scholarly journals Cation-Induced Transcriptional Regulation of the dlt Operon of Staphylococcus aureus

2006 ◽  
Vol 188 (10) ◽  
pp. 3622-3630 ◽  
Author(s):  
Tomaz Koprivnjak ◽  
Vid Mlakar ◽  
Lindsey Swanson ◽  
Benedicte Fournier ◽  
Andreas Peschel ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Envelope ◽  
Divalent Cations ◽  
Northern Blotting ◽  
Start Codon ◽  
Two Component System ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
High Concentrations ◽  
Wall Teichoic Acids

ABSTRACT Lipoteichoic and wall teichoic acids (TA) are highly anionic cell envelope-associated polymers containing repeating polyglycerol/ribitol phosphate moieties. Substitution of TA with d-alanine is important for modulation of many cell envelope-dependent processes, such as activity of autolytic enzymes, binding of divalent cations, and susceptibility to innate host defenses. d-Alanylation of TA is diminished when bacteria are grown in medium containing increased NaCl concentrations, but the effects of increased salt concentration on expression of the dlt operon encoding proteins mediating d-alanylation of TA are unknown. We demonstrate that Staphylococcus aureus transcriptionally represses dlt expression in response to high concentrations of Na+ and moderate concentrations of Mg2+ and Ca2+ but not sucrose. Changes in dlt mRNA are induced within 15 min and sustained for several generations of growth. Mg2+-induced dlt repression depends on the ArlSR two-component system. Northern blotting, reverse transcription-PCR, and SMART-RACE analyses suggest that the dlt transcript begins 250 bp upstream of the dltA start codon and includes an open reading frame immediately upstream of dltA. Chloramphenicol transacetylase transcriptional fusions indicate that a region encompassing the 171 to 325 bp upstream of dltA is required for expression and Mg2+-induced repression of the dlt operon in S. aureus.

scholarly journals Transactivation of Latent Marek's Disease Herpesvirus Genes in QT35, a Quail Fibroblast Cell Line, by Herpesvirus of Turkeys

2000 ◽  
Vol 74 (21) ◽  
pp. 10176-10186 ◽  
Author(s):  
T. Yamaguchi ◽  
S. L. Kaplan ◽  
P. Wakenell ◽  
K. A. Schat
Keyword(s):  
Cell Line ◽  
Fibroblast Cell ◽  
Northern Blotting ◽  
Marek's Disease ◽  
Pcr Analysis ◽  
Marek’S Disease ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Herpesvirus Of Turkeys ◽  
Serotype 1

ABSTRACT The QT35 cell line was established from a methylcholanthrene-induced tumor in Japanese quail (Coturnix coturnix japonica) (C. Moscovici, M. G. Moscovici, H. Jimenez, M. M. Lai, M. J. Hayman, and P. K. Vogt, Cell 11:95–103, 1977). Two independently maintained sublines of QT35 were found to be positive for Marek's disease virus (MDV)-like genes by Southern blotting and PCR assays. Sequence analysis of fragments of the ICP4, ICP22, ICP27, VP16, meq, pp14, pp38, open reading frame (ORF) L1, and glycoprotein B (gB) genes showed a strong homology with the corresponding fragments of MDV genes. Subsequently, a serotype 1 MDV-like herpesvirus, tentatively name QMDV, was rescued from QT35 cells in chicken kidney cell (CKC) cultures established from 6- to 9-day-old chicks inoculated at 8 days of embryonation with QT35 cells. Transmission electron microscopy failed to show herpesvirus particles in QT35 cells, but typical intranuclear herpesvirus particles were detected in CKCs. Reverse transcription-PCR analysis showed that the following QMDV transcripts were present in QT35 cells: sense and antisense meq, ORF L1, ICP4, and latency-associated transcripts, which are antisense to ICP4. A transcript of approximately 4.5 kb was detected by Northern blotting using total RNA from QT35 cells. Inoculation of QT35 cells with herpesvirus of turkeys (HVT)-infected chicken embryo fibroblasts (CEF) but not with uninfected CEF resulted in the activation of ICP22, ICP27, VP16, pp38, and gB. In addition, the level of ICP4 mRNA was increased compared to that in QT35 cells. The activation by HVT resulted in the production of pp38 protein. It was not possible to detect if the other activated genes were translated due to the lack of serotype 1-specific monoclonal antibodies.

scholarly journals Dual Roles of FmtA in Staphylococcus aureus Cell Wall Biosynthesis and Autolysis

2012 ◽  
Vol 56 (7) ◽  
pp. 3797-3805 ◽  
Author(s):  
Aneela Qamar ◽  
Dasantila Golemi-Kotra
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Wall Stress ◽  
Microscopy Study ◽  
Dual Roles ◽  
Content Type ◽  
Teichoic Acids ◽  
Low Concentrations ◽  
High Concentrations ◽  
Wall Teichoic Acids

ABSTRACTThefmtAgene is a member of theStaphylococcus aureuscore cell wall stimulon. The FmtA protein interacts with β-lactams through formation of covalent species. Here, we show that FmtA has weakd-Ala-d-Ala-carboxypeptidase activity and is capable of covalently incorporating C14-Gly into cell walls. The fluorescence microscopy study showed that the protein is localized to the cell division septum. Furthermore, we show that wall teichoic acids interact specifically with FmtA and mediate recruitment of FmtA to theS. aureuscell wall. Subjection ofS. aureusto FmtA concentrations of 0.1 μM or less induces autolysis and biofilm production. This effect requires the presence of wall teichoic acids. At FmtA concentrations greater than 0.2 μM, autolysis and biofilm formation inS. aureusare repressed and growth is enhanced. Our findings indicate dual roles of FmtA inS. aureusgrowth, whereby at low concentrations, FmtA may modulate the activity of the major autolysin (AtlA) ofS. aureusand, at high concentrations, may participate in synthesis of cell wall peptidoglycan. These two roles of FmtA may reflect dual functions of FmtA in the absence and presence of cell wall stress, respectively.

scholarly journals Transcriptional Organization of the czcHeavy-Metal Homeostasis Determinant from Alcaligenes eutrophus

1999 ◽  
Vol 181 (8) ◽  
pp. 2385-2393 ◽  
Author(s):  
Cornelia Große ◽  
Gregor Grass ◽  
Andreas Anton ◽  
Sylvia Franke ◽  
Alexander Navarrete Santos ◽  
...  
Keyword(s):  
Response Regulator ◽  
Regulatory System ◽  
Alcaligenes Eutrophus ◽  
Northern Blotting ◽  
Open Reading Frames ◽  
Mrna Synthesis ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Reporter Strains ◽  
Reading Frames

ABSTRACT The Czc system of Alcaligenes eutrophus mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB2A cation-proton antiporter. DNA sequencing of the region upstream of the czcNICBADRS determinant located on megaplasmid pMOL30 revealed the 5′ end of czcN and a gene for a MgtC-like protein which is transcribed in the orientation opposite that of czc. Additional open reading frames upstream of czc had no homologs in the current databases. Using oligonucleotide-probed Northern blotting experiments, a 500-nucleotide czcN message and a 400-nucleotideczcI message were found, and the presence of 6,200-nucleotide czcCBA message (D. Van der Lelie et al., Mol. Microbiol. 23:493–503, 1997) was confirmed. Induction ofczcN, czcI, czcCBA, andczcDRS followed a similar pattern: transcription was induced best by 300 μM zinc, less by 300 μM cobalt, and only slightly by 300 μM cadmium. Reverse transcription-PCR gave evidence for additional continuous transcription from czcN toczcC and from czcD to czcS, but not between czcA and czcD nor betweenczcS and a 131-amino-acid open reading frame followingczcS. The CzcR putative response regulator was purified and shown to bind in the 5′ region of czcN. A reporter strain carrying a czcNIC-lacZ-czcBADRS determinant on plasmid pMOL30 was constructed, as were ΔczcR and ΔczcS mutants of this strain and of AE128(pMOL30) wild type. Experiments on (i) growth of these strains in liquid culture containing 5 mM Zn2+, (ii) induction of the β-galactosidase in the reporter strains by zinc, cobalt, and cadmium, and (iii) cDNA analysis of czcCBA mRNA synthesis under inducing and noninducing conditions showed that the CzcRS two-component regulatory system is involved in Czc regulation.

scholarly journals Attenuation and Persistence of and Ability To Induce Protective Immunity to a Staphylococcus aureus aroA Mutant in Mice

2006 ◽  
Vol 74 (6) ◽  
pp. 3498-3506 ◽  
Author(s):  
Fernanda R. Buzzola ◽  
María Sol Barbagelata ◽  
Roberto L. Caccuri ◽  
Daniel O. Sordelli
Keyword(s):  
Staphylococcus Aureus ◽  
Protective Immunity ◽  
Lethal Dose ◽  
Bovine Mastitis ◽  
Start Codon ◽  
Economic Losses ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Wild Type Phenotype ◽  
Th1 And Th2 Responses

ABSTRACT Staphylococcus aureus is the most important etiological agent of bovine mastitis, a disease that causes significant economic losses to the dairy industry. Several vaccines to prevent the disease have been tested, with limited success. The aim of this study was to obtain a suitable attenuated aro mutant of S. aureus by transposon mutagenesis and to demonstrate its efficacy as a live vaccine to induce protective immunity in a murine model of intramammary infection. To do this, we transformed S. aureus RN6390 with plasmid pTV1ts carrying Tn917. After screening of 3,493 erythromycin-resistant colonies, one mutant incapable of growing on plates lacking phenylalanine, tryptophan, and tyrosine was isolated and characterized. Molecular characterization of the mutant showed that the affected gene was aroA and that the insertion occurred 756 bp downstream of the aroA start codon. Complementation of the aroA mutant with a plasmid carrying aroA recovered the wild-type phenotype. The mutant exhibited a 50% lethal dose (1 × 106 CFU/mouse) higher than that of the parental strain (4.3 × 104 CFU/mouse). The aroA mutant showed decreased ability to persist in the lungs, spleens, and mammary glands of mice. Intramammary immunization with the aroA mutant stimulated both Th1 and Th2 responses in the mammary gland, as ascertained by reverse transcription-PCR, and induced significant protection from challenge with either the parental wild-type or a heterologous strain isolated from a cow with mastitis.

scholarly journals Modification of Type IV Pilus-Associated Epithelial Cell Adherence and Multicellular Behavior by the PilU Protein of Neisseria gonorrhoeae

2002 ◽  
Vol 70 (7) ◽  
pp. 3891-3903 ◽  
Author(s):  
Hae-Sun Moon Park ◽  
Matthew Wolfgang ◽  
Michael Koomey
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Membrane Trafficking ◽  
Northern Blotting ◽  
Epithelial Tissue ◽  
Twitching Motility ◽  
Loss Of Function ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Type Iv

ABSTRACT Expression of type IV pili (Tfp) correlates with the ability of Neisseria gonorrhoeae to colonize the human host, as well as with adherence to human epithelial tissue, twitching motility, competence for natural transformation, and autoagglutination. N. gonorrhoeae PilF (required for Tfp biogenesis) and PilT (required for twitching motility and transformation) share significant identities with members of a family of putative ATPases involved in membrane trafficking of macromolecules. An open reading frame downstream of the pilT locus encoding a 408-amino-acid protein with 33% identity with the gonococcal PilT protein and 45% identity with the PilU protein in Pseudomonas aeruginosa was characterized, and the corresponding gene was designated pilU. Unlike N. gonorrhoeae pilT mutants, pilU mutants express twitching motility and are competent for DNA transformation. However, loss-of-function mutations in pilU increased bacterial adherence to ME-180 human epithelial cells eightfold and disrupted in vitro Tfp-associated autoagglutination. Comparative alignment of N. gonorrhoeae PilU with other members of the TrbB-like family of traffic ATPases revealed a conserved carboxy-terminal domain unique to family members which are not essential for Tfp biogenesis but which specifically modify Tfp-associated phenotypes. Studies of the pilT-pilU locus by using Northern blotting, transcriptional fusions, and reverse transcription-PCR showed that the two genes encoding closely related proteins with dissimilar effects on Tfp phenotypes are transcribed from a single promoter.

scholarly journals Forespore-Specific Expression of Bacillus subtilis yqfS, Which Encodes Type IV Apurinic/Apyrimidinic Endonuclease, a Component of the Base Excision Repair Pathway

2003 ◽  
Vol 185 (1) ◽  
pp. 340-348 ◽  
Author(s):  
Norma Urtiz-Estrada ◽  
José M. Salas-Pacheco ◽  
Ronald E. Yasbin ◽  
Mario Pedraza-Reyes
Keyword(s):  
Bacillus Subtilis ◽  
Excision Repair ◽  
Transcriptional Start Site ◽  
Northern Blotting ◽  
Start Codon ◽  
Start Site ◽  
Specific Expression ◽  
Reading Frame ◽  
Type Iv

ABSTRACT The temporal and spatial expression of the yqfS gene of Bacillus subtilis, which encodes a type IV apurinic/apyrimidinic endonuclease, was studied. A reporter gene fusion to the yqfS opening reading frame revealed that this gene is not transcribed during vegetative growth but is transcribed during the last steps of the sporulation process and is localized to the developing forespore compartment. In agreement with these results, yqfS mRNAs were mainly detected by both Northern blotting and reverse transcription-PCR, during the last steps of sporulation. The expression pattern of the yqfS-lacZ fusion suggested that yqfS may be an additional member of the EσG regulon. A primer extension product mapped the transcriptional start site of yqfS, 54 to 55 bp upstream of translation start codon of yqfS. Such an extension product was obtained from RNA samples of sporulating cells but not from those of vegetatively growing cells. Inspection of the nucleotide sequence lying upstream of the in vivo-mapped transcriptional yqfS start site revealed the presence of a sequence with good homology to promoters preceding genes of the σG regulon. Although yqfS expression was temporally regulated, neither oxidative damage (after either treatment with paraquat or hydrogen peroxide) nor mitomycin C treatment induced the transcription of this gene.

scholarly journals The PhoPQ two-component system is the major regulator of cell surface properties, stress responses and plant-derived substrate utilisation during development of Pectobacterium versatile-host plant pathosystem

2020 ◽  
Author(s):  
Uljana Kravchenko ◽  
Natalia Gogoleva ◽  
Anastasia Kolubako ◽  
Alla Kruk ◽  
Julia Diubo ◽  
...  
Keyword(s):  
Host Plant ◽  
Stress Responses ◽  
Cell Envelope ◽  
Large Fraction ◽  
Single Gene ◽  
Carbon Sources ◽  
Two Component System ◽  
Component System ◽  
Two Component ◽  
High Concentrations

SummaryThe PhoPQ two-component system, originally described in pectobacteria as PehRS, was previously shown to regulate a single gene, pehA. Using an insertional phoP mutant of Pectobacterium versatile, we demonstrate that PhoP controls a regulon of at least 116 genes with a large fraction of regulon members specific for pectobacteria. The functions performed by the PhoP controlled genes include transport and metabolism of plant-derived carbon sources (polygalacturonate, arabinose and citrate), modification of bacterial cell envelope and stress resistance. High concentrations of calcium and magnesium ions were found to abolish the PhoPQ-dependent transcription activation. Reduced PhoP expression and minimisation of PhoP dependence of regulon members’ expression in the cells isolated from rotten potato tuber tissues suggest that PhoPQ system may adjust expression levels of multiple virulence-related genes during the course of P. versatile-host plant pathosystem development.

The Effects of Shock Vancomycin Concentrations on the Formation of Heteroresistance in Staphylococcus aureus

2020 ◽  
Vol 65 (9-10) ◽  
pp. 3-7
Author(s):  
V. V. Gostev ◽  
Yu. V. Sopova ◽  
O. S. Kalinogorskaya ◽  
M. E. Velizhanina ◽  
I. V. Lazareva ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
In Vitro Selection ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
High Concentration ◽  
Short Term ◽  
High Concentrations ◽  
Selection Of ◽  
Selection Of Resistance ◽  
Test Cultures

Glycopeptides are the basis of the treatment of infections caused by MRSA (Methicillin-Resistant Staphylococcus aureus). Previously, it was demonstrated that antibiotic tolerant phenotypes are formed during selection of resistance under the influence of high concentrations of antibiotics. The present study uses a similar in vitro selection model with vancomycin. Clinical isolates of MRSA belonging to genetic lines ST8 and ST239, as well as the MSSA (ATCC29213) strain, were included in the experiment. Test isolates were incubated for five hours in a medium with a high concentration of vancomycin (50 μg/ml). Test cultures were grown on the medium without antibiotic for 18 hours after each exposure. A total of ten exposure cycles were performed. Vancomycin was characterized by bacteriostatic action; the proportion of surviving cells after exposure was 70–100%. After selection, there was a slight increase in the MIC to vancomycin (MIC 2 μg/ml), teicoplanin (MIC 1.5–3 μg/ml) and daptomycin (MIC 0.25–2 μg/ml). According to the results of PAP analysis, all strains showed an increase in the area under curve depending on the concentration of vancomycin after selection, while a heteroresistant phenotype (with PAP/AUC 0.9) was detected in three isolates. All isolates showed walK mutations (T188S, D235N, E261V, V380I, and G223D). Exposure to short-term shock concentrations of vancomycin promotes the formation of heteroresistance in both MRSA and MSSA. Formation of VISA phenotypes is possible during therapy with vancomycin.

scholarly journals Open Reading Frame sso2387 from the Archaeon Sulfolobus solfataricus Encodes a Polypeptide with Protein-Serine Kinase Activity

2003 ◽  
Vol 185 (11) ◽  
pp. 3436-3445 ◽  
Author(s):  
Brian H. Lower ◽  
Peter J. Kennelly
Keyword(s):  
Protein Kinases ◽  
Catalytic Domain ◽  
Sulfolobus Solfataricus ◽  
Open Reading Frame ◽  
Serine Kinase ◽  
Eukaryotic Protein Kinase ◽  
Reading Frame ◽  
Eukaryotic Protein ◽  
High Concentrations ◽  
Eukaryotic Protein Kinases

ABSTRACT The predicted polypeptide product of open reading frame sso2387 from the archaeon Sulfolobus solfataricus, SsoPK2, displayed several of the sequence features conserved among the members of the “eukaryotic” protein kinase superfamily. sso2387 was cloned, and its polypeptide product was expressed in Escherichia coli. The recombinant protein, rSsoPK2, was recovered in insoluble aggregates that could be dispersed by using high concentrations (5 M) of urea. The solubilized polypeptide displayed the ability to phosphorylate itself as well as several exogenous proteins, including mixed histones, casein, bovine serum albumin, and reduced carboxyamidomethylated and maleylated lysozyme, on serine residues. The source of this activity resided in that portion of the protein displaying homology to the catalytic domain of eukaryotic protein kinases. By use of mass spectrometry, the sites of autophosphorylation were found to be located in two areas, one immediately N terminal to the region corresponding to subdomain I of eukaryotic protein kinases, and the second N terminal to the presumed activation loop located between subdomains VII and VIII. Autophosphorylation of rSsoPK2 could be uncoupled from the phosphorylation of exogenous proteins by manipulation of the temperature or mutagenic alteration of the enzyme. Autophosphorylation was detected only at temperatures ≥60°C, whereas phosphorylation of exogenous proteins was detectable at 37°C. Similarly, replacement of one of the potential sites of autophosphorylation, Ser548, with alanine blocked autophosphorylation but not phosphorylation of an exogenous protein, casein.

Sign in / Sign up

Export Citation Format

Share Document