Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jun 25;200(14):e00136-18.
doi: 10.1128/JB.00136-18. Print 2018 Jul 15.

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

Affiliations
Free PMC article

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

Alyssa N King et al. J Bacteriol. .
Free PMC article

Abstract

In Staphylococcus aureus, 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 (GNs) on S. aureus physiology and CodY activity by constructing a guaA null mutant (ΔguaA strain). De novo biosynthesis of guanine monophosphate is abolished due to the guaA mutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked out guaA, 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 ΔguaA mutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, ΔguaA cells failed to form robust biofilms when limited for guanine or guanosine. Transcriptome sequencing (RNA-Seq) analysis of the S. aureus transcriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alterations of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting that S. aureus 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.IMPORTANCE Staphylococcus aureus infections impose a serious economic burden on health care facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing fitness and virulence of S. aureus in response to environmental cues is critical for developing efficient diagnostics and treatments. De novo purine biosynthesis is essential for both fitness and virulence in S. aureus since inhibiting production cripples S. aureus's ability to cause infection. Here, we corroborate these findings and show that blocking guanine nucleotide synthesis severely affects S. aureus 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 S. aureus infections.

Keywords: Agr; GTP; SigB; biofilms; proteases.

Figures

FIG 1
FIG 1
Pathway for guanine nucleotide synthesis. IMP is synthesized from successive enzymatic steps catalyzed by the Pur enzymes. IMP is then converted to xanthine monophosphate (XMP) by IMP dehydrogenase (GuaB). XMP can also be synthesized by xanthine phosphoribosyltransferase (Xpt) during xanthine uptake. GMP synthetase (GuaA) converts XMP to GMP, which is then phosphorylated to GTP by guanylate kinase (SAR1185, similar to B. subtilis YloD) and nucleoside diphosphate kinase (SAR1478, similar to B. subtilis Ndk). GMP can also be formed via the guanosine salvage pathway by the concerted action of guanosine/deoxyguanosine-inosine/deoxyinosine phosphorylase (PupG) and hypoxanthine-guanine phosphoribosyltransferase (Hpt).
FIG 2
FIG 2
Blocking de novo guanine nucleotide synthesis limits growth and stimulates staphyloxanthin synthesis. (A) UAMS-1 (WT) and ΔguaA mutant cells were streaked to TSA or TSA supplemented with 160 μM guanosine (GUO), as indicated. (B) UAMS-1 and LAC strains were pregrown to exponential phase in TSB and then rediluted into TSB medium for 24 h. Cells were pelleted and washed, and carotenoid pigments were extracted using hot methanol as described in Materials and Methods. Data shown indicate the means ± standard errors of the means of three independent experiments. Statistical significance was assessed using one-way analysis of variance with Tukey's posttest (*, P < 0.05; ***, P < 0.001; ****, P < 0.0001). (C) In the UAMS-1 background, staphyloxanthin pigment accumulation was assessed after 24 h of growth in TSB medium (black bars) or in TSB medium supplemented with 160 μM guanosine (gray bars) for the indicated strains. Data shown indicate the mean ± standard errors of the means of three independent experiments. Statistical significance was assessed using two-way analysis of variance with Tukey's posttest (**, P < 0.01, results for the guaA strain without guanosine supplementation compared to all other conditions). (D) pAH12 was used to measure asp23 promoter activity in UAMS-1 and ΔguaA mutant cells (SRB1219 and SRB1225, respectively). Fluorescence was read after 24 h of growth in TSB medium and normalized to CFU counts. Data are the means ± standard errors of the means of three independent experiments. Statistical significance was assessed using Welch's t test (*, P < 0.05).
FIG 3
FIG 3
Guanine nucleotide limitation increases Agr activity and Agr-dependent protease activity. (A) UAMS-1 WT (SRB1081) and ΔguaA (SRB1093) cells carrying pAH08 (PagrP3-mCherry) were grown overnight in TSB medium and then diluted into fresh TSB for 24 h. Cells were pelleted, washed, and resuspended in PBS, and then mCherry fluorescence was measured. Data shown indicate the means ± standard errors of the means of three biological replicates. Statistical significance was assessed using Student's t test (*, P < 0.05). (B) UAMS-1 WT (SRB1220) and ΔguaA cells (SRB1226) carrying the Paur-gfp reporter fusion (plasmid pCM13) were grown and processed as described for panel A, and GFP fluorescence was measured. Welch's t test was used to evaluate statistical significance (*, P < 0.05). (C) UAMS-1 WT (SRB337), guaA (SRB1081), sigB (SRB923), and guaA agr (SRB1061) cells were grown overnight in TSB medium and spotted onto TSB plates containing 2% skim milk. Protease activity (indicated by a zone of clearing) was assessed after 48 h of growth. Images are representative of two independent experiments.
FIG 4
FIG 4
Guanine nucleotide-limited cells are unable to form mature biofilm structures during S. aureus biofilm development. S. aureus (UAMS-1) wild-type and ΔguaA mutant cells were grown in a BioFlux 1000 microfluidic flow cell system. Bright-field microscopic images were acquired at ×200 magnification after 4, 9, and 14 h of biofilm development. Images are representative of multiple experiments. Scale bar, 50 μm. GUO, supplementation with 160 μM guanosine.
FIG 5
FIG 5
Multidimensional scaling (MDS) analysis of RNA-Seq samples reveals distinct genotype and treatment differences during steady-state growth. The UAMS-1 ΔguaA strain (open symbols) and the ΔguaA ΔcodY strain (filled symbols) were grown exponentially under GN-replete (160 μM guanosine; squares) and GN-limited (40 μM guanosine; circles) conditions, and RNA-Seq libraries were subjected to Illumina sequencing en masse. Each symbol represents a single biological replicate.
FIG 6
FIG 6
Metabolic and virulence genes show altered CodY-dependent regulation in guanosine-limited chemostat cultures. Mean RNA-Seq gene expression ratios (ΔguaA ΔcodY transcript levels relative to ΔguaA transcript levels) from three independent experiments in the UAMS-1 background are shown for the indicated genes. Black bars, GN-replete conditions; gray bars, GN-limited conditions. It is important to note that limiting GN increases gene expression. For instance, mean normalized counts for gltB in the guaA strain were 53 under replete conditions and 90 under limited conditions, whereas the normalized counts for the ΔguaA ΔcodY strain were 200 and 156, respectively.
FIG 7
FIG 7
Expression patterns for msaB, sspA, QV15_09215, and fnbA during growth in chemically defined medium. The UAMS-1 ΔguaA and ΔguaA ΔcodY strains were grown to exponential phase in CDM supplemented with 160 μM guanosine and rediluted into CDM with 160 μM guanosine (replete) or 40 μM guanosine (limited), as indicated. Data are the means ± standard deviations of normalized counts from three biological replicates. The effects of strain (Strain), environment (Env), and strain-by-environment (Strain×Env) are modeled as defined in the text. Significant differences by factor are as indicated on each figure (Benjamini-Hochberg adjusted P value; *, P < 0.05; ***, P < 0.001; ns, not significant).
FIG 8
FIG 8
Select genes differentially regulated during guanine nucleotide limitation. Heat maps displaying the RNA-Seq fold change of selected genes under GN-limited/GN-replete conditions are shown. Log2 changes in transcript abundance are color-coded, with blue and red indicating a decrease and increase, respectively. The RNA-Seq data shown are from three independent experiments in the UAMS-1 background and are significantly different (Benjamini-Hochberg adjusted P value, <0.05). Full data are available in Table S4 in the supplemental material.

Similar articles

See all similar articles

Cited by 3 articles

Publication types

MeSH terms

LinkOut - more resources

Feedback