Characterization and genomic analysis of an efficient dibutyl phthalate degrading bacterium Microbacterium sp. USTB-Y.

A promising bacterial strain for biodegrading dibutyl phthalate (DBP) was successfully isolated from activated sludge and characterized as a potential novel Microbacterium sp. USTB-Y based on 16S rRNA sequence analysis and whole genome average nucleotide identity (ANI). Initial DBP of 50 mg/L could be completely biodegraded by USTB-Y both in mineral salt medium and in DBP artificially contaminated soil within 12 h at the optimal culture conditions of pH 7.5 and 30 â„ƒ, which indicates that USTB-Y has a strong ability in DBP biodegradation. Phthalic acid (PA) was identified as the end-product of DBP biodegraded by USTB-Y using GC/MS. The draft genome of USTB-Y was sequenced by Illumina NovaSeq and 29 and 188 genes encoding for putative esterase/carboxylesterase and hydrolase/alpha/beta hydrolase were annotated based on NR (non redundant protein sequence database) analysis, respectively. Gene3781 and gene3780 from strain USTB-Y showed 100% identity with dpeH and mpeH from Microbacterium sp. PAE-1. But no phthalate catabolic gene (pht) cluster was found in the genome of strain USTB-Y. The results in the present study are valuable for obtaining a more holistic understanding on diverse genetic mechanisms of PAEs biodegrading Microbacterium sp. strains.

Microbacterium stercoris sp. nov., an indole acetic acid-producing actinobacterium isolated from cow dung.

A novel growth-promoting and indole acetic acid-producing strain, designated NEAU-LLBT, was isolated from cow dung collected from Shangzhi, Heilongjiang Province, PR China. Cells of strain NEAU-LLBT were Gram-stain-positive, non-motile, aerobic and non-spore-forming. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain NEAU-LLBT belonged to the genus Microbacterium. Strain NEAU-LLBT had high 16S rRNA sequence similarities of 98.81 and 98.41 % to Microbacterium paludicola DSM 16915T and Microbacterium marinilacus DSM 18904T, and less than 98 % to other members of the genus Microbacterium. Chemotaxonomic characteristics showed that MK-11 and MK-12 were detected as the predominant menaquinones. The peptidoglycan contained glutamic acid, aspartic acid, glycine, ornithine and a small amount of alanine, with ornithine as the diagnostic diamino acid. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. The major fatty acids were identified as anteiso-C15 : 0, iso-C16 : 0 and iso-C17 : 0. The genomic DNA G+C content of strain NEAU-LLBT was 70.2 mol%. In addition, the average nucleotide identity values between strain NEAU-LLBT and its reference strains, M. paludicola DSM 16915T, M. marinilacus DSM 18904T and M. album SYSU D8007T, were found to be 81.1, 79.4 and 78.7 %, respectively, and the level of digital DNA-DNA hybridization between them were 23.8, 22.6 and 21.8 %, respectively. Based on the phenotypic, phylogenetic and genotypic data, strain NEAU-LLBT is considered to represent a novel species of the genus Microbacterium, for which the name Microbacterium stercoris sp. nov is proposed, with NEAU-LLBT (=CCTCC AA 2018028T=JCM 32660T) as the type strain.

Microbacterium paulum sp. nov., isolated from microfiltered milk.

A novel Gram-stain-positive, strictly aerobic, short rod-shaped bacterium, designated 2CT, was isolated from freshly packaged microfiltered milk. This strain was able to grow within the NaCl concentration range of 0-5 % (w/v), temperature range of 8-37 °C (optimally at 30 °C) and at pH 6.0-10.0. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 2CT was closely related to species of the genus Microbacterium, with the highest sequence similarity (99.2 %) to Microbacterium lacticum DSM 20427T as well as Microbacterium flavum DSM 18909T (=YM18-098T). The phylogenetic tree based on 16S rRNA genes showed that strain 2CT clustered with M. flavum DSM 18909T. However, the phylogenetic tree based on concatenated 16S rRNA and four housekeeping genes showed that strain 2CT clustered with M. lacticum DSM 20427T. Furthermore, the phylogenomic tree showed that strain 2CT clustered with M. lacticum DSM 20427T and M. flavum DSM 18909T. The major respiratory quinones were MK-10, MK-11 and MK-12. The predominant cellular fatty acids were anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The polar lipid composition of strain 2CT consisted of diphosphatidylglycerol, phosphatidylglycerol, three unidentified glycolipids and two unidentified lipids. The cell-wall peptidoglycan type was a variant of B1α {Gly} [l-Lys] d-Glu-l-Lys, with the amino acids lysine, glycine, alanine and glutamic acid. The whole-cell sugars consisted of galactose, glucose, ribose and minor amounts of rhamnose. In addition, strain 2CT showed a glycolyl-type cell wall. The genomic DNA G+C content was 69.8mol%, while the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values with the closely related Microbacterium species were below the recognized thresholds of 95-96 % ANI and 70 % DDH for species definition. Based on the phenotypic and genotypic data, strain 2CT (=LMG 32277T=CECT 30329T) is considered to represent a new species, for which the name Microbacterium paulum sp. nov. is proposed.

Description of Microbacterium luteum sp. nov., Microbacterium cremeum sp. nov., and Microbacterium atlanticum sp. nov., three novel C50 carotenoid producing bacteria.

We have identified three Microbacterium strains, A18JL200T, NY27T, and WY121T, that produce C50 carotenoids. Taxonomy shows they represent three novel species. These strains shared < 98.5% 16S rRNA gene sequence identity with each other and were closely related to Microbacterium aquimaris JCM 15625T, Microbacterium yannicii JCM 18959T, Microbacterium ureisolvens CFH S00084T, and Microbacterium hibisci CCTCC AB 2016180T. Digital DNA-DNA hybridization (dDDH) values and average nucleotide identity (ANI) showed differences among the three strains and from their closest relatives, with values ranging from 20.4% to 34.6% and 75.5% to 87.6%, respectively. These values are below the threshold for species discrimination. Both morphology and physiology also differed from those of phylogenetically related Microbacterium species, supporting that they are indeed novel species. These strains produce C50 carotenoids (mainly decaprenoxanthin). Among the three novel species, A18JL200T had the highest total yield in carotenoids (6.1 mg/L or 1.2 mg/g dry cell weight). Unusual dual isoprenoid biosynthetic pathways (methylerythritol phosphate and mevalonate pathways) were annotated for strain A18JL200T. In summary, we found strains of the genus Microbacterium that are potential producers of C50 carotenoids, but their genome has to be investigated further.

Microbacterium chengjingii sp. nov. and Microbacterium fandaimingii sp. nov., isolated from bat faeces of Hipposideros and Rousettus species.

Four aerobic, Gram-stain-positive, rod-shaped bacteria (HY60T, HY54, HY82T and HY89) were isolated from bat faeces of Hipposideros and Rousettus species collected in PR China. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the four novel strains formed two separate but adjacent subclades close to Microbacterium agarici CGMCC 1.12260T (97.6-97.7 % similarity), Microbacterium humi JCM 18706T (97.3-97.5 %) and Microbacterium lindanitolerans JCM 30493T (97.3-97.4 %). The 16S rRNA gene sequence similarity was 98.3 % between strains HY60T and HY82T, and identical within strain pairs HY60T/HY54 and HY82T/HY89. The DNA G+C contents of strains HY60T and HY82T were 61.9 and 63.3 mol%, respectively. The digital DNA-DNA hybridization and average nucleotide identity values between each novel strain and their closest relatives were all below the 70 % and 95-96 % thresholds for species delimitation, respectively. All four novel strains contained anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0 as the main fatty acids, MK-11 and MK-12 as the major respiratory quinones, and diphosphatidylglycerol, phosphatidylglycerol and one unidentified glycolipid as the predominant polar lipids. The cell-wall peptidoglycan was of B type and contained alanine, glutamate, glycine and ornithine. The acyl type of the muramic acid was glycolyl. The whole-cell sugars were rhamnose and ribose. Based on the foregoing polyphasic analyses, it was concluded that the four uncharacterized strains represented two novel species of the genus Microbacterium, for which the names Microbacterium chengjingii sp. nov. [type strain HY60T (=CGMCC 1.17468T=GDMCC 1.1951T=KACC 22102T)] and Microbacterium fandaimingii sp. nov. [type strain HY82T (=CGMCC 1.17469T=GDMCC 1.1949T=KACC 22101T)] are proposed, respectively.

Microbacterium helvum sp. nov., a novel actinobacterium isolated from cow dung.

A Gram-positive, aerobic, non-motile, non-spore-forming, short rod-shaped strain, NEAU-LLCT, was isolated from cow dung in Shangzhi City, Heilongjiang Province, Northeast China and identified by a polyphasic taxonomic study. Colonies was light yellow, round, with entire margin. Strain NEAU-LLCT was grown at 15-45 â„ƒ and pH 6.0-10.0. NaCl concentration ranged from 0 to 5% (W/V). The 16S rRNA gene sequence of NEAU-LLCT showed the high similarities with Microbacterium kyungheense JCM 18735T (98.5%), Microbacterium trichothecenolyticum JCM 1358T (98.3%) and Microbacterium jejuense JCM 18734T (98.2%). The whole-cell sugars were glucose, rhamnose and ribose. The menaquinones contained MK-12 and MK-13. Ornithine, glutamic acid, lysine and a small amount of alanine and glycine were the amino acids in the hydrolyzed products of the cell wall. The major fatty acids were iso-C16:0, iso-C18:0, anteiso-C15:0 and anteiso-C17:0. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. The genome of NEAU-LLCT was 4,369,375 bp and G + C content is 70.28 mol%. A combination of DNA-DNA hybridization result and some phenotypic characteristics demonstrated that strain NEAU-LLCT could be distinguished from its closely related strains. Therefore, the strain NEAU-LLCT was considered to represent a novel species, which was named Microbacterium helvum sp. (Type strain NEAU-LLCT = CCTCC AA 2018026T = JCM 32661T).

Comparative assessment of multi-trait plant growth-promoting endophytes associated with cultivated and wild Oryza germplasm of Assam, India.

This paper presents a comparative study of endophytic bacteria from cultivated (Oryza sativa) and wild rice (Oryza rufipogon) plants and their functional traits related to plant growth promotion. A total of 70 bacterial isolates were characterized by both biochemical and molecular identification methods. Taxonomic classification showed dominance of three major phyla, viz, Firmicutes (57.1%), Actinobacteria (20.0%) and Proteobacteria (22.8%). Screening for in vitro plant growth-promoting activities revealed a hitherto unreported endophytic bacterium from wild rice germplasm, Microbacterium laevaniformans RS0111 with highest indole acetic acid (28.39 ± 1.39 µg/ml) and gibberellic acid (67.23 ± 1.83 µg/ml) producing efficiency. Few other endophytic isolates from cultivated rice germplasm such as Bacillus tequilensis RHS01 showed highest phosphate solubilizing activity (81.70 ± 1.98 µg/ml), while Microbacterium testaceum MKLS01 and Microbacterium enclense MI03 L05 showed highest potassium (53.42 ± 0.75 µg/ml) and zinc solubilizing activity (157.50%). Fictibacillus aquaticus LP20 05 produced highest siderophore (64.8%). In vivo evaluation of plant growth-promoting efficiencies of the isolates showed that Microbacterium laevaniformans RS0111, Microbacterium testaceum MKLS01 and Bacillus tequilensis RHS 01 could increase rice grain yield by 3.4-fold when compared to the control group. This study indicates the potentiality of rice endophytes isolates as an effective bioinoculants.

Microbacterium karelineae sp. nov. isolated from a halophyte plant in the Taklamakan desert.

A bacterial strain, designated TRM 80801T, was isolated from the Karelinea in Taklamakan desert, Xinjiang Uygur Autonomous Region, north-west China. Cells were Gram-stain-positive, aerobic, non-motile, short rods. Strain TRM 80801T grew at 4-50 °C, with optimum growth at 28 °C, and grew at pH 6.0-11.0 and 1-15 % (w/v) NaCl. Phylogenetic analyses of the 16S rRNA gene sequences placed strain TRM 80801T within the genus Microbacterium with the highest similarities to Microbacterium suaedae YZYP 306T (98.97 %) and Microbacterium indicum BBH6T (98.17 %), respectively. The DNA G+C content of TRM 80801T is 69.38 mol%. The cell-wall peptidoglycan contained the amino acids ornithine, glutamic acid, glycine and alanine, the diagnostic diamino acid was ornithine. The acyl type of the peptidoglycan was glycolyl. Whole-cell sugars were ribose, mannose, glucose, rhamnose and galactose. The major cellular fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The predominant menaquinones were MK-10, MK-11 and MK-12. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol. The whole-genome average nucleotide identity (ANI) value between strain TRM 80801T and Microbacterium suaedae YZYP 306T is 70.2 %. On the basis of the evidence presented in this study, strain TRM 80801T is representative of a novel species in the genus Microbacterium, for which the name Microbacterium karelineae sp. nov. is proposed. The type strain is TRM 80801T (=CCTCC AB 2019248T=KCTC 49357T).

Microbacterium caowuchunii sp. nov. and Microbacterium lushaniae sp. nov., isolated from plateau pika (Ochotona curzoniae) on the Qinghai-Tibet Plateau of PR China.

Four novel bacterial strains (ST-M6T, L-033, L-031T and Z-333) were isolated from the intestinal contents of plateau pikas (Ochotona curzoniae) collected on the Qinghai-Tibet Plateau, PR China. Cells were aerobic, non-motile, Gram-stain-positive, catalase-positive, oxidase-negative, capsuled and short-rod-shaped. Phylogenetic analyses based on the 16S rRNA gene sequences and 387 core genes indicated that the four isolates belong in the genus Microbacterium and clearly separate from recognized species. The two type strains (ST-M6T and L-031T) shared low 16S rRNA similarity, average nucleotide identity values and digital DNA-DNA hybridization relatedness with their phylogenetic neighbours (Microbacterium ginsengisoli DSM 18659T, Microbacterium hatanonis DSM 19179T, Microbacterium rhizomatis JCM 30598T, Microbacterium radiodurans CCTCC M208212T, Microbacterium oleivorans DSM 16091T and Microbacterium arborescens DSM 20754T). The genomic DNA G+C contents of strains ST-M6T and L-031T were 70.4 and 70.7 mol%, respectively. The major cellular fatty acids of strain ST-M6T were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0, in contrast to anteiso-C17 : 0, anteiso-C15 : 0 and anteiso-C17 : 1 ω9c of strain L-031T. Both type strains (ST-M6T and L-031T) were glycolate test positive and shared the following common features: MK-11 and MK-12 as major menaquinones; rhamnose, ribose, mannose and galactose as major cell-wall sugars; diphosphatidylglycerol, phosphatidylglycerol and two glycolipids as polar lipids; and ornithine, alanine, glycine and glutamic acid as cell-wall amino acids. Comparing the phenotypic, phylogenetic and chemotaxonomic features of the four strains and their related taxa, strains ST-M6T and L-031T represent two novel species of the genus Microbacterium, for which the names Microbacterium caowuchunii sp. nov. (type strain ST-M6T=CGMCC 1.16364T=DSM 104058T) and Microbacterium lushaniae sp. nov. (type strain L-031T =CGMCC 1.16363T=DSM 106170T) are proposed.

Microbacterium algeriense sp. nov., a novel actinobacterium isolated from Algerian oil production waters.

A non-motile, straight-rod-shaped, Gram-stain-positive and facultative anaerobic bacterium (i.e., strain G1T) was isolated from production waters from an Algerian oilfield. Growth was observed in the presence of 0.3-3.5 % (w/v) NaCl, at 20-50 °C and at pH 6.0-9.0. Results of phylogenetic analyses based on 16S rRNA gene sequences showed that strain G1T belonged to the genus Microbacterium. Strain G1 T was closely related to Microbacterium oxydans (DSM 20578T) and Microbacterium maritypicum (DSM 12512T) with 99.8 % sequence similarity and to Microbacterium saperdae (DSM 20169T) with 99.6 % sequence similarity. Strain G1 T contained MK9, MK10, MK11, MK12 and MK13 as respiratory quinones, and phosphatidylglycerol, diphosphatidylglycerol and glycolipid as the major polar lipids. The major cellular fatty acids were anteiso-C15:0, iso-C16:0 and anteiso-C17:0. The estimated DNA G+C content was 69.57 mol% based on its draft genome sequence. Genome annotation of strain G1T predicted the presence of 3511 genes, of which 3483 were protein-coding and 47 were tRNA genes. The DNA-DNA hybridization (DDH) and average nucleotide identity (ANI) values between strain G1T and M. oxydans (DSM 20578T) and M. maritypicum (DSM 12512T) were in both cases far below the respective species boundary thresholds (27.5 and 28.0 % for DDH; and 84.40 and 84.82% for ANI, respectively). Based on the data presented above, strain G1T was considered to represent a novel species for which the name Microbacterium algeriense is proposed with the type strain G1T (=DSM 109018T=LMG 31276T).

Microbacterium excoecariae sp. nov., a novel endophytic actinobacterium isolated from bark of Excoecaria agallocha Linn.

A Gram-stain-positive, aerobic actinobacterium, designated strain CBS5P-1T, was isolated from bark of Excoecaria agallocha Linn collected from Guangxi Zhuang Autonomous Region, PR China. Cells were short rods. Colonies were light yellow, circular and had entire margins. Strain CBS5P-1T grew at 10-37 °C (optimum, 30 °C) and pH 6.0-12.0 (optimum, pH 7.0-8.0). Its nearest phylogenetic neighbour was Microbacterium amylolyticum DSM 24221T with 97.1 % 16S rRNA gene sequence similarity. The genomic DNA G+C content of strain CBS5P-1T was 71.8 mol%. Anteiso-C15  : 0, anteiso-C17 : 0, iso-C16 : 0 and C16:0 were predominant cellular fatty acids. Major menaquinones were MK-11 and MK-10. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, an unidentified glycolipid and an unidentified phospholipid. The combination of chemotaxonomic, phylogenetic and phenotypic data clearly distinguished strain CBS5P-1T from its phylogenetic neighbour. Accordingly, the name Microbacterium excoecariae sp. nov. is proposed to accommodate this new member of the genus Microbacterium. The type strain is CBS5P-1T (=KCTC 49239T=CGMCC 1.13862T).