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Hu D.,Nankai University | Liu B.,Nankai University | Liu B.,The Key Laboratory of Molecular Microbiology and Technology | Dijkshoorn L.,Leiden University | And 4 more authors.
PLoS ONE | Year: 2013

We have sequenced the gene clusters for type strains of the Acinetobacter baumannii serotyping scheme developed in the 1990s, and used the sequences to better understand diversity in surface polysaccharides of the genus. We obtained genome sequences for 27 available serovar type strains, and identified 25 polysaccharide gene cluster sequences. There are structures for 12 of these polysaccharides, and in general the genes present are appropriate to the structure where known. This greatly facilitates interpretation. We also find 53 different glycosyltransferase genes, and for 7 strains can provisionally allocate specific genes to all linkages. We identified primers that will distinguish the 25 sequence forms by PCR or microarray, or alternatively the genes can be used to determine serotype by "molecular serology". We applied the latter to 190 Acinetobacter genome-derived gene-clusters, and found 76 that have one of the 25 gene-cluster forms. We also found novel gene clusters and added 52 new gene-cluster sequence forms with different wzy genes and different gene contents. Altogether, the strains that have one of the original 25 sequence forms include 98 A. baumannii (24 from our strains) and 5 A. nosocomialis (3 from our strains), whereas 32 genomes from 12 species other than A. baumannii or A. nosocomialis, all have new sequence forms. One of the 25 serovar type sequences is found to be in European clone I (EC I), 2 are in EC II but none in EC III. The public genome strains add an additional 52 new sequence forms, and also bring the number found in EC I to 5, in EC II to 9 and in EC III to 2. © 2013 Hu et al.


Liu B.,Nankai University | Liu B.,The Key Laboratory of Molecular Microbiology and Technology | Knirel Y.A.,RAS N. D. Zelinsky Institute of Organic Chemistry | Feng L.,Nankai University | And 9 more authors.
FEMS Microbiology Reviews | Year: 2014

This review covers the structures and genetics of the 46 O antigens of Salmonella, a major pathogen of humans and domestic animals. The variation in structures underpins the serological specificity of the 46 recognized serogroups. The O antigen is important for the full function and virulence of many bacteria, and the considerable diversity of O antigens can confer selective advantage. Salmonella O antigens can be divided into two major groups: those which have N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc) and those which have galactose (Gal) as the first sugar in the O unit. In recent years, we have determined 21 chemical structures and sequenced 28 gene clusters for GlcNAc-/GalNAc-initiated O antigens, thus completing the structure and DNA sequence data for the 46 Salmonella O antigens. The structures and gene clusters of the GlcNAc-/GalNAc-initiated O antigens were found to be highly diverse, and 24 of them were found to be identical or closely related to Escherichia coli O antigens. Sequence comparisons indicate that all or most of the shared gene clusters were probably present in the common ancestor, although alternative explanations are also possible. In contrast, the better-known eight Gal-initiated O antigens are closely related both in structures and gene cluster sequences. In this review, we systematically analyzed and summarized Salmonella O-antigen diversity including the chemical structures, gene cluster sequences, and evolutionary aspects. © 2013 Federation of European Microbiological Societies.


Reeves P.R.,University of Sydney | Liu B.,Nankai University | Liu B.,The Key Laboratory of Molecular Microbiology and Technology | Zhou Z.,Nankai University | And 13 more authors.
PLoS ONE | Year: 2011

Although over 50 complete Escherichia coli/Shigella genome sequences are available, it is only for closely related strains, for example the O55:H7 and O157:H7 clones of E. coli, that we can assign differences to individual evolutionary events along specific lineages. Here we sequence the genomes of 14 isolates of a uropathogenic E. coli clone that persisted for 3 years within a household, including a dog, causing a urinary tract infection (UTI) in the dog after 2 years. The 20 mutations observed fit a single tree that allows us to estimate the mutation rate to be about 1.1 per genome per year, with minimal evidence for adaptive change, including in relation to the UTI episode. The host data also imply at least 6 host transfer events over the 3 years, with 2 lineages present over much of that period. To our knowledge, these are the first direct measurements for a clone in a well-defined host community that includes rates of mutation and host transmission. There is a concentration of non-synonymous mutations associated with 2 transfers to the dog, suggesting some selection pressure from the change of host. However, there are no changes to which we can attribute the UTI event in the dog, which suggests that this occurrence after 2 years of the clone being in the household may have been due to chance, or some unknown change in the host or environment. The ability of a UTI strain to persist for 2 years and also to transfer readily within a household has implications for epidemiology, diagnosis, and clinical intervention. © 2011 Reeves et al.


Li D.,Nankai University | Li D.,Tianjin Key Laboratory of Microbial Functional Genomics | Liu B.,Nankai University | Liu B.,Tianjin Key Laboratory of Microbial Functional Genomics | And 14 more authors.
Journal of Microbiological Methods | Year: 2010

Urinary tract infections (UTIs) are one of the most common bacterial infections and are predominantly caused by uropathogenic Escherichia coli (UPEC). E. coli strains belonging to 14 serogroups, including O1, O2, O4, O6, O7, O8, O15, O16, O18, O21, O22, O25, O75 and O83, are the most frequently detected UPEC strains in a diverse range of clinical urine specimens. In the current study, the O-antigen gene clusters of E. coli serogroups O1, O2, O18 and O75 were characterized. A multiplex PCR method based on O-antigen-specific genes was developed for the simultaneous detection of all 14 E. coli serogroups. The multiplex PCR method was shown to be highly specific and reproducible when tested against 186 E. coli and Shigella O-serogroup reference strains, 47 E. coli clinical isolates and 10 strains of other bacterial species. The sensitivity of the multiplex PCR method was analyzed and shown to detect O-antigen-specific genes in samples containing 25. ng of genomic DNA or in mock urine specimens containing 40 colony-forming units (CFUs) per ml. Five urine specimens from hospital were examined using this multiplex PCR method, and the result for one sample was verified by the conventional serotyping methods. The multiplex PCR method developed herein can be used for the detection of relevant E. coli strains from clinical and/or environmental samples, and it is particularly useful for epidemiologic analysis of urine specimens from patients with UTIs. © 2010 Elsevier B.V.


Jiang L.,Nankai University | Jiang L.,The Key Laboratory of Molecular Microbiology and Technology | Ni Z.,Nankai University | Ni Z.,The Key Laboratory of Molecular Microbiology and Technology | And 8 more authors.
Current Microbiology | Year: 2015

Salmonella, a genus that is closely related to Escherichia coli, includes many pathogens of humans and other animals. A notable feature that distinguishes Salmonella from E. coli is lactose negativity, because the lac operon is lost in most Salmonella genomes. Here, we expressed the lac operon in Salmonella enterica serovar Typhimurium and compared the virulence of the Lac+ strain to that of the wild-type strain in a murine model, invasion assays, and macrophage replication assays. We showed that the Lac+ strain is attenuated in vivo and the attenuation of virulence is caused by its defect in epithelial cell invasion. However, the invasion-defective phenotype is unrelated to lactose utilization. Through sequencing and the comparison of the transcriptome profile between the Lac+ and wild-type strains during invasion, we found that most flagellar genes were markedly downregulated in the Lac+ strain, while other genes associated with invasion, such as the majority of genes encoded in Salmonella pathogenicity island 1, were not differentially expressed. Moreover, we discovered that lacA is the major repressor of flagellar gene expression in the lac operon. In conclusion, these data demonstrate that the lac operon decreases Salmonella invasion of epithelial cells through repression of flagellar biosynthesis. As the ability to invade epithelial cells is a critical virulence determinant of Salmonella, our results provide important evidence that the loss of the lac operon contributes to the evolution of Salmonella pathogenicity. © 2014, Springer Science+Business Media New York.


Li P.,The Key Laboratory of Molecular Microbiology and Technology | Wang L.,Nankai University | Feng L.,Tianjin Key Laboratory of Microbial Functional Genomics
Journal of Bacteriology | Year: 2013

The cold-tolerant bacterium Pusillimonas sp. strain T7-7 is able to utilize diesel oils (C5 to C30 alkanes) as a sole carbon and energy source. In the present study, bioinformatics, proteomics, and real-time reverse transcriptase PCR approaches were used to identify the alkane hydroxylation system present in this bacterium. This system is composed of a Rieske-type monooxygenase, a ferredoxin, and an NADH-dependent reductase. The function of the monooxygenase, which consists of one large (46.711 kDa) and one small (15.355 kDa) subunit, was further studied using in vitro biochemical analysis and in vivo heterologous functional complementation tests. The purified large subunit of the monooxygenase was able to oxidize alkanes ranging from pentane (C5) to tetracosane (C24) using NADH as a cofactor, with greatest activity on the C15 substrate. The large subunit also showed activity on several alkane derivatives, including nitromethane and methane sulfonic acid, but it did not act on any aromatic hydrocarbons. The optimal reaction condition of the large subunit is pH 7.5 at 30°C. Fe2+ can enhance the activity of the enzyme evidently. This is the first time that an alkane monooxygenase system belonging to the Rieske non-heme iron oxygenase family has been identified in a bacterium. © 2013, American Society for Microbiology.


Wang H.,Nankai University | Wang H.,Tianjin Key Laboratory of Microbial Functional Genomics | Liu B.,Nankai University | Liu B.,Tianjin Key Laboratory of Microbial Functional Genomics | And 5 more authors.
PLoS ONE | Year: 2013

Fis, one of the most important nucleoid-associated proteins, functions as a global regulator of transcription in bacteria that has been comprehensively studied in Escherichia coli K12. Fis also influences the virulence of Salmonella enterica and pathogenic E. coli by regulating their virulence genes, however, the relevant mechanism is unclear. In this report, using combined RNA-seq and chromatin immunoprecipitation (ChIP)-seq technologies, we first identified 1646 Fis-regulated genes and 885 Fis-binding targets in the S. enterica serovar Typhimurium, and found a Fis regulon different from that in E. coli. Fis has been reported to contribute to the invasion ability of S. enterica. By using cell infection assays, we found it also enhances the intracellular replication ability of S. enterica within macrophage cell, which is of central importance for the pathogenesis of infections. Salmonella pathogenicity islands (SPI)-1 and SPI-2 are crucial for the invasion and survival of S. enterica in host cells. Using mutation and overexpression experiments, real-time PCR analysis, and electrophoretic mobility shift assays, we demonstrated that Fis regulates 63 of the 94 Salmonella pathogenicity island (SPI)-1 and SPI-2 genes, by three regulatory modes: i) binds to SPI regulators in the gene body or in upstream regions; ii) binds to SPI genes directly to mediate transcriptional activation of themselves and downstream genes; iii) binds to gene encoding OmpR which affects SPI gene expression by controlling SPI regulators SsrA and HilD. Our results provide new insights into the impact of Fis on SPI genes and the pathogenicity of S. enterica. © 2013 Wang et al.


Feng J.,Hubei University | Liu B.,Nankai University | Zhang Z.,Hubei University | Ren Y.,Nankai University | And 9 more authors.
PLoS ONE | Year: 2012

Natrinema sp. J7-2 is an extreme haloarchaeon capable of growing on synthetic media without amino acid supplements. Here we report the complete genome sequence of Natrinema sp. J7-2 which is composed of a 3,697,626-bp chromosome and a 95,989-bp plasmid pJ7-I. This is the first complete genome sequence of a member of the genus Natrinema. We demonstrate that Natrinema sp. J7-2 can use gluconate, glycerol, or acetate as the sole carbon source and that its genome encodes complete metabolic pathways for assimilating these substrates. The biosynthetic pathways for all 20 amino acids have been reconstructed, and we discuss a possible evolutionary relationship between the haloarchaeal arginine synthetic pathway and the bacterial lysine synthetic pathway. The genome harbors the genes for assimilation of ammonium and nitrite, but not nitrate, and has a denitrification pathway to reduce nitrite to N2O. Comparative genomic analysis suggests that most sequenced haloarchaea employ the TrkAH system, rather than the Kdp system, to actively uptake potassium. The genomic analysis also reveals that one of the three CRISPR loci in the Natrinema sp. J7-2 chromosome is located in an integrative genetic element and is probably propagated via horizontal gene transfer (HGT). Finally, our phylogenetic analysis of haloarchaeal genomes provides clues about evolutionary relationships of haloarchaea. © 2012 Feng et al.


PubMed | Nankai University, The Key Laboratory of Molecular Microbiology and Technology and RAS N. D. Zelinsky Institute of Organic Chemistry
Type: | Journal: Carbohydrate research | Year: 2014

The O-polysaccharide (O-antigen) of Escherichia coli O68 was studied by sugar analysis, partial solvolysis with anhydrous trifluoroacetic acid, and 1D and 2D (1)H and (13)C NMR spectroscopies. The following structure of the branched heptasaccharide repeating unit was established: [structure: see text]. The O-antigen gene cluster of E. coli O68 was sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in full agreement with the O-antigen structure.


PubMed | The Key Laboratory of Molecular Microbiology and Technology
Type: Journal Article | Journal: Journal of proteomics | Year: 2012

Geobacillus thermodenitrificans NG80-2 is a crude oil-degrading thermophilic bacterium isolated from an oil reservoir in China. In this study, the gene clusters and pathways for the degradation of benzoate (via benzoyl-CoA), phenylacetate (via phenylacetyl-CoA), 4-hydroxyphenylacetate (via 3,4-dihydroxyphenylacetate) and anthranilate (via 3-hydroxyanthranilate) were confirmed using combined in silico analysis and proteomics approaches. It was observed that synthesis of the enzymes catalyzing initial activation, ring oxidation and ring cleavage reactions were generally induced specifically by their respective substrates, while many of the enzymes catalyzing downstream reactions exhibited broader substrate specificities. Novel genes encoding benzoyl-CoA epoxidase and 3,4-dihydroxyphenylacetate 2,3-dioxygenase, and a paaX homologue that serves as a positive regulator of benzoate degradation were proposed. Downregulation of the glycolysis pathway, along with upregulation of the gluconeogenesis pathway and the glyoxylate bypass (phenylacetate) were detected in association with the utilization of the aromatics. This novel proteomics approach confirmed the presence of multiple metabolic pathways for aromatic compounds in NG80-2, which is highly advantageous to the survival of this thermophilic bacterium under reservoir conditions.

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