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

2019 ◽  
Vol 112 (4) ◽  
pp. 1083-1099 ◽  
Author(s):  
Daniela Keinhörster ◽  
Andrea Salzer ◽  
Alejandra Duque‐Jaramillo ◽  
Shilpa E. George ◽  
Gabriella Marincola ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Capsular Polysaccharide ◽  
Biosynthesis Gene Cluster ◽  
Polysaccharide Biosynthesis ◽  
Biosynthesis Gene

scholarly journals Downregulated Expression of Virulence Factors Induced by Benzyl Isothiocyanate in Staphylococcus Aureus: A Transcriptomic Analysis

2019 ◽  
Vol 20 (21) ◽  
pp. 5441 ◽  
Author(s):  
Xiaoning Wang ◽  
Hongyan Wu ◽  
Tongxin Niu ◽  
Jingran Bi ◽  
Hongman Hou ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Pathogenic Bacteria ◽  
Capsular Polysaccharide ◽  
Protein A ◽  
Foodborne Pathogen ◽  
Control Group ◽  
Benzyl Isothiocyanate ◽  
Polysaccharide Biosynthesis ◽  
Polysaccharide Synthesis ◽  
Transcriptional Changes

Staphylococcus aureus (S. aureus) is a common foodborne pathogen that leads to various diseases; therefore, we urgently need to identify different means to control this harmful pathogen in food. In this study, we monitored the transcriptional changes of S. aureus by RNA-seq analysis to better understand the effect of benzyl isothiocyanate (BITC) on the virulence inhibition of S. aureus and determined the bacteriostatic effect of BITC at subinhibitory concentrations. Our results revealed that, compared with the control group (SAC), the BITC-treated experimental group (SAQ_BITC) had 708 differentially expressed genes (DEGs), of which 333 genes were downregulated and the capsular polysaccharide (cp) was significantly downregulated. Furthermore, we screened five of the most virulent factors of S. aureus, including the capsular polysaccharide biosynthesis protein (cp5D), capsular polysaccharide synthesis enzyme (cp8F), thermonuclease (nuc), clumping factor (clf), and protein A (spa), and verified the accuracy of these significantly downregulated genes by qRT-PCR. At the same time, we used light microscopy, scanning electron microscopy (SEM) and inverted fluorescence microscopy (IFM) to observe changes in biofilm associated with the cp5D and cp8F. Therefore, these results will help to further study the basis of BITC for the antibacterial action of foodborne pathogenic bacteria.

scholarly journals CdSe QD Biosynthesis in Yeast Using Tryptone-Enriched Media and Their Conjugation with a Peptide Hecate for Bacterial Detection and Killing

Nanomaterials ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 1463 ◽  
Author(s):  
Vishma Pratap Sur ◽  
Marketa Kominkova ◽  
Zaneta Buchtova ◽  
Kristyna Dolezelikova ◽  
Ondrej Zitka ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Synthesis Process ◽  
Synthesis Methods ◽  
Bacterial Cells ◽  
Cdse Qds ◽  
Yeast Saccharomyces Cerevisiae ◽  
Shape Distribution ◽  
Transmission Electron ◽  
A Cell ◽  
Physical And Chemical

The physical and chemical synthesis methods of quantum dots (QDs) are generally unfavorable for biological applications. To overcome this limitation, the development of a novel “green” route to produce highly-fluorescent CdSe QDs constitutes a promising substitute approach. In the present work, CdSe QDs were biosynthesized in yeast Saccharomyces cerevisiae using a novel method, where we showed for the first time that the concentration of tryptone highly affects the synthesis process. The optimum concentration of tryptone was found to be 25 g/L for the highest yield. Different methods were used to optimize the QD extraction from yeast, and the best method was found to be by denaturation at 80 °C along with an ultrasound needle. Multiple physical characterizations including transmission electron microscopy (TEM), dynamic light scattering (DLS), energy-dispersive X-ray spectroscopy (EDX), and spectrophotometry confirmed the optical features size and shape distribution of the QDs. We showed that the novel conjugate of the CdSe QDs and a cell-penetrating peptide (hecate) can detect bacterial cells very efficiently under a fluorescent microscope. The conjugate also showed strong antibacterial activity against vancomycin-resistant Staphylococcus aureus (VRSA), methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli, which may help us to cope with the problem of rising antibiotic resistance.

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