Spreading versus biomass production by colonies of the fish pathogen Flavobacterium psychrophilum: role of the nutrient concentration

Colonies of the fish pathogen Flavobacterium psychrophilum have gliding motility in media with low agar concentrations. Although gliding motility, particularly in Flavobacterium johnsoniae, has been well-studied, little is known about its regulation by environmental factors. The work described here shows that the ability of F. psychrophilum to spread over surfaces depends on nutrient availability. In fact, as the nutrient contents of the medium decreased, spreading was favored and the diameter of the colonies increased. Macroscopy examination revealed modifications in colony morphology as nutrient depletion increased: from a dense and defined colony to the formation of microcolonies inside a general colony structure. Additionally, colony expansion dynamics and population density across the colony radius varied inversely with bacterial biomass production. Motility was an immediate response when bacteria were transferred from a rich to a more diluted medium. Our results suggest that, when nutrients are limiting, F. psychrophilum activates a specific growth mode that enables it to colonize surfaces by means of gliding motility. The use of diluted media allowed the differentiation, among previously isolated F. psychrophilum non-gliding mutants, of those completely unable to glide and those with only partially impaired gliding ability. [Int Microbiol 2009; 12(4):207-214

The yctCBA Operon of Yersinia ruckeri, Involved in In Vivo Citrate Uptake, Is Not Required for Virulence▿

A three-gene operon, named yctCBA (Yersinia citrate transporter), induced by citrate and repressed by glucose was identified from a previously selected in vivo-induced (ivi) clone in the fish pathogen Yersinia ruckeri. Interestingly, despite being an ivi clone, the drastic growth reduction of the yctC mutant in the presence of citrate, and the relatively high content of this compound in rainbow trout serum, the operon was not required for virulence

A Novel cdsAB Operon Is Involved in the Uptake of l-Cysteine and Participates in the Pathogenesis of Yersinia ruckeri▿

Application of in vivo expression technology (IVET) to Yersinia ruckeri, an important fish pathogen, allowed the identification of two adjacent genes that represent a novel bacterial system involved in the uptake and degradation of l-cysteine. Analysis of the translational products of both genes showed permease domains (open reading frame 1 [ORF1]) and amino acid position identities (ORF2) with the l-cysteine desulfidase from Methanocaldococcus jannaschii, a new type of enzyme involved in the breakdown of l-cysteine. The operon was named cdsAB (cysteine desulfidase) and is found widely in anaerobic and facultative bacteria. cdsAB promoter analysis using lacZY gene fusion showed highest induction in the presence of l-cysteine. Two cdsA and cdsB mutant strains were generated. The limited toxic effect and the low utilization of l-cysteine observed in the cdsA mutant, together with radiolabeled experiments, strongly suggested that CdsA is an l-cysteine permease. Fifty percent lethal dose (LD50) and competence index experiments showed that both the cdsA and cdsB loci were involved in the pathogenesis of the bacteria. In conclusion, this study has shown for the first time in bacteria the existence of an l-cysteine uptake system that together with an additional l-cysteine desulfidase-encoding gene constitutes a novel operon involved in bacterial virulence

Genome-Scale Mapping Reveals Complex Regulatory Activities of RpoN in Yersinia pseudotuberculosis

RpoN, an alternative sigma factor commonly known as σ54, is implicated in persistent stages of Yersinia pseudotuberculosis infections in which genes associated with this regulator are upregulated. We here combined phenotypic and genomic assays to provide insight into its role and function in this pathogen. RpoN was found essential for Y. pseudotuberculosis virulence in mice, and in vitro functional assays showed that it controls biofilm formation and motility. Mapping genome-wide associations of Y. pseudotuberculosis RpoN using chromatin immunoprecipitation coupled with next-generation sequencing identified an RpoN binding motif located at 103 inter- and intragenic sites on both sense and antisense strands. Deletion of rpoN had a large impact on gene expression, including downregulation of genes encoding proteins involved in flagellar assembly, chemotaxis, and quorum sensing. There were also clear indications of cross talk with other sigma factors, together with indirect effects due to altered expression of other regulators. Matching differential gene expression with locations of the binding sites implicated around 130 genes or operons potentially activated or repressed by RpoN. Mutagenesis of selected intergenic binding sites confirmed both positive and negative regulatory effects of RpoN binding. Corresponding mutations of intragenic sense sites had less impact on associated gene expression. Surprisingly, mutating intragenic sites on the antisense strand commonly reduced expression of genes carried by the corresponding sense strand

Comparative analysis and mutation effects of fpp2-fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum

International audienceFlavobacterium psychrophilum is a very significant fish pathogen that secretes two biochemically characterized extracellular proteolytic enzymes, Fpp1 and Fpp2. The genes encoding these enzymes are organized as an fpp2-fpp1 tandem in the genome of strain F psychrophilum THCO2/90. Analysis of the corresponding encoded proteins showed that they belong to two different protease families. For gene function analysis, new genetic tools were developed in F psychrophilum by constructing stable isogenic fpp1 and fpp2 mutants via single-crossover homologous recombination. RT-PCR analysis of wild-type and mutant strains suggested that both genes are transcribed as a single mRNA from the promoter located upstream of the fpp2 gene. Phenotypic characterization of the fpp2 mutant showed lack of caseinolytic activity and higher colony spreading compared with the wild-type strain. Both characteristics were recovered in the complemented strain. One objective of this work was to assess the contribution to virulence of these proteolytic enzymes. LD50 experiments using the wild-type strain and mutants showed no significant differences in virulence in a rainbow trout challenge model, suggesting instead a possible nutritional role. The gene disruption procedure developed in this work, together with the knowledge of the complete genome sequence of F psychrophilum, open new perspectives for the study of gene function in this bacterium