Host specificity and virulence of Flavobacterium psychrophilum: a comparative study in ayu (Plecoglossus altivelis) and rainbow trout (Oncorhynchus mykiss) hosts.

Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease, is a devastating, worldwide distributed, fish pathogen causing significant economic loss in inland fish farms. Previous epidemiological studies showed that prevalent clonal complexes (CC) differ in fish species affected with disease such as rainbow trout, coho salmon and ayu, indicating significant associations between particular F. psychrophilum genotypes and host species. Yet, whether the population structure is driven by the trade of fish and eggs or by host-specific pathogenicity is uncertain. Notably, all F. psychrophilum isolates retrieved from ayu belong to Type-3 O antigen (O-Ag) whereas only very few strains retrieved from other fish species possess this O-Ag, suggesting a role in outbreaks affecting ayu. Thus, we investigated the links between genotype and pathogenicity by conducting comparative bath infection challenges in two fish hosts, ayu and rainbow trout, for a collection of isolates representing different MLST genotypes and O-Ag. Highly virulent strains in one host species exhibited low to no virulence in the other. F. psychrophilum strains associated with ayu and possessing Type-3 O-Ag demonstrated significant variability in pathogenicity in ayu, ranging from avirulent to highly virulent. Strikingly, F. psychrophilum strains retrieved from rainbow trout and possessing the Type-3 O-Ag were virulent for rainbow trout but not for ayu, indicating that Type-3 O-Ag alone is not sufficient for pathogenicity in ayu, nor does it prevent pathogenicity in rainbow trout. This study revealed that the association between a particular CC and host species partly depends on the pathogen's adaptation to specific host species.

Phylogenomic characterization of Flavobacterium psychrophilum isolates retrieved from Turkish rainbow trout farms.

Flavobacterium psychrophilum, a devastating fish pathogen, is responsible for bacterial cold-water disease (BCWD), also known as rainbow trout fry syndrome. F. psychrophilum is the main causative agent of outbreaks in rainbow trout farms, especially at early live stages. In the present study, we aimed to characterize F. psychrophilum Turkish isolates. Eighteen isolates were retrieved from BCWD outbreaks between 2014 and 2021. In vitro phenotypic characterization showed gelatin and casein hydrolysis capacities and in vitro adhesion for all isolates, whereas elastinolytic activity was present for 16 of 18 isolates. We used complete genome sequencing to infer MLST-type, serotype and phylogenetic reconstruction. Strikingly, one strain isolated from Coruh trout (FP-369) belongs to ST393, a previously undescribed ST, and is phylogenetically distant from the other isolates. However, all strains retrieved from rainbow trout belong to the well-characterized clonal complex CC-ST10, 12 of 17 were tightly connected in a single cluster. Several serotypes (Types -1, -2 and -3) were represented among isolates, but no correlation was observed with geographic origins. This analysis suggests a regional dissemination of an epidemic, disease-producing bacterial population. This study provides a basis for epidemiological surveillance of isolates circulating in Turkey and phenotypic data for future molecular studies of virulence traits of this important fish pathogen.

Investigation of the Genus Flavobacterium as a Reservoir for Fish-Pathogenic Bacterial Species: the Case of Flavobacterium collinsii.

Bacteria of the genus Flavobacterium are recovered from a large variety of environments. Among the described species, Flavobacterium psychrophilum and Flavobacterium columnare cause considerable losses in fish farms. Alongside these well-known fish-pathogenic species, isolates belonging to the same genus recovered from diseased or apparently healthy wild, feral, and farmed fish have been suspected to be pathogenic. Here, we report the identification and genomic characterization of a Flavobacterium collinsii isolate (TRV642) retrieved from rainbow trout spleen. A phylogenetic tree of the genus built by aligning the core genome of 195 Flavobacterium species revealed that F. collinsii stands within a cluster of species associated with diseased fish, the closest one being F. tructae, which was recently confirmed as pathogenic. We evaluated the pathogenicity of F. collinsii TRV642 as well as of Flavobacterium bernardetii F-372T, another recently described species reported as a possible emerging pathogen. Following intramuscular injection challenges in rainbow trout, no clinical signs or mortalities were observed with F. bernardetii. F. collinsii showed very low virulence but was isolated from the internal organs of survivors, indicating that the bacterium is able to survive inside the host and may provoke disease in fish under compromised conditions such as stress and/or wounds. Our results suggest that members of a phylogenetic cluster of fish-associated Flavobacterium species may be opportunistic fish pathogens causing disease under specific circumstances. IMPORTANCE Aquaculture has expanded significantly worldwide in the last decades and accounts for half of human fish consumption. However, infectious fish diseases are a major bottleneck for its sustainable development, and an increasing number of bacterial species from diseased fish raise a great concern. The current study revealed phylogenetic associations with ecological niches among the Flavobacterium species. We also focused on Flavobacterium collinsii, which belongs to a group of putative pathogenic species. The genome contents revealed a versatile metabolic repertoire suggesting the use of diverse nutrient sources, a characteristic of saprophytic or commensal bacteria. In a rainbow trout experimental challenge, the bacterium survived inside the host, likely escaping clearance by the immune system but without provoking massive mortality, suggesting opportunistic pathogenic behavior. This study highlights the importance of experimentally evaluating the pathogenicity of the numerous bacterial species retrieved from diseased fish.

Dynamic insights on transcription initiation and RNA processing during bacterial adaptation.

Transcription initiation and RNA processing govern gene expression and enable bacterial adaptation by reshaping the RNA landscape. The aim of this study was to simultaneously observe these two fundamental processes in a transcriptome responding to an environmental signal. A controlled σE system in E. coli was coupled to our previously described tagRNA-seq method to yield process kinetics information. Changes in transcription initiation frequencies (TIF) and RNA processing frequencies (PF) were followed using 5' RNA tags. Changes in TIF showed a binary increased/decreased pattern that alternated between transcriptionally activated and repressed promoters, providing the bacterial population with transcriptional oscillation. PF variation fell into three categories of cleavage activity: (i) constant and independent of RNA levels, (ii) increased once RNA has accumulated, and (iii) positively correlated to changes in TIF. This work provides a comprehensive and dynamic view of major events leading to transcriptomic reshaping during bacterial adaptation. It unveils an interplay between transcription initiation and the activity of specific RNA cleavage sites. This study utilized a well-known genetic system to analyze fundamental processes and can serve as a blueprint for comprehensive studies that exploit the RNA metabolism to decipher and understand bacterial gene expression control.

Regulation of alginate catabolism involves a GntR family repressor in the marine flavobacterium Zobellia galactanivorans DsijT.

Marine flavobacteria possess dedicated Polysaccharide Utilization Loci (PULs) enabling efficient degradation of a variety of algal polysaccharides. The expression of these PULs is tightly controlled by the presence of the substrate, yet details on the regulatory mechanisms are still lacking. The marine flavobacterium Zobellia galactanivorans DsijT digests many algal polysaccharides, including alginate from brown algae. Its complex Alginate Utilization System (AUS) comprises a PUL and several other loci. Here, we showed that the expression of the AUS is strongly and rapidly (<30 min) induced upon addition of alginate, leading to biphasic substrate utilization. Polymeric alginate is first degraded into smaller oligosaccharides that accumulate in the extracellular medium before being assimilated. We found that AusR, a GntR family protein encoded within the PUL, regulates alginate catabolism by repressing the transcription of most AUS genes. Based on our genetic, genomic, transcriptomic and biochemical results, we propose the first model of regulation for a PUL in marine bacteria. AusR binds to promoters of AUS genes via single, double or triple copies of operator. Upon addition of alginate, secreted enzymes expressed at a basal level catalyze the initial breakdown of the polymer. Metabolic intermediates produced during degradation act as effectors of AusR and inhibit the formation of AusR/DNA complexes, thus lifting transcriptional repression.

The Type IX Secretion System Is Required for Virulence of the Fish Pathogen Flavobacterium psychrophilum.

Flavobacterium psychrophilum causes bacterial cold-water disease in wild and aquaculture-reared fish and is a major problem for salmonid aquaculture. The mechanisms responsible for cold-water disease are not known. It was recently demonstrated that the related fish pathogen, Flavobacterium columnare, requires a functional type IX protein secretion system (T9SS) to cause disease. T9SSs secrete cell surface adhesins, gliding motility proteins, peptidases, and other enzymes, any of which may be virulence factors. The F. psychrophilum genome has genes predicted to encode components of a T9SS. Here, we used a SacB-mediated gene deletion technique recently adapted for use in the Bacteroidetes to delete a core F. psychrophilum T9SS gene, gldN The ΔgldN mutant cells were deficient for secretion of many proteins in comparison to wild-type cells. Complementation of the mutant with wild-type gldN on a plasmid restored secretion. Compared to wild-type and complemented strains, the ΔgldN mutant was deficient in adhesion, gliding motility, and extracellular proteolytic and hemolytic activities. The ΔgldN mutant exhibited reduced virulence in rainbow trout and complementation restored virulence, suggesting that the T9SS plays an important role in the disease.IMPORTANCE Bacterial cold-water disease, caused by F. psychrophilum, is a major problem for salmonid aquaculture. Little is known regarding the virulence factors involved in this disease, and control measures are inadequate. A targeted gene deletion method was adapted to F. psychrophilum and used to demonstrate the importance of the T9SS in virulence. Proteins secreted by this system are likely virulence factors and targets for the development of control measures.

The conserved regulatory RNA RsaE down-regulates the arginine degradation pathway in Staphylococcus aureus.

RsaE is a regulatory RNA highly conserved amongst Firmicutes that lowers the amount of mRNAs associated with the TCA cycle and folate metabolism. A search for new RsaE targets in Staphylococcus aureus revealed that in addition to previously described substrates, RsaE down-regulates several genes associated with arginine catabolism. In particular, RsaE targets the arginase rocF mRNA via direct interactions involving G-rich motifs. Two duplicated C-rich motifs of RsaE can independently downregulate rocF expression. The faster growth rate of ΔrsaE compared to its parental strain in media containing amino acids as sole carbon source points to an underlying role for RsaE in amino acid catabolism. Collectively, the data support a model in which RsaE acts as a global regulator of functions associated with metabolic adaptation.

Oral delivery of pancreatitis-associated protein by Lactococcus lactis displays protective effects in dinitro-benzenesulfonic-acid-induced colitis model and is able to modulate the composition of the microbiota.

Antimicrobial peptides secreted by intestinal immune and epithelial cells are important effectors of innate immunity. They play an essential role in the maintenance of intestinal homeostasis by limiting microbial epithelium interactions and preventing unnecessary microbe-driven inflammation. Pancreatitis-associated protein (PAP) belongs to Regenerating islet-derived III proteins family and is a C-type (Ca+2 dependent) lectin. PAP protein plays a protective effect presenting anti-inflammatory properties able to reduce the severity of colitis, preserving gut barrier and epithelial inflammation. Here, we sought to determine whether PAP delivered at intestinal lumen by recombinant Lactococcus lactis strain (LL-PAP) before and after chemically induced colitis is able to reduce the severity in two models of colitis. After construction and characterization of our recombinant strains, we tested their effects in dinitro-benzenesulfonic-acid (DNBS) and Dextran sulfate sodium (DSS) colitis model. After the DNBS challenge, mice treated with LL-PAP presented less severe colitis compared with PBS and LL-empty-treated mice groups. After the DSS challenge, no protective effects of LL-PAP could be detected. We determined that after 5 days administration, LL-PAP increase butyrate producer's bacteria, especially Eubacterium plexicaudatum. Based on our findings, we hypothesize that a treatment with LL-PAP shifts the microbiota preventing the severity of colon inflammation in DNBS colitis model. These protective roles of LL-PAP in DNBS colitis model might be through intestinal microbiota modulation.

Correction to: Quantitative trait loci for resistance to Flavobacterium psychrophilum in rainbow trout: effect of the mode of infection and evidence of epistatic interactions.

After publication of this work [1], we noted that there was an error in Table 3 Line 4.

Quantitative trait loci for resistance to Flavobacterium psychrophilum in rainbow trout: effect of the mode of infection and evidence of epistatic interactions.

BACKGROUND: Bacterial cold-water disease, which is caused by Flavobacterium psychrophilum, is one of the major diseases that affect rainbow trout (Oncorhynchus mykiss) and a primary concern for trout farming. Better knowledge of the genetic basis of resistance to F. psychrophilum would help to implement this trait in selection schemes and to investigate the immune mechanisms associated with resistance. Various studies have revealed that skin and mucus may contribute to response to infection. However, previous quantitative trait loci (QTL) studies were conducted by using injection as the route of infection. Immersion challenge, which is assumed to mimic natural infection by F. psychrophilum more closely, may reveal different defence mechanisms. RESULTS: Two isogenic lines of rainbow trout with contrasting susceptibilities to F. psychrophilum were crossed to produce doubled haploid F2 progeny. Fish were infected with F. psychrophilum either by intramuscular injection (115 individuals) or by immersion (195 individuals), and genotyped for 9654 markers using RAD-sequencing. Fifteen QTL associated with resistance traits were detected and only three QTL were common between the injection and immersion. Using a model that accounted for epistatic interactions between QTL, two main types of interactions were revealed. A "compensation-like" effect was detected between several pairs of QTL for the two modes of infection. An "enhancing-like" interaction effect was detected between four pairs of QTL. Integration of the QTL results with results of a previous transcriptomic analysis of response to F. psychrophilum infection resulted in a list of potential candidate immune genes that belong to four relevant functional categories (bacterial sensors, effectors of antibacterial immunity, inflammatory factors and interferon-stimulated genes). CONCLUSIONS: These results provide new insights into the genetic determinism of rainbow trout resistance to F. psychrophilum and confirm that some QTL with large effects are involved in this trait. For the first time, the role of epistatic interactions between resistance-associated QTL was evidenced. We found that the infection protocol used had an effect on the modulation of defence mechanisms and also identified relevant immune functional candidate genes.

An assessment of bacterial small RNA target prediction programs.

Most bacterial regulatory RNAs exert their function through base-pairing with target RNAs. Computational prediction of targets is a busy research field that offers biologists a variety of web sites and software. However, it is difficult for a non-expert to evaluate how reliable those programs are. Here, we provide a simple benchmark for bacterial sRNA target prediction based on trusted E. coli sRNA/target pairs. We use this benchmark to assess the most recent RNA target predictors as well as earlier programs for RNA-RNA hybrid prediction. Moreover, we consider how the definition of mRNA boundaries can impact overall predictions. Recent algorithms that exploit both conservation of targets and accessibility information offer improved accuracy over previous software. However, even with the best predictors, the number of true biological targets with low scores and non-targets with high scores remains puzzling.

Genome-wide identification of genes directly regulated by the pleiotropic transcription factor Spx in Bacillus subtilis.

The transcriptional regulator Spx plays a key role in maintaining the redox homeostasis of Bacillus subtilis cells exposed to disulfide stress. Defects in Spx were previously shown to lead to differential expression of numerous genes but direct and indirect regulatory effects could not be distinguished. Here we identified 283 discrete chromosomal sites potentially bound by the Spx-RNA polymerase (Spx-RNAP) complex using chromatin immunoprecipitation of Spx. Three quarters of these sites were located near Sigma(A)-dependent promoters, and upon diamide treatment, the fraction of the Spx-RNAP complex increased in parallel with the number and occupancy of DNA sites. Correlation of Spx-RNAP-binding sites with gene differential expression in wild-type and Δspx strains exposed or not to diamide revealed that 144 transcription units comprising 275 genes were potentially under direct Spx regulation. Spx-controlled promoters exhibited an extended -35 box in which nucleotide composition at the -43/-44 positions strongly correlated with observed activation. In vitro transcription confirmed activation by oxidized Spx of seven newly identified promoters, of which one was also activated by reduced Spx. Our study globally characterized the Spx regulatory network, revealing its role in the basal expression of some genes and its complex interplay with other stress responses.

Interplay between a bacterial pathogen and its host in rainbow trout isogenic lines with contrasted susceptibility to cold water disease.

Infectious diseases are a major constraint on aquaculture. Genetic lines with different susceptibilities to diseases are useful models to identify resistance mechanisms to pathogens and to improve prophylaxis. Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum represents a major threat for freshwater salmonid farming worldwide. A collection of rainbow trout (Oncorhynchus mykiss) isogenic lines was previously produced from a French domestic population. Here, we compared BCWD resistance phenotypes using a subset of isogenic lines chosen for their contrasted susceptibilities to F. psychrophilum. We applied individual monitoring to document the infection process, including time-course quantification of bacteremia and innate immune response. Strikingly, BCWD resistance was correlated with a lower bacterial growth rate in blood. Several immune genes were expressed at higher levels in resistant fish regardless of infection: the Type II arginase (arg2), a marker for M2 macrophages involved in anti-inflammatory responses and tissue repair, and two Toll-like receptors (tlr2/tlr7), responsible for pathogen detection and inflammatory responses. This study highlights the importance of innate and intrinsic defense mechanisms in determining the outcome of F. psychrophilum infections, and illustrates that non-lethal time-course blood sampling for individual monitoring of bacteremia is a powerful tool to resolve within-host pathogen behavior in bacterial fish diseases.

Proteomic analysis of spontaneous mutants of Lactococcus lactis: Involvement of GAPDH and arginine deiminase pathway in H2O2 resistance.

Lactococcus lactis, one of the most commonly used dairy starters, is often subjected to oxidative stress in cheese manufacturing. A comparative proteomic analysis was performed to identify the molecular modifications responsible for the robustness of three spontaneous H(2)O(2)-resistant (SpOx) strains. In the parental strain, glyceraldehyde-3-phosphate deshydrogenase (GAPDH) activity is ensured by GapB and the second GAPDH GapA is not produced in standard growth conditions. We showed that GapA was overproduced in the highly resistant SpOx2 and SpOx3 mutants. Its overproduction in the MG1363 strain led to an increased H(2)O(2) resistance of exponential growing cells. Upon H(2)O(2) exposure, GapB was fully inactivated by oxidation in the parental strain. In SpOx mutants, it partly remained in the reduced form sustaining partially GAPDH activity. The analysis of gapA disruption in these SpOx strains indicated that additional unraveled mechanisms likely contribute to the resistance phenotype. In the SpOx1 mutant, the arginine deiminase pathway was found to be upregulated and disruption of arcA or arcB genes abolished H(2)O(2) resistance. We concluded that arginine consumption was directly responsible for the SpOx1 phenotype. Finally, these results suggest that sustaining energy supply is a major way of leading to oxidative stress resistance in L. lactis.

Two functionally distinct heme/iron transport systems are virulence determinants of the fish pathogen Flavobacterium psychrophilum.

Bacterial pathogens have a critical impact on aquaculture, a sector that accounts for half of the human fish consumption. Flavobacterium psychrophilum (phylum Bacteroidetes) is responsible for bacterial cold-water disease in salmonids worldwide. The molecular factors involved in host invasion, colonization and haemorrhagic septicaemia are mostly unknown. In this study, we identified two new TonB-dependent receptors, HfpR and BfpR, that are required for adaptation to iron conditions encountered during infection and for virulence in rainbow trout. Transcriptional analyses revealed that their expression is tightly controlled and upregulated under specific iron sources and concentrations. Characterization of deletion mutants showed that they act without redundancy: BfpR is required for optimal growth in the presence of high haemoglobin level, while HfpR confers the capacity to acquire nutrient iron from haem or haemoglobin under iron scarcity. The gene hfpY, co-transcribed with hfpR, encodes a protein related to the HmuY family. We demonstrated that HfpY binds haem and contributes significantly to host colonization and disease severity. Overall, these results are consistent with a model in which both BfpR and Hfp systems promote haem uptake and respond to distinct signals to adapt iron acquisition to the different stages of pathogenesis. Our findings give insight into the molecular basis of pathogenicity of a serious pathogen belonging to the understudied family Flavobacteriaceae and point to the newly identified haem receptors as promising targets for antibacterial development.

Sustainable plant-based diets promote rainbow trout gut microbiota richness and do not alter resistance to bacterial infection.

BACKGROUND: Farmed fish food with reduced fish-derived products are gaining growing interest due to the ecological impact of fish-derived protein utilization and the necessity to increase aquaculture sustainability. Although different terrestrial plant proteins could replace fishmeal proteins, their use is associated with adverse effects. Here, we investigated how diets composed of terrestrial vegetal sources supplemented with proteins originating from insect, yeast or terrestrial animal by-products affect rainbow trout (Onchorynchus mykiss) gut microbiota composition, growth performance and resistance to bacterial infection by the fish pathogen Flavobacterium psychrophilum responsible for frequent outbreaks in aquaculture settings. RESULTS: We showed that the tested regimes significantly increased gut bacterial richness compared to full vegetal or commercial-like diets, and that vegetal diet supplemented with insect and yeast proteins improves growth performance compared to full vegetal diet without altering rainbow trout susceptibility to F. psychrophilum infection. CONCLUSION: Our results demonstrate that the use of insect and yeast protein complements to vegetal fish feeds maintain microbiota functions, growth performance and fish health, therefore identifying promising alternative diets to improve aquaculture's sustainability.

Transcriptome architecture and regulation at environmental transitions in flavobacteria: the case of an important fish pathogen.

The family Flavobacteriaceae (phylum Bacteroidetes) is a major component of soil, marine and freshwater ecosystems. In this understudied family, Flavobacterium psychrophilum is a freshwater pathogen that infects salmonid fish worldwide, with critical environmental and economic impact. Here, we report an extensive transcriptome analysis that established the genome map of transcription start sites and transcribed regions, predicted alternative sigma factor regulons and regulatory RNAs, and documented gene expression profiles across 32 biological conditions mimicking the pathogen life cycle. The results link genes to environmental conditions and phenotypic traits and provide insights into gene regulation, highlighting similarities with better known bacteria and original characteristics linked to the phylogenetic position and the ecological niche of the bacterium. In particular, osmolarity appears as a signal for transition between free-living and within-host programs and expression patterns of secreted proteins shed light on probable virulence factors. Further investigations showed that a newly discovered sRNA widely conserved in the genus, Rfp18, is required for precise expression of proteases. By pointing proteins and regulatory elements probably involved in host-pathogen interactions, metabolic pathways, and molecular machineries, the results suggest many directions for future research; a website is made available to facilitate their use to fill knowledge gaps on flavobacteria.

Combining Multiple Approaches and Models to Dissect the Genetic Architecture of Resistance to Infections in Fish.

Infectious diseases represent a major threat for the sustainable development of fish farming. Efficient vaccines are not available against all diseases, and growing antibiotics resistance limits the use of antimicrobial drugs in aquaculture. It is therefore important to understand the basis of fish natural resistance to infections to help genetic selection and to develop new approaches against infectious diseases. However, the identification of the main mechanisms determining the resistance or susceptibility of a host to a pathogenic microbe is challenging, integrating the complexity of the variation of host genetics, the variability of pathogens, and their capacity of fast evolution and adaptation. Multiple approaches have been used for this purpose: (i) genetic approaches, QTL (quantitative trait loci) mapping or GWAS (genome-wide association study) analysis, to dissect the genetic architecture of disease resistance, and (ii) transcriptomics and functional assays to link the genetic constitution of a fish to the molecular mechanisms involved in its interactions with pathogens. To date, many studies in a wide range of fish species have investigated the genetic determinism of resistance to many diseases using QTL mapping or GWAS analyses. A few of these studies pointed mainly toward adaptive mechanisms of resistance/susceptibility to infections; others pointed toward innate or intrinsic mechanisms. However, in the majority of studies, underlying mechanisms remain unknown. By comparing gene expression profiles between resistant and susceptible genetic backgrounds, transcriptomics studies have contributed to build a framework of gene pathways determining fish responsiveness to a number of pathogens. Adding functional assays to expression and genetic approaches has led to a better understanding of resistance mechanisms in some cases. The development of knock-out approaches will complement these analyses and help to validate putative candidate genes critical for resistance to infections. In this review, we highlight fish isogenic lines as a unique biological material to unravel the complexity of host response to different pathogens. In the future, combining multiple approaches will lead to a better understanding of the dynamics of interaction between the pathogen and the host immune response, and contribute to the identification of potential targets of selection for improved resistance.

Assessment of Bona Fide sRNAs in Staphylococcus aureus.

Bacterial regulatory RNAs have been extensively studied for over a decade, and are progressively being integrated into the complex genetic regulatory network. Transcriptomic arrays, recent deep-sequencing data and bioinformatics suggest that bacterial genomes produce hundreds of regulatory RNAs. However, while some have been authenticated, the existence of the others varies according to strains and growth conditions, and their detection fluctuates with the methodologies used for data acquisition and interpretation. For example, several small RNA (sRNA) candidates are now known to be parts of UTR transcripts. Accurate annotation of regulatory RNAs is a complex task essential for molecular and functional studies. We defined bona fide sRNAs as those that (i) likely act in trans and (ii) are not expressed from the opposite strand of a coding gene. Using published data and our own RNA-seq data, we reviewed hundreds of Staphylococcus aureus putative regulatory RNAs using the DETR'PROK computational pipeline and visual inspection of expression data, addressing the question of which transcriptional signals correspond to sRNAs. We conclude that the model strain HG003, a NCTC8325 derivative commonly used for S. aureus genetic regulation studies, has only about 50 bona fide sRNAs, indicating that these RNAs are less numerous than commonly stated. Among them, about half are associated to the S. aureus sp. core genome and a quarter are possibly expressed in other Staphylococci. We hypothesize on their features and regulation using bioinformatic approaches.

Genomic Diversity and Evolution of the Fish Pathogen Flavobacterium psychrophilum.

Flavobacterium psychrophilum, the etiological agent of rainbow trout fry syndrome and bacterial cold-water disease in salmonid fish, is currently one of the main bacterial pathogens hampering the productivity of salmonid farming worldwide. In this study, the genomic diversity of the F. psychrophilum species is analyzed using a set of 41 genomes, including 30 newly sequenced isolates. These were selected on the basis of available MLST data with the two-fold objective of maximizing the coverage of the species diversity and of allowing a focus on the main clonal complex (CC-ST10) infecting farmed rainbow trout (Oncorhynchus mykiss) worldwide. The results reveal a bacterial species harboring a limited genomic diversity both in terms of nucleotide diversity, with ~0.3% nucleotide divergence inside CDSs in pairwise genome comparisons, and in terms of gene repertoire, with the core genome accounting for ~80% of the genes in each genome. The pan-genome seems nevertheless "open" according to the scaling exponent of a power-law fitted on the rate of new gene discovery when genomes are added one-by-one. Recombination is a key component of the evolutionary process of the species as seen in the high level of apparent homoplasy in the core genome. Using a Hidden Markov Model to delineate recombination tracts in pairs of closely related genomes, the average recombination tract length was estimated to ~4.0 Kbp and the typical ratio of the contributions of recombination and mutations to nucleotide-level differentiation (r/m) was estimated to ~13. Within CC-ST10, evolutionary distances computed on non-recombined regions and comparisons between 22 isolates sampled up to 27 years apart suggest a most recent common ancestor in the second half of the nineteenth century in North America with subsequent diversification and transmission of this clonal complex coinciding with the worldwide expansion of rainbow trout farming. With the goal to promote the development of tools for the genetic manipulation of F. psychrophilum, a particular attention was also paid to plasmids. Their extraction and sequencing to completion revealed plasmid diversity that remained hidden to classical plasmid profiling due to size similarities.