Engineering and Research Center for Microbial Functional Genomics and Detection Technology

Tianjin, China

Engineering and Research Center for Microbial Functional Genomics and Detection Technology

Tianjin, China
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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 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.


Maharjan R.,University of Sydney | Zhou Z.,Nankai University | Zhou Z.,Tianjin Key Laboratory of Microbial Functional Genomics | Ren Y.,Nankai University | And 13 more authors.
Journal of Bacteriology | Year: 2010

Beneficial mutations in diversifying glucose-limited Escherichia coli populations are mostly unidentified. The genome of an evolved isolate with multiple differences from that of the ancestor was fully assembled. Remarkably, a single mutation in hfq was responsible for the multiple benefits under glucose limitation through changes in at least five regulation targets. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


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 | Shao Z.,Chinese National Institute for Communicable Disease Control and Prevention | And 13 more authors.
Infection and Immunity | Year: 2010

Neisseria meningitidis is a leading cause of septicemia and meningitis worldwide. N. meningitidis capsular polysaccharides have been classified into 13 distinct serogroups which are defined by antibody reactivity and structural analysis, and the capsule plays an important role in virulence. Serogroups A, B, C, W135, and Y have been reported to be clinically important. Several newly identified serogroup C isolates belonging to the unique sequence type 7 (ST-7) were identified in China. Since most ST-7 isolates from China belonged to serogroup A, the newly identified ST-7 serogroup C strains were proposed to have arisen from those belonging to ST-7 serogroup A. In this study, six ST-7 serogroup C and three ST-7 serogroup A isolates were analyzed by pulsed-field gel electrophoresis to confirm their sequence type. In order to clarify the genetic basis of capsular switching between ST-7 serogroup A and C strains, the whole capsular gene clusters and surrounding genes of the two representative ST-7 strains belonging to serogroups A and C, respectively, were sequenced and compared. Potential recombination sites were analyzed using the RDP3 beta software, and recombination-related regions in two other ST-7 serogroup A and five ST-7 serogroup C strains were also sequenced and compared to the representative ST-7 serogroup A and C strain sequences. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


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 | Wang S.,Nankai University | And 14 more authors.
Journal of Clinical Microbiology | Year: 2010

Enterotoxigenic Escherichia coli (ETEC) is a common pathogen worldwide causing infectious diarrhea, especially traveler's diarrhea. Traditional physiological assays, immunoassays, and PCR-based methods for the detection of ETEC target the heat-labile enterotoxin and/or the heat-stable enterotoxin. Separate serotyping methods using antisera are required to determine the ETEC serogroup. In this study, we developed a DNA microarray that can simultaneously detect enterotoxin genes and the 19 most common O serogroup genes in ETEC strains. The specificity and reproducibility of this approach were verified by hybridization to 223 strains: 50 target reference or clinical strains and 173 other strains, including those belonging to other E. coli O serogroups and closely related species. The sensitivity of detection was determined to be 50 ng of genomic DNA or 108 CFU per ml of organisms in pure culture. The random PCR strategy used in this study with minimal bias provides an effective alternative to multiplex PCR for the detection of pathogens using DNA microarrays. The assay holds promise for applications in the clinical diagnosis and epidemiological surveillance of pathogenic microorganisms. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Dong Y.,Nankai University | Dong Y.,Tianjin Key Laboratory of Microbial Functional Genomics | Dong Y.,Tianjin Research Center for Functional Genomics and Biochip | Yan J.,Nankai University | And 15 more authors.
Applied Microbiology and Biotechnology | Year: 2012

LadA, a monooxygenase catalyzing the oxidation of n-alkanes to 1-alkanols, is the key enzyme for the degradation of long-chain alkanes (C15-C36) in Geobacillus thermodenitrificans NG80-2. In this study, random- and sitedirected mutagenesis were performed to enhance the activity of the enzyme. By screening 7,500 clones from randommutant libraries for enhanced hexadecane hydroxylation activity, three mutants were obtained: A102D, L320V, and F146C/N376I. By performing saturation site-directed mutagenesis at the 102, 320, 146, and 376 sites, six more mutants (A102E, L320A, F146Q/N376I, F146E/N376I, F146R/N376I, and F146N/N376I) were generated. Kinetic studies showed that the hydroxylation activity of purified LadA mutants on hexadecane was 2-3.4-fold higher than that of the wild-type enzyme, with the activity of F146N/N376I being the highest. Effects of the mutations on optimum temperature, pH, and heat stability of LadA were also investigated. A complementary study showed that Pseudomonas fluorescens KOB2Δ1 strains expressing the LadA mutants grew more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutants in vivo. Structural changes resulting from the mutations were analyzed and the correlation between structural changes and enzyme activity was discussed. The mutants generated in this study are potentially useful for the treatment of environmental oil pollution and in other bioconversion processes. © Springer-Verlag 2012.


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.


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.


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.


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.


Xu Y.-Y.,Nankai University | Dong C.-Y.,Nankai University | Zhou D.-W.,Nankai University | Zhou D.-W.,Engineering and Research Center for Microbial Functional Genomics and Detection Technology
Journal of Chinese Mass Spectrometry Society | Year: 2016

Considering the importance of carbohydrate moieties on infectivity and host mimicry, there is a need to better understand the biosynthetic pathways of these unusual sugars in order to identify key targets involved in bacterial pathogenesis. Since the in vitro biochemical characterization of the biosynthetic pathway of unusual sugars is currently hindered by the demand for more accurate, sensitive, and rapid analytical methods for characterizing unusual sugar structures, information about the structure and biosynthesis pathway of these compounds is fragmentary. Mass spectrometry is a rapid, sensitive, and accurate approach for the direct monitoring of enzyme-catalyzed reactions that does not require a chromophore or radiolabeling and thus provides aviable alternative to existing analytical techniques. The objective of this study is to demonstrate the use of electrospray ionization-tandem mass spectrometry (ESI-MS/MS) as a powerful technique for the characterization of enzymatic products in the biosynthetic pathway of deoxythymidine 5'-diphosphate-D-rhamnose (dTDP-L-Rha) in E. coliO7. The dTDP-d-glucose 4,6-dehydratase (RmlB), dTDP-4-keto-6-deoxy-glucose-3,5 epimerase (RmlC), and dTDP-4-keto-rhamnose reductase (RmlD) catalyzed reactions were directly monitored by ESI-MS, followed by detailed structural characterization of the final enzymatic products using ESI-MS/MS in the negative-ion mode after minimal cleanup. The biosynthetic pathway of dTDP-L-Rha, beginning from dTDP-L-Rha in three reaction steps catalyzed by RmlB, RmlC, and RmlD, was characterized by ESI-MS/MS. The results obtained were in good agreement with that of traditional high-performance liquid chromatography (HPLC) monitoring and preparation, as well as NMR and ESI-MS structural characterization. Collectively, these data demonstrate that a CID-ESI-MSn based platform is applicable to the facile characterization of the biosynthetic pathway of important unusual dTNP-sugar in the O-chain and offers significant advantages over current methods in terms of speed, sensitivity, reproducibility, automation and reagent costs. © 2016, Editorial Board of Journal of Chinese Mass Spectrometry Society. All right reserved.

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