Phylogenetic analysis of the genus Microbacterium based on 16S rRNA gene sequences

Abstract 16S rRNA gene (rDNA) studies of the six species of the genus Microbacterium, M. lacticum, M. laevaniformans, M. dextranolyticum, M. imperiale, M. arborescens and M. aurum , were performed and the primary structures were compared with those of 29 representative actinobacteria and related organisms. Phylogenetic analysis indicated that six species of the genus Microbacterium and representative four species of the genus Aureobacterium appear to be phylogenetically coherent as was suggested by Rainey et al., although the peptidoglycan types of these two genera are different (peptidoglycan type B1 or B2). Thus, the phylogenetical analyses revealed that members of actinobacteria with group B-peptidoglycan do not cluster according to their peptidoglycan types, but form compact cluster different from actinobacteria or actinomycetes with group A-peptidoglycan.     

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)).     

Microbacterium suwonense sp. nov., isolated from cow dung

An actinomycete strain, designated M1T8B9^T, was isolated from cow dung in Suwon, Republic of Korea. The isolate was a Gram-positive, nonmotile, and non-spore-forming bacterium. Phylogenetic evaluation based on 16S rRNA gene sequence similarity showed that this isolate belongs to the genus Microbacterium, with its closest neighbors being Microbacterium soli DCY17^T (98.2%) and Microbacterium esteraromaticum DSM 8609^T (98.0%). The polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, and one unknown glycolipid. Strain M1T8B9^T contained the major fatty acids C_15:0 anteiso, C_16:0 iso, C _17:0 anteiso, and C_15:0 iso, and the cell-wall peptidoglycan was of type B2β. According to DNA-DNA hybridization studies, strain M1T8B9^T showed 42% and 26% relatedness with M. soli DCY17^T and M. esteraromaticum DSM 8609^T, respectively. On the basis of the data presented, strain M1T8B9^T is considered to represent a novel species of the genus Microbacterium, for which the name Microbacterium suwonense sp. nov. is proposed. The type strain is M1T8B9^T (=KACC 14058^T =NBRC 106310^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。     

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.     

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)).     

Papaya shoot tip associated endophytic bacteria isolated from in vitro cultures and host-endophyte interaction in vitro and in vivo

Fourteen distinct bacterial clones were isolated from surface-sterilized shoot tips (approximately 1 cm) of papaya (Carica papaya L. 'Surya') planted on Murashige and Skoog (MS)-based papaya culture medium (23/50 nos.) during the 2-4 week period following in vitro culturing. These isolates were ascribed to six Gram-negative genera, namely Pantoea (P. ananatis), Enterobacter (E. cloacae), Brevundimonas (B. aurantiaca), Sphingomonas, Methylobacterium (M. rhodesianum), and Agrobacterium (A. tumefaciens) or two Gram-positive genera, Microbacterium (M. esteraromaticum) and Bacillus (B. benzoevorans) based on 16S rDNA sequence analysis. Pantoea ananatis was the most frequently isolated organism (70% of the cultures) followed by B. benzoevorans (13%), while others were isolated from single stocks. Bacteria-harboring in vitro cultures often showed a single organism. Pantoea, Enterobacter, and Agrobacterium spp. grew actively on MS-based normal papaya medium, while Microbacterium, Brevundimonas, Bacillus, Sphingomonas, and Methylobacterium spp. failed to grow in the absence of host tissue. Supplying MS medium with tissue extract enhanced the growth of all the organisms in a dose-dependent manner, indicating reliance of the endophyte on its host. Inoculation of papaya seeds with the endophytes (20 h at OD550=0.5) led to delayed germination or slow seedling growth initially. However, the inhibition was overcome by 3 months and the seedlings inoculated with Pantoea, Microbacterium, or Sphingomonas spp. displayed significantly better root and shoot growths.     

<Emphasis Type="Italic">Microbacterium rhizosphaerae</Emphasis> sp. nov., isolated from a Ginseng field,South Korea

A novel Gram-stain positive, aerobic, short rod-shaped, non-motile bacterium, designated strain CHO1^T, was isolated from rhizosphere soil from a ginseng agriculture field. Strain CHO1^T was observed to form yellow colonies on R2A agar medium. The cell wall peptidoglycan was found to contain alanine, glycine, glutamic acid, d-ornithine and serine. The cell wall sugars were identified as galactose, mannose, rhamnose and ribose. Strain CHO1^T was found to contain MK-11, MK-12, MK-13 as the predominant menaquinones and anteiso-C_15:0, iso-C_16:0, and anteiso-C_17:0 as the major fatty acids. Diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipid, an unidentified phospholipid and three unidentified glycolipids were found to be present in strain CHO1^T. Based on 16S rRNA gene sequence analysis, strain CHO1^T was found to be closely related to Microbacterium mangrovi DSM 28240^T (97.81 % similarity), Microbacterium immunditiarum JCM 14034^T (97.45 %), Microbacterium oryzae JCM 16837^T (97.33 %) and Microbacterium ulmi KCTC 19363^T (97.10 %) and to other species of the genus Microbacterium. The DNA G+C content of CHO1^T was determined to be 70.1 mol %. The DNA–DNA hybridization values of CHO1^T with M. mangrovi DSM 28240^T, M. immunditiarum JCM 14034^T, M. oryzae JCM 16837^T and M. ulmi KCTC 19363^T were 46.7 ± 2, 32.4 ± 2, 32.0 ± 2 and 29.2 ± 2 %, respectively. On the basis of genotypic, phenotypic and phylogenetic properties, it is concluded that strain CHO1^T represents a novel species within the genus Microbacterium, for which the name Microbacterium rhizosphaerae sp. nov. is proposed. The type strain of M. rhizosphaerae is CHO1^T (= KEMB 7306-513^T = JCM 31396^T).     

Genome sequence of Microbacterium testaceum StLB037, an N-acylhomoserine lactone-degrading bacterium isolated from potato leaves

Microbacterium testaceum is an endophytic Gram-positive bacterium that resides within plant hosts. M. testaceum StLB037 was isolated from potato leaves and shows N-acylhomoserine lactone-degrading activity. Here, we present the 3.98-Mb complete genome sequence of StLB037, with an average GC content of 70.28%.     

The ERK MAP kinase cascade mediates tail swelling and a protective response to rectal infection in C. elegans

The nematode Caenorhabditis elegans is proving to be an attractive model organism for investigating innate immune responses to infection. Among the known pathogens of C. elegans is the bacterium Microbacterium nematophilum, which adheres to the nematode rectum and postanal cuticle, inducing swelling of the underlying hypodermal tissue and causing mild constipation. We find that on infection by M. nematophilum, an extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase cascade mediates tail swelling and protects C. elegans from severe constipation, which would otherwise arrest development and cause sterility. Involvement in pathogen defense represents a new role for ERK MAP kinase signaling in this organism.     

Some Observations on the Taxonomy of the Genus Microbacterium. II. Cell Wall Analysis, Gel Electrophoresis and Serology

S ummary . The chemical composition of the cell wall mucopeptide, the esterases and catalases of the cell free extracts, and the serology of the 25 species of microbacteria whose morphological and cultural characteristics had been investigated previously, were examined. Suggestions are made about the relationships of the species within the genus Microbacterium and with other genera.     

The Development of the Anaerobic Spoilage Flora of Meat Stored at Chill Temperatures

In meat juice medium under anaerobic conditions, spoilage bacteria utilized the following substrates: Microbacterium thermosphactum , glucose; Enterobacter , glucose and glucose-6-phosphate; Lactobacillus , glucose and arginine. On meat stored anaerobically. Lactobacillus grew faster than the other species at all temperatures between 2° and 15 °C. Enterobacter had a greater affinity for glucose than M. thermosphactum. As a result, high numbers of Enterobacter inhibited growth of M. thermosphactum under the glucose limiting conditions at the meat surface. High numbers of Lactobacillus inhibited growth of both competing species, apparently by producing an inhibitory substance.     

Microbacterium isolates from the vicinity of a radioactive waste depository and their interactions with uranium

Three oligotrophic bacterial strains were cultured from the ground water of the deep-well monitoring site S15 of the Siberian radioactive waste depository Tomsk-7, Russia. They were affiliated with Actinobacteria from the genus Microbacterium. The almost fully sequenced 16S rRNA genes of two of the isolates, S15-M2 and S15-M5, were identical to those of cultured representatives of the species Microbacterium oxydans. The third isolate, S15-M4, shared 99.8% of 16S rRNA gene identity with them. The latter isolate possessed a distinct cell morphology as well as carbon source utilization pattern from the M. oxydans strains S15-M2 and S15-M5. The three isolates tolerated equal amounts of uranium, lead, copper, silver and chromium but they differed in their tolerance of cadmium and nickel. The cells of all three strains accumulated high amounts of uranium, i.e. up to 240 mg U (g dry biomass)(-1) in the case of M. oxydans S15-M2. X-ray absorption spectroscopy (XAS) analysis showed that this strain precipitated U(VI) at pH 4.5 as a meta-autunite-like phase. At pH 2, the uranium formed complexes with organically bound phosphate groups on the cell surface. The results of the XAS studies were consistent with those obtained by transmission electron microscopy (TEM) and energy dispersive X-ray analysis (EDX).     

Substrate induction and statistical optimization for the production of chitosanase from Microbacterium sp. OU01

The chitosanase production was markedly enhanced by substrate induction, statistical optimization of medium composition and culture conditions by Microbacterium sp. OU01 in shake-flask. A significant influence of (NH(4))(2)SO(4), MgSO(4).7H(2)O and initial pH on chitosanase production was noted with Plackett-Burman design. It was then revealed with the method of steepest ascent and response surface methodology (RSM) that 19.0g/L (NH(4))(2)SO(4), 1.3g/L MgSO(4) and an initial pH of 2.0 were optimum for the production of chitosanase; colloidal chitosan appeared to be the best inducer for chitosanase production by Microbacterium sp. OU01. This optimization strategy led to the enhancement of chitosanase from 3.6U/mL to 118U/mL.     

Genome characterization, analysis of virulence and transformation of Microbacterium nematophilum, a coryneform pathogen of the nematode Caenorhabditis elegans

A coryneform bacterium designated Microbacterium nematophilum has previously been reported to act as a pathogen for Caenorhabditis elegans. This bacterium is able to colonize the rectum of infected worms and cause localized swelling, constipation and slowed growth. Additional isolates and analysis of this bacterium are described here. Tests of pathogenicity on other Caenorhabditis nematodes show that M. nematophilum infection is lethal to most species in the genus, in contrast to its relatively mild effects on C. elegans. The size and geometry of the pathogen genome have been determined as a closed circular molecule of 2.85 Mb with high G+C content. Bacteria also harbor a 55 kb plasmid, pMN1, which is largely composed of a lysogenic bacteriophage genome. Mutagenesis experiments have yielded stable avirulent mutants of M. nematophilum. As a first step towards molecular genetic analysis, methods for low-efficiency transformation of M. nematophilum have been developed.     

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)).     

Sources of Psychrotrophic Bacteria on Meat at the Abattoir

An export abattoir was examined to determine the sources and types of psychrotrophic bacteria which gain access to meat. Gram negative psychrotrophs were recovered from the hides, from structural and work surfaces within the abattoir, and from carcasses and meat at all stages of processing. Microbacterium thermosphactum was recovered intermittently at all sampling sites. Psychrotrophic and total counts and the incidence of M. thermosphactum on carcasses and meat increased during processing through the abattoir. Structural and work surfaces may be as important as the hide as sources of bacterial contaminants on meat. Seasonal variation was observed in psychrotrophic counts which correlated positively with rainfall and negatively with temperature.     

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.     

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.