[show abstract][hide abstract] ABSTRACT: The study of the fish pathogen Flavobacterium psychrophilum has been drastically hampered by the difficulty to perform genetic manipulation of this organism. Although recent publications described the successful transfer of genetic material into this bacterium by transformation and conjugation, additional tools are still needed. This paper reports the construction of vector pCP23-G, which permits for the first time to monitor transcriptional regulation in this pathogen by using a promoterless gfpmut3 gene as a reporter. Additionally, use of pCP23-G enabled the trancriptional analysis of three putative promoter regions of F. psychrophilum, corresponding to genes fpp2-fpp1, pdhB and gldJ, under different growth conditions. Overall, the construction of pCP23-G facilitates genetic analysis in F. psychrophilum, by enabling the determination of gene expression both in vitro and in vivo. Furthermore, this would also open the possibility for studies on the location of this bacterium in the fish tissues.
[show abstract][hide abstract] ABSTRACT: Lactococcus garvieae is the causative microbial agent of lactococcosis, an important and damaging fish disease in aquaculture. This bacterium has also been isolated from vegetables, milk, cheese, meat and sausages, from cow and buffalo as a mastitis agent, and even from humans, as an opportunistic infectious agent. In this work pathogenicity experiments were performed in rainbow trout and mouse models with strains isolated from human (L. garvieae HF) and rainbow trout (L. garvieae UNIUDO74; henceforth referred to as 074). The mean LD(50) value in rainbow trout obtained for strain 074 was 2.1 × 10(2) ± 84 per fish. High doses of the bacteria caused specific signs of disease as well as histological alterations in mice. In contrast, strain HF did not prove to be pathogenic either for rainbow trout or for mice. Based on these virulence differences, two suppressive subtractive hybridizations were carried out to identify unique genetic sequences present in L. garvieae HF (SSHI) and L. garvieae 074 (SSHII). Differential dot-blot screening of the subtracted libraries allowed the identification of 26 and 13 putative ORFs specific for L. garvieae HF and L. garvieae 074, respectively. Additionally, a PCR-based screening of 12 of the 26 HF-specific putative ORFs and the 13 074-specific ones was conducted to identify their presence/absence in 25 L. garvieae strains isolated from different origins and geographical areas. This study demonstrates the existence of genetic heterogeneity within L. garvieae isolates and provides a more complete picture of the genetic background of this bacterium.
[show abstract][hide abstract] ABSTRACT: Flavobacterium 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 THC02/90. 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. LD(50) 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.
[show abstract][hide abstract] ABSTRACT: 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.
Applied and environmental microbiology 02/2011; 77(3):1107-10. · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 (LD(50)) 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.
Journal of bacteriology 02/2011; 193(4):944-51. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: The dltABCD cluster is involved in the d-alanylation of teichoic acids in gram positive bacteria. In order to determine the role of this alanylation in the physiology and virulence of Lactococcus garvieae, a previously isolated dltA Delta Tn917 signature tagged mutagenesis (STM) clone was analyzed. RT-PCR results revealed that dltABCD genes form an operon. No major differences could be established between the parental and mutant strains with respect to growth rate, autolytic properties, and susceptibility to acid conditions, lysozyme treatment, anionic detergents, or oxidant agents. However, the dltA mutant was more susceptible to nisin than the parental strain, with minimum inhibitory concentration (MIC) values of 8 and 16 microg/ml, respectively. Less proliferation of the mutant was observed in in vivo competence index experiments (CI=0.08). Furthermore, the mutant strain had a 50% lethal dose (LD(50)) 3-fold that of the parental strain. These results, together with the fact that the dltA Delta Tn917 mutant was isolated as a STM clone, reveal that the dltA locus of Lactococcus garvieae is required for full growth and pathogenesis on rainbow trout.
[show abstract][hide abstract] ABSTRACT: 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.
International Microbiology 12/2009; 12(4):207-14. · 2.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nucleotide sequence analysis of the region surrounding the pIVET8 insertion site in Yersinia ruckeri 150RiviXII, previously selected by in vivo expression technology (IVET), revealed the presence of eight genes (traHIJKCLMN [hereafter referred to collectively as the tra operon or tra cluster]), which are similar both in sequence and organization to the tra operon cluster found in the virulence-related plasmid pADAP from Serratia entomophila. Interestingly, the tra cluster of Y. ruckeri is chromosomally encoded, and no similar tra cluster has been identified yet in the genomic analysis of human pathogenic yersiniae. A traI insertional mutant was obtained by homologous recombination. Coinfection experiments with the mutant and the parental strain, as well as 50% lethal dose determinations, indicate that this operon is involved in the virulence of this bacterium. All of these results suggest the implication of the tra cluster in a virulence-related type IV secretion/transfer system. Reverse transcriptase PCR studies showed that this cluster is transcribed as an operon from a putative promoter located upstream of traH and that the mutation of traI had a polar effect. A traI::lacZY transcriptional fusion displayed higher expression levels at 18 degrees C, the temperature of occurrence of the disease, and under nutrient-limiting conditions. PCR detection analysis indicated that the tra cluster is present in 15 Y. ruckeri strains from different origins and with different plasmid profiles. The results obtained in the present study support the conclusion, already suggested by different authors, that Y. ruckeri is a very homogeneous species that is quite different from the other members of the genus Yersinia.
Applied and environmental microbiology 01/2009; 75(4):937-45. · 3.69 Impact Factor