scholarly journals The Staphylococcus aureus lrgAB Operon Modulates Murein Hydrolase Activity and Penicillin Tolerance

2000 ◽  
Vol 182 (7) ◽  
pp. 1794-1801 ◽  
Author(s):  
Kajetan H. Groicher ◽  
Brian A. Firek ◽  
David F. Fujimoto ◽  
Kenneth W. Bayles
Keyword(s):  
Staphylococcus Aureus ◽  
Regulatory System ◽  
Cell Lysis ◽  
Negative Control ◽  
Exponential Phase ◽  
Hydrolase Activity ◽  
Wild Type ◽  
Early Exponential Phase ◽  
Associated Proteins ◽  
Murein Hydrolase

ABSTRACT Previous studies in our laboratory have shown that theStaphylococcus aureus LytSR two-component regulatory system affects murein hydrolase activity and autolysis. A LytSR-regulated dicistronic operon has also been identified and shown to encode two potential membrane-associated proteins, designated LrgA and LrgB, hypothesized to be involved in the control of murein hydrolase activity. In the present study, a lrgAB mutant strain was generated and analyzed to test this hypothesis. Zymographic and quantitative analysis of murein hydrolase activity revealed that thelrgAB mutant produced increased extracellular murein hydrolase activity compared to that of the wild-type strain. Complementation of the lrgAB defect by providing thelrgAB genes in trans restored the wild-type phenotype, indicating that these genes confer negative control on extracellular murein hydrolase activity. In addition to these effects, the influence of the lrgAB mutation on penicillin-induced lysis and killing was examined. These studies demonstrated that thelrgAB mutation enhanced penicillin-induced killing of cells approaching the stationary phase of growth, the time at which thelrgAB operon was shown to be maximally expressed. This effect of the lrgAB mutation on penicillin-induced killing was shown to be independent of cell lysis. In contrast, thelrgAB mutation did not affect penicillin-induced killing of cells growing in early-exponential phase, a time in whichlrgAB expression was shown to be minimal. However, expression of the lrgAB operon in early-exponential-phase cells inhibited penicillin-induced killing, again independent of cell lysis. The data generated by this study suggest that penicillin-induced killing of S. aureus involves a novel regulator of murein hydrolase activity.

scholarly journals The Staphylococcus aureus cidAB Operon: Evaluation of Its Role in Regulation of Murein Hydrolase Activity and Penicillin Tolerance

2003 ◽  
Vol 185 (8) ◽  
pp. 2635-2643 ◽  
Author(s):  
Kelly C. Rice ◽  
Brian A. Firek ◽  
Jeremy B. Nelson ◽  
Soo-Jin Yang ◽  
Toni G. Patton ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Exponential Growth ◽  
Parental Strain ◽  
Positive Influence ◽  
Hydrolase Activity ◽  
Gene Products ◽  
Wild Type ◽  
Wild Type Phenotype ◽  
Quantitative Analyses ◽  
Murein Hydrolase

ABSTRACT Recent studies have shown that expression of the Staphylococcus aureus lrgAB operon inhibits murein hydrolase activity and decreases sensitivity to penicillin-induced killing. It was proposed that the lrgAB gene products function in a manner analogous to an antiholin, inhibiting a putative holin from transporting murein hydrolases out of the cell. In the present study the cidAB operon was identified and characterized based on the similarity of the cidA and cidB gene products to the products of the lrgAB operon. Zymographic and quantitative analyses of murein hydrolase activity revealed that mutation of the cidA gene results in decreased extracellular murein hydrolase activity compared to that of S. aureus RN6390, the parental strain. Complementation of cidA expression restored the wild-type phenotype, indicating that expression of the cidAB operon has a positive influence on extracellular murein hydrolase activity. The cidA mutant also displayed a significant decrease in sensitivity to the killing effects of penicillin. However, complementation of the cidA defect did not restore penicillin sensitivity to wild-type levels. Reverse transcriptase PCR also revealed that cidAB is maximally expressed during early exponential growth, opposite of what was previously observed for lrgAB expression. Based on these results, we propose that the cidAB operon encodes the holin-like counterpart of the lrgAB operon and acts in a manner opposite from that of lrgAB by increasing extracellular murein hydrolase activity and increasing sensitivity to penicillin-induced killing.

scholarly journals The Staphylococcus aureus LytSR Two-Component Regulatory System Affects Biofilm Formation

2009 ◽  
Vol 191 (15) ◽  
pp. 4767-4775 ◽  
Author(s):  
Batu K. Sharma-Kuinkel ◽  
Ethan E. Mann ◽  
Jong-Sam Ahn ◽  
Lisa J. Kuechenmeister ◽  
Paul M. Dunman ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Regulatory System ◽  
Hydrolase Activity ◽  
Antibiotic Tolerance ◽  
Gene Products ◽  
Knockout Mutant ◽  
Two Component ◽  
Murein Hydrolase ◽  
Biofilm Development

ABSTRACT Studies of the Staphylococcus aureus LytSR two-component regulatory system have led to the identification of the cid and lrg operons, which affect murein hydrolase activity, stationary-phase survival, antibiotic tolerance, and biofilm formation. The cid gene products enhance murein hydrolase activity and antibiotic tolerance whereas the lrg gene products inhibit these processes in a manner believed to be analogous to bacteriophage-encoded holins and antiholins, respectively. Importantly, these operons have been shown to play significant roles in biofilm development by controlling the release of genomic DNA, which then becomes an important structural component of the biofilm matrix. To determine the role of LytSR in biofilm development, a lytS knockout mutant was generated from a clinical S. aureus isolate (UAMS-1) and the effects on gene expression and biofilm formation were examined. As observed in laboratory isolates, LytSR was found to be required for lrgAB expression. Furthermore, the lytS mutant formed a more adherent biofilm than the wild-type and complemented strains. Consistent with previous findings, the increased adherence of the mutant was attributed to the increased prevalence of matrix-associated eDNA. Transcription profiling studies indicated that the lrgAB operon is the primary target of LytSR-mediated regulation but that this regulatory system also impacts expression of a wide variety of genes involved in basic metabolism. Overall, the results of these studies demonstrate that the LytSR two-component regulatory system plays an important role in S. aureus biofilm development, likely as a result of its direct influence on lrgAB expression.

scholarly journals Identification of sarV (SA2062), a New Transcriptional Regulator, Is Repressed by SarA and MgrA (SA0641) and Involved in the Regulation of Autolysis in Staphylococcus aureus

2004 ◽  
Vol 186 (16) ◽  
pp. 5267-5280 ◽  
Author(s):  
Adhar C. Manna ◽  
Susham S. Ingavale ◽  
MaryBeth Maloney ◽  
Willem van Wamel ◽  
Ambrose L. Cheung
Keyword(s):  
Staphylococcus Aureus ◽  
Target Genes ◽  
Transcriptional Regulator ◽  
Analysis Data ◽  
Hydrolase Activity ◽  
Northern Analysis ◽  
Wild Type ◽  
Expression Of Genes ◽  
Murein Hydrolase

ABSTRACT The expression of genes involved in the pathogenesis of Staphylococcus aureus is known to be controlled by global regulatory loci, including agr, sarA, sae, arlRS, lytSR, and sarA-like genes. Here we described a novel transcriptional regulator called sarV of the SarA protein family. The transcription of sarV is low or undetectable under in vitro conditions but is significantly augmented in sarA and mgrA (norR or rat) (SA0641) mutants. The sarA and mgrA genes act as repressors of sarV expression, as confirmed by transcriptional fusion and Northern analysis data. Purified SarA and MgrA proteins bound specifically to separate regions of the sarV promoter as determined by gel shift and DNase I footprinting assays. The expression of 19 potential target genes involved in autolysis and virulence, phenotypes affected by sarA and mgrA, was evaluated in an isogenic sarV mutant pair. Our data indicated that the sarV gene product played a role regulating some virulence genes and more genes involved in autolysis. The sarV mutant was more resistant to Triton X-100 and penicillin-induced lysis compared to the wild type and the sarA mutant, whereas hyperexpression of sarV in the parental strain or the sarV mutant rendered the resultant strain highly susceptible to lysis. Zymographic analysis of murein hydrolase activity revealed that inactivation of the sarV gene results in decreased extracellular murein hydrolase activity compared to that of wild-type S. aureus. We propose that sarV may be part of the common pathway by which mgrA and sarA gene products control autolysis in S. aureus.

scholarly journals Transcription of the Staphylococcus aureus cid and lrg Murein Hydrolase Regulators Is Affected by Sigma Factor B

2004 ◽  
Vol 186 (10) ◽  
pp. 3029-3037 ◽  
Author(s):  
Kelly C. Rice ◽  
Toni Patton ◽  
Soo-Jin Yang ◽  
Alexis Dumoulin ◽  
Markus Bischoff ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Northern Blot ◽  
Exponential Growth ◽  
Sigma Factor ◽  
Positive Impact ◽  
Consensus Sequence ◽  
Hydrolase Activity ◽  
Factor B ◽  
Sigma Factor B ◽  
Murein Hydrolase

ABSTRACT The Staphylococcus aureus lrg and cid loci are homologous operons that have been shown to regulate murein hydrolase activity and affect sensitivity to penicillin. Although the mode of action of these operons has not been demonstrated, a model based on the similarities of the lrgA and cidA gene products to the bacteriophage holin family of proteins has been proposed. In this study, the transcription organization and regulation of these operons were examined by Northern blot analyses. Unexpectedly, cidB and a gene located immediately downstream, designated cidC, were found to be cotranscribed on a 2.7-kb transcript. Maximal cidBC transcription occurred during early exponential growth, and high-level transcription of cidBC was dependent on the rsbU-mediated activation of the alternative sigma factor B (σB). In contrast, lrgAB transcription in stationary phase was negatively regulated by σB. Although cidABC transcription was not detected by Northern blot analysis, reverse transcriptase PCR revealed that these genes are also cotranscribed as a single RNA message in early exponential growth. Primer extension analysis revealed the presence of two cidBC transcription start sites, but no apparent σB-dependent promoter consensus sequence was identified in these regions. The rsbU gene was also shown to have a positive impact on murein hydrolase activity but a negligible effect on sensitivity to penicillin-induced killing. These results suggest that the lrgAB and cidBC genes may be part of the S. aureus σB-controlled stress regulon.

scholarly journals Impaired respiration elicits SrrAB-dependent programmed cell lysis and biofilm formation in Staphylococcus aureus

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Ameya A Mashruwala ◽  
Adriana van de Guchte ◽  
Jeffrey M Boyd
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Regulatory System ◽  
Cell Lysis ◽  
Cellular Respiration ◽  
Terminal Electron Acceptor ◽  
Etiologic Agents ◽  
System Data ◽  
Chemical Inhibition ◽  
Wall Teichoic Acids

Biofilms are communities of microorganisms attached to a surface or each other. Biofilm-associated cells are the etiologic agents of recurrent Staphylococcus aureus infections. Infected human tissues are hypoxic or anoxic. S. aureus increases biofilm formation in response to hypoxia, but how this occurs is unknown. In the current study we report that oxygen influences biofilm formation in its capacity as a terminal electron acceptor for cellular respiration. Genetic, physiological, or chemical inhibition of respiratory processes elicited increased biofilm formation. Impaired respiration led to increased cell lysis via divergent regulation of two processes: increased expression of the AtlA murein hydrolase and decreased expression of wall-teichoic acids. The AltA-dependent release of cytosolic DNA contributed to increased biofilm formation. Further, cell lysis and biofilm formation were governed by the SrrAB two-component regulatory system. Data presented support a model wherein SrrAB-dependent biofilm formation occurs in response to the accumulation of reduced menaquinone.

scholarly journals Interconnections between Sigma B, agr, and Proteolytic Activity in Staphylococcus aureus Biofilm Maturation

2009 ◽  
Vol 77 (4) ◽  
pp. 1623-1635 ◽  
Author(s):  
Katherine J. Lauderdale ◽  
Blaise R. Boles ◽  
Ambrose L. Cheung ◽  
Alexander R. Horswill
Keyword(s):  
Staphylococcus Aureus ◽  
Hydrolase Activity ◽  
Extracellular Proteases ◽  
Sigma B ◽  
Genes Encoding ◽  
Usa300 Strain ◽  
Sigma Factor B ◽  
Murein Hydrolase ◽  
Host Tissues

ABSTRACT Staphylococcus aureus is a proficient biofilm former on host tissues and medical implants. We mutagenized S. aureus strain SH1000 to identify loci essential for ica-independent mechanisms of biofilm maturation and identified multiple insertions in the rsbUVW-sigB operon. Following construction and characterization of a sigB deletion, we determined that the biofilm phenotype was due to a lack of sigma factor B (SigB) activity. The phenotype was conserved in a sigB mutant of USA300 strain LAC, a well-studied community-associated methicillin-resistant S. aureus isolate. We determined that agr RNAIII levels were elevated in the sigB mutants, and high levels of RNAIII expression are known to have antibiofilm effects. By introducing an agr mutation into the SH1000 or LAC sigB deletion strain, S. aureus regained biofilm capacity, indicating that the biofilm phenotype was agr dependent. Protease activity is linked to agr activity and ica-independent biofilm formation, and we observed that the protease inhibitors phenylmethylsulfonyl fluoride and α-macroglobulin could reverse the sigB biofilm defect. Similarly, inactivating genes encoding both the aureolysin and Spl extracellular proteases in the sigB mutant restored biofilm capacity. Due to the growing link between murein hydrolase activity and biofilm maturation, autolysin zymography was performed, which revealed an altered profile in the sigB mutant; again, the phenotype could be repaired through protease inactivation. These findings indicate that the lack of SigB activity results in increased RNAIII expression, thus elevating extracellular protease levels and altering the murein hydrolase activity profile. Altogether, our observations demonstrate that SigB is an essential regulator of S. aureus biofilm maturation.

scholarly journals ThemecAHomologmecCConfers Resistance against β-Lactams in Staphylococcus aureus Irrespective of the Genetic Strain Background

2014 ◽  
Vol 58 (7) ◽  
pp. 3791-3798 ◽  
Author(s):  
Britta Ballhausen ◽  
André Kriegeskorte ◽  
Nina Schleimer ◽  
Georg Peters ◽  
Karsten Becker
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistance ◽  
Regulatory System ◽  
Wild Type ◽  
Beta Lactam ◽  
Beta Lactams ◽  
Content Type ◽  
Genetic Strain ◽  
Mrsa Strains ◽  
Strain Background

ABSTRACTIn staphylococci, methicillin resistance is mediated bymecA-encoded penicillin-binding protein 2a (PBP2a), which has a low affinity for beta-lactams. Recently, a novel PBP2a homolog was described as being encoded bymecC, which shares only 70% similarity tomecA. To prove thatmecCis the genetic determinant that confers methicillin resistance inStaphylococcus aureus, amecCknockout strain was generated. TheS. aureusΔmecCstrain showed considerably reduced oxacillin and cefoxitin MICs (0.25 and 4 μg/ml, respectively) compared to those of the corresponding wild-type methicillin-resistantS. aureus(MRSA) strain (8 and 16 μg/ml, respectively). Complementing the mutant intranswith wild-typemecCrestored the resistance to oxacillin and cefoxitin. By expressingmecCandmecAin differentS. aureusclonal lineages, we found thatmecCmediates resistance irrespective of the genetic strain background, yielding oxacillin and cefoxitin MIC values comparable to those withmecA. In addition, we showed thatmecCexpression is inducible by oxacillin, which supports the assumption that a functional beta-lactam-dependent regulatory system is active in MRSA strains possessing staphylococcal cassette chromosomemec(SCCmec) type XI. In summary, we showed thatmecCis inducible by oxacillin and mediates beta-lactam resistance in SCCmectype XI-carrying strains as well as in differentS. aureusgenetic backgrounds. Furthermore, our results could explain the comparatively low MICs for clinicalmecC-harboringS. aureusisolates.

scholarly journals The Spl Serine Proteases Modulate Staphylococcus aureus Protein Production and Virulence in a Rabbit Model of Pneumonia

mSphere ◽  
2016 ◽  
Vol 1 (5) ◽  
Author(s):  
Alexandra E. Paharik ◽  
Wilmara Salgado-Pabon ◽  
David K. Meyerholz ◽  
Mark J. White ◽  
Patrick M. Schlievert ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Serine Proteases ◽  
Rabbit Model ◽  
Human Infection ◽  
Lung Damage ◽  
Wild Type ◽  
Content Type ◽  
Associated Proteins ◽  
Human Proteins

ABSTRACT Staphylococcus aureus is a versatile human pathogen that produces an array of virulence factors, including several proteases. Of these, six proteases called the Spls are the least characterized. Previous evidence suggests that the Spls are expressed during human infection; however, their function is unknown. Our study shows that the Spls are required for S. aureus to cause disseminated lung damage during pneumonia. Further, we present the first example of a human protein cut by an Spl protease. Although the Spls were predicted not to cut staphylococcal proteins, we also show that an spl mutant has altered abundance of both secreted and surface-associated proteins. This work provides novel insight into the function of Spls during infection and their potential ability to degrade both staphylococcal and human proteins. The Spl proteases are a group of six serine proteases that are encoded on the νSaβ pathogenicity island and are unique to Staphylococcus aureus. Despite their interesting biochemistry, their biological substrates and functions in virulence have been difficult to elucidate. We found that an spl operon mutant of the community-associated methicillin-resistant S. aureus USA300 strain LAC induced localized lung damage in a rabbit model of pneumonia, characterized by bronchopneumonia observed histologically. Disease in the mutant-infected rabbits was restricted in distribution compared to that in wild-type USA300-infected rabbits. We also found that SplA is able to cleave the mucin 16 glycoprotein from the surface of the CalU-3 lung cell line, suggesting a possible mechanism for wild-type USA300 spreading pneumonia to both lungs. Investigation of the secreted and surface proteomes of wild-type USA300 and the spl mutant revealed multiple alterations in metabolic proteins and virulence factors. This study demonstrates that the Spls modulate S. aureus physiology and virulence, identifies a human target of SplA, and suggests potential S. aureus targets of the Spl proteases. IMPORTANCE Staphylococcus aureus is a versatile human pathogen that produces an array of virulence factors, including several proteases. Of these, six proteases called the Spls are the least characterized. Previous evidence suggests that the Spls are expressed during human infection; however, their function is unknown. Our study shows that the Spls are required for S. aureus to cause disseminated lung damage during pneumonia. Further, we present the first example of a human protein cut by an Spl protease. Although the Spls were predicted not to cut staphylococcal proteins, we also show that an spl mutant has altered abundance of both secreted and surface-associated proteins. This work provides novel insight into the function of Spls during infection and their potential ability to degrade both staphylococcal and human proteins.

scholarly journals σB and the σB-Dependent arlRS and yabJ-spoVG Loci Affect Capsule Formation in Staphylococcus aureus

2007 ◽  
Vol 75 (9) ◽  
pp. 4562-4571 ◽  
Author(s):  
Stefan Meier ◽  
Christiane Goerke ◽  
Christiane Wolz ◽  
Kati Seidl ◽  
Dagmar Homerova ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Capsular Polysaccharide ◽  
Regulatory System ◽  
Strong Impact ◽  
Wild Type ◽  
Capsule Formation ◽  
Native Promoter ◽  
Alternative Transcription ◽  
In The Wild ◽  
Two Component Signal Transduction

ABSTRACT The alternative transcription factor σB of Staphylococcus aureus affects the transcription of the cap gene cluster, required for the synthesis of capsular polysaccharide (CP), although this operon is lacking an apparent σB-dependent promoter. Regulation of cap expression and CP production in S. aureus strain Newman was shown here to be influenced by σB, the two-component signal transduction regulatory system ArlRS, and the yabJ-spoVG locus to different extents. Inactivation of arlR or deletion of the sigB operon strongly suppressed capA (CP synthesis enzyme A) transcription. Deletion of spoVG had a polar effect on yabJ-spoVG transcription and resulted in a two- to threefold decrease in capA transcription. Interestingly, immunofluorescence showed that CP production was strongly impaired in all three mutants, signaling that the yabJ-spoVG inactivation, despite its only partial effect on capA transcription, abolished capsule formation. trans-Complementation of the ΔspoVG mutant with yabJ-spoVG under the control of its native promoter restored CP-5 production and capA expression to levels seen in the wild type. Northern analyses revealed a strong impact of σB on arlRS and yabJ-spoVG transcription. We hypothesize that ArlR and products of the yabJ-spoVG locus may serve as effectors that modulate σB control over σB-dependent genes lacking an apparent σB promoter.

scholarly journals REPRESSOR AND ANTIREPRESSOR IN THE REGULATION OF STAPHYLOCOCCAL PENICILLINASE SYNTHESIS

Genetics ◽  
1973 ◽  
Vol 75 (1) ◽  
pp. 1-17
Author(s):  
John Imsande
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Site ◽  
Regulatory Protein ◽  
Positive Control ◽  
Negative Control ◽  
Biochemical Data ◽  
Wild Type ◽  
Data Support ◽  
Operator Binding ◽  
Effector Binding

ABSTRACT 5-methyltryptophan (5MT) induces penicillinase synthesis in Staphylococcus aureus. The analog is incorporated into protein by both wild-type and tryptophan-starved cells. Since normal penicillinase repressor appears to contain tryptophan even though penicillinase itself does not, it is concluded that 5MT induces penicillinase synthesis by becoming incorporated into the penicillinase repressor and thereby inactivating the repressor. Thus biochemical data support the existence of a penicillinase repressor and indicate that penicillinase synthesis is regulated by negative control and not by positive control.—In the absence of exogenous tryptophan, staphylococcal penicillinase induction can be inhibited by 7-azatryptophan (7azaT). Because 7azaT is incorporated into protein by tryptophan-starved cells, it is concluded that 7azaT blocks penicillinase induction by inactivating a penicillinase regulatory protein into which the analog has been incorporated. Incorporation of 7azaT does not appear to inactivate the operator binding site or the effector binding site on the penicillinase repressor. Therefore, it appears that 7azaT blocks penicillinase induction by inactivating the penicillinase antirepressor, a protein required for inactivation of the penicillinase repressor and, hence, required for penicillinase induction.

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