Isoptericola Variabilis Research Articles

Isoptericola luteus sp. nov., a novel yellow bacterium isolated from soil.

An aerobic, non-motile, Gram-stain-positive bacterium, designated strain NEAU-Y5T, was isolated from a soil sample collected from Northeast Agricultural University, Heilongjiang province. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain NEAU-Y5T belonged to the genus and showed high 16S rRNA sequence similarity to Isoptericola variabilis (98.9 %), Isoptericola nanjingensis (98.9 %), Isoptericola cucumis (98.5 %), Isoptericola hypogeus (98.5 %), Isoptericola dokdonensis (98.5 %), Isoptericola jiangsuensis (98.3 %), and Isoptericola halalbus (98.1 %), followed by other members of the genus Isoptericola (<98 %), and phylogenetically clustered with I. dokdonensis and I. jiangsuensis. Strain NEAU-Y5T was found to grow at 4-40 °C (optimum, 28 °C), pH 6.0-12.0 (optimum, pH 7.0), and tolerated 0-6 % NaCl (w/v). The cell-wall peptidoglycan type was l-Lys-d-Asp. The whole-cell hydrolysates contained glucose, galactose, and ribose. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, and glucosamine unknown phospholipid. Major fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0. The predominant menaquinone was MK-9(H4). The DNA G+C content was 73.4 mol%. The digital DNA-DNA hybridization and average nucleotide identity values between strain NEAU-Y5T and the type strains of the genus Isoptericola ranged from 18.6 to 23.5 % and from 77.3 to 81.6 %, respectively. Based on morphological, physiological, chemotaxonomic, and phylogenetic data, as well as digital DNA-DNA hybridization and average nucleotide identity values, the novel strain NEAU-Y5T could be differentiated from its closest relatives. Therefore, the strain represents a novel species of the genus Isoptericola, for which the name Isoptericola luteus sp. nov. is proposed. The type strain is NEAU-Y5T (=CCTCC AA 2019087T=DSM 110637T).

Coexpression of I. variabilis-EPSPS* and WBceGO-B3S1 Genes Contributes to High Glyphosate Tolerance and Low Glyphosate Residues in Transgenic Rice.

Weeds are one of the main factors that affect the yield and quality of rice. The combination of glyphosate-resistant transgenic crops and glyphosate is regarded as an important strategy for weed management in modern agriculture. In this study, a codon-optimized glyphosate oxidase gene WBceGO-B3S1 from a variant BceGO-B3S1 and a glyphosate-tolerant gene I. variabilis-EPSPS* from the bacterium Isoptericola variabilis were transformed into an Oryza sativa subsp. geng rice variety Zhonghua11 by Agrobacterium-mediated genetic transformation. Molecular detection and field agronomic trait analysis contributed to the selection of three homozygous lines with stable expression of a single copy of the transferred genes integrated into the intergenic region. Under the treatment of glyphosate at a test amount in the field, transgenic lines exhibited no differences in agronomic traits. Under the treatment by 3600 g ha-1 glyphosate, the glyphosate residues in the aboveground tissues of the three candidate transgenic homozygous lines were significantly lower than those in the transgenic homozygous line with I. variabilis-EPSPS* alone at 1, 5, and 10 days. The transgenic line coexpressing I. variabilis-EPSPS* and WBceGO-B3S1 has great application value in breeding of transgenic rice varieties with high glyphosate resistance and low glyphosate residues. This study is a step forward in solving the problem of herbicide residues in food crops by taking advantage of genes that degrade glyphosate.

Statistical augmentation of polyhydroxybutyrate production by Isoptericola variabilis: Characterization, moulding, in vitro cytocompatibility and biodegradability evaluation

This study aimed to explore the production of polyhydroxybutyrate (PHB), a polyhydroxyalkanoate (PHA), which has been widely considered as a potential substitute for the synthetic polymers. Among 53 actinomycete isolates, 11 of them were found to be PHB positive and the quantity of PHB from the positive isolates varied from 10.5 to 29.82 wt% on a dry cell weight basis. A strain designated as PPLAT 012, accumulated relatively higher PHB and has been identified as Isoptericola variabilis by 16S rRNA gene sequence analysis. An effort has also been made to optimize the PHB production by the hyper-producing strain using the conventional, one-factor-at-a-time, and statistical response surface methodologies and the maximum PHB production (46.18%) in DSMZ medium, amended with 12% glucose and 9% potassium nitrate with a pH of 7.0. Further, the characteristic properties such as processability, cytocompatibility and biodegradability of the extracted PHB was also demonstrated. The physical properties of the recovered PHB was further improved by blending with PLA and the resultant blends were characterized. The present investigation has demonstrated that the isolate, Isoptericola variabilis, could be utilized as a potential source for the production of PHB with desirable characteristics, suitable for biomedical applications.

Lactococcus protaetiae sp. nov. and Xylanimonas protaetiae sp. nov., isolated from gut of larvae of Protaetia brevitarsis seulensis.

Two bacterial strains, designated 2DFWM-2T and FW10M-9T, were isolated from gut of larva of Protaetia brevitarsis seulensis grown at the National Institute of Agricultural Sciences, Wanju-gun, South Korea. 16S rRNA and rpoB gene sequences showed that strain 2DFWM-2T formed a separate branch with Lactococcus allomyrinae 1JSPR-7T in the genus Lactococcus, adjacent to a group of Lactococcus lactis subspecies. ANI and dDDH values between 2DFWM-2T and Lactococcus allomyrinae 1JSPR-7T were 93.30% and 53.20%, respectively. Based on the 16S rRNA gene sequence analysis, strain FW10M-9T was classified into the genus Xylanimonas revealing 96.9-98.5% sequence similarities with the Xylanimonas species. The ANI values of strain FW10M-9T with the closely species Xylanimonas pachnodae NBRC 107786T, Xylanimonas allomyrinae 2JSPR-7T, Isoptericola variabilis JCM 11754T and Xylanimonas cellulosilytica DSM 15894T was 81.5%, 81.2%, 81.0% and 84.1%, respectively, and the dDDH values estimated by GGDC was 24.3%, 24.3%, 29.3% and 28.1%, respectively. On the basis of the phenotypic and genotypic data, it is proposed that strain 2DFWM-2T represents a novel species of the genus Lactococcus, for which the name Lactococcus protaetiae sp. nov. is proposed, and the type strain is 2DFWM-2T (= KACC 19320T = NBRC 113069T). And, strain FW10M-9T represents a novel species of the genus Xylanimonas, for which the name Xylanimonas protaetiae sp. nov. is proposed, and the type strain is FW10M-9T (= KACC 19331T = NBRC 113053T).

Compatibility testing and enhancing the pulp bleaching process by hydrolases of the newly isolated thermophilic Isoptericola variabilis strain UD-6

In the present study, Isoptericola variabilis strain UD-6 isolated from alkaline hot spring of Unapdev, Maharashtra, India was assessed for its biobleaching activity by hydrolytic enzymes on rice straw pulp. Results of primary and secondary screening manifested that it was a multi-enzyme producer, competent to produce amylase, cellulase, mannanase, pectinase, and xylanase at 9.73, 4.11, 6.26, 8.42, and 6.61 IU ml−1 in fermentation conditions, respectively. Maximum activity of all enzymes was gained at thermal temperature (50–55 °C), alkaline condition (pH 8–9), under 5 mM KCl and 5 mM NaCl salt concentration. In compatibility testing, activities of all enzymes were spectacularly reduced when they utilized with chemicals of pulp bleaching. Results of rice straw pulp bleaching was effectual when pulp was initially bleached with mannanase, pectinase, and xylanase enzymes (Es) for 90 min and then with diluted chemicals (DC) for further 90 min instead of their separate use. Treatment of rice straw pulp with Es + DC, enhanced the release of reducing sugars, hydrophobic compounds, and phenolic compounds, whereas Kappa number was reduced. Overall, the results of the present study indicated that pre-bleaching of pulp with hydrolytic enzymes obtained from I. variabilis strain UD-6 helps to minimize chemicals used in the bleaching process and make it more sustainable for pulp and paper industries as well as for the environment.

Development of Novel Glyphosate-Tolerant Japonica Rice Lines: A Step Toward Commercial Release.

Glyphosate is the most widely used herbicide for its low cost and high efficiency. However, it is rarely applied directly in rice field due to its toxicity to rice. Therefore, glyphosate-tolerant rice can greatly decrease the cost of rice production and provide a more effective weed management strategy. Although, several approaches to develop transgenic rice with glyphosate tolerance have been reported, the agronomic performances of these plants have not been well evaluated, and the feasibility of commercial production has not been confirmed yet. Here, a novel glyphosate-tolerant gene cloned from the bacterium Isoptericola variabilis was identified, codon optimized (designated as I. variabilis-EPSPS*), and transferred into Zhonghua11, a widely used japonica rice cultivar. After systematic analysis of the transgene integration via PCR, Southern blot and flanking sequence isolation, three transgenic lines with only one intact I. variabilis-EPSPS* expression cassette integrated into intergenic regions were identified. Seed test results showed that the glyphosate tolerance of the transgenic rice was about 240 times that of wild type on plant medium. The glyphosate tolerance of transgenic rice lines was further evaluated based on comprehensive agronomic performances in the field with T3 and T5generations in a 2-year assay, which showed that they were rarely affected by glyphosate even when the dosage was 8400 g ha−1. To our knowledge, this is the first demonstration of the development of glyphosate-tolerant rice lines based on a comprehensive analysis of agronomic performances in the field. Taken together, the results suggest that the selected glyphosate-tolerant rice lines are highly tolerant to glyphosate and have the possibility of commercial release. I. variabilis-EPSPS* also can be a promising candidate gene in other species for developing glyphosate-tolerant crops.

Isoptericola cucumis sp. nov., isolated from the root tissue of cucumber (Cucumis sativus).

A Gram-stain-positive, aerobic organism, showing an irregular cell morphology, was isolated from the root tissue of cucumber (Cucumis sativus) and investigated in detail for its taxonomic position. On the basis of the 16S rRNA gene sequence analysis, strain AP-38T was shown to be most closely related to Isoptericola variabilis (99.1 %) and Isoptericola nanjingensis (98.9 %). The 16S rRNA gene sequence similarity to all other species of the genus Isoptericola was ≤98.5 %. DNA-DNA relatedness to Isoptericola variablis DSM 10177T and Isoptericola nanjingensis DSM 24300T was 31(reciprocal 41 %) and 34 (reciprocal 34 %), respectively. The diagnostic diamino acid of the peptidoglycan was l-lysine. The quinone system contained predominantly menaquinones MK-9(H4) and MK-9(H2). In the polar lipid profile, major compounds were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two phosphatidylinositol mannosides. The polyamine pattern contained the major components spermidine and spermine and significant amounts of tyramine. In the fatty acid profile, anteiso-C15 : 0 and iso-C15 : 0 were present in major amounts. These data support the allocation of the strain to the genus Isoptericola. The results of physiological and biochemical characterization additionally provide phenotypic differentiation of strain AP-38T from I. variabilis and I. nanjingensis. AP-38T represents a novel species of the genus Isoptericola, for which we propose the name Isoptericola cucumis sp. nov., with AP-38T (= LMG 29223T=CCM 8653T) as the type strain.

Characterization of a thermostable endoglucanase produced by Isoptericola variabilis sp. IDAH9

This study aimed to isolate and evaluate the cellulase activity of cellulolytic bacteria in hot springs of Dehloran, Ilam province, Iran. Water and sludge samples were collected from the hot springs and the bacterial enrichment was performed in a medium containing rice barn and carboxymethyl cellulose (CMC). The cultures were incubated at 50 °C in aerobic conditions. The bacteria were isolated on CMC agar (1%) medium. Cellulase assay of the isolates was measured by the evaluation of endoglucanase enzyme activity, which is also called as carboxymethyl cellulase (CMCase). The isolated thermotolerant bacteria were then identified and optimized for the production of CMCase. Moreover, stabilizing elements of the enzyme were identified with in silico approach. The chosen isolate was identified as Isoptericola variabilis sp. IDAH9. The identified strain produced the most thermostable CMCase at a concentration of 5.6 g/L of ammonium sulfate, 9 g/L CMCase or 12 g/L rice bran, 0/6% Tween-80, and 0.2% sucrose. The produced enzyme showed 80% of the residual activity after 1 h of incubation at 65 °C. In silico data indicated that the remaining residual activity was due to the redundant stabilizing elements in the protein structure. Consequently, I. variabilis can be isolated from the extreme environment and has a thermostable endoglucanase which may be used for various applications after studying them.

Exploring the microbiota dynamics related to vegetable biomasses degradation and study of lignocellulose-degrading bacteria for industrial biotechnological application.

The aims of this study were to evaluate the microbial diversity of different lignocellulosic biomasses during degradation under natural conditions and to isolate, select, characterise new well-adapted bacterial strains to detect potentially improved enzyme-producing bacteria. The microbiota of biomass piles of Arundo donax, Eucalyptus camaldulensis and Populus nigra were evaluated by high-throughput sequencing. A highly complex bacterial community was found, composed of ubiquitous bacteria, with the highest representation by the Actinobacteria, Proteobacteria, Bacteroidetes and Firmicutes phyla. The abundances of the major and minor taxa retrieved during the process were determined by the selective pressure produced by the lignocellulosic plant species and degradation conditions. Moreover, cellulolytic bacteria were isolated using differential substrates and screened for cellulase, cellobiase, xylanase, pectinase and ligninase activities. Forty strains that showed multienzymatic activity were selected and identified. The highest endo-cellulase activity was seen in Promicromonospora sukumoe CE86 and Isoptericola variabilis CA84, which were able to degrade cellulose, cellobiose and xylan. Sixty-two percent of bacterial strains tested exhibited high extracellular endo-1,4-ß-glucanase activity in liquid media. These approaches show that the microbiota of lignocellulosic biomasses can be considered an important source of bacterial strains to upgrade the feasibility of lignocellulose conversion for the ‘greener' technology of second-generation biofuels.

Characterization of a new type of glyphosate-tolerant 5-enolpyruvyl shikimate-3-phosphate synthase from Isoptericola variabilis

The 5-enolpyruvylshikimate-3-phosphate synthases (EPSPSs) encoded by aroA genes are traditionally divided into two classes, and only the class II EPSPS is naturally insensitive to herbicide glyphosate. Here, a novel EPSPS from Isoptericola variabilis, designated as I. variabilis EPSPS, was characterized. Its aroA gene contained 1374bp with 77% of GC contents, encoding a peptide of 458 amino acids. The EPSPS carried a highly conserved region involved in substrate bindings in EPSPSs and displayed a high sequence and structure similarity to class I EPSPS. However, this EPSPS had high naturally glyphosate-tolerant capability and enzymatic kinetics constants similar to that of class II EPSPSs. Escherichia coli AB2829 harboring the aroA gene grew well in medium containing 150mM glyphosate. The purified EPSPS had the highest enzymatic activity at pH 8.0 and 30°C, and retained its activity at pH 6–10 and 20–60°C. This enzyme had a Km value of 28μM for substrate phosphoenolpyruvate and 111μM for shikimate-3-phosphate, respectively, with a rather high Ki value for glyphosate. Cations increased substrate affinity but slightly reduced glyphosate tolerance. The I. variabilis EPSPS may be used for generating transgenic glyphosate-tolerant plants and dissecting mechanisms underlying the glyphosate tolerance.

Isolation and Characterization of a Nitrile-Hydrolysing Bacterium Isoptericola variabilis RGT01.

A nitrile-hydrolysing bacterium, identified as Isoptericola variabilis RGT01, was isolated from industrial effluent through enrichment culture technique using acrylonitrile as the carbon source. Whole cells of this microorganism exhibited a broad range of nitrile-hydrolysing activity as they hydrolysed five aliphatic nitriles (acetonitrile, acrylonitrile, propionitrile, butyronitrile and valeronitrile), two aromatic nitriles (benzonitrile and m-Tolunitrile) and two arylacetonitriles (4-Methoxyphenyl acetonitrile and phenoxyacetonitrile). The nitrile-hydrolysing activity was inducible in nature and acetonitrile proved to be the most efficient inducer. Minimal salt medium supplemented with 50mM acetonitrile, an incubation temperature of 30°C with 2% v/v inoculum, at 200rpm and incubation of 48h were found to be the optimal conditions for maximum production (2.64±0.12 U/mg) of nitrile-hydrolysing activity. This activity was stable at 30°C as it retained around 86% activity after 4h at this temperature, but was thermolabile with a half-life of 120min and 45min at 40°C and 50°C respectively.

Diversity of Xylanolytic Bacteria Isolated from Thai Sources

Twenty-three xylanolytic bacteria were isolated from soils, sediments and buffalo faeces collected in Thailand. They were divided into 10 groups based on the phenotypic and chemotaxonomic characteristics including 16S rRNA gene sequence analyses. Eleven isolates were Gram-positive, facultatively anaerobic, spore-forming, rod-shaped bacteria. They contained meso-diaminopimelic in cell wall peptidoglycan. Two isolates (Group IA) were identified as Bacillus subtilis, 4 isolates (Group IB) were B. licheniformis, 2 isolates (Group IC) were B. niabensis, one (Group ID) was B. nealsonii, and 2 isolates (Group IE) were B. cereus. Seven isolates were Gram-positive, non-spore-forming, rod-shaped bacteria and were identified as Isoptericola variabilis (2 isolates in Group II), as Jonesia denitrificans (2 isolates in Group III), as Microbacterium natoriense (2 isolates in Group IV), and one isolate as Nocardioides simplex (Group V). Five isolates were Gram-negative; facultatively anaerobic, non-spore-forming, rod-shaped bacteria and each of them were identified respectively as Acinetobacter junii (Group VI), Aeromonas enteropelogenes (Group VII), Pseudomonas stutzeri (Group VIII), Stenotrophomonas maltophilia (Group IX) and Zobellella denitrificans (Group X). The isolates produced xylanase activity ranged from 1.03 to 17.65±0.25 unit/ml.

Amylolytic Microorganism from São Paulo Zoo Composting: Isolation, Identification, and Amylase Production

Composting is a way of transforming the organic waste into fertilizer, minimizing the use of inorganic compounds that may contaminate the environment. This transformation is the result of the microorganism action, converting complex carbon sources into energy. Enzymes that are exported by the microorganisms to the surrounding environment mediate this process. The aiming of the present work is to prospect the compost produced by the organic composting unit (OCU) of the Fundação Parque Zoológico de São Paulo (FPZSP) to find novel starch hydrolyzing organisms (SHO) that secrete large amounts of amylases under harsh conditions, such as high temperature. We found five bacterial isolates that have amylolytic activity induced by soluble starch and 39°C temperature of growth. These bacterial strains were identified by MALDI-TOF (Matrix-assisted laser desorption/ionization-Time of Flight) analysis, a rapid and efficient methodology for microbe identification in large scale. Our results present amylolytic strains that belong to diverse taxonomic groups (Solibacillus silvestris, Arthrobacter arilaitensis, Isoptericola variabilis, and Acinetobacter calcoaceticus); some of them have never been associated with this kind of hydrolytic activity before. The information regarding enzyme induction will be important to optimize the production by the bacterial isolates, which may be a great value for biotechnological applications.

Reclassification of Promicromonospora pachnodae Cazemier et al. 2004 as Xylanimicrobium pachnodae gen. nov., comb. nov.

The recently described facultatively anaerobic Promicromonospora pachnodae is phylogenetically only moderately related to authentic members of Promicromonospora. P. pachnodae is closely related to Xylanibacterium ulmi and slightly less closely related to Xylanimonas cellulosilytica and Isoptericola variabilis (basonym Cellulosimicrobium variabile). Members of the different genera of Promicromonosporaceae have similar chemotaxonomic properties; they share the same peptidoglycan type (A4alpha) and have similar profiles of polar lipids, menaquinones, fatty acids and whole cell sugars. However, they differ from each other in the detailed amino acid composition of peptidoglycan, a taxonomically significant character that has previously been used in the delineation of actinobacterial genera. Recognized Promicromonospora species and Xylanibacterium ulmi exhibit the L-Lys-L-Ala-D-Glu type, Xylanimonas cellulosilytica and I. variabilis show the L-Lys-D-Asp type, whereas P. pachnodae has the L-Lys-L-Ser-D-Glu type. This property, together with the distinct phylogenetic position of Promicromonospora pachnodae, suggests a novel genus for the xylanolytic organism Xylanimicrobium pachnodae (Cazemier et al. 2004) gen. nov., comb. nov.

Reclassification of Cellulosimicrobium variabile Bakalidou et al. 2002 as Isoptericola variabilis gen. nov., comb. nov.

As already depicted in the original publication, the type strain of the species Cellulosimicrobium variabile Bakalidou et al. 2002, DSM 10177(T), does not cluster unambiguously with the type species, Cellulosimicrobium cellulans DSM 43879(T), in phylogenetic analysis. Strain DSM 10177(T) is moderately related to the recently described species Xylanimonas cellulosilytica, Promicromonospora pachnodae and Xylanibacterium ulmi, forming a lineage that branches between C. cellulans and members of the genus Promicromonospora in most dendrograms generated on the basis of different algorithms and reference strains. The type strains of the two Cellulosimicrobium species resemble each other in morphology, composition of fatty acids, DNA G+C content, phospholipids and the presence of lysine in position 3 of the peptide subunit of peptidoglycan. However, the two strains differ from each other in cell-wall sugars and in the amino acid composition of the A4alpha-type peptidoglycan, which contains serine and aspartic acid in C. cellulans, whereas only aspartic acid is present in the interpeptide bridge of C. variabile. This type is also present in Xylanimonas cellulosilytica XIL07(T), but not in the neighbouring species P. pachnodae DSM 12657(T), which exhibits the L-lys-L-ser-D-Glu type. On the basis of distinct phylogenetic position and the amino acid composition of peptidoglycan, a novel genus and combination for C. variabile, Isoptericola variabilis gen. nov., comb. nov., is proposed.