BackgroundBacterial taxonomy and phylogeny based on rrs (16S rDNA) sequencing is being vigorously pursued. In fact, it has been stated that novel biological findings are driven by comparison and integration of massive data sets. In spite of a large reservoir of rrs sequencing data of 1,237,963 entries, this analysis invariably needs supplementation with other genes. The need is to divide the genetic variability within a taxa or genus at their rrs phylogenetic boundaries and to discover those fundamental features, which will enable the bacteria to naturally fall within them. Within the large bacterial community, Clostridium represents a large genus of around 110 species of significant biotechnological and medical importance. Certain Clostridium strains produce some of the deadliest toxins, which cause heavy economic losses. We have targeted this genus because of its high genetic diversity, which does not allow accurate typing with the available molecular methods.ResultsSeven hundred sixty five rrs sequences (> 1200 nucleotides, nts) belonging to 110 Clostridium species were analyzed. On the basis of 404 rrs sequences belonging to 15 Clostridium species, we have developed species specific: (i) phylogenetic framework, (ii) signatures (30 nts) and (iii) in silico restriction enzyme (14 Type II REs) digestion patterns. These tools allowed: (i) species level identification of 95 Clostridium sp. which are presently classified up to genus level, (ii) identification of 84 novel Clostridium spp. and (iii) potential reduction in the number of Clostridium species represented by small populations.ConclusionsThis integrated approach is quite sensitive and can be easily extended as a molecular tool for diagnostic and taxonomic identification of any microbe of importance to food industries and health services. Since rapid and correct identification allows quicker diagnosis and consequently treatment as well, it is likely to lead to reduction in economic losses and mortality rates.
To explore bacterial diversity for elucidating genetic variability in acylhomoserine lactone (AHL) lactonase structure, we screened 800 bacterial strains. It revealed the presence of a quorum quenching (QQ) AHL-lactonase gene (aiiA) in 42 strains. These 42 strains were identified using rrs (16S rDNA) sequencing as Bacillus strains, predominantly B. cereus. An in silico restriction endonuclease (RE) digestion of 22 AHL lactonase gene (aiiA) sequences (from NCBI database) belonging to 9 different genera, along with 42 aiiA gene sequences from different Bacillus spp. (isolated here) with 14 type II REs, revealed distinct patterns of fragments (nucleotide length and order) with four REs; AluI, DpnII, RsaI, and Tru9I. Our study reflects on the biodiversity of aiiA among Bacillus species. Bacillus sp. strain MBG11 with polymorphism (115Alanine > Valine) may confer increased stability to AHL lactonase, and can be a potential candidate for heterologous expression and mass production. Microbes with ability to produce AHL-lactonases degrade quorum sensing signals such as AHL by opening of the lactone ring. The naturally occurring diversity of QQ molecules provides opportunities to use them for preventing bacterial infections, spoilage of food, and bioremediation.
Pseudomonas is a highly versatile bacterium at the species level with great ecological significance. These genetically and metabolically diverse species have undergone repeated taxonomic revisions. We propose a strategy to identify Pseudomonas up to species level, based on the unique features of their 16S rDNA (rrs) gene sequence, such as the frame work of sequences, sequence motifs and restriction endonuclease (RE) digestion patterns. A species specific phylogenetic framework composed of 31 different rrs sequences, allowed us to segregate 1,367 out of 2,985 rrs sequences of this genus, which have been classified at present only up to genus (Pseudomonas) level, as follows: P. aeruginosa (219 sequences), P. fluorescens (463 sequences), P. putida (347 sequences), P. stutzeri (197 sequences), and P. syringae (141 sequences). These segregations were validated by unique 30-50 nucleotide long motifs and RE digestion patterns in their rrs. A single gene thus provides multiple makers for identification and surveillance of Pseudomonas.
Microbial classification is based largely on the 16S rRNA (rrs) gene sequence, which is conserved throughout the prokaryotic domain. The Ribosomal Database Project (RDP) has become a reference point for almost all practical purposes. The use of this gene is limited by the fact that it can be used to identify only to the extent to what has been known and is available in the RDP. In order to identify an organism whose rrs is not present in the RDP database, we need to generate novel markers to place the unknown on the evolutionary map. Here, sequenced genomes of 27 Clostridium strains belonging to 9 species have been used to identify two sets of genes: (1) common to most of the species, and (2) unique to a species. Combinations of genes (recN, dnaJ, secA, mutS, and/or grpE) and their unique restriction endonuclease digestion (AluI, BfaI and/or Tru9I) patterns have been established to rapidly identify Clostridium species. This strategy for identifying novel markers can be extended to all other organisms and diagnostic applications.
Diversity analysis of Clostridium botulinum strains is complicated by high microheterogeneity caused by the presence of 9-22 copies of rrs (16S rRNA gene). The need is to mine genetic markers to identify very closely related strains. Multiple alignments of the nucleotide sequences of the 212 rrs of 13 C. botulinum strains revealed intra- and inter-genomic heterogeneity. Low intragenomic heterogeneity in rrs was evident in strains 230613, Alaska E43, Okra, Eklund 17B, Langeland, 657, Kyoto, BKT015925, and Loch Maree. The most heterogenous rrs sequences were those of C. botulinum strains ATCC 19397, Hall, H04402065, and ATCC 3502. In silico restriction mapping of these rrs sequences was observable with 137 type II Restriction endonucleases (REs). Nucleotide changes (NC) at these RE sites resulted in appearance of distinct and additional sites, and loss in certain others. De novo appearances of RE sites due to NC were recorded at different positions in rrs gene. A nucleotide transition A>G in rrs of C. botulinum Loch Maree and 657 resulted in the generation of 4 and 10 distinct RE sites, respectively. Transitions A>G, G>A, and T>C led to the loss of RE sites. A perusal of the entire NC and in silico RE mapping of rrs of C. botulinum strains provided insights into their evolution. Segregation of strains on the basis of RE digestion patterns of rrs was validated by the cladistic analysis involving six house keeping genes: dnaN, gyrB, metG, prfA, pyrG, and Rho.
Esophageal squamous cell carcinoma (ESCC) has a complex, multifactorial etiology in which environmental, geographical, and genetic factors play major roles. It is the second most common cancer among men and the fourth most common among women in India, with a particularly high prevalence in Northeast India. In this study, an integrative in silico [DAVID, NCG5.0, Oncomine, Cancer Cell Line Encyclopedia, and The Cancer Genome Atlas (TCGA)] approach was used to identify the potential biomarkers by using the available three genomic datasets on ESCC from Northeast India followed by its in vitro functional validation. Fibroblast Growth Factor 12 (FGF12) gene was overexpressed in ESCC. The upregulation of FGF12 was also observed on ESCC of TCGA OncoPrint portal, whereas very low expression of FGF12 gene was mapped in normal esophageal tissue on the GTEx database. Silencing of FGF12 showed significant inhibition in activity of tumor cell proliferation, colony formation, and cell migration. The upregulation of FGF12 showed significantly reduced survival in ESCC patients. The protein interaction analysis of FGF12 found the binding with MAPK8IP2 and MAPK13. High expression of FGF12 along with MAPK8IP2, and MAPK13 proteins correlate with poor survival in ESCC patients. Tissue microarray also showed expression of these proteins in patients with ESCC. These results indicate that FGF12 has a potential role in ESCC and suggest that cancer genomic datasets with application of in silico approaches are instrumental for biomarker discovery research broadly and specifically, for the identification of FGF12 as a putative biomarker in ESCC.
Based on our article, we believe that, soon, the scientific community will come up with certain azole which will work against cancer at large rather than a specific type of cancer.
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