Factors influencing bacterial production of inducers of settlement behavior of larvae of the oysterCrassostrea gigas

Dissolved chemical inducers of settlement behavior of veliger larvae of the oysterCrassostrea gigas are found in supernatants of both pigmented species of bacteria (Alteromonas colwelliana, Vibrio cholerae strain HTX) as well as nonpigmented bacteria (Excherichia coli, Vibrio cholerae strain 596-B). Usually less than 10% of veligers exhibited settlement behavior in response to supernatants from the early bacterial growth phases, whereas 30–90% of larvae responded when exposed to supernatant from late-log and stationary phase cultures. Percentages of larvae exhibiting settlement behavior were inversely correlated with oxygen levels in the culture. Furthermore, the behavioral response decreased with pigment formation, suggesting that quantities of noxious compounds such as quinones may build up in the supernatants of cultures of pigmented bacteria. Tyrosinase, an enzyme that converts L-tyrosine to L-DOPA in the first step of melanogenesis, was detected both in the bacterial pellet and the supernatant during growth of the pigmented species. The enzyme is not required for the production of settlement inducer as the nonpigmented speciesE. coli andV. cholerae (596-B) also released inducer into the supernatant and had no detectable tyrosinase. The data suggest either that there is more than one inducer of settlement behavior found in bacterial supernatants or that the inducer is not L-DOPA or an L-DOPA-mimetic associated with the melanin biochemical pathway.     

The protein encoded by the Shewanella colwellianamelA gene is a p-hydroxyphenylpyruvate dioxygenase

Abstract The identity of the product of the melA gene from Shewanella colelliana with the enzyme p -hydroxyphenylpyruvic dioxygenase is shown. Cloning of the melA gene endowed Escherichia coli with the capacity to synthesize melanin-like pigments from L-tyrosine. E. coli contained transaminases that transforms L-tyrosine into p -hydroxyphenylpyruvate. This keto acid was detected in the cultures. On the other hand, E. coli containing melA was able to go further in the catabolic pathway, releasing a great amount of homogentisic acid. This acid can spontaneously polymerize giving the pigment. Furthermore, p -hydroxyphenylpyruvate dioxygenase activity was detected in this system. Analysis of the deduced amino acid sequence revealed a high homology with the p -hydroxyphenylpyruvate deoxygenase enzyme from different organisms.     

Methyl directed DNA mismatch repair inVibrio cholerae

Mismatches in DNA occur either due to replication error or during recombination between homologous but non-identical DNA sequences or due to chemical modification of bases. The mismatch in DNA, if not repaired, result in high spontaneous mutation frequency. The repair has to be in the newly synthesized strand of the DNA molecule, otherwise the error will be fixed permanently. Three distinct mechanisms have been proposed for the repair of mismatches in DNA in prokaryotic cells and gene functions involved in these repair processes have been identified. The methyl-directed DNA mismatch repair has been examined inVibrio cholerae, a highly pathogenic gram negative bacterium and the causative agent of the diarrhoeal disease cholera. The DNA adenine methyltransferase encoding gene (dam) of this organism which is involved in strand discrimination during the repair process has been cloned and the complete nucleotide sequence has been determined.Vibrio cholerae dam gene codes for a 21.5 kDa protein and can substitute for theEscherichia coli enzyme. Overproduction ofVibrio cholerae Dam protein is neither hypermutable nor lethal both in Escherichia coli andVibrio cholerae. WhileEscherichia coli dam mutants are sensitive to 2-aminopurine,Vibrio cholerae 2-aminopurine sensitive mutants have been isolated with intact GATC methylation activity. The mutator genesmutS andmutL involved in the recognition of mismatch have been cloned, nucleotide sequence determined and their products characterized. Mutants ofmutS andmutL ofVibrio cholerae have been isolated and show high rate of spontaneous mutation frequency. ThemutU gene ofVibrio cholerae, the product of which is a DNA helicase II, codes for a 70 kDa protein. The deduced amino acid sequence of themutU gene hs all the consensus helicase motifs. The DNA cytosine methyltransferase encoding gene (dam) ofVibrio cholerae has also been cloned. Thedcm gene codes for a 53 kDa protein. This gene product might be involved in very short patch (VSP) repair of DNA mismatches. The vsr gene which is directly involved in VSP repair process codes for a 23 kDa protein. Using these information, the status of DNA mismatch repair inVibrio cholerae will be discussed.     

L-tyrosine regulation and biosynthesis via arogenate dehydrogenase in suspension-cultured cells of Nicotiana silvestris Speg. et Comes

The biosynthetic route to L-tyrosine was identified in isogenic suspension-cultured cells of N. silvestris. Arogenate (NADP⁺) dehydrogenase, the essential enzyme responsible for the conversion of L-arogenato L-tyrosine, was readily observed in crude extracts. In contrast, prephenate dehydrogenase (EC 1.3.1.13) activity with either NAD⁺ or NADP⁺ was absent altogether. Therefore, it seems likely that this tobacco species utilizes the arogenate pathway as the exclusive metabolic route to L-tyrosine. L-Tyrosine (but not L-phenylalanine) was a very effective endproduct inhibitor of arogenate dehydrogenase. In addition, analogs of L-tyrosine (m-fluoro-DL-tyrosine [MFT], D-tyrosine and N-acetyl-DL-tyrosine), but not of L-phenylalanine (o-fluoro-DL-phenylalanine and p-fluoro-DL-phenylalanine), were able to cause inhibition of arogenate dehydrogenase. The potent antimetabolite of L-tryptophan, 6-fluoro-DL-tryptophan, had no effect upon arogenate dehydrogenase activity. Of the compounds tested, MFT was actually more effective as an inhibitor of arogenate dehydrogenase than was L-tyrosine. Since MFT was found to be a potent antimetabolite inhibitor of growth in N. silvestris and since inhibition was specifically and effectively reversed by L-tyrosine, arogenate dehydrogenase is an outstanding candidate as the in vivo target of analog action. Although chorismate mutase (EC 5.4.99.5) cannot be the prime target of MFT action, MFT can mimick L-tyrosine in partially inhibiting this enzyme activity. The activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was insensitive to L-phenylalanine or L-tyrosine. The overall features of this system indicate that MFT should be a very effective analog mimick for selection of feedback-insensitive regulatory mutants L-tyrosine biosynthesis.Abbreviations DAHP synthase 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase - 6FT 6-fluoro-DL-tryptophan - MFT m-fluoro-DL-tyrosine - OFP o-fluoro-DL-phenylalanine - PFP p-fluoro-DL-phenylalanine     

The Autophagic Pathway: A Cell Survival Strategy Against the Bacterial Pore-Forming Toxin Vibrio Cholerae Cytolysin

Vibrio cholerae is the causative agent of cholera in humans. In addition to the critical virulence factors cholera toxin and toxin co-regulated pilus, V. cholerae secretes V. cholerae cytolysin (VCC), a pore-forming exotoxin able to induce cell lysis and extensive vacuolation. We have shown that this vacuolation is related to the activation of autophagy in response to VCC action. Furthermore, we found that the autophagic pathway was required to protect cells upon VCC intoxication. Based on additional data presented here, we propose a model aimed to explain the mechanism of cell protection. We postulate that VCC-induced autophagic vacuoles, which display features of multivesicular bodies and enclose the toxin, are implicated in cell defense through VCC degradation involving fusion with lysosomes.

Addendum to:

Protective Role of Autophagy Against Vibrio cholerae Cytolysin, a Pore-Forming Toxin from V. cholerae

M.G. Gutierrez, H.A. Saka, I. Chinen, F.C.M. Zoppino, T. Yoshimori, J.L. Bocco and M.I. Colombo

Proc Natl Acad Sci USA 2007; 104:1829-34     

Brown Pigments Produced by Yarrowia lipolytica Result from Extracellular Accumulation of Homogentisic Acid

Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid, p-hydroxyphenylethanol, and p-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol and p-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1). p-Hydroxyphenylethanol and p-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde, p-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in Y. lipolytica involves the formation of p-hydroxyphenylacetaldehyde, which is oxidized to p-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn²⁺ accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn²⁺. Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.     

L-Tyrosine oxidative metabolism byStreptomyces aureofaciens

Summary L-Tyrosine metabolism found in normal mammalian system via homogentisic acid was investigated inStreptomyces aureofaciens S-834 as L-tyrosine was shown to possess the stimulatory role in the production of chlortetracycline antibiotic. L-Tyrosine as well as the substrates of the intermediary oxidative enzymes-p-hydroxyphenylpyruvate (p-HPPA) hydroxylase and homogentisate (HGA) oxygenase were incubated with the washed, resting cells of the culture and by adding sodium diethyldithiocarbamate and,-dipyridyl, the specific inhibitors of these enzymes to the culture, the oxidative intermediates-p-HPPA and HGA were blocked from further utilisation and were accumulated and isolated by extraction with the appropriate solvent system. The intermediates consisted of keto acids and aromatic phenolic acids were separated by adsorption and elution on silica gel and sephadex gel columns. p-HPPA and acetoacetate (AA) were identified as keto acids by obtaining their 2,4-dinitrophenylhydrazone derivatives. HGA as well as p-HPPA were identified as phenolic acids along with HGA lactone using diazotized reagents. These investigations helped to establish L-tyrosine oxidative pathway inStreptomyces aureofaciens via p-HPPA, HGA and AA leading to the biosynthesis of chlortetracycline antibiotic.     

Demonstration of viable but nonculturable Vibrio cholerae O1 in fresh water environment of India using ciprofloxacin DFA-DVC method

Aim: To demonstrate the presence of culturable and nonculturable viable pathogenic Vibrio cholerae O1 in fresh water environments of a cholera‐endemic region in India. Methods and Results: Conventional culture and ciprofloxacin DFA–DVC were utilized to investigate the existence of V. cholerae O1. We isolated pathogenic culturable V. cholerae O1 from water samples collected from cholera‐affected areas. No culturable V. cholerae O1 was isolated from water and plankton samples from natural fresh water bodies. Ciprofloxacin was used for DFA–DVC as V. cholerae O1 are 100% resistant to nalidixic acid in our region. The viable but nonculturable O1 cells were demonstrated in 2·21 and 40·69% samples from natural water bodies and cholera‐affected areas, respectively. Conclusion: Vibrio cholerae O1 VBNC could be demonstrated using modified DFA–DVC technique. Ciprofloxacin is preferable to nalidixic acid for DVC in view of existing high‐level resistance to nalidixic acid in cholera‐endemic areas. Significance and Impact of the study: We endorse that for public health surveillance, cholera outbreak investigation and disease control water samples in addition to culture should be tested for V. cholerae using DFA–DVC.     

Activation studies of the α- and β-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae with amines and amino acids

The α- and β-class carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae, VchCAα, and VchCAβ, were investigated for their activation with natural and non-natural amino acids and amines. The most effective VchCAα activators were L-tyrosine, histamine, serotonin, and 4-aminoethyl-morpholine, which had K_As in the range of 8.21–12.0?µM. The most effective VchCAβ activators were D-tyrosine, dopamine, serotonin, 2-pyridyl-methylamine, 2-aminoethylpyridine, and 2-aminoethylpiperazine, which had K_As in the submicromolar – low micromolar range (0.18–1.37?µM). The two bacterial enzymes had very different activation profiles with these compounds, between each other, and in comparison to the human isoforms hCA I and II. Some amines were selective activators of VchCAβ, including 2-pyridylmethylamine (K_A of 180?nm for VchCAβ, and more than 20?µM for VchCAα and hCA I/II). The activation of CAs from bacteria, such as VchCAα/β has not been considered previously for possible biomedical applications. It would be of interest to study in more detail the extent that CA activators are implicated in the virulence and colonisation of the host by such pathogenic bacteria, which for Vibrio cholerae, is highly dependent on the bicarbonate concentration and pH in the surrounding tissue.     

Competence and natural transformation in vibrios

Natural transformation is a major mechanism of horizontal gene transfer in bacteria. By incorporating exogenous DNA elements into chromosomes, bacteria are able to acquire new traits that can enhance their fitness in different environments. Within the past decade, numerous studies have revealed that natural transformation is prevalent among members of the Vibrionaceae, including the pathogen Vibrio cholerae. Four environmental factors: (i) nutrient limitation, (ii) availability of extracellular nucleosides, (iii) high cell density and (iv) the presence of chitin, promote genetic competence and natural transformation in Vibrio cholerae by co‐ordinating expression of the regulators CRP, CytR, HapR and TfoX respectively. Studies of other Vibrionaceae members highlight the general importance of natural transformation within this bacterial family.     

Identification and Initial Characterization of Elastase Activity Associated with Vibrio cholerae

Strains of Vibrio cholerae O1 (Ogawa, Inaba) and non-O1 serogroups have been found to produce an elastolytic protease that can be detected on 0.3% elastin agar plates or in broth cultures. The elastase enzyme appears to be maximally expressed in late log phase (14–18 h postinoculation) and has optimum activity at a pH range between 7 and 8. Comparative studies indicate that more than 60% of V. cholerae strains analyzed quantitatively produce more elastase in broth (two- to fourfold higher) than other elastase-positive Vibrio species such as Vibrio vulnificus. The V. cholerae elastase enzyme was not inhibited by trypsin, serine-protease, or thiol-protease inhibitors, but was inhibited by phosphoramidon. Ultrafiltration studies indicate the V. cholerae elastase enzyme has a molecular weight >30,000, and a 34K protein with possible elastase activity has been detected by SDS-PAGE for one non-O1 isolate (strain 2396). Cumulative results suggest that the V. cholerae elastase is probably a member of the N-type metalloprotease family and shares similar properties with other elastase enzymes described for pathogenic and nonpathogenic species in this genus. Received: 26 February 1999 / Accepted: 29 March 1999     

Gibberellic acid and indole acetic acid compete in ethylene promoted abscission

Summary Gibberellic acid inhibition of betacyanin biosynthesis has been studied in Amaranthus caudatus L. using the pigment precursors L-tyrosine and L-dihydroxyphenylalanine. Precursors fed to gibberellic acid (GA) treated seedlings completely recovered betacyanin synthesis while the GA induced growth enhancement remained unaltered. Inhibition of betacyanin biosynthesis by GA is related to depletion of metabolites/amino acids and their diversion to support changed pattern of metabolism leading to growth.Abbreviations GA gibberellic acid - L-DOPA L-dihydroxyphenylalanine - Pr phytochrome red absorbing form - Pfr phytochrome, far-red absorbing form     

Cellular localization and export of the soluble haemolysin of Vibrio cholerae El Tor

Summary The cellular location of the haemolysin of Vibrio cholerae El Tor strain 017 has been analyzed. This protein is found both in the periplasmic space and the extracellular medium in Vibrio cholerae. However, when the cloned gene, present on plasmid pPM431, is introduced into E. coli K-12 this protein remains localized predominantly in the periplasmic space with no activity detected in the extracellular medium. Mutants of E. coli K-12 (tolA and tolB) which leak periplasmic proteins mimic excretion and release the haemolysin into the growth medium. Secretion of haemolysin into the periplasm is independent of perA (envZ) and in fact, mutants in perA (envZ) harbouring pPM431 show hyperproduction of periplasmic haemolysin. These results in conjunction with those for other V. cholerae extracellular proteins suggest that although E. coli K-12 can secrete these proteins into the periplasm, it lacks a specific excretion mechanism, present in V. cholerae, for the release of soluble proteins into the growth medium.     

The Sulphation of p-hydroxyphenylpyruvic acid and related compounds by the rat liver cytosol.

Cytosol preparations of rat liver and kidney were examined for their ability to transfer sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to p-hydroxyphenylpyruvic acid. Little activity towards this substrate was observed, and the main product detected in the reaction mixtures was identified as p-hydroxybenzyl alcohol O-sulphate. This was not formed from p-hydroxybenzaldehyde, a spontaneous oxidation product of p-hydroxyphenylpyruvic acid, by sulphation followed by a rapid enzyme-catalysed reduction of the intermediate phydroxybenzaldehyde O-sulphate. This product was formed mainly by this sequence of reactions, but the reverse, reduction followed by sulphation, also appeared possible. p-Hydroxybenzyl alcohol itself was very readily sulphated by both preparations, and the liver also produced a sulpho-conjugate of homogentisic acid. These observations are important in calculating the turnover of L-tyrosine O-sulphate in the mammalian system, and establish that p-hydroxyphenylpyruvic acid O-sulphate is an end product of its metabolism, rather than an intermediate in its synthesis by reversed transamination.     

霍乱弧菌检测技术研究进展

随着社会的发展,公共卫生及水体质量已得到明显提升,但到目前为止在发展中国家由霍乱疫情导致的死亡率依旧非常高,而霍乱弧菌即为霍乱疫情爆发的病原菌,且霍乱在我国被列为甲类传染病。目前国内外针对霍乱弧菌已经建立了多种有效的检测技术,对控制霍乱暴发作用显著。本文综述了近年来霍乱弧菌检测技术研究新进展,包括微生物学、免疫学、分子生物学等较成熟的检测方法,生物传感器、快速检测技术等新兴检测方法,并总结各种技术优缺点,展望未来霍乱弧菌检测的市场需求。     

The DHICA Oxidase Activity of the Melanosomal Tyrosinases LEMT and HEMT

Although melanins can be formed in vitro by the unique action of tyrosinase on L-tyrosine, it is now well accepted that other enzymes termed tyrosinase-related proteins are involved in mammalian melanogenesis. However, some aspects of their roles in the regulation of the pathway are still unknown. The action of dopachrome tautomerase on L-dopachrome yields DHICA, a stable dihydroxyindole with a low rate of spontaneous oxidation. However, DHICA is efficiently incorporated to the pigment, as judged by the high content of carboxylated indole units in natural melanins. Therefore, the fate of this melanogenic intermediate and the mechanisms of its incorporation to the melanin polymer are major issues in the study of melanogenesis. We have recently shown that mouse melanosomes contain two electrophoretically distinguishable tyrosinase isoenzymes, LEMT and HEMT, that can be purified and completely resolved (Jiménez-Cervantes et al., 1993a). Herein, we have compared the ability of these tyrosinases to catalyze DHICA oxidation. Although highly purified LEMT shows a very low specific activity for dopa oxidation in comparison to HEMT, it is able to catalyze DHICA oxidation. However, the DHICA oxidase activity of HEMT was very low, if significant. The ability of purified LEMT to catalyze DHICA oxidation was abolished by heat, trypsin, or phenylthiourea treatments. LEMT acting on DHICA caused the formation of a brownish soluble color similar to DHICA-melanin. Immunoprecipitation of the DHICA oxidase activity of LEMT by specific antibodies suggests that this activity corresponds to TRP1. These results indicate that LEMT, most probably identical to the product of the b locus, is a tyrosinase having a specific DHICA oxidase activity. Opposite to HEMT, the true tyrosinase encoded by the albino locus, its role in melanogenesis would be related to the incorporation of DHICA into eumelanin rather than to the first steps of the pathway.     

Do Protozoa Control the Elimination of Vibrio choleraein Brackish Water?

Elimination of inoculated Vibrio cholerae (≥10⁷ cells ml⁻¹) within a brackish water bacteria assemblage (Mecoacán Lagoon, State of Tabasco, Mexico) was studied in laboratory microcosms with filtration‐fractionated water. Feeding of a ciliate, Cyclidium glaucoma was evaluated using fluorescently labelled V. cholerae o1. Even though V. cholerae was not exploited as the major food source, ciliates were able to eliminate it efficiently. An addition of chitin directly supported the growth of bacteria, although not so much of V. cholerae, and indirectly the growth of the protistan assemblage. Generally, the changes in a bacterial assemblage structure were the most important in V. cholerae elimination.     

Binding efficiencies of carbohydrate ligands with different genotypes of cholera toxin B: molecular modeling, dynamics and docking simulation studies

Vibrio cholerae produces cholera toxin (CT) that consists of two subunits, A and B, and is encoded by a filamentous phage CTXΦ. The A subunit carries enzymatic activity that ribosylates ADP, whereas the B subunit binds to monosialoganglioside (GM1) receptor in epithelial cells. Molecular analysis of toxigenic V. cholerae strains indicated the presence of multiple ctxB genotypes. In this study, we employed a comparative modeling approach to define the structural features of all known variants of ctxB found in O139 serogroup V. cholerae. Modeling, molecular dynamics and docking simulations studies suggested subtle variations in the binding ability of ctxB variants to carbohydrate ligands of GM1 (galactose, sialic acid and N-acetyl galactosamine). These findings throw light on the molecular efficiencies of pathogenic isolates of V. cholerae harboring natural variants of ctxB in causing the disease, thus suggesting the need to consider ctxB variations when designing vaccines against cholera.     

NaCl concentration determines the outcome of competition experiments betweenVibrio pelagius andVibrio cholerae in chemostat cultures

The effect of NaCl concentration gradients on the growth of a marine species,Vibrio pelagius, and two freshwater and estuarine species,Vibrio cholerae andAeromonas hydrophila, was determined in chemostat cultures containing a mineral medium withd-glucose as the sole source of carbon and energy. Complementary cell density profiles were obtained with the marine and the freshwater-estuarine species at the extreme ends of the NaCl concentration gradients. At the lower NaCl concentrations,V. cholerae andA. hydrophila attained maximal cell densities, while no growth or submaximal cell yields were characteristic ofV. pelagius; the reverse was true at the higher NaCl concentrations. At the intermediate concentrations (100–400 mM) all three organisms were able to grow with cell densities ranging from 80%–100% of the maximum. Competition experiments betweenV. cholerae andV. pelagius within this region (100, 150, and 300 mM) indicated that the lower NaCl concentrations favoredV. cholerae while the higher concentrations favoredV. pelagius. The results suggest that the responses ofV. pelagius, V. cholerae, andA. hydrophila to different concentrations of NaCl are important in determining the environmental distribution of these species.