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Chemistry (Weinheim An Der Bergstrasse,... Sep 2020Defined sialoglycoconjugates are important molecular probes for studying the role of sialylated glycans in biological systems. We show that the α2,3-sialyltransferase...
Defined sialoglycoconjugates are important molecular probes for studying the role of sialylated glycans in biological systems. We show that the α2,3-sialyltransferase from Photobacterium phosphoreum JT-ISH-467 (2,3SiaT ) tolerates a very broad substrate scope for modifications in the sialic acid part, including bulky amide variation, C5/C9 substitution, and C5 stereoinversion. To reduce the enzyme's hydrolytic activity, which erodes the product yield, an extensive structure-guided mutagenesis study identified three variants that show up to five times higher catalytic efficiency for sialyltransfer, up to ten times lower efficiency for substrate hydrolysis, and drastically reduced product hydrolysis. Variant 2,3SiaT (A151D) displayed the best performance overall in the synthesis of the GM3 trisaccharide (α2,3-Neu5Ac-Lac) from lactose in a one-pot, two-enzyme cascade. Our study demonstrates that several complementary solutions can be found to suppress the common problem of undesired hydrolysis activity of microbial GT80 sialyltransferases. The new enzymes are powerful catalysts for the synthesis of a wide variety of complex natural and new-to-nature sialoconjugates for biological studies.
Topics: Hydrolysis; N-Acetylneuraminic Acid; Photobacterium; Sialyltransferases; Substrate Specificity
PubMed: 32596832
DOI: 10.1002/chem.202002277 -
Journal of Bacteriology Nov 1949
Topics: Aliivibrio fischeri; Bacteria; Photobacterium; Purines
PubMed: 15393696
DOI: 10.1128/jb.58.5.653-657.1949 -
Current Microbiology Nov 2014A facultative anaerobe, alkalitolerant, gram-negative marine bacterium strain LBS5(T), was isolated from eggs carried on the pleopods of female spiny lobster (Panulirus...
A facultative anaerobe, alkalitolerant, gram-negative marine bacterium strain LBS5(T), was isolated from eggs carried on the pleopods of female spiny lobster (Panulirus penicillatus) in Andaman Sea from a depth of 3.5 m. Heterotrophic growth was observed at 15-38 °C and pH 5.5-11. Optimum growth occurred at 28 °C and pH 7.5. It can grow in the presence of 0.5-7 % NaCl (w/v), and the optimal NaCl required for growth was 2-4 %. 16S rRNA gene sequence analysis revealed the strain LBS5(T) belongs to the genus Photobacterium and showed 99.6 % similarity with P. aquae AE6(T), 98.2 % with P. aphoticum M46(T), 97 % with P. rosenbergii CC1(T), 96.9 % with P. lutimaris DF-42(T), and 96.6 % with P. halotolerans MACL01(T). The DNA-DNA similarities between strains LBS5(T) with other closely related strains were well below 70 %. The DNA G + C content was 50.52 (±0.9) mol%. The major fatty acids were C16:1w7c/w6c, C18:1w6c/w7c, C16:0, C15:0 iso, C16:0 10-methyl/17:1 iso w9c, C17:0 iso. Polar lipids included a phosphatidylglycerol, a diphosphatidylglycerol, a phosphatidylethanolamine, and one unidentified lipid. Based on the polyphasic evidences, strain LBS5(T) represents a novel species of the genus Photobacterium for which Photobacterium panuliri sp. nov. is proposed. The type strain is LBS5(T) (=DSM 27646(T) = LMG 27617(T) = JCM 19199(T)).
Topics: Animals; Bacterial Typing Techniques; Base Composition; Cluster Analysis; Cytosol; DNA, Bacterial; DNA, Ribosomal; Fatty Acids; Hydrogen-Ion Concentration; Molecular Sequence Data; Nucleic Acid Hybridization; Palinuridae; Phospholipids; Photobacterium; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Sodium Chloride; Temperature; Zygote
PubMed: 24962598
DOI: 10.1007/s00284-014-0638-0 -
MicrobiologyOpen Aug 2023Gene inactivation studies are critical in pathogenic bacteria, where insights into species biology can guide the development of vaccines and treatments. Allelic exchange...
Transforming the untransformable with knockout minicircles: High-efficiency transformation and vector-free allelic exchange knockout in the fish pathogen Photobacterium damselae.
Gene inactivation studies are critical in pathogenic bacteria, where insights into species biology can guide the development of vaccines and treatments. Allelic exchange via homologous recombination is a generic method of targeted gene editing in bacteria. However, generally applicable protocols are lacking, and suboptimal approaches are often used for nonstandard but epidemiologically important species. Photobacterium damselae subsp. piscicida (Pdp) is a primary pathogen of fish in aquaculture and has been considered hard to transform since the mid-1990s. Consequently, conjugative transfer of RK2/RP4 suicide vectors from Escherichia coli S17-1/SM10 donor strains, a system prone to off-target mutagenesis, was used to deliver the allelic exchange DNA in previous studies. Here we have achieved efficient electrotransformation in Pdp using a salt-free highly concentrated sucrose solution, which performs as a hypertonic wash buffer, cryoprotectant, and electroporation buffer. High-efficiency transformation has enabled vector-free mutagenesis for which we have employed circular minimalistic constructs (knockout minicircles) containing only allelic exchange essentials that were generated by Gibson assembly. Preparation of competent cells using sucrose and electroporation/integration of minicircles had virtually no detectable off-target promutagenic effect. In contrast, a downstream sacB selection apparently induced several large deletions via mobilization of transposable elements. Electroporation of minicircles into sucrose-treated cells is a versatile broadly applicable approach that may facilitate allelic exchange in a wide range of microbial species. The method permitted inactivation of a primary virulence factor unique to Pdp, apoptogenic toxin AIP56, demonstrating the efficacy of minicircles for difficult KO targets located on the high copy number of small plasmids.
Topics: Animals; Photobacterium; Electroporation; Fishes
PubMed: 37642481
DOI: 10.1002/mbo3.1374 -
Microbial Genomics Apr 2021Despite the recent advances in sequencing technologies, the complete assembly of multi-chromosome genomes of the , often containing several plasmids, remains...
Despite the recent advances in sequencing technologies, the complete assembly of multi-chromosome genomes of the , often containing several plasmids, remains challenging. Using a combination of Oxford Nanopore MinION long reads and short Illumina reads, we fully sequenced, closed and curated the genomes of two strains of a primary aquatic pathogen subsp. isolated in Australia. These are also the first genome sequences of subsp. isolated in Oceania and, to our knowledge, in the Southern hemisphere. We also investigated the phylogenetic relationships between Australian and overseas isolates, revealing that Australian subsp. are more closely related to the Asian and American strains rather than to the European ones. We investigated the mobilome and present new evidence showing that a host specialization process and progressive adaptive evolution to fish are ongoing in subsp. , and are largely mediated by transposable elements, predominantly in chromosome 2, and by plasmids. Finally, we identified two novel potential virulence determinants in subsp. - a chorismate mutase gene, which is ubiquitously retained and co-localized with the AIP56 apoptogenic toxin-encoding gene on the pPHDP10 plasmid, and transfer-messenger RNA gene located on the main chromosome, homologous to a critical-to-virulence determinant in . Our study describes, to our knowledge, the only fully closed and manually curated genomes of subsp. available to date, offering new insights into this important fish pathogen and its evolution.
Topics: Bacterial Proteins; Chromosome Mapping; DNA Transposable Elements; Evolution, Molecular; Genome, Bacterial; Photobacterium; Phylogeny; Virulence Factors
PubMed: 33885359
DOI: 10.1099/mgen.0.000562 -
Journal of Fish Diseases Dec 2020Mass mortality has occurred among cultured Nile tilapia, Oreochromis niloticus, on fish farms in Manzala, Dakahlia province, Egypt, in the summer season, 2019. Moribund...
Mass mortality has occurred among cultured Nile tilapia, Oreochromis niloticus, on fish farms in Manzala, Dakahlia province, Egypt, in the summer season, 2019. Moribund fish were reported with deep ulcers, septicaemic lesions and sampled for bacterial isolation. In this study, most isolates were subjected to bacteriological examination, antibiotic sensitivity test, 16S rRNA gene sequencing and histopathological examination. Following isolate identification, intraperitoneal challenge of Nile tilapia with a bacterial suspension 2 × 10 CFU/ml was performed. Samples from liver, spleen and kidney were collected for histological and biochemical analysis. The results showed a high similarity (99%) to Photobacterium damselae strains using phylogenetic analysis of 16S rRNA. P. damselae exhibited resistance to amoxicillin and erythromycin, as well it was highly sensitive to chloramphenicol and doxycycline. Moreover, haemorrhage, oedema, hemosiderosis and melanomacrophage activation in the liver and head kidney of infected fish were detected by light and electron microscopy. Also, significant higher levels of CAT and SOD in the spleen and head kidney, as well as the serum levels of NO were observed in experimentally challenged O. niloticus, compared to the control fish. Our data identified P. damselae for the first time from infected Nile tilapia, describing its sensitivity to a variety of antibiotics, histopathological alterations and oxidative stress impact, and it could be useful indicators for understanding P. damselae pathogenesis, which might provide a preventive efficacy for P. damselae.
Topics: Animals; Aquaculture; Cichlids; Drug Resistance, Bacterial; Egypt; Fish Diseases; Gram-Negative Bacterial Infections; Microbial Sensitivity Tests; Photobacterium; RNA, Ribosomal, 16S
PubMed: 32984991
DOI: 10.1111/jfd.13251 -
Environmental Microbiology Sep 2021The marine bacterium Photobacterium damselae subsp. damselae (Pdd) causes disease in marine animals and humans. Previous studies demonstrated that mutation of the...
The marine bacterium Photobacterium damselae subsp. damselae (Pdd) causes disease in marine animals and humans. Previous studies demonstrated that mutation of the two-component system RstAB strongly impacts virulence of this pathogen, but the RstAB regulon has not been thoroughly elucidated. We here compared the transcriptomes of Pdd RM-71 and ΔrstA and ΔrstB derivatives using RNA-seq. In accordance with previous studies, RstAB positively regulated cytotoxins Dly, PhlyP and PhlyC. This analysis also demonstrated a positive regulation of outer membrane proteins, resistance against antimicrobials and potential virulence factors by this system. Remarkably, RstAB positively regulated two hitherto uncharacterised gene clusters involved in the synthesis of a polysaccharide capsule. Presence of a capsular layer in wild-type cells was confirmed by transmission electron microscopy, whereas rstA and rstB mutants were non-capsulated. Mutants for capsule synthesis genes, wza and wzc exhibited acapsular phenotypes, were impaired in resistance against the bactericidal action of fish serum and mucus, and were strongly impaired in virulence for fish, indicating a major role of capsule in virulence. Collectively, this study demonstrates that RstAB is a major positive regulator of key virulence factors including a polysaccharide capsule essential for full virulence in a pathogenic Photobacterium.
Topics: Animals; Fish Diseases; Humans; Photobacterium; Polysaccharides; Virulence
PubMed: 34423883
DOI: 10.1111/1462-2920.15731 -
Infection and Immunity Nov 2015Photobacterium damselae subsp. damselae, an important pathogen of marine animals, may also cause septicemia or hyperaggressive necrotizing fasciitis in humans. We...
Photobacterium damselae subsp. damselae, an important pathogen of marine animals, may also cause septicemia or hyperaggressive necrotizing fasciitis in humans. We previously showed that hemolysin genes are critical for virulence of this organism in mice and fish. In the present study, we characterized the hlyA gene product, a putative small β-pore-forming toxin, and termed it phobalysin P (PhlyP), for "photobacterial lysin encoded on a plasmid." PhlyP formed stable oligomers and small membrane pores, causing efflux of K(+), with no significant leakage of lactate dehydrogenase but entry of vital dyes. The latter feature distinguished PhlyP from the related Vibrio cholerae cytolysin. Attack by PhlyP provoked a loss of cellular ATP, attenuated translation, and caused profound morphological changes in epithelial cells. In coculture experiments with epithelial cells, Photobacterium damselae subsp. damselae led to rapid hemolysin-dependent membrane permeabilization. Unexpectedly, hemolysins also promoted the association of P. damselae subsp. damselae with epithelial cells. The collective observations of this study suggest that membrane-damaging toxins commonly enhance bacterial adherence.
Topics: Amino Acid Sequence; Animals; Bacterial Adhesion; Bacterial Toxins; Epithelial Cells; Erythrocytes; Hemolysin Proteins; Hemolysis; Humans; Molecular Sequence Data; Photobacterium; Rabbits; Sequence Alignment
PubMed: 26303391
DOI: 10.1128/IAI.00277-15 -
MSystems Jun 2023Facultative marine bacterial pathogens sense environmental signals so that the expression of virulence factors is upregulated on entry into hosts and downregulated...
Facultative marine bacterial pathogens sense environmental signals so that the expression of virulence factors is upregulated on entry into hosts and downregulated during the free-living lifestyle in the environment. In this study, we utilized transcriptome sequencing to compare the transcriptional profiles of subsp. , a generalist pathogen that causes disease in diverse marine animals and fatal infections in humans at NaCl concentrations that mimic the free-living lifestyle or host internal milieu, respectively. We here show that NaCl concentration constitutes a major regulatory signal that shapes the transcriptome and uncover 1,808 differentially expressed genes (888 upregulated and 920 downregulated in response to low-salt conditions). Growth at 3% NaCl, a salinity that mimics the free-living lifestyle, upregulated genes involved in energy production, nitrogen metabolism, transport of compatible solutes, utilization of trehalose and fructose, and carbohydrate and amino acid metabolism with strong upregulation of the arginine deiminase system (ADS). In addition, we observed a marked increase in resistance to antibiotics at 3% NaCl. On the contrary, the low salinity conditions (1% NaCl) that mimic those encountered in the host triggered a virulence gene expression profile that maximized the production of the type 2 secretion system (T2SS)-dependent cytotoxins damselysin, phobalysin P, and a putative PirAB-like toxin, observations that were corroborated by the analysis of the secretome. Low salinity also upregulated the expression of iron-acquisition systems, efflux pumps, and other functions related to stress response and virulence. The results of this study greatly expand our knowledge of the salinity-responsive adaptations of a generalist and versatile marine pathogen. IMPORTANCE Pathogenic species experience continuous shifts of NaCl concentration in their life cycles. However, the impact of salinity changes in gene regulation has been studied in a small number of species. In this study, we analyzed the transcriptional response of subsp. (), a generalist and facultative pathogen, to changes in salinity, and demonstrate that growth at 1% NaCl in comparison to 3% NaCl triggers a virulence program of gene expression, with a major impact in the T2SS-dependent secretome. The decrease in NaCl concentration encountered by bacteria on entry into a host is proposed to constitute a regulatory signal that upregulates a genetic program involved in host invasion and tissue damage, nutrient scavenging (notably iron), and stress responses. This study will surely inspire new research on pathobiology, as well as on other important pathogens of the family and related taxa whose salinity regulons still await investigation.
Topics: Humans; Animals; Virulence; Sodium Chloride; Salinity; Photobacterium; Iron
PubMed: 37288979
DOI: 10.1128/msystems.01253-22 -
Southern Medical Journal Apr 2009Photobacterium damsela (P damsela) is a common marine bacterium. Rare reports of P damsela causing septicemia and wound infection have been described in children. We...
Photobacterium damsela (P damsela) is a common marine bacterium. Rare reports of P damsela causing septicemia and wound infection have been described in children. We report a case of Photobacterium damsela isolated from a wound culture taken from a 14-year-old male injured after a minor trauma while surfing. We alert physicians to this infection and the importance of early diagnosis and appropriate antimicrobial therapy for wound infection following seawater injury.
Topics: Adolescent; Anti-Infective Agents; Combined Modality Therapy; Debridement; Diagnosis, Differential; Humans; Male; Photobacterium; Therapeutic Irrigation; Vibrio Infections
PubMed: 19279534
DOI: 10.1097/SMJ.0b013e31819b9491