Devi Lal

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.

Novosphingobium panipatense sp. nov. and Novosphingobium mathurense sp. nov., from oil-contaminated soil

In an ongoing attempt to analyse the diversity of culturable micro-organisms from oil-contaminated soil, two yellow-pigmented, Gram-negative, halophilic bacterial strains (SM16T and SM117T) were isolated. These two strains were characterized using a polyphasic approach. Strains SM16T and SM117T showed a range of phenotypic and chemotaxonomic properties that were consistent with those of members of the genus Novosphingobium. Both strains contained sphingoglycolipids, thus confirming that they belong to the Alphaproteobacteria. Furthermore, the polar lipid profile consisted of phosphatidylglycerol, phosphatidyldimethylethanolamine and phosphatidylethanolamine, with minor amounts of phosphatidyldimethylethanolamine, phosphatidylcholine and phosphatidylmonomethylethanolamine. Spermidine was the major polyamine in the cell wall, a characteristic feature of members of the genus Novosphingobium. Fatty acid analysis revealed the presence of C(18 : 1)omega7c, C(16 : 0) and C(14 : 0) 2-OH in both isolates, a characteristic feature of sphingomonads. 16S rRNA gene sequence similarities with the type strains of the most closely related species of the genus Novosphingobium (Novosphingobium pentaromativorans and Novosphingobium resinovorum) were less than 98.8 % for both strains. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains SM16T and SM117T fall in the clade represented by the genus Novosphingobium. Strains SM16T and SM117T shared 98.8 % similarity in their 16S rRNA gene sequences and their mean level of DNA-DNA relatedness was 8.5 %. Strains SM16T and SM117T differed from each other with respect to their morphological, physiological and chemotaxonomic properties. Thus, these results indicate that strains SM16T and SM117T belong to separate species of the genus Novosphingobium, for which the names Novosphingobium panipatense sp. nov. and Novosphingobium mathurense sp. nov. are proposed; the type strains are SM16T (=CCM 7472T =MTCC9019T) and SM117T (=CCM 7473T =MTCC9020T), respectively.

Microbacterium lindanitolerans sp. nov., isolated from hexachlorocyclohexane-contaminated soil

A Gram-stain-positive, aerobic, rod-shaped, yellow actinobacterium, designated MNA2(T), was isolated from a hexachlorocyclohexane-contaminated soil in North India. Strain MNA2(T) showed 95 % 16S rRNA gene sequence similarity with Microbacterium halotolerans YIM 70130(T). Phylogenetic analysis of 16S rRNA gene sequences showed that strain MNA2(T) belonged to a clade represented by the genus Microbacterium of the family Microbacteriaceae. Strain MNA2(T) contained anteiso-C(15 : 0), anteiso-C(17 : 0), iso-C(16 : 0) and iso-C(15 : 0) as the predominant fatty acids and diphosphatidylglycerol, phosphatidylglycerol and two unknown polar lipids. The menaquinones were MK-12, MK-11, MK-13 and MK-10, in an approximate molar ratio of 45 : 40 : 13 : 3, respectively. The DNA G+C content was 65.3 mol%. The peptidoglycan was of the B type of cross-linkage with ornithine as the diagnostic diamino acid. The results of the phylogenetic, phenotypic and chemotaxonomic analysis indicate that strain MNA2(T) belongs to a previously unrecognized species of the genus Microbacterium, for which the name Microbacterium lindanitolerans sp. nov. is proposed. The type strain is MNA2(T) (=DSM 22422(T) =CCM 7585(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.

Phylogenetic analyses of phylum Actinobacteria based on whole genome sequences

Actinobacteria constitute one of the largest and ancient taxonomic phylum within the domain bacteria and are well known for their secondary metabolites. Considerable variation in the metabolic properties, genome size and GC content of the members of this phylum has been observed. Therefore, the placement of new or existing species based on 16S rRNA gene sometimes becomes problematic due to the low congruence level. In the present study, phylogeny of ninety actinobacterial genomes was reconstructed using single gene and whole genome based data. Where alignment-free phylogenetic method was found to be more robust, the concatenation of 94 proteins improved the resolution which all single gene based phylogenies failed to resolve. The comprehensive analysis of 94 conserved proteins resulted in a total of 42,447 informative sites, which is so far the largest meta-alignment obtained for this phylum. But the ultimate resolved phylogeny was obtained by generating a consensus tree by combining the information from single gene and genome based phylogenies. The present investigation clearly revealed that the consensus approach is a useful tool for phylogenetic inference and the taxonomic affiliations must be based on this approach. The consensus approach suggested that there is a need for taxonomic amendments of the orders Frankiales and Micrococcales.

Flavobacterium ummariense sp. nov., isolated from hexachlorocyclohexane-contaminated soil, and emended description of Flavobacterium ceti Vela et al. 2007.

A Gram-negative, strictly aerobic, yellow bacterial strain, designated DS-12(T), was isolated from hexachlorocyclohexane-contaminated soil in Lucknow, Uttar Pradesh, India. Strain DS-12(T) showed the highest 16S rRNA gene sequence similarity with Flavobacterium ceti 454-2(T) (94.2%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DS-12(T) belonged to the genus Flavobacterium. Strain DS-12(T) produced flexirubin-type pigments. Gliding motility was not observed. The major fatty acids of strain DS-12(T) were iso-C(15:0) (48.0%), summed feature 9 (comprising iso-C(17:1)ω9c and/or C(16:0) 10-methyl; 19.3%), iso-C(17:0) 3-OH (8.5%) and summed feature 3 (comprising one or more of C(16:1)ω7c, C(16:1)ω6c and iso-C(15:0) 2-OH; 7.2%). The only respiratory quinone was menaquinone-6 and the major polyamine was homospermidine. Strain DS-12(T) contained phosphatidyldimethylethanolamine, phosphatidylserine, phosphatidylethanolamine, one unknown phospholipid and one unknown aminolipid. The DNA G+C content was 37.4 mol%. Phylogenetic inference and phenotypic properties indicated that strain DS-12(T) represents a novel species of the genus Flavobacterium, for which the name Flavobacterium ummariense sp. nov. is proposed. The type strain is DS-12(T) (=CCM 7847(T) =MTCC 10766(T)). An emended description of Flavobacterium ceti is also given.

Evolution of mercuric reductase ( merA ) gene: A case of horizontal gene transfer

In the present study the role of horizontal gene transfer events in providing the mercury resistance is depicted. merA gene is key gene in mer operon and has been used for this swtudy. Phylogenetic analysis of aligned merA gene sequences shows broad similarities to the established 16S rRNA gene phylogeny. But there is no separation of bacterial merA gene from archael merA gene which suggests that merA gene in both these groups share considerable sequence homology. However, inconsistencies between merA gene and 16S rRNA gene phylogenetic trees are apparent for some taxa. These discrepancies in the phylogenetic trees for merA gene and 16S rRNA gene have lead to the suggestion that horizontal gene transfer (HGT) is a major contributor for its evolution. The close association among members of different groups in merA gene tree, as supported by high bootstrap values, deviations in GC content and codon usage pattern indicate the possibility that horizontal gene transfer events might have taken place during the evolution of this gene.

Codon usage bias in phylum Actinobacteria: relevance to environmental adaptation and host pathogenicity.

Actinobacteria are Gram-positive bacteria commonly found in soil, freshwater and marine ecosystems. In this investigation, bias in codon usages of ninety actinobacterial genomes was analyzed by estimating different indices of codon bias such as Nc (effective number of codons), SCUO (synonymous codon usage order), RSCU (relative synonymous codon usage), as well as sequence patterns of codon contexts. The results revealed several characteristic features of codon usage in Actinobacteria, as follows: 1) C- or G-ending codons are used frequently in comparison with A- and U ending codons; 2) there is a direct relationship of GC content with use of specific amino acids such as alanine, proline and glycine; 3) there is an inverse relationship between GC content and Nc estimates, 4) there is low SCUO value (<0.5) for most genes; and 5) GCC-GCC, GCC-GGC, GCC-GAG and CUC-GAC are the frequent context sequences among codons. This study highlights the fact that: 1) in Actinobacteria, extreme GC content and codon bias are driven by mutation rather than natural selection; (2) traits like aerobicity are associated with effective natural selection and therefore low GC content and low codon bias, demonstrating the role of both mutational bias and translational selection in shaping the habitat and phenotype of actinobacterial species.

Changes in the bacterial community and lin genes diversity during biostimulation of indigenous bacterial community of hexachlorocyclohexane (HCH) dumpsite soil

In this study hexachlorocyclohexane (HCH) contaminated soil (with HCH level 84 g/kg of soil) from HCH dumpsite (Ummari village, Lucknow, India) was used to demonstrate biostimulation approach for HCH bioremediation. Different nutrients (molasses and ammonium phosphate) were used in different pits having contaminated soil to stimulate the indigenous microbial community. There was a substantial reduction in the total HCH content of the soil in 12 months long experiment. Maximum reduction was seen in the pit that received a combination of molasses and ammonium phosphate. A change in the microbial community concomitant with degradation of HCH was observed. Sphingomonads, which are known degraders of HCH, were found to dominate the experimental pits. Moreover changes in linA and linB gene (primary genes involved in HCH degradation) diversity and number were also seen as revealed by T-RFLP and RT-PCR respectively. The study suggests the prospects of biostimulation in decontaminating soils heavily contaminated with HCH.