Effect of nutrient deprivation on lipid, carbohydrate, DNA, RNA, and protein levels in Vibrio cholerae

The response of Vibrio cholerae to low nutrient levels was determined by measuring the concentrations of lipids, carbohydrates, DNA, RNA, and proteins over a 30-day starvation period. Ultrastructural integrity was observed by transmission electron microscopy. Total lipids and carbohydrates declined rapidly within the first 7 days, while DNA and protein exhibited a more constant decline over the 30 days of starvation. In contrast, RNA showed little decrease upon starvation. Although neutral lipids were lost, the percentage of neutral lipids did not decline as rapidly as the phospholipids. Detectable levels of poly-beta-hydroxybutyrate disappeared completely by 7 days. Carbohydrate profiles revealed the relative loss of the five-carbon sugar ribose and N-acetylglucosamine and a relative increase in the total six-carbon sugars, especially glucose. Morphologically, ribosomes appeared to exhibit no structural change, while inclusion bodies and mesosomelike structures disappeared completely, and cell wall and membrane integrity was lost. The data suggest that V. cholerae differs somewhat from other marine vibrios in its response to low nutrients but shares some characteristics in common with them. The data also suggest that certain lipids and carbohydrates may provide the endogenous energy sources needed for dormancy preparation and cell maintenance under nutrient starvation.     

Effects of nutrient deprivation on Vibrio cholerae.

Environmental and clinical strains of Vibrio cholerae were exposed to nutrient-free artificial seawater and filtered natural seawater microcosms for selected time intervals and examined for changes in cell morphology and number. Cells observed by transmission electron and epifluorescence microscopy were found to undergo gross alterations in cell morphology with time of exposure. The vibroid cells decreased in volume by 85% and developed into small coccoid forms surrounded by remnant cell walls. The initial number of cells inoculated into nutrient-free microcosms (culturable count and direct viable count) increased 2.5 log10 within 3 days, and even after 75 days the number of viable cells was still 1 to 2 log10 higher than the initial inoculum size. Nutrient-depleted coccoid-shaped cells were restored to normal size and assumed a bacillary shape within 3 h and began to divide within 5 h after nutrient supplementation. The increase in cell number and decrease in cell volume under nutrient-depleted conditions, as well as the rapid growth response after nutrient supplementation, may describe some of the survival mechanisms of V. cholerae in the aquatic environment.     

Effects of nutrient deprivation on Vibrio cholerae

Environmental and clinical strains of Vibrio cholerae were exposed to nutrient-free artificial seawater and filtered natural seawater microcosms for selected time intervals and examined for changes in cell morphology and number. Cells observed by transmission electron and epifluorescence microscopy were found to undergo gross alterations in cell morphology with time of exposure. The vibroid cells decreased in volume by 85% and developed into small coccoid forms surrounded by remnant cell walls. The initial number of cells inoculated into nutrient-free microcosms (culturable count and direct viable count) increased 2.5 log10 within 3 days, and even after 75 days the number of viable cells was still 1 to 2 log10 higher than the initial inoculum size. Nutrient-depleted coccoid-shaped cells were restored to normal size and assumed a bacillary shape within 3 h and began to divide within 5 h after nutrient supplementation. The increase in cell number and decrease in cell volume under nutrient-depleted conditions, as well as the rapid growth response after nutrient supplementation, may describe some of the survival mechanisms of V. cholerae in the aquatic environment.     

In-vitro interaction between nitrofurantoin and Vibrio cholerae DNA.

In-vitro interaction of nitrofurantoin with V. cholerae DNA resulted in a quenching and red spectral shift of the drug absorption pattern. Scatchard analysis revealed that the drug binding involved more than one processes and that the strongest mode of binding was characterised by an association constant (k) of 5.04 x 10(6) M-1 and the number of binding sites per nucleotide (n) of 0.015. Based on viscosity measurements, the mode of drug binding to DNA appeared to be through intercalation, the helix unwinding angle of supercoiled plasmid pBR322 DNA being 10 degrees. Nitrofurantoin binding to DNA resulted in an elevation of the thermal melting temperature (Tm) of DNA by 6 degrees C and inhibition of the action of DNase on DNA.     

Effect of dietary protein and carbohydrate levels on growth,nutrient utilization and body composition in fingerling rohu,Labeo rohita (Hamilton)

Twelve experimental diets (D‐1 to D‐12) in a 4 × 3 factorial design having four protein levels (25, 35, 40 and 45%) and three carbohydrate levels (15, 25 and 35%) were formulated and fed to fingerling rohu, Labeo rohita (5.48 ± 0.02 g) for 60 days in three replicates at 2% BW per day. The best performance of fish in terms of weight gain (%), specific growth rate (SGR; % per day), feed conversion ratio (FCR) and protein efficiency ratio (PER) was recorded with diet D‐9 containing 40% protein and 35% dextrin as a source of dietary carbohydrate. In general, lower protein consumption per kilogram BW was observed at all protein levels with the rise of the dextrin level. The apparent digestibilities of protein and lipid were not affected by the dietary treatments. At the end of the experiment the body composition of animals from all treatments showed lower percentages of moisture and higher percentages of protein as compared to the initial values. A consistent rise in protein retention efficiency was noted in fish fed diets with increasing dextrin levels. The highest protein sparing effect was found in fish fed the diet containing 40% protein and 35% dextrin.     

Analysis of protein,carbohydrate and lipid in rotifers

Protein, carbohydrate and lipid amounts were determined for several rotifer species collected directly from the field. Brachionus calyciflorus was the most abundant species; therefore making possible more data for it. An increase in protein content of this species occurred when its concentration in food (µg protein/ml) also increased. Keratella tropica showed a similar pattern, but Asplanchna brightwelli did not.Carbohydrate proved to be the main form of storage in rotifers. In Brachionus calyciflorus females bearing no egg, 8% of the total biomass was carbohydrate; in females bearing one egg, 15% carbohydrate was found. Lipid does not appear to be used for storage since no increase in the amount of lipid was detected in females bearing eggs or embryos. This suggests that lipid has a structural function. Finally, a relationship between rotifer body volume and protein content at a given food concentration was obtained. The cladoceran Daphnia magna follows the same pattern.     

Detection of an OmpA-like protein in Vibrio cholerae

Abstract Rhodopseudomonas marina/agilis was enriched from a natural microbial mat by using conditions that favor growth of anoxygenic photoheterotrophs able to fix N₂ rapidly. The isolated bacterium grows more readily on fructose or mannitol than on organic acid carbon sources, requires preformed biotin and thiamine as growth factors, and is extraordinarily motile; growth occurs up to a temperature of approx. 44°C. The photosynthetic pigments of R. marina/agilis are housed in intracytoplasmic lamellar membranes which show the in vivo absorbance characteristics of bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series. In common with other non-sulfur purple bacteria, R. marina/agilis can also grow as an aerobic heterotroph in darkness. Under these conditions, photopigment synthesis is severely repressed. R. marina/agilis requires 1–5% NaCl for optimal growth, and cells grown on N₂ showed nitrogenase activity of >1000 nmol acetylene reduced h/mg dry wt.     

Regulation of ribosomal protein synthesis in Vibrio cholerae

We have investigated the regulation of the S10 and spc ribosomal protein (r-protein) operons in Vibrio cholerae. Both operons are under autogenous control; they are mediated by r-proteins L4 and S8, respectively. Our results suggest that Escherichia coli-like strategies for regulating r-protein synthesis extend beyond the enteric members of the gamma subdivision of proteobacteria.     

Changes in cathepsin D, acid autolytic activity, RNA, DNA in skeletal muscle and liver of mouse kept on high protein, carbohydrate and lipid diets

The influence was studied of different diets on the activity of cathepsin D (PSCatD), pepstatin (PIA) and leupeptin (LIA) insensitive acid autolytic activity (AAA), RNA, DNA and protein in skeletal leg muscle (LM) and liver of 37 mice. The diets affected the weight of the liver and content of protein in the liver and LM. The protein:DNA ratio was lowest on high carbohydrate (HC) and commercial (C) diets in both tissues and about 3 times higher in LM than in the liver. The RNA:protein ratio was highest in the high protein-fat (HPF) and recommended (R) diet fed groups. The RNA:DNA ratio was lowest on HC and C diets. In the liver, PSCatD, AAA, LIA were lowest on HPF, and highest on HC diets, but for PIA on high fat-protein (HFP) and C diets, respectively. The highest activities were correlated with the lowest percentage of protein and fat in the diets (low energy diets). For LM, the highest activities were found on a C diet and lowest for PSCatD on HEP but for AAA, PIA, LIA on HC diets. Cathepsin D accounted for about 70% of hemoglobin degradation in the liver and 66% in LM. In AAA, cathepsin D participates in 58.5% and 50.5% in the liver and LM inhibition, respectively, but leupeptin accounted for about 15% and 27% (in the presence of Mg++) of inhibition.     

The ToxR protein of Vibrio cholerae forms homodimers and heterodimers.

The ToxR protein of Vibrio cholerae regulates the expression of several virulence factors that play important roles in the pathogenesis of cholera. Previous experiments with ToxR-alkaline phosphatase (ToxR-PhoA) fusion proteins suggested a model for gene regulation in which the inactive form of ToxR was a monomer and the active form of ToxR was a dimer (V. L. Miller, R. K. Taylor, and J. J. Mekalanos, Cell 48:271-279, 1987). In order to examine whether ToxR exists in a dimeric form in vivo, biochemical cross-linking analyses were carried out. Different dimeric cross-linked species were detected depending on the expression level of ToxR: when overexpressed, ToxR+ToxR homodimers and ToxR+ToxS heterodimers were detected, and when ToxR was expressed at normal levels, exclusively ToxR+ToxS heterodimers were detected. The amount of overexpression was quantitated by using ToxR-PhoA fusion proteins and was found to correspond to 2.7-fold the normal level of ToxR. The formation of both homodimeric ToxR species and heterodimeric ToxR+ToxS species is consistent with previously reported genetic data that suggested that both types of ToxR oligomeric interactions occur. However, variation in the amount of either the homodimeric or heterodimeric form detectable by this cross-linking analysis was not observed to correlate with laboratory culture conditions known to modulate ToxR activity. Thus, genetic and biochemical data indicate that ToxR is able to interact with both itself and ToxS but that these interactions may not explain mechanistically the observed changes in ToxR activity that occur in response to environmental conditions.     

Influence of salinity and organic nutrient concentration on survival and growth of Vibrio cholerae in aquatic microcosms.

Laboratory microcosms were employed to evaluate the influence of selected environmental parameters, organic nutrient concentration, and salinity on the growth and survival of a toxigenic strain of Vibrio cholerae LA4808. Over the range conditions tested, this strain of V. cholerae showed maximum response as determined by increased plate counts and direct microscopic counts in microcosms prepared with a chemically defined sea salts solution at a salinity of 25%, but with lower or higher salinity levels, the maximum population size declined. When added organic concentrations of less than 1,000 micrograms/liter were present, a marked salinity effect on the growth of V. cholerae was detected. However, at or above an organic nutrient concentration of 1,000 micrograms/liter, the need for an optimum salinity level was spared. From the results of this study, it is concluded that V. cholerae can grow under conditions of organic nutrient concentration and salinity typical of estuaries. Results obtained support the hypothesis that V. cholerae is an autochthonous member of the estuarine microbial community.     

Effect of prostaglandins on protein, RNA, DNA and collagen synthesis in experimental wounds.

The effect of exogenous prostaglandins E1, E2 and F2alpha (PGE1, PGE2 and PGF2alpha) on 3H-leucine, 3H-uridine, 3H-thymidine and 3H-proline incorporation in experimental cutaneous wounds has been studied in rats. Prostaglandins E1 and E2 markedly stimulate the incorporation of these tritiated precursors, into protein, RNA, DNA and collagen synthesis, whereas F2 inhibits it. All tested prostaglandins exhibit their maximum effect within the first hours following administration. Most active is PGE1. These observations indicate that application of prostaglandins significantly stimulate incorporation with protein, RNA, DNA and collagen synthesis in the skin of wounded rats and thus, may play a role in epidermal cell growth and division as well as in scar-forming tissue.     

Genetic transformation of Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio cholerae non O-1 with plasmid DNA by electroporation

An electroporation procedure for the plasmid-mediated transformation of the genus Vibrio was performed, as part of an effort to develop recombinant DNA techniques for genetic manipulation of the genus Vibrio. Vibrio parahaemolyticus, V. alginolyticus, and V. cholerae non O-1 (9 different strains) were transformed with 3 vector plasmids (pACYC184, pHSG398, and pBR325). The efficiency of transformation was highly dependent on three parameters: the concentration of plasmid DNA; the strength of the electric field; and the combination of plasmid DNA and recipient strain. The drug-resistance genes on the vector plasmid were expressed in the Vibrio strains.     

Characterization of lipid A and polysaccharide moieties of the lipopolysaccharides from Vibrio cholerae.

Lipid A and polysaccharide moieties obtained by mild acid hydrolysis of the lipopolysaccharides from Vibrio cholerae 569 B (Inaba) and Vibrio el-tor (Inaba) were characterized. Heterogeneity of lipid A fractions was indicated by t.l.c. and by gel filtration of the de-O-acylated products from mild alkaline methanolysis of the lipids. Presumably lipid A contains a glucosamine backbone, and the fatty acids are probably bound to the hydroxyl and amino groups of glucosamine residues. Approximately equal amounts of fatty acids C16:0, C18:1 and 3-hydroxylauric acid were involved in ester linkages, but 3-hydroxymyristic acid was the only amide-linked fatty acid. Sephadex chromatography of the polysaccharide moiety showed the presence of a high-molecular-weight heptose-free fraction and a low-molecular-weight heptose-containing fraction. Haemagglutination-inhibition assays of these fractions showed the heptose-free fraction to be an O-specific side-chain polysaccharide, whereas the heptose-containing fraction was the core polysaccharide region of the lipopolysaccharides. Identical results were obtained for both organisms.     

RNA Colony Blot Hybridization Method for Enumeration of Culturable Vibrio cholerae and Vibrio mimicus Bacteria

A species-specific RNA colony blot hybridization protocol was developed for enumeration of culturable Vibrio cholerae and Vibrio mimicus bacteria in environmental water samples. Bacterial colonies on selective or nonselective plates were lysed by sodium dodecyl sulfate, and the lysates were immobilized on nylon membranes. A fluorescently labeled oligonucleotide probe targeting a phylogenetic signature sequence of 16S rRNA of V. cholerae and V. mimicus was hybridized to rRNA molecules immobilized on the nylon colony lift blots. The protocol produced strong positive signals for all colonies of the 15 diverse V. cholerae-V. mimicus strains tested, indicating 100% sensitivity of the probe for the targeted species. For visible colonies of 10 nontarget species, the specificity of the probe was calculated to be 90% because of a weak positive signal produced by Grimontia (Vibrio) hollisae, a marine bacterium. When both the sensitivity and specificity of the assay were evaluated using lake water samples amended with a bioluminescent V. cholerae strain, no false-negative or false-positive results were found, indicating 100% sensitivity and specificity for culturable bacterial populations in freshwater samples when G. hollisae was not present. When the protocol was applied to laboratory microcosms containing V. cholerae attached to live copepods, copepods were found to carry approximately 10,000 to 50,000 CFU of V. cholerae per copepod. The protocol was also used to analyze pond water samples collected in an area of cholera endemicity in Bangladesh over a 9-month period. Water samples collected from six ponds demonstrated a peak in abundance of total culturable V. cholerae bacteria 1 to 2 months prior to observed increases in pathogenic V. cholerae and in clinical cases recorded by the area health clinic. The method provides a highly specific and sensitive tool for monitoring the dynamics of V. cholerae in the environment. The RNA blot hybridization protocol can also be applied to detection of other gram-negative bacteria for taxon-specific enumeration.Vibrio cholerae is autochthonous to the aquatic environment, but some strains produce enterotoxins and are capable of causing epidemics of the human disease cholera. Strains of V. cholerae are classified by their O antigen, with over 210 serogroups recognized to date. Seven cholera pandemics have occurred since 1832: while microbiologic data on the earlier pandemics are not available, the last two are known to have been caused by strains within serogroup O1, with the major pathogenic factor being production of cholera toxin. The genes encoding cholera toxin and other pathogenic factors have been shown to reside in a mobile genetic element of phage origin, designated CTXΦ (20).Standard microbiologic methods for isolation of V. cholerae present in natural waters rely primarily on a method originally developed for clinical diagnosis, namely, enrichment in alkaline peptone water, followed by subculture on selective media and confirmation using selected biochemical and immunological tests (7). The alkaline nature of the enrichment broth allows differential multiplication of Vibrio species but renders this method inappropriate for enumeration. PCR methods and oligonucleotide hybridization have been used to detect and enumerate toxigenic V. cholerae bacteria (3, 11, 12, 14, 15, 21). These methods typically rely on amplification of or hybridization to pathogenic markers, such as O1/O139 wbe, tcpA, and ctxA DNA sequences.However, occasional localized outbreaks of cholera have been caused by non-O1, non-O139 V. cholerae, which may be toxigenic or nontoxigenic. Conversely, many environmental V. cholerae O1 strains isolated from areas of endemicity do not harbor ctx genes (9). It has also been shown that CTXΦ is capable of lysogenic conversion of strains that are CTXΦ negative (20). Additionally, the cholera toxin (CTX) prophage has also been detected in clinical strains of V. mimicus, and V. mimicus has been proposed as a natural reservoir for CTXΦ (2). Furthermore, ecological studies of V. cholerae are often hampered by the fact that toxigenic strains represent only a small percentage of the total V. cholerae population in the environment, especially in areas where cholera is not endemic. These facts underline the need for a method of detection of the total number of V. cholerae bacteria present in environmental samples.The many copies of 16S rRNA molecules in each V. cholerae cell offer appropriate targets for species-specific enumeration. In this study, the probe Vchomim1276, previously described by Heidelberg et al. (4-6), was employed in an RNA colony blot hybridization protocol. The specificity and sensitivity of the probe were tested using type strains and environmental and clinical isolates. The method was evaluated using laboratory microcosms to which cells of V. cholerae were added, and the protocol was used to enumerate V. cholerae bacteria in samples collected from ponds in a region of cholera endemicity in Bangladesh.