Inhibition ofd-alanyl-d-alanine ligase in different bacterial species by amino phosphonic acids

A comparative study was performed on the kinetic properties and the specificity ofd-alanyl-d-alanine ligases fromPseudomonas aeruginosa, Streptococcus faecalis, andStaphylococcus aureus, using some aminophosphonic acids and related compounds.dl-I-Aminoethylphosphonic acid was shown to be a competitive inhibitor of theP. aeruginosa andS. faecalis ligases; assuming ad-form stereospecificity, its activity was nearly equal to that ofd-cycloserine. 2-Aminoethylphosphonate was found to be a weak inhibitor of the ligases, in contrast to the carboxylic analog, β-alanine. γ-Aminobutyric acid and phosphoethanolamine also exhibited some inhibitory properties.     

Effects of fumarate,l-malate,and anAspergillus oryzae fermentation extract ond-lactate Utilization by the ruminal bacteriumSelenomonas ruminantium

Growth ofSelenomonas ruminantium HD4 in medium that contained 21mm d-lactate was stimulated to varying degrees by 10mm l-malate, 10mm fumarate, and 2% (v/v)Aspergillus oryzae fermentation extract (Amaferm). Amaferm treatment caused the greatest growth stimulation. Initial uptake rates (30s) and long-term uptake rates (30 min) ofd-lactate by whole cells ofS. ruminantium were increased in the presence of 10mm l-malate. Amaferm (25 l/ml) also stimulated long-term uptake rates ofd-lactate, whereas fumarate had no effect. Initial uptake ofd-lactate was depressed in the presence of fumarate or Amaferm. When eitherl-malate, fumarate, or Amaferm was included in thed-lactate growth medium, a homosuccinate fermentation resulted and an inverse relationship was observed between growth (protein synthesis) and succinate production. Recent research demonstrated that Amaferm containsl-malate, and this dicarboxylic acid may be involved in stimulatingd-lactate utilization byS. ruminantium.     

Regulation of biosynthesis of monensins on chemically defined medium

Addition ofL-valine andDL-isoleucine to the cultivation medium ofStreptomyces cinnamonensis was found to affect the ratio of synthesized monensins A and B. In the presence ofL-valine monensin A is synthesized predominantly, whereas in the presence ofDL-isoleucine the production of monensin B increases.     

Occurrence of L-DOPA and dopamine in plants and cell cultures ofMucuna pruriens and effects of 2,4-d and NaCl on these compounds

The development of the L-DOPA-content of roots, stems and leaves ofMucuna pruriens during growth of the plants is described. Besides L-DOPA, the leaves, but not the stems and the roots, also contain the related catechol dopamine. The time course of dopamine accumulation is compared to that of L-DOPA. In cell suspension cultures ofM. pruriens dopamine can be detected as well. Its level is strongly increased by addition of the growth regulator 2,4-d to the medium, a condition that suppresses cell growth and L-DOPA-accumulation. Dopamine induction appears to be a specific metabolic effect of 2,4-d. Salt stress, as caused by the addition of NaCl, gives no induction of dopamine formation, whereas L-DOPA is released into the medium.     

Utilization of oligosaccharides by three pathogenic isolates of colletotrichum gloeosporioides

Summary Utilization of six oligosaccharides (viz., sucrose, maltose, cellobiose, lactose, raffinose and melezitose) by the isolates ofColletotrichum gloeosporioides Penz. causing leaf-spot diseases ofCarissa carandas L.,Eucalyptus robusta Sm. andBougainvillaea glabra Choizy has been studied. Daily chromatographic analysis of the culture medium revealed that the utilization of sucrose, maltose, cellobiose and raffinose was through a hydrolytic pathway. In case of lactose and melezitose no hydrolytic product could be detected. During the utilization of sucrose two oligosaccharides (Rf 0.27 and 0.2) made their appearance. TheBougainvillaea-isolate synthesized two oligosaccharides (Rf 0.18 and 0.1) in a maltose medium although no glucose could be traced, while in case of the other two isolates, in addition to these synthetic oligosaccharides, glucose also appeared. When theCarissa-isolate ofColletotrichum gloeosporioides was grown on a cellobiose medium, in addition to glucose, two synthetic oligosaccharides (Rf 0.15 and 0.11) were formed. Other two isolates showed the formation of one oligosaccharide (Rf 0.15) and no glucose. The dry weight of mycelium recorded an increase on oligosaccharides with slow rate of consumption, while on oligosaccharides with rapid rate of utilization it tended to become constant or showed a decline towards the end of the incubation.     

Utilization of oligosaccharides by three imperfect fungi

Summary The utilization of oligosaccharides (maltose, sucrose, lactose and raffinose) by three imperfect fungi viz.Alternaria tenuis Auct.,Colletotrichum capsici (Syd.)Butler &Bisby andColletotrichum gloeosporioides Penz. isolated from diseased leaves of tomato,Scindapsus pictus andPolyscias balfuriana respectively, was studied chromatographically. Except for lactose, all the sugars were utilizzed through a hydrolytic pathway and almost all proved to be a suitable source of carbon for the growth of fungi. Along with the utilization of sucrose, a simultaneous synthesis of an oligosaccharide was also observed in case of two species ofColletotrichum. C. gloeosporioides, however, also synthesized an oligosaccharide, maltotriose if it is grown in the medium containing maltose. During the utilization of raffinose, melibiose and fructose were detectted in the culture medium ofA. tenuis. Melibiose was further broken down into glucose and galactose by the two species ofColletotrichum.All the sugars were consumed from the media within 15 days except for lactose which persisted in the media even after 15 days.     

Regulation of biosynthesis of thiolutin and aureothricin inStreptomyces kasugaensis

l-Methionine anddl-ethionine decreased production of thiolutin and aureothricin inStreptomyces kasugaensis. In the presence ofl-methionine the culture also produced 3-methylthioacrylic acid, 3-methylthiopropionic acid and 3,6-bis-(2-methylthioethyl)-2,5-dioxopiperazine. Production of the metabolites depended on the concentration ofl-methionine in the medium.     

Nutritional requirements ofXanthomonas phaseoli var.fuscans

As found by Starr (1946),l-glutamic acid is necessary for the growth ofXanthomonas phaseoli var.fuscans. According to our results, the growth is stimulated byl-asparagine in the presence ofl-glutamic acid;l-asparagine itself, however, does not serve as a source of carbon and nitrogen.Xanthomonas phaseoli var.fuscans grew well in a medium containing tryptone. Some peptides of the acidic fraction isolated from tryptone affected the growth as much as tryptone itself. Vitamins and plant growth substances did not affect the growth of the bacteria; proteins appeared to be a poor carbon and nitrogen source. On substituting glucose in a glutamic acid-containing medium with another saccharide, the growth of the bacteria was found equal or better in media containing mannose, sucrose, fructose, maltose or starch. The bacteria grew less satisfactorily in media containing galactose and cellobiose as compared with media containing glucose.     

Plantlet formation from isolated protoplasts ofSolanum melongena L.

Summary Mesophyll protoplasts were isolated from axenic shoot cultures ofSolanum melongena by the one-step enzymatic method. Of the different media employed for the culture of protoplasts, a medium modified fromKao andMichayluk (1975) supported sustained mitotic cycles most effectively. Organogenesis from protoplast-derived callus was achieved on transfer toMurashige andSkoog'S (1962) medium supplemented with an appropriate auxin and a cytokinin.     

Utilization ofL- andDL-cystine by the fungusMicrosporum gypseum

Growth of the fungusMicrosporum gypseum and utilization of cystine during this growth was studied in a glucose-arginine medium containing either sodium sulphate, andL-cystine orDL-cystine. Replacement of sulphate withL-cystine brought about no significant changes in the growth of the microorganism. Utilization ofL-cystine as a source of carbon and nitrogen was rapid and complete and excess sulphur was excreted into the medium in the form of sulphate. Similarly excreted were also minute amounts of sulphite which immediately reacted with the remaining cystine to formS. sulphocysteine. Growth ofM. gypseum in a medium withDL-cystine was slow. Although this substance was not utilized as readily asL-cystine, its utilization was still complete and excess sulphur was similarly excreted in the form of sulphate and sulphite. The initial step in the utilization of theD-isomer is probably its extracellular deamination.     

An assessment of the inhibitory activity of rat serum on staphylococci

A study has been made on the effect of rat serum on staphylococci. By following the respiration and growth of 5 strains ofStaphylococcus aureus and an equal number ofS. epidermidis in fresh, normal rat serum, we found thatS. aureus grew in, and oxidized rat serum better thanS. epidermidis. Difference in growth was not correlated with nutritional requirements. The antibacterial agent of fresh, normal, undiluted rat serum was stable to heating at 56 C for 1 hr, but its activity was completely destroyed after heating at 60 C for 2 hr. A 50 per cent dilution of rat serum with nutrient broth significantly reduced the antibacterial activity and treatment of rat serum with 0.4m solutions of sodium citrate reduced it drastically. Once the serum had been treated with sodium citrate, or oxalate, addition of equimolar solutions of calcium chloride or magnesium chloride failed to restore the antibacterial activity. Addition of ferric ions in high concentrations allowed the coagulase-negative strains of staphylococci to grow well in this serum. The antibacterial agent of rat serum was absorbed by heat-killed cells ofStaphylococcus aureus andS. epidermidis but not byStreptococcus pyogenes andEscherichia coli. Treatment of rat serum with bentonite at a concentration of 100 mg per ml decreased its antibacterial activity.     

Plant regeneration from cotyledonary protoplasts of cauliflower (Brassica oleracea L. var.botrytis L.)

Summary Protoplasts isolated enzymatically from precultured cotyledonary leaves ofB. oleracea var.botrytis and cultured in KM8p medium (Kao andMichayluk 1975) underwent sustained divisions in about 0.1% population to eventually produce callus, whereas mesophyll protoplasts from either field grown orin vitro raised plants failed to divide. The callus readily differentiated on Murashige-Skoog medium as modified for shoot culture (Binding 1974) to give rise to shoot and roots.     

Stimulation of L-asparaginase production inEscherichia coli by organic and amino acids

The effect of 18 amino acids and 7 organic acids on the production ofl-asparaginase EC-2 by a strain ofEscherichia coli in a chemically defined medium was investigated under moderate aeration. All the amino acids and some of the organic acids stimulated the enzyme production. The specific activity without stimulants was about 0.16 nkat per mg dry weight, with stimulants it lay between 1 and 6 nkat per mg dry weight but withl-leucine andl-methionine the values were 12 nkat and 17 nkat per mg, respectively. When two organic or amino acids were added simultaneously at concentrations that were suboptimal for stimulation, the stimulating effects were cumulative in most cases. When cells were grown under conditions approaching anaerobiosis, the specific activity reached, even in the absence of stimulants, values as high as 5 nkat per mg; under these conditions, a further substantial increase in specific activity was only caused byl-leucine andl-methionine. Stimulating effects ofdl-lactate and of some amino acids were also found in other strains ofEscherichia coli. The ability to grow on a medium withl-asparagine as the sole source of both nitrogen and carbon was found in two strains; growth took place even when there was no measurable activity ofl-asparaginase EC-2.     

Metabolic design in the amino-acid-producing bacteriumCorynebacterium glutamicum

The Gram-positive bacteriumCorynebacterium glutamicum is used for the industrial production of amino acids,e.g. ofl-glutamate andl-lysine. By cloning and expressing the various genes of thel-lysine pathway inC. glutamicum we could demonstrate that an increase of the flux ofl-4-aspartaldehydate tol-lysine could be obtained in strains with increased dihydro-dipicolinate synthase activity. Recently we detected that inC. glutamicum two pathways exist for the synthesis ofdl-2,6-diaminopimelate andl-lysine. Mutants defective in one pathway are still able to synthesize enoughl-lysine for growth but thel-lysine secretion is reduced to 50–70%. Using NMR-spectroscopy we could calculate how much of thel-lysine secreted into the medium is synthesizedvia the one and the other pathway. Amplification of the feedback-inhibition-insensitive-homoserine dehydrogenase and homoserine kinase in a highl-lysine-overproducing strain made it possible to channell of the carbon flow from the intermediate 4-aspartaldehydate toward homoserine, resulting in a high accumulation ofl-threonine. For a further flux froml-threonine tol-isoleucine the allosteric control of threonine dehydratase was eliminated. Dedicated to Dr. Z. Vaněk on the occasion of his 70th birthday Presented at theIUMS Congresses '94-7th International Congress of Bacteriology and Applied Microbiology Division, Prague, July 3–8, 1994 (Bacteriological Symposium BS-12Regulation of Microbial Product Overproduction).     

Influence of monosaccharides on aerobactin production byAerobacter aerogenes 62-1

Supplementation of cultures ofAerobacter aerogenes 62-1, 43/4 h after initiation of growth withd-glucose (20 mM), resulted in a threefold increase in the production of aerobactin. Administration ofl-lysine under similar conditions led to a twofold incrasse in the yield of the siderophore. Studies with a cell-free system ofAerobacter aerogenes 62-1 revealed considerable stimulation of lysine-N⁶-hydroxylase activity by glucose and several of its derivatives. Inclusion of ferric chloride (0.1 mM) in the growth medium led to the repression of both lysine-N⁶-hydroxylase and aerobactin synthetase.     

Indolyl-3-acetic acid formation byAzotobacter chroococcum

Summary Small amounts of indolyl-3-acetic acid (IAA) were detected in aerated cultures ofAzotobacter chroococcum grown with or withoutl-tryptophane in the medium, but IAA was detected in agar cultures only whenl-tryptophane was present. Most IAA was found in 7-day-old cultures and less in older cultures. Washed cells did not convert tryptophane enzymically to IAA. The time course of IAA formation byA. chroococcum strain A6 has been described and the effect of adding tryptophane to the medium has been studied. In contrast to results elsewhere strain A6 produced traces of IAA in aerated cultures with or without added tryptophane. IAA was detected only after the end of exponential growth when cells had begun to autolyse. The amount of IAA declined as cultures aged. The slight effect ofl- but not ofd-tryptophane in promoting IAA formation in ageing cultures suggests some kind of biological transformation but it seems unlikely that IAA formation is part of the normal metabolic processes of intact Azotobacter cells.     

Growth inhibition ofMicrococcus pyogenes by manganese and riboflavin

Summary Riboflavin, in the presence of manganese, inhibited the growth ofMicrococcus pyogenes var.aureus. The inhibitory action was shown to be dependent upon aerobiosis, and was characterized predominately as a prolongation of the lag phase. Increasing concentrations of either manaanese or riboflavin, in the presence of a constant quantity of the other, enhanced the inhibitory effect. Ultra-violet irradiation of riboflavin reduced its toxic capacity in proportion to the amount of riboflavin degraded. Riboflavin-5-phosphate was equally as inhibitory as riboflavin. Ferrous iron was capable of a non-stoichiometric antagonism at low concentrations of riboflavin and/or manganese. Suported, in part, by a research grant (E-605) from the National Institutes of Health, U.S. Public Health Service. A preliminary report has appeared (Ramsey andWilson, 1956).     

Comparative media studies in the isolation ofCandida albicans from pregnant women

Summary Rice agar and corn meal agar, with and without Tween 80, were evaluated clinically as directly inoculable selective and differential media for the isolation ofC. albicans from vulvovaginal specimens taken from pregnant women. Chlamydospore formation on these media was investigated as a criterion for the identification ofC. albicans.Of 301 patients cultured, 118 (39.2 %) gave positive cultures for yeast-like fungi of the genusCandida. Of 118 strains for which fermentation patterns were determined, 69 (58.5 %) gave the pattern forC. albicans. Of these, 56 (81.1 %) formed chlamydospores.Tween 80 was found to exert a very stimulating influence on chlamydospore production. Rice agar with Tween 80 appeared to be the most efficient medium for eliciting chlamydospores. However, since strains of 4 out of 6 species ofCandida isolated were found to sporulate it was concluded that chlamydospore formation is not a reliable criterion for the speciation ofC. albicans.Each of the 4 media served satisfactorily as a directly inoculable selective medium for the isolation of yeast-like fungi of the genusCandida from vulvovaginal specimens. None of the media appeared to preferentially stimulate chlamydospore production inC. albicans.Dr.Smith is Associate Professor in the Department of Microbiology; Dr.Taubert is a Fellow in the Department of Obstetrics and Gynecology; Mr.Towns is Laboratory Assistant in the Department of Microbiology.Supported in part by a grant from the Lederle Medical Faculty Awards Committee and in part by United States Public Health Service Grant E-3068.     

Breeding anl-isoleucine producer by protoplast fusion ofCorynebacterium glutamicum

Summary Corynebacterium glutamicum R-18 is a strain forl-isoleucine production. Polyethylene glycol (PEG)-induced protoplast fusion was applied to improve the strain of thisl-isoleucine producer. Strain R-18 was fused with anl-lysine producerC. glutamicum S-37, becausel-isoleucine andl-lysine are synthesized from a common intermediate, aspartate--semialdehyde. Two thousand fusants were checked for their phenotypes. Most of the fusants accumulatedl-lysine, and only 0.9% of the fusants accumulatedl-isoleucine. Two strains, F-28 and F-91, were selected and cultivated in production medium. Fusant F-28 accumulated 12.1 g/l ofl-isoleucine and 4.8 g/l ofl-lysine, and fusant F-91 accumulated 4.8 g/l ofl-isoleucine and 13.0 g/l ofl-lysine, while the parental strains R-18 and S-37 accumulated 9.5 g/l ofl-isoleucine and 26.8 g/l ofl-lysine, respectively. Sugar consumption activity was improved by protoplast fusion, and thel-isoleucine production rate of F-28 was 2.4 times higher than that of R-18.     

Osservazioni sui corpi ifici di Delacroixia coronata (Cost.) Sacc. et P. Syd. in coltura

Summary The author follows the formation, the resting features and the further development of the peculiar hyphal bodies ofDelacroixia coronata. By the microscopical examination of colonies grown in Couch medium on cover-slips, he distinguishes various schemes of development and septation in the hyphal bodies recorded above and, after having mentioned the uncertainty of some other denominations (azygospores, parthenospores), he states that they are to be considered chlamidospores in the sense ofLangeron.