Bacterial breakdown of benomyl. I. Pure cultures

With different soil and water samples as inoculum and the benzimidazole fungicides benomyl (either as Benlate or as the pure compound) and thiabendazole as selective agents, a large number of, mainly fluorescent, Pseudomonas strains were isolated which nearly all were able to grow in a mineral medium with benomyl as the sole source of carbon. However, no growth occurred with any of a series of other benzimidazole compounds, viz. benzimidazole, 2-aminobenzimidazole (2-AB), thiabendazole and fuberidazole. Although benomyl—or rather its non-enzymatic breakdown product methyl benzimidazol-2-yl carbamate (MBC)—was partially degraded to 2-AB, most probably n-butylamine, which arises after splitting off of the butylcarbamoyl side chain, was the actual carbon source for the Pseudomonas isolates.When incorporated in a lactate medium, 2-AB markedly inhibited the growth of Pseudomonas spp. at a concentration of 250 g/ml, with complete inhibition being attained at 500 g/ml. For Bacillus spp. grown in liquid peptone media benzimidazole compounds were inhibitory at concentrations of 500–1000 g/ml, with a toxicity increasing in the order: benzimidazole 相似文献   

Distribution of ultramicrobacteria in a gulf coast estuary and induction of ultramicrobacteria

The abundance of ultramicrobacteria (i.e., bacteria that pass through a 0.2m filter) in a subtropical Alabama estuary was determined during a 1-year period. Although phenotypic and molecular characterization indicated that the population of ultramicrobacteria was dominated byVibrio species, species ofListonella andPseudomonas were also abundant. Vibrios occurred with the greatest frequency in waters whose salinities were less than 14, and were the most abundant species of the total ultramicrobacterial population year-round, whilePseudomonas species were absent or considerably reduced during the winter months. The total number of ultramicrobacteria showed an inverse relationship to total heterotrophic bacteria as measured by colony-forming units (CFU)/ml and to water quality as measured by several parameters. Analysis by generic composition indicated that both salinity and temperature significantly affected the distribution of these organisms. Laboratory studies revealed that strains of vibrios under starvation in both static and continuous-flow microcosms could be induced to form cells that passed through 0.2 and/or 0.4m filters. Cells exposed to low nutrients became very small; some grew on both oligotrophic (5.5 mg carbon/liter) and eutrophic (5.5 g carbon/liter) media; and some few cells grew only on oligotrophic media. By passing selected vibrio strains on progressively diluted nutrient media, cells were also obtained that were small, that passed through 0.4m filters, and that could grow in oligotrophic media. These results suggest that ultramicrobacteria in estuaries (at least some portion of the population) may be nutrientstarved or low nutrient-induced forms of certain heterotrophic, eutrophic, autochthonous, estuarine bacteria.     

Enhancement of symbiotic nitrogen fixation by vitamin-secreting fluorescent Pseudomonas

Fluorescent Pseudomonas sp. strain 267 promotes growth of nodulated clover plants under gnotobiotic conditions. In the growth conditions (60 M FeCl₃), the production of siderophores of the pseudobactin-pyoverdin group was repressed. Plant growth enhancement results from secretion of B vitamins by Pseudomonas sp. strain 267. This was proven by stimulation of clover growth by naturally auxotrophic strains of Rhizobium leguminosarum bv. trifolii and marker strains E. coli thi^- and R. meliloti pan^- in the presence of the supernatant of Pseudomonas sp. strain 267. The addition of vitamins to the plant medium increased symbiotic nitrogen fixation by the clover plants.     

Metabolism of dodecyldimethylamine by Pseudomonas MA3

Pseudomonas MA3 was isolated from activated sludge on the basis of its capacity to use dodecyldimethylamine as a sole carbon (C) and energy source. Dodecylamine, dodecanal, dodecanoic acid and acetic acid also supported growth of Pseudomonas MA3. Dodecyldimethylamine-grown cells oxidized a wide range of alkylamine derivatives, dodecanal, dodecanoic acid and acetic acid. Degradation of the alkyl chain of dodecyldimethylamine by Pseudomonas MA3 appeared from the stoichiometric liberation of dimethylamine. A dehydrogenase catalysed the cleavage of the C_alkyl-N bond. The first intermediate of the proposed degradation pathway, dodecanal, accumulated in the presence of decanal used as a competitive inhibitor. The second intermediate,dodecanoic acid, was formed in the presence of acrylic acid during the degradation of dodecyldimethylamine. Dodecanal was converted into dodecanoic acid by a dehydrogenase and dodecanoic acid was then degraded via the oxidation pathway.     

Hydrophobicity of ruminant mastitisStaphylococcus aureus in relation to bacterial aging and slime production

Hydrophobicity of 72 bovine and 53 ovine mastitisStaphylococcus aureus strains was studied throughout the logarithmic growth phase with a water-xylene two-phase system. Hydrophobicity increased during this growth phase. Freshly isolated strains were more hydrophobic than old strains (p<0.01). Old (bovine) strains became more hydrophobic (i.e., refreshed) after passage through the mouse mammary gland (p<0.01). Bovine strains were more hydrophobic than ovine strains (p<0.01). For the majority of strains, bacteria became more hydrophilic (p<0.001) after growth in exopolysaccharide-inducing media (Columbia and modifiedStaphylococcus 110 broth). This could be expected, since exopolysaccharides are hydrophilic. However, in these media, the strains that were able to produce slime in Congo red agar or in tryptic soy broth supplemented with 2% glucose (w/v) did not become more hydrophilic.It is proposed that different mechanisms may be involved in triggering exopolysaccharide production when different exopolysaccharide-inducing media are used.     

Microbial utilization of the industrial wastewater pollutants 2-ethylhexylthioglycolic acid and <Emphasis Type="Italic">iso</Emphasis>-octylthioglycolic acid by aerobic Gram-negative bacteria

Industrial wastewater from the production of sulfur containing esters and the resulting products of this synthesis, 2-ethylhexylthioglycolic acid (EHTG) and iso-octylthioglycolic acid (IOTG), were deployed in this study to enrich novel bacterial strains, since no wastewater and EHTG or IOTG degrading microorganisms were hitherto described or available. In addition, nothing is known about the biodegradation of these thiochemicals. The effect of this specific wastewater on the growth behaviour of microorganisms was investigated using three well-known Gram-negative bacteria (Escherichia coli, Pseudomonas putida, and Ralstonia eutropha). Concentrations of 5% (v/v) wastewater in complex media completely inhibited growth of these three bacterial strains. Six bacterial strains were successfully isolated, characterized and identified by sequencing their 16S rRNA genes. Two isolates referred to as Achromobacter sp. strain MT-E3 and Pseudomonas sp. strain MT-I1 used EHTG or IOTG, respectively, as well as the wastewater as sole source of carbon and energy for weak growth. More notably, both isolates removed these sulfur containing esters in remarkable amounts from the cultures supernatant. One further isolate was referred to as Klebsiella sp. strain 58 and exhibited an unusual high tolerance against the wastewater’s toxicity without utilizing the contaminative compounds. If cultivated with gluconic acid as additional carbon source, the strain grew even in presence of more than 40% (v/v) wastewater. Three other isolates belonging to the genera Bordetella and Pseudomonas tolerated these organic sulfur compounds but showed no degradation abilities.     

Effects of iron(III) analogs on growth and pseudobactin synthesis in a chromiumtolerantPseudomonas isolate

Summary The growth and siderophore production of a fluorescentPseudomonas species isolated from soil contaminated with chromium was found to be influenced by the presence of trivalent cations. Overproduction of pseudobactin occurred when the isolate was grown in media containing 1 mM Cr(III) under ironlimited conditions but not when Fe(III) was added at 10 M Pseudobactin synthesis was derepressed in iron-limited cultures containing 1 mM Sc(III) or Y(III), examples of group III-B elements. We found that AI(III), Ga(III) or In(III), representative metals from group III-A, repressed synthesis of pseudobactin under iron-deficient conditions. Analogs of Fe(III) were found to inhibit growth of thePseudomonas isolate in iron-limited media and the trivalent metals listed in order of decreasing toxicity were as follows: Ga > In > Sc > Cr > Y > Al. The inhibition of growth by 1 mM In(III), Sc(III) and Ga(III) was greater during iron-limited growth than in media containing 10 M Fe(III). These data show that, although the metal analogs of Fe(III) have similar chemical and physical characteristics, the physiological response of the fluorescent pseudomonad when grown in the presence of these metals varied markedly.     

Mycelial growth of strains of the genera <Emphasis Type="Italic">Suillus</Emphasis> and <Emphasis Type="Italic">Boletinus</Emphasis> in media with a wide range of concentrations of carbon and nitrogen sources

Suillus and Boletinus were studied using Ohta medium. In media with glucose or trehalose, all tested strains grew well. With mannose and cellobiose, strains generally grew well, except for one strain of Suillus. Utilization of dextrin and soluble starch differed with each strain, and that of sucrose and glycerol was low for all strains. Utilization of four amino acids, arginine, glutamic acid, aspartic acid, and alanine, was similar to that of ammonium tartrate for Suillus strains, but mycelial growth with amino acids was clearly lower than with ammonium tartrate for the Boletinus strain. The effect of glucose and ammonium tartrate concentrations for nine strains of the genera Suillus and Boletinus was studied with ranges for glucose of 1–100 and 200g/l, respectively, and for ammonium tartrate of 0.2–5 and 20g/l, respectively. Six strains showed maximal growth at a glucose concentration greater than 25g/l, and one strain showed maximal growth at 70g/l. The results indicate that these fungi are adapted to relatively high concentrations of carbon sources. In general, glucose concentration at mycelial growth maximum decreased as ammonium tartrate concentration increased, and at higher concentrations of glucose, mycelial growth decreased more rapidly in higher concentrations of ammonium tartrate.     

Fluorescent pseudomonad mixtures mediate disease resistance in rice plants against sheath rot (<Emphasis Type="Italic">Sarocladium oryzae</Emphasis>) disease

Plant growth-promoting rhizobacterial (PGPR) strains were isolated from different agro-ecosystems of Tamil Nadu, India, and were tested for their efficacy against the sheath rot pathogen Sarocladium oryzae under in vitro, glasshouse and field conditions. Vigour and a relative performance index (RPI) were used to assay the growth promotion and antagonistic activity of Pseudomonas strains against S. oryzae under in vitro conditions. The results revealed the significant performance by strains Pf1, TDK1 and PY15 compared to other strains. Further, the combination of Pseudomonas strains Pf1, TDK1 and PY15 was more effective in reducing sheath rot disease in rice plants compared to individual strains under glasshouse and field conditions. Quantitative and native polyacrylamide gel electrophoresis (PAGE) analysis of peroxidase (PO), polyphenol oxidase (PPO) and chitinase activity in rice plants showed an increased accumulation of defence enzymes in the treatment with a combination of Pf1, TDK1 and PY15 compared to the treatment with individual strains and untreated controls. The present study revealed the probable influence of antagonism, plant growth promotion and induced systemic resistance (ISR) by the mixture of Pseudomonas bioformulations in enhancing the disease resistance in rice plants against sheath rot disease.

Enantioselective hydrolysis of O-acetylmandelonitrile to O-acetylmandelic acid by bacterial nitrilases

Bacteria were enriched from soil samples, using benzylcyanide, -methyl-, -ethyl- or -methoxybenzyl-cyanide as the sole source of nitrogen. All isolated strains belonged to the genus Pseudomonas. Resting cells of the isolates hydrolysed O-acetylmandelonitrile to O-acetylmandelic acid, O-acetylmandelic acid amide and mandelic acid. From racemic O-acetylmandelonitrile all isolates preferentially formed R(–)-acetylmandelic acid ( = d-acetylmandelic acid). The enantioselective hydrolysis of O-acetylmandelonitrile could also be demonstrated in vitro. Crude extracts did not hydrolyse O-acetylmandelic acid amide indicating an enantioselective nitrilase rather than a nitrile hydratase/amidase system.     

Autotrophic growth of four Sulfolobus strains on tetrathionate and the effect of organic nutrients

Autotrophic growth yields of four strains of Sulfolobus using tetrathionate as sole energy substrate fell in the range 6.2–7.8 g dry weight (mol tetrathionate oxidized)^-1. Autotrophic organisms lacked ribulose 1,5-bis-phosphate carboxylase, but contained pyruvate and phosphoenolpyruvate carboxylases. S. brierleyi and strains B6-2 and LM exhibited mixotrophic growth, with tetrathionate oxidation, CO₂-fixation and organic substrate assimilation occurring concurrently, using media containing glucose or acetate. Yeast extract or succinate supported heterotrophic growth and showed strain-dependent repression of one or both of tetrathionate oxidation and CO₂-fixation resulting in biphasic growth. All four carbon atoms of succinate were assimilated to cell-carbon during growth. Acetate was the major source of cell-carbon during mixotrophic growth. These observations are not inconsistent with the possibility of a reductive carboxylic acid cycle in these organisms. Radiorespirometric analysis of glucose oxidation indicated CO₂ release to occur by means of an Entner-Doudoroff pathway (followed by pyruvate decarboxylation) and oxidative pentose phosphate pathway reactions. There was little evidence from the glucose radiorespirometry of the large-scale use of an oxidative tricarboxylic acid cycle for terminal oxidation of acetate derived from pyruvate. These results demonstrate the considerable metabolic versatility of Sulfolobus strains and show that there is significant variation among them.Abbreviations PIPES Piperazine-N,N-bis (2-ethane sulphonic acid)     

Differential sensitivity of plant-associated bacteria to sulfonylurea and imidazolinone herbicides

The side effects of sulfonylurea and imidazolinone herbicides on plant-associated bacteria were investigated under pure culture conditions. Eighteen isolates, belonging to the genera Azotobacter, Azospirillum, Bacillus, Enterobacter Pseudomonas and Serratia, were exposed to four active compounds at concentration ranges similar to those in field soil. The sulfonylureas chlorsulfuron and rimsulfuron inhibited the growth of one of two Azospirillum and one of four Pseudomonas strains, while the imidazolinones imazapyr and imazethapyr were effective on two out of five Bacillus isolates. Surfactants in commercial formulation significantly enhanced rimsulfuron toxicity. With the exception of one Azospirillum strain, the differential tolerance of rhizobacteria to these herbicides was related to a differential sensitivity of their target, the activity of the first enzyme in branched-chain amino acid biosynthesis, acetohydroxyacid synthase (AHAS).Greenhouse pot studies were performed to assess the occurrence of inhibitory effects on bacterial growth in field conditions. Maize seedlings were bacterized with the two strains which had shown in vitro sensitivity to sulfonylureas. Following the application to the soil of a commercial formulation of rimsulfuron at rates of 0, 0.2 and 0.5 mol a.i. kg^–1, significative differences in the resulting degree of bacterial root colonization were found. Moreover, upon co-inoculation with two strains, one tolerant and one sensitive to the herbicide, the presence of rimsulfuron significantly enhanced root occupancy by resistant bacteria, suggesting that shifts in the microbial community structure of crop rhizosphere could indeed result as a consequence of weed control by AHAS inhibitors.Abbreviations AHAS acetohydroxyacid synthase - CETAB cetyltrimethylammonium bromide - ID₅₀ concentration causing 50% inhibition of enzyme activity - LD₅₀ concentration causing 50% decrease of growth constant value     

Production of 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) and Its Derivatives by Vibrio tyrosinaticus

Six strains of bacteria belonging to Vibrio and Pseudomonas were selected as good producers of L-DOPA from L-tyrosine out of various bacteria. The condition for the formation of L-DOPA by Vibrio tyrosinaticus ATCC 19378 was examined and the following results were obtained. (1) Intermittent addition of L-tyrosine in small portions gave higher titer of L-DOPA than single addition of L-tyrosine. (2) Higher amount of L-DOPA was produced in stationary phase of growth than in logarithmic phase. (3) Addition of antioxidant, chelating agent or reductant such as L-ascorbic acid, araboascorbic acid, hydrazine, citric acid and 5-ketofructose increased the amount of L-DOPA formed. (4) L-Tyrosine derivatives such as N-acetyl-L-tyrosine amide, N-acetyl-L-tyrosine, L-tyrosine amide, L-tyrosine methyl ester and L-tyrosine benzyl ester were converted to the corresponding L-DOPA derivatives.

In the selected condition about 4 mg/ml of L-DOPA was produced from 4.3 mg/ml of L-tyrosine.     

Effect of Plant Growth-Promoting Rhizobacteria and Culture Filtrate of <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis> on Phenolic and Salicylic Acid Contents in Chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis>)

Two plant growth-promoting rhizobacteria (PGPR), viz., Pseudomonas fluorescens strain Pf4 and P. aeruginosa strain Pag, protected chickpea (Cicer arietinum) plants from Sclerotium rolfsii infection when applied singly or in combination as seed treatment. Pag gave the best protection to the seedlings, applied either singly (mortality 16%) or in combination with Pf4 (mortality 17%) compared with 44% and 24% mortality in control and Pf4 treatment, respectively. The two PGPR strains induced the synthesis of specific phenolic acids, salicylic acid (SA), as well as total phenolics at different growth stages of chickpea seedlings with varied amount. The maximum amount of total phenolics was recorded in all the aerial parts of 4-week-old plants. Gallic, ferulic, chlorogenic, and cinnamic acids were the major phenolic acids detected in high-performance liquid chromatography (HPLC) analysis. Induction of such phenolic acids in the seedlings was observed up to 6 weeks in comparison with control. Salicylic acid (SA) was induced frequently during the first 3 weeks of growth only. Between the two strains, Pag was more effective in inducing phenolic acid synthesis applied either singly or in combination with strain Pf4 during the entire 6 weeks of growth of chickpea. In the presence of a culture filtrate of S. rolfsii, the two Pseudomonas strains induced more phenolic acids in treated than in non-treated and control plants. The occurrence of salicylic acid was frequent in the first 24 h, but infrequent at 48 and 96 h. Foliar spray of Pseudomonas strains also enhanced the phenolic acid content as well as total phenolics within 24 h of application. Gallic, chlorogenic, and cinnamic acids were consistently discerned in the treated leaves, whereas SA was absent even up to 96 h of application. Resistance in chickpea plants by Pseudomonas strains through induction of phenolic compounds as well as induced systemic resistance via SA-dependent pathway was evident. Received: 1 April 2002 / Accepted: 4 May 2002     

Isolation and identification of the antialgal compound, harmane (1-methyl-β-carboline), produced by the algicidal bacterium, Pseudomonas sp. K44-1

Twelve strains of algicidal bacteria were isolated from the surfacewater of the pond Shinobazu and the moat Ote-bori (Tokyo, Japan). Nine of thesestrains were considered to be in the Pseudomonas group byanalyses of 16S rDNA sequences. The methanol extract ofPseudomonas sp. K44-1 showed marked antialgal activity bythe paper disk method. Harmane (1-methyl--carboline) was isolated fromtheethyl acetate extract of the whole culture broth ofPseudomonas sp. K44-1 by using silica gel columnchromatography and high performance liquid chromatography (HPLC). Harmaneshowedantagonistic activities against several cyanobacterial strains at aconcentration of 30 g disk^–1.     

Pediocin production by recombinant lactic acid bacteria

Production of the anti-listerial bacteriocin, pediocin, by lactic acid bacteria (LAB) transformed with the cloning vector pPC418 (Ped⁺, 9.1 kb) was influenced by composition of media and incubation temperature. Maximum pediocin production, tested against Listeria innocua, by electrotransformants of Lactococcus lactis ssp. lactis was measured in tryptone/lactose/yeast extract medium after 24 h growth at 30 °C, while incubation at 40 °C was optimum for Ped⁺ transformants of Streptococcus thermophilus and Enterococcus faecalis. The amount of pediocin produced by S. thermophilus in skim milk and cheese whey supplemented with 0.5% yeast extract was estimated as 51000 units ml^–1 and 25000 units ml^–1, respectively. Pediocin production remained essentially unchanged in reconstituted skim milk or whey media diluted up to 10-fold. The results demonstrate the capacity of recombinant strains of LAB to produce pediocin in a variety of growth media including skim milk and inexpensive cheese whey-based media, requiring minimum nutritional supplementation.     

Screening and characterization of microorganisms with glutaryl-7 ADCA acylase activity

A screening of microorganisms producing glutaryl-7 ADCA acylase, an enzyme able to hydrolyse glutaric acid selectively from glutaryl-3-deacetoxy-7-aminocephalosporanic acid (glutaryl-7 ADCA), has been carried out in soil samples. Five microorganisms expressing acylase activity were isolated and classified as Bacillus cereus, Achromobacter xylosooxidans, Bacillus sp., Pseudomonas sp. and Pseudomonas paucimobilis. The screening was carried out by preparing enrichment cultures containing glutaryl-7-ADCA or cephalosporin C as the selective carbon source. Four model compounds (adipoyl-, glutamyl- and glutaryl-p-nitroanilide and glutarylcoumarin), mimicking the glutaryl-7 ADCA -lactam moiety, were synthesized as substrates suitable for the rapid screening of the microorganisms (2500) isolated from the enrichment cultures. A total of 300 strains were active on the model substrates and only 5 displayed acylase activity on glutaryl-7 ADCA. The fermentation parameters, such as pH and inducer concentration, for the optimal acylase expression and acylase specificity towards the model substrates were different for each strain.     

Selection of Lactobacillus acidophilus strains for use in “acidophilus products”

Recent studies by DNA-DNA hybridization revealed that strains now designated as L. acidophilus, can be divided into several groups and only one group should be classified as L. acidophilus. We studied several phenotypic characteristics in representative strains from the six DNA-homology groups of L. acidophilus. No group specific pattern was observed among the strains for fermentation of eight carbohydrates, growth at 15 and 45°C, resistance to 0.2% oxgall, lysis by lysozyme or sensitivity to 17 antibiotics. However, some differences among groups were observed in -galactosidase (-gal) activity and surface layer (s-layer) protein. Strains in B1 do not have a s-layer or -gal while B2 strains also lack a s-layer but do possess -gal. All strains in groups A1, A2, A3 and A4, capable of growing in lactose, have -gal activity and also have a s-layer composed of protein subunits of different molecular weights (MW). Strains in A1 homology group have a s-layer with 46 Kd protein subunits while strains in other A groups have s-layer protein subunits that varied in MW within each group. On the basis of these two traits several isolates of unknown homology groups have been tentatively placed in A1, B1 or B2 groups. L. acidophilus from A1 group showed strain variation in -gal specific activity and rate of acid production and growth. For use in dietary adjuncts, L. acidophilus strains should be selected for these three and other desirable traits. They should be maintained and grown in media containing lactose.     

Antibiotic effect of linolenic acid fromChlorococcum strain HS-101 andDunaliella primolecta on methicillin-resistantStaphylococcus aureus

Methanol extracts fromChlorococcum strain HS-101 andDunaliella primolecta strongly inhibited the growth of a strain of methicillin-resistantStaphylococcus aureus (MRSA), which is causing serious problems in Japanese hospitals. So that the anti-MRSA substance(s) could be purified and identified, the growth medium was improved for antibiotic production. When the two strains were cultured in their improved media, antibiotic production byChlorococcum strain HS-101 was 1.8-fold that in the standard BG-11 medium, and production byD. primolecta was 2.3-fold. The activity pattern of fractions eluted by silica-gel or gel-permeation chromatography suggested that both strains produced two antibiotic substances. Identification of the purified substances by NMR and GC-MS showed that one of the active substances in both strains was-linolenic acid. Ten fatty acids from other sources were tested, and it was found that unsaturated fatty acids had antibiotic activity against MRSA, with the highest activity that of -linolenic acid.     

Glutamic Acid Production in Biotin-rich Media by Temperature-sensitive Mutants of Brevibacterium lactofermentum,a Novel Fermentation Process

Temperature-sensitive mutants were derived from Brevibacterium lactofermentum strain 2256 in a search for mutants which would produce a large amount of L-glutamic acid in biotin- rich media at the nonpermissive temperature. A total of 159 mutant strains was selected which showed adequate growth at 30°C but showed little or no growth at 37°C on minimal medium. Twenty of these were found to produce glutamic acid in a biotin-rich medium after a temperature shift from 30°C to 37°C, while the wild-type strain 2256 did not produce it under the same cultural condition.

One of the typical mutant strains, Ts-88, produced approximately 2g/dl of glutamic acid from beet molasses (the yield > 55%) in the presence of 33 µg/liter of biotin when tempera- , ture was shifted from 30°C to 40°C during the cultivation. It was concluded that, by controlling only temperature during fermentation, glutamic acid production could be realized in media containing biotin-rich natural carbon sources, without any chemical control such as the addition of expensive surface-active agents or antibiotics. Characteristics and merits of the novel fermentation process are discussed.