Mansi Verma

Polyphasic approach of bacterial classification — An overview of recent advances

Classification of microorganisms on the basis of traditional microbiological methods (morphological, physiological and biochemical) creates a blurred image about their taxonomic status and thus needs further clarification. It should be based on a more pragmatic approach of deploying a number of methods for the complete characterization of microbes. Hence, the methods now employed for bacterial systematics include, the complete 16S rRNA gene sequencing and its comparative analysis by phylogenetic trees, DNA-DNA hybridization studies with related organisms, analyses of molecular markers and signature pattern(s), biochemical assays, physiological and morphological tests. Collectively these genotypic, chemotaxonomic and phenotypic methods for determining taxonomic position of microbes constitute what is known as the ‘polyphasic approach’ for bacterial systematics. This approach is currently the most popular choice for classifying bacteria and several microbes, which were previously placed under invalid taxa have now been resolved into new genera and species. This has been possible owing to rapid development in molecular biological techniques, automation of DNA sequencing coupled with advances in bioinformatic tools and access to sequence databases. Several DNA-based typing methods are known; these provide information for delineating bacteria into different genera and species and have the potential to resolve differences among the strains of a species. Therefore, newly isolated strains must be classified on the basis of the polyphasic approach. Also previously classified organisms, as and when required, can be reclassified on this ground in order to obtain information about their accurate position in the microbial world. Thus, current techniques enable microbiologists to decipher the natural phylogenetic relationships between microbes.

Devosia chinhatensis sp. nov., isolated from a hexachlorocyclohexane (HCH) dump site in India

A Gram-negative, motile, rod-shaped and non-spore-forming bacterium was isolated from a soil sample collected from the area adjoining an India Pesticide Limited plant, Lucknow, India. Strain IPL18T was characterized on the basis of phenotypic and genotypic data. Based on 16S rRNA gene sequence analysis, this strain was shown to belong to genus Devosia, with highest sequence similarity of 97.5 % to Devosia riboflavina DSM 7230T. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequence analysis revealed that strain IPL18T shows an evolutionary relationship with Devosia neptuniae J1T. Strain IPL18T contains C18 : 1 omega 7c, 11-methyl C18 : 1 omega 7c and C16 : 0 as the major fatty acids along with 3-OH C18 : 0, characteristic of the genus Devosia. The branched-chain fatty acid iso-H C18 : 1 (4.69 %), not present in any of the Devosia species known so far, has been reported in strain IPL18T. The results of DNA-DNA hybridization experiments (13 % relatedness with D. riboflavina DSM 7230T and 11 % with D. neptuniae J1T), phylogenetic analysis and biochemical tests confirm that strain IPL18T represents a novel species of the genus Devosia for which the name Devosia chinhatensis sp. nov. is proposed. The type strain is IPL18T (=CCM 7426T=MTCC 8593T).

Comparative Metagenomic Analysis of Soil Microbial Communities across Three Hexachlorocyclohexane Contamination Levels

This paper presents the characterization of the microbial community responsible for the in-situ bioremediation of hexachlorocyclohexane (HCH). Microbial community structure and function was analyzed using 16S rRNA amplicon and shotgun metagenomic sequencing methods for three sets of soil samples. The three samples were collected from a HCH-dumpsite (450 mg HCH/g soil) and comprised of a HCH/soil ratio of 0.45, 0.0007, and 0.00003, respectively. Certain bacterial; (Chromohalobacter, Marinimicrobium, Idiomarina, Salinosphaera, Halomonas, Sphingopyxis, Novosphingobium, Sphingomonas and Pseudomonas), archaeal; (Halobacterium, Haloarcula and Halorhabdus) and fungal (Fusarium) genera were found to be more abundant in the soil sample from the HCH-dumpsite. Consistent with the phylogenetic shift, the dumpsite also exhibited a relatively higher abundance of genes coding for chemotaxis/motility, chloroaromatic and HCH degradation (lin genes). Reassembly of a draft pangenome of Chromohalobacter salaxigenes sp. (∼8X coverage) and 3 plasmids (pISP3, pISP4 and pLB1; 13X coverage) containing lin genes/clusters also provides an evidence for the horizontal transfer of HCH catabolism genes.

Proposal of biostimulation for hexachlorocyclohexane (HCH)‐decontamination and characterization of culturable bacterial community from high‐dose point HCH‐contaminated soils

Aims:  To locate a high‐dose point hexachlorocyclohexane (HCH)‐contaminated site, to identify HCH‐degrading bacteria in it and assay HCH‐decontamination by biostimulation.

Rhizobium rosettiformans sp. nov., isolated from a hexachlorocyclohexane dump site, and reclassification of Blastobacter aggregatus Hirsch and Muller 1986 as Rhizobium aggregatum comb. nov.

A Gram-negative, rod-shaped, motile, aerobic bacterial strain, W3(T), was isolated from hexachlorocyclohexane (HCH)-contaminated groundwater from Lucknow, India, and its taxonomic position was determined using a polyphasic approach. Strain W3(T) shared highest 16S rRNA gene sequence similarity of 97.8 % with Rhizobium selenitireducens B1(T), followed by Rhizobium daejeonense L61(T) (97.7 %), Rhizobium radiobacter ATCC 19358(T) (97.5 %) and Blastobacter aggregatus IFAM 1003(T) (97.2 %). Strain W3(T) formed a monophyletic clade with Blastobacter aggregatus IFAM 1003(T) ( = DSM 1111(T)) in the cluster of species of the genus Rhizobium. Phylogenetic analyses of strain W3(T) using atpD and recA gene sequences confirmed the phylogenetic arrangements obtained by using 16S rRNA gene sequences. Hence, the taxonomic characterization of B. aggregatus DSM 1111(T) was also undertaken. Strains W3(T) and B. aggregatus DSM 1111(T) contained summed feature 8 (18 : 1ω7c and/or 18 : 1ω6c; 65.4 and 70.8 %, respectively) as the major fatty acid, characteristic of the genus Rhizobium. DNA-DNA relatedness of strain W3(T) with Rhizobium selenitireducens LMG 24075(T), Rhizobium daejeonense DSM 17795(T), Rhizobium radiobacter DSM 30147(T) and B. aggregatus DSM 1111(T) was 42, 34, 30 and 34 %, respectively. The DNA G+C contents of strain W3(T) and B. aggregatus DSM 1111(T) were 62.3 and 62.7 mol%, respectively. A nifH gene encoding dinitrogenase reductase was detected in strain W3(T) but not in B. aggregatus DSM 1111(T). Based on the results obtained by phylogenetic and chemotaxonomic analyses, phenotypic characterization and DNA-DNA hybridization, it is concluded that strain W3(T) represents a novel species of the genus Rhizobium, for which the name Rhizobium rosettiformans sp. nov. is proposed (type strain W3(T)  = CCM 7583(T)  = MTCC 9454(T)). It is also proposed that Blastobacter aggregatus Hirsch and Müller 1986 be transferred to the genus Rhizobium as Rhizobium aggregatum comb. nov. (type strain IFAM 1003(T)  = DSM 1111(T)  = ATCC 43293(T)).

Exploring internal features of 16S rRNA gene for identification of clinically relevant species of the genus Streptococcus

BackgroundStreptococcus is an economically important genus as a number of species belonging to this genus are human and animal pathogens. The genus has been divided into different groups based on 16S rRNA gene sequence similarity. The variability observed among the members of these groups is low and it is difficult to distinguish them. The present study was taken up to explore 16S rRNA gene sequence to develop methods that can be used for preliminary identification and can supplement the existing methods for identification of clinically-relevant isolates of the genus Streptococcus.Methods16S rRNA gene sequences belonging to the isolates of S. dysgalactiae, S. equi, S. pyogenes, S. agalactiae, S. bovis, S. gallolyticus, S. mutans, S. sobrinus, S. mitis, S. pneumoniae, S. thermophilus and S. anginosus were analyzed with the purpose to define genetic variability within each species to generate a phylogenetic framework, to identify species-specific signatures and in-silico restriction enzyme analysis.ResultsThe framework based analysis was used to segregate Streptococcus spp. previously identified upto genus level. This segregation was validated using species-specific signatures and in-silico restriction enzyme analysis. 43 uncharacterized Streptococcus spp. could be identified using this approach.ConclusionsThe markers generated exploring 16S rRNA gene sequences provided useful tool that can be further used for identification of different species of the genus Streptococcus.

Pseudomonas sp. to Sphingobium indicum: a journey of microbial degradation and bioremediation of Hexachlorocyclohexane

The unusual process of production of hexachlorocyclohexane (HCH) and extensive use of technical HCH and lindane has created a very serious problem of HCH contamination. While the use of technical HCH and lindane has been banned all over the world, India still continues producing lindane. Bacteria, especially Sphingomonads have been isolated that can degrade HCH isomers. Among all the bacterial strains isolated so far, Sphingobium indicum B90A that was isolated from HCH treated rhizosphere soil appears to have a better potential for HCH degradation. This conclusion is based on studies on the organization of lin genes and degradation ability of B90A. This strain perhaps can be used for HCH decontamination through bioaugmentation.

Sphingopyxis ummariensis sp. nov., isolated from a hexachlorocyclohexane dump site.

A Gram-negative, motile, rod-shaped, yellow-pigmented bacterium, strain UI2(T), was isolated from a hexachlorocyclohexane (HCH) dump site located in Ummari, in northern India. 16S rRNA gene sequence analysis revealed that strain UI2(T) belongs to the genus Sphingopyxis and showed highest 16S rRNA gene sequence similarity with Sphingopyxis terrae IFO 15098(T) (98.6 %). DNA-DNA relatedness values between strain UI2(T) and Sphingopyxis terrae IFO 15098(T) and other related strains were found to be less than 46 %. The major cellular fatty acids of strain UI2(T) were C(18 : 1)omega7c (28.3 %), C(16 : 0) (14.4 %), 11-methyl C(18 : 1)omega7c (10.9 %), C(17 : 1)omega6c (9.6 %) and summed feature 3 (consisting of C(16 : 1)omega7c and/or C(15 : 0) iso 2-OH; 14.7 %). The DNA G+C content of strain UI2(T) was 68 mol%. On the basis of DNA-DNA hybridization, phenotypic characteristics and phylogenetic analysis, strain UI2(T) is found to represent a novel species of the genus Sphingopyxis, for which the name Sphingopyxis ummariensis sp. nov. is proposed. The type strain is UI2(T) (=CCM 7428(T) =MTCC 8591(T)).

Devosia albogilva sp. nov. and Devosia crocina sp. nov., isolated from a hexachlorocyclohexane dump site.

Two bacterial strains, IPL15(T) and IPL20(T), isolated from a hexachlorocyclohexane dump site in India, were characterized by using a polyphasic approach. Based on 16S rRNA gene sequence analysis, both strains belonged to the genus Devosia; highest sequence similarities of strain IPL15(T) were observed with Devosia neptuniae J1(T) and Devosia geojensis BD-c194(T) (96.2 % in each case) and the highest sequence similarity of strain IPL20(T) was observed with Devosia soli GH2-10(T) (98.6 %). Phylogenetic analysis showed the distinct lineages of strains IPL15(T) and IPL20(T) among members of the genus Devosia. The presence of C(18 : 0) 3-OH and C(10 : 0) 3-OH fatty acids supported their respective positions within the genus Devosia. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA hybridization results, it is concluded that strains IPL15(T) and IPL20(T) represent two distinct species of the genus Devosia for which the names Devosia albogilva sp. nov. and Devosia crocina sp. nov., respectively, are proposed. The type strains are Devosia albogilva IPL15(T) (=CCM 7427(T)=MTCC 8594(T)) and Devosia crocina IPL20(T) (=CCM 7425(T)=MTCC 8590(T)).

Genome Sequence of Sphingobium indicum B90A, a Hexachlorocyclohexane-Degrading Bacterium

Sphingobium indicum B90A, an efficient degrader of hexachlorocyclohexane (HCH) isomers, was isolated in 1990 from sugarcane rhizosphere soil in Cuttack, India. Here we report the draft genome sequence of this bacterium, which has now become a model system for understanding the genetics, biochemistry, and physiology of HCH degradation.