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 Revisiting the regulation of the capsular polysaccharide biosynthesis gene cluster inStaphylococcus aureus

2019 ◽  
Author(s):  
Daniela Keinhörster ◽  
Andrea Salzer ◽  
Alejandra Duque-Jaramillo ◽  
Shilpa E. George ◽  
Gabriella Marincola ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Expression Pattern ◽  
Inverted Repeat ◽  
Capsular Polysaccharide ◽  
Gene Expression Pattern ◽  
Upstream Region ◽  
Transcriptional Level ◽  
Bacterial Cells ◽  
Biosynthesis Gene Cluster ◽  
Polysaccharide Biosynthesis

AbstractInStaphylococcus aureus, the capsular polysaccharide (CP) protects against phagocytosis, but also hinders adherence to endothelial cells and matrix proteins. Its biosynthesis is tightly controlled resulting in a heterogeneous phenotype within a population and CP being mainly detectable in non-growing cells. Capsular biosynthesis genes are encoded by a conservedcapA-Poperon whose expression is driven by an upstream promoter element (Pcap) in front ofcapA. The organization of Pcapis poorly understood, as is the interplay of different regulators that influence the early-Off/late-Heterogeneouscaptranscription pattern. Here, we demonstrate that Pcapcontains a main SigB-dependent promoter. The SigB consensus motif overlaps with a previously described inverted repeat that is crucial forcapexpression. The essentiality of the inverted repeat is derived from this region acting as a SigB binding site rather than as an operator site for the proposedcapactivators RbsR and MsaB. Furthermore, Pcapcontains an extensive upstream region harboring a weak SigA-dependent promoter and binding sites for thecaprepressors SaeR, CodY and Rot. We show that heterogeneous CP synthesis is determined by the combination of SigB activity and repressor binding to the upstream region. The direct SigB dependency and the upstream repressors are also sufficient to explain the temporal gene expression pattern at the transcriptional level. However, CP synthesis remains growth phase-dependent even whencapAtranscription is rendered constitutive, suggesting additional post-transcriptional regulatory circuits. Thus, the interference of multiple repressors with SigB-dependent promoter activity as well as post-transcriptional mechanisms ensure the appropriate regulation of CP synthesis.ImportanceThe majority of bacterial pathogens produce an array of polysaccharides on their surface which are important virulence factors and thus serve as attractive vaccine candidates. However, the synthesis and assembly of these structures is highly variable and tightly regulated at various levels. In the human pathogenStaphylococcus aureus, the synthesis of the capsular polysaccharide (CP) is dependent on a complex regulatory network which ensures that CP is produced only in a fraction of stationary phase cells. Here, we determined main regulators that drive the peculiar CP expression pattern. We found that the interplay of the transcriptional repressors Sae, CodY and Rot with the alternative Sigma factor B is responsible for early-Off/late-Heterogeneous expression at the transcriptional level. The data also implicates post-transcriptional mechanisms that may act to avoid conflict in precursor usage by machineries involved in either synthesis of CP or other glycopolymers in growing bacterial cells.

scholarly journals mgr, a Novel Global Regulator in Staphylococcus aureus

2003 ◽  
Vol 185 (13) ◽  
pp. 3703-3710 ◽  
Author(s):  
Thanh T. Luong ◽  
Steven W. Newell ◽  
Chia Y. Lee
Keyword(s):  
Staphylococcus Aureus ◽  
Capsular Polysaccharide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Binding Motif ◽  
Transcriptional Level ◽  
Virulence Determinants ◽  
Alpha Toxin ◽  
Extracellular Protein Production

ABSTRACT The virulence determinants of Staphylococcus aureus are coordinately controlled by several unlinked chromosomal loci. Here, we report the identification of CYL5614, derived from strain Becker, with a mutation that affects the expression of type 8 capsular polysaccharide (CP8), nuclease, alpha-toxin, coagulase, protease, and protein A. This novel locus, named mgr, was linked by transposon Tn917 and mapped by three-factorial transduction crosses. The region containing the mgr locus was cloned and sequenced. Deletion mutagenesis and genetic complementation showed that the locus consisted of one gene, mgrA. Interestingly, mgrA-null mutants exhibited a phenotype opposite to that of CYL5614. This was due to a T-to-C mutation upstream of mgrA that resulted in a four- to eightfold increase in mgrA transcription in strain CYL5614. Thus, these results indicate that mgrA is an activator of CP8 and nuclease but a repressor of alpha-toxin, coagulase, protease, and protein A. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the mgr locus profoundly affected extracellular protein production, suggesting that the locus may regulate many other genes as well. The translated MgrA protein has a region of significant homology, which includes the helix-turn-helix DNA-binding motif, with the Escherichia coli MarR family of transcriptional regulators. Northern slot blot analyses suggested that mgr affected CP8, alpha-toxin, nuclease, and protein A at the transcriptional level.

scholarly journals Regulation of Staphylococcus aureus Type 5 and Type 8 Capsular Polysaccharides by CO2

2001 ◽  
Vol 183 (15) ◽  
pp. 4609-4613 ◽  
Author(s):  
Silvia Herbert ◽  
Steven W. Newell ◽  
Chia Lee ◽  
Karsten-Peter Wieland ◽  
Bruno Dassy ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Genetic Background ◽  
Capsular Polysaccharide ◽  
Promoter Sequence ◽  
Transcriptional Level ◽  
Marginal Effect ◽  
Promoter Regions ◽  
Sequence Variations ◽  
Derivatives Of

ABSTRACT Staphylococcus aureus expression of capsular polysaccharide type 5 (CP5) has been shown to be downregulated by CO2. Here we show that CO2 reduces CP5 expression at the transcriptional level and that CO2regulates CP8 expression depending on the genetic background of the strains. Growth in the presence of air supplemented with 5% CO2 caused a significant decrease in CP8 expression in fourS. aureus strains, a marginal effect in four strains, and higher CP8 expression in strain Becker. Absolute CP8 expression in the nine S. aureus strains differed largely from strain to strain. Four groups of strains were established due to sequence variations in the promoter region of cap5 andcap8. To test whether these sequence variations are responsible for the different responses to CO2, promoter regions from selected strains were fused to the reporter genexylE in pLC4, and the plasmids were electrotransformed into strains Becker and Newman. XylE activity was negatively regulated by CO2 in all derivatives of strain Newman and was always positively regulated by CO2 in all derivatives of strain Becker. Differences in promoter sequences did not influence the pattern of CP8 expression. Therefore, the genetic background of the strains rather than differences in the promoter sequence determines the CO2 response. trans-acting regulatory molecules may be differentially expressed in strain Becker versus strain Newman. The strain dependency of the CP8 expression established in vitro was also seen in lung tissue sections of patients with cystic fibrosis infected with CP8-positive S. aureus strains.

scholarly journals Regulation of Staphylococcus aureus Capsular Polysaccharide Expression by agr and sarA

2002 ◽  
Vol 70 (2) ◽  
pp. 444-450 ◽  
Author(s):  
Thanh Luong ◽  
Subrata Sau ◽  
Marisa Gomez ◽  
Jean C. Lee ◽  
Chia Y. Lee
Keyword(s):  
Staphylococcus Aureus ◽  
Reporter Gene ◽  
Capsular Polysaccharide ◽  
Logarithmic Phase ◽  
Transcriptional Level ◽  
Slight Reduction ◽  
Mrna Synthesis ◽  
Global Regulators ◽  
In The Wild ◽  
Isogenic Mutants

ABSTRACT This study addresses the regulation of Staphylococcus aureus type 8 capsular polysaccharide (CP8) expression by the global regulators agr and sarA. We analyzed CP8 production, cap8-specific mRNA synthesis, and blaZ reporter gene activities of the transcriptional and translational fusions in strain Becker and its agr, sarA, and agr-sarA isogenic mutants during different phases of bacterial growth. In the wild-type strain, cap8 mRNA was undetectable until the mid-logarithmic phase of growth, whereas CP8 production was undetectable until 2 h later, at the onset of stationary phase. The delay most likely reflects the time needed for completing CP8 synthesis resulting from translation of cap8 mRNA. The agr mutation caused drastic reductions in CP8 production and cap8 gene transcription, suggesting that agr is a major positive regulator of CP8 expression. The results of gene fusion studies indicated that regulation by agr is exerted at the transcriptional level. In contrast, the sarA mutation caused only a slight reduction in cap8 mRNA synthesis and reporter gene activities. By comparing CP8 production and cap8 transcription, we observed that sarA affected CP8 production both trancriptionally and posttranslationally. We showed that agr was a major activator for cap gene expression not only in type 8 strain Becker but also in strains representing the four agr groups.

scholarly journals Binding Sequences for RdgB, a DNA Damage-Responsive Transcriptional Activator, and Temperature-Dependent Expression of Bacteriocin and Pectin Lyase Genes in Pectobacterium carotovorum subsp. carotovorum

2008 ◽  
Vol 74 (19) ◽  
pp. 6017-6025 ◽  
Author(s):  
Kazuteru Yamada ◽  
Jun Kaneko ◽  
Yoshiyuki Kamio ◽  
Yoshifumi Itoh
Keyword(s):  
Binding Affinity ◽  
Inverted Repeat ◽  
Regulatory Protein ◽  
Pectin Lyase ◽  
Transcriptional Level ◽  
Pectobacterium Carotovorum ◽  
Knockout Mutant ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Gel Mobility Shift

ABSTRACT Pectobacterium carotovorum subsp. carotovorum strain Er simultaneously produces the phage tail-like bacteriocin carotovoricin (Ctv) and pectin lyase (Pnl) in response to DNA-damaging agents. The regulatory protein RdgB of the Mor/C family of proteins activates transcription of pnl through binding to the promoter. However, the optimal temperature for the synthesis of Ctv (23�C) differs from that for synthesis of Pnl (30�C), raising the question of whether RdgB directly activates ctv transcription. Here we report that RdgB directly regulates Ctv synthesis. Gel mobility shift assays demonstrated RdgB binding to the P0, P1, and P2 promoters of the ctv operons, and DNase I footprinting determined RdgB-binding sequences (RdgB boxes) on these and on the pnl promoters. The RdgB box of the pnl promoter included a perfect 7-bp inverted repeat with high binding affinity to the regulator (K d [dissociation constant] = 150 nM). In contrast, RdgB boxes of the ctv promoters contained an imperfect inverted repeat with two or three mismatches that consequently reduced binding affinity (K d = 250 to 350 nM). Transcription of the rdgB and ctv genes was about doubled at 23�C compared with that at 30�C. In contrast, the amount of pnl transcription tripled at 30�C. Thus, the inverse synthesis of Ctv and Pnl as a function of temperature is apparently controlled at the transcriptional level, and reduced rdgB expression at 30�C obviously affected transcription from the ctv promoters with low-affinity RdgB boxes. Pathogenicity toward potato tubers was reduced in an rdgB knockout mutant, suggesting that the RdgAB system contributes to the pathogenicity of this bacterium, probably by activating pnl expression.

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 The sbcDC Locus Mediates Repression of Type 5 Capsule Production as Part of the SOS Response in Staphylococcus aureus

2007 ◽  
Vol 189 (20) ◽  
pp. 7343-7350 ◽  
Author(s):  
Zhongyi Chen ◽  
Thanh T. Luong ◽  
Chia Y. Lee
Keyword(s):  
Staphylococcus Aureus ◽  
Promoter Activity ◽  
Capsular Polysaccharide ◽  
Survival Rates ◽  
Sos Response ◽  
Northern Blotting ◽  
Exponential Growth Phase ◽  
Genetic Studies ◽  
Capsule Production ◽  
Gene Transcripts

ABSTRACT Most strains of Staphylococcus aureus produce one type of capsular polysaccharide that belongs to either type 5 or type 8. The production of these capsules has been shown to be regulated by various regulators. Here we report that the sbcD and sbcC genes are involved in the repression of type 5 capsule production. Chromosomal deletions in the sbcDC genes resulted in increased capsule promoter activity, capsule gene transcripts, and capsule production. The survival rates of the sbcDC deletion mutant were reduced upon UV irradiation compared to those for the wild-type strain Newman, suggesting that the genes are involved in DNA repair in S. aureus. The two genes were organized as an operon and were expressed very early in the exponential growth phase. A subinhibitory concentration of ciprofloxacin or mitomycin C induced sbcDC transcription but repressed the capsule promoter activity, suggesting that the sbcDC genes and the capsule genes are part of the SOS regulon. By reporter gene fusion and Northern blotting, we found that sbcDC regulated capsule by downregulating arl and mgr. Further genetic studies indicate that sbcDC functions upstream of arl and mgr in capsule regulation. Collectively, our results indicate that sbcDC, upon the SOS response, represses type 5 capsule production through an arl-mgr pathway. To our knowledge, this is the first demonstration that an SbcDC homolog was involved in transcriptional regulation.

UBE2L6 is Involved in Cisplatin Resistance by Regulating the Transcription of ABCB6

2020 ◽  
Vol 20 (12) ◽  
pp. 1487-1496 ◽  
Author(s):  
Midori Murakami ◽  
Hiroto Izumi ◽  
Tomoko Kurita ◽  
Chiho Koi ◽  
Yasuo Morimoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Anticancer Agent ◽  
Cisplatin Resistance ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Molecular Target ◽  
Transcriptional Level ◽  
And Control ◽  
Resistant Cells

Background: Cisplatin is an important anticancer agent in cancer chemotherapy, but when resistant cells appear, treatment becomes difficult, and the prognosis is poor. Objective: In this study, we investigated the gene expression profile in cisplatin sensitive and resistant cells, and identified the genes involved in cisplatin resistance. Methods: Comparison of gene expression profiles revealed that UBE2L6 mRNA is highly expressed in resistant cells. To elucidate whether UBE2L6 is involved in the acquisition of cisplatin resistance, UBE2L6- overexpressing cells established from cisplatin-sensitive cells and UBE2L6-silenced cells developed from cisplatin- resistant cells were generated, and the sensitivity of cisplatin was examined. Results: The sensitivity of the UBE2L6-overexpressing cells did not change compared with the control cells, but the UBE2L6-silenced cells were sensitized to cisplatin. To elucidate the mechanism of UBE2L6 in cisplatin resistance, we compared the gene expression profiles of UBE2L6-silenced cells and control cells and found that the level of ABCB6 mRNA involved in cisplatin resistance was decreased. Moreover, ABCB6 promoter activity was partially suppressed in UBE2L6-silenced cells. Conclusion: These results suggest that cisplatin-resistant cells have upregulated UBE2L6 expression and contribute to cisplatin resistance by regulating ABCB6 expression at the transcriptional level. UBE2L6 might be a molecular target that overcomes cisplatin resistance.

Transcriptional regulation of Pseudomonas aeruginosa rhlR, encoding a quorum-sensing regulatory protein

Microbiology ◽  
2003 ◽  
Vol 149 (11) ◽  
pp. 3073-3081 ◽  
Author(s):  
Gerardo Medina ◽  
Katy Juárez ◽  
Rafael Díaz ◽  
Gloria Soberón-Chávez
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcriptional Regulation ◽  
Quorum Sensing ◽  
Regulatory Protein ◽  
Culture Conditions ◽  
Transcriptional Level ◽  
Coding Region ◽  
Upstream Gene ◽  
Transcription Start Sites ◽  
Response Systems

The Pseudomonas aeruginosa rhlR gene encodes the transcriptional regulator RhlR which has a central role in the quorum-sensing response. Different gene products involved in bacterial pathogenesis are regulated at the transcriptional level by two quorum-sensing response systems, Las and Rhl. The expression of rhlR has been reported to be under the control of the Las system, but its transcriptional regulation has not been studied in detail. Here, the rhlR promoter region has been characterized and shown to present four different transcription start sites, two of which are included in the upstream gene (rhlB) coding region. It was found that rhlR expression is not only dependent on LasR but also on different regulatory proteins such as Vfr and RhlR itself, and also on the alternative sigma factor σ 54. It is reported that rhlR expression is partially LasR-independent under certain culture conditions and is strongly influenced by environmental factors.

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