Fermentation Studies with Streptomyces griseus: II. Synthetic Media for the Production of Streptomycin

Synthetic media for streptomycin fermentation were studied to determine which media gave highest yields of streptomycin. The effect of salts on streptomycin production by Streptomyces griseus was examined, and a suitable combination of salts was established in a glucose-casein medium. This medium yielded 3,000 μg/ml of the antibiotic with an inoculum of 1.6%. Substitution of amino acids for casein was examined. Of 17 amino acids tested, best results were obtaind with sodium aspartate. Substitution of ammonium salts was tried, and an excellent streptomycin yield was obtained with a medium containing ammonium citrate.     

In vitro inhibition of diphtheria toxin action by ammonium salts and amines

Kim, K. (University of Washington, Seattle), and N. B. Groman. In vitro inhibition of diphtheria toxin action by ammonium salts and amines. J. Bacteriol. 90:1552-1556. 1965.-An inhibitor for diphtheria toxin action on HeLa cells was demonstrated in the growth supernatant fractions of both toxinogenic and nontoxinogenic strains of Corynebacterium diphtheriae and in the Mueller and Miller medium in which these organisms were grown. The inhibitor in the growth supernatant fractions of the nontoxinogenic strain was dialyzable, stable to autoclaving, and stable on storage in the refrigerator for a period of many months, but was destroyed by ashing. When the components of Mueller and Miller medium were analyzed, only the Casamino Acids proved inhibitory. Further study with artificial mixtures of amino acids revealed that glutamine alone inhibited toxin. It was subsequently shown that ammonium salts and the aliphatic amines, glycamine and prolamine, could also function as inhibitors. Histamine and 16 amino acids tested individually were ineffective. The effectiveness of the amines and the ineffectiveness of sodium or potassium ions indicates that there is a specific requirement for inhibition.     

The cellular role of nitrogen in the biosynthesis of alkaloids by submerged culture of Claviceps purpurea (Fr.) Tul.

Asparagine was a superior nitrogen source for clavine-alkaloid production in Claviceps purpurea. Its transport into the cell excedded the cell's biosynthetic need for this amino acid. Asparagine entered the cell without degradation. This disturbed the relative pool sizes of various amino acids resulting in a change in the genetically determined ratio at which amino acids were utilized for protein synthesis. Overproduction of alkaloids (4500 mug.ml-1) may be associated with increased availability of tryptophan because of the enhanced assimilation of asparagine-derived ammonia via glutamine synthetase (EC 6.3.1.2). However, ammonium salts in the fermentation broth led to a depression of the alkaloid yield. Partial replacement of the ammonium salt by aspartic acid elevated alkaloid production.     

Toxin profiles of Vibrio cholerae non-O1 from environmental sources in Calcutta, India

A collection of Vibrio cholerae non-O1 isolated from the aquatic environs of Calcutta, a cholera-hyperendemic area, were examined for the production of cholera toxin (CT), Shiga-like toxins (Vero toxins), heat-stable enterotoxin, and hemolysins. Two (0.5%) V. cholerae non-O1 isolates produced CT. The DNA from both these isolates also hybridized with a DNA probe containing sequences encoding the A subunit of CT. None of the strains produced Shiga-like toxins or heat-stable enterotoxin. Hemolytic activity was observed in 89.7% of the strains, of which 36.1% exhibited biological activity in the suckling mouse. However, none of them produced a hemolysin that cross-reacted with the thermostable direct hemolysin of Vibrio parahaemolyticus. It appears from this study that a small percentage of environmental V. cholerae non-O1 strains do possess the potential for causing cholera-like diarrhea.     

Toxin profiles of Vibrio cholerae non-O1 from environmental sources in Calcutta, India.

A collection of Vibrio cholerae non-O1 isolated from the aquatic environs of Calcutta, a cholera-hyperendemic area, were examined for the production of cholera toxin (CT), Shiga-like toxins (Vero toxins), heat-stable enterotoxin, and hemolysins. Two (0.5%) V. cholerae non-O1 isolates produced CT. The DNA from both these isolates also hybridized with a DNA probe containing sequences encoding the A subunit of CT. None of the strains produced Shiga-like toxins or heat-stable enterotoxin. Hemolytic activity was observed in 89.7% of the strains, of which 36.1% exhibited biological activity in the suckling mouse. However, none of them produced a hemolysin that cross-reacted with the thermostable direct hemolysin of Vibrio parahaemolyticus. It appears from this study that a small percentage of environmental V. cholerae non-O1 strains do possess the potential for causing cholera-like diarrhea.     

Molecular genetic features of Vibrio cholerae classica strains, that caused the Asiatic cholera epidemic in Russian in 1942

Molecular genetic features of Vibrio cholerae classical strains which caused an epidemic of Asian cholera in Russia in 1942 have been studied for the first time. These strains had a high level of choleric toxin production and toxin-coregulated adhesion piles, the main virulence factors; all the strains were auxotrophs and needed purine and/or amino acids for growth in minimal medium. Moreover, having hapA structural gene in the chromosome (according to polymerase chain reaction), they did not produce soluble hemagglutinin protease promoting propagation of vibrios in the environment. These features of natural V. cholerae classical strains are apparently responsible for the peculiar infectious and epidemic processes in the cholera epidemic.     

Cholera enterotoxin production in Vibrio cholerae O1 strains isolated from the environment and from humans in Japan.

Vibrio cholerae O1 strains isolated from various sources in Japan over the years 1977 through 1987 were examined to confirm the presence or absence of the cholera enterotoxin (CT) gene and production of CT and to determine the kappa-phage type. The CT gene was detected in none of 225 isolates from natural waters but was present in all of the 10 isolates from environmental waters implicated in domestic cholera cases, in 64 strains (26.6%) of the 241 isolates from imported seafoods, in 43 strains (95.6%) of the 45 isolates from domestic cholera cases, and in 119 strains (93.7%) of the 127 isolates from imported cholera cases. The results suggest that the CT gene-positive strains of V. cholerae O1 have been imported into Japan through seafoods and/or by travelers. Sporadic cholera cases have resulted in contamination of the surrounding environment, but the CT gene-positive strains may not have persisted in natural waters to serve as a reservoir for epidemic cholera. The commercially available VET-RPLA kit (a latex agglutination kit for immunological detection of CT) detected production of CT in all of the CT gene-positive strains, indicating that there was no silent CT gene in the test strains. There was a strong correlation between the kappa-phage type and the presence or absence of the CT gene, suggesting a significant clonal difference between CT gene-positive and -negative strains. Five CT gene-negative strains isolated from imported cholera cases (travelers with mild diarrhea) induced a considerable amount of fluid accumulation in rabbit and/or suckling mouse intestines, indicating production of an enterotoxic factor(s) other than CT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholera enterotoxin production in Vibrio cholerae O1 strains isolated from the environment and from humans in Japan.

Vibrio cholerae O1 strains isolated from various sources in Japan over the years 1977 through 1987 were examined to confirm the presence or absence of the cholera enterotoxin (CT) gene and production of CT and to determine the kappa-phage type. The CT gene was detected in none of 225 isolates from natural waters but was present in all of the 10 isolates from environmental waters implicated in domestic cholera cases, in 64 strains (26.6%) of the 241 isolates from imported seafoods, in 43 strains (95.6%) of the 45 isolates from domestic cholera cases, and in 119 strains (93.7%) of the 127 isolates from imported cholera cases. The results suggest that the CT gene-positive strains of V. cholerae O1 have been imported into Japan through seafoods and/or by travelers. Sporadic cholera cases have resulted in contamination of the surrounding environment, but the CT gene-positive strains may not have persisted in natural waters to serve as a reservoir for epidemic cholera. The commercially available VET-RPLA kit (a latex agglutination kit for immunological detection of CT) detected production of CT in all of the CT gene-positive strains, indicating that there was no silent CT gene in the test strains. There was a strong correlation between the kappa-phage type and the presence or absence of the CT gene, suggesting a significant clonal difference between CT gene-positive and -negative strains. Five CT gene-negative strains isolated from imported cholera cases (travelers with mild diarrhea) induced a considerable amount of fluid accumulation in rabbit and/or suckling mouse intestines, indicating production of an enterotoxic factor(s) other than CT.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of AmtR on growth and amino acids production in Corynebacterium glutamicum

AmtR, the master regulator of nitrogen control in Corynebacterium glutamicum, plays important roles in nitrogen metabolism. To investigate the influence of AmtR on amino acids production in C. glutamicum ATCC 13032, the amtR deletion strain C. glutamicum Q1 was constructed and cultured in modified CGXII minimal medium for 60 h. The ammonium consumption rates as well as amino acids production of both strains cultured in modified CGXII minimal medium were determined. The amtR deletion in C. glutamicum caused an obvious growth defect in the exponential growth phase, but both strains had the same biomass in the stationary phases. Maybe the less alpha-oxoglutarate was used for the tricarboxylic acid cycle to influence the growth of strains. During 12 h, the rate of ammonium consumption and the concentration of Glu, Pro, Arg and Ser were higher but Asp, Gly, Ile, Leu, Lys were lower in the mutation strain. During 48 h, the Q1 had higher levels of Asp, Lys, Pro, Ala and Val,and lower levels of Glu, Arg, Leu and Ile, compared to the wild. The more Glu was synthesized by the activated GS/GOGAT pathway in Q1, and then the accumulation of relative amino acids (Pro, Arg and Ser) were up-regulated within 12 h growth. After 48 h growth, the amtR deletion obviously influenced accumulation of Ala, Asp and Pro. The amtR deletion could influence the growth and amino acids production, which could be useful to the production of amino acids.     

Nutritional Studies on Xanthan Production by Xanthomonas campestris NRRL B1459

The nutritional requirements of Xanthomonas campestris NRRL B1459 for optimal xanthan production were studied in a chemically defined medium. Of the carbon sources tested, a 4% sucrose or glucose medium yielded the highest xanthan titers. The further addition of certain organic acids, such as succinate, pyruvate, and α-ketoglutarate, stimulated xanthan production; excess concentrations of these organic acids inhibited xanthan formation. Certain amino acids (e.g., glutamate) and nitrate salts were superior to ammonium salts for xanthan production. Concentrations of these nitrogen sources higher than the optimal levels inhibited xanthan production while stimulating growth. Xanthan production was also sensitive to high concentrations of inorganic phosphate. High xanthan potencies, up to 30 g/kg of broth, were achieved in these shake-flask studies, in which completely defined media were used.     

Nitrogen nutrition and regulation of cephalosporin production in Streptomyces clavuligerus.

When used as sole nitrogen source, certain amino acids (e.g., proline, asparagine) supported both growth and sporulation by Streptomyces clavuligerus streaked onto solid defined medium. Ammonium supported growth but suppressed sporulation. Amino nitrogen was best for cephalosporin production in liquid defined medium, although urea was almost as useful. A comparison of amino acids showed asparagine and glutamine to be the best nitrogen sources and arginine to be almost as good. Ammonium salts supported a somewhat lower growth rate than asparagine, but antibiotic production was very poor on these inorganic nitrogen sources. Addition of ammonium to asparagine did not affect growth rate but increased mycelial mass; cephalosporin production was reduced by about 75%. Antibiotic production was more closely associated with growth in the absence of ammonium than in its presence, indicating a strong inhibitory and (or) repressive effect of NH4+ on antibiotic production. Ammonium exerted its negative effect when added at 24h or earlier, i.e. before antibiotic formation began.     

ompU genes in non-toxigenic Vibrio cholerae associated with aquaculture

AIMS: The study was undertaken with the objective of understanding the virulence-associated genes of the CTX and TCP gene clusters in environmental isolates of Vibrio cholerae, an important human pathogen, isolated from the aquaculture environment. The involvement of the ompU gene in conferring bile resistance in these isolates was also evaluated. METHODS AND RESULTS: The V. cholerae isolates were tested by PCR and fluorescent antibody test for O1 (Ogawa and Inaba) and O139 serotypes. All isolates were found to be non-toxigenic V. cholerae confirmed by their positive PCR reaction for toxR but negative for ctx, zot and tcp gene. The hlyA gene was detected in 85% of the strains and ompU in 77%. The results on the bactericidal effect of bile salts suggest that ompU may play a role in conferring bile resistance in non-O1/non-O139 strains. CONCLUSION: The results of the study indicate that most environmental strains lacked the CTX and TCP gene clusters. However, most isolates had the hlyA gene indicating the potential of these environmental strains to cause mild gastroenteritis. It was also observed that strains lacking ompU showed less tolerance to bile salts. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on virulence factors of V. cholerae associated with aquaculture environment and products would be of value in risk assessment for human health.     

Lysophospholipase L2 of Vibrio cholerae O1 affects cholera toxin production

Abstract The implication in cholera toxin (CT) production of the newly identified gene, lypA , that encodes the lysophospholipase L₂ of Vibrio cholerae , was investigated. Introduction of lypA into the V. cholerae O1 mutant (NF404), which has a Tn5-insertion in lypA and has lost CT as well as haemolysin production, restored the lysophospholipase activity and CT production but not the haemolytic activity. Inactivation of the lypA gene of the wild-type strain by chromosomal integration of a plasmid containing a portion of the lypA gene decreased the lysophospholipase L₂ activity and the production of CT but not the haemolytic activity. Furthermore, constructed mutants of El Tor-biotype and Classical-biotype strains which have a defective lypA failed to produce CT and exhibited decreased enterotoxicity in the ligated rabbit ileal loop test. These results suggest that lypA is possibly required for the expression of CT and may play a role in pathogenicity of V. cholerae .     

Comparative study of different methods for detection of toxic and other enzymatic factors in Vibrio cholerae strains

The purpose of this work was to characterize the toxin profile and the presence of other virulence factors involved in the pathogenesis and biology of 13 V. cholerae O1 (11 clinical cases and 2 waters) and 6 V. cholerae non O1 strains (4 clinical cases and 2 waters) using genetic (PCR), immunological (RPLA), biochemical (NAD degradation, haemolysis, Kanagawa phenomenon, caseinase, lecithinase, mucinase, amylase, esculine hydrolysis) and cell culture (Vero E6, HEp-2) assays. The results indicated a concordance between PCR-RPLA (84%), PCR-NAD (73%) and RPLA-NAD (84%) methods. The sensitivity of RPLA and NAD degradation methods were comparable to PCR in detecting CT in Vibrio cholerae O1 strains. Although NAD degradation method was not exclusively specific for the CT detection, it proved its usefulness in screening certain virulent, CT-negative clones of V. cholerae. The cytotoxic effect on Vero E6 cells, enzyme production (Kanagawa haemolysins, lecithinase, caseinase, esculine hydrolysis) as well as adherence ability on inert substrate proved to be much more constant in V. cholerae non O1 (CT- negative) than in V. cholerae O1 (CT-positive). All V. cholerae non O1 strains isolated in diarrheal cases were Kanagawa positive. This complex of virulence factors detected in V. cholerae non O1 strains could probably contribute during interepidemic periods to human-to-human transmission and to greater resistance as compared to O1 strains in the environment.     

Vibrio cholerae VexH encodes a multiple drug efflux pump that contributes to the production of cholera toxin and the toxin co-regulated pilus

The resistance-nodulation-division (RND) efflux systems are ubiquitous transporters that function in antimicrobial resistance. Recent studies showed that RND systems were required for virulence factor production in Vibrio cholerae. The V. cholerae genome encodes six RND efflux systems. Three of the RND systems (VexB, VexD, and VexK) were previously shown to be redundant for in vitro resistance to bile acids and detergents. A mutant lacking the VexB, VexD, and VexK RND pumps produced wild-type levels of cholera toxin (CT) and the toxin co-regulated pilus (TCP) and was moderately attenuated for intestinal colonization. In contrast, a RND negative mutant produced significantly reduced amounts of CT and TCP and displayed a severe colonization defect. This suggested that one or more of the three uncharacterized RND efflux systems (i.e. VexF, VexH, and VexM) were required for pathogenesis. In this study, a genetic approach was used to generate a panel of V. cholerae RND efflux pump mutants in order to determine the function of VexH in antimicrobial resistance, virulence factor production, and intestinal colonization. VexH contributed to in vitro antimicrobial resistance and exhibited a broad substrate specificity that was redundant with the VexB, VexD, and VexK RND efflux pumps. These four efflux pumps were responsible for in vitro antimicrobial resistance and were required for virulence factor production and intestinal colonization. Mutation of the VexF and/or VexM efflux pumps did not affect in vitro antimicrobial resistance, but did negatively affect CT and TCP production. Collectively, our results demonstrate that the V. cholerae RND efflux pumps have redundant functions in antimicrobial resistance and virulence factor production. This suggests that the RND efflux systems contribute to V. cholerae pathogenesis by providing the bacterium with protection against antimicrobial compounds that are present in the host and by contributing to the regulated expression of virulence factors.     

The effect of AmtR on growth and amino acids production in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

AmtR, the master regulator of nitrogen control in Corynebacterium glutamicum, plays important roles in nitrogen metabolism. To investigate the influence of AmtR on amino acids production in C. glutamicum ATCC 13032, the amtR deletion strain C. glutamicum Q1 was constructed and cultured in modified CGXII minimal medium for 60 h. The ammonium consumption rates as well as amino acids production of both strains cultured in modified CGXII minimal medium were determined. The amtR deletion in C. glutamicum caused an obvious growth defect in the exponential growth phase, but both strains had the same biomass in the stationary phases. Maybe the less α-oxoglutarate was used for the tricarboxylic acid cycle to influence the growth of strains. During 12 h, the rate of ammonium consumption and the concentration of Glu, Pro, Arg and Ser were higher but Asp, Gly, He, Leu, Lys were lower in the mutation strain. During 48 h, the Q1 had higher levels of Asp, Lys, Pro, Ala and Val, and lower levels of Glu, Arg, Leu and Ile, compared to the wild. The more Glu was synthesized by the activated GS/GOGAT pathway in Q1, and then the accumulation of relative amino acids (Pro, Arg and Ser) were up-regulated within 12 h growth. After 48 h growth, the amtR deletion obviously influenced accumulation of Ala, Asp and Pro. The amtR deletion could influence the growth and amino acids production, which could be useful to the production of amino acids.     

Nutrient regulation of epothilone biosynthesis in heterologous and native production strains

Fermentation media with different initial concentrations of ammonium and phosphate salts were used to study the inhibitory effects of those ions on growth and production of epothilone in Sorangium cellulosum and Myxococcus xanthus. The native epothilone producer, S. cellulosum was more sensitive to ammonium and phosphate than the heterologous producer, M. xanthus. An ammonium concentration of 12 mM reduced epothilone titers by 90% in S. cellulosum but by only 40% in M. xanthus. When 5 mM phosphate was added to the medium, production in both strains was 60% lower. Higher phosphate concentrations had little additional effect on M. xanthus titers, but epothilone production with 17 mM extra-cellular phosphate in S. cellulosum was 95% lower than in the control condition. The effect of iron supplementation to the fermentation medium was also investigated. Both strains showed best production with 20 microM iron added to the medium.     

Chemotaxis of cholera vibrios: a study method and the relation to different substances

The chemotaxis of V. cholerae in response to 56 different substances (amino acids, carbohydrates, salts, etc.) has been studied by the methods of visual observation and quantitative determination. Attractants, neutral substances and one repellent have been revealed. Adler's method (1973) has been modified with regard to the requirements for the working procedures in handling the causative agents of highly dangerous infections.     

Cholera toxin gene-positive Vibrio cholerae O1 Ogawa and Inaba strains produce the new cholera toxin

Abstract Two strains of cholera toxin (CT) gene-positive Vibrio cholerae O1, Ogawa, isolated from patients with diarrhoea and the hypertoxigenic V. cholerae O1, Inaba (569B), were found to produce the new cholera toxin that has earlier been demonstrated to be elaborated by CT gene-negative human and environmental isolates of V. cholerae O1. The CT gene-positive strains produce the new cholera toxin simultaneously with CT, indicating that they contain the gene coding for the new cholera toxin in addition to that of CT.