Broad-spectrum of conventional antibiotics is one of the key factors that cause antibiotics-resistance of many bacteria. Bacteriocins are regarded as the next generation of antibiotics on account of their narrow-spectrum bactericidal activities. Many attentions have been paid to colicins because they are believed to be safe in regard to human body. In this paper, the genes encoding colicin S4 and its immunity protein were cloned into pQE30 to produce colicin S4 expression vector pQE30-Col S4. Colicin S4 was highly expressed as soluble form in gE colig M15 containing pQE30-Col S4. The yields ranged from 30 mg/L to 50 mg/L. The recombinant colicin S4 with an additional 6 His-tag at its N-terminus was found being similar to the natural colicin S4 in antibacterial activity. It only showed bactericidal activity against E. coli strains, thus makig it attractive to develop this protein as a novel antibiotic with narrow spectrum.
Anti-Bacterial Agents ; biosynthesis ; Bacteriocins ; biosynthesis ; genetics ; Cloning, Molecular ; Colicins ; biosynthesis ; genetics ; Escherichia coli ; drug effects ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo Investigate the differences of sorbitol fermentation related genes and optimize molecular analysis method for distinguishing an epidemic with nonepidemic strains of Vibrio cholerae.

METHODSSequence analysis on four genes of sugar fermentation stimulation protein, periplasmic maltose-binding protein, periplasmic phosphate-binding protein and periplasmic amino acid-binding protein.

RESULTSIn this study, the following data was noticed: for O1 serogroup El Tor biotype V. cholerae, twenty-four epidemic and eight nonepidemic strains were chosen; For O139 serogroup V. cholerae, five epidemic and four nonepidemic strains were chosen. With those genes of sugar fermentation stimulation protein, there were three point mutations. The 106th, 150th, 378th oligonucleotide in epidemic strains were A, A and T, comparing to the nonepidemic strains which were G, G and C. When comparing the protein sequences, epidemic strains had a Threonine at 36th amino acid, whereas nonepidemic strains had an Alanine. The results in O139 serogroup were consistent with those in O1 serogroup El Tor biotype strains. Another two point mutations were found in the genes of periplasmic maltose-binding protein. The 999th, 1003rd oligonucleotides in epidemic strains were A and C, while in nonepidemic which were G and T. For the gene of periplasmic amino acid-binding protein, two point mutations were noticed. The 504th and 690th oligonucleotides in epidemic strains were T and C, but were C and T in nonepidemic. However, no amino acid differences were found in periplasmic maltose-binding protein and periplasmic amino acid-binding protein. For periplasmic amino acid-binding protein gene, there was no difference on oligonucleotide between epidemic and nonepidemic strains.

CONCLUSIONResults suggested that SNPs in these genes might serve as a useful tool to distinguish the epidemic strains from nonepidemic strains. The 36th amino acid mutation of sugar fermentation stimulation protein in epidemic and nonepidemic strains might change the activity of the protein which might be associated with sorbitol fermentation.

Amino Acid Sequence ; Bacterial Proteins ; genetics ; metabolism ; Base Sequence ; Carrier Proteins ; genetics ; metabolism ; Fermentation ; Maltose-Binding Proteins ; Molecular Sequence Data ; Periplasmic Binding Proteins ; genetics ; metabolism ; Phosphate-Binding Proteins ; genetics ; metabolism ; Point Mutation ; Sequence Analysis, Protein ; Sorbitol ; Vibrio cholerae ; genetics ; metabolism

Animals ; Antibodies, Bacterial ; blood ; Bacterial Proteins ; genetics ; immunology ; Brucella Vaccine ; immunology ; Brucella abortus ; immunology ; Female ; Immunization ; Interferon-gamma ; biosynthesis ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Periplasmic Binding Proteins ; genetics ; immunology ; Ribosomal Proteins ; genetics ; immunology ; Vaccines, DNA ; immunology

Anti-Infective Agents ; pharmacology ; Bacterial Outer Membrane Proteins ; genetics ; physiology ; Ciprofloxacin ; pharmacology ; DNA Gyrase ; genetics ; Drug Resistance, Bacterial ; Membrane Proteins ; genetics ; physiology ; Membrane Transport Proteins ; genetics ; physiology ; Mutation ; Pseudomonas aeruginosa ; drug effects

OBJECTIVETo analyze factors related to the virulence associated genes of Leptospires.

METHODSTwelve putative virulence associated genes were detected by polymerase chain reaction (PCR) method in 38 reference strains, 81 field strains of Leptospira interrogans isolated from patients or animals, and 12 avirulent strains of Leptospira biflexa.

RESULTSThese putative virulent genes were widely distributed among the strains of Leptospira interrogans, but only few of them were detected in Leptospira biflexa. Gene lipL32 was detected in all strains of Leptospira interrogans. Distribution of gene lipL36 was varied significantly with detected rates from 0 to 90.91%. Gene la1608 had a positive rate of 87.50% for strains of serogroup Icterohaemorrhagiae, but was only detected in few strains of other serogroups with a range from 0 to 25.00%. Rate of detection on gene sphA was 17.65% in Leptospira interrogans, and was absent in serovar hardjo reference strain.

CONCLUSIONResults indicated that these genes might be of importance for the virulence and pathogenicity of Leptospira interrogans, while gene lipL32 might be one of the common antigens. Gene lipL36 might be involved in serogroup specificity with genetic diversity, but gene la1608 was as one of the genes with specificity for serogroup Icterohaemorrhagiae. However, serovar hadjo might hold quite different genetic characteristics when compared with the other serovars of Leptospires.

Bacterial Outer Membrane Proteins ; genetics ; Bacterial Proteins ; genetics ; Carbohydrate Dehydrogenases ; genetics ; Flagellin ; genetics ; Genes, Bacterial ; genetics ; Hemolysin Proteins ; genetics ; Leptospira ; genetics ; pathogenicity ; Lipoproteins ; genetics ; Polymerase Chain Reaction ; Virulence ; genetics ; Virulence Factors ; genetics

Designed a pair of primers through modifying N-terminal bases (5bps) of gene after ATG but not changing amino acid, and amplified a smaller mutated gene sequnce (468bp) containing two protective antigenic determinants from pBlue-BoNTaHc, N-terminal codon of mutated gene fragment is changed from low to high frenqence in E. coli. Mutated gene was ligated into pGEM-T vector and sequenced, then, cloned into a expression plasmid pBV220. As a result, cloned gene was expressed in insoluble form by temperature inducing (from 30 degrees C to 42 degrees C) in E. coli. Expression product is 40% of total proteins and is of specific binding activity to antibody in ELISA. The successful modification and high level expression of protective fragment of botulinum neurotoxin serotype A(BoNTaHc468) gene is conducive to further study on antitoxin and vaccine.
Bacterial Vaccines ; immunology ; Botulinum Toxins, Type A ; genetics ; immunology ; Clostridium botulinum type A ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis

NB-Cl gene is a potential class IIa bacteriocin gene. To obtain its soluble expression, NB-C1 was fused with the green fluorescent protein (GFP) gene and a recombinant expression vector plVEX 2.4d-GFP-NB-C1 was constructed, which was transformed into Escherichia coli BL21(DE3) pLysS. The expressed fusion protein GFP-NB-CI was purified by Ni-NTA affinity chromatography and the bioactivity was examined using Listeria monocytogenes as the indicator bacteria. The results showed that the expressed fusion protein GFP-NB-C1 was soluble and the final concentration of the purified fusion protein was 36.1 mg/L E. coli culture and had the purity above 95%. The antimicrobial assay of GFP-NB-C1 was analyzed and showed its high activity against Listeria monocytogenes.
Amino Acid Sequence ; Anti-Bacterial Agents ; pharmacology ; Bacteriocins ; biosynthesis ; genetics ; Chromatography, Affinity ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Listeria monocytogenes ; drug effects ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

Cholesterol-dependent cytolysins (CDC) are pore forming toxins. A prototype of the CDC family members is perfringolysin O (PFO), which directly binds to the cell membrane enriched in cholesterol, causing cell lysis. However, an exception of this general observation is intermedilysin (ILY) of Streptococcus intermedius, which requires human CD59 as a receptor in addition to cholesterol for its hemolytic activity. A possible explanation of this functional difference is the conformational variation between the C-terminal domains of the two toxins, particularly in the highly conserved undecapeptide termed tryptophan rich motif. Here, we present the crystal structure of suilysin, a CDC toxin from the infectious swine pathogen Streptococcus suis. Like PFO, suilysin does not require a host receptor for hemolytic activity; yet the crystal structure of suilysin exhibits a similar conformation in the tryptophan rich motif to ILY. This observation suggests that the current view of the structure-function relationship between CDC proteins and membrane association is far from complete.
Amino Acid Sequence ; Animals ; Bacterial Toxins ; chemistry ; Bacteriocins ; chemistry ; Cholesterol ; chemistry ; Crystallography, X-Ray ; Cytotoxins ; chemistry ; Hemolysin Proteins ; chemistry ; genetics ; Molecular Sequence Data ; Point Mutation ; Protein Structure, Tertiary ; Sequence Alignment ; Streptococcus suis ; metabolism ; Swine

The bacterial flagellar structure can be divided into the basal body, the hook and the filament. Three minor components called hook associated proteins (HAP1, HAP2 and HAP3) form a junction between the hook and the filament (HAP1 and HAP3) and a capping structure at the distal end of flagellar filament (HAP2). Vibrio vulnificus is a halophilic pathogenic bacterium that is locomotive by means of a polar flagellum. From a V. vulnificus genome sequencing project, we obtained sequences of V. vulnificus flgK (Vv-flgK), flgL (Vv-flgL), and flaH (Vv-flaH) genes that encode HAP1, HAP3, and HAP2, respectively. To investigate roles of the HAP proteins, deletion mutants of the Vv-flgK, Vv-flgL and Vv-flaH were constructed. Electron microscopic analysis showed that the Vv-flgK or Vv-flgL mutant did not produce an intact polar flagellum while the Vv-flaH mutant produced a fragile flagellar structure. Western blot analysis against a major polar flagellin proposed that the null HAP1 and HAP3 mutations resulted in a failure of normal flagellar assembly since flagellins produced by the mutants were secreted out in the culture supernatants without long flagellar filaments. Motility was completely abolished by a single mutation in HAP1 or HAP3, and the HAP2 mutant showed a decreased motility. Also each of the mutants showed an impaired cytotoxicity and adherence to HeLa cell compared with the isogenic wild type strain. LD(50) increased by 10- and 11-fold in the V. vulnificus HAP3 and HAP2 mutant, respectively. These results suggest that the HAP proteins play important roles in polar flagellation and the virulence of V. vulnificus.
Bacterial Proteins ; Blotting, Western ; Electrons ; Flagella ; Flagellin ; Genome ; HeLa Cells ; Humans ; Proteins ; Sprains and Strains ; Vibrio ; Vibrio vulnificus

OBJECTIVETo investigate the functional relations between the putative proteins YpCD1.08, YpCD1.09, YpCD1.16 encoded in pCD1 plasmid of Yersinia pestis and its type III secretion system (T3SS).

METHODSMutants of YpCD1.08, YpCD1.09, YpCD1.16 were constructed using λ-Red recombinant system. The growth curves of the mutant strains cultivated in TMH medium with or without calcium at 26 °C and 37 °C were determined to analyze the low calcium response phenotype. The transcription levels of ΔYpCD1.08, ΔYpCD1.09, ΔYpCD1.16 in Yersinia pestis and the dependence to temperature were determined using real time RT-PCR after cultivation at 26 °C and 37 °C and extraction of RNA. A β-lactamases reporter system was adopted to study the influence of these genes on the translocation of effector YopE of T3SS.

RESULTSWhen grown in TMH medium without calcium at 26 °C and 37 °C, the growth curve of the YpCD1.08, YpCD1.09, YpCD1.16 mutants were similar to that of the wild-type strain, indicating that the low calcium response of all the mutants were normal. The ratios of YpCD1.08, YpCD1.09, YpCD1.16 gene transcriptional level at 37 °C and 26 °C were 2.3 ± 0.3, 2.3 ± 0.5 and 3.2 ± 0.7, respectively, indicating that these genes were transcribed in Yersinia pestis and their transcription regulations showed a temperature-dependence that was consistent with the well established temperature-dependent expression of Yersinia T3SS genes. The β-lactamases reporter assays demonstrated that ΔYpCD1.08 could translocate much higher level of YopE into HeLa cells, since that the light intensity ratio of 477/520 nm at 140 min was 2.5, whereas it was 1.8 for the wild-type strain, and the values in ΔYpCD1.09 and ΔYpCD1.16 were similar to the wild-type strain.

CONCLUSIONYpCD1.08, YpCD1.09, YpCD1.16 gene are likely to be the new members of T3SS, and the putative protein YpCD1.08 could play some roles in YopE secretion and translocation.

Bacterial Outer Membrane Proteins ; secretion ; Bacterial Secretion Systems ; genetics ; Genes, Bacterial ; Plasmids ; Protein Interaction Mapping ; Yersinia pestis ; genetics ; metabolism ; pathogenicity