(1/769) Small subunit rRNA gene sequences of Aeromonas salmonicida subsp. smithia and Haemophilus piscium reveal pronounced similarities with A. salmonicida subsp. salmonicida.

The small subunit ribosomal RNA (SSU rRNA) encoding genes from reference strains of Aeromonas salmonicida subsp. smithia and Haemophilus piscium were amplified by polymerase chain reaction and cloned into Escherichia coli cells. Almost the entire SSU rRNA gene sequence (1505 nucleotides) from both organisms was determined. These DNA sequences were compared with those previously described from A. salmonicida subsp. salmonicida, subsp. achromogenes and subsp. masoucida. This genetic analysis revealed that A. salmonicida subsp. smithia and H. piscium showed 99.4 and 99.6% SSU rRNA gene sequence identity, respectively, with A. salmonicida subsp. salmonicida.  (+info)

(2/769) Purification and characterization of cold-active L-glutamate dehydrogenase independent of NAD(P) and oxygen.

L-Glutamate dehydrogenase (GLDH) independent of NAD(P) and oxygen was first obtained from the psychrotrophic bacterium Aeromonas sp. L101, originally isolated from the organs of salmon (Oncorhynchus keta). GLDH was purified by a series of chromatography steps on DEAE-Sepharose, Superdex 200pg, Q-Sepharose, CM-Sepharose, and Phenyl-Sepharose. The purified protein was determined to have a molecular mass of 110 kDa and a pI of 5.7. Maximum activity was obtained at 55 degrees C and pH 8.5. The activity of GLDH at 4 and 20 degrees C was 38 and 50%, respectively, of that at 50 degrees C. GLDH was coupled to cytochrome c and several redox dyes including 1-methoxy-5-methylphenazinium methylsulfate (1-Methoxy PMS), 2, 6-dichlorophenylindophenol (DCIP), 9-dimethylaminobenzo[alpha]phenoxazin-7-ium chloride (meldola's blue), 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4, 4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H tetrazolium chloride] (nitroblue tetrazolium; NBT), and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H tetrazolium (INT). The presence of NAD(P) and oxygen gave no oxidation activity to GLDH. Spectroscopic profile and ICP data indicated a b-type cytochrome containing iron.  (+info)

(3/769) Fish macrophages express a cyclo-oxygenase-2 homologue after activation.

In mammals, the increased generation of prostaglandins (PG) during the onset of inflammatory responses and activation of immune cell types has been attributed to the induction of a novel cyclo-oxygenase (COX) isoform, termed COX-2, which is distinct from the well-characterized constitutive activity (COX-1). Goldfish (Carassius auratus) macrophages exposed to bacterial lipopolysaccharide and leucocyte-derived macrophage-activating factor(s) showed a significant increase in the generation of the major COX product, PGE2, within the first 6 h of stimulation. The selective COX-2 inhibitor, NS398, inhibited this elevated generation of PGE, whereas the basal level of this product synthesized by unstimulated macrophages was unaffected by such exposure. PGE generation by goldfish macrophages was similarly inhibited by the glucocorticoid, dexamethasone, and an inhibitor of protein synthesis, cycloheximide, suggesting that this stimulation may be due to an inducible enzyme equivalent to mammalian COX-2. The complete coding sequence of rainbow trout (Oncorhynchus mykiss) COX-2 was obtained by PCR. The gene contains a 61 bp 5'-untranslated region (UTR), a 1821 bp open reading frame and a 771 bp 3'UTR containing multiple copies of an mRNA instability motif (ATTTA). The predicted translation product had high homology to known mammalian and chicken COX-2 (83-84%) and COX-1 (77%) sequences. Reverse-transcriptase PCR with cDNA from control and bacterially challenged fish revealed that trout COX-2 expression was not constitutive but could be induced. Overall, these studies show for the first time that the inducible isoform of COX has a long evolutionary history, probably dating back to the evolution of fish over 500 million years ago.  (+info)

(4/769) Intramolecular chaperone and inhibitor activities of a propeptide from a bacterial zinc aminopeptidase.

An aminopeptidase from Aeromonas caviae T-64 was translated as a preproprotein consisting of three domains; a signal peptide (19 amino acid residues), an N-terminal propeptide (101 residues) and a mature region (273 residues). We demonstrated that a proteinase, which was isolated from the culture filtrate of A. caviae T-64, activated the recombinant pro-aminopeptidase by removal of the majority of the propeptide. Using L-Leu-p-nitroanilide as a substrate, the processed aminopeptidase showed a large increase in kcat when compared with the unprocessed enzyme, whereas the Km value remained relatively unchanged. The similar Km values for the pro-aminopeptidase and the mature aminopeptidase indicated that the N-terminal propeptide of the pro-aminopeptidase did not influence the formation of the enzyme-substrate complex, suggesting the absence of marked conformational changes in the active domain. In contrast, the marked difference in kcat suggests a significant decrease in the energy of one or more of the transition states of the enzyme-substrate reaction coordinate. Moreover, we showed that the activity of the urea-denatured pro-aminopeptidase could be recovered by dialysis, whereas the activity of the urea-denatured mature aminopeptidase, which lacked the propeptide, could not. Further to this, the propeptide-deleted aminopeptidase formed an inclusion body in the cytoplasmic space in Escherichia coli and was not secreted at all. These results suggested that the propeptide of the pro-aminopeptidase acted as an intramolecular chaperone that was involved with the correct folding of the enzyme in vitro and was required for extracellular secretion in E. coli.  (+info)

(5/769) Structure elucidation of Sch 20562, a glucosidic cyclic dehydropeptide lactone--the major component of W-10 antifungal antibiotic.

A novel bacterium designated as Aeromonas sp. W-10 produces the antibiotic W-10 complex which comprises of two major and several minor components. The two major components from this complex, Sch 20562 (1) and Sch 20561 (1a), are of biological interest in view of their potent antifungal activity. The chemical degradation studies utilized for the assignment of structure 1 for Sch 20562 are described here. Some of the noteworthy diversity of structural features in this glucosidic cyclic dehydrononapeptide lactone 1 are: an N-terminal (D)-beta-hydroxymyristyl unit, three D-amino acid units, two (E)-alpha-aminocrotonyl units, and an O-alpha-D-glucosyl-N-methyl-L-allo-threonine unit. The structure determination of 1 utilized the selective cleavage of the dehydropeptide units by ozonolysis to form fragments that were sequenced by mass spectrometry. The stereochemistry of the amino acid units were assigned by isolation of the free amino acids from the hydrolysates of the fragments. The stereochemistry of the alpha-aminocrotonyl units and the glucosidic linkage were assigned by nmr spectroscopy and molecular rotation data.  (+info)

(6/769) Structure elucidation of Sch 20561, a cyclic dehydropeptide lactone--a major component of W-10 antifungal antibiotic.

Antibiotic W-10 is a fermentation complex produced by the bacterium Aeromonas sp. W-10. The cyclic dehydropeptide lactones Sch 20562 (1) and Sch 20561 (2) are the major components of this fermentation complex and are of biological interest in view of their unique structural features and potent antifungal activity. The chemical degradation studies that were utilized in the assignment of structure 2 for Sch 20561 are described here. The structure determination of 2 made use of the ozonolytic cleavage of the dehydropeptide units to form fragments that were sequenced by mass spectrometry. The cyclic dehydropeptide lactone Sch 20561 (2) was found to be the aglycone of Sch 20562 (1) and these two natural products were correlated by a chemical transformation involving the deglucosidation of 1 to form 2.  (+info)

(7/769) Cloning, sequencing, and role in virulence of two phospholipases (A1 and C) from mesophilic Aeromonas sp. serogroup O:34.

Two different representative recombinant clones encoding Aeromonas hydrophila lipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophila AH-3 (serogroup O34) introduced into Escherichia coli DH5alpha. Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 (pla) and C (plc) genes code for an 83-kDa putative lipoprotein and a 65-kDa protein, respectively. Defined insertion mutants of A. hydrophila AH-3 defective in either pla or plc genes were defective in phospholipase A1 and C activities, respectively. Lecithinase (phospholipase C) was shown to be cytotoxic but nonhemolytic or poorly hemolytic. A. hydrophila AH-3 plc mutants showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the plc single gene on these mutants abolished this effect, suggesting that Plc protein is a virulence factor in the mesophilic Aeromonas sp. serogroup O:34 infection process.  (+info)

(8/769) Efficacy of orally administered oxolinic acid and Vetoquinol, an oxolinic acid ester, for the treatment of furunculosis in Atlantic salmon Salmo salar held in seawater.

This study was performed to determine the efficacy of orally administered oxolinic acid and Vetoquinol, an oxolinic acid ester, in the treatment of experimental induced furunculosis in Atlantic salmon Salmo salar held in seawater. Two strains of the causative bacterium Aeromonas salmonicida subsp. salmonicida, 1 sensitive (VI-88/09/03175) and 1 resistant (3475/90) to oxolinic acid, were used. In 2 trials, cohabitational challenges were performed by introducing 8 fish challenged in advance by an intraperitoneal injection of 2.2 x 10(4) colony forming units of strain 3475/90 (Trial 1) or strain VI-88/09/03175 (Trial 2) to 10 aquaria each containing 40 healthy fish. The treatment groups in both trials consisted of 4 groups receiving either oxolinic acid (2 groups) or Vetoquinol (2 groups) and 1 control group. An unchallenged, unmedicated group was used to determine the natural mortality in the population. The recommended therapeutic dose of 25 mg oxolinic acid kg-1 fish at Days 1, 2, 4, 6, 8 and 10 following initiation of treatment was used. Oral medication initiated at Day 10 (Trial 1) or Day 11 (Trial 2) following challenge significantly (p < 0.05) lowered the specific mortality in all drug-treated groups compared to the untreated control groups. Mortality in Vetoquinol-treated groups was significantly (p < 0.05) lower than in oxolinic acid-treated groups in Trial 1 whereas no significant (p < 0.05) difference in survival rate was found between the medicated groups in Trial 2.  (+info)

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