Description of a Novel Actinobacterium Microbacterium assamensis sp. nov., Isolated from Water Sample Collected from the River Brahmaputra, Assam, India

A Gram-positive, yellow pigmented actinobacterium, strain S2-48(T) was isolated from water sample collected from the river Brahmaputra, Assam, India and subjected to a polyphasic taxonomic study. Most of the physiological and biochemical properties, major fatty acids (C(15:0) Anteiso, iso C(16:0) and C(17:0) Anteiso), estimated DNA G+C content (70.2 mol%) and 16S rRNA gene sequence analysis showed that strain S2-48(T) belonged to the genus Microbacterium. Strain S2-48(T) exhibited highest 16S rRNA gene sequence similarity with Microbacterium testaceum (97.0%); however, the DNA-DNA relatedness value between strain S2-48(T) and M. testaceum was 9.1%. On the basis of differential phenotypic characteristics and genotypic distinctiveness, strain S2-48(T) should be classified within the genus Microbacterium as a novel species, for which the name Microbacterium assamensis is proposed. The type strain is S2-48(T) (=MTCC 10486(T) = DSM 23998(T)).     

一株抑制黄曲霉毒素的深海微杆菌的分离与鉴定

从南大西洋深海海水中分离到一株放线菌菌株R104,该菌株的发酵无细胞上清液对黄曲霉毒素合成的抑制率高达96.2%,经16S rDNA序列分析,初步将该菌株鉴定为微杆菌（Microbacterium）,这是首次报道深海来源的微杆菌具有抑制黄曲霉毒素合成的能力。     

Characterization of a protease of a feather-degrading Microbacterium species

AIMS: To characterize a new feather-degrading bacterium. METHODS AND RESULTS: The strain kr10 producing a high keratinolytic activity when cultured on native feather broth was identified as Microbacterium sp., based on phenotypical characteristics and 16S rDNA sequence. The bacterium presented optimum growth and feather-degrading activity at pH 7.0 and 30 degrees C. Complete feather degradation was achieved during cultivation. The keratinase was partially purified by gel filtration chromatography. It was optimally active at pH 7.0 and 55 degrees C. The enzyme was inhibited by 1,10-phenanthroline, EDTA, p-chloromercuribenzoic acid, 2-mercaptoethanol and metal ions like Hg(2+), Cu(2+) and Zn(2+). SIGNIFICANCE AND IMPACT OF THE STUDY: A new Microbacterium sp. strain was characterized presenting high feather-degrading activity, which appears to be associated to a metalloprotease-type keratinase. This micro-organism has enormous potential for use in biotechnological processes involving keratin hydrolysis.     

Microbacterium terricolae sp. nov., isolated from soil in Japan

The taxonomic positions of two novel strains isolated from a soil sample collected in Japan using Glucose-Peptone-Meat extract (GPM) agar plates supplemented with superoxide dismutase or superoxide dismutase plus catalase were investigated based on the results of chemotaxonomic, phenotypic and genotypic characteristics. Strains were Gram-positive, catalase-positive, non-motile bacteria with L-ornithine as a diagnostic diamino acid of the peptidoglycan. The acyl type of the peptidoglycan was N-glycolyl. The major menaquinones were MK-12 and 13. Mycolic acids were not detected. The G+C content of the DNA was 70 mol%. Comparative 16S rRNA studies on the two isolated strains revealed that they belong to the genus Microbacterium. DNA-DNA relatedness data revealed that KV-448(T) and KV-769 are a new species of the genus Microbacterium. From these results, we propose that these bacteria should be classified in the genus Microbacterium as Microbacterium terricolae sp. nov. The type strain of Microbacterium terricolae is KV-448(T) (=NRRL B-24468(T), NBRC 101801(T)).     

黑粉虫与黄粉虫幼虫肠道细菌的比较

在黑粉虫和黄粉虫肠道中分别分离获得5株细菌,对其菌体形态、培养性状、染色反应、生理生化反应等进行了系统研究。鉴定结果表明,黑粉虫的5个细菌菌株分别属于金杆菌属(Aureobacterium)、李斯特氏菌属(Listeria)、微杆菌属(M i-crobacterium)、莫拉氏菌属(M oraxella)、短小杆菌属(Curtobacterium);黄粉虫的5个细菌菌株分别属于金杆菌属(Aureobacte-rium)、球形芽孢杆菌(Bacillus sphaericus)、微杆菌属(M icrobacterium)、巨大芽孢杆菌(B.m egaterium)、短小杆菌属(Curtobac-terium)。金杆菌属(Aureobacterium)、微杆菌属(M icrobacterium)和短小杆菌属(Curtobacterium)均在2种昆虫肠道中出现。     

A new aerobic gram-positive bacterium with a unique ability to degrade ortho- and para-chlorinated biphenyls

Strain B51 capable of degrading polychlorinated biphenyls (PCB) was isolated from soil contaminated with wastes from the chemical industry. Based on its morphological and chemotaxonomic characteristics, the strain was identified as a Microbacterium sp. Experiments with washed cells showed that strain B51 is able to degrade ortho- and para-substituted mono-, di-, and trichlorinated biphenyls (MCB, DCB, and TCB, respectively). Unlike the known PCB degraders, Microbacterium sp. B51 is able to oxidize the ortho-chlorinated ring of 2,2'-DCB and 2,4'-DCB and the para-chlorinated ring of 4.4'-DCB. The degradation of 2,4'-DCB and 4,4'-DCB was associated with the accumulation of 4-chlorobenzoic acid (4-CBA) in the medium in amounts comprising 80-90% of the theoretical yield. The strain was able to utilize 2-MCB, 2,2'-DCB, and their intermediate 2-CBA and to oxidize the mono(ortho)-chlorinated ring of 2,4,2'-TCB and the di(ortho-para)-chlorinated ring of 2,4,4'-TCB. A mixed culture of Microbacterium sp. B51 and the 4-CBA-degrading bacterium Arthrobacter sp. H15 was found to grow well on 1 g/l 2,4'-DCB as the sole source of carbon and energy.     

Some Observations on the Taxonomy of the Genus Microbacterium. I. Cultural and Physiological Reactions and Heat Resistance

S ummary . The cultural and physiological reactions and the heat resistance of 25 organisms of the genus Microbacterium were examined. They could be differentiated into 3 groups corresponding with the previously described species, Microbacterium flavum, M. lacticum and M. liquefaciens.     

Isolation and characterization of a xanthan-degrading Microbacterium sp. strain XT11 from garden soil

AIMS: Isolation and characterization of the xanthan-degrading Microbacterium sp. XT11. METHODS AND RESULTS: The bacterial isolate XT11, capable of fragmenting xanthan, has been isolated from soil sample. Morphological and biochemical analyses, as well as 16S rRNA gene sequence comparisons, demonstrated that strain XT11 should be grouped in the genus Microbacterium, and represented a new member in this family. Xanthan could be degraded by the xanthan-degrading enzyme released from strain XT11. It has been shown that xantho-oligosaccharides fragmented from xanthan had both elicitor activity and antibacterial effect against Xanthomonas campestris pv. campestris. CONCLUSIONS: The xanthan-degrading enzyme produced by the newly isolated XT11 could fragment xanthan to form oligosaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: Xanthan-degrading products would be useful for potential application in the control of black rot of cruciferous plants caused by X. campestris pv. campestris and, as an oligosaccharide elicitor, in making these plants resistant to disease.     

Purification and properties of a keratinolytic metalloprotease from Microbacterium sp

AIMS: This study was developed to purify and to characterize a keratinolytic protease from the bacterium Microbacterium sp. strain kr10. METHODS AND RESULTS: Enzyme purification was carried out by sequential liquid chromatography on Sephadex G-100 and Q-Sepharose columns. The purification was about 255-fold, with a yield of 34%, as determined with azocasein as substrate. The molecular weight of the enzyme was estimated as 42,000 Da by SDS-PAGE. The enzyme had pH and temperature optima of 7.5 and 50 degrees C respectively. This keratinase was inhibited by EDTA and 1,10-phenanthroline, and analysis of metal content indicates that Zn(2+) and Mg(2+) are present. A 2(2) factorial design was developed to investigate the effect of keratinase and mercaptoacetate concentration on feather keratinolysis. Statistical analysis showed that both variables have a significant effect on hydrolysis of keratin. CONCLUSIONS: A new keratinase produced by Microbacterium sp. was purified and characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: This keratinolytic enzyme offers an interesting potential for the hydrolysis of keratin wastes to be used as feed supplement or bioconversion to added-value products.     

Microbial metabolism of quinoline and related compounds. XI. Degradation of quinoline-4-carboxylic acid by Microbacterium sp. H2, Agrobacterium sp. 1B and Pimelobacter simplex 4B and 5B.

From soil enrichment cultures four strains, using quinoline-4-carboxylic acid as sole source of energy and carbon, have been isolated. According to their physiological properties these bacteria have been identified as Microbacterium sp. designated H2, as Agrobacterium sp. designated 1b and Pimelobacter simplex designated 4B and 5B. Metabolites of the degradation pathway of quinoline-4-carboxylic acid have been isolated and identified. With Pimelobacter simplex 4B and 5B 2-oxo-1,2-dihydroquinoline-4-carboxylic acid and 8-hydroxycoumarin-4-carboxylic acid were isolated. The Agrobacterium strain accumulated 2-oxo-1,2-dihydroquinoline-4-carboxylic acid and 2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid in the media during growth; with Microbacterium sp. H2 we only found 8-hydroxycoumarin-4-carboxylic acid. With mutants of Microbacterium sp. H2 which were induced with N-methyl-N'-nitro-N-nitrosoguanidine we found 2-oxo-1,2-dihydroquinoline-4-carboxylic acid, 8-hydroxy-coumarin-4-carboxylic acid and 2,3-dihydroxyphenyl-succinic acid.     

Draft genome sequence for Microbacterium laevaniformans strain OR221, a bacterium tolerant to metals, nitrate, and low pH

Microbacterium laevaniformans strain OR221 was isolated from subsurface sediments obtained from the Field Research Center (FRC) in Oak Ridge, TN. It was characterized as a bacterium tolerant to heavy metals, such as uranium, nickel, cobalt, and cadmium, as well as nitrate and low pH. We present its draft genome sequence.     

Microbacterium marinum sp. nov., isolated from deep-sea water

Two Gram-positive, rod-shaped bacterial strains, H101(T) and H207, were isolated from deep sea water collected from South-West Indian Ocean. Phylogenetic analysis of 16S rRNA gene sequences showed that the two strains were closely related to one another (100% similarity), and had the closest relationship with Microbacterium hominis NBRC 15708(T) and Microbacterium insulae KCTC 19247(T) (98.2-98.3% similarities). DNA-DNA hybridization value between strains H101(T) and H207 was 87.2 ± 3.7%, and the values between the two strains and the closely related type strains were well below 70%. The two strains also shared a number of physiological and biochemical characteristics that were distinct from the closely related species, and grew at 2-37 ° C, pH 5-11 and 0-8% (w/v) NaCl. Both strains contained MK-12, MK-13 and MK-11 as the detected menaquinones. The peptidoglycan was of type B1γ with an interpeptide bridge D-Glu(Hyg)→ Gly(2)→ l-Lys. The major cellular fatty acids were anteiso-C(15:0), anteiso-C(17:0), and iso-C(16:0). Based on the genetic and phenotypic properties, it is proposed that strains H101(T) and H207 be classified as representatives of a novel species of the genus Microbacterium, with the name Microbacterium marinum sp. nov. The type strain is H101(T) (= CGMCC 4.6941(T) = DSM 24947(T)).     

Expression and purification of a recombinant enantioselective amidase

Microbacterium sp. AJ115 metabolises a wide range of nitriles using the two-step nitrile hydratase/amidase pathway. In this study, the amidase gene of Microbacterium sp. AJ115 has been inserted into the pCal-n-EK expression vector and expressed in Escherichia coli BL21(DE3)pLysS. The expressed protein is active in E. coli and expression of the amidase gene allows E. coli to grow on acetamide as sole carbon and/or nitrogen source. Expression of active amidase in E. coli was temperature dependent with high activity found when cultures were grown between 20 and 30 degrees C but no activity at 37 degrees C. On induction, the amidase represents 28% of the total soluble protein in E. coli. The expressed amidase has been purified in a single step from the crude lysate using the calmodulin-binding peptide (CBP) affinity tag. The V(max) and K(m) of the purified enzyme with acetamide (50 mM) were 4.4 micromol/min/mg protein and 4.5mM, respectively. The temperature optimum was found to be 50 degrees C. Purified enzyme demonstrated enantioselectivity with the ability to preferentially act on the S enantiomer of racemic (R,S)-2-phenylpropionamide. S-2-phenylpropionic acid is produced with an enantiomeric excess of >82% at 50% conversion of the parent amide.     

山东半岛农药污染点源放线菌多样性及抑菌活性

【目的】探究山东半岛农药污染点源放线菌多样性及非链霉菌抑菌活性,试图发现新放线菌和新抗生素。【方法】通过16S rDNA序列对已分离得到的154株纯培养放线菌进行初步分类鉴定并进行系统发育分析;采用管碟法和菌丝生长速率法检测10株非链霉菌的抑菌活性。【结果】154株放线菌覆盖7个科,8个属,有一株非链霉菌(205)可能为潜在的新种。10株非链霉菌的发酵液均对供试的植物病原真菌和细菌有不同程度的抑制作用,尤其是菌株Microbacterium oxydans JN853773和Kocuria rosea JN192402对所有供试病原菌都有强烈的抑制作用。【结论】农药污染点源可以作为开发新型抗生素产生菌的重要来源。     

Biocatalytic deprotection of a cetraxate ester by Microbacterium sp. strain 7-1W cells

Enzymatic deprotection of the terminal ester bond of a cetraxate methyl ester was done with resting cells of Microbacterium sp. strain 7-1W, which produces an esterase catalyzing a regioselective hydrolysis reaction, as the catalyst. When 20 g of cetraxate methyl ester in 50 ml of a reaction mixture was incubated with 5 g of wet cells for 17 h, 96% of the substrate was converted to the desired product, cetraxate, quantitatively.     

Molecular and enzymatic characterization of a levan fructotransferase from Microbacterium sp. AL-210

Microbacterium sp. AL-210 producing a novel levan fructotransferase (LFTase) was screened from soil samples. The LFTase was purified to homogeneity by (NH4)2SO4 fractionation, column chromatography on Resource Q, and Superdex 200HR. The molecular weight of the purified enzyme was estimated to be approximately 46 kDa by both SDS-PAGE and gel filtration, and the enzyme's isoelectric point was pH 4.8. The major product produced from the levan hydrolysis by the enzyme reaction was identified by atmospheric pressure ionization mass spectrometry and NMR analysis as di-D-fructose-2,6':6,2'-dianhydride (DFA IV). The optimum pH and temperature for DFA IV production were 7.0 and 40 degrees C, respectively. The enzyme was stable at a pH range 7.0-8.0 and up to 40 degrees C. The enzyme activity was inhibited by FeCl2 and AgNO3. The enzyme converted the levan to DFA IV, with a conversion yield of approximately 44%. A gene encoding the LFTase (lftM) from Microbacterium sp. AL-210 was cloned and sequenced. The nucleotide sequence included an ORF of 1593 nucleotides, which is translated into a protein of 530 amino acid residues. The predicted amino acid sequence of the enzyme shared 79% of the identity and 86% of the homology with that of Arthrobacter nicotinovorans GS-9.     

Reduction of Cr(VI) by immobilized cells of Desulfovibrio vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776

Hexavalent chromium, a carcinogen and mutagen, can be reduced to Cr(III) by Desulfovibrio vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776. This study examined Cr(VI) reduction by immobilized cells of the two strains in a common solution matrix using various entrapment matrices. Chitosan and PVA-borate beads did not retain integrity and supported low or no reduction of Cr(VI) by the cells. A commercial preparation (Lentikats) was stable but also did not support Cr(VI) reduction. K-carrageenan beads were stable in batch suspensions but gel integrity was lost after only 5 h in a flow-through system in the presence of 100 microM Cr(VI). The best immobilization matrices were agar and agarose, where the initial rates of reduction of Cr(VI) (from 500 microM solution) for D. vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776 were 127 (agar) and 130 (agarose), and 15 (agar) and 12 (agarose) nmol h(-1) mg dry cell wt(-1), respectively. The higher removal of Cr(VI) by D. vulgaris was also seen in 14-mL packed-bed flow-through columns, where, at a flow rate of 2.4 mL h(-1), the percentage removal of Cr(VI) was approximately 95% and 60% for D. vulgaris and Microbacterium sp., respectively (agar-immobilized cells). The Cr(VI) reducing activities of D. vulgaris and Microbacterium sp. were lost after 159 and 140 h, respectively. Examination of the beads for structural integrity within the columns in situ using magnetic resonance imaging after 24 and 100 h of continuous operation against Cr(VI) (with negligible Cr retained within the columns) showed that agar beads were more stable with time. The most appropriate system for development of a continuous bioprocess is thus the use of D. vulgaris NCIMB 8303 immobilized in an agar gel matrix.     

Purification and properties of a new type of protease produced by Microbacterium liquefaciens

A bacterium, identified as Microbacterium liquefaciens MIM-CG-9535-I, was isolated from a soil sample taken from the industrial site of a gelatin manufacturer. A new type of protease, which restrictively decomposes gelatin at one or two positions, was purified from the bacterial culture. The molecular mass of the purified enzyme was 21 kDa by SDS-polyacrylamide gel electrophoresis. The purified enzyme specifically degraded the alpha-chain of gelatin with a molecular weight of 100 kDa into two peptides of 60 kDa and 40 kDa. Native collagen was not a substrate for the enzyme.     

Enhanced removal of dimethyl sulfide from a synthetic waste gas stream using a bioreactor inoculated with <Emphasis Type="Italic">Microbacterium</Emphasis> sp. NTUT26 and <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

The removal of dimethyl sulfide (DMS) from industrial gas streams has received a high priority due to its very low odorous threshold value and relatively low biodegradability compared to other reduced sulfur compounds. A variety of bacteria that utilize DMS as a carbon/energy source have been studied and the degradation pathway elucidated. However, to date, there have been few reports on the industrial application of such bacteria inoculated into a bioreactor for DMS treatment. An additional problem of such systems is the accumulation of intermediate metabolites that strongly impact on DMS removal by the microbe. The results reported here were obtained using a bioreactor inoculated with the H(2)S-degrader Pseudomonas putida and the DMS-degrader Microbacterium sp. NTUT26 to facilitate removal of metabolic intermediates and DMS. This bioreactor performed well (1.71 g-S/day/kg-dry packing material) in terms of DMS gas removal, based on an evaluation of the apparent kinetics and maximal removal capacity of the system. Under varying conditions (changes in start-up, inlet loading, shutdown, and re-start), the bioreactor inoculated with Microbacterium sp. NTUT26 and P. putida enhanced removal of high concentrations of DMS. Our results suggest that this type of bioreactor system has significant potential applications in treating (industrial) DMS gas streams.