Versatile enzymatic system for the production of guanosine polyphosphates

Posters - ED02 Signalling and systems biology: abstract no. ED02/20During periods of environmental stress, bacteria synthesize guanosine tetraphosphate (ppGpp, magic spot I) and guanosine pentaphosphate (pppGpp, magic spot II) in a process known as the stringent response. These intracellular allarmone molecules ‘reprogramme’ the transcriptional and translational machinery to help the cell conserve scarce resources. Existing methods for the production of guanosine polyphosphates are either technically difficult or inefficient, hindering investigations into their biological roles. We have developed a simple and efficient one-step enzymatic method for the production of guanosine polyphosphates using a recombinant protein cloned from Staphylococcus aureus. The purified enzyme efficiently catalyses the formation of pppGpp (and AMP) from GTP + ATP; and ppGpp (and AMP) from GDP + ATP. Notably, it also catalyses the synthesis of pGpp (guanosine 5’-monophosphate 3’-diphosphate, and AMP) from GMP + ATP; albeit with reduced efficiency. The reverse reactions are not catalysed, leading to high conversion rates. Guanosine polyphosphate products can be obtained in a homogeneous form using a combination of anion exchange chromatography followed by desalting. Our approach can be used to produce guanosine polyphosphates on a multi-milligram scale. Furthermore, our results also suggest that a third ‘magic spot’ allarmone may be formed within certain bacterial species.postprintThe Apring 2010 Meeting of the Society for General Microbiology (SGM), Edinburgh, U.K., 29 March-1 April 2010. In Abstract Book of the SGM Spring 2010 Meeting, 2010, p. 9

Loops, hairpins and flipped bases: a DNA aptamer that discriminates Plasmodium lactate dehydrogenase from the blind watchmaker

Poster Presentation: no. 113The Oligo MeetingLittle is known about how aptamers achieve their specificities in binding and discriminating between closely related targets. Under the pretext of investigating the potential for aptamers in malaria diagnostics, here we solve the crystal structure of a new DNA aptamer which was selected and evolved to bind specifically to the Plasmodium falciparum lactate dehydrogenase (PfLDH) and not bind to human lactate dehydrogenase. The structure reveals two aptamers bind per Plasmodium lactate dehydrogenase tetramer with opposite apical geometry, whereby each aptamer has a distorted hairpin structure. The aptamer comprises a B-helix stem, an asymmetric internal loop involved in target discrimination and an apical loop involved in binding interactions. Each loop contains a critical flipped base. Isothermal titration calorimetry, surface plasmon resonance and electrophoretic mobility shift assay all provide evidence for binding with a dissociation constant in the range 40-70 …postprin

Prevalence and diversity of Synergistetes taxa in periodontal health and disease

postprin

Determination of the functions of the putative metal-binding domain of the SCV helicase.

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Pseudomonas aeruginosa inhibits in-vitro Candida biofilm development

<p>Abstract</p> <p>Background</p> <p>Elucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as <it>Candida spp</it>. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of <it>Pseudomonas aeruginosa </it>and six different species of <it>Candida </it>comprising <it>C. albicans</it>, <it>C. glabrata, C. krusei</it>, <it>C. tropicalis</it>, <it>C. parapsilosis</it>, and <it>C. dubliniensis </it>in dual species biofilm development.</p> <p>Results</p> <p>A significant reduction in colony forming units (CFU) of <it>C. parapsilosis </it>(90 min), <it>C. albicans </it>and <it>C. tropicalis </it>(90 min, 24 h and 48 h), <it>C. dubliniensis </it>and <it>C. glabrata</it>, (24 h and 48 h) was noted when co-cultured with <it>P. aeruginosa </it>in comparison to their monospecies counterparts (P < 0.05). A simultaneous significant reduction in <it>P. aeruginosa </it>numbers grown with <it>C. albicans </it>(90 min and 48 h), <it>C. krusei </it>(90 min, 24 h and 48 h),<it>C. glabrata</it>, (24 h and 48 h), and an elevation of <it>P. aeruginosa </it>numbers co-cultured with <it>C. tropicalis </it>(48 h) was noted (P < 0.05). When data from all <it>Candida spp</it>. and <it>P. aeruginosa </it>were pooled, highly significant mutual inhibition of biofilm formation was noted (<it>Candida </it>P < 0.001, <it>P. aeruginosa </it>P < 0.01). Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) analyses confirmed scanty architecture in dual species biofilm in spite of dense colonization in monospecies counterparts.</p> <p>Conclusions</p> <p><it>P. aeruginosa </it>and <it>Candida </it>in a dual species environment mutually suppress biofilm development, both quantitatively and qualitatively. These findings provide a foundation to clarify the molecular basis of bacterial-fungal interactions, and to understand the pathobiology of mixed bacterial-fungal infections.</p

pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

Background The ethanol-producing bacterium Zymomonas mobilis has attracted considerable scientific and commercial interest due to its exceptional physiological properties. Shuttle vectors derived from native plasmids have previously been successfully used for heterologous gene expression in this bacterium for a variety of purposes, most notably for metabolic engineering applications. Results A quantitative PCR (qPCR) approach was used to determine the copy numbers of two endogenous double stranded DNA plasmids: pZMO1A (1,647 bp) and pZMO7 (pZA1003; 4,551 bp) within the NCIMB 11163 strain of Z. mobilis. Data indicated pZMO1A and pZMO7 were present at ca. 3-5 and ca. 1-2 copies per cell, respectively. A ca. 1,900 bp fragment from plasmid pZMO7 was used to construct two Escherichia coli - Z. mobilis shuttle vectors (pZ7C and pZ7-184). The intracellular stabilities and copy numbers of pZ7C and pZ7-184 were characterized within the NCIMB 11163, ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains of Z. mobilis. Both shuttle vectors could be stably maintained within the ATCC 29191 strain (ca. 20-40 copies per cell), and the CU1 Rif2 strain (ca. 2-3 copies per cell), for more than 50 generations in the absence of an antibiotic selectable marker. A selectable marker was required for shuttle vector maintenance in the parental NCIMB 11163 strain; most probably due to competition for replication with the endogenous pZMO7 plasmid molecules. N-terminal glutathione S-transferase (GST)-fusions of four endogenous proteins, namely the acyl-carrier protein (AcpP); 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA); DNA polymerase III chi subunit (HolC); and the RNA chaperone protein Hfq; were successfully expressed from pZ7C-derived shuttle vectors, and their protein-protein binding interactions were analyzed in Z. mobilis ATCC 29191. Using this approach, proteins that co-purified with AcpP and KdsA were identified. Conclusions We show that a shuttle vector-based protein affinity 'pull-down' approach can be used to probe protein interaction networks in Z. mobilis cells. Our results demonstrate that protein expression plasmids derived from pZMO7 have significant potential for use in future biological or biotechnological applications within Z. mobilis.published_or_final_versio

Effects of creatine supplementation on housekeeping genes in human skeletal muscle using real-time RT-PCR.

The present study examined the validity and reliability of measuring the expression of various genes in human skeletal muscle using quantitative real-time RT-PCR on a GeneAmp 5700 sequence detection system with SYBR Green 1 chemistry. In addition, the validity of using some of these genes as endogenous controls (i.e., housekeeping genes) when human skeletal muscle was exposed to elevated total creatine levels and exercise was also examined. For all except 28S, linear relationships between the logarithm of the starting RNA concentrations and the cycle threshold (C(T)) values were established for beta-actin, beta2-microglobulin (beta2M), cyclophilin (CYC), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a linear response between C(T) values and the logarithm of a given amount of starting cDNA for all the genes tested. The overall intra-assay coefficient of variance for these genes was 1.3% and 21% for raw C(T) values and the linear value of 2(-C(T)), respectively. Interassay variability was 2.3% for raw C(T) values and 34% for the linear value of 2(-C(T)). We also examined the expression of various housekeeping genes in human skeletal muscle at days 0, 1, and 5 following oral supplementation with either creatine or a placebo employing a double-blind crossover study design. Treatments were separated by a 5-wk washout period. Immediately following each muscle sampling, subjects performed two 30-s all-out bouts on a cycle ergometer. Creatine supplementation increased (P &lt; 0.05) muscle total creatine content above placebo levels; however, there were no changes (P &gt; 0.05) in C(T) values across the supplementation periods for any of the genes. Nevertheless, 95% confidence intervals showed that GAPDH was variable, whereas beta-actin, beta2M, and CYC were the least varying genes. Normalization of the data to these housekeeping genes revealed variable behavior for beta2M with more stable expressions for both beta-actin and CYC. We conclude that, using real-time RT-PCR, beta-actin or CYC may be used as housekeeping genes to study gene expression in human muscle in experiments employing short-term creatine supplementation combined with high-intensity exercise

Purification, crystallization and X-ray crystallographic analysis of a putative exopolyphosphatase from Zymomonas mobilis

Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 Å resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 122.0, b = 47.1, c = 89.5 Å, α = γ = 90, β = 124.5°. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 Å resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate.published_or_final_versio

Arginine deiminase pathway is far more important than urease for acid resistance and intracellular survival in Laribacter hongkongensis: a possible result of arc gene cassette duplication

Title in Final Programme: Arginine deiminase pathway is far more important than urease for acid resistance in Laribacter hongkongensis: result of arc gene cassette duplicationPoster Session - Pathogenesis and animal models of bacterial infections: abstract no. P1122INTRODUCTION AND PURPOSE: Laribacter hongkongensis is a Gram-negative, urease-positive bacillus associated with invasive bacteremic infections in liver cirrhosis patients and fish-borne community- acquired gastroenteritis and traveler’s diarrhea (1-2). Its mechanisms of acid resistance are unknown. a complete urease cassette and two adjacent arc gene cassettes (encoding enzymes of ADI pathway) were found in the genome (3). In this study, we investigated the mechanism for resisting acidic environment in vitro, in macrophages and in a mouse model ...published_or_final_versio

Arginine deiminase pathway is far more important than urease for acid resistance and intracellular survival in Laribacter hongkongensis: a possible result of arc gene cassette duplication

BACKGROUND: Laribacter hongkongensis is a Gram-negative, urease-positive bacillus associated with invasive bacteremic infections in liver cirrhosis patients and fish-borne community-acquired gastroenteritis and traveler's diarrhea. Its mechanisms of adaptation to various environmental niches and host defense evasion are largely unknown. During the process of analyzing the L. hongkongensis genome, a complete urease cassette and two adjacent arc gene cassettes were found. We hypothesize that the urease cassette and/or the arc gene cassettes are important for L. hongkongensis to survive in acidic environment and macrophages. In this study, we tested this hypothesis by constructing single, double and triple non-polar deletion mutants of the urease and two arc gene cassettes of L. hongkongensis using the conjugation-mediated gene deletion system and examining their effects in acidic environment in vitro, in macrophages and in a mouse model. RESULTS: HLHK9ureA, HLHK9ureC, HLHK9ureD and HLHK9ureE all exhibited no urease activity. HLHK9arcA1 and HLHK9arcA2 both exhibited arginine deiminase (ADI) activities, but HLHK9arcA1/arcA2 double deletion mutant exhibited no ADI activity. At pH 2 and 3, survival of HLHK9arcA1/arcA2 and HLHK9ureA/arcA1/arcA2 were markedly decreased (p < 0.001) but that of HLHK9ureA was slightly decreased (p < 0.05), compared to wild type L. hongkongensis HLHK9. Survival of HLHK9ureA/arcA1/arcA2 and HLHK9arcA1/arcA2 in macrophages were also markedly decreased (p < 0.001 and p < 0.01 respectively) but that of HLHK9ureA was slightly decreased (p < 0.05), compared to HLHK9, although expression of arcA1, arcA2 and ureA genes were all upregulated. Using a mouse model, HLHK9ureA exhibited similar survival compared to HLHK9 after passing through the murine stomach, but survival of HLHK9arcA1/arcA2 and HLHK9ureA/arcA1/arcA2 were markedly reduced (p < 0.01). CONCLUSIONS: In contrast to other important gastrointestinal tract pathogens, ADI pathway is far more important than urease for acid resistance and intracellular survival in L. hongkongensis. The gene duplication of the arc gene cassettes could be a result of their functional importance in L. hongkongensis.published_or_final_versio