Recoding UAG to selenocysteine in Saccharomyces cerevisiae.

Unique chemical and physical properties are introduced by inserting selenocysteine (Sec) at specific sites within proteins. Recombinant and facile production of eukaryotic selenoproteins would benefit from a yeast expression system; however, the selenoprotein biosynthetic pathway was lost in the evolution of the kingdom Fungi as it diverged from its eukaryotic relatives. Based on our previous development of efficient selenoprotein production in bacteria, we designed a novel Sec biosynthesis pathway in Saccharomyces cerevisiae using Aeromonas salmonicida translation components. S. cerevisiae tRNASer was mutated to resemble A. salmonicida tRNASec to allow recognition by S. cerevisiae seryl-tRNA synthetase as well as A. salmonicida selenocysteine synthase (SelA) and selenophosphate synthetase (SelD). Expression of these Sec pathway components was then combined with metabolic engineering of yeast to enable the production of active methionine sulfate reductase enzyme containing genetically encoded Sec. Our report is the first demonstration that yeast is capable of selenoprotein production by site-specific incorporation of Sec.

Role of cold shock proteins B and D in Aeromonas salmonicida subsp. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus).

Cold shock proteins (Csp) are pivotal nucleic acid binding proteins known for their crucial roles in the physiology and virulence of various bacterial pathogens affecting plant, insect, and mammalian hosts. However, their significance in bacterial pathogens of teleost fish remains unexplored. Aeromonas salmonicida subsp. salmonicida (hereafter A. salmonicida) is a psychrotrophic pathogen and the causative agent of furunculosis in marine and freshwater fish. Four csp genes (cspB, cspD, cspA, and cspC) have been identified in the genome of A. salmonicida J223 (wild type). Here, we evaluated the role of DNA binding proteins, CspB and CspD, in A. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus). A. salmonicida ΔcspB, ΔcspD, and the double ΔcspBΔcspD mutants were constructed and characterized. A. salmonicida ΔcspB and ΔcspBΔcspD mutants showed a faster growth at 28°C, and reduced virulence in lumpfish. A. salmonicida ΔcspD showed a slower growth at 28°C, biofilm formation, lower survival in low temperatures and freezing conditions (-20°C, 0°C, and 4°C), deficient in lipopolysaccharide synthesis, and low virulence in lumpfish. Additionally, ΔcspBΔcspD mutants showed less survival in the presence of bile compared to the wild type. Transcriptome analysis revealed that 200, 37, and 921 genes were differentially expressed in ΔcspB, ΔcspD, and ΔcspBΔcspD, respectively. In ΔcspB and ΔcspBΔcspD virulence genes in the chromosome and virulence plasmid were downregulated. Our analysis indicates that CspB and CspD mostly act as a transcriptional activator, influencing cell division (e.g., treB), virulence factors (e.g., aexT), and ultimately virulence.

Detection of Aeromonas salmonicida subsp. salmonicida infection in zebrafish by labelling bacteria with GFP and a fluorescent probe based on the siderophore amonabactin.

Zebrafish (Danio rerio) is an excellent model to study bacterial infections in fish and their treatment. We used zebrafish as a model of infection for Aeromonas salmonicida subsp. salmonicida (hereinafter A. salmonicida), the causative agent of fish furunculosis. The infection process of A. salmonicida was studied by immersion of zebrafish larvae in 2 different doses of the bacteria and the fish mortality was monitored for three days. The bacterium caused a high mortality (65 %) in zebrafish larvae only when they were exposed to a high bacterial concentration (107 bacterial cells/mL). To evaluate the use of fluorescence microscopy to follow A. salmonicida infection in vivo, two different fluorescent strains generated by labeling an A. salmonicida strain with either, the green fluorescent protein (GFP), or with a previously reported siderophore amonabactin-sulforhodamine B conjugate (AMB-SRB), were used. The distribution of both labeled bacterial strains in the larvae tissues was evaluated by conventional and confocal fluorescence microscopy. The fluorescent signal showed a greater intensity with the GFP-labeled bacteria, so it could be observed using conventional fluorescence microscopy. Since the AMB-SRB labeled bacteria showed a weaker signal, the larvae were imaged using a laser scanning confocal microscope after 48 h of exposure to the bacteria. Both fluorescent signals were mainly observed in the larvae digestive tract, suggesting that this is the main colonization route of zebrafish for waterborne A. salmonicida. This is the first report of the use of a siderophore-fluorophore conjugate to study a bacterial infection in fish. The use of a siderophore-fluorophore conjugate has the advantage that it is a specific marker and that does not require genetic manipulation of the bacteria.

Isolation of Aeromonas salmonicida subspecies salmonicida from aquaculture environment in India: Polyphasic identification, virulence characterization, and antibiotic susceptibility.

This study reports the polyphasic identification, characterization of virulence potential, and antibiotic susceptibility of Aeromonas salmonicida subspecies salmonicida COFCAU_AS, isolated from an aquaculture system in India. The physiological, biochemical, 16s rRNA gene sequencing and PAAS PCR test identified the strain as Aeromonas salmonicida. The MIY PCR tests established the subspecies as 'salmonicida'. The in vitro tests showed the isolated bacterium as haemolytic with casein, lipid, starch, and gelatin hydrolysis activity, indicating its pathogenic attributes. It also showed the ability to produce slime and biofilm, and additionally, it possessed an A-layer surface protein. In vivo pathogenicity test was performed to determine the LD50 dose of the bacterium in Labeo rohita fingerlings (14.42 ± 1.01 g), which was found to be 106.9 cells fish-1. The bacteria-challenged fingerlings showed skin lesions, erythema at the base of the fins, dropsy, and ulcer. Almost identical clinical signs and mortalities were observed when the same LD50 dose was injected into other Indian major carp species, L. catla and Cirrhinus mrigala. Out of the twelve virulent genes screened, the presence of nine genes viz., aerA, act, ast, alt, hlyA, vapA, exsA, fstA, and lip were detected, whereas ascV, ascC, and ela genes were absent. The A. salmonicida subsp. salmonicida COFCAU_AS was resistant to antibiotics such as penicillin G, rifampicin, ampicillin, and vancomycin while highly sensitive to amoxiclav, nalidixic acid, chloramphenicol, ciprofloxacin, and tetracycline. In summary, we have isolated a virulent A. salmonicida subsp. salmonicida from a tropical aquaculture pond which can cause significant mortality and morbidity in Indian major carp species.

Aeromonas salmonicida isolates from Canada demonstrate wide distribution and clustering among mesophilic strains.

All the 36 known species to date of the genus Aeromonas are mesophilic except the species Aeromonas salmonicida, which includes both psychrophilic and mesophilic subspecies. For 20 years, more and more mesophilic A. salmonicida strains have been discovered. Only A. salmonicida subsp. pectinolytica has officially been classified as a mesophilic subspecies. Most mesophiles have been isolated in hot countries. We present, for the first time, the characterization of two new mesophilic isolates from Quebec (Canada). Phenotypic and genomic characterizations were carried out on these strains, isolated from dead fish from a fish farm. Isolates 19-K304 and 19-K308 are clearly mesophiles, virulent to the amoeba Dictyostelium discoideum, a surrogate host, and close to strain Y577, isolated in India. To our knowledge, this is the first time that mesophilic strains isolated from different countries are so similar. The major difference between the isolates is the presence of plasmid pY47-3, a cryptic plasmid that sometimes presents in mesophilic strains. More importantly, our extensive phylogenetic analysis reveals two well-defined clades of mesophilic strains with psychrophiles associated with one of these clades. This helps to have a better understanding of the evolution of this species and the apparition of psychrophilic subspecies.

Isolation of vB_AsaM_LPM4 reveals the dynamics of Prophage 3 in Aeromonas salmonicida subsp. salmonicida.

Aeromonas salmonicida subsp. salmonicida causes furunculosis, a major infection that affects fish farms worldwide. We isolated phage vB_AsaM_LPM4 (LPM4) from a diseased fish. Based on its DNA sequence, LPM4 is identical to the uncharacterized Prophage 3, a prophage present mostly in North American A. salmonicida subsp. salmonicida isolates that bear the genomic island AsaGEI2a. Prophage 3 and AsaGEI2a are inserted side by side in the bacterial chromosome. The LPM4/Prophage 3 sequence is similar to that of other prophages found in various members of the genus Aeromonas. LPM4 specifically infects A. salmonicida subsp. salmonicida strains that do not already bear Prophage 3. The presence of an A-layer on the surface of the bacteria is not necessary for the adsorption of phage LPM4 but seems to facilitate its infection process. We also successfully produced lysogenic strains that bear Prophage 3 using sensitive strains with different genetic backgrounds, suggesting that there is no interdependency between LPM4 and AsaGEIs. PCR analysis of the excision dynamics of Prophage 3 and AsaGEIs revealed that these genetic elements can spontaneously excise themselves from the bacterial chromosome independently of one another. Through the isolation and characterization of LPM4, this study reveals new facets of Prophage 3 and AsaGEIs.

Genome-wide integrated analysis reveals functions of lncRNA-miRNA-mRNA interactions in Atlantic salmon challenged by Aeromonas salmonicida.

Aeromonas salmonicida (A. salmonicida) is a pathogenic bacterium that causes serious problems in the global Atlantic salmon aquaculture industry. In this study, we comprehensively analyzed the profiles of lncRNAs, miRNAs and mRNAs in gills of Atlantic salmon at high-dose A. salmonicida infection (3.06 × 108 CFU/mL), low-dose A. salmonicida infection (3.06 × 105 CFU/mL), and a PBS (100 µL) control. We identified 65 differentially expressed lncRNAs, 41 miRNAs, and 512 mRNAs between the control group and infection groups. Functional analysis showed that these genes were significantly enriched in the p53 signaling pathway, Wnt signaling pathway, mTOR signaling pathway, JAK-STAT signaling pathway, and Toll-like receptor signaling pathway. In addition, we predicted key genes in immune-related pathways and constructed a lncRNA-miRNA-mRNA network based on whole transcriptomic analysis. We further predicted three lncRNA-miRNA-mRNA axes as potential novel biomarkers in regulating the immune response of Atlantic salmon against A. salmonicida infection.

The Sigma Factor AsbI Is Required for the Expression of Acinetobactin Siderophore Transport Genes in Aeromonas salmonicida.

Aeromonas salmonicida subsp. salmonicida (A. salmonicida), a Gram-negative bacterium causing furunculosis in fish, produces the siderophores acinetobactin and amonabactins in order to extract iron from its hosts. While the synthesis and transport of both systems is well understood, the regulation pathways and conditions necessary for the production of each one of these siderophores are not clear. The acinetobactin gene cluster carries a gene (asbI) encoding a putative sigma factor belonging to group 4 σ factors, or, the ExtraCytoplasmic Function (ECF) group. By generating a null asbI mutant, we demonstrate that AsbI is a key regulator that controls acinetobactin acquisition in A. salmonicida, since it directly regulates the expression of the outer membrane transporter gene and other genes necessary for Fe-acinetobactin transport. Furthermore, AsbI regulatory functions are interconnected with other iron-dependent regulators, such as the Fur protein, as well as with other sigma factors in a complex regulatory network.

CC and CXC chemokines in turbot (Scophthalmus maximus L.): Identification, evolutionary analyses, and expression profiling after Aeromonas salmonicida infection.

Chemokines are a superfamily of structurally related cytokines, which exert essential roles in guiding cell migration in development, homeostasis, and immunity. CC and CXC chemokines are the two major subfamilies in teleost species. In this study, a total of seventeen CC and CXC chemokines, with inclusion of twelve CC and five CXC chemokines, were systematically identified from the turbot genome, making turbot the teleost harboring the least number of CC and CXC chemokines among all teleost species ever reported. Phylogeny, synteny, and genomic organization analyses were performed to annotate these genes, and multiple chemokine genes were identified in the turbot genome, due to the tandem duplications (CCL19 and CCL20), the whole genome duplications (CCL20, CCL25, and CXCL12), and the teleost-specific members (CCL34-36, CCL44, and CXCL18). In addition, chemokines were ubiquitously expressed in nine examined healthy tissues, with high expression levels observed in liver, gill, and spleen. Moreover, most chemokines were significantly differentially expressed in gill and spleen after Aeromonas salmonicida infection, and exhibited tissue-specific and time-dependent manner. Finally, protein-protein interaction network (PPI) analysis indicated that turbot chemokines interacted with a few immune-related genes such as interleukins, cathepsins, stats, and TLRs. These results should be valuable for comparative immunological studies and provide insights for further functional characterization of chemokines in teleost.

Genome-Wide Identification, Evolutionary Analysis, and Expression Patterns of Cathepsin Superfamily in Black Rockfish (Sebastes schlegelii) following Aeromonas salmonicida Infection.

Cathepsins are lysosomal cysteine proteases belonging to the papain family and play crucial roles in intracellular protein degradation/turnover, hormone maturation, antigen processing, and immune responses. In the present study, 18 cathepsins were systematically identified from the fish S. schlegelii genome. Phylogenetic analysis indicated that cathepsin superfamilies are categorized into eleven major clusters. Synteny and genome organization analysis revealed that whole-genome duplication led to the expansion of S. schlegelii cathepsins. Evolutionary rate analyses indicated that the lowest Ka/Ks ratios were observed in CTSBa (0.13) and CTSBb (0.14), and the highest Ka/Ks ratios were observed in CTSZa (1.97) and CTSZb (1.75). In addition, cathepsins were ubiquitously expressed in all examined tissues, with high expression levels observed in the gill, intestine, head kidney, and spleen. Additionally, most cathepsins were differentially expressed in the head kidney, gill, spleen, and liver following Aeromonas salmonicida infection, and their expression signatures showed tissue-specific and time-dependent patterns. Finally, protein-protein interaction network (PPI) analyses revealed that cathepsins are closely related to a few immune-related genes, such as interleukins, chemokines, and TLR genes. These results are expected to be valuable for comparative immunological studies and provide insights for further functional characterization of cathepsins in fish species.

Transcriptional Signatures of Immune, Neural, and Endocrine Functions in the Brain and Kidney of Rainbow Trout (Oncorhynchus mykiss) in Response to Aeromonas salmonicida Infection.

Rainbow trout (Oncorhynchus mykiss) serves as one of the most important commercial fish with an annual production of around 800,000 tonnes. However, infectious diseases, such as furunculosis caused by Aeromonas salmonicida infection, results in great economic loss in trout culture. The brain and kidney are two important organs associated with "sickness behaviors" and immunomodulation in response to disease. Therefore, we worked with 60 trout and investigated transcriptional responses and enrichment pathways between healthy and infected trout. We observed that furunculosis resulted in the activation of toll-like receptors with neuroinflammation and neural dysfunction in the brain, which might cause the "sickness behaviors" of infected trout including anorexia and lethargy. We also showed the salmonid-specific whole genome duplication contributed to duplicated colony stimulating factor 1 (csf-1) paralogs, which play an important role in modulating brain immunomodulation. Enrichment analyses of kidneys showed up-regulated immunomodulation and down-regulated neural functions, suggesting an immune-neural interaction between the brain and kidney. Moreover, the kidney endocrine network was activated in response to A. salmonicida infection, further convincing the communications between endocrine and immune systems in regulating internal homeostasis. Our study provided a foundation for pathophysiological responses of the brain and kidney in response to furunculosis and potentially offered a reference for generating disease-resistant trout strains.

Microbe profile: Aeromonas salmonicida: an opportunistic pathogen with multiple personalities.

The bacterial species Aeromonas salmonicida is a fish pathogen. Feared by fish farmers everywhere on Earth over the past century, this species has turned out to be more diverse than initially suspected. While some psychrophilic subspecies cannot grow at temperatures above 25 °C or 30 °C, other mesophilic strains growing up to 37 °C and above are now characterized. Adding to the surprising diversity of this species, some of the mesophilic strains infect mammals and birds. The remarkable diversity is explained in part by the presence of numerous mobile genetic elements, which sculpt and modify the genome of the various strains of this species.

Rapid visual detection of Aeromonas salmonicida by loop-mediated isothermal amplification with hydroxynaphthol blue dye.

To make crucial prevention, reduce fish losses and minimize the economic damage of diseases on the fish farm owners, a rapid detection of fish pathogens is mandatory. In this study, a loop-mediated isothermal amplification assay combined with hydroxynaphthol blue dye (LAMP-HNB) was developed and used for the rapid detection of Aeromonas salmonicida that caused significant economic losses in fish farming. Firstly, a pair of outer and inner primers specific for conserved fragment of vapA gene in A. salmonicida were designed and synthesized. Secondly, by optimizing the reaction conditions including reaction temperature, time, Mg2+ concentration, dNTP concentration and primer ratio, a LAMP-HNB assay was successfully established for the detection of A. salmoncida. Thirdly, the assay showed good specificity with no false-positive and false-negative results, and good sensitivity with the detection limit of 3.077 × 10-6  ng/µl, which was 102 times more sensitive than the conventional PCR. Finally, the LAMP-HNB assay was validated by the fish samples inoculated with different concentrations of A. salmoncida. This is the first development of rapid visual detection of A. salmonicida based on LAMP-HNB assay, which has great application prospect and market for diagnostic testing, health certification and active surveillance programmers.

Beyond the A-layer: adsorption of lipopolysaccharides and characterization of bacteriophage-insensitive mutants of Aeromonas salmonicida subsp. salmonicida.

Aeromonas salmonicida subsp. salmonicida is a fish pathogen that causes furunculosis. Antibiotherapy used to treat furunculosis in fish has led to resistance. Virulent phages are increasingly seen as alternatives or complementary treatments against furunculosis in aquaculture environments. For phage therapy to be successful, it is essential to study the natural mechanisms of phage resistance in A. salmonicida subsp. salmonicida. Here, we generated bacteriophage-insensitive mutants (BIMs) of A. salmonicida subsp. salmonicida, using a myophage with broad host range and characterized them. Phage plaques were different depending on whether the A-layer surface array protein was expressed or not. The genome analysis of the BIMs helped to identify mutations in genes involved in the biogenesis of lipopolysaccharides (LPS) and on an uncharacterized gene (ASA_1998). The characterization of the LPS profile and gene complementation assays identified LPS as a phage receptor and confirmed the involvement of the uncharacterized protein ASA_1998 in phage infection. In addition, we confirmed that the presence of an A-layer at the bacterial surface could act as protection against phages. This study brings new elements into our understanding of the phage adsorption to A. salmonicida subsp. salmonicida cells.

The determination of antimicrobial susceptibility by MIC and epidemiological cut-off values and the detection of resistance genes in Aeromonas species isolated from cultured fish.

The present study was aimed at determining antimicrobial susceptibility by a CLSI standard microdilution testing protocol and detecting the resistance genes of motile Aeromonas species isolated from cultured fish. The importance of the minimum inhibitory concentrations was assessed based on statistically determined epidemiological cut-off values calculated by normalized resistance analysis. Unfortunately, CLSI epidemiological cut-off values are available only for Aeromonas salmonicida, and there is no further detailed data on Aeromonas isolated from aquatic animals. The antimicrobial susceptibilities of pre-identified motile Aeromonas species to florfenicol, tetracycline and sulfamethoxazole were determined by calculating epidemiological cut-off values with fully automated and freely available Excel spreadsheets, applying the normalized resistance interpretation (NRI) method. Furthermore, the presence of the antimicrobial resistance genes floR, tetA, tetB, tetC, tetD, tetE, tetH, sulI, sulII and sulIII was detected by PCR analysis and confirmed by sequence analysis. The presence of up to six different genes (multiple antimicrobial resistance) was determined in the Aeromonas isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: In this study, we investigated phenotypic and genotypic antimicrobial resistance characteristics by a novel method based on epidemiological cut-off values. This is the second comprehensive study on the antimicrobial susceptibility characteristics of Aeromonas species using NRI and epidemiological cut-off values. The present research is related to our previous researches focussed on the identification of motile Aeromonads, their prevalence in relation to different fish lengths, seasons and regions, and covered the investigation of Lactococcus garvieae, Yersinia ruckeri, Flavobacterium spp., Enterobacter spp. and Citrobacter spp.

4-Hydroxyphenylpyruvate Dioxygenase Thermolability Is Responsible for Temperature-Dependent Melanogenesis in Aeromonas salmonicida subsp. salmonicida.

Aeromonas salmonicida subsp. salmonicida is a major pathogen affecting fisheries worldwide and is a well-known pigmented member of the Aeromonas genus. This subspecies produces melanin at ≤22°C. However, melanogenesis decreases as the culture temperature increases and is completely suppressed at 30°C to 35°C, while bacterial growth is unaffected. The mechanism and biological significance of this temperature-dependent melanogenesis remain unclear. Heterologous expression of an A. salmonicida subsp. salmonicida 4-hydroxyphenylpyruvate dioxygenase (HppD), the most critical enzyme in the homogentisic acid (HGA)-melanin synthesis pathway, results in thermosensitive pigmentation in Escherichia coli, suggesting that HppD plays a key role in this process. In this study, we demonstrated that the thermolability of HppD is responsible for the temperature-dependent melanization of A. salmonicida subsp. salmonicida Substitutions of three residues, S18T, P103Q, and L119P, in A. salmonicida subsp. salmonicida HppD increased the thermostability of this enzyme and resulted in temperature-independent melanogenesis. Moreover, the replacement of the corresponding residues in HppD from Aeromonas media strain WS, which forms pigment independent of temperature, with those of A. salmonicida subsp. salmonicida HppD resulted in thermosensitive melanogenesis. A structural analysis suggested that mutations at these sites, especially at position P103, strengthen the secondary structure of HppD and greatly improve its thermal stability. Additionally, we found that the HppD sequences of all A. salmonicida subsp. salmonicida isolates were identical and that two of the three residues were clearly distinct from those of other Aeromonas strains.IMPORTANCEAeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a bacterial septicemia of cold-water fish of the Salmonidae family. Although other Aeromonas species can produce melanin, A. salmonicida subsp. salmonicida is the only member of this genus that has been reported to exhibit temperature-dependent melanization. Here, we demonstrated that thermosensitive melanogenesis in A. salmonicida subsp. salmonicida strains is due to the thermolability of 4-hydroxyphenylpyruvate dioxygenase (HppD). Additionally, we confirmed that this thermolabile HppD exhibited higher activity at low temperatures than its mesophilic homologues, suggesting this as an adaptive strategy of this enzyme to the psychrophilic lifestyle of A. salmonicida subsp. salmonicida The strictly conserved hppD sequences among A. salmonicida subsp. salmonicida isolates and the specific possession of P103 and L119 residues could be used as a reference for the identification of A. salmonicida subsp. salmonicida isolates.

A recombinant adenovirus targeting typical Aeromonas salmonicida induces an antibody-mediated adaptive immune response after immunization of rainbow trout.

Aeromonas salmonicida, the oldest known fish pathogen and currently endemic throughout most of the world in both fresh and marine waters, causes severe economic losses to the salmon farming industry. Although there have been many studies on the prevention of furunculosis over the past few decades, it is still prevalent in many fisheries. In this study, a recombinant adenovirus vaccine candidate harboring the highly immunogenic Vapa gene (pAd-easy-cmv-Vapa) was successfully constructed and tested. The immune protection rate and specific antibody levels in the peripheral blood were then determined after immunizing rainbow trout. In addition, relative levels of IgM and IgT in the head kidney and hindgut before and after immunization were measured by quantitative reverse transcription PCR. Western blotting results indicated that the recombinant adenovirus could infect HEK-293 cells and express the A layer protein (encoded by Vapa). Further, survival analysis of fish 28 days after challenge showed that immunization significantly lowered the mortality rate (40%) compared to that in the control group (76.6%) and empty vector group (73.6%). This also led to an increase in specific antibodies in peripheral serum. In addition, levels of IgM and IgT in the head kidney and hindgut were increased to varying degrees. In conclusion, our research provides a candidate vaccine for the prevention of Aeromonas salmonicida A450 infection in rainbow trout and lays the foundation for future research on adaptive immune mechanisms associated with rainbow trout antibodies.

[Enzymatic activity of recombinant metallopeptidase for further using in meat industry].

Enzymatic modification of meat with a high content of connective tissue is an effective mean, allowing to improve its properties and expand its use. Microbial enzymes have been extensively investigated as meat tenderizers. Compliance with safety requirements in terms of forecasting the development of various risks is essential for the use of these enzymes in food industry. The method of producing recombinant protease as a potential candidate for applications on meat tenderization was described in the article. The aim of this study was the production of recombinant Pichia pastoris with M9 peptidase gene from Aeromonas salmonicida. Material and methods. Objects: peptidase gene M9 (GenBank: CP000644.1 ASA_3723) Aeromonas salmonicida (strain of laboratory collection, isolated from the surface of raw meat), the vector plasmid pPic9K, competent E. coli DH5α cells, competent Pichia pastoris GS115 cells, culture fluid (QOL) from recombinant Pichia pastoris clones, beef shank samples. To obtain a recombinant strain, genetic engineering methods, the PCR method, and the bacteriological method were used. Polyacrylamide gel electrophoresis was used to separate and analyze the components of the supernatant. Enzyme activity was evaluated by HPLC-MS/MS using synthesized peptides. The impact of the supernatant from recombinant clones on the connective tissue of raw meat was assessed by histological method. Results and discussion. A metalloprotease M9 gene was cloned from the Aeromonas salmonicida (2748 bp) and expressed in Pichia pastoris. The molecular mass of the recombinant protein was estimated to be 120 kDa by SDS-PAGE. Histological analyses of the control and enzyme treated beef samples showed degradation intramuscular connective tissue, suggesting its effectiveness on meat tenderization. Conclusion. The recombinant strain Pichia pastoris, which produces the recombinant M9 peptide of Aeromonas salmonicida, has a specific enzymatic activity against collagen, the main component of the connective tissue of meat. The obtained recombinant peptidase M9 can be used as an enzyme softener of raw meat with a high content of connective tissue.

The complete and fully assembled genome sequence of Aeromonas salmonicida subsp. pectinolytica and its comparative analysis with other Aeromonas species: investigation of the mobilome in environmental and pathogenic strains.

BACKGROUND: Due to the predominant usage of short-read sequencing to date, most bacterial genome sequences reported in the last years remain at the draft level. This precludes certain types of analyses, such as the in-depth analysis of genome plasticity. RESULTS: Here we report the finalized genome sequence of the environmental strain Aeromonas salmonicida subsp. pectinolytica 34mel, for which only a draft genome with 253 contigs is currently available. Successful completion of the transposon-rich genome critically depended on the PacBio long read sequencing technology. Using finalized genome sequences of A. salmonicida subsp. pectinolytica and other Aeromonads, we report the detailed analysis of the transposon composition of these bacterial species. Mobilome evolution is exemplified by a complex transposon, which has shifted from pathogenicity-related to environmental-related gene content in A. salmonicida subsp. pectinolytica 34mel. CONCLUSION: Obtaining the complete, circular genome of A. salmonicida subsp. pectinolytica allowed us to perform an in-depth analysis of its mobilome. We demonstrate the mobilome-dependent evolution of this strain's genetic profile from pathogenic to environmental.

Quorum sensing asaI mutants affect spoilage phenotypes, motility, and biofilm formation in a marine fish isolate of Aeromonas salmonicida.

Microbial spoilage is associated with the regulation of quorum sensing (QS). A. salmonicida AE03 with QS mediated acylated homoserine lactones (AHLs) activity was isolated from spoiled large yellow croaker (Pseudosciaena crocea). In this study the activity and role of AHLs in spoilage phenotypes, motility and biofilm formation of AE03 were investigated. The strain AE03 could induce Chromobacterium violaceum CV026 to produce the violacein pigment both at 28 °C and 4 °C in a density-dependent manner. Five types of AHLs were detected in AE03 culture by LC-MS/MS analysis, and N-butanoyl-l-homoserine lactone (C4-HSL) was a major signal molecule, reaching the highest concentration when incubated for 30 h at 28 °C. An asaI-mutant, constructed by a suicide plasmid, failed to produce short chain AHLs signal. Compared with wild type (WT) strain, the production of trimethylamine (TMA), biogenic amino and protease significantly increased in asaI-mutant during the exponential and stationary phase, while the growth rate did not differ. Swimming motility in asaI-mutant was comparatively stronger than that of WT strain, whereas, asaI-mutant resulted in the decrease of maturing biofilm. Furthermore, supplementation of exogenous C4-HSL restored the production of spoilage metablites, protease and biofilm formation in mutant. In accordance with the effect of asaI deletion on the spoilage phenotypes and motility, asaI-mutant was showed to significantly up-regulate the transcript levels of torA, cadA and fliR, as well as asaR, indicating that C4-HSL could be involved in the modulation of the spoilage related enzymes and flagella. Indeed, the asaI-mutant promoted the spoilage progress of fish fillets stored at 4 °C, while exogenous C4-HSL repressed the sensory change and TVB-N accumulation. The present study highlighted that AsaI/C4-HSL was an important regulator in spoilage, motility and biofilm formation of A. salmonicida, and spoilage potential was under the negative control of AsaI/AsaR-type system.