scholarly journals Effect of Promoter Region Mutations and mgrA Overexpression on Transcription of norA, Which Encodes a Staphylococcus aureus Multidrug Efflux Transporter

2005 ◽  
Vol 49 (1) ◽  
pp. 161-169 ◽  
Author(s):  
Glenn W. Kaatz ◽  
Rama V. Thyagarajan ◽  
Susan M. Seo
Keyword(s):  
Staphylococcus Aureus ◽  
Inverted Repeat ◽  
Promoter Region ◽  
Regulatory Protein ◽  
Multidrug Efflux ◽  
Full Effect ◽  
Repressive Effect ◽  
Dose Dependent Fashion ◽  
Dose Dependent ◽  
Promoter Mutant

ABSTRACT NorA is a Staphylococcus aureus multidrug transporter that confers resistance to structurally distinct compounds. The MgrA global regulatory protein is reported to augment norA expression when mgrA is overexpressed from an undefined plasmid-based promoter. Further details about norA regulatory mechanisms are scant. A chromosomal norA::lacZ transcriptional fusion was constructed in different S. aureus strains, and allele replacement was used to define the relevance of promoter region sequences to norA expression. The effect of mgrA overexpression in wild-type and mutant backgrounds was also determined. Contrary to existing data, overexpression of mgrA repressed norA transcription in all parent and selected norA promoter mutant strains in a dose-dependent fashion. Disruption of a near-perfect inverted repeat or other putative regulatory protein binding sites did not affect norA transcription, but the repressive effect of mgrA overexpression was blunted in these mutants. This result, and the conservation of all of these motifs in S. aureus, suggests that their presence is required for the full effect of MgrA, or other regulatory proteins, on norA expression. Mutations at the +5 nucleotide of norA mRNA (flqB mutations) had a major impact; all resulted in markedly increased norA expression that was significantly reversed by mgrA overexpression. The flqB position of norA mRNA is part of a conserved imperfect inverted repeat; it is feasible that this motif could be a binding site for a norA regulatory protein.

scholarly journals MepR, a Repressor of the Staphylococcus aureus MATE Family Multidrug Efflux Pump MepA, Is a Substrate-Responsive Regulatory Protein

2006 ◽  
Vol 50 (4) ◽  
pp. 1276-1281 ◽  
Author(s):  
Glenn W. Kaatz ◽  
Carmen E. DeMarco ◽  
Susan M. Seo
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Regulatory Protein ◽  
Inducible Promoter ◽  
Multidrug Efflux ◽  
Recognition Sequence ◽  
Multidrug Efflux Pump ◽  
Mobility Shift ◽  
Repressive Effect ◽  
Operator Interaction

ABSTRACT The mepRAB gene cluster of Staphylococcus aureus encodes a MarR family repressor (MepR; known to repress mepA expression), a MATE family multidrug efflux pump (MepA), and a protein of unknown function (MepB). In this report, we show that MepR also is autoregulatory, repressing the expression of its own gene. Exposure of strains containing a mepR::lacZ fusion with mepR provided in trans under the control of an inducible promoter, or a mepA::lacZ fusion alone, to subinhibitory concentrations of MepA substrates resulted in variably increased expression mainly of mepA. Mobility shift assays revealed that MepR binds upstream of mepR and mepA, with an apparently higher affinity for the mepA binding site. MepA substrates abrogated MepR binding to each site in a differential manner, with the greatest effect observed on the MepR-mepA operator interaction. DNase I footprinting identified precise binding sites which included promoter motifs, inverted repeats, and transcription start sites for mepR and mepA, as well as a conserved GTTAG motif, which may be a signature recognition sequence for MepR. Analogous to other multidrug efflux pump regulatory proteins such as QacR, the substrate-MepR interaction likely results in its dissociation from its mepA, and in a more limited fashion its mepR, operator sites and relief of its repressive effect. The enhanced effect of substrates on mepA compared to mepR expression, and on the MepR-mepA operator interaction, results in significant relief of mepA and relative maintenance of mepR repression, leading to increased MepA protein unimpeded by MepR when the need for detoxification exists.

scholarly journals Inhibition of Penicillinase by Epigallocatechin Gallate Resulting in Restoration of Antibacterial Activity of Penicillin against Penicillinase-Producing Staphylococcus aureus

2002 ◽  
Vol 46 (7) ◽  
pp. 2266-2268 ◽  
Author(s):  
Wei-Hua Zhao ◽  
Zhi-Qing Hu ◽  
Yukihiko Hara ◽  
Tadakatsu Shimamura
Keyword(s):  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Inhibitory Concentration ◽  
Epigallocatechin Gallate ◽  
Clinical Isolates ◽  
Main Constituent ◽  
Tea Catechins ◽  
Dose Dependent Fashion ◽  
Dose Dependent

ABSTRACT The combination of epigallocatechin gallate (EGCg, a main constituent of tea catechins) with penicillin showed synergism against 21 clinical isolates of penicillinase-producing Staphylococcus aureus. Besides binding directly to peptidoglycan, the inhibition of penicillinase activity by EGCg is responsible for the synergism. EGCg inhibited the penicillinase activity in a dose-dependent fashion, with a 50% inhibitory concentration of 10 μg/ml.

scholarly journals Mutations within themepAOperator Affect Binding of the MepR Regulatory Protein and Its Induction by MepA Substrates in Staphylococcus aureus

2015 ◽  
Vol 197 (6) ◽  
pp. 1104-1114 ◽  
Author(s):  
Bryan D. Schindler ◽  
Susan M. Seo ◽  
Ivan Birukou ◽  
Richard G. Brennan ◽  
Glenn W. Kaatz
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Sites ◽  
Regulatory Protein ◽  
Titration Calorimetry ◽  
Multidrug Efflux ◽  
Mobility Shift ◽  
Content Type ◽  
Operator Mutant ◽  
Current Sequence

The expression ofmepA, encoding theStaphylococcus aureusMepA multidrug efflux protein, is repressed by the MarR homologue MepR. Repression occurs through binding of two MepR dimers to an operator with two homologous and closely approximated pseudopalindromic binding sites (site 1 [S1] and site 2 [S2]). MepR binding is impeded in the presence of pentamidine, a MepA substrate. The effects of variousmepAoperator mutations on MepR binding were determined using electrophoretic mobility shift assays and isothermal titration calorimetry, and anin vivoconfirmation of the effects observed was established for a fully palindromic operator mutant. Altering the S1-S2 spacing by 1 to 4 bp severely impaired S2 binding, likely due to a physical collision between adjacent MepR dimers. Extension of the spacing to 9 bp eliminated the S1 binding-mediated DNA allostery required for efficient S2 binding, consistent with positive cooperative binding of MepR dimers. Binding of a single dimer to S1 was maintained when S2 was disrupted, whereas disruption of S1 eliminated any significant binding to S2, also consistent with positive cooperativity. Palindromization of binding sites, especially S2, enhanced MepR affinity for themepAoperator and reduced MepA substrate-mediated MepR induction. As a result, the on-off equilibrium between MepR and its binding sites was shifted toward the on state, resulting in less free MepR being available for interaction with inducing ligand. The selective pressure(s) under whichmepAexpression is advantageous likely contributed to the accumulation of mutations in themepAoperator, resulting in the current sequence from which MepR is readily induced by MepA substrates.

scholarly journals Promoter Analysis of the cap8 Operon, Involved in Type 8 Capsular Polysaccharide Production in Staphylococcus aureus

1999 ◽  
Vol 181 (8) ◽  
pp. 2492-2500 ◽  
Author(s):  
Shu Ouyang ◽  
Subrata Sau ◽  
Chia Y. Lee
Keyword(s):  
Staphylococcus Aureus ◽  
Yeast Extract ◽  
Inverted Repeat ◽  
Promoter Activity ◽  
Capsular Polysaccharide ◽  
Regulatory Protein ◽  
Biological Significance ◽  
Growth Media ◽  
Transcriptional Level ◽  
Promoter Deletion Analysis

ABSTRACT The production of type 8 capsular polysaccharide (CP8) inStaphylococcus aureus is regulated in response to a variety of environmental factors. The cap8 genes required for the CP8 production in strain Becker are transcribed as a single large transcript by a primary promoter located within a 0.45-kb region upstream of the first gene of the cap8 gene cluster. In this study, we analyzed the primary cap8 promoter region in detail. We determined the transcription initiation site of the primary transcript by primer extension and identified the potential promoter sequences. We found several inverted and direct repeats upstream of the promoter. Deletion analysis and site-directed mutagenesis showed that a 10-bp inverted repeat of one of the repeats was required for promoter activity. We showed that the distance but not the specific sequences between the inverted repeat and the promoter was critical to the promoter activity. However, insertion of a DNA sequence with two or four helix turns in this intervening region had a slight effect on promoter activity. To demonstrate the biological significance of the 10-bp inverted repeat, we constructed a strain with a mutation in the repeat in the S. aureus Becker chromosome and showed that the repeat affected CP8 production mostly at the transcriptional level. By gel mobility shift assay, we demonstrated that strain Becker produced at least one protein capable of specific binding to the 10-bp inverted repeat, indicating that the repeat serves as a positive regulatory protein binding site. In addition, reporter gene fusion analysis showed that the cap8 promoter activity was influenced by various growth media and affected most by yeast extract. Our results suggest that yeast extract may exert its profound inhibitory effect on cap8 gene expression through the 10-bp inverted repeat element.

scholarly journals Novel Ciprofloxacin-Resistant, Nalidixic Acid-Susceptible Mutant of Staphylococcus aureus

2002 ◽  
Vol 46 (7) ◽  
pp. 2276-2278 ◽  
Author(s):  
Laura J. V. Piddock ◽  
Yu Fang Jin ◽  
Mark A. Webber ◽  
Martin J. Everett
Keyword(s):  
Staphylococcus Aureus ◽  
Nalidixic Acid ◽  
Promoter Region ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump

ABSTRACT A ciprofloxacin-resistant, nalidixic acid-susceptible mutant of Staphylococcus aureus (F145) contained no mutations within gyrA, gyrB, grlA, and grlB or within norA or its promoter region. MICs and accumulation studies suggest the role of a novel multidrug efflux pump.

scholarly journals Insulin exerts actions through a distinct species of guanine nucleotide regulatory protein: inhibition of adenylate cyclase

1983 ◽  
Vol 214 (2) ◽  
pp. 547-552 ◽  
Author(s):  
C M Heyworth ◽  
M D Houslay
Keyword(s):  
Adenylate Cyclase ◽  
Regulatory Protein ◽  
Distinct Species ◽  
Inhibitory Effect ◽  
Guanine Nucleotide ◽  
Cellular Effects ◽  
Dose Dependent Fashion ◽  
Guanine Nucleotide Regulatory Protein ◽  
Median Effective Concentration ◽  
Dose Dependent

Insulin failed to exert an effect on the basal and glucagon- and guanosine 5′-[beta, gamma-imido]-triphosphate-stimulated adenylate cyclase activities of hepatocyte membranes. In the presence of high GTP (0.1 mM) concentrations, however, insulin was shown to inhibit adenylate cyclase activity. This effect was dose-dependent, exhibiting an EC50 (median effective concentration) of 3 microM for GTP. Elevated glucagon concentrations blocked the inhibitory effect of insulin in a dose-dependent fashion, with an EC50 of 1 nM. The insulin inhibition was dose-dependent (EC50 = 90 pM). The inhibitory effects of insulin were abolished using membranes from either glucagon-desensitized hepatocytes or cholera-toxin-treated hepatocytes. If either Mn2+ replaced Mg2+ in adenylate cyclase assays or Na+ was removed from the assay mixtures then insulin failed to exert any inhibitory effect. It is suggested that insulin exerts its action on adenylate cyclase through an inhibitory guanine nucleotide protein. This is integrated with the proposal [Heyworth, Rawal & Houslay (1983) FEBS Lett. 154, 87-91; Heyworth, Wallace & Houslay (1983) Biochem. J. in the press] that insulin mediates a variety of cellular effects through a specific guanine nucleotide regulatory protein and associated protein kinase(s).

The Staphylococcus aureus surface protein SasG and its homologues promote bacterial adherence to human desquamated nasal epithelial cells

Microbiology ◽  
2003 ◽  
Vol 149 (10) ◽  
pp. 2759-2767 ◽  
Author(s):  
Fiona M. Roche ◽  
Mary Meehan ◽  
Timothy J. Foster
Keyword(s):  
Staphylococcus Aureus ◽  
Epithelial Cells ◽  
Surface Protein ◽  
Bacterial Adherence ◽  
Homologous Proteins ◽  
Nasal Epithelial Cells ◽  
Dose Dependent Fashion ◽  
Dose Dependent ◽  
Reduced Adherence

Staphylococcus aureus binds to human desquamated nasal epithelial cells, a phenomenon likely to be important in nasal colonization. ClfB was identified previously as one staphylococcal adhesin that promoted binding to nasal epithelia. In this study, it is shown that the S. aureus surface protein SasG, identified previously by in silico analysis of genome sequences, and two homologous proteins, Pls of S. aureus and AAP of Staphylococcus epidermidis, also promote bacterial adherence to nasal epithelial cells. Conditions for in vitro expression of SasG by S. aureus were not found. Adherence assays were therefore performed with S. aureus and Lactococcus lactis expressing SasG from an expression plasmid. These studies showed that SasG did not bind several ligands typically bound by S. aureus. Significantly, SasG and Pls did promote bacterial adherence to nasal epithelial cells. Furthermore, pre-incubation of epithelial cells with purified recombinant proteins revealed that the N-terminal A regions of SasG, Pls and AAP, but not the B repeats of SasG, inhibited adherence of L. lactis expressing SasG in a dose-dependent fashion. These results suggest that SasG, Pls and AAP bind to the same as-yet-unidentified receptor on the surface of nasal epithelial cells. In addition, expression of SasG, like Pls, reduced adherence of S. aureus to fibronectin and fibrinogen.

Plasminogen Interactions with Immobilized Fibrinogen

1989 ◽  
Vol 62 (04) ◽  
pp. 1078-1082 ◽  
Author(s):  
Burt Adelman ◽  
Patricia Ouynn
Keyword(s):  
Immunosorbent Assay ◽  
Aminocaproic Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Regions ◽  
Dose Dependent Fashion ◽  
Epsilon Aminocaproic Acid ◽  
Dose Dependent

SummaryThis report describes the binding of plasminogen to fibrinogen adsorbed onto polystyrene wells. Binding was determined by enzyme linked immunosorbent assay. Both glu- and lys-plasminogen bound to immobilized fibrinogen in a dose-dependent fashion. However, more lys- than glu-plasminogen bound when equal concentrations of either were added to immobilized fibrinogen. Plasminogen binding was inhibited by epsilon aminocaproic acid indicating that binding was mediated via lysine-binding regions of plasminogen. Soluble fibrinogen added in excess of immobilized fibrinogen did not compete for plasminogen binding but fibrinogen fragments produced by plasmin digestion of fibrinogen did. Treatment of immobilized fibrinogen with thrombin caused a small but significant (p <0.01) increase in plasminogen binding. These studies demonstrate that immobilized fibrinogen binds both glu- and lys-plasminogen and that binding is mediated via lysine-binding regions. These interactions may facilitate plasminogen binding to fibrinogen adsorbed on to surfaces and to cells such as platelets which bind fibrinogen.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

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