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*

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

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

OBJECTIVETo purify a low-temperature hydroxylamine oxidase (HAO) from a heterotrophic nitrifying bacterium Acinetobacter sp. Y16 and investigate the enzyme property.

METHODSA HAO was purified by an anion-exchange and gel-filtration chromatography from strain Y16. The purity and molecular mass were determined by RP-HPLC and SDS-PAGE. The HAO activity was detected by monitoring the reduction of potassium ferricyanide using hydroxylamine as substrate and ferricyanide as electron acceptor. The partial amino acid sequence was determined by mass spectrometry.

RESULTSThe low-temperature HAO with a molecular mass of 61 kDa was purified from strain Y16 by an anion-exchange and gel-filtration chromatography. The enzyme exhibited an ability to oxidize hydroxylamine in wide temperature range (4-40 °C) in vitro using hydroxylamine as substrate and ferricyanide as electron acceptor. It was stable in the temperature range of 4 to 15 °C and pH range of 6.0 to 8.5 with less than 30% change in its activity. The optimal temperature and pH were 15 °C and 7.5, respectively. Three peptides were determined by mass spectrometry which were shown to be not identical to other reported HAOs.

CONCLUSIONThis is the first study to purify a low-temperature HAO from a heterotrophic nitrifier Acinetobacter sp. It differs from other reported HAOs in molecular mass and enzyme properties. The findings of the present study have suggested that the strain Y16 passes through a hydroxylamine-oxidizing process catalyzed by a low-temperature HAO for ammonium removal.

Acinetobacter ; enzymology ; genetics ; metabolism ; Amino Acid Sequence ; Cold Temperature ; Gene Expression Regulation, Bacterial ; physiology ; Gene Expression Regulation, Enzymologic ; physiology ; Hydrogen-Ion Concentration ; Oxidoreductases ; genetics ; metabolism ; Substrate Specificity

The most important factor in the pathogenesis of biomaterial-associated Staphylococcal infections is the formation of bacterial biofilms. Biofilm formation was regulated or influenced by quorum sensing. One of the quorum sensing systems agr is genus specific which controls the expression of a series of toxins and virulence factors and the interaction with the innate immune system. New research indicates that the role of agr during infection is controversial. The research progress will play an important role in the development of novel antibacterial agents and management of device-related infection of Staphylococci.
Bacterial Proteins ; physiology ; Biocompatible Materials ; Biofilms ; growth & development ; Gene Expression Regulation, Bacterial ; Humans ; Signal Transduction ; genetics ; Staphylococcal Infections ; microbiology ; Staphylococcus ; genetics ; physiology ; Trans-Activators ; physiology

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

Pseudomonas aeruginosa is an important pathogenic bacterium of nosocomial infections. The microbe easily produce biofilm which brings us much difficulties in clinical treatment. The formation processes of biofilm, including the stages of early bacteria planting, mushroom-like structure forming and extracellular matrix producing, are regulated by a series of molecules and genes. And quorum sensing system of the microbe is responsible for regulation of the whole process of biofilm formation. According to the process of biofilm formation and the mimitat associated regulation mechanism, several anti-biofilm therapeutic strategies have been applied in clinical medicine, and some novel drugs and methods are developed.
Biofilms ; growth & development ; Gene Expression Regulation, Bacterial ; Polysaccharides, Bacterial ; metabolism ; Pseudomonas Infections ; drug therapy ; microbiology ; Pseudomonas aeruginosa ; genetics ; physiology ; Quorum Sensing ; genetics ; physiology

OBJECTIVEYersinia enterocolitica is an extracellular pathogen and its related antigens interact with the host immune system. We investigated the difference in immunological characteristics between a highly pathogenic and poorly pathogenic strain of Y. enterocolitica.

METHODSWe used SDS-PAGE and western blotting to characterize lipopolysaccharide (LPS), Yersinia outer membrane proteins (Yops), membrane proteins, and whole-cell proteins from poorly pathogenic Y. enterocolitica bio-serotype 2/O:9, isolated from China, and highly pathogenic bio-serotype 1B/O:8, isolated from Japan.

RESULTSThese two strains of Y. enterocolitica had different LPS immune response patterns. Comparison of their Yops also showed differences that could have accounted for their differences in pathogenicity. The membrane and whole-cell proteins of both strains were similar; immunoblottting showed that the 35 kD and perhaps the 10 kD proteins were immunogens in both strains.

CONCLUSIONThe major antigens of the two strains eliciting the host immune response were the LPS and membrane proteins, as shown by comparing protein samples with reference and purified preparations.

Animals ; Antigens, Bacterial ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Female ; Gene Expression Regulation, Bacterial ; physiology ; Lipopolysaccharides ; metabolism ; Rabbits ; Yersinia enterocolitica ; classification ; metabolism

OBJECTIVETo investigate the relationship between the expression of the quorum-sensing related genes during Enterococcus faecalis(Ef) biofilm formation.

METHODSEf biofilms model was established in vitro and film formation process was observed by confocal laser scanning microscope at 6, 12, 24 and 48 hours respectively.Quantification of biofilms was achieved by staining with crystal violet.Real-time fluorescence quantitative PCR method was used to detect the expression of fsrB, gelE and sprE genes in the process of Ef biofilm formation.

RESULTSA lot of live and dead bacteria unevenly distributed in Ef biofilm. The quantity of biofilms increased with time within 24 hours and was 0 h:0.00 ± 0.00, 6 h:1.09 ± 0.13, 12 h:2.10 ± 0.79, 24 h:3.30 ± 0.13, which was significantly different among the 4 time period(P < 0.05). The quantity of biofilm at 48 h(3.51 ± 0.01) increased slightly compared with 24 h(3.30 ± 0.13) , but did not show significant difference.Quantitative real-time PCR showed that the expression of quorum-sensing related fsrB increased with time within 24 hours and was 0 h:9.98 ± 0.46, 6 h:23.45 ± 1.13, 12 h:47.30 ± 2.49, 24 h: 331.30 ± 2.18, which was significantly different among the 4 time period(P < 0.05). The expression of gelE was 0 h: 6.54 ± 0.73, 6 h: 14.26 ± 1.24, 12 h: 37.47 ± 2.35, 24 h:264.80 ± 5.10(P < 0.05). The expression of sprE was 0 h: 7.72 ± 0.74, 6 h: 21.15 ± 0.96, 12 h:49.87 ± 3.18, 24 h:441.89 ± 7.74, which was significantly different among the 4 time period(P < 0.05).

CONCLUSIONSThe fsrB, gelE and sprE genes are closely related to the biofilm formation in Ef.

Bacterial Proteins ; metabolism ; Biofilms ; growth & development ; Enterococcus faecalis ; genetics ; metabolism ; physiology ; Gelatinases ; metabolism ; Gene Expression Regulation, Bacterial ; Genes, Bacterial ; Quorum Sensing ; Serine Proteases ; 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