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*

Similar to other studies of bacterial pathogens, current studies of the pathogenesis of Riemerella anatipestifer (RA) are focused mainly on in vitro culture conditions. To elucidate further the pathogenesis of RA in vivo, bacterial RNA was extracted from overnight tryptic soy broth cultures (in vitro) and from the blood of infected ducks (in vivo) for comparative RNA sequencing analysis. In total, 682 upregulated genes were identified in vivo. Among the upregulated genes, a signal transduction response regulator (ArsR) and a signal transduction histidine kinase (SthK) were predicted to be located on the same operon. A mutant was constructed by deletion of both of these genes. Duck infection tests showed that genes ArsR and SthK were related to the virulence of the pathogen in vivo. Differentially expressed genes identified by comparison of in vitro and in vivo conditions provided an insight into the physiological process of RA infection and provided an opportunity to identify additional virulence factors.
Ducks ; Genes, vif ; Histidine ; In Vitro Techniques ; Operon ; Phosphotransferases ; Physiological Processes ; Riemerella ; RNA, Bacterial ; Sequence Analysis, RNA ; Signal Transduction ; Virulence Factors ; Virulence

OBJECTIVES: Biofilm formation is one of the important features of Staphylococcus epidermidis, particularly in nosocomial infections. We aimed to investigate the biofilm production by phenotypic methods and the presence of ica genes in S epidermidis. METHODS: A total of 41 S epidermidis isolates were recovered from different clinical specimens. Biofilm formation was evaluated by microtiter plate, tube method and Congo red agar method. The presence of icaA and icaD genes was investigated by PCR. Validity of methods (sensitivity and specificity), and metrics for test performance (positive/negative predictive value, and positive/negative likelihood ratio) were determined. RESULTS: By both microtiter plate and tube method, 53.6% of S epidermidis isolates were able to produce biofilm, whilst only 24.4% of isolates provided a biofilm phenotype on Congo red agar plates. icaA and icaD genes were found in 100% and 95.1% of isolates, respectively. Biofilm phenotypes accounted for 4.8% by microtiter plate assay, despite the absence of the ica gene. Congo red agar and PCR exhibited a lower sensitivity (18% and 45.5%, respectively) for identifying the biofilm phenotype in comparison to microtiter plate. CONCLUSION: The microtiter plate method remains generally a better tool to screen biofilm production in S epidermidis. In addition, the ability of S epidermidis to form biofilm is not always dependent on the presence of ica genes, highlighting the importance of ica-independent mechanisms of biofilm formation. The use of reliable methods to specifically detect biofilms can be helpful to treat the patients affected by such problematic bacteria.
Agar ; Bacteria ; Biofilms* ; Congo Red ; Cross Infection ; Humans ; Methods ; Operon ; Phenotype ; Polymerase Chain Reaction ; Staphylococcus epidermidis* ; Staphylococcus*

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

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

Vibrio vulnificus causes fatal infections in susceptible individuals. Group 1 capsular polysaccharide (CPS) operon is responsible for CPS expression, which plays an essential role in the pathogenesis of this pathogen. Cyclic AMP (cAMP) and cAMP receptor protein (crp) complex, which responds to glucose availability and functions as a global regulator, has been known to affect CPS production in this pathogen. This study was undertaken to experimentally verify whether cAMP-Crp directly or indirectly affects CPS production. A mutation in cyaA encoding adenylate cyclase, which is required for cAMP biosynthesis, inhibited V. vulnificus growth and changed opaque colonies to translucent colonies, and these changes were recovered by complementing cyaA or by adding exogenous cAMP. A mutation in crp encoding Crp also inhibited V. vulnificus growth and changed opaque colonies to translucent colonies, and these changes were recovered by complementing crp. Moreover, the crp or cyaA mutation decreased the susceptibility of V. vulnificus against NaOCl. The crp mutation reduced the transcription levels of group 1 CPS operon on a per cell basis. Glucose addition in the absence of Crp stimulated V. vulnificus growth, changed translucent colonies to opaque colonies, and increased the transcription levels of group 1 CPS operon. These results indicate that cAMP or Crp is indirectly involved in optimal CPS production by positively affecting metabolism or V. vulnificus growth rather than by directly controlling the expression of group 1 CPS operon.
Adenylyl Cyclases ; Complement System Proteins ; Cyclic AMP Receptor Protein ; Cyclic AMP* ; Glucose ; Metabolism ; Operon ; Vibrio vulnificus

β-carotene belongs to carotenoids family, widely applied in pharmaceuticals, neutraceuticals, cosmetics and food industries. In this study, three key genes (dxs, idi, and crt operon) within β-carotene synthetic pathway in recombinant Escherichia coli strain CAR005 were modulated with RBS Library to improve β-carotene production. There were 7%, 11% and 17% increase of β-carotene yield respectively after modulating dxs, idi and crt operon genes with RBS Library, demonstrating that modulating gene expression with regulatory parts libraries would have more opportunities to obtain optimal production of target compound. Combined modulation of crt operon, dxs and idi genes led to 35% increase of β-carotene yield compared to parent strain CAR005. The optimal gene expression strength identified in single gene modulation would not be the optimal strength when used in combined modulation. Our study provides a new strategy for improving production of target compound through modulation of gene expression.
Escherichia coli ; metabolism ; Gene Expression ; Gene Library ; Operon ; beta Carotene ; biosynthesis

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