Molecular characterization of the PhoPQ-PmrD-PmrAB mediated pathway regulating polymyxin B resistance in Klebsiella pneumoniae CG43

<p>Abstract</p> <p>Background</p> <p>The cationic peptide antibiotic polymyxin has recently been reevaluated in the treatment of severe infections caused by gram negative bacteria.</p> <p>Methods</p> <p>In this study, the genetic determinants for capsular polysaccharide level and lipopolysaccharide modification involved in polymyxin B resistance of the opportunistic pathogen <it>Klebsiella pneumoniae </it>were characterized. The expressional control of the genes responsible for the resistance was assessed by a LacZ reporter system. The PmrD connector-mediated regulation for the expression of <it>pmr </it>genes involved in polymyxin B resistance was also demonstrated by DNA EMSA, two-hybrid analysis and <it>in vitro </it>phosphor-transfer assay.</p> <p>Results</p> <p>Deletion of the <it>rcsB</it>, which encoded an activator for the production of capsular polysaccharide, had a minor effect on <it>K. pneumoniae </it>resistance to polymyxin B. On the other hand, deletion of <it>ugd </it>or <it>pmrF </it>gene resulted in a drastic reduction of the resistance. The polymyxin B resistance was shown to be regulated by the two-component response regulators PhoP and PmrA at low magnesium and high iron, respectively. Similar to the control identified in <it>Salmonella</it>, expression of <it>pmrD </it>in <it>K. pneumoniae </it>was dependent on PhoP, the activated PmrD would then bind to PmrA to prolong the phosphorylation state of the PmrA, and eventually turn on the expression of <it>pmr </it>for the resistance to polymyxin B.</p> <p>Conclusions</p> <p>The study reports a role of the capsular polysaccharide level and the <it>pmr </it>genes for <it>K. pneumoniae </it>resistance to polymyxin B. The PmrD connector-mediated pathway in governing the regulation of <it>pmr </it>expression was demonstrated. In comparison to the <it>pmr </it>regulation in <it>Salmonella</it>, PhoP in <it>K. pneumoniae </it>plays a major regulatory role in polymyxin B resistance.</p

Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis

In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123) was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W84, P95, P110, or V129. The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3) were subsequently dissolved using 6 M urea and then refolded by stepwise dialysis. Compared to the unfolded wild-type antigen, the refolded M3b antigen (W84S, P110S and V129L) exhibited an increase of 66% antigenicity with binding capacity of 0.96 and affinity of 113 μM−1. Moreover, the 33% decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection

Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria

a b s t r a c t Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia coli is approximately 10 4 colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PP i and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PP i -recycling loop was completed using ATP sulfurylase and adenosine 5 0 phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10 CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water. Ó 2011 Elsevier Inc. All rights reserved. Bacteria monitoring is essential for many industrial manufacturing processes, and particularly those involving food, semiconductors, and biopharmaceuticals. The presence of bacteria reduces production yield and may cause serious health problems in humans. Researchers have developed several rapid assays for detecting bacteria in water. These methods include polymerase chain reactions, fluorescence in situ hybridization [1], b-D-glucuronidase activity measurement The ATP luminescence assay is a rapid, sensitive, and easy-toperform method based on the detection of ATP, a molecule ubiquitously present in all living cells. The enzyme luciferase catalyzes the oxidation of the substrate luciferin while transforming the energy derived from ATP into light, which can be quantified by a luminometer. This assay has been widely used in bacteria monitoring for food hygiene [4] and surface cleanliness The current detection limit of the ATP luminescence method for Escherichia coli is approximately 10 4 colony-forming units (CFU) 1 [12,13], which is not sensitive enough for many industrial and medical applications. Several approaches have been adopted to improve the assay sensitivity. The first strategy involves the identification of chemical extractants that can effectively disrupt bacterial cells while not interfering with the luminescence assay. Both dimethyl sulfoxide (DMSO) 0003-2697/$ -see front matter

The MBA degree and Singapore employers' perception on MBA holders and programs.

The objective of this research is to allow readers to gain a better understanding of the MBA degree itself as well as the local employers’ perceptions on MBA programs and holders. Despite the fact that there is a rise in the number of working Singaporeans taking up MBAs, there is limited local literature available on MBAs. The research was conducted through surveys and interviews with established firms in Singapore to shed detailed insights on the employers’ perceptions on MBAs. The results of surveys, interviews and the published appointments, together with the review of re-engineering done by the business schools, formed the framework of this particular literature.ACCOUNTANC

RmpA2, an Activator of Capsule Biosynthesis in Klebsiella pneumoniae CG43, Regulates K2 cps Gene Expression at the Transcriptional Level

The rmpA2 gene, which encodes an activator for capsular polysaccharide (CPS) synthesis, was isolated from a 200-kb virulence plasmid of Klebsiella pneumoniae CG43. Based on the sequence homology with LuxR at the carboxyl-terminal DNA-binding motif, we hypothesized that RmpA2 exerts its effect by activating the expression of cps genes that are responsible for CPS biosynthesis. Two luxAB transcriptional fusions, each containing a putative promoter region of the K. pneumoniae K2 cps genes, were constructed and were found to be activated in the presence of multicopy rmpA2. The activation is likely due to direct binding of RmpA2 to the cps gene promoter through its C-terminal DNA binding motif. Moreover, the loss of colony mucoidy in a K. pneumoniae strain deficient in RcsB, a regulator for cps gene expression, could be recovered by complementing the strain with a multicopy plasmid carrying rmpA2. The CPS production in Lon protease-deficient K. pneumoniae significantly increased, and the effect was accompanied by an increase of RmpA2 stability. The expression of the rmpA2 gene was negatively autoregulated and could be activated when the organism was grown in M9 minimal medium. An IS3 element located upstream of the rmpA2 was required for the full activation of the rmpA2 promoter. In summary, our results suggest that the enhancement of K2 CPS synthesis in K. pneumoniae CG43 by RmpA2 can be attributed to its transcriptional activation of K2 cps genes, and the expression level of rmpA2 is autoregulated and under the control of Lon protease

Identification of amino acid residues important for the phosphomannose isomerase activity of PslB in Pseudomonas aeruginosa PAO1

AbstractPhosphomannose isomerase (PMI) plays a pivotal role in biosynthesis of GDP-mannose, an important precursor of many polysaccharides. We demonstrate in this study that Pseudomonas aeruginosa pslB encodes a protein with GDP-mannose pyrophosphorylase/PMI dual activities. The PMI activity is Co2+-dependent and could be inhibited by GDP-mannose in a competitive manner. Furthermore, the activity could be inactivated by 2,3-butanedione suggesting the presence of a catalytic Arg residue. Site-specific mutations at R373, R472, R479, E410, H411, N433 and E458 increase the KM approximately 8–20-fold. The PMI activity of PslB was completely diminished with a R408K or R408A, reflecting the importance of this residue in catalysis. Overall, these results provide a basis for understanding the catalytic mechanism of PMI

The importance of conserved residues in human liver UDPglucose pyrophosphorylase

Comparison of the amino acid sequences of five eukaryotic UDPglucose pyrophosphorylases has identified a number of conserved residues that may be important for substrate binding or catalysis. Using the cloned cDNA for the human liver enzyme, we have investigated the role of several of these residues by site-directed mutagenesis. Changing the single conserved cysteine (residue 123) to serine resulted in an active enzyme, as did mutating the single conserved histidine (residue 266) to arginine. The two conserved tryptophans were each altered to serine; W218S is active while W333S is not. In the latter case, the enzyme does not appear to fold correctly, and a similar result was obtained by mutation to lysine at one (residue 391) of the four conserved arginines. The other three arginines are not essential, as judged by the observation that R389H, R422Q and R445H are all active. The kinetic properties of each active mutant were investigated and in most cases were found to be similar to those of wild-type. The most dramatic change is a sevenfold increase in the K(m) for magnesium pyrophosphate with C123S. Overall, none of these conserved residues appears to be essential for activity, although such a role cannot be ruled out for W333 and R391 where mutation resulted in defective folding