Phylogenetic Analyses of Microbial Hydrolytic Dehalogenases Reveal Polyphyletic Origin.

Hydrolytic dehalogenases form an important class of dehalogenases that include haloacid dehalogenase, haloalkane dehalogenase, haloacetate dehalogenase, and atrazine chlorohydrolase. These enzymes are involved in biodegradation of various environmental pollutants and therefore it is important to understand their phylogeny. In the present study, it was found that the enzymes haloalkane and haloacetate dehalogenases share a common ancestry with enzymes such as carboxyesterase, epoxide hydrolase, and lipases, which can be traced to ancestral α/ß hydrolase fold enzyme. Haloacid dehalogenases and atrazine chlorohydrolases have probabaly evolved from ancestral enzymes with phosphatase and deaminases activity, respectively. These findings were supported by the similarities in the secondary structure, key catalytic motifs and placement of catalytic residues. The phylogeny of haloalkane dehalogenases and haloacid dehalogenases differs from 16S rRNA gene phylogeny, suggesting spread through horizontal gene transfer. Hydrolytic dehalogenases are polyphyletic and do not share a common evolutionay history, the functional similarities are due to convergent evolution. The present study also identifies key functional residues, mutating which, can help in generating better enzymes for clean up of the persistent environmental pollutants using enzymatic bioremediation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01043-8.

Bacterial diversity and real-time PCR based assessment of linA and linB gene distribution at hexachlorocyclohexane contaminated sites.

The disposal of hexachlorocyclohexane (HCH) muck has created large number of HCH dumpsites all over the world from where the harmful HCH isomers are leaking into the environment. Bacteria have evolved at such contaminated sites that have the ability to degrade HCH. Degradation of various HCH isomers in bacterial strains is mediated primarily by two genes: linA and linB which encode dehydrochlorinase and haloalkane dehalogenase respectively. In this study we explored one such highly contaminated HCH dumpsite located in Lucknow, Uttar Pradesh, India. To assess the biostimulation potential of the contaminated site, microbial diversity study and real-time PCR based quantification of lin genes was carried out. The soil samples from dumpsite and surrounding areas were found to be highly contaminated with HCH residue levels as high as 1.8 × 10(5) mg kg(-1). The residues were detected in areas upto 13 km from the dumpsite. Sphingomonads, Chromohalobacter, and Marinobacter were the dominant genera present at the dump-site. Role of Sphingomonads in HCH degradation has been well documented. The highest copy numbers of linA and linB genes as determined using real-time PCR were 6.2 × 10(4) and 5.3 × 10(5), respectively, were found in sample from the dump site. The presence of Sphingomonads, linA, and linB genes from HCH contaminated soil indicates the presence of indigenous bacterial communities capable of HCH degradation.

Gut microbiota in pathophysiology, diagnosis, and therapeutics of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a multifactorial disease, which is thought to be an interplay between genetic, environment, microbiota, and immune-mediated factors. Dysbiosis in the gut microbial composition, caused by antibiotics and diet, is closely related to the initiation and progression of IBD. Differences in gut microbiota composition between IBD patients and healthy individuals have been found, with reduced biodiversity of commensal microbes and colonization of opportunistic microbes in IBD patients. Gut microbiota can, therefore, potentially be used for diagnosing and prognosticating IBD, and predicting its treatment response. Currently, there are no curative therapies for IBD. Microbiota-based interventions, including probiotics, prebiotics, synbiotics, and fecal microbiota transplantation, have been recognized as promising therapeutic strategies. Clinical studies and studies done in animal models have provided sufficient evidence that microbiota-based interventions may improve inflammation, the remission rate, and microscopic aspects of IBD. Further studies are required to better understand the mechanisms of action of such interventions. This will help in enhancing their effectiveness and developing personalized therapies. The present review summarizes the relationship between gut microbiota and IBD immunopathogenesis. It also discusses the use of gut microbiota as a noninvasive biomarker and potential therapeutic option.

Edaphobacillus lindanitolerans gen. nov., sp. nov., isolated from hexachlorocyclohexane (HCH) contaminated soil.

A Gram-staining-positive, aerobic, cocco-bacilli-shaped, non-motile, non-spore forming, cream colored strain bacterium (strain MNA4(T) ) was isolated from hexachlorocyclohexane (HCH) contaminated soil. Strain MNA4(T) showed the highest 16S rRNA gene sequence similarity of 97.47% with type species of the newly defined genus Bhargavaea cecembensis. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MNA4(T) belonged to a clade represented by Bhargavaea cecembensis, Bacillus beijingensis and Bacillus ginsengi. DNA-DNA hybridization values of the strain MNA4(T) with close relatives were well below the 70% threshold value recommended for delineation of species. The major fatty acids were anteiso C15:0 , anteiso C17:0 , iso C16:0 and iso C15:0 . The strain was found to contain respiratory quinones MK-8, MK-9 and MK-7 in an approximate molar ratio of 85:7:5. Polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol and two unknown phospholipids. The DNA G + C content was 55.6 mol%. Peptidoglycan type was A4α (L-Lys - L-Ala - D-Asp). Phylogenetic analysis, fatty acids profile, phenotypic properties and chemotaxonomic data of strain MNA4(T) indicated that it represents a novel species of a novel genus for which the name Edaphobacillus lindanitolerans gen. nov., sp. nov., is proposed. The type strain is MNA4(T) (=CCM 7584(T) = DSM 22424(T) ).

Gut Microbiota in Colorectal Cancer: Biological Role and Therapeutic Opportunities.

Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths worldwide. While CRC is thought to be an interplay between genetic and environmental factors, several lines of evidence suggest the involvement of gut microbiota in promoting inflammation and tumor progression. Gut microbiota refer to the ~40 trillion microorganisms that inhabit the human gut. Advances in next-generation sequencing technologies and metagenomics have provided new insights into the gut microbial ecology and have helped in linking gut microbiota to CRC. Many studies carried out in humans and animal models have emphasized the role of certain gut bacteria, such as Fusobacterium nucleatum, enterotoxigenic Bacteroides fragilis, and colibactin-producing Escherichia coli, in the onset and progression of CRC. Metagenomic studies have opened up new avenues for the application of gut microbiota in the diagnosis, prevention, and treatment of CRC. This review article summarizes the role of gut microbiota in CRC development and its use as a biomarker to predict the disease and its potential therapeutic applications.

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.

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

BACKGROUND: Streptococcus 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. METHODS: 16S 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. RESULTS: The 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. CONCLUSIONS: The markers generated exploring 16S rRNA gene sequences provided useful tool that can be further used for identification of different species of the genus Streptococcus.

Genome analyses of 174 strains of Mycobacterium tuberculosis provide insight into the evolution of drug resistance and reveal potential drug targets.

Mycobacterium tuberculosis is a known human pathogen that causes the airborne infectious disease tuberculosis (TB). Every year TB infects millions of people worldwide. The emergence of multi-drug resistant (MDR), extensively drug resistant (XDR) and totally drug resistant (TDR) M. tuberculosis strains against the first- and second-line anti-TB drugs has created an urgent need for the development and implementation of new drug strategies. In this study, the complete genomes of 174 strains of M. tuberculosis are analysed to understand the evolution of molecular drug target (MDT) genes. Phylogenomic placements of M. tuberculosis strains depicted close association and temporal clustering. Selection pressure analysis by deducing the ratio of non-synonymous to synonymous substitution rates (dN/dS) in 51 MDT genes of the 174 M. tuberculosis strains led to categorizing these genes into diversifying (D, dN/dS>0.70), moderately diversifying (MD, dN/dS=0.35-0.70) and stabilized (S, dN/dS<0.35) genes. The genes rpsL, gidB, pncA and ahpC were identified as diversifying, and Rv0488, kasA, ndh, ethR, ethA, embR and ddn were identified as stabilized genes. Furthermore, sequence similarity networks were drawn that supported these divisions. In the multiple sequence alignments of diversifying and stabilized proteins, previously reported resistance mutations were checked to predict sensitive and resistant strains of M. tuberculosis. Finally, to delineate the potential of stabilized or least diversified genes/proteins as anti-TB drug targets, protein-protein interactions of MDT proteins with human proteins were analysed. We predict that kasA (dN/dS=0.29), a stabilized gene that encodes the most host-interacting protein, KasA, should serve as a potential drug target for the treatment of TB.

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)).

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.

Large-Scale Sequence Comparison.

There are millions of sequences deposited in genomic databases, and it is an important task to categorize them according to their structural and functional roles. Sequence comparison is a prerequisite for proper categorization of both DNA and protein sequences, and helps in assigning a putative or hypothetical structure and function to a given sequence. There are various methods available for comparing sequences, alignment being first and foremost for sequences with a small number of base pairs as well as for large-scale genome comparison. Various tools are available for performing pairwise large sequence comparison. The best known tools either perform global alignment or generate local alignments between the two sequences. In this chapter we first provide basic information regarding sequence comparison. This is followed by the description of the PAM and BLOSUM matrices that form the basis of sequence comparison. We also give a practical overview of currently available methods such as BLAST and FASTA, followed by a description and overview of tools available for genome comparison including LAGAN, MumMER, BLASTZ, and AVID.

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.

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.

Understanding alternative fluxes/effluxes through comparative metabolic pathway analysis of phylum actinobacteria using a simplified approach.

Actinobacteria are known for their diverse metabolism and physiology. Some are dreadful human pathogens whereas some constitute the natural flora for human gut. Therefore, the understanding of metabolic pathways is a key feature for targeting the pathogenic bacteria without disturbing the symbiotic ones. A big challenge faced today is multiple drug resistance by Mycobacterium and other pathogens that utilize alternative fluxes/effluxes. With the availability of genome sequence, it is now feasible to conduct the comparative in silico analysis. Here we present a simplified approach to compare metabolic pathways so that the species specific enzyme may be traced and engineered for future therapeutics. The analyses of four key carbohydrate metabolic pathways, i.e., glycolysis, pyruvate metabolism, tri carboxylic acid cycle and pentose phosphate pathway suggest the presence of alternative fluxes. It was found that the upper pathway of glycolysis was highly variable in the actinobacterial genomes whereas lower glycolytic pathway was highly conserved. Likewise, pentose phosphate pathway was well conserved in contradiction to TCA cycle, which was found to be incomplete in majority of actinobacteria. The clustering based on presence and absence of genes of these metabolic pathways clearly revealed that members of different genera shared identical pathways and, therefore, provided an easy method to identify the metabolic similarities/differences between pathogenic and symbiotic organisms. The analyses could identify isoenzymes and some key enzymes that were found to be missing in some pathogenic actinobacteria. The present work defines a simple approach to explore the effluxes in four metabolic pathways within the phylum actinobacteria. The analysis clearly reflects that actinobacteria exhibit diverse routes for metabolizing substrates. The pathway comparison can help in finding the enzymes that can be used as drug targets for pathogens without effecting symbiotic organisms within the same host. This may help to prevail over the multiple drug resistance, for designing broad spectrum drugs, in food industries and other clinical research areas.

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.

Evaluation of hexachlorocyclohexane contamination from the last lindane production plant operating in India.

PURPOSE: α-Hexachlorocyclohexane (HCH), ß-HCH, and lindane (γ-HCH) were listed as persistent organic pollutants by the Stockholm Convention in 2009 and hence must be phased out and their wastes/stockpiles eliminated. At the last operating lindane manufacturing unit, we conducted a preliminary evaluation of HCH contamination levels in soil and water samples collected around the production area and the vicinity of a major dumpsite to inform the design of processes for an appropriate implementation of the Convention. METHODS: Soil and water samples on and around the production site and a major waste dumpsite were measured for HCH levels. RESULTS: All soil samples taken at the lindane production facility and dumpsite and in their vicinity were contaminated with an isomer pattern characteristic of HCH production waste. At the dumpsite surface samples contained up to 450 g kg(-1) Σ HCH suggesting that the waste HCH isomers were simply dumped at this location. Ground water in the vicinity and river water was found to be contaminated with 0.2 to 0.4 mg l(-1) of HCH waste isomers. The total quantity of deposited HCH wastes from the lindane production unit was estimated at between 36,000 and 54,000 t. CONCLUSIONS: The contamination levels in ground and river water suggest significant run-off from the dumped HCH wastes and contamination of drinking water resources. The extent of dumping urgently needs to be assessed regarding the risks to human and ecosystem health. A plan for securing the waste isomers needs to be developed and implemented together with a plan for their final elimination. As part of the assessment, any polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDF) generated during HCH recycling operations need to be monitored.

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 is key gene in mer operon and has been used for this study. Phylogenetic analysis of aligned merA sequences shows broad similarities to the established 16S rRNA phylogeny. But there is no separation of bacterial merA from archael merA which suggests that merA gene in both these groups share considerable sequence homology. However, inconsistencies between merA 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.

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.