scholarly journals Roles of CcrA and CcrB in Excision and Integration of Staphylococcal Cassette Chromosome mec, a Staphylococcus aureus Genomic Island

2010 ◽  
Vol 192 (12) ◽  
pp. 3204-3212 ◽  
Author(s):  
Lei Wang ◽  
Gordon L. Archer
Keyword(s):  
Staphylococcus Aureus ◽  
Genomic Island ◽  
Dna Interaction ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Site Specific ◽  
Gel Shift ◽  
The Right ◽  
Electrophoretic Mobility Shift Assays ◽  
Staphylococcal Cassette Chromosome

ABSTRACT The gene encoding resistance to methicillin and other β-lactam antibiotics in staphylococci, mecA, is carried on a genomic island, SCCmec (for staphylococcal cassette chromosome mec). The chromosomal excision and integration of types I to IV SCCmec are catalyzed by the site-specific recombinases CcrA and CcrB, the genes for which are encoded on each element. We sought to identify the relative contributions of CcrA and CcrB in the excision and integration of SCCmec. Purified CcrB but not CcrA was shown to mediate the gel shift of chromosomal target integration sequences (attB) in electrophoretic mobility shift assays. However, preincubation of CcrB-DNA complexes with increasing concentrations of CcrA blocked gel shift. The interaction of CcrB and CcrA was confirmed by Escherichia coli two-hybrid analysis. SCCmec excision mediated by plasmid-encoded and inducible ccrA, ccrB, or both genes was assessed by PCR in Staphylococcus aureus. CcrB alone could mediate excision but excision was at an alternate att site (attR2) within the right extremity of SCCmec. In contrast, both CcrB and CcrA were required to mediate excision at the chromosomal attB site (called attR when SCCmec is integrated). Insertion of a plasmid containing the SCCmec att site (attS) into the chromosome required both CcrA and CcrB, but CcrA overexpression lowered integration frequency. Thus, while CcrB binds DNA, interaction between CcrA and CcrB, in a precise ratio, is required for attB site-specific excision and SCCmec chromosomal insertion.

scholarly journals Transcriptional regulation of Mycobacterium tuberculosis hupB gene expression

Microbiology ◽  
2014 ◽  
Vol 160 (8) ◽  
pp. 1637-1647 ◽  
Author(s):  
Satya Deo Pandey ◽  
Mitali Choudhury ◽  
Manjula Sritharan
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Iron Homeostasis ◽  
Iron Concentration ◽  
Dna Interaction ◽  
Intracellular Iron ◽  
Mobility Shift ◽  
Dna Footprinting ◽  
Sequence Of Events ◽  
Electrophoretic Mobility Shift Assays

The influence of iron levels on the transcription of the hupB gene in Mycobacterium tuberculosis is the focus of this study. Studies in our laboratory showed HupB to be co-expressed with the two siderophores in low-iron organisms. Mycobactin biosynthesis is repressed by the IdeR–Fe2+ complex that binds the IdeR box in the mbtB promoter. Recently, we demonstrated the positive regulatory effect of HupB on mycobactin biosynthesis by demonstrating its binding to a 10 bp HupB box in the mbtB promoter. Earlier, we observed that HupB, expressed maximally in low-iron media (0.02 µg Fe ml−1; 0.36 µM Fe) was still detectable at 8 µg Fe ml−1 (144 µM Fe) when the siderophores were absent and complete repression was seen only at 12 µg Fe ml−1 (216 µM Fe). In this study, we observed elevated levels of hupB transcripts in iron-limited organisms. IdeR, and not FurA, functioned as the iron regulator, by binding to two IdeR boxes in the hupB promoter. Interestingly, the 10 bp HupB box, first reported in the mbtB promoter, was identified in the hupB promoter. Using DNA footprinting and electrophoretic mobility shift assays, we demonstrated the functionality of the HupB box and the two IdeR boxes. The high hupB transcript levels expressed by the organism and the in vitro protein–DNA interaction studies led us to hypothesize the sequence of events occurring in response to changes in the intracellular iron concentration, emphasizing the roles played by IdeR and HupB in iron homeostasis.

scholarly journals A Subset of Staphylococcus aureus Strains Harboring Staphylococcal Cassette Chromosome mec (SCCmec) Type IV Is Deficient in CcrAB-Mediated SCCmec Excision

2006 ◽  
Vol 50 (8) ◽  
pp. 2782-2788 ◽  
Author(s):  
Michael J. Noto ◽  
Gordon L. Archer
Keyword(s):  
Staphylococcus Aureus ◽  
Resistance Gene ◽  
Methicillin Resistance ◽  
Sccmec Type ◽  
Amino Acid Level ◽  
Virulence Gene ◽  
Mobile Element ◽  
Gene Encoding ◽  
Type Iv ◽  
Staphylococcal Cassette Chromosome

ABSTRACT The gene encoding resistance to β-lactam antibiotics in the staphylococci is found on the chromosome in a genomic island designated staphylococcal cassette chromosome mec, or SCCmec. In addition to the resistance gene mecA, SCCmec also contains site-specific recombinase genes that are capable of catalyzing the chromosomal excision and reintegration of SCCmec. SCCmec is found in five major isotypes partially defined by the recombinase genes present, either ccrAB or ccrC. Of these, SCCmec type IV is presumed to be mobile in the environment, and this mobility may be partially responsible for the rise in community-associated methicillin-resistant staphylococcal infections. In this study, we investigate the presumptive first step in type IV SCCmec mobility: chromosomal excision of the element. CcrAB from a panel of six Staphylococcus aureus and four Staphylococcus epidermidis strains were able to catalyze chromosomal excision of SCCmec types I and II, indicating that these proteins maintain recombinase activity despite varying by up to 3.7% at the amino acid level. Excision of type IV SCCmec was not universally seen, as a subset of S. aureus strains with type IV SCCmec did not excise their element. These strains are all highly related and represent a lineage of successful community-associated pathogens. In addition, the inability to excise SCCmec in these strains is associated with the insertion of a presumptive mobile element containing the gene for staphylococcal enterotoxin H (seh) immediately downstream of SCCmec on the chromosome. Acquisition of this mobile element, containing a known virulence gene, appears to have stabilized the chromosomal integration of the methicillin resistance gene in these strains.

scholarly journals Pseudomonas aeruginosa LysR PA4203 Regulator NmoR Acts as a Repressor of the PA4202nmoAGene, Encoding a Nitronate Monooxygenase

2014 ◽  
Vol 197 (6) ◽  
pp. 1026-1039 ◽  
Author(s):  
Ken Vercammen ◽  
Qing Wei ◽  
Daniel Charlier ◽  
Andreas Dötsch ◽  
Susanne Haüssler ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Mobility Shift ◽  
Quinone Oxidoreductase ◽  
Gene Encoding ◽  
Nitropropionic Acid ◽  
Gene Coding ◽  
Intergenic Regions ◽  
Increased Sensitivity ◽  
Electrophoretic Mobility Shift Assays

The PA4203 gene encodes a LysR regulator and lies between theppgLgene (PA4204), which encodes a periplasmic gluconolactonase, and, in the opposite orientation, the PA4202 (nmoA) gene, coding for a nitronate monooxygenase, andddlA(PA4201), encoding ad-alanine alanine ligase. The intergenic regions between PA4203 andppgLand between PA4203 andnmoAare very short (79 and 107 nucleotides, respectively). Here we show that PA4203 (nmoR) represses its own transcription and the expression ofnmoA. A chromatin immunoprecipitation analysis showed the presence of a single NmoR binding site betweennmoAandnmoR, which was confirmed by electrophoretic mobility shift assays (EMSAs) with the purified NmoR protein. Despite this observation, a transcriptome analysis revealed more genes to be affected in annmoRmutant, including genes known to be part of the MexT LysR activator regulon. The PA1225 gene, encoding a quinone oxidoreductase, was the most highly upregulated gene in thenmoRdeletion mutant, independently of MexT. Finally, deletion of thenmoAgene resulted in an increased sensitivity of the cells to 3-nitropropionic acid (3-NPA), confirming the role of the nitronate monooxygenase protein in the detoxification of nitronate.

scholarly journals VraR Binding to the Promoter Region of agr Inhibits Its Function in Vancomycin-Intermediate Staphylococcus aureus (VISA) and Heterogeneous VISA

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Yuanyuan Dai ◽  
Wenjiao Chang ◽  
Changcheng Zhao ◽  
Jing Peng ◽  
Liangfei Xu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Promoter Region ◽  
Reference Strain ◽  
Vancomycin Resistance ◽  
Mobility Shift ◽  
Content Type ◽  
Promoter Sequences ◽  
Dnase I Footprinting ◽  
Resistant Strains ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Acquisition of vancomycin resistance in Staphylococcus aureus is often accompanied by a reduction in virulence, but the mechanisms underlying this change remain unclear. The present study was undertaken to investigate this process in a clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) strain, 10827; an hVISA reference strain, Mu3; and a VISA reference strain, Mu50, along with their respective series of vancomycin-induced resistant strains. In these strains, increasing MICs of vancomycin were associated with increased expression of the vancomycin resistance-associated regulator gene (vraR) and decreased expression of virulence genes (hla, hlb, and coa) and virulence-regulated genes (RNAIII, agrA, and saeR). These results suggested that VraR might have a direct or indirect effect on virulence in S. aureus. In electrophoretic mobility shift assays, VraR did not bind to promoter sequences of hla, hlb, and coa genes, but it did bind to the agr promoter region. In DNase I footprinting assays, VraR protected a 15-nucleotide (nt) sequence in the intergenic region between the agr P2 and P3 promoters. These results indicated that when S. aureus is subject to induction by vancomycin, expression of vraR is upregulated, and VraR binding inhibits the function of the Agr quorum-sensing system, causing reductions in the virulence of VISA/hVISA strains. Our results suggested that VraR in S. aureus is involved not only in the regulation of vancomycin resistance but also in the regulation of virulence.

scholarly journals Identification of a Novel Transposon (Tn6072) and a Truncated Staphylococcal Cassette Chromosome mec Element in Methicillin-Resistant Staphylococcus aureus ST239

2010 ◽  
Vol 54 (8) ◽  
pp. 3347-3354 ◽  
Author(s):  
Liang Chen ◽  
José R. Mediavilla ◽  
Davida S. Smyth ◽  
Kalyan D. Chavda ◽  
Ramona Ionescu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistance ◽  
Prototype Strain ◽  
Complete Sequence ◽  
Gene Complex ◽  
Reading Frame ◽  
Clonal Group ◽  
Type Iii ◽  
The Right ◽  
Staphylococcal Cassette Chromosome

ABSTRACT A novel composite transposon (Tn6072) resembling staphylococcal cassette chromosome mercury (SCCHg) was identified in a collection of sequence type (ST) 239 methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates from Romanian hospitals. Tn6072 is homologous to the 5′ region of SCCHg found in staphylococcal cassette chromosome mec (SCCmec) type III prototype strain 85/2082 but lacks the characteristic mer operon. SCCHg has previously been reported to integrate downstream of orfX, at the same chromosomal location as SCCmec. Tn6072, by contrast, is demarcated by two IS431 elements, flanked by 8-bp direct repeats, and inserted upstream of the origin of replication, within an open reading frame homologous to SAR2700 of S. aureus strain MRSA252. Analysis of a geographically and temporally diverse collection of 111 strains from the ST239 clonal group uncovered 11 additional strains harboring Tn6072, demonstrating a lineage-specific insertion pattern. Complete sequence analysis of the SCCmec regions of two representative Romanian strains (BK16704, BK16691) revealed two additional novel structures derived from a type III SCCmec background. BK16704 possesses an SCCmec 3A.1.4 structure, with an IS256 insertion downstream of the right chromosomal junction. In contrast, the SCCmec element of BK16691 is truncated downstream of the mec gene complex, with a 24-kb deletion encompassing the right chromosomal junction and an inverted downstream IS256 element. This structure, tentatively named “ψSCCmec 16691,” confers methicillin resistance but lacks most of the J1/J2 region, including the ccr gene complex. Taken together, these findings provide evidence for the continuing evolution of SCC elements, as well as the ST239 clonal group.

scholarly journals Mapping the Transcription Start Points of the Staphylococcus aureus eap, emp, and vwb Promoters Reveals a Conserved Octanucleotide Sequence That Is Essential for Expression of These Genes

2007 ◽  
Vol 190 (1) ◽  
pp. 447-451 ◽  
Author(s):  
Niamh Harraghy ◽  
Dagmar Homerova ◽  
Mathias Herrmann ◽  
Jan Kormanec
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Site ◽  
Electrophoretic Mobility ◽  
Transcription Start ◽  
Mobility Shift ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Mapping the transcription start points of the eap, emp, and vwb promoters revealed a conserved octanucleotide sequence (COS). Deleting this sequence abolished the expression of eap, emp, and vwb. However, electrophoretic mobility shift assays gave no evidence that this sequence was a binding site for SarA or SaeR, known regulators of eap and emp.

scholarly journals Involvement of the LuxR-Type Transcriptional Regulator RamA in Regulation of Expression of the gapA Gene, Encoding Glyceraldehyde-3-Phosphate Dehydrogenase of Corynebacterium glutamicum

2008 ◽  
Vol 191 (3) ◽  
pp. 968-977 ◽  
Author(s):  
Koichi Toyoda ◽  
Haruhiko Teramoto ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Corynebacterium Glutamicum ◽  
Sugar Transport ◽  
Transcriptional Regulator ◽  
Phosphotransferase System ◽  
Mobility Shift ◽  
Regulation Of Expression ◽  
Gene Encoding ◽  
Global Regulators ◽  
Gapdh Activity ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT SugR, RamA, GlxR, GntR1, and a MarR-type transcriptional regulator bind to the promoter region of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), essential for glycolysis in Corynebacterium glutamicum. We previously showed that SugR, a transcriptional repressor of phosphotransferase system genes for the sugar transport system, is involved in the downregulation of gapA expression in the absence of sugar. In this study, the role of RamA in the expression of the gapA gene was examined. Comparing the gapA expression and GAPDH activity of a ramA mutant with those of the wild type revealed that RamA is involved in upregulation of gapA expression in glucose-grown cells. DNase I footprint analyses and electrophoretic mobility shift assays revealed that RamA binds with different affinities to three sites in the gapA promoter. lacZ reporter assays with mutated RamA binding sites in the gapA promoter showed that the middle binding site is the most important for RamA to activate gapA expression and that binding of RamA to the gapA promoter activates the gene expression not only in glucose-grown cells but also in acetate-grown cells. Furthermore, RamA also directly activates sugR expression, indicating that two global regulators, RamA and SugR, are coordinately involved in the complex regulation of gapA expression in C. glutamicum.

scholarly journals Control of Directionality in Bacteriophage mv4 Site-Specific Recombination: Functional Analysis of the Xis Factor

2009 ◽  
Vol 192 (3) ◽  
pp. 624-635 ◽  
Author(s):  
Michèle Coddeville ◽  
Paul Ritzenthaler
Keyword(s):  
Binding Sites ◽  
Basic Protein ◽  
Lactobacillus Delbrueckii ◽  
Mobility Shift ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Dna Binding Sites ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The integrase of the temperate bacteriophage mv4 catalyzes site-specific recombination between the phage attP site and the host attB site during Lactobacillus delbrueckii lysogenization. The mv4 prophage is excised during the induction of lytic growth. Excisive site-specific recombination between the attR and attL sites is also catalyzed by the phage-encoded recombinase, but the directionality of the recombination is determined by a second phage-encoded protein, the recombination directionality factor (RDF). We have identified and functionally characterized the RDF involved in site-specific excision of the prophage genome. The mv4 RDF, mv4Xis, is encoded by the second gene of the early lytic operon. It is a basic protein of 56 amino acids. Electrophoretic mobility shift assays demonstrated that mv4Xis binds specifically to the attP and attR sites via two DNA-binding sites, introducing a bend into the DNA. In vitro experiments and in vivo recombination assays with plasmids in Escherichia coli and Lactobacillus plantarum demonstrated that mv4Xis is absolutely required for inter- or intramolecular recombination between the attR and attL sites. In contrast to the well-known phage site-specific recombination systems, the integrative recombination between the attP and attB sites seems not to be inhibited by the presence of mv4Xis.

scholarly journals Characterization of Staphylococcus aureus SarA Binding Sites

2003 ◽  
Vol 185 (15) ◽  
pp. 4410-4417 ◽  
Author(s):  
Kristen M. Sterba ◽  
Samuel G. Mackintosh ◽  
Jon S. Blevins ◽  
Barry K. Hurlburt ◽  
Mark S. Smeltzer
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Site ◽  
Binding Sites ◽  
Target Genes ◽  
Direct Interaction ◽  
Nucleotide Composition ◽  
Dna Fragments ◽  
Mobility Shift ◽  
High Affinity ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The staphylococcal accessory regulator locus (sarA) encodes a DNA-binding protein (SarA) that modulates expression of over 100 genes. Whether this occurs via a direct interaction between SarA and cis elements associated with its target genes is unclear, partly because the definitive characteristics of a SarA binding site have not been identified. In this work, electrophoretic mobility shift assays (EMSAs) were used to identify a SarA binding site(s) upstream of the SarA-regulated gene cna. The results suggest the existence of multiple high-affinity binding sites within the cna promoter region. Using a SELEX (systematic evolution of ligands by exponential enrichment) procedure and purified, recombinant SarA, we also selected DNA targets that contain a high-affinity SarA binding site from a random pool of DNA fragments. These fragments were subsequently cloned and sequenced. Randomly chosen clones were also examined by EMSA. These DNA fragments bound SarA with affinities comparable to those of recognized SarA-regulated genes, including cna, fnbA, and sspA. The composition of SarA-selected DNAs was AT rich, which is consistent with the nucleotide composition of the Staphylococcus aureus genome. Alignment of selected DNAs revealed a 7-bp consensus (ATTTTAT) that was present with no more than one mismatch in 46 of 56 sequenced clones. By using the same criteria, consensus binding sites were also identified upstream of the S. aureus genes spa, fnbA, sspA, agr, hla, and cna. With the exception of cna, which has not been previously examined, this 7-bp motif was within the putative SarA binding site previously associated with each gene.

scholarly journals FxkR Provides the Missing Link in the fixL-fixK Signal Transduction Cascade in Rhizobium etli CFN42

2012 ◽  
Vol 25 (11) ◽  
pp. 1506-1517 ◽  
Author(s):  
David Zamorano-Sánchez ◽  
Alma Reyes-González ◽  
Nicolás Gómez-Hernández ◽  
Patricia Rivera ◽  
Dimitris Georgellis ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Transcriptional Control ◽  
Response Regulator ◽  
Transcriptional Start Site ◽  
Site Directed Mutagenesis ◽  
Rhizobium Etli ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Reading Frame ◽  
Electrophoretic Mobility Shift Assays

Transcriptional control of the fixK gene in Rhizobium etli and R. leguminosarum bv. viciae is governed by a two-component signal transduction system that diverts from the conventional FixL-FixJ cascade that occurs in model rhizobia. Although a fixL gene, encoding a hybrid histidine kinase (hFixL), is present in R. etli, no fixJ, the cognate response regulator, has been identified. In this work, we present evidence that the pRet42f-located open reading frame RHE_PF00530 (fxkR) encodes a novel response regulator indispensable for fixKf activation under microaerobic growth. Moreover, results from complementation assays demonstrate that the activation of fixKf expression requires the presence of both hFixL and FxkR, and that the fxkR ortholog from R. leguminosarum bv. viciae is able to substitute for FxkR transcriptional control in R. etli. In addition, in these two organisms, hFixL- and FxkR-related proteins were identified in other bacteria, located in close proximity to a fixK-related gene. Using reporter fusions, site-directed mutagenesis, and electrophoretic mobility shift assays, we identified the FxkR binding site upstream from the transcriptional start site of fixKf. Similar to our previous observations for fixL and fixKf mutants, a null mutation in fxkR does not affect the symbiotic effectiveness of the strain. Thus, our findings reveal that FxkR is the long-standing missing key regulator that allows the transduction of the microaerobic signal for the activation of the FixKf regulon.

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