Production of antibiotic substances by natural variants of the marine bacterium Vibrio Fischeri

It was shown that under definite conditions there was competition between natural variants of sea bacteria belonging to V. fischeri. Natural variants of V. fischeri, strain 6 differed in their resistance to streptomycin and had different growth rates under conditions of limited aeration. Morphologically all the variants were identical. V. fischeri P-0, V. fischeri P-1 and V. fischeri P-2 were studied. The study revealed that V. fischeri P-0 produced a non-dialysing thermostable trypsin-sensitive substance inhibiting the growth of V. fischeri P-1 and V. fischeri P-2. The maximum activity of the antibacterial substance was observed when V. fischeri P-0 was grown in a liquid medium with peptone and yeast extract without agitation at 26 degrees C. The inhibiting substance was also active against V. fischeri BKM B995 and V. fischeri P-7 isolated as a result of V. fischeri P-0 exposure to ethidium bromide. The substance had no effect on the following bacterial species: Aeromonas liquefaciens 301, Achromobacter liquefaciens, Pseudomonas putida 15, Pseudomonas fluorescence 7, Escherichia coli AH-32 and Staphylococcus aureus.     

Aposymbiotic culture of the sepiolid squid Euprymna scolopes: role of the symbiotic bacterium Vibrio fischeri in host animal growth, development, and light organ morphogenesis

The sepiolid squid Euprymna scolopes forms a bioluminescent mutualism with the luminous bacterium Vibrio fischeri, harboring V. fischeri cells in a complex ventral light organ and using the bacterial light in predator avoidance. To characterize the contribution of V. fischeri to the growth and development of E. scolopes and to define the long-term effects of bacterial colonization on light organ morphogenesis, we developed a mariculture system for the culture of E. scolopes from hatching to adulthood, employing artificial seawater, lighting that mimicked that of the natural environment, and provision of prey sized to match the developmental stage of E. scolopes. Animals colonized by V. fischeri and animals cultured in the absence of V. fischeri (aposymbiotic) grew and survived equally well, developed similarly, and reached sexual maturity at a similar age. Development of the light organ accessory tissues (lens, reflectors, and ink sac) was similar in colonized and aposymbiotic animals with no obvious morphometric or histological differences. Colonization by V. fischeri influenced regression of the ciliated epithelial appendages (CEAs), the long-term growth of the light organ epithelial tubules, and the appearance of the cells composing the ciliated ducts, which exhibit characteristics of secretory tissue. In certain cases, aposymbiotic animals retained the CEAs in a partially regressed state and remained competent to initiate symbiosis with V. fischeri into adulthood. In other cases, the CEAs regressed fully in aposymbiotic animals, and these animals were not colonizable. The results demonstrate that V. fischeri is not required for normal growth and development of the animal or for development of the accessory light organ tissues and that morphogenesis of only those tissues coming in contact with the bacteria (CEAs, ciliated ducts, and light organ epithelium) is altered by bacterial colonization of the light organ. Therefore, V. fischeri apparently makes no major metabolic contribution to E. scolopes beyond light production, and post-embryonic development of the light organ is essentially symbiont independent. J. Exp. Zool. 286:280-296, 2000.     

Laboratory glass fermenter on the basis of the airlift principle

A 1.5-1 minifermenter consisting of RASOTHERM glass on the basis of the airlift principle was constructed and tested in the Institute of Biotechnology. The development of this fermenter bridged the gap between shake flasks and lab-scale fermenters and provided a possibility for the continuous cultivation of extremely halophilic bacteria. It was demonstrated that the fermenter can be used for cultivating bacteria that are sensitive to shear stress. It can also be recommended for purposes of cultivation of plant and animal cells because of this advantage and the possibility of this fermenter to be sterilized.     

Effects of luxCDABEG induction in Vibrio fischeri: enhancement of symbiotic colonization and conditional attenuation of growth in culture

Production of bioluminescence theoretically represents a cost, energetic or otherwise, that could slow Vibrio fischeri growth; however, bioluminescence is also thought to enable full symbiotic colonization of the Euprymna scolopes light organ by V. fischeri. Previous tests of these models have proven inconclusive, partly because they compared nonisogenic strains, or undefined and/or pleiotropic mutants. To test the influence of the bioluminescence-producing lux operon on growth and symbiotic competence, we generated dark luxCDABEG mutants in strains MJ1 and ES114 without disrupting the luxR-luxI regulatory circuit. The MJ1 luxCDABEG mutant out-competed its visibly luminescent parent approximately 26% per generation in a carbon-limited chemostat. Similarly, induction of luminescence in the otherwise dim ES114 strain slowed growth relative to DeltaluxCDABEG mutants. Some culture conditions yielded no detectable effect of luminescence on growth, indicating that luminescence is not always growth limiting; however, luminescence was never found to confer an advantage in culture. In contrast to this conditional disadvantage of lux expression, ES114 achieved approximately fourfold higher populations than its luxCDABEG mutant in the light organ of E. scolopes. These results demonstrate that induction of luxCDABEG can slow V. fischeri growth under certain culture conditions and is a positive symbiotic colonization factor.     

Evolutionary perspectives in a mutualism of sepiolid squid and bioluminescent bacteria: combined usage of microbial experimental evolution and temporal population genetics

The symbiosis between marine bioluminescent Vibrio bacteria and the sepiolid squid Euprymna is a model for studying animal-bacterial Interactions. Vibrio symbionts native to particular Euprymna species are competitively dominant, capable of outcompeting foreign Vibrio strains from other Euprymna host species. Despite competitive dominance, secondary colonization events by invading nonnative Vibrio fischeri have occurred. Competitive dominance can be offset through superior nonnative numbers and advantage of early start host colonization by nonnatives, granting nonnative vibrios an opportunity to establish beachheads in foreign Euprymna hosts. Here, we show that nonnative V. fischeri are capable of rapid adaptation to novel sepiolid squid hosts by serially passaging V. fischeri JRM200 (native to Hawaiian Euprymna scolopes) lines through the novel Australian squid host E. tasmanica for 500 generations. These experiments were complemented by a temporal population genetics survey of V. fischeri, collected from E. tasmanica over a decade, which provided a perspective from the natural history of V. fischeri evolution over 15,000-20,000 generations in E. tasmanica. No symbiont anagenic evolution within squids was observed, as competitive dominance does not purge V. fischeri genetic diversity through time. Instead, abiotic factors affecting abundance of V. fischeri variants in the planktonic phase sustain temporal symbiont diversity, a property itself of ecological constraints imposed by V. fischeri host adaptation.     

Identification of the genes encoding NAD(P)H-flavin oxidoreductases that are similar in sequence to Escherichia coli Fre in four species of luminous bacteria: Photorhabdus luminescens, Vibrio fischeri, Vibrio harveyi, and Vibrio orientalis.

Genes encoding NAD(P)H-flavin oxidoreductases (flavin reductases) similar in both size and sequence to Fre, the most abundant flavin reductase in Escherichia coli, were identified in four species of luminous bacteria, Photorhabdus luminescens (ATCC 29999), Vibrio fischeri (ATCC 7744), Vibrio harveyi (ATCC 33843), and Vibrio orientalis (ATCC 33934). Nucleotide sequence analysis showed Fre-like flavin reductases in P. luminescens and V. fischeri to consist of 233 and 236 amino acids, respectively. As in E. coli Fre, Fre-like enzymes in luminous bacteria preferably used riboflavin as an electron acceptor when NADPH was used as an electron donor. These enzymes also were good suppliers of reduced flavin mononucleotide (FMNH2) to the bioluminescence reaction. In V. fischeri, the Fre-like enzyme is a minor flavin reductase representing < 10% of the total FMN reductase. That the V. fischeri Fre-like enzyme has no appreciable homology in amino acid sequence to the major flavin reductase in V. fischeri, FRase I, indicates that at least two different types of flavin reductases supply FMNH2 to the luminescence system in V. fischeri. Although Fre-like flavin reductases are highly similar in sequence to luxG gene products (LuxGs), Fre-like flavin reductases and LuxGs appear to constitute two separate groups of flavin-associated proteins.     

Design of a nutrient medium for bacteria of the genus Haemophilus--producers of restriction endonucleases

A culture medium for the cultivation of hemophilic bacteria, containing acidic casein hydrolysate, aminopeptide and fodder yeast extract, has been proposed. The growth-stimulating properties of this medium have been studied on 5 strains producing restrictases differing in their specificity. In growing these producer strains in a model ANKUM-2 fermenter with the supply of carbohydrate substrates (glucose, sucrose, glycerin) the yield of biomass, considered to be high for hemophilic bacteria (10-14 g of humid substance from 1 liter of the medium), has been achieved. As shown on H. influenzae Rc B-2297 used as an example, an increase in the yield of microbial biomass leads to a decrease in restrictase specific activity.     

Population dynamics of Vibrio fischeri during infection of Euprymna scolopes

The luminous bacterium Vibrio fischeri colonizes a specialized light-emitting organ within its squid host, Euprymna scolopes. Newly hatched juvenile squid must acquire their symbiont from ambient seawater, where the bacteria are present at low concentrations. To understand the population dynamics of V. fischeri during colonization more fully, we used mini-Tn7 transposons to mark bacteria with antibiotic resistance so that the growth of their progeny could be monitored. When grown in culture, there was no detectable metabolic burden on V. fischeri cells carrying the transposon, which inserts in single copy in a specific intergenic region of the V. fischeri genome. Strains marked with mini-Tn7 also appeared to be equivalent to the wild type in their ability to infect and multiply within the host during coinoculation experiments. Studies of the early stages of colonization suggested that only a few bacteria became associated with symbiotic tissue when animals were exposed for a discrete period (3 h) to an inoculum of V. fischeri cells equivalent to natural population levels; nevertheless, all these hosts became infected. When three differentially marked strains of V. fischeri were coincubated with juvenile squid, the number of strains recovered from an individual symbiotic organ was directly dependent on the size of the inoculum. Further, these results indicated that, when exposed to low numbers of V. fischeri, the host may become colonized by only one or a few bacterial cells, suggesting that symbiotic infection is highly efficient.     

Effect of oxygen supply on the suspension culture of genetically modified tobacco cells.

The effect of oxygen supply on the cultivation of the genetically modified tobacco cells and the formation of a foreign protein, beta-glucuronidase (GUS), was investigated in 250-mL Erlenmeyer flasks, a 5-L stirred tank fermenter, and a 7-L air-lift fermenter. The oxygen supply was varied by using different volumes of medium in the case of the 250-mL Erlenmeyer flask culture or by the different aeration rate in the case of the two types of fermenters tested. Higher oxygen supply stimulated cell growth and increased oxygen consumption rate, the level of phenolics, and GUS productions.     

Dominance of Vibrio fischeri in secreted mucus outside the light organ of Euprymna scolopes: the first site of symbiont specificity

Previous studies of the Euprymna scolopes-Vibrio fischeri symbiosis have demonstrated that, during colonization, the hatchling host secretes mucus in which gram-negative environmental bacteria amass in dense aggregations outside the sites of infection. In this study, experiments with green fluorescent protein-labeled symbiotic and nonsymbiotic species of gram-negative bacteria were used to characterize the behavior of cells in the aggregates. When hatchling animals were exposed to 10(3) to 10(6) V. fischeri cells/ml added to natural seawater, which contains a mix of approximately 10(6) nonspecific bacterial cells/ml, V. fischeri cells were the principal bacterial cells present in the aggregations. Furthermore, when animals were exposed to equal cell numbers of V. fischeri (either a motile or a nonmotile strain) and either Vibrio parahaemolyticus or Photobacterium leiognathi, phylogenetically related gram-negative bacteria that also occur in the host's habitat, the symbiont cells were dominant in the aggregations. The presence of V. fischeri did not compromise the viability of these other species in the aggregations, and no significant growth of V. fischeri cells was detected. These findings suggested that dominance results from the ability of V. fischeri either to accumulate or to be retained more effectively within the mucus. Viability of the V. fischeri cells was required for both the formation of tight aggregates and their dominance in the mucus. Neither of the V. fischeri quorum-sensing compounds accumulated in the aggregations, which suggested that the effects of these small signal molecules are not critical to V. fischeri dominance. Taken together, these data provide evidence that the specificity of the squid-vibrio symbiosis begins early in the interaction, in the mucus where the symbionts aggregate outside of the light organ.     

培养基除菌过滤在百日咳菌苗生产中的应用

经一系列试验证实百日咳菌大罐培养浓度的下降与培养基中不耐热营养因子不足有关,将培养基的高压灭菌方式改为除菌过滤,保留热不稳定营养因子,可以提高百日咳菌大罐培养浓度;优化酸沉淀条件后,提高了菌苗回收率,从而节省了大量培养基,缩短了生产时间,获得了良好的经济效益。     

Physiology of the growth and supersynthesis of DNA-polymerase in Escherichia coli during 2-stage continuous cultivation

The physiology of growth under the conditions of batch and continuous cultivation was studied with the recombinant strain of Escherichia coli CM 5199 capable of DNA polymerase I superproduction. The specific growth rate of the strain is 0.8 h-1 under the conditions of continuous cultivation which is almost 2.5 times greater than that in the exponential phase of batch cultivation. When the strain was cultivated at a flow rate above 0.3 h-1, the biomass concentration in the fermenter decreased and the culture was no more limited by the carbon source in the absence of other growth limiting components of the medium. Apparently, the metabolic product ceased to inhibit high growth rates of the culture under the conditions of continuous cultivation. The rate of DNA polymerase synthesis correlated with the specific growth rate and the respiration activity of the culture when the lambda pol A prophage was induced in the cells. The authors discuss the effectiveness of ribosome operation in the cells at a growth rate of 0.05 to 0.3 h-1 and the content of ribosomes at a higher growth rate in relation to DNA polymerase I synthesis.     

Continuous culture of photobacterium

The design and performance characteristics of a small volume (20 ml) continuous culture device for the cultivation of luminous bacteria are described. This simply constructed device can be used to supply luminescent bacteria with constant properties for either laboratory use or the assay of environmental pollutants. Furthermore, bacteria can be deployed to make sensitive (<1 nM) oxygen measurements. The culture device may be configured, alone or in combination, as a chemostat, turbidostat or a "bioluminostat" where bacterial bioluminescence becomes the controlling variable. Over extended periods (>1 week) it was possible to maintain steady-state luminescence within 5% of a pre-set value, although occasionally a non-bioluminescent "mutant" became dominant; in this case light emission was irreversibly lost. A secondary chamber provided additional flexibility and easy manipulation of the cultivated bacteria for testing. The continuous culture system is also suitable for the growth of recombinant microorganisms that either constitutively express luciferase, or do so in response to stress promoter activity. The non-standard control methodologies reported may have important research and industrial applications, for example in providing immediate process control or as an inferential method to optimize biomass: product yield ratios.     

Optimization of culture conditions for production of the anti-tubercular alkaloid hirsutellone A by Trichoderma gelatinosum BCC 7579

AIMS: This work aimed to optimize the culture conditions for production of a novel and potent anti-tubercular alkaloid, hirsutellone A, by the saprophytic soil fungus Trichoderma gelatinosum BCC 7579. METHODS AND RESULTS: The fungus was initially cultured in shake flasks at 25 degrees C in the potato dextrose broth (PDB) supplemented with various carbon and nitrogen sources and mineral salts to select suitable medium for mycelial growth and hirsutellone A production. Cultivation conditions were further optimized by adjusting initial pH and changing temperature levels to maximize the production of hirsutellone A. The optimal condition that increased the production of hirsutellone A from 19.04 mg l(-1), obtained from basal condition, to 610.55 mg l(-1) and reduced the cultivation time from 40 to 6 days was to cultivate in a shaker at 200 rev min(-1) at 25 degrees C in PDB plus 20 g l(-1) soluble starch, 10 g l(-1) peptone and 2.5% (v/v) salt solution with initial pH of 7. Production of hirsutellone A in larger-scale using a 5-l batch fermenter was also completed yielding 958 mg l(-1) of hirsutellone A within 6 days. CONCLUSIONS: The suitable culture conditions for hirsutellone A production by T. gelatinosum BCC 7579 was the cultivation in 5-l fermenter at 25 degrees C in PDB plus 20 g l(-1) soluble starch, 10 g l(-1) peptone and 2.5% (v/v) salt solution with an initial pH of 7. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of hirsutellone A in a fermenter to obtain a high yield and reduce an incubation period will become very useful in anti-tubercular drug development process in the future.     

The regulating action of oxygen on the nutritional requirements of Neisseria meningitidis

The study has revealed regularities in changing nutritional requirements of Neisseria meningitidis with changes in the degree of the oxygen saturation of the culture medium in a fermenter under the conditions of the controlled cultivation of N. meningitidis in a synthetic culture medium in the process of batch, semicontinuous and continuous flow cultivation. As shown in this study, when oxygen supply is limited, the consumption of carbohydrates prevails, while in the presence of surplus oxygen the prevalence of the consumption of amino nitrogen is observed.     

Small molecule-mediated density-dependent control of gene expression in prokaryotes: bioluminescence and the biosynthesis of carbapenem antibiotics.

Sophisticated signal transduction systems enable prokaryotes to sense their growth environment and mount an appropriate adaptive response. Signal transduction and gene regulation through the phosphorylation of two regulatory components is now recognised as one of the major global regulatory networks in bacteria. However, not all types of sensor-regulator circuits relay information via phosphoryl transfer. The Vibrio fischeri LuxR protein which has previously been characterised as a member of the response-regulator superfamily responds to a small diffusible signal molecule N-(3-oxohexanoyl)homoserine lactone (HSL). Biosynthesis of HSL in V. fischeri is dependent on the expression of the luxI gene. Until recently, the role of HSL as an 'autoinducer' was thought to be restricted to V. fischeri and a few related marine bacteria in which it controls the onset of bioluminescence. However, we have discovered that a diverse group of terrestrial bacteria: (1) produce HSL; (2) possess genes analogous to luxI; and (3) exhibit cell density-dependent induction of bioluminesence when transformed with a recombinant plasmid carrying V. fischeri lux genes but lacking luxI. In one of these, Erwinia carotovora, HSL is shown to mediate the cell density-dependent biosynthesis of a carbapenem antibiotic.