Chitinolytic enzymes contribute to the pathogenicity of Aliivibrio salmonicida LFI1238 in the invasive phase of cold-water vibriosis.

BACKGROUND: Aliivibrio salmonicida is the causative agent of cold-water vibriosis in salmonids (Oncorhynchus mykiss and Salmo salar L.) and gadidae (Gadus morhua L.). Virulence-associated factors that are essential for the full spectrum of A. salmonicida pathogenicity are largely unknown. Chitin-active lytic polysaccharide monooxygenases (LPMOs) have been indicated to play roles in both chitin degradation and virulence in a variety of pathogenic bacteria but are largely unexplored in this context. RESULTS: In the present study we investigated the role of LPMOs in the pathogenicity of A. salmonicida LFI238 in Atlantic salmon (Salmo salar L.). In vivo challenge experiments using isogenic deletion mutants of the two LPMOs encoding genes AsLPMO10A and AsLPMO10B, showed that both LPMOs, and in particular AsLPMO10B, were important in the invasive phase of cold-water vibriosis. Crystallographic analysis of the AsLPMO10B AA10 LPMO domain (to 1.4 Å resolution) revealed high structural similarity to viral fusolin, an LPMO known to enhance the virulence of insecticidal agents. Finally, exposure to Atlantic salmon serum resulted in substantial proteome re-organization of the A. salmonicida LPMO deletion variants compared to the wild type strain, indicating the struggle of the bacterium to adapt to the host immune components in the absence of the LPMOs. CONCLUSION: The present study consolidates the role of LPMOs in virulence and demonstrates that such enzymes may have more than one function.

The Fish Pathogen Aliivibrio salmonicida LFI1238 Can Degrade and Metabolize Chitin despite Gene Disruption in the Chitinolytic Pathway.

The fish pathogen Aliivibrio (Vibrio) salmonicida LFI1238 is thought to be incapable of utilizing chitin as a nutrient source, since approximately half of the genes representing the chitinolytic pathway are disrupted by insertion sequences. In the present study, we combined a broad set of analytical methods to investigate this hypothesis. Cultivation studies revealed that A. salmonicida grew efficiently on N-acetylglucosamine (GlcNAc) and chitobiose [(GlcNAc)2], the primary soluble products resulting from enzymatic chitin hydrolysis. The bacterium was also able to grow on chitin particles, albeit at a lower rate than on the soluble substrates. The genome of the bacterium contains five disrupted chitinase genes (pseudogenes) and three intact genes encoding a glycoside hydrolase family 18 (GH18) chitinase and two auxiliary activity family 10 (AA10) lytic polysaccharide monooxygenases (LPMOs). Biochemical characterization showed that the chitinase and LPMOs were able to depolymerize both α- and ß-chitin to (GlcNAc)2 and oxidized chitooligosaccharides, respectively. Notably, the chitinase displayed up to 50-fold lower activity than other well-studied chitinases. Deletion of the genes encoding the intact chitinolytic enzymes showed that the chitinase was important for growth on ß-chitin, whereas the LPMO gene deletion variants only showed minor growth defects on this substrate. Finally, proteomic analysis of A. salmonicida LFI1238 growth on ß-chitin showed expression of all three chitinolytic enzymes and, intriguingly, also three of the disrupted chitinases. In conclusion, our results show that A. salmonicida LFI1238 can utilize chitin as a nutrient source and that the GH18 chitinase and the two LPMOs are needed for this ability. IMPORTANCE The ability to utilize chitin as a source of nutrients is important for the survival and spread of marine microbial pathogens in the environment. One such pathogen is Aliivibrio (Vibrio) salmonicida, the causative agent of cold water vibriosis. Due to extensive gene decay, many key enzymes in the chitinolytic pathway have been disrupted, putatively rendering this bacterium incapable of chitin degradation and utilization. In the present study, we demonstrate that A. salmonicida can degrade and metabolize chitin, the most abundant biopolymer in the ocean. Our findings shed new light on the environmental adaption of this fish pathogen.

Features and structure of a cold active N-acetylneuraminate lyase.

N-acetylneuraminate lyases (NALs) are enzymes that catalyze the reversible cleavage and synthesis of sialic acids. They are therefore commonly used for the production of these high-value sugars. This study presents the recombinant production, together with biochemical and structural data, of the NAL from the psychrophilic bacterium Aliivibrio salmonicida LFI1238 (AsNAL). Our characterization shows that AsNAL possesses high activity and stability at alkaline pH. We confirm that these properties allow for the use in a one-pot reaction at alkaline pH for the synthesis of N-acetylneuraminic acid (Neu5Ac, the most common sialic acid) from the inexpensive precursor N-acetylglucosamine. We also show that the enzyme has a cold active nature with an optimum temperature for Neu5Ac synthesis at 20°C. The equilibrium constant for the reaction was calculated at different temperatures, and the formation of Neu5Ac acid is favored at low temperatures, making the cold active enzyme a well-suited candidate for use in such exothermic reactions. The specific activity is high compared to the homologue from Escherichia coli at three tested temperatures, and the enzyme shows a higher catalytic efficiency and turnover number for cleavage at 37°C. Mutational studies reveal that amino acid residue Asn 168 is important for the high kcat. The crystal structure of AsNAL was solved to 1.65 Å resolution and reveals a compact, tetrameric protein similar to other NAL structures. The data presented provides a framework to guide further optimization of its application in sialic acid production and opens the possibility for further design of the enzyme.

Differential expression profiling of ΔlitR and ΔrpoQ mutants reveals insight into QS regulation of motility, adhesion and biofilm formation in Aliivibrio salmonicida.

BACKGROUND: The coordination of group behaviors in bacteria is achieved by a cell-cell signaling process called quorum sensing (QS). QS is an intercellular communication system, which synchronously controls expression of a vast range of genes in response to changes in cell density and is mediated by autoinducers that act as extracellular signals. Aliivibrio salmonicida, the causative agent of cold-water vibrosis in marine aquacultures, uses QS to regulate several activities such as motility, biofilm formation, adhesion and rugose colony morphology. However, little is known about either genes or detailed mechanisms involved in the regulation of these phenotypes. RESULTS: Differential expression profiling allowed us to define the genes involved in controlling phenotypes related to QS in A. salmonicida LFI1238. RNA sequencing data revealed that the number of expressed genes in A. salmonicida, ΔlitR and ΔrpoQ mutants were significantly altered due to changes in cell density. These included genes that were distributed among the 21 functional groups, mainly presented in cell envelope, cell processes, extrachromosomal/foreign DNA and transport-binding proteins functional groups. The comparative transcriptome of A. salmonicida wild-type at high cell density relative to low cell density revealed 1013 genes to be either up- or downregulated. Thirty-six downregulated genes were gene clusters encoding biosynthesis of the flagellar and chemotaxis genes. Additionally we identified significant expression for genes involved in acyl homoserine lactone (AHL) synthesis, adhesion and early colonization. The transcriptome profile of ΔrpoQ compared to the wild-type revealed 384 differensially expressed genes (DEGs) that allowed us to assign genes involved in regulating motility, adhesion and colony rugosity. Indicating the importance of RpoQ in controlling several QS related activities. Furthermore, the comparison of the transcriptome profiles of ΔlitR and ΔrpoQ mutants, exposed numerous overlapping DEGs that were essential for motility, exopolysaccharide production via syp operon and genes associated with tad operon. CONCLUSION: Our findings indicate previously unexplained functional roles for LitR and RpoQ in regulation of different phenotypes related to QS. Our transcriptome data provide a better understanding of the regulation cascade of motility, wrinkling colony morphology and biofilm formation and will offer a major source for further research and analysis on this important field.

The alternative sigma factor RpoQ regulates colony morphology, biofilm formation and motility in the fish pathogen Aliivibrio salmonicida.

BACKGROUND: Quorum sensing (QS) is a cell-to cell communication system that bacteria use to synchronize activities as a group. LitR, the master regulator of QS in Aliivibrio salmonicida, was recently shown to regulate activities such as motility, rugosity and biofilm formation in a temperature dependent manner. LitR was also found to be a positive regulator of rpoQ. RpoQ is an alternative sigma factor belonging to the sigma -70 family. Alternative sigma factors direct gene transcription in response to environmental signals. In this work we have studied the role of RpoQ in biofilm formation, colony morphology and motility of A. salmonicida LFI1238. RESULTS: The rpoQ gene in A. salmonicida LFI1238 was deleted using allelic exchange. We found that RpoQ is a strong repressor of rugose colony morphology and biofilm formation, and that it controls motility of the bacteria. We also show that overexpression of rpoQ in a ΔlitR mutant of A. salmonicida disrupts the biofilm produced by the ΔlitR mutant and decreases its motility, whereas rpoQ overexpression in the wild-type completely eliminates the motility. CONCLUSION: The present work demonstrates that the RpoQ sigma factor is a novel regulatory component involved in modulating motility, colony morphology and biofilm formation in the fish pathogen A. salmonicida. The findings also confirm that RpoQ functions downstream of the QS master regulator LitR. However further studies are needed to elucidate how LitR and RpoQ work together in controlling phenotypes related to QS in A. salmonicida.

Aliivibrio salmonicida requires O-antigen for virulence in Atlantic salmon (Salmo salar L.).

Aliivibrio salmonicida is the causative agent of cold-water vibriosis, a hemorrhagic septicemia of salmonid fish. The bacterium has been shown to rapidly enter the fish bloodstream, and proliferation in blood is seen after a period of latency. Although the pathogenesis of the disease is largely unknown, shedding of high quantities of outer-membrane complex VS-P1, consisting of LPS and a protein moiety, has been suggested to act as decoy and contribute to immunomodulation. To investigate the role of LPS in the pathogenesis, we constructed O-antigen deficient mutants by knocking out the gene encoding O-antigen ligase waaL. As this gene exists in two copies in the Al. salmonicida genome, we constructed single and double in-frame deletion mutants to explore potential effects of copy number variation. Our results demonstrate that the LPS structure of Al. salmonicida is essential for virulence in Atlantic salmon. As the loss of O-antigen did not influence invasive properties of the bacterium, the role of LPS in virulence applies to later stages of the pathogenesis. One copy of waaL was sufficient for O-antigen ligation and virulence in experimental models. However, as a non-significant decrease in mortality was observed after immersion challenge with a waaL single mutant, it is tempting to suggest that multiple copies of the gene are beneficial to the bacterium at lower challenge doses. The loss of O-antigen was not found to affect serum survival in vitro, but quantification of bacteria in blood following immersion challenge suggested a role in in vivo survival. Furthermore, fish challenged with the waaL double mutant induced a more transient immune response than fish challenged with the wild type strain. Whether the reduction in virulence following the loss of waaL is caused by altered immunomodulative properties or impaired survival remains unclear. However, our data demonstrate that LPS is crucial for development of disease.

A unique role of flagellar function in Aliivibrio salmonicida pathogenicity not related to bacterial motility in aquatic environments.

Aliivibrio salmonicida is the causative agent of cold-water vibriosis, a septicemia of farmed salmonid fish. The mechanisms of disease are not well described, and few virulence factors have been identified. However, a requirement for motility in the pathogenesis has been reported. Al. salmonicida is motile by the means of lophotrichous polar flagella, consisting of multiple flagellin subunits that are expressed simultaneously. Here we show that flagellin subunit FlaA, but not FlaD, is of major importance for motility in Al. salmonicida. Deletion of flaA resulted in 62% reduction in motility, as well as a reduction in the fraction of flagellated cells and number of flagella per cell. Similarly, deletion of the gene encoding motor protein motA gave rise to an aflagellate phenotype and cessation of motility. Surprisingly, we found that Al. salmonicida does not require motility for invasion of Atlantic salmon. Nevertheless, in-frame deletion mutants defective of motA and flaA were less virulent in Atlantic salmon challenged by immersion, whereas an effect on virulence after i.p. challenge was only seen for the latter. Our results indicate a complex requirement for motility and/or flagellation in the pathogenesis of cold-water vibriosis, but the mechanisms involved remain unknown. We hypothesize that the differences in virulence observed after immersion and i.p. challenge are related to the immune response of the host.

A comparative study of cold- and warm-adapted Endonucleases A using sequence analyses and molecular dynamics simulations.

The psychrophilic and mesophilic endonucleases A (EndA) from Aliivibrio salmonicida (VsEndA) and Vibrio cholera (VcEndA) have been studied experimentally in terms of the biophysical properties related to thermal adaptation. The analyses of their static X-ray structures was no sufficient to rationalize the determinants of their adaptive traits at the molecular level. Thus, we used Molecular Dynamics (MD) simulations to compare the two proteins and unveil their structural and dynamical differences. Our simulations did not show a substantial increase in flexibility in the cold-adapted variant on the nanosecond time scale. The only exception is a more rigid C-terminal region in VcEndA, which is ascribable to a cluster of electrostatic interactions and hydrogen bonds, as also supported by MD simulations of the VsEndA mutant variant where the cluster of interactions was introduced. Moreover, we identified three additional amino acidic substitutions through multiple sequence alignment and the analyses of MD-based protein structure networks. In particular, T120V occurs in the proximity of the catalytic residue H80 and alters the interaction with the residue Y43, which belongs to the second coordination sphere of the Mg2+ ion. This makes T120V an amenable candidate for future experimental mutagenesis.

The autoinducer synthases LuxI and AinS are responsible for temperature-dependent AHL production in the fish pathogen Aliivibrio salmonicida.

BACKGROUND: Quorum sensing (QS) is a cell-to-cell communication system used by bacteria to regulate activities such as virulence, bioluminescence and biofilm formation. The most common QS signals in Gram-negative bacteria are N-acyl-homoserine lactones (AHLs). Aliivibrio salmonicida is the etiological agent of cold water vibriosis in Atlantic salmon, a disease which occurs mainly during seasons when the seawater is below 12°C. In this work we have constructed several mutants of A. salmonicida LFI1238 in order to study the LuxI/LuxR and AinS/AinR QS systems with respect to AHL production and biofilm formation. RESULTS: Using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) we found that LuxI in A. salmonicida LFI1238 is responsible for producing seven of the different AHLs, whereas AinS is responsible for producing only one. The production of these various AHLs is dependent on both cell density and growth temperature. The AHLs were efficiently produced when wild type LFI1238 was grown at 6 or 12°C, however at 16°C AHL production decreased dramatically, and LFI1238 produced less than 5% of the maximum concentrations observed at 6°C. LitR, the master regulator of QS, was found to be a positive regulator of AinS-dependent AHL production, and to a lesser extent LuxI-dependent AHL production. This implies a connection between the two systems, and both systems were found to be involved in regulation of biofilm formation. Finally, inactivation of either luxR1 or luxR2 in the lux operon significantly reduced production of LuxI-produced AHLs. CONCLUSION: LuxI and AinS are the autoinducer synthases responsible for the eight AHLs in A. salmonicida. AHL production is highly dependent on growth temperature, and a significant decrease was observed when the bacterium was grown at a temperature above its limit for disease outbreak. Numerous AHLs could offer the opportunity for fine-tuning responses to changes in the environment.

The immediate global responses of Aliivibrio salmonicida to iron limitations.

BACKGROUND: Iron is an essential micronutrient for all living organisms, and virulence and sequestration of iron in pathogenic bacteria are believed to be correlated. As a defence mechanism, potential hosts therefore keep the level of free iron inside the body to a minimum. In general, iron metabolism is well studied for some bacteria (mostly human or animal pathogens). However, this area is still under-investigated for a number of important bacterial pathogens. Aliivibrio salmonicida is a fish pathogen, and previous studies of this bacterium have shown that production of siderophores is temperature regulated and dependent on low iron conditions. In this work we studied the immediate changes in transcription in response to a sudden decrease in iron levels in cultures of A. salmonicida. In addition, we compared our results to studies performed with Vibrio cholerae and Vibrio vulnificus using a pan-genomic approach. RESULTS: Microarray technology was used to monitor global changes in transcriptional levels. Cultures of A. salmonicida were grown to mid log phase before the iron chelator 2,2'-dipyridyl was added and samples were collected after 15 minutes of growth. Using our statistical cut-off values, we retrieved thirty-two differentially expressed genes where the most up-regulated genes belong to an operon encoding proteins responsible for producing the siderophore bisucaberin. A subsequent pan-transcriptome analysis revealed that nine of the up-regulated genes from our dataset were also up-regulated in datasets from similar experiments using V. cholerae and V. vulnificus, thus indicating that these genes are involved in a shared strategy to mitigate low iron conditions. CONCLUSIONS: The present work highlights the effect of iron limitation on the gene regulatory network of the fish pathogen A. salmonicida, and provides insights into common and unique strategies of Vibrionaceae species to mitigate low iron conditions.

Properties and distribution of a metallo-ß-lactamase (ALI-1) from the fish pathogen Aliivibrio salmonicida LFI1238.

OBJECTIVES: To characterize the chromosome-encoded metallo-ß-lactamase (MBL) from the psychrophilic, marine fish-pathogenic bacterium Aliivibrio salmonicida LFI1238 and check for the presence of the gene in other Aliivibrio isolates both connected to the fish-farming industry and from the environment. METHODS: The MBL gene was cloned and intracellularly expressed in Escherichia coli. Kinetic parameters, NaCl dependence, pH optimum and temperature optimum were determined using purified enzyme. The VIM-2 enzyme from a Pseudomonas aeruginosa hospital isolate was used as a counterpart in comparative analysis. PCRs with degenerate MBL primers were used to screen different A. salmonicida isolates for the presence of the gene. RESULTS: A. salmonicida MBL (ALI-1) is an Ambler class B ß-lactamase sharing 39% and 29% amino acid identity with IMP-1 and VIM-2, respectively. ALI-1 hydrolysed all ß-lactam antibiotics tested, except for the monobactam aztreonam and the penicillin piperacillin. A profound increase in activity was observed when adding NaCl to the assay mixture (60% active without addition of NaCl, increasing to 100% at 0.5 M NaCl). The increase was less noticeable for VIM-2 (100% active at 0.2 M NaCl). ALI-1 appears to be ubiquitous in nature as it is found in Aliivibrio isolates not affected by human activity. CONCLUSIONS: This work provides more data for the ever-expanding MBL group of enzymes. These periplasmic enzymes are activated by addition of NaCl, and the marine enzyme is highly salt tolerant and cold active. The observed enzyme properties very likely reflect the conditions that the enzymes face in situ.

IS elements in Aliivibrio salmonicida LFI1238: occurrence, variability and impact on adaptability.

Insertion sequence (IS) elements are short, self-replicating DNA sequences that are capable of efficiently spreading over the host genome. Possessing varied integration specificity IS elements are capable of the irreversible inactivation of genes, which diversifies the pool of intact genetic determinants in host populations. In the current study, we performed a complex analysis of IS elements (Vsa IS) in the previously sequenced genome of Aliivibrio salmonicida LFI1238 and proposed a model of the spread of the Vsa IS elements over the genome of this microorganism. Along with the prediction of the integration sites for Vsa IS elements, the current study provides an overview of the properties of A. salmonicida IS elements, as well as information regarding their occurrence in different bacterial classes. An analysis of individual alleles of the IS elements has allowed us to depict a history of the accumulation of mutations and to describe distinctive microevolution lines for actively transposing Vsa IS elements in the genome of A. salmonicida LFI1238. Our results demonstrate the high importance of the dead end microevolution of actively transposing Vsa IS elements for the inactivation of genes in A. salmonicida LFI1238.

LitR is a repressor of syp genes and has a temperature-sensitive regulatory effect on biofilm formation and colony morphology in Vibrio (Aliivibrio) salmonicida.

Vibrio (Aliivibrio) salmonicida is the etiological agent of cold water vibriosis, a disease in farmed Atlantic salmon (Salmo salar) that is kept under control due to an effective vaccine. A seawater temperature below 12°C is normally required for disease development. Quorum sensing (QS) is a cell density-regulated communication system that bacteria use to coordinate activities involved in colonization and pathogenesis, and we have previously shown that inactivation of the QS master regulator LitR attenuates the V. salmonicida strain LFI1238 in a fish model. We show here that strain LFI1238 and a panel of naturally occurring V. salmonicida strains are poor biofilm producers. Inactivation of litR in the LFI1238 strain enhances medium- and temperature-dependent adhesion, rugose colony morphology, and biofilm formation. Chemical treatment and electron microscopy of the biofilm identified an extracellular matrix consisting mainly of a fibrous network, proteins, and polysaccharides. Further, by microarray analysis of planktonic and biofilm cells, we identified a number of genes regulated by LitR and, among these, were homologues of the Vibrio fischeri symbiosis polysaccharide (syp) genes. The syp genes were regulated by LitR in both planktonic and biofilm lifestyle analyses. Disruption of syp genes in the V. salmonicida ΔlitR mutant alleviated adhesion, rugose colony morphology, and biofilm formation. Hence, LitR is a repressor of syp transcription that is necessary for expression of the phenotypes examined. The regulatory effect of LitR on colony morphology and biofilm formation is temperature sensitive and weak or absent at temperatures above the bacterium's upper threshold for pathogenicity.

Recombinant expression and purification of an ATP-dependent DNA ligase from Aliivibrio salmonicida.

The genome of the psychrophilic fish-pathogen Aliivibrio salmonicida encodes a putative ATP-dependent DNA ligase in addition to a housekeeping NAD-dependent enzyme. In order to study the structure and activity of the ATP dependent ligase in vitro we have undertaken its recombinant production and purification from an Escherichia coli based expression system. Expression and purification of this protein presented two significant challenges. First, the gene product was moderately toxic to E. coli cells, second it was necessary to remove the large amounts of E. coli DNA present in bacterial lysates without contamination of the protein preparation by nucleases which might interfere with future assaying. The toxicity problem was overcome by fusion of the putative ligase to large solubility tags such as maltose-binding protein (MBP) or Glutathione-S-transferase (GST), and DNA was removed by treatment with a nuclease which could be inhibited by reducing agents. As the A. salmonicida ATP-dependent DNA ligase gene encodes a predicted leader peptide, both the full-length and mature forms of the protein were produced. Both possessed ATP-dependent DNA ligase activity, but the truncated form was significantly more active. Here we detail the first reported production, purification and preliminary characterization of active A. salmonicida ATP-dependent DNA ligase.

LitR of Vibrio salmonicida is a salinity-sensitive quorum-sensing regulator of phenotypes involved in host interactions and virulence.

Vibrio (Aliivibrio) salmonicida is the causal agent of cold-water vibriosis, a fatal bacterial septicemia primarily of farmed salmonid fish. The molecular mechanisms of invasion, colonization, and growth of V. salmonicida in the host are still largely unknown, and few virulence factors have been identified. Quorum sensing (QS) is a cell-to-cell communication system known to regulate virulence and other activities in several bacterial species. The genome of V. salmonicida LFI1238 encodes products presumably involved in several QS systems. In this study, the gene encoding LitR, a homolog of the master regulator of QS in V. fischeri, was deleted. Compared to the parental strain, the litR mutant showed increased motility, adhesion, cell-to-cell aggregation, and biofilm formation. Furthermore, the litR mutant produced less cryptic bioluminescence, whereas production of acylhomoserine lactones was unaffected. Our results also indicate a salinity-sensitive regulation of LitR. Finally, reduced mortality was observed in Atlantic salmon infected with the litR mutant, implying that the fish were more susceptible to infection with the wild type than with the mutant strain. We hypothesize that LitR inhibits biofilm formation and favors planktonic growth, with the latter being more adapted for pathogenesis in the fish host.

Expression profiling reveals Spot 42 small RNA as a key regulator in the central metabolism of Aliivibrio salmonicida.

BACKGROUND: Spot 42 was discovered in Escherichia coli nearly 40 years ago as an abundant, small and unstable RNA. Its biological role has remained obscure until recently, and is today implicated in having broader roles in the central and secondary metabolism. Spot 42 is encoded by the spf gene. The gene is ubiquitous in the Vibrionaceae family of gamma-proteobacteria. One member of this family, Aliivibrio salmonicida, causes cold-water vibriosis in farmed Atlantic salmon. Its genome encodes Spot 42 with 84% identity to E. coli Spot 42. RESULTS: We generated a A. salmonicida spf deletion mutant. We then used microarray and Northern blot analyses to monitor global effects on the transcriptome in order to provide insights into the biological roles of Spot 42 in this bacterium. In the presence of glucose, we found a surprisingly large number of ≥ 2X differentially expressed genes, and several major cellular processes were affected. A gene encoding a pirin-like protein showed an on/off expression pattern in the presence/absence of Spot 42, which suggests that Spot 42 plays a key regulatory role in the central metabolism by regulating the switch between fermentation and respiration. Interestingly, we discovered an sRNA named VSsrna24, which is encoded immediately downstream of spf. This new sRNA has an expression pattern opposite to that of Spot 42, and its expression is repressed by glucose. CONCLUSIONS: We hypothesize that Spot 42 plays a key role in the central metabolism, in part by regulating the pyruvat dehydrogenase enzyme complex via pirin.

Prediction, microarray and northern blot analyses identify new intergenic small RNAs in Aliivibrio salmonicida.

Bacterial small RNAs (sRNAs) are trans-encoded regulatory RNAs that typically bind mRNAs by short-sequence complementarities and change the expression of the corresponding proteins. Some of the well-characterized sRNAs serve critical steps in the regulation of important cellular processes, such as quorum sensing (Qrr), iron homeostasis (RyhB), oxidative stress (OxyS), and carbon metabolism (Spot 42). However, many sRNAs remain to be identified, and the functional roles of sRNAs are known for only a small fraction. For example, of the hundreds of candidate sRNAs from members of the bacterial family Vibrionaceae, the function is known for only 9. We have in this study significantly contributed to the discovery and verification of new sRNAs in a representative of Vibrionaceae, i.e. the Aliivibrio salmonicida, which causes severe disease in farmed Atlantic salmon and other fishes. A computational search for intergenic non-coding (nc) RNAs in the 4.6-Mb genome identified a total of 252 potential ncRNAs (including 233 putative sRNAs). Depending on the set threshold value for fluorescence signal in our microarray approach, we identified 50-80 putative ncRNAs, 12 of which were verified by Northern blot analysis. In total, we identified 9 new sRNAs.

Vibrio salmonicida pathogenesis analyzed by experimental challenge of Atlantic salmon (Salmo salar).

Cold-water vibriosis (CV) is a bacterial septicemia of farmed salmonid fish and cod caused by the Gram-negative bacterium Vibrio (Aliivibrio) salmonicida. To study the pathogenesis of this marine pathogen, Atlantic salmon was experimentally infected by immersion challenge with wild type V. salmonicida and the bacterial distribution in different organs was investigated at different time points. V. salmonicida was identified in the blood as early as 2 h after challenge demonstrating a rapid establishment of bacteremia without an initial period of colonization of the host. Two days after immersion challenge, only a few V. salmonicida were identified in the intestines, but the amount increased with time. In prolonged CV cases, V. salmonicida was the dominating bacterium of the gut microbiota causing a release of the pathogen to the water. We hypothesize that V. salmonicida uses the blood volume for proliferation during the infection of the fish and the salmonid intestine as a reservoir that favors survival and transmission. In addition, a motility-deficient V. salmonicida strain led us to investigate the impact of motility in the CV pathogenesis by comparing the virulence properties of the mutant with the wild type LFI1238 strain in both i.p. and immersion challenge experiments. V. salmonicida was shown to be highly dependent on motility to gain access to the fish host. After invasion, motility was no longer required for virulence, but the absence of normal flagellation delayed the disease development.

Identification and cloning of immunogenic Aliivibrio salmonicida Pal-like protein present in profiled outer membrane and secreted subproteome.

Aliivibrio salmonicida is the aetiological agent of cold water vibriosis affecting farmed fish species, a disease that today is fully controlled by vaccination. However, the molecular mechanisms behind the successful vaccine are largely unknown. In order to gain insight into the possible mechanisms of A. salmonicida vaccines, we report here the profiles of both the outer membrane and secreted subproteomes of A. salmonicida LFI315. The 2 subproteomes were resolved by 2-dimensional electrophoresis that identified a total of 82 protein entries. Monoclonal antibodies specific to an unidentified protein antigen were utilized in the immunoproteomic analysis of both outer membrane proteins and extracellular proteins. The immunogenic protein was located in both subproteomes and identified as a 20 kDa peptidoglycan-associated lipoprotein (Pal). The identity of the antigen was verified by heterologous expression of the cloned A. salmonicida pal gene (VSAL_I1899). It is likely that the immunogenic Pal-like protein is among the constituents that act as a protective antigen in the successful vaccine used today. In view of this, it may be considered a potentially useful component in future vaccine development and pathogenicity studies.

Experimental and computational characterization of the ferric uptake regulator from Aliivibrio salmonicida (Vibrio salmonicida).

The Ferric uptake regulator (Fur) is a global transcription factor that affects expression of bacterial genes in an iron-dependent fashion. Although the Fur protein and its iron-responsive regulon are well studied, there are still important questions that remain to be answered. For example, the consensus Fur binding site also known as the "Fur box" is under debate, and it is still unclear which Fur residues directly interact with the DNA. Our long-term goal is to dissect the biological roles of Fur in the development of the disease cold-water vibriosis, which is caused by the psychrophilic bacteria Aliivibrio salmonicida (also known as Vibrio salmonicida). Here, we have used experimental and computational methods to characterise the Fur protein from A. salmonicida (AS-Fur). Electrophoretic mobility shift assays show that AS-Fur binds to the recently proposed vibrio Fur box consensus in addition to nine promoter regions that contain Fur boxes. Binding appears to be dependent on the number of Fur boxes, and the predicted "strength" of Fur boxes. Finally, structure modeling and molecular dynamics simulations provide new insights into potential AS-Fur-DNA interactions.