Basis Of 16S rRNA Research Articles

Faecalibacterium taiwanense sp. nov., isolated from human faeces.

Two Gram-stain-negative, straight rods, non-motile, asporogenous, catalase-negative and obligately anaerobic butyrate-producing strains, HLW78T and CYL33, were isolated from faecal samples of two healthy Taiwanese adults. Phylogenetic analyses of 16S rRNA and DNA mismatch repair protein MutL (mutL) gene sequences revealed that these two novel strains belonged to the genus Faecalibacterium. On the basis of 16S rRNA and mutL gene sequence similarities, the type strains Faecalibacterium butyricigenerans AF52-21T(98.3-98.1 % and 79.0-79.5 % similarity), Faecalibacterium duncaniae A2-165T(97.8-97.9 % and 70.9-80.1 %), Faecalibacterium hattorii APC922/41-1T(97.1-97.3 % and 80.3-80.5 %), Faecalibacterium longum CM04-06T(97.8-98.0% and 78.3 %) and Faecalibacterium prausnitzii ATCC 27768T(97.3-97.4 % and 82.7-82.9 %) were the closest neighbours to the novel strains HLW78T and CYL33. Strains HLW78T and CYL33 had 99.4 % both the 16S rRNA and mutL gene sequence similarities, 97.9 % average nucleotide identity (ANI), 96.3 % average amino acid identity (AAI), and 80.5 % digital DNA-DNA hybridization (dDDH) values, indicating that these two strains are members of the same species. Phylogenomic tree analysis indicated that strains HLW78T and CYL33 formed an independent robust cluster together with F. prausnitzii ATCC 27768T. The ANI, AAI and dDDH values between strain HLW78T and its closest neighbours were below the species delineation thresholds of 77.6-85.1 %, 71.4-85.2 % and 28.3-30.9 %, respectively. The two novel strains could be differentiated from the type strains of their closest Faecalibacterium species based on their cellular fatty acid compositions, which contained C18 : 1 ω7c and lacked C15 : 0 and C17 : 1 ω6c, respectively. Phenotypic, chemotaxonomic and genotypic test results demonstrated that the two novel strains HLW78T and CYL33 represented a single, novel species within the genus Faecalibacterium, for which the name Faecalibacterium taiwanense sp. nov. is proposed. The type strain is HLW78T (=BCRC 81397T=NBRC 116372T).

Isolation and Characterization of Glyphosate Degrading Bacteria from Kubau Local Govt. Area, Kaduna State

Strains of bacteria with the ability of utilizing glyphosate as the sole source of carbon were isolated from soil exposed to glyphosate herbicide by culture enrichment method and identified through partial 16S rRNA gene sequence analysis. Sixteen different microbial strains were isolated from glyphosate-contaminated sites in Kubau local govt. area, Kaduna state. Two of the microbial strains: BBDD3 and BBD4 were found to be the best degraders and were able to withstand up to 16.4 mg/ml of the glyphosate concentration. On the basis of 16s rRNA and biochemical tests, BBDD3 and BBDD4 strains were identified as Escherichia coli and Sporanaerobacter acetigenes respectively. These strains demonstrate laboratory utilizing ability of 92% and 98% for 16.4 mg/ml of the glyphosate formulation, respectively. The best optimum conditions for degradation were observed at pH 6.0 for the strains and at the temperature of 35oC for strain BBDD3 and BBDD4. Based on these results, the two strains displayed their potentials to be used in the bioremediation of glyphosate-contaminated environment.

Screening of antibacterial activities of Bacillus spp. isolated from the Parangkusumo coastal sand dunes, Indonesia

Abstract Background: The emergence of multidrug-resistant bacteria because of poor understanding of the issue and the misuse of antibiotics has become global health concern. Therefore, the discovery of novel antibacterial drugs is urgently needed. New antibacterial compounds may be found in the Bacillus species, which are abundant in sand dune ecosystems. Herein, we examined samples from the Parangkusumo coastal sand dunes in Indonesia. Methods: Samples were collected from three areas in the sand dunes (the area closest to the sea, the core area of sand dunes, and the area farthest from the sea). The samples were inoculated on Luria Bertani agar. Morphological and molecular identification was performed on the basis of 16S rRNA. The samples’ antimicrobial activity was evaluated with the disc diffusion method and compared with that of opportunistic pathogenic bacteria. Results: Five species of Bacillus were successfully isolated from the Parangkusumo coastal sand dunes. To our knowledge, this is the first report of the isolation of Bacillus aryabhattai in Indonesia. All samples showed antimicrobial activity against pathogenic bacteria. B. velezensis and B. subtilis showed antibacterial activity against Gram-positive bacteria, whereas B. aryabhattai and B. megaterium showed antibacterial activity against Gram-negative bacteria, and B. spizizenii showed antibacterial activity toward Gram-positive and Gram-negative bacteria. Conclusion: Five Bacillus species were successfully isolated from the Parangkusumo coastal sand dunes, Indonesia, and all samples showed antimicrobial activity toward opportunistic pathogenic bacteria. The crude antimicrobial compounds from B. megaterium, B. aryabhattai, B. subtilis, and B. spizizenii showed the highest growth-inhibition activity against E. coli, P. aeruginosa, B. cereus, and S. aureus, respectively.

Microbial Diversity of Upland Rice Roots and Their Influence on Rice Growth and Drought Tolerance.

Among abiotic stresses, drought is one of the most important factors limiting plant growth. To increase their drought tolerance and survival, most plants interact directly with a variety of microbes. Upland rice (Oryza sativa L.) is a rice ecotype that differs from irrigated ecotype rice; it is adapted to both drought-stress and aerobic conditions. However, its root microbial resources have not been explored. We isolated bacteria and fungi from roots of upland rice in Xishuangbanna, China. Four hundred sixty-two endophytic and rhizospheric isolates (337 bacteria and 125 fungi) were distributed. They were distributed among 43 genera on the basis of 16S rRNA and internal transcribed spacer (ITS) gene sequence analysis. Notably, these root microbes differed from irrigated rice root microbes in irrigated environments; for example, members of the Firmicutes phylum were enriched (by 28.54%) in the roots of the upland plants. The plant growth-promoting (PGP) potential of 217 isolates was investigated in vitro. The PGP ability of 17 endophytic and 10 rhizospheric isolates from upland rice roots was evaluated under well-irrigated and drought-stress conditions, and 9 fungal strains increased rice seedling shoot length, shoot and root fresh weight (FW), antioxidant capability, and proline (Pro) and soluble sugar contents. Our work suggests that fungi from upland rice roots can increase plant growth under irrigated and drought-stress conditions and can serve as effective microbial resources for sustainable agricultural production in arid regions.

Salinigranum halophilum sp. nov., isolated from marine solar salterns.

Three halophilic archaeal strains, YJ-53T, ZS-5 and DYF38, were isolated from marine solar salterns located in different provinces of China. The three strains formed a single cluster (99.7-99.8 and 97.9-99.2 % similarities, respectively) that was separate from the current two members of Salinigranum (96.7-98.0 and 89.8-92.9 % similarities, respectively) on the basis of 16S rRNA and rpoB' gene sequence comparisons and phylogenetic analysis. Diverse phenotypic characteristics differentiated strains YJ-53T, ZS-5 and DYF38 from Salinigranum rubrum GX10T and Salinigranum salinum YJ-50-S2T. The major polar lipids of isolated strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and two major glycolipids chromatographically identical to mannosyl glucosyl diether and sulfated mannosyl glucosyl diether, detected in the current members of Salinigranum. The OrthoANI and in silico DNA-DNA hybridization (DDH) values between the three strains were in the range of 97.7-98.4 % and 80.3-86.1 %, respectively, much higher than the threshold values proposed as species boundaries (average nucleotide identity 95-96 % and in silico DDH 70 %), revealing that the three strains represent one species. Results of comparative OrthoANI and in silico DDH analyses of the strains described in this study with validly described members of the genus Salinigranum supported that strains YJ-53T (=CGMCC 1.12860T=JCM 30238T), ZS-5 (=CGMCC 1.12867=JCM 30240) and DYF38 (=CGMCC 1.13779=JCM 33557) represent a novel species of the genus Salinigranum, for which the name Salinigranum halophilum sp. nov. is proposed.

A newly isolated Pseudomonas sp. can degrade endosulfan via hydrolytic pathway

Endosulfan an organochlorinated pesticide was used extensively throughout the world. Its enormous and inadequate use creates environmental as well as health problems. A bacterial strain capable to utilize endosulfan as a sole source of sulfur was isolated from pesticide contaminated soil and identified as Pseudomonas sp. on the basis of 16S rRNA. Batch experiments were conducted at various initial concentrations of endosulfan, i.e. 5, 25, 50, 75 and 100 mg/l to study its rate of degradation. After three days of incubation, 70–80% of each initial concentration was degraded by the isolated strain as compared to the control. Degradation of endosulfan increased with the time of incubation and maximum degradation was observed after 5 days of incubation. GC–MS revealed that the major metabolite was endosulfan lactone, which accumulated after 5 days of incubation. Kinetic studies at various initial concentrations also revealed that the bacterium has very promising attitude to utilize endosulfan as sole source of sulfur. It was observed that the addition of auxiliary sulfur Fe(SO4)3 in any concentration (0.05, 0.01 and 0.1%) decreased the rate of degradation of endosulfan. The ratio of μmax/ Ks was high (0.03 mg/l) when endosulfan was single sulfur source as compared to the value recorded when Fe(SO4)3 was added alongwith the endosulfan. This indicates that the newly isolated bacterium attacks sulfur moiety for its degradation.

Biodegradation of N-methylated carbamates by free and immobilized cells of newly isolated strain Enterobacter cloacae strain TA7

ABSTRACTA bacterial strain (TA7) capable of consuming three N-methylated carbamates as sole nitrogen and carbon source was isolated and identified as “Enterobacter cloacae” on the basis of 16S rRNA, from carbamate contaminated agricultural soil by enrichment culture technique. The agar entrapment was used to immobilize the bacterial cells. Both the free as well as the immobilized cells were used to study the degradation of three carbamets viz. aldicarb, carbofuran, and carbaryl. The immobilized cells degraded all the three carbamates much faster than their free cell counterparts. The biodegradation kinetics of aldicarb, carbaryl, and carbofuran was studied using 50 ppm as initial concentration in the presence of free cells. The average values of Ks for aldicarb, carbofuran, and carbaryl were 22.6, 17.87, and 8.9 mg/L, respectively, whereas the values for µmax were calculated as 1.35, 1.3, and 1.2 mg/l/h−1. The results indicated that the bacterium has high affinity towards all the three carbamates. However, relatively higher affinity is for carbaryl, in comparison with carbofuran and aldicarb. Results indicate the potential of E. Cloacae TA7 to remediate N-methylated carbamates polluted water and soil.

Metagenome-Derived Draft Genome Sequence of Acidithiobacillus ferrooxidans RV1 from an Abandoned Gold Tailing in Neuquén, Argentina

In this work we report the metagenome-derived draft genomic sequence of an enrichment culture dominated by A. ferrooxidans obtained from an airlift bioreactor inoculated with the microbial consortium recovered from the “Relave Viejo” tailing. The genome of this culture was assembled de-novo and by reference, generating a consensus assembly of 3.0 Mb. On the basis of 16S rRNA (100 % identity), average nucleotide identity analysis (99.33% identity) and in silico DNA-DNA hybridization against A. ferrooxidans ATCC 23270T (97.9%), the recovered genome is confirmed to pertain to A. ferrooxidans species. Comparative genomics results are presented to uncover the genetic traits of the variant surviving lime treatment and to further explore the genomic diversity of these model iron oxidizing species.

A new insight to adsorption and accumulation of high lead concentration by exopolymer and whole cells of lead-resistant bacterium Acinetobacter junii L. Pb1 isolated from coal mine dump

A lead-resistant bacterial strain was isolated from coal mine dump and identified as Acinetobacter junii Pb1 on basis of 16S rRNA (ribosomal ribonucleic acid) gene sequencing. The minimum inhibitory concentration of lead for the strain was 16,000mgl-1 and it showed antibiotic and multi metal resistance. In aqueous culture, at an initial lead (Pb(II)) concentration of 100 and 500mgl-1, lead adsorption and accumulation by the isolate was 100 and 60%, at pH 7 at 30°C after 48 and 120h, respectively. The two fractions of exopolysaccharide (EPS), loosely associated EPS (laEPS) and bound EPS (bEPS), and whole cells (devoid of EPS) showed high binding affinity towards Pb(II). The binding affinity of laEPS towards Pb(II) (1071mgPbg-1) was three times higher than that of bEPS (321.5mgPbg-1) and 6.5 times higher than that of whole cells (165mgPbg-1). The binding affinity of EPS and whole cells with Pb(II), reported in the current study, is considerably higher as compared to that reported in the literature, till date. SEM analysis, showed an increase in thickness of cells on exposure to Pb(II) and TEM analysis, revealed its accumulation (interior of cell) and its adsorption (with the external cell surface). The isolate was also found to be positive for indole acetic acid (IAA) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase production which helps in promoting plant growth. Thus, this study provides a new understanding towards Pb(II) uptake by A. junii Pb1, highlighting its potential on the restoration of Pb(II) contaminated repositories.

Advanced Microbial Taxonomy Combined with Genome-Based-Approaches Reveals that Vibrio astriarenae sp. nov., an Agarolytic Marine Bacterium, Forms a New Clade in Vibrionaceae.

Advances in genomic microbial taxonomy have opened the way to create a more universal and transparent concept of species but is still in a transitional stage towards becoming a defining robust criteria for describing new microbial species with minimum features obtained using both genome and classical polyphasic taxonomies. Here we performed advanced microbial taxonomies combined with both genome-based and classical approaches for new agarolytic vibrio isolates to describe not only a novel Vibrio species but also a member of a new Vibrio clade. Two novel vibrio strains (Vibrio astriarenae sp. nov. C7T and C20) showing agarolytic, halophilic and fermentative metabolic activity were isolated from a seawater sample collected in a coral reef in Okinawa. Intraspecific similarities of the isolates were identical in both sequences on the 16S rRNA and pyrH genes, but the closest relatives on the molecular phylogenetic trees on the basis of 16S rRNA and pyrH gene sequences were V. hangzhouensis JCM 15146T (97.8% similarity) and V. agarivorans CECT 5085T (97.3% similarity), respectively. Further multilocus sequence analysis (MLSA) on the basis of 8 protein coding genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) obtained by the genome sequences clearly showed the V. astriarenae strain C7T and C20 formed a distinct new clade protruded next to V. agarivorans CECT 5085T. The singleton V. agarivorans has never been included in previous MLSA of Vibrionaceae due to the lack of some gene sequences. Now the gene sequences are completed and analysis of 100 taxa in total provided a clear picture describing the association of V. agarivorans into pre-existing concatenated network tree and concluded its relationship to our vibrio strains. Experimental DNA-DNA hybridization (DDH) data showed that the strains C7T and C20 were conspecific but were separated from all of the other Vibrio species related on the basis of both 16S rRNA and pyrH gene phylogenies (e.g., V. agarivorans CECT 5085T, V. hangzhouensis JCM 15146T V. maritimus LMG 25439T, and V. variabilis LMG 25438T). In silico DDH data also supported the genomic relationship. The strains C7T also had less than 95% average amino acid identity (AAI) and average nucleotide identity (ANI) towards V. maritimus C210, V. variabilis C206, and V. mediterranei AK1T, V. brasiliensis LMG 20546T, V. orientalis ATCC 33934T, and V. sinaloensis DSM 21326. The name Vibrio astriarenae sp. nov. is proposed with C7 as the type strains. Both V. agarivorans CECT 5058T and V. astriarenae C7T are members of the newest clade of Vibrionaceae named Agarivorans.

Paenibacillus panaciterrae sp. nov., isolated from ginseng-cultivated soil.

A novel bacterium, designated DCY95T, was isolated from ginseng-cultivated soil in Quang Nam province, Vietnam. On the basis of 16S rRNA and gyrB gene sequence analysis, this isolate was assigned to the genus Paenibacillus and found to be closely related to Paenibacillus sacheonensis SY01T (97.1 % 16S rRNA gene sequence similarity) and Paenibacillus taihuensis THMBG22T (96.4 %). The partial gyrB gene of DCY95T possessed 69.6-83.9 % sequence identity to those of other members of the genus Paenibacillus. Strain DCY95T was Gram-reaction-negative, catalase-negative, oxidase-positive, strictly aerobic, rod-shaped and motile by means of peritrichous flagella. Ellipsoidal free spores or subterminal endospores were produced in sporangia. MK-7 was the diagnostic menaquinone. The cell-wall peptidoglycan contained meso-diamonopimelic acid as the diamino acid. Whole-cell sugars comprised ribose, mannose and glucose. The major cellular fatty acids were anteiso-C15 : 0, iso-C16 : 0 and C16 : 0. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, three unidentified aminophospholipids, and two unidentified phospholipids. The genomic DNA G+C content was 60.7 ± 0.9 mol%. Phenotypic and chemotaxonomic results placed strain DCY95T within the genus Paenibacillus. However, DNA-DNA relatedness values between strain DCY95T and P. sacheonensis KACC 14895T or P. taihuensis NBRC 108766T were lower than 36 %. The low DNA relatedness data in combination with phylogenetic and (GTG)5-PCR analyses, as well as biochemical tests, indicated that strain DCY95T could not be assigned to any recognized species. In conclusion, the results in this study support the classification of strain DCY95T as a representative of a novel species within the genus Paenibacillus, for which the name Paenibacillus panaciterrae is proposed. The type strain is DCY95T ( = KCTC 33581T = DSM 29477T).

Identification of strains Bacillus aerophilus MTCC 7304T as Bacillus altitudinis and Bacillus stratosphericus MTCC 7305T as a Proteus sp. and the status of the species Bacillus aeriusShivaji et al. 2006. Request for an Opinion.

On the basis of 16S rRNA, rpoB, gyrB and pycA gene sequence analyses, characterization of biochemical features and other phenotypic traits and pulsed-field gel electrophoresis (PFGE) fingerprinting, it was ascertained that strains Bacillus aerius MTCC 7303T, Bacillus aerophilus MTCC 7304(T) and Bacillus stratosphericus MTCC 7305(T) do not conform to the descriptions of the type strains of the respective species. Strains MTCC 7303(T) and MTCC 7304(T) were indistinguishable from Bacillus altitudinis DSM 21631(T), while strain MTCC 7305(T) should be classified as a representative of a Proteus sp. Our attempts to find other deposits of the type strains of these species were unsuccessful. Therefore, the results support the Request for an Opinion on the status of the species Bacillus aerophilus and Bacillus stratosphericus by Branquinho et al. [Branquinho, R., Klein, G., Kämpfer, P. & Peixe, L. V. (2015). Int J Syst Evol Microbiol 65, 1101]. It is also proposed that the Judicial Commission should place the name Bacillus aerius on the list of rejected names if a suitable replacement type strain cannot be found or a neotype is not proposed within two years following the publication of this Request (Rule 18c).

Aeromonas aquatica sp. nov., Aeromonas finlandiensis sp. nov. and Aeromonas lacus sp. nov. isolated from Finnish waters associated with cyanobacterial blooms

Three groups of Aeromonas strains isolated from Finland lakes experiencing cyanobacterial blooms could not be assigned to any known species of this genus on the basis of 16S rRNA and rpoD gene sequences. The Multilocus Phylogenetic Analysis (MLPA) of the concatenated sequence of seven genes (gyrB, rpoD, recA, dnaJ, gyrA, dnaX and atpD; 4093bp) showed that the three groups of strains did not cluster with any known Aeromonas spp. and formed three independent lineages. This was confirmed by performing the analysis with their closest relatives using 15 genes (the latter 7 and cpn60, dnaK, gltA, mdh, radA, rpoB, tsf, zipA; 8751bp). Furthermore, ANI results between the genomes of the type strains of the three potential new species and those of their close relatives were all <96% which is the previously proposed cutoff value for differentiating species within this genus. The in silico DDH values of the three type strains of the new species also showed a similarity <70% with the most closely related species indicating they belong to different taxa. The three groups of strains could be differentiated from each other and from other known Aeromonas species on the basis of several phenotypic characters. This polyphasic study revealed that the 3 groups of strains represent 3 novel Aeromonas species for which the names Aeromonas aquatica sp. nov. (type strain AE235T=CECT 8025T=LMG 26712T), Aeromonas finlandiensis sp. nov. (type strain 4287DT=CECT 8028T=LMG 26709T) and Aeromonas lacus sp. nov. (type strain AE122T=CECT 8024T=LMG 26710T) are proposed.

Pseudomonas matsuisoli sp. nov., isolated from a soil sample.

An aerobic, Gram-stain-negative, rod-shaped and polar-flagellated bacterium, designated strain CC-MHH0089(T), was isolated from a soil sample taken on Matsu Island (Taiwan). Strain CC-MHH0089(T) grew at 15-30 °C and pH 5.0-10.0 and tolerated ≤8 % (w/v) NaCl. 16S rRNA gene sequence analysis showed high pairwise sequence similarity to Pseudomonas azotifigens 6H33b(T) (97.3 %) and Pseudomonas balearica SP1402(T) (96.7 %) and lower sequence similarity to other strains (<96.0 %). In DNA-DNA reassociation experiments, the relatedness of strain CC-MHH0089(T) to P. azotifigens JCM 12708(T) was 38.3 % (reciprocal value 19.5 %). Evolutionary trees reconstructed on the basis of 16S rRNA, gyrB and rpoB gene sequences revealed a varying phylogenetic neighbourhood of strain CC-MHH0089(T) with regard to the most closely related type strains. The predominant quinone system was ubiquinone 9 (Q-9) and the DNA G+C content was 63.6 mol%. The major fatty acids were C12 : 0, C16 : 0, C17 : 0, C19 : 0 cyclo ω8c and summed features 2 (C14 : 0 3-OH/iso-C16 : 1 I), 3 (C16 : 1ω7c/C16 : 1ω6c) and 8 (C18 : 1ω7c/C18 : 1ω6c). The major polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. According to its distinct phylogenetic, phenotypic and chemotaxonomic features, strain CC-MHH0089(T) is proposed to represent a novel species within the genus Pseudomonas, for which the name Pseudomonas matsuisoli sp. nov. is proposed. The type strain is CC-MHH0089(T) ( = BCRC 80771(T) = JCM 30078(T)).

Modification interference analysis of the ribosome.

RNAs are versatile molecules involved in myriad functions in the cell. To understand how a RNA molecule functions in the cell it is important to identify the nucleotides in the RNA molecule that are important for its structure and function. There are several biochemical methods such as footprinting, cross-linking, and modification interference analysis that can be used to study RNA-RNA and RNA-protein interactions. Ribosome is a classical example of a RNA-protein complex that has been extensively studied using these methods. Here, we describe a modification interference method that was used to identify bases in 16S rRNA that are important for the translocation of the mRNA-tRNA complex by the ribosome.

Whole genome phylogeny of Prochlorococcus marinus group of cyanobacteria: genome alignment and overlapping gene approach

Prochlorococcus is the smallest known oxygenic phototrophic marine cyanobacterium dominating the mid-latitude oceans. Physiologically and genetically distinct P. marinus isolates from many oceans in the world were assigned two different groups, a tightly clustered high-light (HL)-adapted and a divergent low-light (LL-) adapted clade. Phylogenetic analysis of this cyanobacterium on the basis of 16S rRNA and other conserved genes did not show consistency with its phenotypic behavior. We analyzed phylogeny of this genus on the basis of complete genome sequences through genome alignment, overlapping-gene content and gene-order approach. Phylogenetic tree of P. marinus obtained by comparing whole genome sequences in contrast to that based on 16S rRNA gene, corresponded well with the HL/LL ecotypic distinction of twelve strains and showed consistency with phenotypic classification of P. marinus. Evidence for the horizontal descent and acquisition of genes within and across the genus was observed. Many genes involved in metabolic functions were found to be conserved across these genomes and many were continuously gained by different strains as per their needs during the course of their evolution. Consistency in the physiological and genetic phylogeny based on whole genome sequence is established. These observations improve our understanding about the adaptation and diversification of these organisms under evolutionary pressure.

Enterobacter xiangfangensis sp. nov., isolated from Chinese traditional sourdough, and reclassification of Enterobacter sacchari Zhu et al. 2013 as Kosakonia sacchari comb. nov.

A Gram-stain-negative bacterial strain, 10-17(T), was isolated from traditional sourdough in Heilongjiang Province, China. The bacterium was characterized by a polyphasic approach, including 16S rRNA gene sequence analysis, RNA polymerase β subunit (rpoB) gene sequence analysis, DNA gyrase (gyrB) gene sequence analysis, initiation translation factor 2 (infB) gene sequence analysis, ATP synthase β subunit (atpD) gene sequence analysis, fatty acid methyl ester analysis, determination of DNA G+C content, DNA-DNA hybridization and an analysis of phenotypic features. Strain 10-17(T) was phylogenetically related to Enterobacter hormaechei CIP 103441(T), Enterobacter cancerogenus LMG 2693(T), Enterobacter asburiae JCM 6051(T), Enterobacter mori LMG 25706(T), Enterobacter ludwigii EN-119(T) and Leclercia adecarboxylata LMG 2803(T), having 99.5%, 99.3%, 98.7%, 98.5%, 98.4% and 98.4% 16S rRNA gene sequence similarity, respectively. On the basis of polyphasic characterization data obtained in the present study, a novel species, Enterobacter xiangfangensis sp. nov., is proposed and the type strain is 10-17(T) ( = LMG 27195(T) = NCIMB 14836(T) = CCUG 62994(T)). Enterobacter sacchari Zhu et al. 2013 was reclassified as Kosakonia sacchari comb. nov. on the basis of 16S rRNA, rpoB, gyrB, infB and atpD gene sequence analysis and the type strain is strain SP1(T)( = CGMCC 1.12102(T) = LMG 26783(T)).

Isolation, Identification and Optimization of Culture Conditions of Bacillus sp. Strain PM1 for Alkalo-thermostable Amylase Production

Aims: To isolate and optimize the culture conditions for thermo stable and alkaline amylase production from bacteria. Study Design: Optimization of different physiological and nutritional parameters for amylase production and kinetic studies of amylase. Place and Duration of Study: Soil Samples: Herbal garden of Amity University Haryana, Gurgaon (Manesar), India, between April 2012 and September 2012. Methodology: Amylolytic isolates were selected by flooding the nutrient agar plates containing 2% starch with Lugol solution. Isolates were selected on the basis of higher ratio of clear zone to colony size and grown in nutrient broth containing 2% starch. The level of amylase was detected in the culture filtrate. The selected isolate showing maximum amylase production was identified on the basis of 16S rDNA amplification. Results: An Alkalo-thermostable amylase producing bacterial isolate from soil was identified as Bacillus sp. strain PM1 on the basis of 16S rRNA. It yielded 3.5 U/ml of amylase in medium containing (%) starch 2.0, beef extract 0.5, NaCl 0.5 at 50oC, pH 7.0 at 180 rpm after 72 h. The optimum pH and temperature for amylase activity was 8.0 and 50°C, respectively. The enzyme exhibited 67% activity after 60 minute incubation at 50oC. Original Research Article British Microbiology Research Journal, 4(4): 369-380, 2014 370 At pH 8.0, the enzyme retained 78% activity after 4 h. Conclusion: The properties of the isolated enzyme are adequate for its use in starch processing and baking industry.

English

In this work, microbial community structure of two distinct arid plants like ker (Capparis deciduas) and pearl millet (Pennisetum glaucum) was assessed and defined by culture-dependent and culture-independent approaches on the basis of 16S rRNA and random amplified polymorphic DNA (RAPD) analysis. The average Jaccard&rsquo;s similarity coefficient values for cultivated bacteria that is within ker and pearl millet rhizosphere were 0.701 and 0.707, respectively, for non-rhizosphere of ker and pearl millet 0.739 and 0.762, respectively, and for non-cultivable bacteria under ker (0.519) and under pearl millet (0.534). Both culture- dependent and culture-independent methods indicated that in arid crops, microbial diversity is more influenced by soil type rather than plant type and lower Jaccard value for metagenome showed that whole community harbours more diversity because of different microflora than cultivated only. Salinity and temperature tolerance study of bacteria indicated that ker rhizosphere harbours more salinity and temperature tolerant bacteria. &nbsp; Key words: Random amplified polymorphic DNA (RAPD), ribotyping, Thar Desert, microbial diversity, 16S rRNA.

Lactobacillus hokkaidonensis sp. nov., isolated from subarctic timothy grass (Phleum pratense L.) silage

Four strains of Gram-positive, non-spore-forming, rod-shaped, catalase-negative and non-motile lactic acid bacteria, LOOC260(T), LOOC253, LOOC273 and LOOC279, were isolated from timothy grass (Phleum pratense L.) silage produced in Hokkaido, a subarctic region of Japan. These isolates grew at 4-37 °C, indicating the psychrotolerant nature of these strains. Phylogenetic analysis on the basis of 16S rRNA and pheS gene sequences, as well as biochemical and physiological characteristics, indicated that these four strains were members of the genus Lactobacillus. 16S rRNA gene sequence analysis of strain LOOC260(T) demonstrated that the closest neighbours were the type strains of Lactobacillus suebicus (97.7 %), Lactobacillus oligofermentans (96.7 %) and Lactobacillus vaccinostercus (96.7 %). Strain LOOC260(T) showed low levels of DNA-DNA association with Lactobacillus suebicus JCM 9504(T) (14.7 ± 3.5 %), Lactobacillus oligofermentans JCM 16175(T) (15.1 ± 4.8 %) and Lactobacillus vaccinostercus JCM 1716(T) (10.7 ± 3.0 %). The cell wall contained meso-diaminopimelic acid and the major fatty acids were C18 : 1ω9c and C19 : 1 cyclo 9,10. On the basis of phenotypic, physiological and phylogenetic evidence, these isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillus hokkaidonensis sp. nov. is proposed. The type strain is LOOC260(T) ( = JCM 18461(T) = DSM 26202(T)).