Bacillus anthracis and Bacillus cereus PcrA helicases can support DNA unwinding and in vitro rolling-circle replication of plasmid pT181 of Staphylococcus aureus

Replication of rolling-circle replicating (RCR) plasmids in gram-positive bacteria requires the unwinding of initiator protein-nicked plasmid DNA by the PcrA helicase. In this report, we demonstrate that heterologous PcrA helicases from Bacillus anthracis and Bacillus cereus are capable of unwinding Staphylococcus aureus plasmid pT181 from the initiator-generated nick and promoting in vitro replication of the plasmid. These helicases also physically interact with the RepC initiator protein of pT181. The ability of PcrA helicases to unwind noncognate RCR plasmids may contribute to the broad-host-range replication and dissemination of RCR plasmids in gram-positive bacteria.     

Proton transfer in the oxidative half-reaction of pentaerythritol tetranitrate reductase. Structure of the reduced enzyme-progesterone complex and the roles of residues Tyr186, His181, His184

The roles of His181, His184 and Tyr186 in PETN reductase have been examined by mutagenesis, spectroscopic and stopped-flow kinetics, and by determination of crystallographic structures for the Y186F PETN reductase and reduced wild-type enzyme-progesterone complex. Residues His181 and His184 are important in the binding of coenzyme, steroids, nitroaromatic ligands and the substrate 2-cyclohexen-1-one. The H181A and H184A enzymes retain activity in reductive and oxidative half-reactions, and thus do not play an essential role in catalysis. Ligand binding and catalysis is not substantially impaired in Y186F PETN reductase, which contrasts with data for the equivalent mutation (Y196F) in Old Yellow Enzyme. The structure of Y186F PETN reductase is identical to wild-type enzyme, with the obvious exception of the mutation. We show in PETN reductase that Tyr186 is not a key proton donor in the reduction of alpha/beta unsaturated carbonyl compounds. The structure of two electron-reduced PETN reductase bound to the inhibitor progesterone mimics the catalytic enzyme-steroid substrate complex and is similar to the structure of the oxidized enzyme-inhibitor complex. The reactive C1-C2 unsaturated bond of the steroid is inappropriately orientated with the flavin N5 atom for hydride transfer. With steroid substrates, the productive conformation is achieved by orientating the steroid through flipping by 180 degrees , consistent with known geometries for hydride transfer in flavoenzymes. Our data highlight mechanistic differences between Old Yellow Enzyme and PETN reductase and indicate that catalysis requires a metastable enzyme-steroid complex and not the most stable complex observed in crystallographic studies.     

Effect of sulfur on nitrate reductase and ATP sulfurylase Activities in groundnut (Arachis hypogea L.)

Field experiments were conducted to determine the effect of sulfur (S) and Nitrogen (N) on nitrate reductase (NR) and ATP-sulfurylase activities in groundnut cultivars (Arachis hypogea L. cv. Ambar and Kaushal). Two combinations of S (in kg ha^-1): OS (-S) and 20S (+S) were used with 20 kg ha^-1 N. The application of S enhanced the NR and ATP-sulfurylase activities in both the cultivars at all the growth stages. The application of S also increased soluble protein and chlorophyll content in the all growth stages of both the cultivars. NR and ATP-sulfurylase activities in the leaves were measured at various growth stages as the two enzymes catalyze the rate limiting steps of the assimilatory pathways of nitrate and sulfate, respectively.     

Influence of sediment characteristics on density and distribution of Ocypodoid crab burrows (superfamily: Ocypodoidea) along the coastal areas of Pakistan

Crabs belong to the superfamily Ocypodoidea are a significant component of benthic fauna and considered as ecosystem engineers because of their dynamic role as an active burrower in mangrove and estuarine environment. The current investigation was to evaluate the crab burrow density, diameter and total area of burrow opening along the coast of Pakistan. The variations in burrow properties and their relation to sediment characteristics were also evaluated to recognize the most influencing variables of sediments that effects on crab burrows. All crab burrow and sediment characteristics differed significantly (p < 0.05) among the monitoring sites. Regression analysis showed that crab density was significantly correlated with burrow density (P < 0.001). Moreover, burrow density was noticed significantly greater (p < 0.05) than crab density. Pearson correlation analysis reveals that moisture, porosity, organics, sand and mean grain size observed as most influencing the features of sediment to determine the ecological functioning of crab burrows in mangrove and mudflats of Pakistan.     

Incidence of metallo-beta-lactamase-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> isolates from hospital setting in Pakistan

The aim of this study was monitoring and surveillance in different wards of the PIMS hospital, Islamabad, to understand emerging challenges of antibiotic resistance in particular association with most virulent serotypes of Klebsiella pneumoniae. The study was conducted during March 2015 to September 2015. The study showed that rate of isolation of K. pneumoniae was 37% (103 positives out of a total of 277 clinical samples) and 7.7% (8) were phenotypically and genotypically confirmed to be metallo-β-lactamase resistant (carbapenem resistant) and all of them were multidrug resistant (MDR). These carbapenem-resistant isolates were isolated from blood, endotracheal tubes, and pus. Molecular screening for the presence of integrons indicated that distribution of class I integrons (87.5% of carbapenem-resistant K. pneumoniae isolates) was higher than class II integrons (1.25%) among given isolates. The study indicated that exposure of metallo-beta-lactamase-producing strains through hospitalizations increases the chances of spread of MDR pathogens. There is an urgent need for effective surveillance and monitoring strategies to control the spread of extremely resistant K. pneumoniae implicated in nosocomial infections leading to the increased health burden and enforcement of policy guideline on appropriate antibiotics usage.     

Time-course analysis of the phenols in cucumber mosaic virus-resistant, -tolerant and -susceptible tomato genotypes

In this study, changes in quantity and quality of phenolic compounds were compared in cucumber mosaic virus (CMV)-inoculated and -un-inoculated plants of nine resistant, tolerant, susceptible and highly susceptible genotypes at three different time intervals. Total phenolic contents and the number of phenolic compounds were generally increased in CMV-inoculated plants. Maximum per cent increase in total phenolic contents over un-inoculated controls was observed as 77.55% in resistant genotype TMS-1, 84.17% in tolerant genotype L06238 and 82.88% in resistant genotype L02223 after 10, 20 and 30 days of inoculation, respectively. Thin layer chromatography of inoculated and un-inoculated plants indicates that in most of the tested genotypes, the number of phenolic compounds varied from cultivar to cultivar and within the same cultivar, depending upon the status of plants and growth stages. However, the trend of increase in quantity and quality of phenolic compounds in the tested units was not constant to draw a meaningful conclusion.     

Phylogenetic analysis of coat protein gene of BYDV-MAV strain from wheat

Barley yellow dwarf disease is globally the most important viral disease of wheat. The full-length nucleotide sequence of coat protein (CP) gene of 12 isolates revealed the presence of three distinct clusters. Pakistani isolate of MAV (MAV-PK) has maximum similarity of 99.23% with MAV isolate of Morocco and PAV-Australia following 99.22 and 99.22% with PAV-France. Similar degree of similarity was found in comparison of amino acid sequence. The finding of this study is that MAV-PK has similarity with both MAV-France and PAV-Australia, which is due to the reason that both MAV and PAV belong to the same group and both share maximum nucleotide homology. Low genetic diversity was found not only between MAV isolates but also between MAV and PAV isolates because phylogenetic analysis was done on the CP gene which is highly conserved region in genome of Barley yellow dwarf viruses (BYDVs). Divergence in MAV-PK was due to this recombination which is now most prevalent in Pakistan. MAV-PK has maximum similarity with MAV-Morocco followed by MAV-Sweden and MAV-Cz, which seems to indicate that Pakistani isolate of MAV evolved as the result of recombination between MAV isolates of the USA and PAV isolates of Australia and France. At the same time, recombination of MAV-CZ and MAV-Sweden also occur. This work can be successfully utilised in epidemiological studies of MAV isolate in Pakistan. Further analysis of variation level in these isolates will help scientists to formulate appropriate management strategies like incorporation of BdV 2 gene in wheat against BYDVs.