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*

Objective: To investigate reciprocal regulation between Fur and two RyhB homologs in Methods: Regulatory relationships were assessed by a combination of colony morphology assay, primer extension, electrophoretic mobility shift assay and DNase I footprinting. Results: Fur bound to the promoter-proximal DNA regions of Conclusion Fur and the two RyhB homologs exert negative reciprocal regulation, and RyhB homologs have a positive regulatory effect on biofilm formation in
Bacterial Proteins/metabolism* ; Biofilms ; Gene Expression Regulation, Bacterial/physiology* ; Yersinia pestis/physiology*

OBJECTIVETo investigate the relationship between the resuscitation promoting role of resuscitation promoting factor and the initial bacteria amount of dormant Mycobacterium tuberculosis.

METHODSMycobacterium tuberculosis (dormant bacteria) was cultured for 100 days, then diluted into 1 mg/ml concentration with 7H9, and further diluted into 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg/ml. Twelve new tubes added with 5 ml 7H9 and divided into two groups: the first group was added with the resuscitation-promoting factor protein, and the second group as control was added with 7H9. In each group the above diluted solutions were added. The tubes were located at 37 degrees C for culture. Optical density (OD) was detected on day 15, 25, 30, and 35. From each tube 1 microl culture solution was plated on 7H11 medium for colony counting.

RESULTSOD detection showed that bacteria proliferation in each group had positive linear correlation (P < 0.05, P < 0.01), indicating that the resuscitation-promoting factor played a similiar role in solutions with different dilution concentrations. 7H11 results and the OD results show that these two detection methods in each group had linear correlation (P < 0.05, P < 0.01), indicating that these two methods showed consistent test results.

CONCLUSIONThe resuscitation-promoting factor has no effect on the resuscitation of dormant Mycobacterium tuberculosis and its initial bacteria amount.

Bacterial Proteins ; metabolism ; Cytokines ; metabolism ; Mycobacterium tuberculosis ; physiology ; Resuscitation

BACKGROUNDQacA, a main exporter mediating the multidrug-resistance of Staphylococcus aureus to a variety of antiseptics and disinfectants, possesses a topology of 14 alpha-helical transmembrane segments (TMS). Our study aimed to determine the importance and topology of amino acid residues in and flanking the cytoplasmic end of TMS5.

METHODSSite-directed mutagenesis was used to mutate 5 residues, including L146, A147, V148, W149 and S150, into cysteine. A minimum inhibitory concentration (MIC) and transport assay with or without N-ethylmaleimide (NEM) were performed to analyse the function of these mutants.

RESULTSAll of the mutants showed comparable protein expression levels. MIC analysis suggested that mutant W149C showed low resistance levels to the drugs, but the mutations at L146, A147, V148, and S150C had little or no effect on the resistance level. And the results of the fluorimetric transport assay were in agreement with those of MIC analysis, that is to say, W149C did not allow transport to the substrates to be tested, while the other mutants retained significant transport ability. The reaction of the different mutant proteins with Fluorescein-NEM revealed that the mutant L146C was highly reactive with NEM; the W149C and S150C mutants were moderately reactive; A147C was barely reactive and V148C showed no reactivity.

CONCLUSIONSThe study identified that residues W149 and S150 situated at the interface of the aqueous: lipid junction as functionally important residues, probably involved in the substrate binding and translocation of QacA.

Bacterial Proteins ; chemistry ; physiology ; Drug Resistance, Bacterial ; Ethylmaleimide ; pharmacology ; Indoles ; metabolism ; Membrane Transport Proteins ; chemistry ; physiology ; Structure-Activity Relationship

OBJECTIVEThis study is to verify the use of rich BHI medium to substitute synthetic media for gene regulation studies in Yersinia pestis.

METHODSThe transcriptional regulation of rovA by PhoP or via temperature upshift, and that of pla by CRP were investigated when Y. pestis was cultured in BHI. After cultivation under 26 °C, and with temperature shifting from 26 to 37 °C, the wild-type (WT) strain or its phoP or crp null mutant (ΔphoP or Δcrp, respectively) was subject to RNA isolation, and then the promoter activity of rovA or pla in the above strains was detected by the primer extension assay. The rovA promoter-proximal region was cloned into the pRW50 containing a promoterless lacZ gene. The recombinant LacZ reporter plasmid was transformed into WT and ΔphoP to measure the promoter activity of rovA in these two strains with the β-Galactosidase enzyme assay system.

RESULTSWhen Y. pestis was cultured in BHI, the transcription of rovA was inhibited by PhoP and upon temperature upshift while that of pla was stimulated by CRP.

CONCLUSIONThe rich BHI medium without the need for modification to be introduced into the relevant stimulating conditions (which are essential to triggering relevant gene regulatory cascades), can be used in lieu of synthetic TMH media to cultivate Y. pestis for gene regulation studies.

Bacterial Proteins ; genetics ; metabolism ; Bacteriological Techniques ; Culture Media ; pharmacology ; Gene Expression Regulation, Bacterial ; drug effects ; physiology ; Yersinia pestis ; metabolism ; physiology

For recent years, the research has been focused on the ina gene application in the field of biological ice nucleation. This paper reviewed the application of ina genes in bacterial cell surface display, construction of reporter gene systems, killing insect pests through induced freezing, sensitive detection of pathogenic bacteria contaminating foods, breeding of cold resistant varieties. A brief introduction of the ina gene application in killing insect pests in China was also made in this review.
Bacterial Outer Membrane Proteins ; genetics ; physiology ; Bacterial Proteins ; genetics ; physiology ; Freezing ; Insect Control ; methods ; Pseudomonas ; genetics ; Recombinant Fusion Proteins ; genetics ; Research Design

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

The formation of disulfide bonds in secreted proteins of E. coli is a synergetic process depending on a series of Dsb proteins containing DsbA, DsbB, DsbC, DsbD, DsbE and DsbG. DsbA functions as an oxidant to form a disulfide bond between two -SH- in vivo and DsbB reactivates DsbA by reoxidizing it. Both DsbC and DsbG, two periplasmic proteins with isomerase activity, can correct mis-paired disulfide bonds introduced by DsbA although they recognize different substrates. DsbD, an inner membrane protein, plays a role in reducing DsbC and DsbG in vivo. It is regarded that DsbE has the similar function with DsbD. All DsbA, DsbC and DsbG have chaperone activity besides involving in the formation of disulfide bonds. Furthermore, their chaperone activity can promote the formation of protein disulfide bonds. There are a few reports dealing with soluble expression of heterologous proteins containing disulfide bonds assisted by DsbA and DsbC in E. coli. So far there has been no reports about the soluble expression of heterologous proteins promoted by DsbG. Our experiments first demonstrated that both DsbC and DsbG can improve the expression of single chain antibodies as soluble and functional forms in E. coli, and DsbG has additive effects with DsbC.
Bacterial Proteins ; chemistry ; physiology ; Escherichia coli ; genetics ; Escherichia coli Proteins ; Genetic Engineering ; methods ; Membrane Proteins ; chemistry ; physiology ; Molecular Chaperones ; physiology ; Oxidoreductases ; chemistry ; physiology ; Periplasmic Proteins ; Protein Disulfide-Isomerases ; chemistry ; physiology ; Recombinant Proteins ; biosynthesis

Confocal laser scanning microscopy was used to observe the spatio-temporal expression of the pathway-specific gene redD during S. coelicolor cell cultivation. The corresponding mutant S. coelicolor lyqRY1522 carrying redD::eyfp in the chromosome was constructed. The temporal expression results of the fusion protein during submerged cultivation demonstrated that expression of redD began in the transition phase, continuing through the exponential growth phase to the stationary phase, and reached maximum in the stationary phase. On the other hand, redD was expressed only in substrate mycelia during solid-state culture, while aerial mycelia remained essentially non-fluorescent throughout culture. Results demonstrated that the expression pattern of redD coincides with that of the biosynthesis of the antibiotics during culture, revealing a direct correlation between the spatio-temporal distribution of regulatory gene expression and second metabolism.
Anti-Bacterial Agents ; biosynthesis ; Bacterial Proteins ; genetics ; metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Bacterial ; physiology ; Mutation ; Signal Transduction ; physiology ; Streptomyces coelicolor ; genetics ; metabolism ; Trans-Activators ; genetics ; metabolism

We used a proteomic approach to identify IbpA in Cronobacter sakazakii (C. sakazaki), which is related to heat tolerance in this strain. The abundance of IbpA in C. sakazakii strains strongly increased after heat shock. C. sakazakii CMCC 45402 ibpA deletion mutants were successfully constructed. The C. sakazakii CMCC 45402 ΔibpA and wild-type strains could not be distinguished based on colony morphology on LB agar plates or biochemical assays. The growth of the C. sakazakii CMCC 45402 ΔibpA mutant in heat shock conditions was indistinguishable from that of the isogenic wild-type, but showed greater heat resistance than E. coli O157:H7 strain CMCC 44828. This study suggests that the absence of a single ibpA gene has no obvious effect on the phenotype or heat resistance of the strain C. sakazakii CMCC 45402.
Bacterial Proteins ; genetics ; metabolism ; Cronobacter sakazakii ; genetics ; physiology ; Gene Expression Regulation, Bacterial ; physiology ; Genotype ; Heat-Shock Proteins ; genetics ; metabolism ; Hot Temperature ; Stress, Physiological