Bacterial Proteins ; chemistry ; genetics ; metabolism ; DNA, Bacterial ; chemistry ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; physiology ; Glutathione ; metabolism ; Listeria monocytogenes ; chemistry ; genetics ; metabolism ; Peptide Termination Factors ; chemistry ; genetics ; metabolism

DNA stable-isotope probing (DNA-SIP) is a recently developed method with which the incorporation of stable isotope from a labeled substrate is used to identify the function of microorganisms in the environment. The technique has now been used in conjunction with metagenomics to establish links between microbial identity and particular metabolic functions. The combination of DNA-SIP and metagenomics not only permits the detection of rare low-abundance species from metagenomic libraries but also facilitates the detection of novel enzymes and bioactive compounds. We summarize recent progress in SIP-metagenomic techniques and applications and discuss prospects for this combined approach in environmental microbiology and biotechnology.
Animals ; DNA ; genetics ; DNA Probes ; chemistry ; genetics ; metabolism ; DNA, Bacterial ; chemistry ; genetics ; metabolism ; Humans ; Isotope Labeling ; methods ; Metagenomics ; methods ; Molecular Probe Techniques ; Sequence Analysis, DNA ; methods

8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a mutagenic adduct formed by reactive oxygen species. In Escherichia coli, 2,6-dihydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) removes this mutagenic adduct from DNA. In this report, we demonstrate base excision repair (BER) synthesis of DNA containing 8-oxo-G with Fpg in vitro. Fpg cut the oligonucleotide at the site of 8-oxo-G, producing one nucleotide gap with 3' and 5' phosphate termini. Next, 3' phosphatase(s) in the supernatant obtained by precipitating a crude extract of E. coli with 40% ammonium sulfate, removed the 3' phosphate group at the gap, thus exposing the 3' hydroxyl group to prime DNA synthesis. DNA polymerase and DNA ligase then completed the repair. These results indicate the biological significance of the glycosylase and apurinic/ apyrimidinic (AP) lyase activities of Fpg, in concert with 3' phosphatase(s) to create an appropriately gapped substrate for efficient BER synthesis of DNA containing 8-oxo-G.
DNA Glycosylases/metabolism ; *DNA Repair ; DNA, Bacterial/*chemistry/*metabolism ; DNA-Formamidopyrimidine Glycosylase/metabolism ; Escherichia coli/*enzymology/*genetics ; Guanine/*analogs & derivatives/*metabolism

Here we tell a 20-year long story. It began with an easily overlooked DNA degradation (Dnd) phenomenon during electrophoresis and eventually led to the discovery of an unprecedented DNA sulfur modification governed by five dnd genes. This unusual DNA modification, called phosphorothioation, is the first physiological modification identified on the DNA backbone, in which the nonbridging oxygen is replaced by sulfur in a sequence selective and stereo-specific manner. Homologous dnd gene clusters have been identified in diverse and distantly related bacteria and thus have drawn immediate attention of the entire microbial scientific community. Here, we summarize the progress in chemical, genetic, enzymatic, bioinformatical and analytical aspects of this novel postreplicative DNA modification. We also discuss perspectives on the physiological functions of the DNA phosphorothioate modification in bacteria and their implications.
Bacteria ; genetics ; metabolism ; DNA, Bacterial ; chemistry ; history ; metabolism ; Genes, Bacterial ; History, 20th Century ; History, 21st Century ; Multigene Family ; Streptomyces lividans ; genetics ; metabolism ; Sulfur ; chemistry ; history ; metabolism

Genome duplication in E. coli is carried out by DNA polymerase III, an enzyme complex consisting of ten subunits. Investigations of the biochemical and structural properties of DNA polymerase III require the expression and purification of subunits including α, ge, θ, γ, δ', δ, and β separately followed by in vitro reconstitution of the pol III core and clamp loader. Here we propose a new method for expressing and purifying DNA polymerase III components by utilizing a protein co-expression strategy. Our results show that the subunits of the pol III core and those of the clamp loader can be coexpressed and purified based on inherent interactions between the subunits. The resulting pol III core, clamp loader and sliding clamp can be reconstituted effectively to perform DNA polymerization. Our strategy considerably simplifies the expression and purification of DNA polymerase III and provides a feasible and convenient method for exploring other multi-subunit systems.
Cloning, Molecular ; DNA Polymerase III ; chemistry ; genetics ; metabolism ; DNA Replication ; DNA, Bacterial ; biosynthesis ; genetics ; Escherichia coli ; enzymology ; genetics ; Plasmids ; metabolism ; Polymerization ; Protein Engineering ; methods ; Protein Subunits ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; metabolism

The purpose of this study was to develop pneumococcal typing by multiplex PCR and compare it with conventional serotyping by quellung reaction. Pneumococcal strains used in this study included 77 isolates from clinical specimens collected from children at Seoul National University Children's Hospital from 2006 to 2010. These strains were selected as they represented 26 different serotypes previously determined by quellung reaction. Molecular type was determined by 8 sequential multiplex PCR assays. Bacterial DNA extracted from cultured colonies was used as a template for PCR, and primers used in this study were based on cps operon sequences. Types 6A, 6B, 6C, and 6D were assigned based on the presence of wciNbeta and/or wciP genes in 2 simplex PCRs and sequencing. All 77 isolates were successfully typed by multiplex PCR assays. Determined types were as follows: 1, 3, 4, 5, 6A, 6B, 6C, 6D, 7C, 7F, 9V, 10A, 11A, 12F, 13, 14, 15A, 15B/15C, 19A, 19F, 20, 22F, 23A, 23F, 34, 35B, and 37. The results according to the PCR assays were in complete concordance with those determined by conventional quellung reaction. The multiplex PCR assay is highly reliable and potentially reduces reliance upon conventional serotyping.
Child ; DNA Primers/chemistry/metabolism ; DNA, Bacterial/chemistry/genetics ; Humans ; Multiplex Polymerase Chain Reaction ; Pneumococcal Infections/microbiology ; Serotyping ; Streptococcus pneumoniae/*classification/genetics/isolation & purification

To investigate the distribution of the genes of two major metallo-beta-lactamases (MBL; i.e., IMP and VIM) and class 1 integrons (intI) in the clinical imipenem-resistant Pseudomonas aeruginosa, a total of 65 isolates, from a university hospital in Sichuan between December 2004 and April 2005 were screened for MBL genes by PCR using primers specific for bla ( IMP-1 ), bla ( VIM ) and bla ( VIM-2 ) genes. The MBL-positive isolates were further assessed for class 1 integrons by PCR using specific primers. The nucleotide sequences of several PCR products were also determined. The results revealed that the bla ( VIM ) gene was found in 81.5% (53/65) of all isolates, bla ( VIM-2 ) gene was found in only 1 isolate and the intI gene was observed in 45.3% (24/53) of bla ( VIM )-positive isolates. One isolate carried simultaneously both bla ( IMP-1 ) and intI genes, and to the best of our knowledge this is the first report of such isolate in southwest China. These observations highlight that the genes for VIM beta-lactamase and class 1 integrons were predominantly present among the imipenem-resistant P. aeruginosa tested, confirming the current widespread threat of imipenem-resistant, integron-borne P. aeruginosa.
Anti-Bacterial Agents/pharmacology ; China ; DNA Primers/chemistry ; Drug Resistance, Bacterial ; Gene Expression Regulation, Bacterial ; Imipenem/*pharmacology ; Integrons ; Microbial Sensitivity Tests ; Models, Genetic ; Pseudomonas Infections/genetics ; Pseudomonas Infections/*microbiology ; Pseudomonas aeruginosa/*metabolism ; Sequence Analysis, DNA ; beta-Lactamases/*metabolism

We evaluated the performance of a new chromogenic medium for detection of Clostridium difficile, chromID C. difficile agar (CDIF; bioMerieux, France), by comparison with BBL C. difficile Selective Agar (CDSA; Becton Dickinson and Company, USA). After heat pre-treatment (80degrees C, 5 min), 185 diarrheal stool samples were inoculated onto the two media types and incubated anaerobically for 24 hr and 48 hr for CDIF and for 48 hr and 72 hr for CDSA. All typical colonies on each medium were examined by Gram staining, and the gram-positive rods confirmed to contain the tpi gene by PCR were identified as C. difficile. C. difficile was recovered from 36 samples by using a combination of the two media. The sensitivity with CDIF 48 hr was highest (100%) and was significantly higher than that with CDIF 24 hr (58.3%; P<0.001), because samples with a low burden of C. difficile tended to require prolonged incubation up to 48 hr (P<0.001). The specificity of CDIF 24 hr and CDIF 48 hr (99.3% and 90.6%, respectively) was significantly higher than that of CDSA 48 hr and CDSA 72 hr (72.5% and 67.1%, respectively; P<0.001). CDIF was effective for detecting C. difficile in heat-pretreated stool specimens, thus reducing unnecessary testing for toxin production in non-C. difficile isolates and turnaround time.
Agar/chemistry ; Bacterial Proteins/genetics ; Bacteriological Techniques/*methods ; Chromogenic Compounds/chemistry/metabolism ; Clostridium difficile/genetics/*isolation & purification ; Culture Media/chemistry ; DNA, Bacterial/analysis/metabolism ; Diarrhea/microbiology/pathology ; Feces/*microbiology ; Humans ; Polymerase Chain Reaction ; Time Factors

A simple and rapid method for preparation of plasmid DNA from overnight incubation was introduced. It does not require any additional reagents; the incubation mixture containing recombinant plasmid DNA was just mixed with H2O and phenol/chloroform/isoamyl alcohol in certain ratio. After vortexing and spinning of the mixture, the supernatant could be directly loaded onto agarose gel and analyzed using electrophoresis. The whole preparation requires only 3-5 minutes. So to quickly screen recombinant clones, this method is better compared with traditional methods.
Centrifugation, Density Gradient ; Chloroform ; chemistry ; Cloning, Molecular ; methods ; DNA, Bacterial ; genetics ; isolation & purification ; metabolism ; Deoxyribonuclease HindIII ; metabolism ; Electrophoresis ; Escherichia coli ; genetics ; Pentanols ; chemistry ; Phenol ; chemistry ; Plasmids ; chemistry ; genetics ; Reproducibility of Results ; Time Factors ; Water ; chemistry

Xanthomonas campestris pv. campestris ( Xcc), causative agent of the black rot disease of cruciferous crops worldwide, produces large amount of extracellular polysaccharide( EPS), which has found wide applications in industry. In order to clone genes involved in EPS biosynthesis, Xcc wild-type strain 8004 was mutagenized with transposon Tn5gus A5, and a number of EPS-defective mutants were isolated. The Tn5gusA5 insertion sites in the mutants were analyzed by using thermal asymmetric interlaced PCR(TAIL-PCR), and the corresponding genes were identified by homology blast to the completely sequenced genome of Xcc 8004 strain. A novel gene, waxE, identified from the EPS-defective mutant 151D09, was found to be disrupted by the insertion of Tn5gusA5 in the open reading frame(ORF) with genome coordinates 4478998bp to 4479819bp.This gene showed 52% similarity to the kdtX gene of Serratia marcescens and 50% to the waaE of Klebsiella pneumoniae at amino acid level, with characteristics of glycostransferase 2 family domain. In order to identify the function of waxE gene, waxE gene deletion mutant of Xcc 8004 was constructed by gene replacement strategy in which waxE gene of genome was replaced by kanamycin resistant gene kan. The waxE gene deletion mutant strain, named Xcc 8570, was confirmed by both PCR and southern analysis. The growth rate of the deletion mutant 8570 in rich medium was not affected, but the EPS yield reduced by 35% as compared with the wildtype strain 8004. The deletion mutant could be completmented in trans with plasmid pLATC8976 harboring an intact waxE gene, and the EPS yield of the mutant was restored. The combined data showed that waxE gene involved in EPS biosynthesis in Xcc.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; Blotting, Southern ; Cloning, Molecular ; DNA Transposable Elements ; genetics ; Molecular Sequence Data ; Polymerase Chain Reaction ; Polysaccharides, Bacterial ; genetics ; metabolism ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; Xanthomonas campestris ; genetics ; metabolism