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Wang Q.,Nankai University | Wang Q.,Key Laboratory of Molecular Microbiology and Technology | Wang Q.,Engineering and Research Center for Microbial Functional Genomics and Detection Technology | Xu Y.,Nankai University | And 15 more authors.
Journal of Bacteriology | Year: 2012

Streptococcus pneumoniae is a major human pathogen associated with many diseases worldwide. Capsular polysaccharides (CPSs) are the major virulence factor. The biosynthetic pathway of D-arabinitol, which is present in the CPSs of several S. pneumoniae serotypes, has never been identified. In this study, the genes abpA (previously known as abp1) and abpB (previously known as abp2), which have previously been reported to be responsible for nucleoside diphosphate (NDP)-D-arabinitol (the nucleotide- activated form of D-arabinitol) synthesis, were cloned. The enzyme products were overexpressed, purified, and analyzed for their respective activities. Novel products produced by AbpA- and AbpB-catalyzing reactions were detected by capillary electrophoresis, and the structures of the products were elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. As a result, abpA was identified to be a D-xylulose-5-phosphate cytidylyltransferase-encoding gene, responsible for the transfer of CTP to D-xylulose-5-phosphate (D-Xlu-5-P) to form CDP-D-xylulose, and abpB was characterized to be a CDP-D-xylulose reductase-encoding gene, responsible for the conversion of CDP-D-xylulose to CDP-D-arabinitol as the final product. The kinetic parameters of AbpA for the substrates D-Xlu-5-P and CTP and those of AbpB for the substrate CDP-D-xylulose and the cofactors NADH or NADPH were measured, and the effects of temperature, pH, and cations on the two enzymes were analyzed. This study confirmed the involvement of the genes abpA and abpB and their products in the biosynthetic pathway of CDP-D-arabinitol. © 2012, American Society for Microbiology. Source


Wang Q.,Nankai University | Wang Q.,Key Laboratory of Molecular Microbiology and Technology | Wang Q.,Engineering and Research Center for Microbial Functional Genomics and Detection Technology | Xu Y.,Nankai University | And 17 more authors.
Journal of Bacteriology | Year: 2010

Capsule polysaccharide (CPS) plays an important role in the virulence of Streptococcus pneumoniae and is usually used as the pneumococcal vaccine target. Glycerol-2-phosphate is found in the CPS of S. pneumoniae types 15A and 23F and is rarely found in the polysaccharides of other bacteria. The biosynthetic pathway of the nucleotide-activated form of glycerol-2-phosphate (NDP-2-glycerol) has never been identified. In this study, three genes (gtp1, gtp2, and gtp3) from S. pneumoniae 23F that have been proposed to be involved in the synthesis of NDP-2-glycerol were cloned and the enzyme products were expressed, purified, and assayed for their respective activities. Capillary electrophoresis was used to detect novel products from the enzyme-substrate reactions, and the structure of the product was elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Gtp1 was identified as a reductase that catalyzes the conversion of 1,3-dihydroxyacetone to glycerol, Gtp3 was identified as a glycerol-2- phosphotransferase that catalyzes the conversion of glycerol to glycerol-2-phosphate, and Gtp2 was identified as a cytidylyltransferase that transfers CTP to glycerol-2-phosphate to form CDP-2-glycerol as the final product. The kinetic parameters of Gtp1 and Gtp2 were characterized in depth, and the effects of temperature, pH, and cations on these two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-2-glycerol has been identified biochemically; this pathway provides a method to enzymatically synthesize this compound. Copyright © 2010, American Society for Microbiology. All Rights Reserved. Source


Zhu H.,Nankai University | Zhu H.,Key Laboratory of Molecular Microbiology and Technology | Wang Q.,Nankai University | Wang Q.,Key Laboratory of Molecular Microbiology and Technology | And 12 more authors.
Journal of Clinical Microbiology | Year: 2012

Neisseria meningitidis is a leading pathogen of epidemic bacterial meningitis and fulminant sepsis worldwide. Twelve different N. meningitidis serogroups have been identified to date based on antigenic differences in the capsular polysaccharide. However, more than 90% of human cases of N. meningitidis meningitis are the result of infection with just five serogroups, A, B, C, W135, and Y. Efficient methods of detection and genogrouping of N. meningitidis isolates are needed, therefore, in order to monitor prevalent serogroups as a means of disease control and prevention. The capsular gene complex regions have been sequenced from only seven out of the 12 serogroups. In this study, the capsular gene complexes of the remaining five serogroups were sequenced and analyzed. Primers were designed that were specific for N. meningitidis species and for the 12 individual serogroups, and a multiplex PCR assay using these specific primers was developed for N. meningitidis detection and genogrouping. The assay was tested using 15 reference strains covering all 12 serogroups, 143 clinical isolates, and 21 strains from closely related species or from species that cause meningitis. The assay could detect N. meningitidis serogroups and was shown to be specific, with a detection sensitivity of 1 ng of genomic DNA (equivalent to ∼4 × 10 5 genomes) or 3 × 10 5 CFU/ml in noncultured mock cerebrospinal fluid (CSF) specimens. This study, therefore, describes for the first time the development of a molecular protocol for the detection of all N. meningitidis serogroups. This multiplex PCR-based assay may have use for the clinical diagnosis and epidemiological surveillance of N. meningitidis. Copyright © 2012, American Society for Microbiology. All Rights Reserved. Source


Ren Y.,Nankai University | Ren Y.,Engineering and Research Center for Microbial Functional Genomics and Detection Technology | Ren Y.,Key Laboratory of Molecular Microbiology and Technology | Ren Y.,Tianjin Biochip Corporation | And 15 more authors.
Journal of Bacteriology | Year: 2010

Enterobacter cloacae is an important nosocomial pathogen. Here, we report the completion of the genome sequence of E. cloacae ATCC 13047, the type strain of E. cloacae subsp. cloacae. Multiple sets of virulence determinant and heavy-metal resistance genes have been found in the genome. To the best of our knowledge, this is the first complete genome sequence of the E. cloacae species. Copyright © 2010, American Society for Microbiology. All Rights Reserved. Source


Sun Y.,Nankai University | Sun Y.,Key Laboratory of Molecular Microbiology and Technology | Sun Y.,Engineering and Research Center for Microbial Functional Genomics and Detection Technology | Wang M.,Nankai University | And 24 more authors.
Applied and Environmental Microbiology | Year: 2011

Cronobacter sakazakii is an opportunistic pathogen that can cause severe infections. Serotyping provides a basis for the categorization of bacterial strains and is an important tool for epidemiological and surveillance purposes. In this study, of the 135 Cronobacter strains tested initially, 119 were identified as C. sakazakii and used. A serotyping scheme for C. sakazakii that classifies strains based on their different O antigens was developed. Seven antisera that exhibited high agglutinin titers (>640) were produced. O2 and O6 antisera were specific for their homologous strains, O4 and O7 antisera gave heterologous titers with O1 and O6 antigens, respectively, and O1, O3, and O5 antisera cross-reacted with each other and require preabsorption with the other two antigens. All of these 119 C. sakazakii strains were clearly assigned to these seven serotypes. O1 and O2 are the dominant serotypes, comprising 69.7% of the isolates. We also characterized the O-antigen gene clusters using restriction fragment length polymorphism (RFLP). The grouping of C. sakazakii strains based on their RFLP banding patterns correlated well with the grouping of strains based on our serotyping scheme. The serotype scheme presented here could prove to be a useful tool for serotyping C. sakazakii isolates. © 2011, American Society for Microbiology. Source

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