OBJECTIVETo construct plant expression pCAMBIA1301-PMI by substituting PMI for hygromycin resistance gene in pCAMBIA1301 and obtain transgenic Salvia miltiorrhiza f. alba using PMI-mannose selection system.

METHODThe 6-phosphomannose isomerase gene (PMI) of Escherichia coli was amplified by PCR. Sequence analysis showed that it shared 100% amino acids identities with the sequences of PMI genes isolates reported in the NCBI. Based on pCAMBIA1305, the plant expression pCAMBIA1305-PMI was constructed successfully by substituting PMI for hygromycin resistance gene in pCAMBIA1305. pCAMBIA1305-PMI was transformed into Agrobacterium tumefaciens LBA4404, and then the leaves of S. miltiorrhiza f. alba were inoculated in LBA4404 with pCAMBIA1305-PMI.

RESULTPlant expression pCAMBIA1301-PMI was successfully constructed and the leaves of S. miltiorrhiza f. alba inoculated in LBA4404 with pCAMBIA1305-PMI were selected on medium supplemented with a combination of 20 g x L(-1) mannose and 10 g x L(-1) sucrose as a carbon source. The transformation efficiency rate was 23.7%.

CONCLUSIONGenetic transformation was confirmed by PCR, indicating that a new method for obtaining transgenic S. miltiorrhiza f. alba plants was developed using PMI-mannose selection system.

Anti-Bacterial Agents ; pharmacology ; Biomarkers ; Cinnamates ; pharmacology ; Escherichia coli ; enzymology ; genetics ; Escherichia coli Proteins ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Hygromycin B ; analogs & derivatives ; pharmacology ; Mannose-6-Phosphate Isomerase ; genetics ; metabolism ; Plants, Genetically Modified ; drug effects ; genetics ; metabolism ; Salvia miltiorrhiza ; drug effects ; genetics ; metabolism ; Transformation, Genetic

BACKGROUND/AIMS: A worldwide increase in amoxicillin resistance in Helicobacter pylori is having an adverse effect on eradication therapy. In this study, we investigated the mechanism of the amoxicillin resistance of H. pylori in terms of amino acid substitutions in penicillin-binding protein 1 (PBP1). METHODS: In total, 150 H. pylori strains were isolated from 144 patients with chronic gastritis, peptic ulcers, or stomach cancer. The minimum inhibitory concentrations (MICs) of the strains were determined with a serial 2-fold agar dilution method. The resistance breakpoint for amoxicillin was defined as >0.5 microg/mL. RESULTS: Nine of 150 H. pylori strains showed amoxicillin resistance (6%). The MIC values of the resistant strains ranged from 1 to 4 microg/mL. A PBP1 sequence analysis of the resistant strains revealed multiple amino acid substitutions: Val16-->Ile, Val45-->Ile, Ser414-->Arg, Asn562-->Tyr, Thr593-->Ala, Gly595-->Ser, and Ala599-->Thr. The natural transformation of these mutated genes into amoxicillin-sensitive strains was performed in two separate pbp1 gene segments. A moderate increase in the amoxicillin MIC was observed in the segment that contained the penicillin-binding motif of the C-terminal portion, the transpeptidase domain. CONCLUSIONS: pbp1 mutation affects the amoxicillin resistance of H. pylori through the transfer of the penicillin-binding motif.
Adult ; Amino Acid Sequence ; *Amino Acid Substitution ; Amoxicillin/*pharmacology ; Anti-Bacterial Agents/*pharmacology ; Female ; Helicobacter Infections/drug therapy ; Helicobacter pylori/*chemistry/*drug effects/genetics ; Humans ; Male ; Microbial Sensitivity Tests ; Middle Aged ; *Penicillin Resistance/genetics ; Penicillin-Binding Proteins/*chemistry/genetics ; Republic of Korea ; Sequence Analysis, Protein ; Transformation, Genetic

The objective of this study was to characterize the oxygen dependent regulation of pyruvate oxidase (SpxB) gene expression and protein production in Streptococcus sanguinis (S. sanguinis). SpxB is responsible for the generation of growth-inhibiting amounts of hydrogen peroxide (H2O2) able to antagonize cariogenic Streptococcus mutans (S. mutans). Furthermore, the ecological consequence of H2O2 production was investigated in its self-inhibiting ability towards the producing strain. Expression of spxB was determined with quantitative Real-Time RT-PCR and a fluorescent expression reporter strain. Protein abundance was investigated with FLAG epitope engineered in frame on the C-terminal end of SpxB. Self inhibition was tested with an antagonism plate assay. The expression and protein abundance decreased in cells grown under anaerobic conditions. S. sanguinis was resistant against its own produced H2O2, while cariogenic S. mutans was inhibited in its growth. The results suggest that S. sanguinis produces H2O2 as antimicrobial substance to inhibit susceptible niche competing species like S. mutans during initial biofilm formation, when oxygen availability allows for spxB expression and Spx production.
Antibiosis ; physiology ; Bacterial Proteins ; biosynthesis ; genetics ; Epitopes ; genetics ; Gene Expression Regulation, Bacterial ; Hydrogen Peroxide ; metabolism ; pharmacology ; Oligopeptides ; Oxygen ; metabolism ; Peptides ; genetics ; Pyruvate Oxidase ; biosynthesis ; genetics ; Streptococcus mutans ; drug effects ; Streptococcus sanguis ; enzymology ; genetics ; growth & development ; Transformation, Bacterial

OBJECTIVETo discuss the possible mechanism of drug resistance transmission between Staphylococcus and Escherichia coli.

METHODSThe chloramphenicol resistance plasmid of Staphylococcus aureus was extracted to transform the sensitive Escherichia coli, and the drug-resistant Escherichia coli were screened by drug sensitivity test.

RESULTSThe drug-resistant Escherichia coli were successfully obtained.

CONCLUSIONStaphylococcus may have a natural shuttle plasmid of drug resistance, which can transform Escherichia coli under specific conditions.

Drug Resistance, Bacterial ; genetics ; Escherichia coli ; drug effects ; genetics ; Plasmids ; Staphylococcus ; genetics ; Transformation, Bacterial

Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 10(6) conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alternatively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.
Agrobacterium tumefaciens ; genetics ; Base Sequence ; Citrus ; microbiology ; DNA Primers ; genetics ; DNA, Bacterial ; genetics ; DNA, Fungal ; genetics ; DNA, Recombinant ; genetics ; Drug Resistance, Fungal ; genetics ; Hygromycin B ; pharmacology ; Molecular Sequence Data ; Mutagenesis, Insertional ; Penicillium ; drug effects ; genetics ; pathogenicity ; Plant Diseases ; microbiology ; Plasmids ; genetics ; Transformation, Genetic

Obtaining marker-free plants with high efficiency will benefit the environmental release of transgenic crops. To achieve this point, a binary vector pNB35SVIP1 with three T-DNAs was constructed by using several mediate plasmids, in which one copy of bar gene expression cassette and two copies of VIP1 gene expression cassette were included. EHA101 Agrobacterium strain harboring the final construct was applied to transform soybean (Glycine max) cotyledon nodes. Through 2 - 3 months regeneration and selection on 3 - 5mg/L glufosinate containing medium, transgenic soybean plants were confirmed to be obtained at 0.83% - 3.16%, and co-transformation efficiency of both gene in the same individual reached up to 86.4%, based on southern blot test. By the analysis of PCR, southern blot and northern blot combining with leaf painting of herbicide in T1 progenies, 41 plants were confirmed to be eliminated of bar gene with the frequency of 7.6% . Among the T1 populations tested, the loss of the alien genes happened in 22.7% lines, the silence of bar gene took place in 27.3% lines, and VIP1 gene silence existed in 37.1% marker-free plants. The result also suggested that the plasmid with three T-DNAs might be an ideal vector to generate maker-free genetic modified organism.
Aminobutyrates ; pharmacology ; Blotting, Northern ; Blotting, Southern ; Cotyledon ; drug effects ; genetics ; physiology ; DNA, Bacterial ; genetics ; Gene Expression Regulation, Plant ; Genes, Plant ; genetics ; Genetic Vectors ; genetics ; Herbicide Resistance ; genetics ; Herbicides ; pharmacology ; Plant Leaves ; drug effects ; genetics ; physiology ; Plants, Genetically Modified ; drug effects ; genetics ; physiology ; Regeneration ; drug effects ; genetics ; physiology ; Rhizobium ; genetics ; Soybeans ; drug effects ; genetics ; physiology ; Transformation, Genetic

A strain Mortierella isabellina M6-22-4, which was sensitive to hygromycin B, was selected by treating parental spores with N-methyl-N' -Nitro-N-nitrosoguanidine (MNNG). Protoplasts of the strain Mortierella isabellina M6-22-4 were transformed successfully to hygromycin B resistance using the PD4 plasmid, which contains the Escherichia coli hph gene under the control of Mortierella alpina his H4.1 promoter. The PD4 plasmid was introduced by PEG/CaCl2 treatment. Transformation frequencies of 1.6 - 2.8 transformants/microg of DNA were achieved. Then they were successively incubated to non-selected PDA plates for 10 generations. About 31.6% transformants only from digested plasmid were mitotically stable and showed different hygromycin B resistance when they were incubated back to selection plates. The results of PCR and Southern analysis in three transformants indicated that the plasmid PD4 had been integrated into the fungal genome with 1 - 2 copies. This is the first report of Mortierella isabellina transformation system and supplies an important tool for further research into genetic manipulation of this filamentous fungus.
Anti-Bacterial Agents ; pharmacology ; Blotting, Southern ; Drug Resistance, Microbial ; genetics ; Escherichia coli Proteins ; genetics ; Genome, Fungal ; genetics ; Hygromycin B ; pharmacology ; Mortierella ; drug effects ; genetics ; growth & development ; Plasmids ; genetics ; Polymerase Chain Reaction ; Protoplasts ; metabolism ; Transformation, Genetic

Hepcidin is a liver-expressed, small cysteine rich peptide that acts as a regulator of systemic iron homeostasis. In this work, according to the partiality codon of Pichia pastoris, a DNA fragment containing the coding sequence of hepcidin was designed and synthesized, especially a Kex2 signal cleavage site was fused in 5' end of the antibacterial peptide genes. Then the modified hepcidin gene was inserted into the Pichia pastoris expression vector plasmid pPICZalpha-A. After electroporation of the resulting vector, pPICZalpha-A-Hepc, into the yeast host strain GS115, transformants with high copy inserts were selected by 1500 mg/L Zeocin selection. Under the control of the promoter AOX1 (alcohol oxidase 1), recombinant hepcidin secreted from P. pastoris had a molecular weight of 2.7kD. After optimization of the flask-shaking culture fermentation, the yield of hepcidin reached 100 mg/L in the clarified broth. Through antibacterial assay, the recombinant hepcidin displayed obvious antibacterial activity against Bacillus subtilis. But it could not distinctly inhibit the growth of E. coli BL21 (DE3).
Amino Acid Sequence ; Anti-Bacterial Agents ; chemistry ; metabolism ; pharmacology ; Antimicrobial Cationic Peptides ; genetics ; metabolism ; pharmacology ; Bacillus subtilis ; drug effects ; growth & development ; Base Sequence ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Electroporation ; Escherichia coli ; drug effects ; growth & development ; Gene Expression ; Hepcidins ; Humans ; Molecular Sequence Data ; Molecular Weight ; Pichia ; genetics ; Plasmids ; genetics ; Polymerase Chain Reaction ; Recombinant Proteins ; chemistry ; metabolism ; pharmacology ; Transformation, Genetic

Virginiamycin acetyltransferase D (VatD) plays a vital rule in streptogramins resistance by chemically inactivating streptogramin A. Therefore, it is desirable to discover novel small molecular weight inhibitors of VatD via state-of-the-art virtual screening techniques. This "cocktail" strategy by combining VatD inhibitor with streptogramins may provide new therapeutic opportunity for resistant bacteria infections. Structure-based virtual screening method (molecular docking) was applied to rank and score a chemical database containing 300 000 commercially available compounds against the VatD substrate binding site. Twenty six out of the 200 top scored compounds from the docking calculation were selected and submitted to the VatD enzymatic inhibition assay. The plasmid pRSET B/vatD was constructed and transformed into E. coli (trxB) host cells for over-expression, and VatD enzyme was purified and validated by showing acetyltransferase activity to Virginiamycin M1. Three out of these 26 tested compounds showed enzymatic inhibition on VatD with IC50 168.6, 91.0 and 55.2 micromol x L(-1), separately. Other compounds could not be dissolved in the system and/or had little effect on the enzyme (IC50 > 200 micromol x L(-1)). To our knowledge, it is first time that small molecular weight organic compounds were identified as VatD inhibitors. It is expected that the VatD inhibitors identified at present study could serve as lead compounds for the further development of the novel therapeutic agents to overcome streptogramins resistance.
Acetyltransferases ; antagonists & inhibitors ; genetics ; metabolism ; Catalysis ; drug effects ; Drug Design ; Drug Resistance, Bacterial ; Enzyme Inhibitors ; chemistry ; metabolism ; pharmacology ; Escherichia coli ; genetics ; Genetic Vectors ; Kinetics ; Molecular Structure ; Plasmids ; Streptogramin Group A ; chemistry ; metabolism ; pharmacology ; Transformation, Genetic

Base Sequence ; Conjugation, Genetic ; Drug Resistance, Bacterial ; Electrophoresis, Gel, Pulsed-Field ; Escherichia coli ; drug effects ; enzymology ; Humans ; Klebsiella pneumoniae ; drug effects ; enzymology ; Microbial Sensitivity Tests ; Molecular Sequence Data ; Polymerase Chain Reaction ; Transformation, Bacterial ; beta-Lactamases ; genetics