Rhodococcus sp. strain TM1 plays a synergistic role in the degradation of piperidine by Mycobacterium sp. strain THO100

Mycobacterium sp. strain THO100 and Rhodococcus sp. strain TM1 were isolated from a morpholine-containing enrichment culture of activated sewage sludge. Strain THO100, but not strain TM1, was able to degrade alicyclic amines such as morpholine, piperidine, and pyrrolidine. The mixed strains THO100 and TM1 showed a better growth on piperidine as the substrate than the pure strain THO100 because strain TM1 was able to reduce the level of glutaraldehyde (GA) produced during piperidine degradation. GA was toxic to strain THO100 (IC₅₀ = 28.3 μM) but less toxic to strain TM1 (IC₅₀ = 215 μM). Strain THO100 possessed constitutive semialdehyde dehydrogenases, namely Sad1 and Sad2, whose activities toward succinic semialdehyde (SSA) were strongly inhibited by GA. The two isozymes were identified as catalase–peroxidase (KatG = Sad1) and semialdehyde dehydrogenase (Sad2) based on mass spectrometric analyses of tryptic peptides and database searches of the partial DNA sequences of their genes. In contrast, strain TM1 containing another constitutive enzyme Gad1 could oxidize both SSA and GA. This study suggested that strain TM1 possessing Gad1 played a synergistic role in reducing the toxic and inhibitory effects of GA produced in the degradation of piperidine by strain THO100.     

Differentiation of a rapidly growing,scotochromogenic, polycyclic-aromatic-hydrocarbon-metabolizing strain of Mycobacterium sp. from other known Mycobacterium species

A rapidly-growing, acid-alcohol fast, scotochromogenic, polycyclic-aromatic-hydrocarbon-degrading Mycobacterium sp. isolate, Pyr-1, which was different from known Mycobacterium species based on biochemical tests, was further analyzed to compare its mycolic acids, cellular proteins, and nucleic acids with those of known species. Mass spectral analysis of the mycolic acids of Mycobacterium sp. Pyr-1 indicated that its mycolic acids were C₆₀H₁₂₀O₃ and C₆₂H₁₂₄O₃. The mycolic acid pattern from this bacterium was compared to those of 29 rapidly-growing, scotochromogenic species and 31 other species of Mycobacterium by reversed-phase high-performance liquid chromatography (HPLC). The mycolic acid pattern was unique, most closely resembling M. austroafricanum but also resembling M. parafortuitum and M. gilvum. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of soluble cellular proteins also readily differentiated this isolate from other species. The polypeptide pattern of Mycobacterium sp. Pyr-1 most closely resembled that of M. austroafricanum. Restriction enzyme analysis and Southern blot hybridization, however, revealed differences between the chromosomal DNA of our isolate and that of M. austroafricanum. The unique biochemical characteristics, mycolic acid pattern, polypeptide fingerprints, DNA restriction digest patterns, and DNA homology indicate that this strain is different from previously known species of mycobacteria. Since this bacterium is efficient in the metabolism of polycyclic aromatic hydrocarbons, its characteristics and relationships to other Mycobacterium species are reported here.     

Biodegradation of 1,4-dioxane and transformation of related cyclic compounds by a newly isolated Mycobacterium sp. PH-06

A new bacterial strain PH-06 was isolated using enrichment culture technique from river sediment contaminated with 1,4-dioxane, and identified as belonging to the genus Mycobacterium based on 16S rRNA sequencing (Accession No. EU239889). The isolated strain effectively utilized 1,4-dioxane as a sole carbon and energy source and was able to degrade 900 mg/l 1,4-dioxane in minimal salts medium within 15 days. The key degradation products identified were 1,4-dioxane-2-ol and ethylene glycol, produced by monooxygenation. Degradation of 1,4-dioxane and concomitant formation of metabolites were demonstrated by GC/MS analysis using deuterium labeled 1,4-dioxane (1,4-dioxane-d8). In addition to 1,4-dioxane, this bacterium could also transform structural analogues such as 1,3-dioxane, cyclohexane and tetrahydrofuran when pre-grown with 1,4-dioxane as the sole growth substrate. Our results suggest that PH-06 can maintain sustained growth on 1,4-dioxane without any other carbon sources.     

Physiological and biochemical characterization of glyoxalase I,a general marker for cell proliferation,from a soybean cell suspension

Using a strictly auxin-dependent soybean (Glycine max (L.) Merr.) cell suspension, we studied the correlation of auxin-dependent cell proliferation and the activity of glyoxalase I (S-lactoylglutathione-lyase EC 4.4.1.5.), an enzyme generally associated with cell proliferation in animal, microbial and, as reported recently, also plant systems. We found the activity of glyoxalase I to be modulated during the proliferation cycle, with a maximal activity between day 2 and day 4 of culture growth. After starving the culture of auxins for three subsequent periods, both the enzyme activity and cell growth could be re-initiated with auxin. Enzyme activity reached its maximum 1 d before cell number was at a maximum. The enzyme was purified to homogeneity and characterized.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GSH reuced glutathione - Mr relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate The authors thank Dr. K. Palme, Max-Planck-Institute, Cologne, for reverse-phase chromatography. Part of this work was done by C. Paulus at the Department of Biotechnology, New Delhi, India under the Bundesministerium für Forschung und Technologie (BMFT)-funded Indo-FRG collaboration programme. Thanks are due to Professors S. Guha-Mukherjee and S.K. Sopory, New Delhi, for introduction into glyoxalase research. The research was funded by a BMFT-DECHEMA fellowship to C. Paulus, a BMFT grant to H.-J. Jacobsen and a Graduierten Förderung des Landes Nordrhein-Westfalen fellowship to B. Köllner.     

Cloning and characterization of a cyclohexanone monooxygenase gene from Arthrobacter sp. L661

Cyclohexanone monooxygenase (CHMO), a type of Baeyer-Villiger oxidation, catalyzes the oxidation of cyclohexanone into ɛ-caprolactone, which has been utilized as a building block in organic synthesis. A bacterium that is capable of growth on cyclohexanone as a sole carbon source was recently isolated and was identified as Arthrobacter sp. L661. The strain is believed to harbor a CHMO gene (chnB), considering the high degradablity of cyclohexanone. In order to characterize the CHMO, a chnB gene was cloned from Arthrobacter sp. L661. The deduced amino acids of the chnB gene evidenced the highest degree of homology (90% identity) with the CHMO of Arthrobacter sp. BP2 (accession no. AY123972). The CHMO of L661 was shown to be functionally expressed in Escherichia coli cells, purified via affinity chromatography, and characterized. The specific activity of the purified enzyme was 24.75 μmol/min/mg protein. The optimum pH was 7.0 and the enzyme maintained over 70% of its activity for up to 24 h in a pH range of 6.0 to 8.0 at 4°C. The CHMO of L661 readily oxidized cyclobutanone and cyclopentanone whereas less activity was detected with those of Arthrobacter sp. BP2, Rhodococcus sp. Phi1, and Rhodococcus sp. Phi2, thereby suggesting that the CHMO of L661 evidenced the different substrate specificities compared with other CHMOs. These results can provide us with useful information for the development of biocatalysts applicable to commercial organic syntheses, especially because only a few CHMO genes have been identified thus far.     

Physiological characterization of Mycobacterium sp. strain 1B isolated from a bacterial culture able to degrade high-molecular-weight polycyclic aromatic hydrocarbons

AIM: The aim of this study was to further characterize a bacterial culture (VUN 10,010) capable of benzo[a]pyrene cometabolism. METHODS AND RESULTS: The bacterial culture, previously characterized as a pure culture of Stenotrophomonas maltophilia (VUN 10,010), was found to also contain another bacterial species (Mycobacterium sp. strain 1B), capable of degrading a similar range of PAH substrates. Analysis of its 16S rRNA gene sequence and growth characteristics revealed the strain to be a fast-growing Mycobacterium sp., closely related to other previously isolated PAH and xenobiotic-degrading mycobacterial strains. Comparison of the PAH-degrading characteristics of Mycobacterium sp. strain 1B with those of S. maltophilia indicated some similarities (ability to degrade phenanthrene and pyrene), but some differences were also noted (S. maltophilia able to degrade fluorene, but not fluoranthene, whereas Mycobacterium sp. strain 1B can degrade fluoranthene, but not fluorene). Unlike the S. maltophilia culture, there was no evidence of benzo[a]pyrene degradation by Mycobacterium sp. strain 1B, even in the presence of other PAHs (ie pyrene) as co-metabolic substrates. Growth of Mycobacterium sp. strain 1B on other organic carbon sources was also limited compared with the S. maltophilia culture. CONCLUSIONS: This study isolated a Mycobacterium strain from a bacterial culture capable of benzo[a]pyrene cometabolism. The Mycobacterium strain displays different PAH-degrading characteristics to those described previously for the PAH-degrading bacterial culture. It is unclear what role the two bacterial strains play in benzo[a]pyrene cometabolism, as the Mycobacterium strain does not appear to have endogenous benzo[a]pyrene degrading ability. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the isolation and characterization of a novel PAH-degrading Mycobacterium strain from a PAH-degrading culture. Further studies utilizing this strain alone, and in combination with other members of the consortium, will provide insight into the diverse roles different bacteria may play in PAH degradation in mixed cultures and in the environment.     

Isolation and characterization of Arthrobacter sp. strain MCM B-436, an atrazine-degrading bacterium, from rhizospheric soil

Atrazine is one of the most environmentally prevalent s-triazine-ring herbicides. The widespread use of atrazine and its toxicity necessitates search for remediation technology. As atrazine is still used in India as a major herbicide, exploration of atrazine-degrading bacterial community is of immense importance. Considering lack of reports on well characterized atrazine-degrading bacterial cultures from India and wide diversity and density of microorganisms in rhizosphere, soil sample from rhizosphere of atrazine-resistant plant was studied. Arthrobacter sp. strain isolated in this investigation utilizes atrazine as the sole nitrogen source. In addition, the bacterium degrades other triazines such as ametryn, cyanizine, propazine and simazine. PCR analysis confirms the presence of atzBCD and triazine hydrolase (trzN) genes on chromosomal DNA. Sequencing of the trzN gene reveals high sequence similarity with trzN from Nocardioides sp. C190. An inducible and intracellular atrazine chlorohydrolase enzyme was isolated and partially purified from this isolate. This study confirms the presence of atrazine-degrading microbial population in Indian soils and could be used efficiently for remediation of contaminated soils. Presence of trzN gene indicates possible presence of bacterial community with more efficient and novel enzymatic capabilities. Comparison of enzyme and gene structure of this isolate with other geographically distinct atrazine-degrading strains will help us in the better understanding of gene transfer and evolution.     

Production of PHB and P (3HB-co-3HV) biopolymers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strain OU303A isolated from municipal sewage sludge

Bacillus megaterium strain OU303A isolated from municipal sewage sludge was selected for the study of biosynthesis of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate P (HB-co-HV) copolymer. The strain yielded a maximum of 62.43% DCW polymer in the medium containing glycerol as carbon source, which was followed by 58.63% DCW polymer in glucose containing medium. We found that this strain was capable of producing 2.5% hydroxyvalerate copolymer from a single carbon substrate, glucose. The strain showed an increase in the amount of HV monomer content, when the precursor for the copolymer was included in the fermentation medium. The characterization of the biopolymers was carried out using FTIR, GC-MS, H¹ NMR and DSC. This is the first report of B. megaterium strain producing HV copolymer, without the addition of any precursor in the fermentation medium.     

Purification and characterization of an alkaline cellulase from a newly isolated alkalophilic Bacillus sp. HSH-810

An alkaline cellulase from Bacillus sp. HSH-810 was purified 8.7-fold with a 30% yield and a specific activity of 71 U mg^–1 protein. It was optimally active at pH 10 and 50 °C and was stable from pH 6 to 10 with more than 60% activity remaining after heating at 60 °C for 60 min. The molecular mass of cellulase was 80 kDa. It was inhibited by 50% by Fe³⁺ (1 mM) and Mn²⁺ (0.1 mM) but was relatively insensitive to Hg²⁺ and Pb²⁺ at 1 mM.Revisions requested: 8 October 2004/1 December 2004; Revisions received 29 November 2004/5 January 2005     

Isolation, characterization and optimization of culture parameters for production of an alkaline protease isolated from Aspergillus tamarii

Aspergillus tamarii expresses an extracellular alkaline protease that we show to be effective in removing hair from cattle hide. Large quantities of the enzyme will be required for the optimization of the enzymatic dehairing process so the growth conditions for maximum protease expression by A. tamarii were optimized for both solid-state culture on wheat bran and for broth culture. Optimal protease expression occurred, for both cultural media, at initial pH 9; the culture was incubated at 30 °C for 96 h using a 5% inoculum. The crude enzyme was isolated, purified and characterized using MALDI TOF TOF. The alkaline protease was homologous to the alkaline protease expressed by Aspergillus viridinutans. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Physiological characterization of starch-utilizing <Emphasis Type="Italic">Saccharomyces</Emphasis> sp. SK0704 isolated from kimchi

Saccharomyces sp. SK0704 (further defined as SK0704) isolated from long-term-ripening kimchi was identified by a biochemical method with an API kit; its physiology was found to be very similar to that of S. cerevisiae ATCC 26603 (further defined as ATCC 26603), except in terms of starch utilization. SK0704 did not excrete extracellular glucoamylase, but utilized starch as a sole carbon source under only aerobic conditions. Crude enzyme excreted from SK0704 catalyzed the saccharification of starch to glucose, but ATCC 26603 did not. The PCR product obtained using the chromosomal DNA of SK0704 and the primers designed on the basis of the extracellular glucoamylase-coding gene of S. diastaticus was homologous with the intracellular sporulation-specific glucoamylase of S. cerevisiae. SDS-PAGE pattern of soluble protein extracted from yeast cells grown on glucose was greatly different from that on starch. From these results, we proposed that the SK0704 may have a specific physiological function for starch catabolism such as membrane transport system and intracellular sac-charification of starch.     

松萝内生酿酒酵母菌株耐酸生理特性与分子机制探究

【目的】对我国西藏地区来源的不同酵母菌株进行有机酸发酵性能测试,此外,对具有良好产酸性能的分离自松萝内部的酿酒酵母菌株Saccharomy cescerevisiae 2-2进行耐酸性能分析,并探究其耐酸较强的分子机制。【方法】比较不同糖浓度培养基液体发酵培养过程中pH的变化,并比较低pH胁迫条件下菌株的生长,检测酿酒酵母菌株的产酸潜力和耐酸特性;对菌株2-2和模式酵母菌株S288C进行比较基因组分析,并利用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)分析关键基因的转录,探究菌株2-2耐酸分子机制。【结果】松萝内生酿酒酵母2-2在所有检测的菌株中产酸潜力较大,耐酸性能较好。在菌株2-2中与胁迫耐受性相关的基因PDR15、PDR12和SUR1在低pH胁迫条件下存在显著的上调或下调,但这些基因转录变化趋势与菌株S288C相反。【结论】松萝内生酿酒酵母2-2是一株产酸耐酸性能较好的菌株,对其独特的调节机制进行深入分析,有希望选育性能更好的产酸酵母菌株。     

Molecular characterization of Vibrio parahaemolyticus of similar serovars isolated from sewage and clinical cases of diarrhoea in Calcutta,India

Vibrio parahaemolyticus is a seafood-borne halophilic pathogen that causes acute gastroenteritis in humans. During the course of an investigation on the incidence of V. parahaemolyticus in sewage water samples of Calcutta, India, we isolated eight (26.7%) strains of V. parahaemolyticus from 30 samples. Among these strains, five (62.5%) carried the thermostable direct hemolysin (tdh) gene, a major virulence marker of V. parahaemolyticus. Two strains belonged to serovar O5:K3 and the remaining three to O5:KUT, which is common among clinical strains of V. parahaemolyticus isolated from hospitalized patients of Calcutta with acute diarrhoea. The tdh positive sewage strains of V. parahaemolyticus were compared by randomly amplified polymorphic DNA (RAPD)-PCR and pulsed-field gel electrophoresis (PFGE) with strains of similar serovars selected from our culture collection to determine the genetic relatedness. Our results showed that except for sharing the similar serovar, sewage and clinical strains of V. parahaemolyticus were genetically different. In addition, toxRS-targeted group-specific (GS) PCR and open reading frame 8 (ORF-8) PCR showed that the sewage strains did not belong to the pandemic genotype. Since the sewage in Calcutta is directly used for cultivation of vegetables and for pisciculture, the presence of tdh positive V. parahaemolyticus in the sewage highlights the need for constant monitoring of the environment.     

Cloning,expression in yeast,and functional characterization of CYP71D14, a root-specific cytochrome P450 from <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Alkaloids, which are naturally occurring amines, are biosynthesized and accumulated in plant tissues such as roots, leaves, and stems. Many alkaloids have pharmacological effects on humans and animals. Cytochrome P450 (P450 or CYP) monooxygenases are known to play key roles in the biosynthesis of alkaloids in higher plants. A cDNA clone encoding a P450 protein consisting of 502 amino acids was isolated from Petunia hybrida. The deduced amino acid sequence of the cDNA clone showed a high level of similarity with the other P450 species in the CYP71D family; hence, this novel P450 was named CYP71D14. Among plant P450 species, CYP71D14 had 45.7% similarity in its amino acid sequence to CYP71D12, which is involved in the biosynthesis of the indole alkaloids vinblastine and vincristine. Expression of CYP71D14 mRNA in Petunia plants was examined by Northern blot analysis by using a full-length cDNA of CYP71D14 as a probe. CYP71D14 mRNA was expressed most abundantly in the roots. The nucleotide sequence of CYP71D14 has been submitted to the DDBJ, EMBL, and GenBank nucleotide databases under the accession number AB028462. An erratum to this article can be found at     

Production and characterization of xylanases of a Bacillus strain isolated from soil

Xylanase production was performed by growing a Bacillus isolate on agricultural by-products, wheat straw, wheat bran, corn cobs and cotton bagasse. A maximum xylanase activity of 180 U/ml was obtained together with a cellulase activity of 0.03 U/ml on 4 (w/v) corn cobs. Electrophoretic analysis showed the presence of three endo--1, 4-xylanases having molecular weights of about 22, 23 and 40 kDa. Xylanolytic activity was stable up to 50 °C in the pH range of 4.5–10 and the highest activity was observed at 70 °C and pH 6.5.     

Degradation of olestra, a non caloric fat replacer, by microorganisms isolated from activated sludge and other environments

Olestra is a non-caloric fat substitute consisting of fatty acids esterified to sucrose. Previous work has shown that olestra is not metabolized in the gut and is excreted unmodified in human feces. To better understand the fate of olestra in engineered and natural environments, aerobic bacteria and fungi that degrade olestra were enriched from sewage sludges, soils and municipal solid waste compost not previously exposed to olestra. Various mixed and pure cultures were obtained from these sources which were able to utilize olestra as a sole carbon and energy source. The fastest growing enrichment was obtained from activated sludge and later yielded an olestra-degrading pure culture of Pseudomonas aeruginosa. This mixed culture extensively degraded both ¹⁴C-fatty acid labeled olestra and ¹⁴C-sucrose labeled olestra during 8 days of incubation. Longer-term incubation with pure cultures of P. aeruginosa demonstrated that >98% of ¹⁴C-sucrose labeled olestra and >72% of ¹⁴C-fatty acid labeled olestra was mineralized to CO₂ after 69 days. These results indicate that olestra degraders are present in environments not previously exposed to olestra and that olestra can serve as a sole carbon and energy source. Furthermore, a common bacterial species was isolated from activated sludge and shown to have the ability to degrade olestra.     

Phenylobacterium falsum sp. nov., an Alphaproteobacterium isolated from a nonsaline alkaline groundwater, and emended description of the genus Phenylobacterium

A Gram-negative bacterium designated AC-49T was isolated from an alkaline groundwater with a pH 11.4. This organism formed rod-shaped cells, was strictly aerobic, catalase and oxidase positive, with an optimum growth temperature of 35 degrees C and an optimum pH value of 8.0. Strain AC-49T assimilated primarily amino acids and some Krebs cycle metabolites, did not use sugars for growth. The organism did not grow on L-phenylalanine or antipyrin. The G+ C content of DNA was 66.9 mol%. The phylogenetic analyses based on the 16S rRNA sequencing showed that the closest relatives of strain AC-49T were Phenylobacterium lituiforme and Phenylobacterium immobile, indicating that the organism is a member of the order Caulobacterales of the Alphaproteobacteria. Based on the phylogenetic analyses and distinct phenotypic characteristics, we are of the opinion that strain AC-49T, represents a novel species of the genus Phenylobacterium for which we propose the name Phenylobacterium falsum sp. nov.     

Cloning, gene expression and characterization of a novel bacterial NAD-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Neisseria meningitidis strain Z2491

Alignment of the amino acid sequence of some archaeal, bacterial and eukaryotic non-phosphorylating glyceraldehydes-3-phosphate dehydrogenases (GAPNs) and aldehyde dehydrogenases (ALDHs) with the sequence of a putative GAPN present in the genome of the Gram-negative bacterium Neisseria meningitidis strain Z2491 demonstrated the conservation of residues involved in the catalytic activity. The predicted coding sequence of the N. meningitidis gapN gene was cloned in Escherichia coli XL1-blue under the expression of an inducible promoter. The IPTG-induced GAPN was purified ca. 48-fold from E. coli cells using a procedure that sequentially employed conventional ammonium sulfate fractionation as well as anion-exchange and affinity chromatography. The purified recombinant enzyme was thoroughly characterized. The protein is a homotetramer with a 50-kDa subunit, exhibiting absolute specificity for NAD and a broad spectrum of aldehyde substrates. Isoelectric focusing analysis with the purified fraction showed the presence of an acidic polypeptide with an isoelectric point of 6.3. The optimum pH of the purified enzyme was between 9 and 10. Studies on the effect of increasing temperatures on the enzyme activity revealed an optimal value ca. 64 °C. Molecular phylogenetic data suggest that N. meningitidis GAPN has a closer relationship with archaeal GAPNs and glyceraldehyde dehydrogenases than with the typical NADP-specific GAPNs from Gram-positive bacteria and photosynthetic eukaryotes.     

Purification and characterization of a thermostable uricase from Microbacterium sp. strain ZZJ4-1

In order to study the properties of a thermostable uricase produced by Microbacterium sp. strain ZZJ4-1, the enzyme was purified by ammonium sulfate precipitation and DEAE-cellulose ion exchange, hydrophobic and molecular sieve chromatography. The molecular mass of the purified enzyme was estimated to be 34 kDa by SDS-PAGE. The enzyme was stable between pH 7.0 and 10.00. The optimal reaction temperature of the enzyme was 30 °C at pH 8.5. The K _m and K _cat of the enzyme were 0.31 mM and 3.01 s⁻¹, respectively. Fe³⁺ could enhance the enzyme activity, whereas Ag⁺, Hg²⁺, o-phenanthroline and SDS inhibited the activity of the enzyme considerably. After purification, the enzyme was purified 19.7-fold with 31% yield. As compared with uricases from other microbial sources, the purified enzyme showed excellent thermostability and other unique characteristics. The results of this work showed that strains of Microbacterium could be candidates for the production of a thermostable uricase, which has the potential clinical application in measurement of uric acid.