Humans ; Staphylococcus lugdunensis/genetics* ; Peptides, Cyclic ; Chromosomes ; Operon/genetics* ; Staphylococcal Infections/genetics* ; Bacterial Proteins/genetics* ; Anti-Bacterial Agents

Bacterial Proteins/metabolism* ; Operon/genetics* ; Phosphotransferases/metabolism* ; Quorum Sensing/genetics* ; Vibrio cholerae/genetics*

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.
Cloning, Molecular ; Escherichia coli/genetics* ; Genes, Reporter/genetics* ; Genetic Vectors/genetics* ; Lac Operon/genetics* ; Plasmids/genetics* ; beta-Galactosidase/genetics*

OBJECTIVETo understand the mechanism of invasion by Legionella dumoffii.

METHODSThe L. dumoffii strain Tex-KL was mutated using the Tn903 derivative, Tn903dIIlacZ. After screening 799 transposon insertion mutants, we isolated one defective mutant. We then constructed the gene-disrupted mutant, KL16, and studied its invasion of and intracellular growth in HeLa and A549 cells, and in A/J mice survival experiments. The structure of traC-traD operon was analyzed by RT-PCR.

RESULTSThe transposon insertion was in a gene homologous to Salmonella typhi traC, which is required for the assembly of F pilin into the mature F pilus structure and for conjugal DNA transmission. Results from RT-PCR suggested that the traC-traD region formed an operon. We found that when the traC gene was disrupted, invasion and intracellular growth of L. dumoffii Tex-KL were impaired in human epithelial cells. When mice were infected by intranasal inoculation with a traC deficient mutant, their survival significantly increased when compared to mice infected with the wild-type strain..

CONCLUSIONOur results indicated that the traC-traD operon is required for the invasion and intracellular growth abilities of L. dumoffii Tex-KL in epithelial cells.

A549 Cells ; Animals ; Genes, Bacterial ; HeLa Cells ; Humans ; Legionella ; genetics ; physiology ; Male ; Mice ; Mutation ; Operon

The expression of dentin sialophosphoprotein (DSPP) is the marker for cells differentiated into odontoblasts. This study attempted to analyze the DSPP promoter and build the reporter LacZ expression system driven by this promoter, which allows convenient and quick detection of odontoblast cells. First, we separated the human dental mesenchymal cells in which the expression of DSPP can be effectively induced by dexamethasone. Second, four 5' flanking regions of human DSPP gene (- 4 000-+54, -2 500-+54, -1 447-+54 and -1 027-+54) were analyzed, the results showed that the highest promoter activity lied in the -2 500-+54 region. The promoter activity is reduced when the 5' flanking region was extended from -2 500 to -4 000 bp upstream from the transcription start site; The promoter activity are also decreased when the 5' flanking regions were shorted from -2 500 to -1 447 bp and from -1 447 to -1 027 bp, indicating that potential suppresser elements are lied in the region between -4 000 and -2 500 bp and potential activator elements are lied in the region between -2 500 and -1 027 bp. Then we constructed the lentiviral report vector phDSPP-LacZ containing the -2 500-+ 54 promoter region in front of the LacZ gene. The expression of LacZ was detected using X-Gal staining in both human dental mesenchymal cells and immortalized human dental mesenchymal cells infected with phDSPP-LacZ. The phDSPP-LacZ lentiviral vector may provide a more convenient method to detect the expression of DSPP in human odontogenic differentiation, tooth development and tooth regeneration studies.
Cell Differentiation ; Extracellular Matrix Proteins ; genetics ; Genes, Reporter ; Humans ; Lac Operon ; Odontoblasts ; cytology ; Phosphoproteins ; genetics ; Promoter Regions, Genetic ; Sialoglycoproteins ; genetics

Animals ; Bacterial Proteins ; genetics ; metabolism ; Biofilms ; growth & development ; Gene Expression Regulation, Bacterial ; Lipopolysaccharides ; chemistry ; metabolism ; Operon ; Promoter Regions, Genetic ; Protein Binding ; Siphonaptera ; microbiology ; Species Specificity ; Transcription, Genetic ; Transferases ; genetics ; metabolism ; Virulence ; Yersinia pestis ; genetics ; metabolism ; pathogenicity ; Yersinia pseudotuberculosis ; genetics ; metabolism

OBJECTIVEThe complex of the cyclic AMP receptor protein (CRP) and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems (PTS) is regulated by the CRP-cAMP complex. The aim of the study is to investigate how the CRP-cAMP complex acting on the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype.

METHODSThe crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. cholerae El Tor. Electrophoretic mobility shift assays (EMSA) and the reporter plasmid pBBRlux were used to confirm the role that the CRP-cAMP complex playing on the mannitol PTS operon mtl.

RESULTSIn this study, we confirmed that CRP is strictly needed for the activation of the mtl operon. We further experimentally identified five CRP binding sites within the promoter region upstream of the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype and found that these sites display different affinities for CRP and provide different contributions to the activation of the operon.

CONCLUSIONThe five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. cholerae, indicating an elaborate and subtle CRP activation mechanism.

Bacterial Proteins ; genetics ; Base Sequence ; Cyclic AMP ; metabolism ; Cyclic AMP Receptor Protein ; Gene Expression Regulation, Bacterial ; Mannitol ; Molecular Sequence Data ; Operon ; Phosphotransferases ; Vibrio cholerae

Development of controllable hypermutable cells can greatly benefit understanding and harnessing microbial evolution. However, there have not been any similar systems developed for Clostridium, an important bacterial genus. Here we report a novel two-step strategy for developing controllable hypermutable cells of Clostridium acetobutylicum, an important and representative industrial strain. Firstly, the mutS/L operon essential for methyldirected mismatch repair (MMR) activity was inactivated from the genome of C. acetobutylicum to generate hypermutable cells with over 250-fold increased mutation rates. Secondly, a proofreading control system carrying an inducibly expressed mutS/L operon was constructed. The hypermutable cells and the proofreading control system were integrated to form a controllable hypermutable system SMBMutC, of which the mutation rates can be regulated by the concentration of anhydrotetracycline (aTc). Duplication of the miniPthl-tetR module of the proofreading control system further significantly expanded the regulatory space of the mutation rates, demonstrating hypermutable Clostridium cells with controllable mutation rates are generated. The developed C. acetobutylicum strain SMBMutC2 showed higher survival capacities than the control strain facing butanol-stress, indicating greatly increased evolvability and adaptability of the controllable hypermutable cells under environmental challenges.
Butanols ; pharmacology ; Cell Engineering ; methods ; Clostridium acetobutylicum ; cytology ; drug effects ; genetics ; physiology ; DNA Methylation ; genetics ; DNA Mismatch Repair ; genetics ; Evolution, Molecular ; Genome, Bacterial ; genetics ; MutS DNA Mismatch-Binding Protein ; genetics ; Mutation ; Operon ; genetics ; Stress, Physiological ; drug effects ; genetics

OBJECTIVETo construct a red fluorescent shuttle vector controlled by recA operon promoter to transform Streptococcus mutans.

METHODSThe promoter of recA was amplified from Streptococcus mutans UA159, and connected to plasmid pDsRed2-N1 to construct pRred with a red fluorescent coding gene, which was then inserted into the shuttle vector pDL276 to construct pLRred.

RESULTSpLRred was successfully constructed, and Escherichia coli transformed with the pLRred plasmid could express reporter gene DsRed.

CONCLUSIONSThe recombination plasmid pLRred can be used in the further research of the expression of cariogenic virulence factor gene by Streptococcus mutans in biofilm.

Escherichia coli ; genetics ; metabolism ; Fluorescent Dyes ; Genes, Essential ; Genes, Reporter ; Genetic Vectors ; Luminescent Proteins ; genetics ; Operon ; Plasmids ; Promoter Regions, Genetic ; Rec A Recombinases ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Streptococcus mutans ; genetics ; Transformation, Bacterial

In this study, the association between virulence genotypes and phylogenetic groups among Escherichia (E.) coli isolates obtained from pet dogs and cats with cystitis was detected, and fingerprinting methods were used to explore the relationship among strains. Forty uropathogenic E. coli (UPEC) isolated from dogs (n = 30) and cats (n = 10) in Italy were analysed by polymerase chain reaction (PCR) for the presence of virulence factors and their classification into phylogenetic groups. The same strains were characterized by repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR techniques. We found a high number of virulence factors such as fimbriae A, S fimbriae (sfa) and cytotoxic necrotizing factor 1 (cnf1) significantly associated with phylogenetic group B2. We demonstrated a high correlation between alpha-hemolysin A and pyelonephritis C, sfa, and cnf1 operons, confirming the presence of pathogenicity islands in these strains. In addition, UPEC belonging to group B2 harboured a greater number of virulence factors than strains from phylogenetic groups A, B1, and D. REP- and ERIC-PCR grouped the UPEC isolates into two major clusters, the former grouping E. coli strains belonging to phylogenetic group B2 and D, the latter grouping those belonging to groups A and B1. Given the significant genetic variability among the UPEC strains found in our study, it can be hypothesized that no specific genotype is responsible for cystitis in cats or dogs.
Animals ; Bacterial Proteins/analysis ; Bacterial Toxins/analysis ; Cat Diseases/microbiology ; Cats ; Cystitis/*microbiology ; Dog Diseases/microbiology ; Dogs ; Escherichia coli Infections/complications/microbiology/*veterinary ; Escherichia coli Proteins/analysis ; Female ; Genetic Variation ; Hemolysin Proteins/analysis ; Italy ; Male ; Operon ; Phylogeny ; Polymerase Chain Reaction ; Pyelonephritis/*microbiology ; Uropathogenic Escherichia coli/classification/*genetics/i ; Virulence Factors/*genetics