Streptomyces species producing the streptovaricin complex

Morphological, cultural and physiological-biochemical properties ofStreptomyces sp. strain 1000 and its antibiotic production were investigated. Antibiotics 1011 (identical with the streptovaricin complex) and 1012 (with antibacterial action) were isolated from the cultural broth of this strain. The overproducing natural variant 1011 was isolated from the population of a strain producing antibiotic 1011 at a concentration of 1000 mg/L (activity of the parent strain represents 41 mg/L only). Comparative taxonomical characteristic ofStreptomyces sp. strain 1000 with strains fromS. spectabilis showed that the strain 1000 differed in some properties and antibiotic production being considered as a new variant ofS. spectabilis. The strain shows an expressed antibiotic activity against G+ as well as G− bacterial and yeasts.     

Characterization and infectivity of a spontaneous variant isolated fromFrankia sp. WEY 0131391

Summary A spontaneous variant, obtained from aFrankia isolate fromAlnus rubra nodules, was compared with the parent strain with regard to infectivity, nitrogenase activity, and electrophoretic and immunological profiles. Both the parent and the variant strain were equally effective in inducing nodulation in seedlings ofA. rubra. All inoculated plants had an active nitrogenase system as measured by the acetylene reduction assay. Electrophoresis of whole cell homogenates on SDS-polyacrylamide slab gels showed similar electrophoretic profiles; however, the variant strain also exhibited striking differences in protein patterns that distinguish it from the parent strain. Immunological analysis of the originalFrankia strain and its variant revealed shared antigens as well as immunologically distinct antigenic determinants in the two strains. The variant strain exhibits a distinct morphology and growth patterns which remain stable after many passages through culture.     

Influence of cultivation conditions on citrate production by Aspergillus niger in a semi-pilot-scale plant

The influence of some fermentation parameters on the semi-pilot scale (alteration of growth conditions,e.g., sugar concentration, incubation temperature and initial pH) on citrate production was demonstrated in parent and mutant strains ofAspergillus niger. Raw material from sugar industry (cane molasses) was examined as basal fermentation medium in a stirred stainless-steel 15-L fermentor. After growth on medium with 150 g/L sugar, the parent strain produced 51.2 g/L citric acid; the mutant strain achieved production maximum of 96.2 g/L. Comparing the growth, kinetic (volumetric substrate uptake rate, rate of substrate consumption and volumetric productivity rate) and production parameters it was found that the mutant strain grows more rapidly, with slightly changed morphology (intermediate, shiny round pellets with diameter 0.6–0.7 mm), and exhibits a higher citrate production and higher efficiency of sugar utilization.     

Production and characteristics of the exocellular polysaccharide in mutant strains ofXanthomonas fuscans

Production of the exocellular polysaccharide of the phytopathogenic bacteriumXanthomonas fuscans was investigated with respect to its possible use in utilization of industrial wastes containing lactose. Six stablelac ⁺ mutants were obtained after the treatment withN-methyl-N′-nitroso-N′-nitroguanidine. The mutants were compared with the parent strain. Morphological and cultivation characteristics, as well as production of the exooellular polysaccharide were compared. The production was found to be maximal during the stationary phase of growth in strains cultivated under submerged conditions. Gas chromatography revealed that the polysaccharide of the parent strain is formed by α- and β-D-glucose and α- and β-d-mannose with a small amount ofd-ribose and 6-deoxy-l-mannose. Composition of the polysaccharides produced by the mutant strains (lac ⁺) does not qualitatively differ from that of the parent strain. However, they were found to contain a higher quantity ofd-mannose, which is favourable for their industrial utilization.     

Growth rate,sugar consumption,chlortetracycline production and anhydrotetracycline oxygenase activity inStreptomyces aureofaciens

The relationship between the activity of ATC oxygenase, CTC production and growth rate was investigated in a low-producing strain ofStreptomyces aureofaciens, closely related to the wildtype strain, and in a higher-producing variant. Different growth rates were achieved by using glucose, fructose and sucrose as carbon sources. Activity of the enzyme and CTC yield in both strains were inversely proportional to the rate of sugar utilization but in the higherproducing variant sugar utilization was slower than in the low-producing strain. The expression of ATC oxygenase was less sensitive to “catabolite repression” in the higherproduc ing strain. BT, a stimulator of CTC production, markedly inhibited growth of the higher-producing variant in a medium with slowly utilized sugars (fructose and sucrose) but had little effect on growth of the lowproducing strain. It also increased the level of ATC oxygenase in both strains under all experimental conditions. It could be established that there was no obligatory relationship between the increase of antibiotic synthesis and the increase of enzyme activity.     

Studies on histoplasmosis. V protective effect of HIF-coated Histoplasma against challenge

Summary Results of testing a total of 145 hamsters in 2 separate trials (a total of 90 challenged animals) suggest the potentiality of HIF-coatedHistoplasma yeast cells as a vaccine. Animals challenged with 5×10⁵ Histoplasma yeast cells in saline 3 weeks after initial inoculation with 5 × 10⁴ Histoplasma yest cells in 1) saline or 2) crude HIF in kidney homogenate or 3) the active 280 mµ fraction thereof were protected by the 280 mµ fraction > the crude HIF > saline suspensions ofHistoplasma, based on analysis of tissue sections and cultures.It is suggested that since HIF has also been found in experimental blasomycosis it should be sought in other mycoses, perhaps in all infectious diseases, and its potential use in a vaccine preparation should be more extensively tested.Supported in part by Public Health Service Grant No. 1-SO-IFR-05359-01 to the senior author at The George Washington University.     

Intracellular proteolytic activity during sporulation ofBacillus megaterium

Intracellular proteolytic activity increased during incubation of the sporogenic strain ofBacillus megaterium KM in a sporulation medium together with excretion of an extracellular metalloprotease. The exocellular protease activity in a constant volume of the medium reached a 100-fold value with respeot to the intracellular activity. Maximal values of the activity of both the extracellular and intracellular enzyme were reached after 3 – 5 h of incubation. After 7 h 20 – 50% cells formed refractile spores. The intracellular proteolytic system hydrolyzed denatured proteinsin vitro at a rate up to 150 μg mg_-1 h_-1 and native proteins at a rate up to 70 μg mg^-1 h^-1. Degradation of proteinsin vivo proceeded from the beginning of transfer to the sporulation medium at a constant rate of 40 μg mg^-1 h^-1 and the inactivation of beta-galactosidase at a rate of 70 μg mg^-1 h^-1. The intracellular proteolytic activity was inhibited to 65 – 88% by EDTA, to 23 – 76% by PMSF. Proteolysis of denatured proteins was inhibited both by EDTA and PMSF more pronouncedly than proteolysis of native proteins; 50 – 65% of the activity were localized in protoplasts. Another strain ofBacillus megaterium (J) characterized by a high (up to 90%) and synchronous sporulation activity was found to behave in a similar way, but the rate of protein turnover in this strain was almost twice as high. The asporogenic strain ofBacillus megaterium KM synthesized the exocellular protease in the sporulation medium, but its protein turnover was found to decrease substantially after 3 – 4 h. The intraeellular proteolytic system of the sporogenic strain J and the asporogenic strain KM were also inhibited by EDTA and PMSF.     

An ultrastructural comparison of the cell envelopes of selected strains of Nocardia asteroides and Nocardia brasiliensis

Growth curves were determined for three strains each ofNocardia asteroides andNocardia brasiliensis. Two strains ofN. brasiliensis and one strain ofN. asteroides had longer lag periods of growth than the remaining three strains. All strains had generation times of approximately 5.5 hours.The ultrastructure of the cell envelope of eachNocardia strain in early stationary phase growth was also examined. All the strains had typical trilaminar cell walls and cell membranes. The thickness of the cell wall layers, especially the inner peptidoglycan layer, varied from strain to strain. The inner layer of two strains ofN. brasiliensis and one strain ofN. asteroides was 12 nm or more in thickness, while that of the remaining three strains was 7 nm thick. These observed differences in growth patterns and/or thickness of the cell wall layers could be correlated to the varying degress of virulence as well as the divergent pathologies exhibited by these organisms.     

Mutants ofEmmonsia crescens — Their pathogenicity and size of adiasporesin vivo

The pathogenicity of seven morphological mutants ofEmmonsia crescens was tested by means of intraperitoneal inoculation in mice. All mutants caused adiaspiromycosis. Adiaspores were isolated from granulomas after 2 months and their diameters were determined. Adiaspores from granulomas caused by five mutants (M-5, M-6, M-8, M-9 and M-16) were significantly smaller than adiaspores from granulomas caused by the wild strain, from which the mutants were derived. Two mutants (M-6 and M-9) produced adiaspores of the smallest diameter (130.5 and 119.9 μm) with the lowest variance of values, differing thus most from the original wild strain with adiaspores of 230.4 μm in diameter. A positive correlation was found between the size of the adiasporein vivo and growth rate of the mycelial stage ofEmmonsia crescens in vitro. The mutation characterized by the decreased growth rate of the mycelial stage is pheno-typically manifested in the adiasporic stage of the life cycle ofEmmonsia crescens, i.e. by the smaller average size of adiaspores in granulomas.     

Production of a biosurfactant exhibiting excellent emulsification and surface active properties bySerratia marcescens

A biosurfactant exhibiting excellent emulsification activity and surface properties was isolated during growth ofSerratia marcescens on 2% (w/v) sucrose. Reduction in surface tension values and increase in the yield of biosurfactant during the late log phase of growth indicates that the biosurfactant is a secondary microbial metabolite. The biosurfactant formed stable emulsions with a wide variety of hydrocarbons. The isolated surface-active compound has a potential application in enhanced oil recovery and is stable over a wide range of temperatures (10-120‡C) and pH (2-12). This is the first report of effective and stable emulsion formation by a strain ofSerratia marcescens.     

Senescence associated with the over-replication of a mitochondrial retroplasmid in Neurospora crassa

Mitochondrial DNA rearrangements and deletions are a prevailing feature of filamentous fungal cultures that undergo senescence. In Neurospora spp., strains containing the Mauriceville and Varkud mitochondrial retroplasmids routinely senesce at elevated temperatures, a process that is initiated by the integration of variant forms of the plasmids into the mitochondrial genome. Here, we describe a strain that is phenotypically distinguishable from previously characterized senescent strains and show that senescence can occur in the absence of plasmid integration and associated alterations in mitochondrial DNA. The MS4416 strain contains a unique variant of the Mauriceville retroplasmid, and undergoes senescence at highly predictable frequencies at 37°, 25° and 18 °C. Decline in vegetative growth rate correlates with increased levels of the variant plasmid and alterations in the synthesis of mitochondrially encoded proteins, suggesting that plasmid over-replication interferes with mitochondrial translation. We also report the isolation of a mutant strain that escapes senescence yet still maintains high levels of the variant plasmid. Its ability to tolerate a growth-suppressive retroplasmid suggests that there are mechanisms in Neurospora which compensate for the deleterious effects that plasmid over-replication has on mitochondrial function. Received: 12 July 1999 / Accepted: 17 December 1999     

A significant variant of Microsporon gypseum

Summary Detailed morphological characteristics of a variant ofMicrosporon gypseum, which evolved spontaneously in a pure culture of a soil strain, have been described. The outstanding characteristics of this variant includes the production of macroconidia resembling more closely the macroconidia ofTrichophyton than those ofMicrosporon.     

Sterol-side chain cleavage by fusants ofAlkaligenes fœcalis andArthrobacter oxydans

Protoplasts of a strain ofA. fœcalis incapable of utilizing β-sitosterol as carbon source for growth were fused with protoplasts ofA. oxydans — a strain capable of complete degradation of β-sitosterol. Five fusants showing morphology and pattern of transformation of C-19 steroids identical toA. Fœcalis were selected. Analysis of the fermentation broth containing β-sitosterol showed that the fusants were capable of utilizing β-sitosterol for growth but their pattern of metabolite formation from β-sitosterol was different from that ofA. oxydans. The study revealed that the protoplast fusion technique could be used for intergeneric transfer of genetic determinants linked to partial cleavage of β-sitosterol side chain toA. fœcalis fromA. oxydans.     

Metabolism associated with raised metabolic flux to sugar nucleotide precursors of exopolysaccharides in Lactobacillus delbrueckii subsp. bulgaricus

Exopolysaccharide (EPS) metabolism was studied in a galactose-negative strain of Lactobacillus delbrueckii subsp. bulgaricus, using two different approaches. Firstly, using both the parent strain and a chemically induced mutant with higher yield and specific productivity of EPS than the parent, comparative information was obtained relating to enzyme activities and metabolite levels associated with EPS formation when grown on lactose. Under continuous culture conditions (D=0.10 h⁻¹), the higher metabolic flux towards EPS formation in the mutant strain relative to the parent appeared to be mediated by raised levels of UDP-glucose pyrophosphorylase (UGP). Marginally raised UDP-galactose 4-epimerase (UGE) activity in the mutant strain suggested that this enzyme could also play a role in EPS overproduction. The second approach involved investigating the effect of growth rate on sugar nucleotide metabolism in the parent, as it is known that EPS production is growth-associated in this strain. UGE activity in the parent strain appeared to increase when the growth rate was elevated from 0.05 to 0.10 h⁻¹, and further to 0.35 h⁻¹, conditions that can be associated with higher levels of metabolic flux to EPS formation. Concurrent with these increments, intracellular ATP levels in the cell were raised. In both investigations glucose-6-phosphate accumulated pointing to a constriction at this branch-point, and a limitation in the flow of carbon towards fructose-6-phosphate or glucose-1-phosphate. The changes in metabolism associated with enhanced flux to EPS provide guidance as to how the yield of Lactobacillus delbrueckii subsp. bulgaricus EPS can be improved.     

Effect of salinity on growth and toxin production of <Emphasis Type="Italic">Alexandrium minutum</Emphasis> isolated from a shrimp culture pond in northern Vietnam

A clonal culture of a Vietnamese strain of Alexandrium minutum, AlexSp17, was subjected to different salinity treatments to determine the growth and toxin production of this strain that produces a novel toxin analogue, deoxy GTX4-12ol. The experiment was carried out in batch cultures without pre-acclimatization at seven salinity treatments from 5 to 35 psu, under constant temperature of 25°C, illumination of 140 μmol photon m⁻² s⁻¹, and 12:12 light/dark photoperiod. The strain grew in all salinity treatments, with optimum growth at 10–15 psu. However, the specific growth rate (0.2 day⁻¹) was lower than those reported in Malaysian strains and other strains from different geographical areas. The optimum range of salinity for the growth of this species agreed with field observations of the locality of origin. No significant change in toxin profiles was observed at different salinities. The cellular toxin quota, Qt, was not affected by the salinity-dependent growth rate. The toxin GTX4-12ol is presumed to be a transformation product of GTX4 from specific cellular reductase enzymes. Further investigation at the molecular level of toxin biosynthesis and subcellular enzyme activities is needed to provide insight in the production of this unique toxin analogue.     

Characterization of a newly isolatedcis-1,2-dichloroethylene and aliphatic compound-degrading bacterium,Clostridium sp. strain KYT-1

Acis-1,2-dichloroethylene (cis-DCE)-degrading anaerobic bacterium,Clostridium sp. strain KYT-1, was isolated from a sediment sample collected from a landfill site in Nanji-do, Seoul, Korea. The KYT-1 strain is a gram-positive, endospore-forming, motile, rod-shaped anaerobic bacterium, of approximately 2.5∼3.0 μm in length. The degradation ofcis-DCE is closely related with the growth of the KYT-1 strain, and it was stopped when the growth of the KYT-1 strain became constant. Although the pathway ofcis-DCE degradation by strain KYT-1 remains to be further elucidated, no accumulation of the harmful intermediate, vinyl chloride (VC), was observed during anaerobiccis-DCE degradation. Strain KYT-1 proved able to degrade a variety of volatile organic compounds, including VC, isomers of DCE (1,1-dichloroethylene,trans-1,2-dichloroethylene, andcis-DCE), trichloroethylene, tetrachloroethylene, 1,2-dichloroethane, 1,1,1-trichloroethane, and 1,1,2-trichloroethane. Strain KYT-1 degradedcis-DCE at a range of temperatures from 15 to 37°C, with an optimum at 30°C, and at a pH range of 5.5 to 8.5, with an optimum at 7.0.     

Characteristics of Group A Streptococcal Variants Treated with Mitomycin C

Transfers of Streptococcus pyogenes strain T12 in Todd–Hewitt broth containing stepwise increases in amounts of mitomycin C (MC) gave rise to slight changes of their colonial appearances. Variants thus obtained were examined for antibiotic and bile resistances; production of streptolysin-S, -O and deoxyribonuclease; growth in alkaline medium, high salt concentration, and at 10 C and 45 C; sugar fermentations, and precipitin reactions. Four strains retained group A antigen, but some of them lost the ability to produce hemolysins and deoxyribonuclease, and acquired resistance to bile, penicillin and streptomycin as well as MC, and to physical environments. Four other strains lost group A antigen and acquired new antigens common to cells of group C, group D, or highly antibiotic-resistant mutants reported previously. A variant which reacted with group C antiserum contained galactosamine, but not glucosamine, while the parent strain showed the reverse pattern. Many other variants contained both hexosamines. Even a variant, strain TL3-2, reacted strongly only with group A antiserum, but contained glucose and both hexosamines. These strains having galactosamine possessed uridine diphosphate (UDP)-N-acetylglucosamine-4-epimerase activity which converted the substrate into UDP-N-acetylgalactosamine, while the parent strain failed to demonstrate the existence of this enzyme. The variants were discussed with respect to the group A streptococcal variations possessing no more original characteristics.     

Amylolytic activity of yeasts

The total amylolytic activity of 14 selected fermentation type I members of the generaSaccharomyces, Zygosaccharomyces, Candida andTorulopsis was studied. Fractions with α-glucosidase activity, the specificty of which was tested for maltose and sucrose, were isolated on carboxymethyl-cellulose from the intracellular contents of two strains ofCandida albicans and one ofCandida stellatoidea. The fraction from the strainCandida albicans 29-3-109, which is more virulent for mice, displayed the greatest α-glucosidase activity, moderate activity was present in the strainCandida stellatoidea 29-64-1, while the lowest activity was found in the less virulent strain ofCandida albicans, 29-3-19.     

Effect of citrate on growth of Lactococcus lactis subsp. lactis in milk

The effect of citrate on the growth of Lactococcus lactis subsp. lactis var. diacetylactis in milk has been investigated. Five strains of Lactococcus lactis subsp. lactis var. diacetylactis were compared to their citrate-negative variants, which lack the plasmid coding for citrate permease. In most cases, acidification kinetics and the final bacterial concentration of pure cultures of parental and variant strains did not differ significantly. Co-cultures of parental and variant strains, however, systematically tended towards the predominance of parental strains. Citrate metabolism is responsible for this change, since the predominance of citrate-positive strains was not observed in the absence of citrate. Continuous culture in milk enabled the difference in growth rates between the parental strain Lactococcus lactis subsp. lactis var. diacetylactis CDI1 and its citrate-negative variant to be quantified by following changes in the populations of the two co-cultured strains. At 26 °C, the growth rate of the parental strain was 7% higher than that of its citrate-negative variant. These results show that citrate metabolism slightly stimulates the growth of lactococci in milk. Received: 18 February 1997 / Received revision: 2 May 1997 / Accepted: 4 May 1997     

A variant of Saccharomyces cerevisiae pep4 strain with improved oligotrophic proliferation, cell survival and heterologous secretion of α-amylase

A variant of Saccharomyces cerevisiae pep4 strain 20B12, with improved oligotrophic proliferation, cell survival and secretion of heterologous mouse α-amylase, is described. Previously we reported a procedure to enrich NI transformants that are not inhibited by cytotoxic expression of hepatitis B virus surface antigen in the secretion pathway of the protease-A-deficient (pep4) strain. To use the NI cells as a host for heterologous expression, we tried to amend the introduced pYAS/12S vector and obtain a host strain, NI-C, with stable NI phenotype and trp1 marker restored. Southern analysis of genomic DNA of NI-C suggested that the original pYAS/12S was abnormally rearranged and not completely corrected. Further assay showed that the viability and mitotic ability of the NI-C strain were increased. While using the NI-C strain as host for plasmid transformation and heterologous expression of mouse α-amylase, we observed that transformed colonies grew more quickly and secreted more α-amylase than general yeast strains. A further test showed that the NI-C strain was able to use mouse α-amylase as a positive selection marker to form transformed colonies on nitrogen-starved plates that contain starch as the sole carbon source. The results imply that the NI-C variant is an improved pep4 strain that can be used for heterologous expression and for the development of new selective markers in the yeast transformation system. Received: 7 January 1998 / Received last revision: 7 September 1998 / Accepted: 11 October 1998