CidR and CcpA Synergistically Regulate Staphylococcus aureus cidABC Expression

Abstract

The death and lysis of a subpopulation of Staphylococcus aureus cells during biofilm development benefit the whole bacterial population through the release of an important component of the biofilm matrix, extracellular DNA. Previously, we have demonstrated that these processes are affected by the gene products of the cidABC operon, the expression of which is controlled by the LysR-type transcriptional regulator, CidR. In this study, we characterized cis- and trans-acting elements essential for the induction of the cidABC operon. In addition to a CidR-binding site located within the cidABC promoter region, sequence analysis revealed the presence of a putative catabolite responsive element (cre box), suggestive of the involvement of the catabolite control protein A (CcpA) in the regulation of cidABC expression. This was confirmed using electrophoretic mobility shift assays and real-time reverse transcriptase PCR analysis demonstrating the direct positive control of cidABC transcription by the master regulator of carbon metabolism. Furthermore, the importance of CcpA and the identified cre site for the induction of the cidABC operon was demonstrated by examining the expression of PcidABC-lacZ reporter fusions in various mutant strains in which the genes involved in carbon metabolism and carbon catabolite repression were disrupted. Together the results of this study demonstrate the necessity of both transcriptional regulators, CidR and CcpA, for the induction of the cidABC operon and reveal the complexity of molecular interactions controlling its expression. IMPORTANCE This work focuses on the characterization of cis- and trans-acting elements essential for the induction of the cidABC operon in S. aureus. The results of this study are the first to demonstrate the synergistic control of cidABC expression by transcriptional regulators CidR and CcpA during carbohydrate metabolism. We established that the full induction of cidABC expression depends on the metabolic state of bacteria and requires both CidR and CcpA. Together, these findings delineate regulatory control of cidABC expression under different metabolic conditions and provide important new insights into our understanding of cell death mechanisms during biofilm development in S. aureus.

Delineating the Role of the msaABCR Operon in Staphylococcal Overflow Metabolism

“…The SrrAB two-component system, which responds to nitric oxide stress and oxygen availability, appears to repress expression of the cidABC operon (Kinkel et al, 2013;Windham et al, 2016). Furthermore, in agreement with the facts that excess glucose is needed to induce expression of the cidABC operon and that CcpA is a master regulator of carbohydrate metabolism, both CcpA and CidR are required to fully induce expression of cidABC and alsSD (Seidl et al, 2009;Sadykov et al, 2011Sadykov et al, , 2019.…”

“…However, this process is a finely balanced phenomenon, as several bacterial mutants defective in lysis and the release of eDNA are poor biofilm formers (Rice et al, , 2005Patton et al, 2005;Yang et al, 2006;Bayles, 2007;Thomas et al, 2014;Chaudhari et al, 2016;Windham et al, 2016). The LysR-type transcriptional regulator, CidR, is a transcriptional activator of the cidABC, lrgAB, and alsSD operons (i.e., cidR regulon) and has been shown to play an important role in autolysis and cell death during biofilm formation (Rice et al, , 2005Patton et al, 2005;Yang et al, 2006;Thomas et al, 2014;Chaudhari et al, 2016;Windham et al, 2016;Sadykov et al, 2019).…”

“…In addition to the cidA-lrgA holin-antiholin system, other genes, such as cidC and alsSD, regulate PCD by modulating overflow metabolism in S. aureus (Patton et al, 2005;Yang et al, 2006;Thomas et al, 2014;Chaudhari et al, 2016;Sadykov et al, 2019). When grown under aerobic conditions, most of the carbon (including glucose) is directed to substrate-level phosphorylation that is catalyzed by the Pta-AckA pathway, which is important for yielding secondary energy, producing acetate when activity of the TCA cycle is limited by repression of carbon catabolites (known as the Crabtree effect; Sadykov et al, 2013;Marshall et al, 2016).…”

“…While CidR is the main transcriptional activator of the cidABC and alsSD operons, other regulators, such as SrrAB and CcpA, also are involved in the complex regulatory network of these operons (Patton et al, 2005(Patton et al, , 2006Yang et al, 2006;Chaudhari et al, 2016;Windham et al, 2016;Sadykov et al, 2019). The SrrAB two-component system, which responds to nitric oxide stress and oxygen availability, appears to repress expression of the cidABC operon (Kinkel et al, 2013;Windham et al, 2016).…”

The Staphylococcus aureus CidA and LrgA Proteins Are Functional Holins Involved in the Transport of By-Products of Carbohydrate Metabolism

“…More recently, the genes contained within the cidABC operon have been shown to play an important role in regulating cell death during overflow metabolism ( 19 , 20 ). The CidA and CidB proteins have opposing effects on CidC, a pyruvate:menaquinone oxidoreductase ( 21 ), which results in alterations in the extracellular acetate and acetoin levels ( 19 ), suggesting that cidA and cidB encode transport proteins involved in the secretion of these two metabolic by-products ( 19 ).…”