Reclassification of Phycicola gilvus (Lee et al. 2008) and Leifsonia pindariensis (Reddy et al. 2008) as Microterricola gilva comb. nov. and Microterricola pindariensis comb. nov. and emended description of the genus Microterricola.

The taxonomic positions of Microterricola viridarii JCM 15926T, Phycicola gilvus DSM 18319T and Leifsonia pindariensis JCM 15132T were re-examined. Phylogenetic analysis and 16S rRNA gene sequence similarities revealed that all three strains are closely related with each other and form a monophyletic cluster with high sequence similarity (99.2 -99.9 %). A dendrogram constructed based on the protein spectra generated by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy also displayed close clustering of these three strains. The fatty acid profiles of three strains were very similar to each other and contained branched fatty acids (anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0) as the predominant cellular fatty acids. The polar lipid profiles of the three stains were similar and consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine as major polar lipids and an unknown lipid. Comparisons of morphological, chemotaxonomic and physiological data of Microterricola viridarii JCM 15926T, Leifsonia pindariensis JCM 15132T and Phycicola gilvus DSM 18319T are in agreement with the features of a common genus. DNA-DNA hybridization data generated during this study showed less than 70 % reassociation value with each other indicating that they are different at species level. Based on the present study, we conclude that Phycicola gilvus DSM 18319T and Leifsonia pindariensis JCM 15132T should be reclassified under the genus Microterricola, since this genus has the nomenclatural priority, and reclassified as Microterricolagilva comb. nov. (type strain SSWW-21T=DSM 18319T=KCTC 19185T=JCM 30550T) and Microterricolapindariensis comb. nov. (type strain PON10T=LMG 24222T=JCM 15132T=MTCC9128T). An emended description of the genus Microterricola is also presented.

Technicalities and Glitches of Terminal Restriction Fragment Length Polymorphism (T-RFLP).

Terminal restriction fragment length polymorphism (T-RFLP) is a rapid, robust, inexpensive and simple tool for microbial community profiling. Methods used for DNA extraction, PCR amplification and digestion of amplified products have a considerable impact on the results of T-RFLP. Pitfalls of the method skew the similarity analysis and compromise its high throughput ability. Despite a high throughput method of data generation, data analysis is still in its infancy and needs more attention. Current article highlights the limitations of the methods used for data generation and analysis. It also provides an overview of the recent methodological developments in T-RFLP which will assist the readers in obtaining real and authentic profiles of the microbial communities under consideration while eluding the inherent biases and technical difficulties.

Biodegradation of tributyl phosphate by novel bacteria isolated from enrichment cultures.

Tributyl phosphate (TBP) is an organophosphorous compound, used extensively (3000-5000 tonnes/annum) as a solvent for nuclear fuel processing and as a base stock in the formulation of fire-resistant aircraft hydraulic fluids and other applications. Because of its wide applications and relative stability in the natural environment TBP poses the problem of pollution and health hazards. In the present study, fifteen potent bacterial strains capable of using tributyl phosphate (TBP) as sole carbon and phosphorus source were isolated from enrichment cultures. These isolates were identified on the basis of biochemical and morphological characteristics and 16S rRNA gene sequence analysis. Phylogenetic analysis of 16S rRNA gene sequences revealed that two isolates belonged to class Bacilli and thirteen to ß and γ-Proteobacteria. All these isolates were found to be members of genera Alcaligenes, Providencia, Delftia, Ralstonia, and Bacillus. These isolates were able to tolerate and degrade up to 5 mM TBP, the highest concentration reported to date. The GC-MS method was developed to monitor TBP degradation. Two strains, Providencia sp. BGW4 and Delftia sp. BGW1 showed respectively, 61.0 ± 2.8% and 57.0 ± 2.0% TBP degradation within 4 days. The degradation rate constants, calculated by first order kinetic model were between 0.0024 and 0.0099 h(-1). These bacterial strains are novel for TBP degradation and could be used as an important bioresource for efficient decontamination of TBP polluted waste streams.

Generation, annotation, and analysis of ESTs from midgut tissue of adult female Anopheles stephensi mosquitoes.

BACKGROUND: Malaria is a tropical disease caused by protozoan parasite, Plasmodium, which is transmitted to humans by various species of female anopheline mosquitoes. Anopheles stephensi is one such major malaria vector in urban parts of the Indian subcontinent. Unlike Anopheles gambiae, an African malaria vector, transcriptome of A. stephensi midgut tissue is less explored. We have therefore carried out generation, annotation, and analysis of expressed sequence tags from sugar-fed and Plasmodium yoelii infected blood-fed (post 24 h) adult female A. stephensi midgut tissue. RESULTS: We obtained 7061 and 8306 ESTs from the sugar-fed and P. yoelii infected mosquito midgut tissue libraries, respectively. ESTs from the combined dataset formed 1319 contigs and 2627 singlets, totaling to 3946 unique transcripts. Putative functions were assigned to 1615 (40.9%) transcripts using BLASTX against UniProtKB database. Amongst unannotated transcripts, we identified 1513 putative novel transcripts and 818 potential untranslated regions (UTRs). Statistical comparison of annotated and unannotated ESTs from the two libraries identified 119 differentially regulated genes. Out of 3946 unique transcripts, only 1387 transcripts were mapped on the A. gambiae genome. These also included 189 novel transcripts, which were mapped to the unannotated regions of the genome. The EST data is available as ESTDB at http://mycompdb.bioinfo-portal.cdac.in/cgi-bin/est/index.cgi. CONCLUSION: 3946 unique transcripts were successfully identified from the adult female A. stephensi midgut tissue. These data can be used for microarray development for better understanding of vector-parasite relationship and to study differences or similarities with other malaria vectors. Mapping of putative novel transcripts from A. stephensi on the A. gambiae genome proved fruitful in identification and annotation of several genes. Failure of some novel transcripts to map on the A. gambiae genome indicates existence of substantial genomic dissimilarities between these two potent malaria vectors.

Enrichment and identification of cellulolytic bacteria from the gastrointestinal tract of Giant African snail, Achatina fulica.

The cellulolytic bacterial community structure in gastrointestinal (GI) tract of Achatina fulica was studied using culture-independent and -dependent methods by enrichment in carboxymethyl cellulose (CMC). Culture-dependent method indicated that GI tract of snail was dominated by Enterobacteriaceae members. When tested for cellulase activities, all isolates obtained by culture-dependent method showed both or either of CMCase or avicelase activity. Isolate identified as Citrobacter freundii showed highest CMCase and medium avicelase activity. Sequencing of clones from the 16S rRNA gene clone library identified ten operational taxonomic units (OTUs), which were affiliated to Enterobacteriaceae of phylum Gammaproteobacteria. Of these ten OTUs, eight OTUs closely matched with Enterobacter and Klebsiella genera. The most abundant OTU allied to Klebsiella oxytoca accounted for 70 % of the total sequences. The members of Klebsiella and Enterobacter were observed by both methods indicating their dominance among the cellulolytic bacterial community in the GI tract of the snail.

Genome sequencing analysis reveals virulence-related gene content of Ochrobactrum intermedium strain 229E, a urease-positive strain isolated from the human gastric niche.

We report draft genome sequence of Ochrobactrum intermedium strain 229E concurrent with Helicobacter pylori in urease positive gastric biopsy of non-ulcer dyspeptic individual from Southern part of India. Since the role of Ochrobactrum in human gastric environment is poorly understood, comprehensive pathological, microbiological, and genome level understanding are necessary to evaluate its association with H. pylori in the gastric niche. Comparative analysis of O. intermedium 299E strain revealed functional similarities with virulence related gene clusters present in H. pylori genomes, which probably might aid in its ability to persist in the human gastric mucosa. However, H.pylori specific vacuolating cytotoxin (vacA) involved in vacuolization, cytotoxicity, and T-cell inhibition was absent in the O. intermedium 229E genome. Taken together, O. intermedium 229E shared numerous features like secretion system, urease, and flagella with H.pylori genome sequence that might aid concurrence in the gastric niche.

Bacterial diversity in different regions of gastrointestinal tract of Giant African snail (Achatina fulica).

The gastrointestinal (GI) tract of invasive land snail Achatina fulica is known to harbor metabolically active bacterial communities. In this study, we assessed the bacterial diversity in the different regions of GI tract of Giant African snail, A. fulica by culture-independent and culture-dependent methods. Five 16S rRNA gene libraries from different regions of GI tract of active snails indicated that sequences affiliated to phylum γ-Proteobacteria dominated the esophagus, crop, intestine, and rectum libraries, whereas sequences affiliated to Tenericutes dominated the stomach library. On phylogenetic analysis, 30, 27, 9, 27, and 25 operational taxonomic units (OTUs) from esophagus, crop, stomach, intestine, and rectum libraries were identified, respectively. Estimations of the total bacterial diversity covered along with environmental cluster analysis showed highest bacterial diversity in the esophagus and lowest in the stomach. Thirty-three distinct bacterial isolates were obtained, which belonged to 12 genera of two major bacterial phyla namely γ-Proteobacteria and Firmicutes. Among these, Lactococcus lactis and Kurthia gibsonii were the dominant bacteria present in all GI tract regions. Quantitative real-time polymerase chain reaction (qPCR) analysis indicated significant differences in bacterial load in different GI tract regions of active and estivating snails. The difference in the bacterial load between the intestines of active and estivating snail was maximum. Principal component analysis (PCA) of terminal restriction fragment length polymorphism suggested that bacterial community structure changes only in intestine when snail enters estivation state.