No abstract available.
Bacterial Proteins/*metabolism ; Humans ; Stenotrophomonas maltophilia/*genetics

As an important protein structure on the surface of bacteria, type Ⅳ pili (TFP) is the sensing and moving organ of bacteria. It plays a variety of roles in bacterial physiology, cell adhesion, host cell invasion, DNA uptake, protein secretion, biofilm formation, cell movement and electron transmission. With the rapid development of research methods, technical equipment and pili visualization tools, increasing number of studies have revealed various functions of pili in cellular activities, which greatly facilitated the microbial single cell research. This review focuses on the pili visualization method and its application in the functional research of TFP, providing ideas for the research and application of TFP in biology, medicine and ecology.
Fimbriae, Bacterial/metabolism* ; Bacterial Proteins/genetics* ; Bacterial Physiological Phenomena ; Bacterial Adhesion/physiology*

OBJECTIVECorynebacterium crenatum MT, a mutant from C. crenatum AS 1.542 with a lethal argR gene, exhibits high arginine production. To confirm the effect of ArgR on arginine biosynthesis in C. crenatum, an intact argR gene from wild-type AS 1.542 was introduced into C. crenatum MT, resulting in C. crenatum MT. sp, and the changes of transcriptional levels of the arginine biosynthetic genes and arginine production were compared between the mutant strain and the recombinant strain.

METHODSQuantitative real-time polymerase chain reaction was employed to analyze the changes of the related genes at the transcriptional level, electrophoretic mobility shift assays were used to determine ArgR binding with the argCJBDF, argGH, and carAB promoter regions, and arginine production was determined with an automated amino acid analyzer.

RESULTSArginine production assays showed a 69.9% reduction in arginine from 9.01 ± 0.22 mg/mL in C. crenatum MT to 2.71 ± 0.13 mg/mL (P<0.05) in C. crenatum MT. sp. The argC, argB, argD, argF, argJ, argG, and carA genes were down-regulated significantly in C. crenatum MT. sp compared with those in its parental C. crenatum MT strain. The electrophoretic mobility shift assays showed that the promoter regions were directly bound to the ArgR protein.

CONCLUSIONThe arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR, and intact ArgR in C. crenatum MT results in a significant descrease in arginine production.

Arginine ; biosynthesis ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; Corynebacterium ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; Repressor Proteins ; chemistry ; genetics ; metabolism

Spore coat proteins, such as CotB, CotC, CotG et al, are able to efficiently display exogenous protein on spore surface for preparing oral vaccines or enzymes. CotX is another structural protein of spore coats of Bacillus subtilis. To investigate whether CotX could carry target protein onto the spore surface, we constructed a recombinant integrative plasmid, designated as pJS749, which carries a recombinant cotX-gfp gene under the control of the cotX promoter. We transformed pJS749 into Bacillus subtilis 168(trp-), an alpha-amylase inactivated mutant DRJS749 was selected and confirmed to be a double crossover integrant, where cotX-gfp fragment was integrated into the chromosome. After induction of spore formation, significant green fluorescence was observed on spore surface of strain DRJS749 under fluorescent microcopy. This suggests that CotX is associated with the outer part of the coat. CotX can therefore be used as a molecular vehicle for spore surface display of exogenous proteins.
Bacillus subtilis ; genetics ; metabolism ; physiology ; Bacterial Proteins ; biosynthesis ; genetics ; Gene Expression Regulation, Bacterial ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Spores, Bacterial ; genetics ; metabolism

Animals ; Bacterial Proteins ; genetics ; metabolism ; Chickens ; microbiology ; Escherichia coli ; genetics ; metabolism ; Genotype ; beta-Lactamases ; genetics ; metabolism

OBJECTIVETo examine the expression of recombinant cytolethal distending toxin (CDT) produced by Actinobacillus actinomycetemcomitans (Aa).

METHODSCDT encoding gene cdtABC was amplified by PCR. Through TA clone and restriction endonuclease digestion, gene cdtABC and vector pQE60 were ligated to form pQE60-cdtABC expression system which transformed into competent cells. Protein expression was induced by IPTG and examined by SDS-PAGE and Western-blotting.

RESULTSRandom colony PCR of pQE60-cdtABC transformed cells demonstrated that all strains contained cdtABC gene. The DNA sequence was blast with cdtABC gene from GenBank and 99% homology was obtained. SDS-PAGE and Western-blotting confirmed that recombinant CDT was obtained.

CONCLUSIONSCDT protein expression system was reconstructed and recombinant protein was obtained. Actinobacillus actinomycetemcomitans;

Aggregatibacter actinomycetemcomitans ; genetics ; metabolism ; Bacterial Toxins ; genetics ; metabolism ; Genetic Vectors ; Recombinant Proteins ; genetics ; metabolism

Vibrio parahaemolyticus, the main pathogen causing seafood related food poisoning worldwide, has strong biofilm formation ability. ToxR is a membrane binding regulatory protein, which has regulatory effect on biofilm formation of V. parahaemolyticus, but the specific mechanism has not been reported. c-di-GMP is an important second messenger in bacteria and is involved in regulating a variety of bacterial behaviors including biofilm formation. In this study, we investigated the regulation of ToxR on c-di-GMP metabolism in V. parahaemolyticus. Intracellular c-di-GMP in the wild type (WT) and toxR mutant (ΔtoxR) strains were extracted by ultrasonication, and the concentrations of c-di-GMP were then determined by enzyme linked immunosorbent assay (ELISA). Three c-di-GMP metabolism-related genes scrA, scrG and vpa0198 were selected as the target genes. Quantitative real-time PCR (q-PCR) was employed to calculate the transcriptional variation of each target gene between WT and ΔtoxR strains. The regulatory DNA region of each target gene was cloned into the pHR309 plasmid harboring a promoterless lacZ gene. The recombinant plasmid was subsequently transferred into WT and ΔtoxR strains to detect the β-galactosidase activity in the cellular extracts. The recombinant lacZ plasmid containing each of the target gene was also transferred into E. coli 100λpir strain harboring the pBAD33 plasmid or the recombinant pBAD33-toxR to test whether ToxR could regulate the expression of the target gene in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and the over-expressed His-ToxR was purified. The electrophoretic mobility shift assay (EMSA) was applied to verify whether His-ToxR directly bound to the target promoter region. ELISA results showed that the intracellular c-di-GMP level significantly enhanced in ΔtoxR strain relative to that in WT strain, suggesting that ToxR inhibited the production of c-di-GMP in V. parahaemolyticus. qPCR results showed that the mRNA levels of scrA, scrG and vpa0198 significantly increased in ΔtoxR strain relative to those in WT strain, suggesting that ToxR repressed the transcription of scrA, scrG and vpa0198. lacZ fusion assay showed that ToxR was able to repress the promoter activities of scrA, scrG and vpa0198 in both V. parahaemolyticus and E. coli 100λpir. EMSA results showed that His-ToxR was able to bind to the regulatory DNA regions of scrA and scrG, but not to the regulatory DNA region of vpa0198. In conclusion, ToxR inhibited the production of c-di-GMP in V. parahaemolyticus via directly regulating the transcription of enzyme genes associated with c-di-GMP metabolism, which would be beneficial for V. parahaemolyticus to precisely control bacterial behaviors including biofilm formation.
Vibrio parahaemolyticus/metabolism* ; Escherichia coli/metabolism* ; Bacterial Proteins/metabolism* ; Transcription Factors/genetics* ; Gene Expression Regulation, Bacterial

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

The microbial transglutamunase (MTG) gene was amplified from the genomic DNA of Streptoverticillium mobaraensea by using PCR and inserted into pET vector to construct the expression plasmid called pET-MTG. The pET-MTG was transfected into E. coli (Rosetta DE3) and the MTG protein was found to be highly expressed as inclusion bodies. The inclusion bodies were isolated and subjected to denaturation and re-naturation, followed by strong cation ion-exchange chromatography to purify the expressed MTG. The specific activity of purified MTG was close to that of native MTG. Taken together, this study might provide a base for the industrial production of microbial transglutaminase.
Bacterial Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genes, Bacterial ; Inclusion Bodies ; enzymology ; Recombinant Proteins ; genetics ; metabolism ; Streptomycetaceae ; enzymology ; genetics ; Transglutaminases ; genetics ; metabolism

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