Isolation and characterization of a new Achromobacter sp. strain CAR1389 as a carbazole-degrading bacterium

In this work, a bacterial strain with suitable capability to metabolize carbazole (CAR) as a main nitrogen containing compound of petroleum was isolated and characterized. 16S rDNA gene analysis and morphological characteristics of the strain showed that the isolate belonged to the genus Achromobacter and was tentatively named as Achromobacter sp. strain CAR1389. The growth monitoring and biodegradation rate measurements of carbazole in minimal medium supplemented by 6?mM CAR revealed that the strain CAR1389 is able to remove more than 90?% of this compound at 25, 30, and 37?°C during 7?days. The effect of higher concentrations of the carbazole on growth rate and metabolizing activity of the strain exhibited the Achromobacter sp. strain CAR1389 can tolerate increasing levels of CAR concentration up to 21?mM in culture media and degrade 43?% of this toxic material. According to these results and high tolerance of this bacterium in regards to higher concentrations of CAR, we suggest the strain CAR1389 as a suitable isolate to do biorefining of crude oil and also bioremediation processes in highly contaminated area of carbazole.     

New Lindane-Degrading Strains Achromobacter sp. NE1 and Brevundimonas sp. 242

Microbiology - Two strains were isolated, Achromobacter sp. NE1 (GenBank MW132988) and Brevundimonas sp. 242 (GenBank MW132989), which possessed a unique ability to use lindane and the...     

Glyphosate utilization byPseudomonas sp. andAlcaligenes sp. isolated from environmental sources

A total of 21 bacterial cultures were isolated that could utilize glyphosate (N-phosphonomethyl glycine) as a sole source of phosphorus in a mineral salts medium. Sources of inocula for enrichment cultures included aerobic digester liquid, raw sewage, trickling filter effluent, pesticide disposal pit liquid, and soil. Eleven cultures were identified asPseudomonas sp., one asPseudomonas stutzeri, and nine asAlcaligenes sp. Aminomethylphosphonic acid, the major metabolic intermediate of glyphosate degradation in soil, could also serve as a sole phosphorus source for all 21 isolates. Neither glyphosate nor aminomethylphosphonic acid could serve as carbon sources in mineral salts media. Experiments withPseudomonas sp. SG-1 (isolated from aerobic digester liquid) suggested that enzymatic activity responsible for glyphosate degradation was intracellular, inducible, and required the cofactors pyruvate and pyridoxal phosphate. The degradation pathway for glyphosate in this culture may be similar to that previously reported for aminoethylphosphonic acid.     

Achromobacter starkeyi sp. n., a methionine-decomposing bacterium isolated from soil

During a screening for methionine-decomposing bacteria several strains were isolated from soil. One of the most active was studied further. It belongs to the genusAchromobacter. As it could not be identified with any of the known species its morphological and biochemical characteristics are presently given.The facilities given by Dr. W. Buttler from the University of California, San Diego, U.S.A. to use his single beam recording spectrophotometer are appreciated.This work was supported by a fellowship from the Comisión de Operación y Fomento de las Actividades Académicas del Instituto Politécnico Nacional.     

Isolation of a Pseudomonas sp. Which Utilizes the Phosphonate Herbicide Glyphosate

A strain of bacteria has been isolated which rapidly and efficiently utilizes the herbicide glyphosate (N-phosphonomethylglycine) as its sole phosphorus source in a synthetic medium. The strain (PG2982) was isolated by subculturing Pseudomonas aeruginosa ATCC 9027 in a synthetic broth medium containing glyphosate as the sole phosphorus source. Strain PG2982 differs from the culture of P. aeruginosa in that it is nonflagellated, does not produce pyocyanin, and has an absolute requirement for thiamine. Strain PG2982 has been tentatively identified as a Pseudomonas sp. strain by its biochemical activities and moles percent guanine plus cytosine. Measurements of glyphosate with an amino acid analyzer show that glyphosate rapidly disappears from the medium during exponential growth of strain PG2982. In batch culture at 30°C, this isolate completely utilized 1.0 mM glyphosate in 96 h and yielded a cell density equal to that obtained with 1.0 mM phosphate as the phosphorus source. However, a longer lag phase and greater generation time were noted in the glyphosate-containing medium. Strain PG2982 can efficiently utilize glyphosate as an alternate phosphorus source.     

Formylation and acetylation of 4-chloroaniline by a Streptomyces sp.

4-Chloroaniline was metabolized in a liquid growth medium by a Streptomyces sp. which was isolated from soil. After 60 gours of incubation the aniline had disappeared and several metabolites could be detected by thin layer chromatographic analysis. 4-Chloroformylaniline and 4-chloroacetanilide were identified as products. The formation of a formylanilide by the actinomycete indicates a new mechanism of microbial aniline transformation.     

Glyphosate catabolism by Pseudomonas sp. strain PG2982.

The pathway for the degradation of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp. PG2982 has been determined by using metabolic radiolabeling experiments. Radiorespirometry experiments utilizing [3-14C]glyphosate revealed that approximately 50 to 59% of the C-3 carbon was oxidized to CO2. Fractionation of stationary-phase cells labeled with [3-14C]glyphosate revealed that from 45 to 47% of the assimilated label is distributed to proteins and that the amino acids methionine and serine are highly labeled. Adenine and guanine received 90% of the C-3 label found in the nucleic acid fraction, and the only pyrimidine base labeled was thymine. These results indicated that C-3 of glyphosate was at some point metabolized to a C-1 compound whose ultimate fate could be both oxidation to CO2 and distribution to amino acids and nucleic acid bases that receive a C-1 group from the C-1-donating coenzyme tetrahydrofolate. Pulse-labeling of PG2982 cells with [3-14C]glyphosate resulted in the isolation of [3-14C]sarcosine as an intermediate in glyphosate degradation. Examination of crude extracts prepared from PG2982 cells revealed the presence of a sarcosine-oxidizing enzyme that oxidizes sarcosine to glycine and formaldehyde. These results indicate that the first step in glyphosate degradation by PG2982 is cleavage of the carbon-phosphorus bond, resulting in the release of sarcosine and a phosphate group. The phosphate group is utilized as a source of phosphorus, and the sarcosine is degraded to glycine and formaldehyde. This pathway is supported by the results of [1,2-14C]glyphosate metabolism studies, which show that radioactivity in the proteins of labeled cells is found only in the glycine and serine residues.     

Resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase as biocatalyst

Enantioselective acylation employing vinyl alkanoates as acyl donors was exploited for the resolution of 2-(substituted phenoxy)-1-propanols carrying different substituents on the benzene ring using Achromobacter sp. lipase. These primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, based on the enantiomeric ratio E (up to 27), through acylation with vinyl butanoate in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors. Using this procedure, enantiomerically enriched (R)-2-(4-chlorophenoxy)-1-propanol was prepared in 97% e.e. and 33% yield in a gram-scale reaction.     

利用Batch-ANIm快速分析无色杆菌属菌株间的亲缘关系

基因组平均核苷酸相似度(average nucleotide identity,ANI)已成为鉴定细菌种内关系的黄金方法,开发可用于快速分析大量基因组之间ANI值的生物信息学工具具有重要意义.本研究以广泛应用的ANI分析软件JSpecies为基础,采用Perl集成编写了能够根据设置的线程数、自动生成多个JSpecies配置文件以完成基因组序列载入与成对选择,快速完成大量基因组ANI计算与分析的工具Batch-ANIm(下载地址:http://www.microbialgenomic.com/Batch-ANIm.html).采用该工具对已测序的 109株无色杆菌属(Achromobacter)菌株进行ANIm(基于MUMmer算法)计算,共获得5 886个ANIm值.整体上,基于"100-ANIm"进化距离获得的聚类分析结果与核心基因组进化树比较一致,表明ANIm聚类分析可用于快速展示无色杆菌属菌株之间亲缘关系.很多无色杆菌属种内菌株之间的ANIm值小于95％,但种间ANIm值却大于95％,这表明从基因组水平上部分无色杆菌属菌株的分类学命名存在错误,特别是无色杆菌属的代表种A.xylosoxidans,命名为A.xylosoxidans的50个菌株,只有40个菌株真正属于A.xylosoxidans.同时,基于全基因组序列的ANI值比较可以将部分种名不确定的菌株分类学命名精确到种水平.     

利用Batch-ANIm快速分析无色杆菌属菌株间的亲缘关系

基因组平均核苷酸相似度(average nucleotide identity,ANI)已成为鉴定细菌种内关系的黄金方法,开发可用于快速分析大量基因组之间ANI值的生物信息学工具具有重要意义.本研究以广泛应用的ANI分析软件JSpecies为基础,采用Perl集成编写了能够根据设置的线程数、自动生成多个JSpecies配置文件以完成基因组序列载入与成对选择,快速完成大量基因组ANI计算与分析的工具Batch-ANIm(下载地址:http://www.microbialgenomic.com/Batch-ANIm.html).采用该工具对已测序的 109株无色杆菌属(Achromobacter)菌株进行ANIm(基于MUMmer算法)计算,共获得5 886个ANIm值.整体上,基于"100-ANIm"进化距离获得的聚类分析结果与核心基因组进化树比较一致,表明ANIm聚类分析可用于快速展示无色杆菌属菌株之间亲缘关系.很多无色杆菌属种内菌株之间的ANIm值小于95％,但种间ANIm值却大于95％,这表明从基因组水平上部分无色杆菌属菌株的分类学命名存在错误,特别是无色杆菌属的代表种A.xylosoxidans,命名为A.xylosoxidans的50个菌株,只有40个菌株真正属于A.xylosoxidans.同时,基于全基因组序列的ANI值比较可以将部分种名不确定的菌株分类学命名精确到种水平.     

Uptake of Glyphosate by an Arthrobacter sp

The uptake of glyphosate (N-[phosphonomethyl]glycine) by an Arthrobacter sp. which can utilize this herbicide as its sole source of phosphorus was investigated. Orthophosphate suppressed the expression of the uptake system for glyphosate and also competed with glyphosate for uptake. The K_m for glyphosate uptake was 125 μM, and the K_i for orthophosphate was 24 μM. Organophosphonates as well as organophosphates inhibited glyphosate uptake, but only organophosphates and orthophosphate suppressed the uptake system. Glyphosate uptake was energy dependent, had a pH optimum of 6 to 7, and was differentially affected by divalent cations.     

Resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase as biocatalyst

Enantioselective acylation employing vinyl alkanoates as acyl donors was exploited for the resolution of 2-(substituted phenoxy)-1-propanols carrying different substituents on the benzene ring using Achromobacter sp. lipase. These primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, based on the enantiomeric ratio E (up to 27), through acylation with vinyl butanoate in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors. Using this procedure, enantiomerically enriched (R)-2-(4-chlorophenoxy)-1-propanol was prepared in 97% e.e. and 33% yield in a gram-scale reaction.     

Isolation and characterization of a new strain of Achromobacter sp. with beta-lactam antibiotic acylase activity

A bacterial strain producing a -lactam antibiotic acylase, able to hydrolyze ampicillin to 6-aminopenicillanic acid more efficiently than penicillin G, was isolated from soil and characterized. The isolate was identified as Achromobacter sp. using the phenotypic characteristics, composition of cellular fatty acids and 16S rRNA gene sequence. The enzyme synthesis was fully induced by phenylacetic acid (PAA) at a concentration of 2 g l^–1. PAA at concentrations up to 12 g l^–1 had no negative effect on the specific activity of acylase and biomass production, but slowed down the specific growth rate. Benzoic or 4-hydroxyphenylacetic acids can also induce synthesis of the enzyme. The inducers were metabolized in all cases. Acylase activity in cell-free extracts was determined with various substrates; ampicillin, cephalexin and amoxicillin were hydrolyzed 1.5- and 2-times faster than penicillin G. A high stability of acylase activity was observed over a wide range of pH (5.0–8.5) and at temperatures above 55°C.     

Co-metabolism of methyl- and chloro-substituted catechols by an Achromobacter sp. possessing a new meta-cleaving oxygenase

Co-metabolism of 3-methylcatechol, 4-chlorocatechol and 3,5-dichlorocatechol by an Achromobacter sp. was shown to result in the accumulation of 2-hydroxy-3-methylmuconic semialdehyde, 4-chloro-2-hydroxymuconic semialdehyde and 3,5-dichloro-2-hydroxymuconic semialdehyde respectively. Formation of these products indicated that cleavage of the aromatic nucleus of the substituted catechols was accomplished by a new meta-cleaving enzyme, catechol 1,6-oxygenase. This enzyme was equally active on both chloro- and methyl-substituted catechols.     

Bacillus sp. B16 kills nematodes with a serine protease identified as a pathogenic factor

An endospore-forming bacterium, strain B16, was isolated from a soil sample and identified as a Bacillus sp. The strain presented remarkable nematotoxic activity against nematode Panagrellus redivivus. The crude extracellular protein extract from culture supernatant of the bacteria killed about 80% of the tested nematodes within 24 h, suggesting the involvement of extracellular proteases. A homogeneous extracellular protease was purified by chromatography, and the hypothesis of proteinaceous pathogeny in the infection of B16 strain was confirmed by the experiments of killing living nematodes and by the degradation of purified nematode cuticle when treated with the homogenous protease. The gene for the virulence protease was cloned, and the nucleotide sequence was determined. The deduced amino acid sequence showed significant similarity with subtilisin BPN' but low homology with the other cuticle-degrading proteases previously reported in fungi. Characterization of the purified protease revealed the molecular mass of 28 kDa and the optimum activity at pH 10, 50°C. The purified protease can hydrolyze several native proteinaceous substrates, including collagen and nematode cuticle. To our knowledge, this is the first report of a serine protease from a Bacillus genus of bacteria that serves as a pathogenic factor against nematodes, an important step in understanding the relationship between bacterial pathogen and host and in improving the nematocidal activity in biological control. Niu Qiuhong and Huang Xiaowei contributed equally to the work