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A Novel Sortase, SrtC2, from Streptococcus pyogenes Anchors a Surface Protein Containing a QVPTGV Motif to the Cell Wall. Timothy C. Barnett, 2004.The important human pathogen Streptococcus pyogenes (group A streptococcus GAS), requires several surface proteins to interact with its human host . Many of these are covalently linked by a sortase enzyme to the cell wall via a C-terminal LPXTG motif . This motif is followed by a hydrophobic region and charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase . Previously, we identified two sortase enzymes in GAS . SrtA is found in all GAS strains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchors a subset of LPXTG-containing proteins . We now report the presence of a third sortase in most strains of GAS, SrtC . We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobic region and charged tail . We also demonstrate that the QVPTGV sequence is a substrate for anchoring of this protein by SrtC . Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein of GAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA . We conclude that srtC encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS . Evolution of the Antimicrobial Resistance of Staphylococcus spp . in Spain: Five Nationwide Prevalence Studies, 1986 to 2002. Oscar Cuevas, 2004.Data regarding the evolution of Staphylococcus resistance in a whole country have a definite influence on the design of empirical treatment regimens . Nevertheless, incidence studies over long periods of time are expensive and very difficult to carry out . In order to ascertain the present situation of the antimicrobial resistance of Staphylococcus in Spain and the change of this resistance over time, we performed five point prevalence studies (1986 to 2002) in a large group of Spanish hospitals (from 68 institutions in 1986 to 143 in 2002) collecting all Staphylococcus strains isolated on a single selected day . All microorganisms were identified in the five studies at the same laboratory, and antimicrobial susceptibility testing was performed against 17 antimicrobial agents by the agar dilution method and a microdilution method . During this period, there was an overall increase in resistance to most antimicrobials among Staphylococcus aureus/coagulase-negative staphylococci, mainly to oxacillin (1.5%/32.5% in 1986 versus 31.2%/61.3% in 2002) (P < 0.001), erythromycin (7%/41.1% in 1986 versus 31.7%/63% in 2002) (P < 0.001), gentamicin (5.2%/25.4% in 1986 versus 16.9%/27.8% in 2002) (P < 0.001; P = 0.5), and ciprofloxacin (0.6%/1.1% in 1986 versus 33.9%/44.9% in 2002) (P < 0.001) . All of the isolates were uniformly susceptible to glycopeptides, linezolid, and quinupristin/dalfopristin . Resistance of S . aureus to trimethoprim/sulfamethoxazole was very low (from 0.5% to 2.1%) (P = 0.152) . Periodic performance of prevalence studies is a useful, inexpensive, and easy tool to know the nationwide situation of a microorganism and its resistance to antimicrobials; it also helps us assess the emergence and spread of antimicrobial resistance . Construction of a Chimeric Thermostable Pyrophosphatase To Facilitate Its Purification and Immobilization by Using the Choline-Binding Tag. Cristina Moldes, 2004.The thermophilic inorganic pyrophosphatase (Pyr) from Thermus thermophilus has been produced in Escherichia coli fused to the C terminus of the choline-binding tag (ChB tag) derived from the choline-binding domain (ChBD) of pneumococcal LytA autolysin . The chimeric ChBD-Pyr protein retains its thermostable activity and can be purified in a single step by DEAE-cellulose affinity chromatography . Pyr can be further released from the ChBD by thrombin, using the specific protease recognition site incorporated in the C terminus of this tag . Remarkably, the ChB tag provides a selective and very strong thermostable noncovalent immobilization of ChBD-Pyr in the DEAE-cellulose matrix . The binding of choline or choline analogues, such as DEAE, confers a high thermal stability to this tag; therefore, the immobilized chimeric enzyme can be assayed at high temperature without protein leakage, demonstrating the usefulness of the ChB tag for noncovalent immobilization of thermophilic proteins . Moreover, ChBD-Pyr can be purified and immobilized in a single step on commercial DEAE-cellulose paper . The affinity of the ChB tag for this versatile solid support can be very helpful in developing many biotechnological applications . Growth of Campylobacter jejuni Supported by Respiration of Fumarate, Nitrate, Nitrite, Trimethylamine-N-Oxide, or Dimethyl Sulfoxide Requires Oxygen. Michael J. Sellars, 2002.The human gastrointestinal pathogen Campylobacter jejuni is a microaerophilic bacterium with a respiratory metabolism . The genome sequence of C . jejuni strain 11168 reveals the presence of genes that encode terminal reductases that are predicted to allow the use of a wide range of alternative electron acceptors to oxygen, including fumarate, nitrate, nitrite, and N- or S-oxides . All of these reductase activities were present in cells of strain 11168, and the molybdoenzyme encoded by Cj0264c was shown by mutagenesis to be responsible for both trimethylamine-N-oxide (TMAO) and dimethyl sulfoxide (DMSO) reduction . Nevertheless, growth of C . jejuni under strictly anaerobic conditions (with hydrogen or formate as electron donor) in the presence of any of the electron acceptors tested was insignificant . However, when fumarate, nitrate, nitrite, TMAO, or DMSO was added to microaerobic cultures in which the rate of oxygen transfer was severely restricted, clear increases in both the growth rate and final cell density compared to what was seen with the control were obtained, indicative of electron acceptor-dependent energy conservation . The C . jejuni genome encodes a single class I-type ribonucleotide reductase (RNR) which requires oxygen to generate a tyrosyl radical for catalysis . Electron microscopy of cells that had been incubated under strictly anaerobic conditions with an electron acceptor showed filamentation due to an inhibition of cell division similar to that induced by the RNR inhibitor hydroxyurea . An oxygen requirement for DNA synthesis can thus explain the lack of anaerobic growth of C . jejuni . The results indicate that strict anaerobiosis is a stress condition for C . jejuni but that alternative respiratory pathways can contribute significantly to energy conservation under oxygen-limited conditions, as might be found in vivo . Cross-Regulation between a Novel Two-Component Signal Transduction System for Catabolism of Toluene in Pseudomonas mendocina and the TodST System from Pseudomonas putida. María-Isabel Ramos-González, 2002.The tmoABCDEF genes encode the toluene-4-monooxygenase from Pseudomonas mendocina KR1 . Upstream from the tmoA gene an open reading frame, tmoX, encoding a protein 83% identical to TodX (todX being the initial gene in the todXFC1C2BADEGIH operon from Pseudomonas putida DOT-T1E) was found . The tmoX gene is also the initial gene in the tmoXABCDEF gene cluster . The transcription initiation point from the tmoX promoter was mapped, and the sequence upstream revealed striking identity with the promoter of the tod operon of P . putida . The tod operon is regulated by a two-component signal transduction system encoded by the todST genes . Two novel genes from P . mendocina KR1, tmoST, were rescued by complementation of a P . putida DOT-T1E todST knockout mutant, whose gene products shared about 85% identity with TodS-TodT . We show that transcription from PtmoX and PtodX can be mediated by TmoS-TmoT or TodS-TodT, in the presence of toluene, revealing cross-regulation between these two catabolic pathways . Novel Psychrophilic and Thermolabile L-Threonine Dehydrogenase from Psychrophilic Cytophaga sp . Strain KUC-1. Takayuki Kazuoka, 2003.A psychrophilic bacterium, Cytophaga sp . strain KUC-1, that abundantly produces a NAD+-dependent L-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified . The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000 . The enzyme is a homotetramer . Atomic absorption analysis showed that the enzyme contains no metals . In these respects, the Cytophaga enzyme is distinct from other L-threonine dehydrogenases that have thus far been studied . L-Threonine and DL-threo-3-hydroxynorvaline were the substrates, and NAD+ and some of its analogs served as coenzymes . The enzyme showed maximum activity at pH 9.5 and at 45°C . The kinetic parameters of the enzyme are highly influenced by temperatures . The Km for L-threonine was lowest at 20°C . Dead-end inhibition studies with pyruvate and adenosine-5'-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD+ binds to an enzyme prior to L-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH . The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced . The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues . The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily . In contrast, L-threonine dehydrogenase from E . coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme . L-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time . The amino acid residues playing an important role in the catalysis of the E . coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the substrate binding . Microbial Diversity of Biofilms in Dental Unit Water Systems. Ruby Singh, 2003.We investigated the microbial diversity of biofilms found in dental unit water systems (DUWS) by three methods . The first was microscopic examination by scanning electron microscopy (SEM), acridine orange staining, and fluorescent in situ hybridization (FISH) . Most bacteria present in the biofilm were viable . FISH detected the ß and  , but not the
 , subclasses of Proteobacteria. In the second method, 55 cultivated biofilm isolates were identified with the Biolog system, fatty acid analysis, and 16S ribosomal DNA (rDNA) sequencing . Only 16S identified all 55 isolates, which represented 13 genera . The most common organisms, as shown by analyses of 16S rDNA, belonged to the genera Afipia (28%) and Sphingomonas (16%) . The third method was a culture-independent direct amplification and sequencing of 165 subclones from community biofilm 16S rDNA . This method revealed 40 genera: the most common ones included Leptospira (20%), Sphingomonas (14%), Bacillus (7%), Escherichia (6%), Geobacter (5%), and Pseudomonas (5%) . Some of these organisms may be opportunistic pathogens . Our results have demonstrated that a biofilm in a health care setting may harbor a vast diversity of organisms . The results also reflect the limitations of culture-based techniques to detect and identify bacteria . Although this is the greatest diversity reported in DUWS biofilms, other genera may have been missed . Using a technique based on jackknife subsampling, we projected that a 25-fold increase in the number of subclones sequenced would approximately double the number of genera observed, reflecting the richness and high diversity of microbial communities in these biofilms .
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