Differential expression of Zymomonas mobilis sucrase genes (sacB and sacC) in Escherichia coli and sucrase mutants of Zymomonas mobilis

The sacB and sacC genes encoding levansucrase and extracellular sucrase respectively were independently subcloned in pBluescript (high copy number) and in Z. mobilis-E. coli shuttle vector, pZA22 (low copy number). The expression of these genes were compared under identical background of E. coli and Z. mobilis host. The level of sacB gene expression in E. coli was almost ten fold less than the expression of sacC gene, irrespective of the growth medium or the host strain. In Z. mobilis the expression of sacB and sacC genes was shown to be subject to carbon source dependent regulation. The transcript of sacB and sacC was three fold higher in cells grown on sucrose than in cells grown on glucose/fructose. Northern blot analysis revealed that the transcript levels of sacC was approximately 2-3 times higher than that of sacB. These results suggested that the expression of sacC gene was more pronounced than sacB

Identification and characterization of alkaline serine protease from goat skin surface metagenome

Metagenomic DNA isolated from goat skin surface was used to construct plasmid DNA library in Escherichia coli DH10B. Recombinant clones were screened for functional protease activity on skim milk agar plates. Upon screening 70,000 clones, a clone carrying recombinant plasmid pSP1 exhibited protease activity. In vitro transposon mutagenesis and sequencing of the insert DNA in this clone revealed an ORF of 1890 bp encoding a protein with 630 amino acids which showed significant sequence homology to the peptidase S8 and S53 subtilisin kexin sedolisin of Shewanella sp. This ORF was cloned in pET30b and expressed in E. coli BL21 (DE3). Although the cloned Alkaline Serine protease (AS-protease) was overexpressed, it was inactive as a result of forming inclusion bodies. After solubilisation, the protease was purified using Ni-NTA chromatography and then refolded properly to retain protease activity. The purified AS-protease with a molecular mass of ~63 kDa required a divalent cation (Co2+ or Mn2+) for its improved activity. The pH and temperature optima for this protease were 10.5 and 42°C respectively

Superantigen profiles of emm and emm-like typeable and nontypeable pharyngeal streptococcal isolates of South India

<p>Abstract</p> <p>Background</p> <p>The major virulence factors determining the pathogenicity of streptococcal strains include M protein encoded by <it>emm </it>and <it>emm</it>-like (<it>emmL</it>) genes and superantigens. In this study, the distribution of <it>emm, emmL </it>and superantigen genes was analyzed among the streptococcal strains isolated from the patients of acute pharyngitis.</p> <p>Methods</p> <p>The streptococcal strains were isolated from the throat swabs of 1040 patients of acute pharyngitis. The <it>emm </it>and <it>emmL </it>genes were PCR amplified from each strain and sequenced to determine the <it>emm </it>types. The dot-blot hybridization was performed to confirm the pathogens as true <it>emm </it>nontypeable strains. The presence of eleven currently known superantigens was determined in all the strains by multiplex PCR.</p> <p>Results</p> <p>Totally, 124 beta-hemolytic streptococcal strains were isolated and they were classified as group A streptococcus (GAS) [15.3% (19/124)], group C streptococcus (GCS) [59.7% (74/124)] and group G streptococcus (GGS) [25.0% (31/124)]. Among 124 strains, only 35 strains were <it>emm </it>typeable and the remaining 89 strains were <it>emm </it>nontypeable. All GAS isolates were typeable, whereas most of the GCS and GGS strains were nontypeable. These nontypeable strains belong to <it>S. anginosus </it>[75.3% (67/89)] and <it>S. dysgalactiae </it>subsp. <it>equisimilis </it>[24.7% (22/89)]. The <it>emm </it>and <it>emmL </it>types identified in this study include <it>emm12.0 </it>(28.6%), <it>stG643.0 </it>(28.6%), <it>stC46.0 </it>(17.0%), <it>emm30.11 </it>(8.5%), <it>emm3.0 </it>(2.9%), <it>emm48.0 </it>(5.7%), <it>st3343.0 </it>(2.9%), <it>emm107.0 </it>(2.9%) and <it>stS104.2 </it>(2.9%). Various superantigen profiles were observed in typeable as well as nontypeable strains.</p> <p>Conclusions</p> <p>Multiplex PCR analysis revealed the presence of superantigens in all the typeable strains irrespective of their <it>emm </it>types. However, the presence of superantigen genes in <it>emm </it>and <it>emmL </it>nontypeable strains has not been previously reported. In this study, presence of at least one or a combination of superantigen coding genes was identified in all the <it>emm </it>and <it>emmL </it>nontypeable strains. Thus, the superantigens may inevitably play an important role in the pathogenesis of these nontypeable strains in the absence of the primary virulence factor, M protein.</p

Pseudomonas

Controlled drug delivery technology represents one of the most rapidly advancing areas of science. They offer numerous advantages compared to conventional dosage forms including improved efficacy, reduced toxicity, improved patient compliance and convenience. Over the past several decades, many delivery tools or methods were developed such as viral vector, liposome-based delivery system, polymer-based delivery system, and intelligent delivery system. Recently, nonviral vectors, especially those based on biodegradable polymers, have been widely investigated as vectors. Unlike the other polymers tested, polyhydroxyalkanoates (PHAs) have been intensively investigated as a family of biodegradable and biocompatible materials for in vivo applications as implantable tissue engineering material as well as release vectors for various drugs. On the other hand, the direct use of these polyesters has been hampered by their hydrophobic character and some physical shortcomings, while its random copolymers fulfilled the expectation of biomedical researchers by exhibiting significant mechanical and thermal properties. This paper reviews the strategies adapted to make functional polymer to be utilized as delivery system

Identification of an antibacterial protein by functional screening of a human oral metagenomic library

This is a corrigendum to the original article published in 2015, updating the author list to include Philip Warburton

Evaluation of INSeq To Identify Genes Essential for

The reciprocal interaction between rhizosphere bacteria and their plant hosts results in a complex battery of genetic and physiological responses. In this study, we used insertion sequencing (INSeq) to reveal the genetic determinants responsible for the fitness of Pseudomonas aeruginosa PGPR2 during root colonization. We generated a random transposon mutant library of Pseudomonas aeruginosa PGPR2 comprising 39,500 unique insertions and identified genes required for growth in culture and on corn roots. A total of 108 genes were identified as contributing to the fitness of strain PGPR2 on roots. The importance in root colonization of four genes identified in the INSeq screen was verified by constructing deletion mutants in the genes and testing them for the ability to colonize corn roots singly or in competition with the wild type. All four mutants were affected in corn root colonization, displaying 5- to 100-fold reductions in populations in single inoculations, and all were outcompeted by the wild type by almost 100-fold after seven days on corn roots in mixed inoculations of the wild type and mutant. The genes identified in the screen had homology to genes involved in amino acid catabolism, stress adaptation, detoxification, signal transduction, and transport. INSeq technology proved a successful tool to identify fitness factors in P aeruginosa PGPR2 for root colonization

Effect of Butachlor Herbicide on Earthworm Eisenia fetida

With the advent of the Green Revolution, there has been a quantum leap in the use of synthetic herbicides and pesticides throughout the world to sustain high yielding crop varieties. Continuous use of these synthetic chemicals leads to loss of soil fertility and soil organisms. To explore the effect of exposure to commercial herbicide (Butachlor) on the life history parameters (biomass, clitellum development, and cocoon production) and the histological changes in the earthworm Eisenia fetida over 60 days, the dried cow dung was contaminated with 0.2575 mg kg−1, 0.5150 mg kg−1, and 2.5750 mg kg−1 of butachlor based on the LC50 value, and a control was maintained. The mean earthworm biomass was found to be decreased with increasing herbicide concentration. Similarly, cocoon production was also reduced by the increasing herbicide concentration. A possible explanation is an increased demand for energy, needed for the regulation and detoxification of herbicide. All earthworms in the exposed group were found to have glandular cell enlargement and to be vacuolated