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Frontiers in Microbiology 2019The RstB histidine kinase of the two component system RstAB positively regulates the expression of damselysin (Dly), phobalysin P (PhlyP) and phobalysin C (PhlyC)...
The RstAB System Impacts Virulence, Motility, Cell Morphology, Penicillin Tolerance and Production of Type II Secretion System-Dependent Factors in the Fish and Human Pathogen subsp. .
The RstB histidine kinase of the two component system RstAB positively regulates the expression of damselysin (Dly), phobalysin P (PhlyP) and phobalysin C (PhlyC) cytotoxins in the fish and human pathogen subsp. , a marine bacterium of the family . However, the function of the predicted cognate response regulator RstA has not been studied so far, and the role of the RstAB system in other cell functions and phenotypes remain uninvestigated. Here, we analyzed the effect of and mutations in cell fitness and in diverse virulence-related features. Both and mutants were severely impaired in virulence for sea bream and sea bass fish. Mutants in and genes were impaired in hemolysis and in Dly-dependent phospholipase activity but had intact PlpV-dependent phospholipase and ColP-dependent gelatinase activities. and mutants grown at 0.5% NaCl exhibited impaired swimming motility, enlarged cell size and impaired ability to separate after cell division, whereas at 1% NaCl the mutants exhibited normal phenotypes. Mutation of any of the two genes also impacted tolerance to benzylpenicillin. Notably, and mutants showed impaired secretion of a number of type II secretion system (T2SS)-dependent proteins, which included the three major cytotoxins Dly, PhlyP and PhlyC, as well as a putative delta-endotoxin and three additional uncharacterized proteins which might constitute novel virulence factors of this pathogenic bacterium. The analysis of the T2SS-dependent secretome of subsp. also led to the identification of RstAB-independent potential virulence factors as lipoproteins, sialidases and proteases. The RstAB regulon included plasmid, chromosome I and chromosome II-encoded genes that showed a differential distribution among isolates of this subspecies. This study establishes RstAB as a major regulator of virulence and diverse cellular functions in subsp. .
PubMed: 31105680
DOI: 10.3389/fmicb.2019.00897 -
Cryo Letters 2023Chitin is the second largest carbon source on the earth, and chitosan oligosaccharides produced by its degradation have good application prospects in medicine,...
BACKGROUND
Chitin is the second largest carbon source on the earth, and chitosan oligosaccharides produced by its degradation have good application prospects in medicine, cosmetics, and agricultural production.
OBJECTIVE
The discovery of a chitinase with high efficiency, high stability and clear degradation mechanism is of great help to promote the research of chitin derivatives and the development of the industrial chain.
MATERIALS AND METHODS
In this experiment, a low-temperature chitinase-producing strain Photobacterium sp. LG-29 was isolated from deep-sea mud in the Bohai Sea, and studied by means of molecular biology, biochemistry and bioinformatics.
RESULTS
Purification of chitinase yielded an enzyme solution with a concentration of 0.918 mg per mL and a specific activity of 21.036 U per mg. The optimum action temperature is 35 degree C, and it is still active at 4 degree C, showing low-temperature enzymatic activity, and also has certain thermal stability. The optimum pH is 8.0, and it maintains more than 70% of the enzyme activity at pH 11, which is very stable in an alkaline environment. Mn, Ca, and Mg are the main activators of enzymes, while Fe, Zn, etc. have extremely significant inhibitory effects on enzymes. The Km and Kcat of chitinase were determined to be 269.05 μmol/L and 0.49 min, respectively. Chitinase PbCHI5 has both endonuclease and exonuclease activity. The theoretical pI of the enzyme is 4.16, which is a stable hydrophilic protein.
CONCLUSION
This experiment laid a theoretical foundation for the development and utilization of new low-temperature chitinases. Doi.org/10.54680/fr23510110212.
Topics: Chitinases; Temperature; Photobacterium; Cryopreservation; Chitin; Cloning, Molecular
PubMed: 38032308
DOI: No ID Found -
MicrobiologyOpen Mar 2021Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics that can be manufactured sustainably and represent a promising green alternative to petrochemical-based...
Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics that can be manufactured sustainably and represent a promising green alternative to petrochemical-based plastics. Here, we describe the complete genome of a new marine PHA-producing bacterium-Photobacterium ganghwense (strain C2.2), which we have isolated from the Black Sea seashore. This new isolate is psychrotolerant and accumulates PHA when glycerol is provided as the main carbon source. Transmission electron microscopy, specific staining with Nile Red visualized via epifluorescence microscopy and gas chromatography analysis confirmed the accumulation of PHA. This is the only PHA-producing Photobacterium for which we now have a complete genome sequence, allowing us to investigate the pathways for PHA production and other secondary metabolite synthesis pathways. The de novo assembly genome, obtained using open-source tools, comprises two chromosomes (3.5, 2 Mbp) and a megaplasmid (202 kbp). We identify the entire PHA synthesis gene cluster that encodes a class I PHA synthase, a phasin, a 3-ketothiolase, and an acetoacetyl-CoA reductase. No conventional PHA depolymerase was identified in strain C2.2, but a putative lipase with extracellular amorphous PHA depolymerase activity was annotated, suggesting that C2.2 is unable to degrade intracellular PHA. A complete pathway for the conversion of glycerol to acetyl-CoA was annotated, in accordance with its ability to convert glycerol to PHA. Several secondary metabolite biosynthetic gene clusters and a low number of genes involved in antibiotic resistance and virulence were also identified, indicating the strain's suitability for biotechnological applications.
Topics: Acetyl Coenzyme A; Acetyl-CoA C-Acyltransferase; Acyltransferases; Alcohol Oxidoreductases; Aquatic Organisms; Biosynthetic Pathways; Drug Resistance, Bacterial; Genome, Bacterial; Glycerol; Photobacterium; Plant Lectins; Plasmids; Polyhydroxyalkanoates; Soil Microbiology; Virulence; Whole Genome Sequencing
PubMed: 33970538
DOI: 10.1002/mbo3.1182 -
International Journal of Systematic and... Dec 2019A Gram-stain-negative, aerobic, motile, rod-shaped bacterium, designated strain LAM9072, was isolated from a sample of a sulfonylurea herbicide-degrading consortium...
A Gram-stain-negative, aerobic, motile, rod-shaped bacterium, designated strain LAM9072, was isolated from a sample of a sulfonylurea herbicide-degrading consortium enriched with saline soil. The optimal temperature and pH for the growth of strain LAM9072 were 35 °C and 7.0, respectively. Strain LAM9072 could grow in the presence of NaCl up to 9 % (w/v). Comparative analysis of the 16S rRNA gene sequences revealed that strain LAM9072 was closely related to members of the family , with the highest similarities to MACL01 (97.7 %) and S2753 (97.7 %). Strain LAM9072 formed a distinct phylogenetic subclade within the genus in the 16S rRNA gene phylogenetic trees. The results of multi-locus sequence analysis revealed a distinct lineage with MACL01 as its closest relative. The genomic G+C content was 50.2 mol%. The DNA-DNA hybridization values between strain LAM9072 and LMG 22194 and LMG 28894 were 41.6 and 22.2 %, respectively. The average nucleotide identity values were 90.9 and 78.8 %, respectively, by comparing the draft genome sequences of strain LAM9072 and LMG 22194 and LMG 28894. The major fatty acids were summed feature 3 (Cω6 and/or Cω7), C and summed feature 8 (Cω7 and/or Cω6). Ubiquinone 8 was detected as the predominant respiratory quinone. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, aminophospholipid and four unidentified lipids. Based on its phenotypic characteristics and the results of genotypic analyses, we propose that strain LAM9072 represents a novel species, for which the name sp. nov. is proposed. The type strain is LAM9072 (=ACCC 19961=JCM 30852).
Topics: Bacterial Typing Techniques; Base Composition; Biodegradation, Environmental; China; DNA, Bacterial; Fatty Acids; Herbicides; Nucleic Acid Hybridization; Phospholipids; Photobacterium; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Soil Microbiology; Sulfonylurea Compounds; Ubiquinone
PubMed: 31693472
DOI: 10.1099/ijsem.0.003705 -
Angewandte Chemie (International Ed. in... Dec 2021Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients. The in-vivo activity of EPO is...
Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients. The in-vivo activity of EPO is carbohydrate-dependent with the number of sialic acid residues regulating its circulatory half-life. EPO carries three N-glycans and thus obtaining pure glycoforms provides a major challenge. We have developed a robust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids. EPO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments. The glycopeptides were obtained by pseudoproline-assisted Lansbury aspartylation. Enzymatic introduction of the sialic acids was readily accomplished at the level of the glycopeptide segments but even more efficiently on the refolded glycoprotein. Biological recognition of the synthetic EPOs was shown by formation of 1:1 complexes with recombinant EPO receptor.
Topics: Erythropoietin; Glycosylation; Humans; Molecular Structure; N-Acetylneuraminic Acid; Photobacterium; Sialyltransferases; beta-D-Galactoside alpha 2-6-Sialyltransferase
PubMed: 34523784
DOI: 10.1002/anie.202110013 -
International Journal of Food... Nov 2016The processing environment of salmon processing plants represents a potential major source of bacteria causing spoilage of fresh salmon. In this study, we have...
The processing environment of salmon processing plants represents a potential major source of bacteria causing spoilage of fresh salmon. In this study, we have identified major contamination routes of important spoilage associated species within the genera Pseudomonas, Shewanella and Photobacterium in pre-rigor processing of salmon. Bacterial counts and culture-independent 16S rRNA gene analysis on salmon fillet from seven processing plants showed higher levels of Pseudomonas spp. and Shewanella spp. in industrially processed fillets compared to salmon processed under strict hygienic conditions. Higher levels of Pseudomonas spp. and Shewanella spp. were found on fillets produced early on the production day compared to later processed fillets. The levels of Photobacterium spp. were not dependent on the processing method or time of processing. In follow-up studies of two plants, bacterial isolates (n=2101) from the in-plant processing environments (sanitized equipment/machines and seawater) and from salmon collected at different sites in the production were identified by partial 16S rRNA gene sequencing. Pseudomonas spp. dominated in equipment/machines after sanitation with 72 and 91% of samples from the two plants being Pseudomonas-positive. The phylogenetic analyses, based on partial 16S rRNA gene sequencing, showed 48 unique sequence profiles of Pseudomonas of which two were dominant. Only six profiles were found on both machines and in fillets in both plants. Shewanella spp. were found on machines after sanitation in the slaughter department while Photobacterium spp. were not detected after sanitation in any parts of the plants. Shewanella spp. and Photobacterium spp. were found on salmon in the slaughter departments. Shewanella was frequently present in seawater tanks used for bleeding/short term storage. In conclusion, this study provides new knowledge on the processing environment as a source of contamination of salmon fillets with Pseudomonas spp. and Shewanella spp., while Photobacterium spp. most likely originate from the live fish and seawater. The study show that strict hygiene during processing is a prerequisite for optimal shelf life of salmon fillets and that about 90% reductions in the initial levels of bacteria on salmon fillets can be obtained using optimal hygienic conditions.
Topics: Animals; Bacterial Load; Fish Products; Food Contamination; Food Handling; Food Industry; Food Microbiology; Food Preservation; Norway; Photobacterium; Phylogeny; Pseudomonas; RNA, Ribosomal, 16S; Salmon; Seawater; Shewanella
PubMed: 27552347
DOI: 10.1016/j.ijfoodmicro.2016.08.016 -
Frontiers in Microbiology 2021Chondroitin sulfate (CS)/dermatan sulfate (DS) is a kind of sulfated polyanionic, linear polysaccharide belonging to glycosaminoglycan. CS/DS sulfatases, which...
Chondroitin sulfate (CS)/dermatan sulfate (DS) is a kind of sulfated polyanionic, linear polysaccharide belonging to glycosaminoglycan. CS/DS sulfatases, which specifically hydrolyze sulfate groups from CS/DS oligo-/polysaccharides, are potential tools for structural and functional studies of CD/DS. However, only a few sulfatases have been reported and characterized in detail to date. In this study, two CS/DS sulfatases, PB_3262 and PB_3285, were identified from the marine bacterium sp. QA16 and their action patterns were studied in detail. PB_3262 was characterized as a novel 4--endosulfatase that can effectively and specifically hydrolyze the 4--sulfate group of disaccharide GlcUAβ1-3GalNAc(4--sulfate) but not GlcUAβ1-3GalNAc(4,6--sulfate) and IdoUAα1-3GalNAc(4--sulfate) in CS/DS oligo-/polysaccharides, which is very different from the identified 4--endosulfatases in the substrate profile. In contrast, PB_3285 specifically hydrolyzes the 6--sulfate groups of GalNAc(6--sulfate) residues located at the reducing ends of the CS chains and is the first recombinantly expressed 6--exosulfatase to effectively act on CS oligosaccharides.
PubMed: 35140691
DOI: 10.3389/fmicb.2021.775124 -
Journal of Fish Diseases Jul 2020Photobacteriosis, caused by Photobacterium damselae subsp. piscicida (Phdp), is a serious disease in marine fish species worldwide. To date, the epidemiological...
Photobacteriosis, caused by Photobacterium damselae subsp. piscicida (Phdp), is a serious disease in marine fish species worldwide. To date, the epidemiological characterization of this pathogen in Taiwan remains limited. In this study, we collected 39 Phdp isolates obtained from different farmed fish for phenotypic and genotypic analysis. Phenotype bioassays using API-20E and API-20NE systems showed that the Phdp is a homogeneous group. However, genotyping using the pulsed-field gel electrophoresis (PFGE) technique revealed genetic variability among Phdp isolates when 13 and 11 different PFGE band patterns were obtained with SmaI and NotI as restriction enzymes, respectively. Phylogenetic analysis using 16S rDNA and the Fur gene clustered Taiwanese isolates and other species of P. damselae in the same clade. In contrast, the ToxR phylogenetic tree, a powerful discriminatory marker, separated the two subspecies. Furthermore, the virulence-associated genes, AIP56, P55, PDP_0080, Sod and Irp1, were detected from all isolates. Virulence testing with nine representative isolates in cobia (Rachycentron canadum) and Asian sea bass (Lates calcarifer) showed that some were highly pathogenic with 80%-100% mortality rates. This study provides epidemiological data of Phdp infections in farmed fish in Taiwan, which is necessary to develop comprehensive prevention and control strategies for the disease.
Topics: Animals; Fish Diseases; Fishes; Genetic Variation; Genotype; Gram-Negative Bacterial Infections; Phenotype; Photobacterium; Phylogeny; Taiwan; Virulence
PubMed: 32419196
DOI: 10.1111/jfd.13173 -
Fish & Shellfish Immunology Apr 2021Photobacterium damselae subsp. piscicida (Phdp) is the causative agent of photobacteriosis in marine fish and is responsible for huge losses to marine aquaculture...
Photobacterium damselae subsp. piscicida (Phdp) is the causative agent of photobacteriosis in marine fish and is responsible for huge losses to marine aquaculture worldwide. Efforts have been made to develop a vaccine against this disease. Heat-shock proteins (HSPs) are a family of proteins that are ubiquitous in cellular life. Bacteria produce elevated levels of HSPs as a survival strategy when exposed to stressful environments in a host during infection. This group of proteins are also important antigens that can induce both humoral and cellular immune responses. In this study, four HSPs of Phdp, HSP90, HSP33, HSP70, and DnaJ, were selected for cloning and recombinant expression. Western blotting with rabbit anti-Phdp helped identify rHSP70 and rHSP33 as immunogenic proteins. Asian seabass (Lates calcarifer) immunised with rHSP90, rHSP33, rHSP70, and rDnaJ showed 48.28%, 62.07%, 51.72%, and 31.03% relative percent survival, respectively, after being challenged with Phdp strain AOD105021. High expression levels of immune-related genes and high antibody titres were observed in the rHSP33 group, and the sera of this group also exhibited a high level of bactericidal activity against Phdp. Collectively, our results suggest that HSP33 is a potential candidate for vaccine development against Phdp infection.
Topics: Animals; Bacterial Vaccines; Bass; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Heat-Shock Proteins; Photobacterium; Vaccines, Synthetic
PubMed: 33556554
DOI: 10.1016/j.fsi.2021.02.002 -
Foods (Basel, Switzerland) Jul 2021Improved quality control and prolonged shelf life are important actions in preventing food waste. To get an overview of the bacterial diversity of fillets from live...
Improved quality control and prolonged shelf life are important actions in preventing food waste. To get an overview of the bacterial diversity of fillets from live stored mature Atlantic cod, bacterial isolates were identified before and after storage (air and vacuum) and freezing/thawing. Based on the load of dominating bacteria, the effect of different packaging methods and a short freezing/thawing process on prolonged shelf-life was evaluated (total viable counts, bacteriota, sensory attributes, and volatile components). Hand filleted (strict hygiene) cod fillets had a low initial bacterial load dominated by the spoilage organism , whereas industrially produced fillets had higher bacterial loads and diversity (, , , ). The identified bacteria after storage in vacuum or air were similar to the initially identified bacteria. Bacteriota analysis showed that a short time freezing/thawing process reduced while modified atmosphere packaging (MAP; 60% CO/40% O or 60% CO/40% N) inhibited the growth of important spoilage bacteria () and allowed the growth of / and Despite being dominated by fresh fillets stored in MAP 60% CO/40% N demonstrated better sensory quality after 13 days of storage than fillets stored in MAP 60% CO/40% O (dominated by ). spp. or other members of may therefore be potential spoilage organisms in cod when other spoilage bacteria are reduced or inhibited.
PubMed: 34441531
DOI: 10.3390/foods10081754