Sadhana Lal

Phylogeny in aid of the present and novel microbial lineages: diversity in Bacillus.

Bacillus represents microbes of high economic, medical and biodefense importance. Bacillus strain identification based on 16S rRNA sequence analyses is invariably limited to species level. Secondly, certain discrepancies exist in the segregation of Bacillus subtilis strains. In the RDP/NCBI databases, out of a total of 2611 individual 16S rDNA sequences belonging to the 175 different species of the genus Bacillus, only 1586 have been identified up to species level. 16S rRNA sequences of Bacillus anthracis (153 strains), B. cereus (211 strains), B. thuringiensis (108 strains), B. subtilis (271 strains), B. licheniformis (131 strains), B. pumilus (83 strains), B. megaterium (47 strains), B. sphaericus (42 strains), B. clausii (39 strains) and B. halodurans (36 strains) were considered for generating species-specific framework and probes as tools for their rapid identification. Phylogenetic segregation of 1121, 16S rDNA sequences of 10 different Bacillus species in to 89 clusters enabled us to develop a phylogenetic frame work of 34 representative sequences. Using this phylogenetic framework, 305 out of 1025, 16S rDNA sequences presently classified as Bacillus sp. could be identified up to species level. This identification was supported by 20 to 30 nucleotides long signature sequences and in silico restriction enzyme analysis specific to the 10 Bacillus species. This integrated approach resulted in identifying around 30% of Bacillus sp. up to species level and revealed that B. subtilis strains can be segregated into two phylogenetically distinct groups, such that one of them may be renamed.

Mining genomic databases to identify novel hydrogen producers

The realization that fossil fuel reserves are limited and their adverse effect on the environment has forced us to look into alternative sources of energy. Hydrogen is a strong contender as a future fuel. Biological hydrogen production ranges from 0.37 to 3.3 moles H(2) per mole of glucose and, considering the high theoretical values of production (4.0 moles H(2) per mole of glucose), it is worth exploring approaches to increase hydrogen yields. Screening the untapped microbial population is a promising possibility. Sequence analysis and pathway alignment of hydrogen metabolism in complete and incomplete genomes has led to the identification of potential hydrogen producers.

In Search of Drug Targets for Mycobacterium tuberculosis.

Mycobacterium tuberculosis is the etiological agent for tuberculosis in humans. The studies related to survival of this pathogen in the human host and development of drugs against reveal that the organism uses a complex physiology to adapt to the host environment. Many studies were targeted to key enzymes that allow this pathogen to either survive or remain latent within the host. Most of the models, which address the survival of pathogen, have evaluated limited dissolved oxygen and prevailing stress conditions. Hence, the truncated citric acid cycle, with the glyoxylate shunt was suggested as an option for survival of the pathogen and pathogenesis. We propose that the precursors to support this pathway could also be generated via enzymatic conversion involving poly-beta-hydroxybutyrate (PHB). We have used available genome sequence data and analyzed for the possible enzymatic conversions that can generate glyoxylate, acetyl CoA, and other enolases that can also be useful for various fatty acid transformations. The enzymes for accumulation and further hydrolysis of PHB were examined in sequence data analysis. The target enzymes were searched for in the genome using identified conserved domains. Using M. tuberculosis H37Rv as a model bacterium a supportive pathway has been envisaged and integrated with glyoxylate cycle to provide a complete option to pathogen for sustainable consumption of available carbon source(s). The study proposes that the enzymes of PHB synthesis and hydrolysis are possible targets for drug design, and that this should be considered when evaluating isocitrate lyase and malate synthase as targets.

Combing databases reveals potential antibiotic producers

The large-scale, persistent use of antibiotics has provoked microbes to evolve mechanisms to evade them. Pharmaceutical companies have found this to be counterproductive to their business economics. To maintain company interest to invest in this sector, innovative alternatives are needed. The availability of metabolic and genomic databases has opened up an avenue for such discoveries. Using these databases, potential producers of penicillin and cephalosporin have been traced. In addition, organisms that can be transformed from their present ‘non’-producer status to antibacterial producers by supplementing their missing gene(s) by recombinant DNA technology have been revealed.

Phylogeny vs genome reshuffling: horizontal gene transfer.

The evolutionary events in organisms can be tracked to the transfer of genetic material. The inheritance of genetic material among closely related organisms is a slow evolutionary process. On the other hand, the movement of genes among distantly related species can account for rapid evolution. The later process has been quite evident in the appearance of antibiotic resistance genes among human and animal pathogens. Phylogenetic trees based on such genes and those involved in metabolic activities reflect the incongruencies in comparison to the 16S rDNA gene, generally used for taxonomic relationships. Such discrepancies in gene inheritance have been termed as horizontal gene transfer (HGT) events. In the post-genomic era, the explosion of known sequences through large-scale sequencing projects has unraveled the weakness of traditional 16S rDNA gene tree based evolutionary model. Various methods to scrutinize HGT events include atypical composition, abnormal sequence similarity, anomalous phylogenetic distribution, unusual phyletic patterns, etc. Since HGT generates greater genetic diversity, it is likely to increase resource use and ecosystem resilience.

Investigating the Phylogeny of Hydrogen Metabolism by Comparative Genomics: Horizontal Gene Transfer

The phylogenetic analysis based on molecular characteristics indicates that lithotrophic metabolism was followed by phototrophy. Hydrogen (H2) metabolism is a signature of such environments. This property is prominent among organisms found in geothermal conditions and in deep aquifers. H2 is generated readily by abiotic mechanisms where the terminal electron acceptor is likely to be the limiting factor. In the post-fossil fuel era, H2 has in fact emerged as a strong contender for future fuel. It is thus important to understand the molecular mechanisms which lead to H2 production and associated biological systems. These can help to comprehend issues such as sustainability, environmental emissions and energy security. Comparative genomic analysis reveals events of horizontal transfer of genes of H2 metabolism among taxonomically diverse organisms. This offers an opportunity to identify those genomes which can be tailored for transforming presently ‘non’-H2 producers into producers. This also suggests that naturally occurring events can be mimicked to provide future fuel H2.

In Silico Reconstitution of Novel Routes for Microbial Plastic

Polyhydroxyalkanoate (PHA) biosynthesis is a phenotype expressed under environmental stress. PHA synthesis relies on important intermediates tricarboxylic acid (TCA) cycle and fatty acid metabolism especially acetyl CoA. Acetyl CoA is in demand by a host of metabolisms and is not easily available under normal “unstressed” conditions. This work was done to find out alternative routes for copolymer polyhydroxybutyrate (PHB), which may exist in nature but not explored so far. Using metabolic pathway database (KEGG), we have devised in silico novel metabolic pathways, which are independent of acetyl CoA, i.e., may operate under different conditions. Moreover, organisms possessing enzymes for these simulated PHB production routes have also been identified. In this comparative analysis, we found some potential organisms such as Brucella, Deinococcus, Homo sapiens, Mus musculus, Rattus, Thermoanaerobacter, and Xanthomonas which have the necessary genetic machinery for one or more of these novel pathways. Incidentally, they have not been reported previously as PHB producers. On the other hand, these novel routes have also been observed in certain known PHB producers such as Clostridium, Ralstonia, Pseudomonas, and Mesorhizobium. The advantage of these novel routes in known PHB producers is expanding the source of starting material for PHB synthesis.