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Modeling Bacterial Evolution with Comparative-Genome-Based Marker Systems: Application to Mycobacterium tuberculosis Evolution and Pathogenesis. David Alland, 2003.The comparative-genomic sequencing of two Mycobacterium tuberculosis strains enabled us to identify single nucleotide polymorphism (SNP) markers for studies of evolution, pathogenesis, and epidemiology in clinical M . tuberculosis . Phylogenetic analysis using these "comparative-genome markers" (CGMs) produced a highly unusual phylogeny with a complete absence of secondary branches . To investigate CGM-based phylogenies, we devised computer models to simulate sequence evolution and calculate new phylogenies based on an SNP format . We found that CGMs represent a distinct class of phylogenetic markers that depend critically on the genetic distances between compared "reference strains." Properly distanced reference strains generate CGMs that accurately depict evolutionary relationships, distorted only by branch collapse . Improperly distanced reference strains generate CGMs that distort and reroot outgroups . Applying this understanding to the CGM-based phylogeny of M . tuberculosis, we found evidence to suggest that this species is highly clonal without detectable lateral gene exchange . We noted indications of evolutionary bottlenecks, including one at the level of the PHRI "C" strain previously associated with particular virulence characteristics . Our evidence also suggests that loss of IS6110 to fewer than seven elements per genome is uncommon . Finally, we present population-based evidence that KasA, an important component of mycolic acid biosynthesis, develops G312S polymorphisms under selective pressure . Molecular Basis of Resistance to Macrolides and Other Antibiotics in Commensal Viridans Group Streptococci and Gemella spp . and Transfer of Resistance Genes to Streptococcus pneumoniae. Paula Cerdá Zolezzi, 2004.We assessed the mechanisms of resistance to macrolide-lincosamide-streptogramin B (MLSB) antibiotics and related antibiotics in erythromycin-resistant viridans group streptococci (n = 164) and Gemella spp . (n = 28) . The macrolide resistance phenotype was predominant (59.38%); all isolates with this phenotype carried the mef(A) or mef(E) gene, with mef(E) being predominant (95.36%) . The erm(B) gene was always detected in strains with constitutive and inducible MLSB resistance and was combined with the mef(A/E) gene in 47.44% of isolates . None of the isolates carried the erm(A) subclass erm(TR), erm(A), or erm(C) genes . The mel gene was detected in all but four strains carrying the mef(A/E) gene . The tet(M) gene was found in 86.90% of tetracycline-resistant isolates and was strongly associated with the presence of the erm(B) gene . The catpC194 gene was detected in seven chloramphenicol-resistant Streptococcus mitis isolates, and the aph(3')-III gene was detected in four viridans group streptococcal isolates with high-level kanamycin resistance . The intTn gene was found in all isolates with the erm(B), tet(M), aph(3')-III, and catpC194 gene . The mef(E) and mel genes were successfully transferred from both groups of bacteria to Streptococcus pneumoniae R6 by transformation . Viridans group streptococci and Gemella spp . seem to be important reservoirs of resistance genes . Novel DNA-Binding Proteins in the Cyanobacterium Anabaena sp . Strain PCC 7120. Olga A. Koksharova, 2002.As an approach towards elucidation of the biochemical regulation of the progression of heterocyst differentiation in Anabaena sp . strain PCC 7120, we have identified proteins that bind to a 150-bp sequence upstream from hepC, a gene that plays a role in the synthesis of heterocyst envelope polysaccharide . Such proteins were purified in four steps from extracts of vegetative cells of Anabaena sp . Two of these proteins (Abp1 and Abp2) are encoded by neighboring genes in the Anabaena sp . chromosome . The genes that encode the third (Abp3) and fourth (Abp4) proteins are situated at two other loci in that chromosome . Insertional mutagenesis of abp2 and abp3 blocked expression of hepC and hepA and prevented heterocyst maturation and aerobic fixation of N2 . Mycobacterium bovis BCG Response Regulator Essential for Hypoxic Dormancy. Calvin Boon, 2002.Obligately aerobic tubercle bacilli are capable of adapting to survive hypoxia by developing into a nonreplicating or dormant form . Dormant bacilli maintain viability for extended periods . Furthermore, they are resistant to antimycobacterials, and hence, dormancy might play a role in the persistence of tuberculosis infection despite prolonged chemotherapy . Previously, we have grown dormant Mycobacterium bovis BCG in an oxygen-limited Wayne culture system and subjected the bacilli to proteome analysis . This work revealed the upregulation of the response regulator Rv3133c and three other polypeptides (  -crystallin and two "conserved hypothetical" proteins) upon entry into dormancy . Here, we replaced the coding sequence of the response regulator with a kanamycin resistance cassette and demonstrated that the loss-of-function mutant died after oxygen starvation-induced termination of growth . Thus, the disruption of this dormancy-induced transcription factor resulted in loss of the ability of BCG to adapt to survival of hypoxia . Two-dimensional gel electrophoresis of protein extracts from the gene-disrupted strain showed that the genetic loss of the response regulator caused loss of the induction of the other three dormancy proteins . Thus, the upregulation of these dormancy proteins requires the response regulator . Based on these two functions, dormancy survival and regulation, we named the Rv3133c gene dosR for dormancy survival regulator . Our results provide conclusive evidence that DosR is a key regulator in the oxygen starvation-induced mycobacterial dormancy response .
Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts. Haitham A. Hussain, 2003.The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels . Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed . Analysis of the expression levels of the P450 proteins in several different E . coli expression hosts identified E . coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra . Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E . coli tRNA species . The level of its expression was improved in the modified forms of E . coli BL21(DE3), which contain extra copies of rare codon E . coli tRNA genes . The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins . Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160 . S . lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin . A Multiplex Reverse Transcription-PCR Method for Detection of Human Enteric Viruses in Groundwater. G. Shay Fout, 2003.Untreated groundwater is responsible for about half of the waterborne disease outbreaks in the United States . Human enteric viruses are thought to be leading etiological agents of many of these outbreaks, but there is relatively little information on the types and levels of viruses found in groundwater . To address this problem, monthly samples from 29 groundwater sites were analyzed for 1 year for enteroviruses, hepatitis A virus, Norwalk virus, reoviruses, and rotaviruses by multiplex reverse transcription-PCR (RT-PCR) . A procedure with which to remove environmental RT-PCR inhibitors from groundwater samples was developed . The procedure allowed an average of 71 liters of the original groundwater to be assayed per RT-PCR, with an average virus recovery rate of 74%, based on seeded samples . Human enteric viruses were detected in 16% of the groundwater samples analyzed, with reoviruses being the most frequently detected virus group . Transformation of Fatty Acids Catalyzed by Cytochrome P450 Monooxygenase Enzymes of Candida tropicalis. William H. Eschenfeldt, 2003.Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in ß-oxidation convert these substrates to long-chain  , -dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992) . The initial step in the formation of these diacids, which is thought to be rate limiting, is
-hydroxylation by a cytochrome P450 (CYP) monooxygenase . C . tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C18:1), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue) . To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C . tropicalis cytochrome P450 reductase (NCP) . Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids . CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to
-hydroxy acids . CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C14:0) much more effectively . Both enzymes, in particular CYP52A17, also oxidized
-hydroxy fatty acids, ultimately generating the
,-diacid . Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for ß-oxidation to enhance the production of dicarboxylic acids . The activity spectrum also identified other potential oxidation targets for commercial development .
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