Isolation and characterization of a new osmotolerant, non-virulent Klebsiella pneumoniae strain SAP for biosynthesis of succinic acid

In the present study, isolation of anaerobic bacteria from 24 different eco-niches was carried out. A total number of 300 bacterial isolates, including 230 obligate and 70 facultative anaerobes were obtained using anaerobic techniques. All the isolates were initially screened for succinic acid production by Fluorescein test and TLC method. During screening, 10 isolates found to produce succinic acid were further examined by HPLC and then finally confirmed for succinic acid by LC-MS analysis. Amongst 10 isolates, isolate SAP, a facultative anaerobe isolated from buffalo rumen fluid, showed maximum yield of 2.1 g/l of succinic acid from 10 g of glucose in 24 hr under anaerobic condition. This isolate was identified as Klebsiella pneumoniae strain SAP by 16S rDNA sequence and signature sequence analysis. Mouse lethality test for the strain SAP showed LD50 value of 3.3 x 10(8) CFU/ml, which shows non-virulent nature of the strain. This strain may become a candidate strain for succinic acid production because of its osmotolerant nature and higher succinate:acetate ratio.     

Selection for early and late adult emergence alters the rate of pre-adult development in Drosophila melanogaster

Background  

Circadian clocks have been implicated in the regulation of pre-adult development of fruit flies Drosophila melanogaster. It is believed that faster clocks speed up development and slower clocks slow it down. We established three sets of D. melanogaster populations (early, control and late). The early and late populations were raised by selecting for flies that emerged either in the morning or in the evening under 12:12 hr light/dark (LD) cycles. After 75 generations of selection, the time course and waveform of the adult emergence and activity rhythms of the early and the late populations diverged from each other as well as from the controls. In this paper, we report the consequence of this selection on the rate of pre-adult development.     

Heat shock protein 90 from neglected protozoan parasites

Significant advances have been made in our understanding of heat shock protein 90 (Hsp90) in terms of its structure, biochemical characteristics, post-translational modifications, interactomes, regulation and functions. In addition to yeast as a model several new systems have now been examined including flies, worms, plants as well as mammalian cells. This review discusses themes emerging out of studies reported on Hsp90 from infectious disease causing protozoa. A common theme of sensing and responding to host cell microenvironment emerges out of analysis of Hsp90 in Malaria, Trypanosmiasis as well as Leishmaniasis. In addition to their functional roles, the potential of Hsp90 from these infectious disease causing organisms to serve as drug targets and the current status of this drug development endeavor are discussed. Finally, a unique and the only known example of a split Hsp90 gene from another disease causing protozoan Giardia lamblia and its evolutionary significance are discussed. Clearly studies on Hsp90 from protozoan parasites promise to reveal important new paradigms in Hsp90 biology while exploring its potential as an anti-infective drug target. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

RAPD Profile Analysis of Betel Vine Cultivars

RAPD analysis in selected cultivars of Kapoori and Bangla betel vines (Piper betle L.) were carried out in order to ascertain the relatedness of the two to each other. On the basis of the data from 10 RAPD primers, it was found that the Kapoori cultivars were more heterogeneous (mean SI = 0.521) while the Bangla cultivars were mostly similar to each other (mean SI = 0.884). Within each type, the overall polymorphism of RAPD bands was more than 70 %. When RAPD band data for both types of cultivars were considered cumulatively, the two were clearly separated from each other. In fact only six bands out of a total of 60 bands were found to be common to cultivars of both types. Bands specific to only one of the two types have potential for developing betel vine cultivar-specific probes and SCAR-markers.     

Isolation of propionate degrading bacterium in co-culture with a methanogen from a cattle dung biogas plant

Various anaerobic hydrolytic and methanogenic bacteria active in cattle dung biogas plants are reported in the literature. Anaerobic bacteria with ability to use volatile fatty acids constitute a vital bridge between hydrolytic bacteria and methanogenic bacteria. The present paper describes the isolation ofSyntrophobacter wolinii a propionate degrading bacterium in co-culture with a hydrogen utilizing methanogenviz.,Methanobacterium formicicum from the fermenting slurry of cattle dung biogas plant. Earlier studies on propionate and butyrate degradation indicatedMethanospirillum hungatei as the hydrogen utilizing partner of the co-culture whereas in the present studies this was not the case. Temperature 35° C, pH 7.5 and 20 mM of propionate were found optimal for growth and activity of co-culture.     

Genome analysis of amaranths: Determination of inter- and intra-species variations

Amaranths are an important group of plants and include grain, vegetable and ornamental types. Despite the economic importance of the amaranths, there is very little information available about the extent and nature of genetic diversity present in the genus Amaranthus at molecular level. We now report the randomly amplified polymorphic DNA (RAPD) profiles of different species of Amaranthus as well as different accessions of the species. These RAPD analyses have been carried out using 65 arbitrary sequence decamer primers. From the RAPD data, an UPGMA dendrogram illustrating the inter-as well as intra-species relationships has been computed. The putative hybrid origin of A.dubious from A. hybridus and A. spinosus is also ruled out by the RAPD data. The trends of species relationships amongst the amaranths determined by RAPDs is consistent with their cytogenetic and evolutionary relationships that have already been determined. NBRI Communication No:464 (N.S.).     

Analysis of variations in RAPD profiles among accessions ofProsopis

The genetic variability among accessions ofProsopis was determined using randomly amplified polymorphic DNA (RAPD) profiles. Similarities of profiles were determined using the algorithm of Jaccard, and UPGMA and neighbour joining trees were generated from the similarity data. The average similarity was highest among the accessions ofP. glandulosa (0.52 ± 0.18) and least in the accessions ofP. juliflora (0.37 ± 0.15), indicating that the latter species has greater diversity among accessions. Our observations suggest that RAPD analysis could help in identifying genetic variations among different accessions ofProsopis.     

A molecular insight into papaya leaf curl—a severe viral disease

Papaya leaf curl disease (PaLCuD) caused by papaya leaf curl virus (PaLCuV) not only affects yield but also plant growth and fruit size and quality of papaya and is one of the most damaging and economically important disease. Management of PaLCuV is a challenging task due to diversity of viral strains, the alternate hosts, and the genomic complexities of the viruses. Several management strategies currently used by plant virologists to broadly control or eliminate the viruses have been discussed. In the absence of such strategies in the case of PaLCuV at present, the few available options to control the disease include methods like removal of affected plants from the field, insecticide treatments against the insect vector (Bemisia tabaci), and gene-specific control through transgenic constructs. This review presents the current understanding of papaya leaf curl disease, genomic components including satellite DNA associated with the virus, wide host and vector range, and management of the disease and suggests possible generic resistance strategies.     

Neutralizing a Surface Charge on the FMN Subdomain Increases the Activity of Neuronal Nitric-oxide Synthase by Enhancing the Oxygen Reactivity of the Enzyme Heme-Nitric Oxide Complex

Nitric-oxide synthases (NOSs) are calmodulin-dependent flavoheme enzymes that oxidize l-Arg to nitric oxide (NO) and l-citrulline. Their catalytic behaviors are complex and are determined by their rates of heme reduction (k_r), ferric heme-NO dissociation (k_d), and ferrous heme-NO oxidation (k_ox). We found that point mutation (E762N) of a conserved residue on the enzyme''s FMN subdomain caused the NO synthesis activity to double compared with wild type nNOS. However, in the absence of l-Arg, NADPH oxidation rates suggested that electron flux through the heme was slower in E762N nNOS, and this correlated with the mutant having a 60% slower k_r. During NO synthesis, little heme-NO complex accumulated in the mutant, compared with ∼50–70% of the wild-type nNOS accumulating as this complex. This suggested that the E762N nNOS is hyperactive because it minimizes buildup of an inactive ferrous heme-NO complex during NO synthesis. Indeed, we found that k_ox was 2 times faster in the E762N mutant than in wild-type nNOS. The mutational effect on k_ox was independent of calmodulin. Computer simulation and experimental measures both indicated that the slower k_r and faster k_ox of E762N nNOS combine to lower its apparent K_m,O2 for NO synthesis by at least 5-fold, which in turn increases its V/K_m value and enables it to be hyperactive in steady-state NO synthesis. Our work underscores how sensitive nNOS activity is to changes in the k_ox and reveals a novel means for the FMN module or protein-protein interactions to alter nNOS activity.Nitric oxide (NO)² is a biological mediator that is produced in animals by three NO synthase isozymes (NOS, EC 1.14.13.39): inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) (1, 2). The NOS are modular enzymes composed of an N-terminal oxygenase domain and a C-terminal flavoprotein domain, with a calmodulin (CaM)-binding site connecting the two domains (3). During NO synthesis, the flavoprotein domain transfers NADPH-derived electrons through its FAD and FMN cofactors to a heme located in the oxygenase domain. The FMN-to-heme electron transfer enables heme-dependent oxygen activation and a stepwise conversion of l-Arg to NO and citrulline (4, 5). Heme reduction also requires that CaM be bound to NOS and is rate-limiting for NO biosynthesis (6–9).NOS enzymes operate under the constraint of having their newly made NO bind to the ferric heme before it can exit the enzyme (10). How this intrinsic heme-NO binding event impacts NOS catalytic cycling is shown in Fig. 1 and has previously been discussed in detail (10–13). The l-Arg to NO biosynthetic reaction (Fe^III to Fe^IIINO in Fig. 1) is limited by the rate of ferric heme reduction (k_r), because all biosynthetic steps downstream are faster than k_r. However, once the ferric heme-NO complex forms at the end of each catalytic cycle, it can either dissociate to release NO into the medium (at a rate k_d as shown in Fig. 1) or become reduced by the flavoprotein domain (at a rate k′_r in Fig. 1; equal to k_r) to form the enzyme ferrous heme-NO species (Fe^IINO), which releases NO very slowly (11, 12). Consequently, two cycles compete during steady-state NO synthesis (Fig. 1); NO dissociation from the ferric heme (k_d) is part of a “productive cycle” that releases NO and is essential for NOS bioactivity, whereas reduction of the ferric heme-NO complex (k_r′) channels the enzyme into a “futile cycle” that actually represents a NO dioxygenase activity. The rate of futile cycling is also determined by the rate of O₂ reaction with the ferrous heme-NO complex (at a rate k_ox in Fig. 1), which regenerates the ferric enzyme. Surprisingly, NOS enzymes have evolved to have a broad range of k_r (varies 40×), k_ox (varies 15×), and k_d (varies 30×) values (Table S1) (12). This causes each NOS to distribute quite differently during steady-state NO synthesis and gives each NOS a unique catalytic profile (12).Open in a separate windowFIGURE 1.Global kinetic model for NOS catalysis. Ferric enzyme reduction (k_r) is rate-limiting for the biosynthetic reactions (central linear portion). k_cat1 and k_cat2 are the conversion rates of the enzyme Fe^IIO₂ species to products in the l-Arg and N^ω-hydroxy-l-arginine (NOHA) reactions, respectively. The ferric heme-NO product complex (Fe^IIINO) can either release NO (k_d) or become reduced (k′_r) to a ferrous heme-NO complex (Fe^IINO), which reacts with O₂ (k_ox) to regenerate ferric enzyme. Enzyme partitioning and NO release are determined by the relative rates of k_r, k_ox, and k_d. This figure is adapted from Ref. 12.The enzyme physical and electronic factors that may set and regulate each of the three kinetic parameters (k_r, k_ox, and k_d) in NOS enzymes remain to be fully described. At present, the composition of the NOS flavoprotein domain and CaM appear to be primarily responsible for determining the k_r (14–17), whereas the composition of the NOS oxygenase domain is presumed to determine the k_d and k_ox (18, 19). Indeed, our recent point mutagenesis study identified a patch of electronegative residues on the FMN subdomain that are required to maintain a normal k_r and NO synthesis activity in nNOS, suggesting that subdomain electrostatic interactions are important in the process (20). We found particularly large effects when the negative charge at Glu⁷⁶² was neutralized via mutation to Asn. Remarkably, the NO synthesis activity of E762N nNOS was double that of wild-type nNOS, despite the mutant displaying a slow k_r that was half of wild type. In the current report, we show that the E762N mutation has an additional, unsuspected effect on the k_ox kinetic parameter of nNOS. How this effect alters distribution of the nNOS enzyme during steady-state catalysis, impacts the apparent K_m,O2, and leads to hyperactive NO synthesis is described. Our finding that the nNOS flavoprotein domain can tune a key kinetic parameter that defines the rate of a heme-based reaction in the nNOS oxygenase domain is unusual and suggests a means by which protein-protein interactions could regulate the catalytic behavior of nNOS.