Metabolism of Inulin via Difructose Anhydride I Pathway in Microbacterium flavum.

Difructose anhydride I (DFA-I) can be produced from inulin, with DFA-I-forming inulin fructotransferase (IFTase-I). However, the metabolism of inulin through DFA-I remains unclear. To clarify this pathway, several genes of enzymes related to this pathway in the genome of Microbacterium flavum DSM 18909 were synthesized, and the corresponding enzymes were encoded, purified, and investigated in vitro. After inulin is decomposed to DFA-I by IFTase-I, DFA-I is hydrolyzed to inulobiose by DFA-I hydrolase. Inulobiose is then hydrolyzed by ß-fructofuranosidase to form fructose. Finally, fructose enters glycolysis through fructokinase. A ß-fructofuranosidase (MfFFase1) clears the byproducts (sucrose and fructo-oligosaccharides), which might be partially hydrolyzed by fructan ß-(2,1)-fructosidase/1-exohydrolase and another fructofuranosidase (MfFFase2). Exploring the DFA-I pathway of inulin and well-studied enzymes in vitro extends our basic scientific knowledge of the energy-providing way of inulin, thereby paving the way for further investigations in vivo and offering a reference for further nutritional investigation of inulin and DFA-I in the future.

Structural and functional insights of a cold-adaptive ß-glucosidase with very high glucose tolerance from Microbacterium sp. CIAB417.

A gene glu1 (WP_243232135.1) coding for ß-glucosidase from the genome of Microbacterium sp. CIAB417 was characterized for its cold adaptive nature and tolerance to high levels of glucose and ethanol. The phylogenetic analysis suggested the close association of glu1 with a similar gene from a mesophilic bacterium Microbacterium indicum. The purified recombinant GLU1 displayed its optimal activity and stability at pH 5 and temperature 30á´¼C. Additionally, the presence of L3 loop in GLU1 suggested its cold adaptive nature. The glucose tolerant Gate keeper residues (Leu 174 & Trp 169) with a distance of ∼ 6.953 Å between them was also predicted in GLU1. The GLU1 enzyme showed ≥ 95% and ≥ 40% relative activity in the presence of 5 M glucose and 20% ethanol. The Vmax, Km, and Kcat values of GLU1 for cellobiose substrate were observed to be 45.22 U/mg, 3.5 mM, and 41.0157 s-1, respectively. The GLU1 was found to be highly efficient in hydrolysis of celloologosaccharides (C2-C5), lactose and safranal picrocrocin into glucose. Hence, cold adaptive GLU1 with very high glucose and ethanol tolerance could be very useful in bio-refinery, dairy, and flavor industries.

Effect of subtherapeutic and therapeutic sulfamethazine concentrations on transcribed genes and translated proteins involved in Microbacterium sp. C448 resistance and degradation.

Microbacterium sp. C448, isolated from a soil regularly exposed to sulfamethazine (SMZ), can use various sulphonamide antibiotics as the sole carbon source for growth. The basis for the regulation of genes encoding the sulphonamide metabolism pathway, the dihydropteroate synthase sulphonamide target (folP), and the sulphonamide resistance (sul1) genes is unknown in this organism. In the present study, the response of the transcriptome and proteome of Microbacterium sp. C448 following exposure to subtherapeutic (33 µM) or therapeutic (832 µM) SMZ concentrations was evaluated. Therapeutic concentration induced the highest sad expression and Sad production, consistent with the activity of SMZ degradation observed in cellulo. Following complete SMZ degradation, Sad production tended to return to the basal level observed prior to SMZ exposure. Transcriptomic and proteomic kinetics were concomitant for the resistance genes and proteins. The abundance of Sul1 protein, 100-fold more abundant than FolP protein, did not change in response to SMZ exposure. Moreover, non-targeted analyses highlighted the increase of a deaminase RidA and a putative sulphate exporter expression and production. These two novel factors involved in the 4-aminophenol metabolite degradation and the export of sulphate residues formed during SMZ degradation, respectively, provided new insights into the Microbacterium sp. C448 SMZ detoxification process.

Characterization of 17ß-estradiol-degrading enzyme from Microbacterium sp. MZT7 and its function on E2 biodegradation in wastewater.

BACKGROUND: 17ß-estradiol (E2) residues exhibit harmful effects both for human and animals and have got global attention of the scientific community. Microbial enzymes are considered as one of the effective strategies having great potential for removal E2 residues from the environment. However, limited literature is available on the removal of E2 from wastewater using short-chain dehydrogenase. RESULTS: In this study, 17ß-estradiol degrading enzyme (17ß-HSD-0095) was expressed and purified from Microbacterium sp. MZT7. The optimal pH and temperature for reaction was 7 and 40 °C, respectively. Molecular docking studies have shown that the ARG215 residue form a hydrogen bond with oxygen atom of the substrate E2. Likewise, the point mutation results have revealed that the ARG215 residue play an important role in the E2 degradation by 17ß-HSD-0095. In addition, 17ß-HSD-0095 could remediate E2 contamination in synthetic livestock wastewater. CONCLUSIONS: These findings offer some fresh perspectives on the molecular process of E2 degradation and the creation of enzyme preparations that can degrade E2.

Metabolomics Analyses Reveal Metabolites Affected by Plant Growth-Promoting Endophytic Bacteria in Roots of the Halophyte Mesembryanthemum crystallinum.

Mesembryanthemum crystallinum L. (common ice plant) is an edible halophyte. However, if ice plants are used to phytoremediate salinity soil, there are problems of slow initial growth, and a long period before active NaCl uptake occurs under higher salinity conditions. Application of endophytic bacteria may improve the problem, but there remain gaps in our understanding of how endophytic bacteria affect the growth and the biochemical and physiological characteristics of ice plants. The aims of this study were to identify growth-promoting endophytic bacteria from the roots of ice plants and to document the metabolomic response of ice plants after application of selected endophytic bacteria. Two plant growth-promoting endophytic bacteria were selected on the basis of their ability to promote ice plant growth. The two strains putatively identified as Microbacterium spp. and Streptomyces spp. significantly promoted ice plant growth, at 2-times and 2.5-times, respectively, compared with the control and also affected the metabolome of ice plants. The strain of Microbacterium spp. resulted in increased contents of metabolites related to the tricarboxylic acid cycle and photosynthesis. The effects of salt stress were alleviated in ice plants inoculated with the endobacterial strains, compared with uninoculated plants. A deeper understanding of the complex interplay among plant metabolites will be useful for developing microbe-assisted soil phytoremediation strategies, using Mesembryanthemum species.

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.

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.

FAD-dependent C-glycoside-metabolizing enzymes in microorganisms: Screening, characterization, and crystal structure analysis.

C-glycosides have a unique structure, in which an anomeric carbon of a sugar is directly bonded to the carbon of an aglycone skeleton. One of the natural C-glycosides, carminic acid, is utilized by the food, cosmetic, and pharmaceutical industries, for a total of more than 200 tons/y worldwide. However, a metabolic pathway of carminic acid has never been identified. In this study, we isolated the previously unknown carminic acid-catabolizing microorganism and discovered a flavoenzyme "C-glycoside 3-oxidase" named CarA that catalyzes oxidation of the sugar moiety of carminic acid. A Basic Local Alignment Search Tool (BLAST) search demonstrated that CarA homologs were distributed in soil microorganisms but not intestinal ones. In addition to CarA, two CarA homologs were cloned and heterologously expressed, and their biochemical properties were determined. Furthermore, a crystal structure of one homolog was determined. Together with the biochemical analysis, the crystal structure and a mutagenesis analysis of CarA revealed the mechanisms underlying their substrate specificity and catalytic reaction. Our study suggests that CarA and its homologs play a crucial role in the metabolism of C-glycosides in nature.

Structural basis of the conformational changes in Microbacterium hydrocarbonoxydans IclR transcription factor homolog due to ligand binding.

Microbacterium hydrocarbonoxydans has been isolated using an unnatural acylhydrazide compound as the sole carbon source. The compound is hydrolyzed by bacterial hydrazidase, and the gene expression of the enzyme is considered to be controlled by a transcription factor of the Isocitrate lyase Regulator (IclR) family, belonging to the one-component signaling systems. Recently, we reported the crystal structure of an unliganded IclR homolog from M. hydrocarbonoxydans, named putative 4-hydroxybenzoate response regulator (pHbrR), which has a unique homotetramer conformation. In this study, we report the crystal structure of pHbrR complexed with 4-hydroxybenzoic acid, the catalytic product of hydrazidase, at 2.0 Å resolution. pHbrR forms a homodimer with multimeric rearrangement in the unliganded state. Gel filtration column chromatography results suggested dimer-tetramer rearrangement. We observed conformational change in the loop region covering the ligand-binding site, and domain rearrangements in the monomer. This study reports the first liganded IclR family protein structure that demonstrates large structural rearrangements between liganded and unliganded proteins, which may represent a general model for IclRs.

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.

A cyclic tetrasaccharide, cycloisomaltotetraose, was enzymatically produced from dextran and its crystal structure was determined.

Two bacterial strains isolated from soil, namely Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006, were found to produce a novel oligosaccharide. The oligosaccharide was enzymatically produced from dextran using the culture supernatant of Agreia sp. D1110 or M. trichothecenolyticum D2006. LC-MS and NMR analysis identified the novel oligosaccharide as cyclo-{→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→}, which was named cycloisomaltotetraose, and abbreviated as CI4. CI4 was subsequently crystalized and its X-ray crystallographic structure was determined. CI4 crystals were shown to be pentahydrate, with the CI4 molecules in the crystal structure displaying a unique 3D structure, in which two glucosyl residues in the molecule were facing each other. This unique 3D structure was quite different from the 3D structure of known cyclic tetrasaccharides. This is the first report of CI4 molecules and their unique crystal structure.

Characterization of a thermotolerant and acidophilic mannanase producing Microbacterium sp. CIAB417 for mannooligosachharide production from agro-residues and dye decolorization.

Mannanases are ubiquitous enzymes and are being explored for diverse industrial applications. In this study, a novel bacterial strain Microbacterium sp. CIAB417 was identified and characterized for extracellular production of mannanase. Microbacterium sp. CIAB417 was found to produce maximum mannanase after 36 h of incubation at 37 °C. Mannanase produced by the isolate was observed for maximum activity at optimum pH of 6 and optimum temperature of 50 °C. Crude mannanase was found to be capable of producing mannooligosachharides (MOS) by hydrolyzing hemicellulose from locust bean gum and Aloe vera. The produced MOS was characterized and found to be mixture of mannobiose to mannohexose units. Mannanase was also explored for decolorization of dyes. Bromophenol blue and coomassie blue R-250 were observed to be decolorized to the extent of 45.40 and 42.75%, respectively. Hence, the identified bacterial strain producing mannanase could be of great significance for applications in food and textile industry.

Identification and Characterization of Lipopeptide Biosurfactant Producing Microbacterium sp Isolated from Brackish River Water.

BACKGROUND: Bioremediation has taken its call for removing pollutants for years. The oilcontaminated surroundings are majorly hazardous for sustaining life, but a great contribution to nature in the form of microorganisms. The complex carbon-hydrogen chain has served as classic raw material to chemical industries, which has perked up the hydrocarbon waste. Microbial remediation has been thus, focused to deal with the lacuna, where the new addition to this category is Microbacterium species. OBJECTIVES: The identification and characterization of lipopeptide biosurfactant producing Microbacterium spp. isolated from brackish river water. METHODS: The strain was isolated from an oil-contaminated lake. The strain was tested with all the other isolated species for oil degradation using screening protocols such as haemolysis, oil spread assay, BATH, E24, etc. The produced biosurfactant was extracted by acid precipitation, followed by solvent recovery. The strain with maximum potential was sequenced and was subjected to phylogeny assessment using in silico tools. RESULTS: Novel Microbacterium species produce the extracellular biosurfactant. The surface tension of Microbacterium was found to be 32mN/m, indicates its powerful surface tension-reducing property. The strain was optimized for the production of biosurfactant and the best results were obtained with sucrose (2%) and yeast extract (3%) medium at 7 pH and 40°C temperature. CONCLUSION: The isolate was confirmed to be a novel Microbacterium species that could produce 0.461 gm biosurfactant in 100 ml of the medium throughout a life cycle and novel strain of isolate was deposited to NCBI as Microbacterium spp. ANSKSLAB01 using an accession number: KU179507.

Symbiotic riboflavin degradation by Microbacterium and Nocardioides bacteria.

Unlike its biosynthetic mechanisms and physiological function, current understanding of riboflavin degradation in soil is limited to a few bacteria that decompose it to lumichrome. Here, we isolated six Microbacterium and three Nocardioides strains. These strains utilized riboflavin and lumichrome, respectively, as carbon sources. Among these strains, we identified Microbacterium paraoxydans R16 (R16) and Nocardioides nitrophenolicus L16 (L16), which were isolated form the same enrichment culture. Co-cultured R16 and L16 reconstituted a riboflavin-degrading interspecies consortium, in which the R16 strain degraded riboflavin to lumichrome and ᴅ-ribose. The L16 strain utilized the lumichrome as a carbon source, indicating that R16 is required for L16 to grow in the consortium. Notably, rates of riboflavin degradation and growth were increased in co-cultured, compared with monocultured R16 cells. These results indicated that a beneficial symbiotic interaction between M. paraoxydans R16 and N. nitrophenolicus L16 results in the ability to degrade riboflavin.

Proteomic mechanism of decabromodiphenyl ether (BDE-209) biodegradation by Microbacterium Y2 and its potential in remediation of BDE-209 contaminated water-sediment system.

The investigation of BDE-209 degradation by Microbacterium Y2 under different condition was conducted. Cell membrane permeability, cell surface hydrophobicity (CSH), membrane potential (MP) and reactive oxygen species (ROS) production were altered under BDE-209 stress. Eleven debrominated congeners were identified, suggesting that BDE-209 biodegradation by Microbacterium Y2 was dominantly a successive debromination process. Proteome analysis showed that the overexpression of haloacid dehalogenases, glutathione S-transferases (GSTs) and ATP-binding cassette (ABC) transporters might occupy important roles in BDE-209 biotransformation. Meanwhile, heat shock proteins (HSPs), ribonuclease E, oligoribonuclease (Orn) and ribosomal protein were activated to counter the BDE-209 toxicity. The up-regulated pyruvate dehydrogenase E1 component beta subunit and dihydrolipoamide dehydrogenase suggested that the pyruvate metabolism pathway was activated. Bioaugmentation of BDE-209 polluted water-sediments system with Microbacterium Y2 could efficiently improve BDE-209 removal. The detection of total 16S rRNA genes in treatment system suggested that Microbacterium (25.6 %), Luteimonas (14.3 %), Methylovorus (12.6 %), Hyphomicrobium (9.2 %) were the dominant genera and PICRUSt results further revealed that the diminution of BDE-209 was owed to cooperation between the introduced bacteria and aboriginal ones.