhibited a hypervirulent phenotype by comparison with the isogenic parent strain, demonstrating that sarA also positively regulates production of virulence factors, some of which are subject to protease‐mediated degradation. We propose a model in which attenuation of sarA mutants is defined by their inability to produce critical factors and simultaneously repress production of extracellular proteases that would otherwise limit accumulation of virulence factors.     

The c‐di‐GMP phosphodiesterase BifA is involved in the virulence of bacteria from the Pseudomonas syringae complex

In a recent screen for novel virulence factors involved in the interaction between Pseudomonas savastanoi pv. savastanoi and the olive tree, a mutant was selected that contained a transposon insertion in a putative cyclic diguanylate (c‐di‐GMP) phosphodiesterase‐encoding gene. This gene displayed high similarity to bifA of Pseudomonas aeruginosa and Pseudomonas putida. Here, we examined the role of BifA in free‐living and virulence‐related phenotypes of two bacterial plant pathogens in the Pseudomonas syringae complex, the tumour‐inducing pathogen of woody hosts, P. savastanoi pv. savastanoi NCPPB 3335, and the pathogen of tomato and Arabidopsis, P. syringae pv. tomato DC3000. We showed that deletion of the bifA gene resulted in decreased swimming motility of both bacteria and inhibited swarming motility of DC3000. In contrast, overexpression of BifA in P. savastanoi pv. savastanoi had a positive impact on swimming motility and negatively affected biofilm formation. Deletion of bifA in NCPPB 3335 and DC3000 resulted in reduced fitness and virulence of the microbes in olive (NCPPB 3335) and tomato (DC3000) plants. In addition, real‐time monitoring of olive plants infected with green fluorescent protein (GFP)‐tagged P. savastanoi cells displayed an altered spatial distribution of mutant ΔbifA cells inside olive knots compared with the wild‐type strain. All free‐living phenotypes that were altered in both ΔbifA mutants, as well as the virulence of the NCPPB 3335 ΔbifA mutant in olive plants, were fully rescued by complementation with P. aeruginosa BifA, whose phosphodiesterase activity has been demonstrated. Thus, these results suggest that P. syringae and P. savastanoi BifA are also active phosphodiesterases. This first demonstration of the involvement of a putative phosphodiesterase in the virulence of the P. syringae complex provides confirmation of the role of c‐di‐GMP signalling in the virulence of this group of plant pathogens.