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Functionally Critical Elements of CooA-Related CO Sensors. Hwan Youn, 2004.CooA is a heme-containing transcriptional activator that enables Rhodospirillum rubrum to sense and grow on CO as a sole energy source . We have identified a number of CooA homologs throughdatabase searches, expressed these heterologously in Escherichiacoli, and monitored their ability to respond to CO in vivo.Further in vitro analysis of two CooA homologs from Azotobactervinelandii and Carboxydothermus hydrogenoformans corroboratedthe in vivo data by revealing the ability of CO to bind to thesehemoproteins and stimulate their binding at specific DNA sequences.These data, as well as the patterns of conserved residues inthe homologs, are compared to what is already known about functionallyimportant residues in the CooA protein of R . rubrum . The results identify critical regions of CooA and indicate features that distinguish CooAs from the general family of cyclic AMP receptor proteins. Distribution, Genetic Diversity, and Variable Expression of the Gene Encoding Hyaluronate Lyase within the Streptococcus suis Population. Samantha J. King, 2004.Although Streptococcus suis is an economically important pathogen of pigs and an occasional cause of zoonotic infections of humans knowledge of crucial virulence factors, and as a consequence targets for therapeutic or prophylactic intervention, remains limited . Here we describe a detailed study of the distribution, diversity, and in vitro expression of hyaluronate lyase, a protein implicated as a virulence factor of many mucosal pathogens . The gene encoding hyaluronate lyase, hyl, was present in all 309 bona fide S . suis isolates examined representing diverse serotypes, geographic sources, and clinical backgrounds . Examination of the genetic diversity of hyl by RFLP and sequence analysis indicated a pattern of diversity shared by many gram-positive surface proteins with a variable 5' region encoding the most distal cell surface-exposed regions of the protein and a much more conserved 3' region encoding domains more closely associated with the bacterial cell . Variation occurs by several mechanisms, including the accumulation of point mutations and deletion and insertion events, and there is clear evidence that genetic recombination has contributed to molecular variation in this gene . Despite the ubiquitous presence of hyl, the corresponding enzyme activity was detected in fewer than 30% of the 309 isolates . In several cases this lack of activity correlates with the presence of mutations (either sequence duplications or point mutations) within hyl that result in a truncated polypeptide . There is a striking absence of hyaluronate lyase activity in a large majority of isolates from classic S . suis invasive disease, indicating that this protein is probably not a crucial virulence factor, although activity is present in significantly higher numbers of isolates associated with pneumonia . Disposition of Artesunate and Dihydroartemisinin after Administration of Artesunate Suppositories in Children from Papua New Guinea with Uncomplicated Malaria. Harin A. Karunajeewa, 2004.A detailed pharmacokinetic analysis was performed with 47 children from Papua New Guinea with uncomplicated falciparum or vivax malaria treated with artesunate (ARTS) suppositories (Rectocaps) given in two doses of approximately 13 mg/kg of body weight 12 h apart . Following an intensive sampling protocol, samples were assayed for ARTS and its primary active metabolite, dihydroartemisinin (DHA), by liquid chromatography-mass spectrometry . A population pharmacokinetic model was developed to describe the data . Following administration of the first dose, the mean maximal concentrations of ARTS and DHA were 1,085 nmol/liter at 0.9 h and 2,525 nmol/liter at 2.3 h, respectively . The absorption half-life for ARTS was 2.3 h, and the conversion half-life (ARTS to DHA) was 0.27 h, while the elimination half-life of DHA was 0.71 h . The mean common volumes of distribution for ARTS and DHA relative to bioavailability were 42.8 and 2.04 liters/kg, respectively, and the mean clearance values relative to bioavailability were 6 and 2.2 liters/h/kg for ARTS and DHA, respectively . Substantial interpatient variability was observed, and the bioavailability of the second dose relative to that of the first was estimated to be 0.72 . The covariates age, sex, and  -thalassemia genotype were not influential in the pharmacokinetic model development; but the inclusion of weight as a covariate significantly improved the performance of the model . An ARTS suppositories dose of 10 of 20 mg/kg is appropriate for use in children with uncomplicated malaria .
Yersinia enterocolitica Type III Secretion: Mutational Analysis of the yopQ Secretion Signal. Kumaran S. Ramamurthi, 2002.Pathogenic Yersinia spp . secrete Yop proteins via the type III pathway . yopQ codons 1 to 15 were identified as a signal necessary and sufficient for the secretion of a fused reporter protein . Frameshift mutations that alter codons 2 to 15 with little alteration of yopQ mRNA sequence do not abolish type III transport, suggesting a model in which yopQ mRNA may provide a signal for secretion (D . M . Anderson and O . Schneewind, Mol . Microbiol . 31:1139-1148, 2001) . In a recent study, the yopE signal was truncated to codons 1 to 12 . All frameshift mutations introduced within the first 12 codons of yopE abolished secretion . Also, multiple synonymous mutations that changed the mRNA sequence of yopE codons 1 to 12 without altering the amino acid sequence did not affect secretion . These results favor a model whereby an N-terminal signal peptide initiates YopE into the type III pathway (S . A . Lloyd et al., Mol . Microbiol . 39:520-531, 2001) . It is reported here that codons 1 to 10 of yopQ act as a minimal secretion signal . Further truncation of yopQ, either at codon 10 or at codon 2, abolished secretion . Replacement of yopQ AUG with either of two other start codons, UUG or GUG, did not affect secretion . However, replacement of AUG with CUG or AAA and initiating translation at the fusion site with npt did not permit Npt secretion, suggesting that the translation of yopQ codons 1 to 15 is a prerequisite for secretion . Frameshift mutations of yopQ codons 1 to 10, 1 to 11, and 1 to 12 abolished secretion signaling, whereas frameshift mutations of yopQ codons 1 to 13, 1 to 14, and 1 to 15 did not . Codon changes at yopQ positions 2 and 10 affected secretion signaling when placed within the first 10 codons but had no effect when positioned in the larger fusion of yopQ codons 1 to 15 . An mRNA mutant of yopQ codons 1 to 10, generated by a combination of nine synonymous mutations, was defective in secretion signaling, suggesting that the YopQ secretion signal is not proteinaceous . A model is discussed whereby the initiation of YopQ polypeptide into the type III pathway is controlled by properties of yopQ mRNA . A DNA Methyltransferase Can Protect the Genome from Postdisturbance Attack by a Restriction-Modification Gene Complex. Noriko Takahashi, 2002.In prokaryotic genomes, some DNA methyltransferases form a restriction-modification gene complex, but some others are present by themselves . Dcm gene product, one of these orphan methyltransferases found in Escherichia coli and related bacteria, methylates DNA to generate 5'-CmCWGG just as some of its eukaryotic homologues do . Vsr mismatch repair function of an adjacent gene prevents C-to-T mutagenesis enhanced by this methylation but promotes other types of mutation and likely has affected genome evolution . The reason for the existence of the dcm-vsr gene pair has been unclear . Earlier we found that several restriction-modification gene complexes behave selfishly in that their loss from a cell leads to cell killing through restriction attack on the genome . There is also increasing evidence for their potential mobility . EcoRII restriction-modification gene complex recognizes the same sequence as Dcm, and its methyltransferase is phylogenetically related to Dcm . In the present work, we found that stabilization of maintenance of a plasmid by linkage of EcoRII gene complex, likely through postsegregational cell killing, is diminished by dcm function . Disturbance of EcoRII restriction-modification gene complex led to extensive chromosome degradation and severe loss of cell viability . This cell killing was partially suppressed by chromosomal dcm and completely abolished by dcm expressed from a plasmid . Dcm, therefore, can play the role of a "molecular vaccine" by defending the genome against parasitism by a restriction-modification gene complex . Arsenic Resistance in Halobacterium sp . Strain NRC-1 Examined by Using an Improved Gene Knockout System. Gejiao Wang, 2004.The genome sequence of Halobacterium sp . strain NRC-1 encodes genes homologous to those responsible for conferring resistance to arsenic . These genes occur on both the large extrachromosomal replicon pNRC100 (arsADRC and arsR2M) and on the chromosome (arsB) . We studied the role of these ars genes in arsenic resistance genetically by construction of gene knockouts . Deletion of the arsADRC gene cluster in a Halobacterium NRC-1  ura3 strain resulted in increased sensitivity to arsenite and antimonite but not arsenate . In contrast, knockout of the chromosomal arsB gene did not show significantly increased sensitivity to arsenite or arsenate . We also found that knockout of the arsM gene produced sensitivity to arsenite, suggesting a second novel mechanism of arsenic resistance involving a putative arsenite(III)-methyltransferase . These results indicate that Halobacterium sp . strain NRC-1 contains an arsenite and antimonite extrusion system with significant differences from bacterial counterparts . Deletion analysis was facilitated by an improved method for gene knockouts/replacements in Halobacterium that relies on both selection and counterselection of ura3 using a uracil dropout medium and 5-fluoroorotic acid . The arsenite and antimonite resistance elements were shown to be regulated, with resistance to arsenic in the wild type inducible by exposure to a sublethal concentration of the metal . Northern hybridization and reverse transcription-PCR analyses showed that arsA, arsD, arsR, arsM, arsC, and arsB, but not arsR2, are inducible by arsenite and antimonite . We discuss novel aspects of arsenic resistance in this halophilic archaeon and technical improvements in our capability for gene knockouts in the genome .
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