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Multilocus Sequence Typing of Streptococcus pyogenes Representing Most Known emm Types and Distinctions among Subpopulation Genetic Structures. Karen F. McGregor, 2004.A long-term goal is to characterize the full range of genetic diversity within Streptococcus pyogenes as it exists in the world today . Since the emm locus is subject to strong diversifying selection, emm type was used as a guide for identifying a genetically diverse set of strains . This report contains a description of multilocus sequence typing based on seven housekeeping loci for 495 isolates representing 158 emm types, yielding 238 unique combinations of sequence type and emm type . A genotypic marker for tissue site preference (emm pattern) revealed that only 17% of the emm types displayed the marker representing strong preference for infection at the throat and that 39% of emm types had the marker for skin tropism, whereas 41% of emm types harbored the marker for no obvious tissue site preference . As a group, the emm types bearing the emm pattern marker indicative of no obvious tissue site preference were far less likely to have two distinct emm types associated with the same sequence type than either of the two subpopulations having markers for strong tissue tropisms (P < 0.002) . In addition, all genetic diversification events clearly ascribed to a recombinational mechanism involved strains of only two of the emm pattern-defined subpopulations, those representing skin specialists and generalists . The findings suggest that the population genetic structure differs for the tissue-defined subpopulations of S . pyogenes . The observed differences may partly reflect differential host immune selection pressures . Degradation and Turnover of Extracellular DNA in Marine Sediments: Ecological and Methodological Considerations. Antonio Dell'Anno, 2004. Identification of a New Class of Cytochrome P450 from a Rhodococcus sp.. Gareth A. Roberts, 2002.A degenerate set of PCR primers were used to clone a gene encoding a cytochrome P450 (the P450RhF gene) from Rhodococcus sp . strain NCIMB 9784 which is of unique primary structural organization . Surprisingly, analysis of the translation product revealed that the P450 is fused to a reductase domain at the C terminus which displays sequence conservation for dioxygenase reductase proteins . The reductase partner comprises flavin mononucleotide- and NADH-binding motifs and a [2Fe2S] ferredoxin-like center . The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a soluble form . A recombinant strain of E . coli was able to mediate the O dealkylation of 7-ethoxycoumarin in good yield, despite the absence of any recombinant redox proteins . This unprecedented finding leads us to propose that P450RhF represents the first example of a new class of cytochromes P450 in which the reducing equivalents are supplied by a novel reductase in a fused arrangement . cis-Acting Sequences of Bacillus subtilis pyrG mRNA Essential for Regulation by Antitermination. Qi Meng, 2002.Expression of the Bacillus subtilis pyrG gene, which encodes CTP synthetase, is repressed by cytidine nucleotides . Regulation involves a termination-antitermination mechanism acting at a transcription terminator located within the 5' untranslated pyrG leader sequence . Deletion and substitution mutagenesis of a series of pyrG'-lacZ transcriptional fusions integrated into the B . subtilis chromosome demonstrated that only the terminator stem-loop and two specific 4- to 6-nucleotide RNA sequences were required for derepression of pyrG by starvation for cytidine nucleotides . The first sequence, GGGC/U, comprises the first four nucleotides at the 5' end of the pyrG transcript, and the second, GCUCCC, forms the first six nucleotides of the 5' strand of the terminator stem . All of the nucleotides lying between the two required RNA sequences can be deleted without loss of regulation . We propose that an as-yet-unidentified regulatory protein binds to these two RNA segments and prevents termination of transcription in the pyrG leader region when intracellular CTP levels are low . A Bacterial TrwC Relaxase Domain Contains a Thermally Stable  -Helical Core.José-Luis R. Arrondo, 2003.The TrwC protein is the relaxase-helicase responsible for the initiation and termination reactions of DNA processing during plasmid R388 conjugation . The TrwC-N275 fragment comprises the 275-amino-acid N-terminal domain of the protein that contains the DNA cleavage and strand transfer activities (the relaxase domain) . It can be easily purified by keeping a cell lysate at 90°C for 10 min . Infrared spectroscopy shows that this domain has a predominantly /ß structure with some amount of unordered structure . Fast heating and cooling does not change the secondary structure, whereas slow heating produces two bands in the infrared spectrum characteristic of protein aggregation . The denaturation temperature is increased in the protein after the fast-heating thermal shock . Two-dimensional infrared correlation spectroscopy shows that thermal unfolding is a very cooperative two-state process without any appreciable steps prior to aggregation . After aggregation, the
-helix percentage is not altered and
-helix signal does not show in the correlation maps, meaning that the helices are not affected by heating . The results indicate that the domain has an
-helix core resistant to temperature and responsible for folding after fast heating and an outer layer of ß-sheet and unordered structure that aggregates under slow heating . The combination of a compact core and a flexible outer layer could be related to the structural requirements of DNA-protein binding .
The CroRS Two-Component Regulatory System Is Required for Intrinsic ß-Lactam Resistance in Enterococcus faecalis. Yannick Comenge, 2003.Enterococcus faecalis produces a specific penicillin-binding protein (PBP5) that mediates high-level resistance to the cephalosporin class of ß-lactam antibiotics . Deletion of a locus encoding a previously uncharacterized two-component regulatory system of E . faecalis (croRS) led to a 4,000-fold reduction in the MIC of the expanded-spectrum cephalosporin ceftriaxone . The cytoplasmic domain of the sensor kinase (CroS) was purified and shown to catalyze ATP-dependent autophosphorylation followed by transfer of the phosphate to the mated response regulator (CroR) . The croR and croS genes were cotranscribed from a promoter (croRp) located in the rrnC-croR intergenic region . A putative seryl-tRNA synthetase gene (serS) located immediately downstream from croS did not appear to be a target of CroRS regulation or to play a role in ceftriaxone resistance . A plasmid-borne croRp-lacZ fusion was trans-activated by the CroRS system in response to the presence of ceftriaxone in the culture medium . The fusion was also induced by representatives of other classes of ß-lactam antibiotics and by inhibitors of early and late steps of peptidoglycan synthesis . The croRS null mutant produced PBP5, and expression of an additional copy of pbp5 under the control of a heterologous promoter did not restore ceftriaxone resistance . Deletion of croRS was not associated with any defect in the synthesis of the nucleotide precursor UDP-MurNAc-pentapeptide or of the D-Ala4  L-Ala-L-Ala-Lys3
peptidoglycan cross-bridge . Thus, the croRS mutant was
susceptible to ceftriaxone despite the production of PBP5 and the
synthesis of wild-type peptidoglycan precursors . These observations
constitute the first description of regulatory genes essential for
PBP5-mediated ß-lactam resistance in
enterococci .
Cloning, Characterization, and Functional Expression of the Klebsiella oxytoca Xylodextrin Utilization Operon (xynTB) in Escherichia coli. Yilei Qian, 2003.Escherichia coli is being developed as a biocatalyst for bulk chemical production from inexpensive carbohydrates derived from lignocellulose . Potential substrates include the soluble xylodextrins (xyloside, xylooligosaccharide) and xylobiose that are produced by treatments designed to expose cellulose for subsequent enzymatic hydrolysis . Adjacent genes encoding xylobiose uptake and hydrolysis were cloned from Klebsiella oxytoca M5A1 and are functionally expressed in ethanologenic E . coli . The xylosidase encoded by xynB contains the COG3507 domain characteristic of glycosyl hydrolase family 43 . The xynT gene encodes a membrane protein containing the MelB domain (COG2211) found in Na+/melibiose symporters and related proteins . These two genes form a bicistronic operon that appears to be regulated by xylose (XylR) and by catabolite repression in both K . oxytoca and recombinant E . coli . Homologs of this operon were found in Klebsiella pneumoniae, Lactobacillus lactis, E . coli, Clostridium acetobutylicum, and Bacillus subtilis based on sequence comparisons . Based on similarities in protein sequence, the xynTB genes in K . oxytoca appear to have originated from a gram-positive ancestor related to L . lactis . Functional expression of xynB allowed ethanologenic E . coli to metabolize xylodextrins (xylosides) containing up to six xylose residues without the addition of enzyme supplements . 4-O-methylglucuronic acid substitutions at the nonreducing termini of soluble xylodextrins blocked further degradation by the XynB xylosidase . The rate of xylodextrin utilization by recombinant E . coli was increased when a full-length xynT gene was included with xynB, consistent with xynT functioning as a symport . Hydrolysis rates were inversely related to xylodextrin chain length, with xylobiose as the preferred substrate . Xylodextrins were utilized more rapidly by recombinant E . coli than K . oxytoca M5A1 (the source of xynT and xynB) . XynB exhibited weak arabinosidase activity, 3% that of xylosidase .
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