OBJECTIVEExpress a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription.

METHODSWe express kod-ssb with the Transrtta (DE3), and kod-ssb was purified by affinity chromatography on a Ni2+ Sepharose column, detected by SDS-PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction.

RESULTSThe plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod) was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15.

CONCLUSIONSkod-ssb may in future be used to enhance DNA and cDNA amplification.

Archaeal Proteins ; genetics ; isolation & purification ; metabolism ; Chromatography, Affinity ; DNA, Bacterial ; genetics ; metabolism ; DNA, Complementary ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; isolation & purification ; metabolism ; Gene Expression ; Thermococcus ; genetics ; metabolism

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

A site-directed mutant DNA fragment was synthesized and transfected into clinical Neisseria gonorrhoeae (NG) stains to construct the transformants that contained the corresponding mutagenesis of regulation region of mtrR gene. According to the technique of gene splicing by overlap extension (SOEing), a DNA segment with specific mutagenesis was constructed by two-step polymerase chain reaction (PCR). The mutation fragments EF could be used for the next experiment in which the mutation NG strains were induced. By comparing the recombinant EF fragments to the corresponding DNA fragments of clinical NG strains, 2 of these were not compatible completely. The results of sequencing revealed that there was a 9 bp deletion between the 45 to 54 inverted repeat sequence localized within the mtrR promoter. It can be confirmed that the fragments EF are the specifically designed mutant fragments.
Bacterial Proteins/*genetics ; DNA Fragmentation ; DNA, Bacterial/genetics ; Mutagenesis, Site-Directed ; Neisseria gonorrhoeae/*genetics ; Neisseria gonorrhoeae/metabolism ; Recombination, Genetic ; Repressor Proteins/*genetics ; Sequence Deletion ; Transfection ; Transformation, Bacterial

Bacterial Proteins ; genetics ; metabolism ; Biofilms ; DNA-Binding Proteins ; genetics ; metabolism ; Flagella ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; Transcription Factors ; genetics ; metabolism ; Vibrio parahaemolyticus ; cytology ; genetics ; growth & development ; physiology

OBJECTIVETo study the relation between the absence of one IS100 in the 102 kb pgm locus of Yersinia pestis and the stability of pigmentation phenotype (pgm(+)).

METHODSWe amplified the segment including IS100 in 102 kb pgm locus of Yersinia pestis that isolated from all ecotypes in China by polymerase chain reaction (PCR). There were 171 strains isolated from 18 ecotypes in this study. One strain was chosen to be cloned and sequenced.

RESULTSBesides the type of Microtus brandti, the types of East-North Tianshan, A and B of West-North Tianshan, Microtus Qinghai had one band with about 2560 bp. These strains lost one IS100 in 102 kb pgm locus of Yersinia pestis. Their pgm(+) phenotype was stable. Some strains of ecotypes from Qilian Mountain, Qinghai-Tibet Plateau, Gangdisi Mountain, West Yunnan Mountain had no bands in the PCR products. Negative strains would lose the whole 102 kb pgm locus. The others had one band with 4492 bp. These strains had two IS100 which flanked the 102 kb pgm locus but the pgm(+) phenotype was unstable.

CONCLUSIONYersinia pestis which had only one IS100 would flank the 102 kb pgm locus and had stable pgm(+) phenotype while the Yersinia pestis that having two IS100 flanked the 102 kb pgm locus would have unstable pgm(+) phenotype.

Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; DNA, Bacterial ; genetics ; Genetic Variation ; Genomic Instability ; Phenotype ; Pigmentation ; genetics ; Yersinia pestis ; genetics

In order to clarify dynamic change of microbial community composition and to identify key functional bacteria in the cellulose degradation consortium, we studied several aspects of the biodegradation of filter papers and rice straws by the microbial consortium, the change of substrate degradation, microbial biomass and pH of fermentation broth. We extracted total DNA of the microbial consortium in different degradation stages for high-throughput sequencing of amplicons of bacterial 16 S rRNA genes. Based on the decomposition characteristics test, we defined the 12th, 72nd and 168th hours after inoculation as the initial stage, peak stage and end stage of degradation, respectively. The microbial consortium was mainly composed of 1 phylum, 2 classes, 2 orders, 7 families and 11 genera. With cellulose degradation, bacteria in the consortium showed different growth trends. The relative abundance of Brevibacillus and Caloramator decreased gradually. The relative abundance of Clostridium, Bacillus, Geobacillus and Cohnella increased gradually. The relative abundance of Ureibacillus, Tissierella, Epulopiscium was the highest in peak stage. The relative abundance of Paenibacillus and Ruminococcus did not change obviously in each stage. Above-mentioned 11 main genera all belonged to Firmicutes, which are thermophilic, broad pH adaptable and cellulose or hemicellulose degradable. During cellulose degradation by the microbial consortium, aerobic bacteria were dominant functional bacteria in the initial stage. However, the relative abundance of anaerobic bacteria increased gradually in middle and end stage, and replaced aerobic bacteria to become main bacteria to degrade cellulose.
Bacteria ; classification ; metabolism ; Biodegradation, Environmental ; Cellulose ; metabolism ; DNA, Bacterial ; genetics ; Microbial Consortia ; RNA, Ribosomal, 16S ; genetics

To develop a genetic transformation method of Aureobasidium pullulans and T-DNA insertion for high-efficient screening of polymalic acid (PMA) producing strain. Agrobacterium tumefaciens-AGL1, containing the selection genes encoding hygromycin B phosphotase or phosphinothricin acetyltranferase, was used to transform Aureobasidium pullulans CCTCC M2012223 and transformants were confirmed by colony PCR method. Transferred DNA (T-DNA) insertional mutants were cultured in microwell plate, and screened for high-titer PMA producing strain according to the pH response model. DNA walking was used to detect the insertion sites in the mutant. Results show that the selection markers could stably generated in the transformants, and 80 to 120 transformants could be found per 10(7) single cells. A high-titer PMA mutant H27 was obtained, giving a good PMA production caused by the disruption of phosphoglycerate mutase, that increased by 24.5% compared with the control. Agrobacterium tumefaciens-mediated transformation and high-efficient screening method were successfully developed, which will be helpful for genetic transformation of Aureobasidium pullulans and its functional genes discovery.
Agrobacterium tumefaciens ; Ascomycota ; genetics ; metabolism ; DNA, Bacterial ; Malates ; metabolism ; Polymerase Chain Reaction ; Polymers ; metabolism ; Transformation, Genetic

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

Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.
Chromosomes, Bacterial ; genetics ; DNA ; Endonucleases ; metabolism ; Escherichia coli ; genetics ; Genetic Engineering ; methods ; Recombination, Genetic ; Sequence Deletion