Guanine Limitation Results in CodY-Dependent and -Independent Alteration of Staphylococcus aureus Physiology and Gene Expression

Abstract

In , the global transcriptional regulator CodY modulates the expression of hundreds of genes in response to the availability of GTP and the branched-chain amino acids isoleucine, leucine, and valine (ILV). CodY DNA-binding activity is high when GTP and ILV are abundant. When GTP and ILV are limited, CodY's affinity for DNA drops, altering expression of CodY regulated targets. In this work, we investigated the impact of guanine nucleotides on physiology and CodY activity by constructing a null mutant (Δ). biosynthesis of guanine monophosphate is abolished due to the mutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked out , activating the Agr two-component system and increasing secreted protease activity. Notably, in a rich, complex medium, we detected an increase in alternative sigma factor B activity in the Δ mutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, Δ cells failed to form robust biofilms when limited for guanine or guanosine. RNA-seq analysis of transcriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alteration of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting that exhibits a more invasive lifestyle when limited for guanosine. Further, gene-products upregulated under GN limitation, including those necessary for lipoic acid biosynthesis and sugar transport, may prove to be useful drug targets for treating Gram-positive infections. infections impose a serious economic burden on healthcare facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing fitness and virulence of in response to environmental cues is critical for developing efficient diagnostics and treatments. purine biosynthesis is essential for both fitness and virulence in , since inhibiting production cripples's ability to cause infection. Here, we corroborate these findings and show that blocking guanine nucleotide synthesis severely affects fitness by altering metabolic and virulence gene expression. Characterizing pathways and gene products upregulated in response to guanine limitation can aid in the development of novel adjuvant strategies to combat infections.

Genetic and Biochemical Analysis of CodY-Mediated Cell Aggregation in Staphylococcus aureus Reveals an Interaction between Extracellular DNA and Polysaccharide in the Extracellular Matrix

“…A ΔguaA mutant of S. aureus is a guanine nucleotide auxotroph. TSB provides this required nutrient (77), suggesting that the DNA incorporated into the biofilm matrix might be derived from the medium. Consistent with this observation, ΔcodY mutant cells failed to aggregate during growth in chemically defined medium (CDM) lacking DNA (see Fig.…”

MsaB and CodY Interact To Regulate Staphylococcus aureus Capsule in a Nutrient-Dependent Manner

“…However, when nutrient concentrations begin to decline, CodY undergoes a conformational change that results in CodY not binding to its DNA targets (46,47). Moreover, CodY senses the availability of nutrients, specifically GTP and branched-chain amino acids (BCAAs), for its regulatory function (48)(49)(50)(51). Previously, we used the prediction tool NsitePred to analyze the MsaB sequence and found that MsaB contains predicted nutrient-sensing domains (15 phenylalanine, 29 histidine, and 30 phenylalanine residues) for nucleotides, including GTP, ADP, and AMP, respectively (data not shown).…”

“…The presence or absence of these nutrients results in a conformational change within the CodY protein structure (46,47,52). This conformational change of the protein results in a decrease in the DNA-binding affinity of CodY for its targets under conditions of low nutrient concentrations (43,48,49,51). CodY is part of a four-gene operon that produces a large, 4.1-kb transcript (43).…”

Revealing 29 sets of independently modulated genes inStaphylococcus aureus, their regulators and role in key physiological responses

“…Concurrent activation of the CodY, PurR, and GR i-modulons in R10LB indicates that this media presents a guanine-limited environment, as activity of all three transcription factors decrease in response to falling cellular concentrations of various forms of guanine derivatives [13][14][15][16] . Consistent with this hypothesis, we also saw decreased activity of the Translation i-modulon in R10LB.…”

“…Consistent with this hypothesis, we also saw decreased activity of the Translation i-modulon in R10LB. Downregulation of translation machinery often occurs during the stringent response, where cellular GTP is depleted as it is rapidly converted to ppGpp 13,[17][18][19] . Similarly, activation of the MntR i-modulon points to manganese starvation in R10LB, and the decreased activity of two i-modulons associated with carbon catabolite repressor CcpA (CcpA-1 and CcpA-2) likely reflects a glucose replete environment 20 .…”