Amphibians are rapidly declining worldwide, but recent studies have shown that their diversity may be heavily underestimated, and many new species have been recently reported from biodiversity hotspots. For successful conservation and management strategies to be implemented within such hotspots, a better understanding of the species diversity and their evolutionary relationships is required. We used three mitochondrial (16S, 12S and CO1) and two nuclear (rag1 and rhodopsin) gene fragments to investigate the genetic diversity within the endemic Indirana genus from the Western Ghats biodiversity hotspot. The species diversity within Indirana was found to be much higher than previously anticipated. Instead of the expected six species within this region, our analyses identified 11 clades with high (4.2-17.1%) sequence divergence. Each of these clades is likely to represent a distinct species. Particularly pronounced polyphyly was found within Indirana beddomii, which consisted of four highly supported monophyletic clades with high genetic divergence. Similarly, Indirana diplosticta was divided into two highly divergent monophyletic clades. We also report a new candidate species within the genus from Vellarimala in Kerala, which we believe is yet to be described. Our results suggest the existence of multiple unrecognized cryptic lineages within Indirana, all of which are likely to have more narrow distribution ranges and lower abundances than the taxonomic units into which they are currently assigned. Hence, the International Union for Conservation of Nature (IUCN) Red List statuses of Indirana frogs are likely to be in need of substantial revision, and detailed genetic studies across the Western Ghats might uncover additional new candidate species from this poorly studied endemic genus. Together with several earlier amphibian studies describing cryptic species from tropics, our results highlight the importance of proper species identification efforts before it is possible to reliably determine the IUCN conservation status of tropical amphibians described on the basis of morphological criteria. bs_bs_banner Animal Conservation. Print ISSN 1367-9430 Animal Conservation 15 (2012) 489-498

The porinACj is an α-helical porin that spans the mycolic acid outer membrane of Gram-positive mycolate, Corynebacterium jeikeium. Here, we report that a 40-amino acid, synthetic peptide, pPorA corresponding to porin PorACj, inserts into the lipid bilayers and forms well-defined pores. By electrical recordings, we measured the single-channel properties that revealed the autonomous assembly of large conductance ion-selective synthetic pores. Further, we characterized the functional properties by blocking the peptide pores by cyclodextrins of different charge and symmetry. We deduced the subunit stoichiometry and putative structure of the pore by site-specific chemical modification in single-channel electrical recordings and gel electrophoresis. On the basis of these findings, we suggest that this is a large functional uniform transmembrane pore built entirely from short synthetic α-helical peptides. Accordingly, we propose a model demonstrating structural assembly of large α-helix-based peptide pores for understanding the action of antimicrobial peptides and for the design of pores with applications in biotechnology.

Membrane protein pores have demonstrated applications in nanobiotechnology and single-molecule chemistry for effective detection of biomolecules. Here, we define the molecular basis of carbohydrate polymers translocation through a substratespecific bacterial nanopore, CymA, which has a 15-residue N terminus segment inside the pore, restricting its diameter. Using single-channel recordings, we determined the kinetics of cationic cyclic oligosaccharide binding and elucidated the translocation mechanism across the pore in real-time. The cationic cyclic hexasaccharide binds to the densely packed negatively charged residues at the extracellular side of the pore with high affinity, facilitating its entry into the pore driven by the applied voltage. Further, the dissociation rate constant increased with increasing voltages, indicating unidirectional translocation toward the pore exit. Specifically, a larger cationic cyclic octasaccharide rapidly blocked the pore more effectively, resulting in the complete closure of the pore with increasing voltage, implying only strong binding. Further, we show that uncharged oligosaccharides exclusively bind to the extracellular side of the pore and the electroosmotic flow most likely drives their translocation. We propose that CymA favors selective translocation of cyclic hexasaccharide and linear maltooligosaccharides due to an asymmetrical charge pattern and the N terminus that regulates the substrate transport. We suggest that this substrate-specific nanopore with sophisticated geometry will be useful for complex biopolymer characterization.

All life forms are equipped with rapidly acting, evolutionally conserved components of an innate immune defense system that consists of a group of unique and diverse molecules known as host defense peptides (HDPs). A Systematic and Modular Modification and Deletion (SMMD) approach was followed to analyse the structural requirement of B1CTcu5, a brevinin antibacterial peptide amide identified from the skin secretion of frog Clinotarsus curtipes, India, to show antibacterial activity and to explore the active core region. Seventeen SMMD-B1CTcu5 analogs were designed and synthesised by C and N-terminal amino acid substitution or deletion. Enhancement in cationicity by N-terminal Lys/Arg substitution or hydrophobicity by Trp substitution produced no drastic change in bactericidal nature against selected bacterial strains except S. aureus. But the sequential removal of N-terminal amino acids had a negative effect on bactericidal potency. Analog B1CTcu5-LIAG obtained by the removal of four N-terminal amino acids displayed bactericidal effect comparable to, or in excess of, the parent peptide with reduced hemolytic character. Its higher activity was well correlated with the improved inner membrane permeabilisation capacity. This region may act as the active core of B1CTcu5. Presence of C-terminal disulphide bond was not a necessary condition to display antibacterial activity but helped to promote hemolytic nature. Removal of the C-terminal rana box region drastically reduced antibacterial and hemolytic activity of the peptide, showing that this region is important for membrane targeting. The bactericidal potency of the D-peptide (DB1CTcu5) helped to rule out the stereospecific interaction with the bacterial membrane. Our data suggests that both the C and N-terminal regions are necessary for bactericidal activity, even though the active core region is located near the N-terminal of B1CTcu5. A judicious modification at the N-terminal region may produce a short SMMD analog with enhanced bactericidal activity and low toxicity against eukaryotic cells.

Chrysomycin A isolated from Streptomyces sp. OA161 is bactericidal to Mycobacterium tuberculosis, methicillin resistant Staphylococcus aureus and vancomycin resistant Enterococcus faecalis.