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. Author manuscript; available in PMC: 2016 Jun 10.
Published in final edited form as: Cell Host Microbe. 2015 May 28;17(6):788–798. doi: 10.1016/j.chom.2015.05.006

The PAMP c-di-AMP is essential for Listeria monocytogenes growth in rich but not minimal media due to a toxic increase in (p)ppGpp

Aaron T Whiteley 1, Alex J Pollock 2, Daniel A Portnoy 1,2,*
PMCID: PMC4469362  NIHMSID: NIHMS690804  PMID: 26028365

SUMMARY

Cyclic di-adenosine monophosphate (c-di-AMP) is a widely distributed second messenger that appears to be essential in multiple bacterial species, including the Gram-positive facultative intracellular pathogen Listeria monocytogenes. In this study, the only L. monocytogenes diadenylate cyclase gene, dacA, was deleted using a Cre-lox system activated during infection of cultured macrophages. All ΔdacA strains recovered from infected cells harbored one or more suppressor mutations that allowed growth in the absence of c-di-AMP. Suppressor mutations in the synthase domain of the bi-functional (p)ppGpp synthase/hydrolase led to reduced (p)ppGpp levels. A genetic assay confirmed that dacA was essential in wild-type but not strains lacking all three (p)ppGpp synthases. Further genetic analysis suggested that c-di-AMP was essential because accumulated (p) ppGpp altered GTP concentrations, thereby inactivating the pleiotropic transcriptional regulator CodY. We propose that c-di-AMP is conditionally essential for metabolic changes that occur in growth in rich medium and host cells but not minimal medium.

Graphical Abstract

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INTRODUCTION

Listeria monocytogenes is a hardy and ubiquitous Gram-positive, facultative intracellular, foodborne pathogen that thrives as an environmental saprophyte, yet is capable of causing serious, often fatal, disease in a wide range of animals including humans (Cossart, 2011). Its broad growth range is accompanied by a largely prototrophic metabolism, capable of utilizing an array of carbon and nitrogen sources and requiring only a few essential vitamins and amino acids (Tsai and Hodgson, 2003). Remarkably, L. monocytogenes grows in the cytosol of host cells at a similar rate to rich medium (doubling in approximately 40 min) (Joseph and Goebel, 2007). To accomplish such efficient intracellular growth, L. monocytogenes remodels its transcriptional program upon entering host cells by activation of the master virulence regulator PrfA (Freitag et al., 2009). PrfA is required for the expression of many determinants of pathogenesis, but it also contributes to metabolic adaptations. For instance, transcription of hpt, encoding a hexose phosphate transporter, is PrfA regulated, allowing growth on glucose-1-phosphate in the host cell cytosol (Chico-Calero et al., 2002). L. monocytogenes also uses non-PrfA-mediated mechanisms for remodeling its metabolism. For example, pyruvate carboxylase (PycA) is dispensable in rich medium but required for pathogenesis and is regulated allosterically by cyclic diadenosine monophosphate (c-di-AMP) (Schär et al., 2010; Sureka et al., 2014).

c-di-AMP is member of the cyclic dinucleotide family of second messengers that includes cyclic diguanosine monophosphate (c-di-GMP) and cyclic-AMP-GMP produced by bacteria and cy-clic[G(2′–5′)pA(3′–5′)p] (cGAMP) produced by some metazoans (Danilchanka and Mekalanos, 2013). The role of c-di-AMP during infection was identified as a result of biochemical characterization of L. monocytogenes mutants that triggered diminished or enhanced activation of the cytosolic surveillance pathway (CSP) (Crimmins et al., 2008; Woodward et al., 2010). The CSP is characterized by the robust induction of host type I interferon and has implications for both innate and adaptive immunity (Archer et al., 2014; O’Riordan et al., 2002). L. monocytogenes secretes c-di-AMP through multidrug efflux pumps; however, the effect of secreted c-di-AMP on the bacterium is not known and remains an active area of investigation (Kaplan Zeevi et al., 2013; Tadmor et al., 2014). c-di-AMP differs from c-di-GMP, the most extensively characterized bacterial cyclic dinucleotide, in that bacteria usually encode a single diadenylate cyclase that is often essential (Corrigan and Gründling, 2013). The DAC protein domain (Pfam: DisA_N, PF02457) is the only identified protein domain capable of c-di-AMP synthesis in vivo and is widely distributed among Archaea, Gram-positive bacteria, and some Gram-negative bacteria (Witte et al., 2008). L. monocytogenes encodes only one DAC, dacA, which cannot be deleted by conventional methods and is therefore also predicted to be essential (Witte et al., 2013). In addition, high-throughput and targeted studies have identified c-di-AMP as essential in Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Mycoplasma genitalium, and Mycoplasma pulmonis (Corrigan and Gründling, 2013).

Many c-di-AMP-associated phenotypes have been observed in bacterial mutants containing inactivating mutations in c-di-AMP-degrading phosphodiesterases. Genetic screens in multiple organisms established that inactivating mutations in homologs of the conserved phosphodiesterase gdpP increase intracellular c-di-AMP levels, increase resistance to acid stress, suppressmutations in lipotechoic acid biosynthesis, and increase resistance to β-lactam antibiotics (Corrigan et al., 2011; Luo and Helmann, 2012; Rallu et al., 2000; Witte et al., 2013). Likewise, bacterial mutants depleted for DAC expression exhibit increased sensitivity to β-lactam antibiotics (Mehne et al., 2013; Witte et al., 2013). A diverse set of proteins interact with c-di-AMP, and a conserved c-di-AMP interacting riboswitch regulates translation of a wide array of genes in many organisms (Corrigan et al., 2013; Nelson et al., 2013; Sureka et al., 2014). However, none of the identified c-di-AMP receptors are conserved among all c-di-AMP-producing organisms, despite conservation of many c-di-AMP-related phenotypes (Corrigan and Gründling, 2013). Here we report the isolation and characterization of suppressor mutations that allow L. monocytogenes to grow in the absence of c-di-AMP. Our findings may help unify divergent c-di-AMP-related phenotypes and support a model in which L. monocytogenes requires intracellular c-di-AMP for metabolic adaptations during growth in rich medium and in host cells.

RESULTS

Generation of L. monocytogenes ΔdacA Mutants and Identification of Suppressor Mutations

There is mounting evidence that c-di-AMP is an essential molecule in many Firmicutes, including L. monocytogenes (Corrigan and Gründling, 2013). Accordingly, we were unable to generate ΔdacA mutants in wild-type L. monocytogenes but were successful in generating a dacA deletion in a strain that contained a second copy of dacA expressed from an inducible promoter (Witte et al., 2013; Woodward et al., 2010).We sought an alternative method to delete dacA based on an inducible Cre-lox system (Reniere et al., 2015). loxP sites were inserted into the L. monocytogenes chromosome flanking dacA (dacAfl). Codonoptimized cre recombinase was expressed from the actA promoter (PactA-cre) and cloned into a temperature-sensitive (Ts) plasmid. The actA promoter was chosen because it is not expressed in broth but is highly active during growth inside mammalian cells (Shetron-Rama et al., 2002). The dacAfl PactA-cre strain grew normally in broth but resulted in deletion of dacA upon infection of cultured macrophages (Figure 1A). Wild-type bacteria from infected macrophages formed colonies on rich medium agar in approximately 14 hr, whereas ΔdacA mutants formed visible colonies between days 2 and 5. The ΔdacA mutants, cured of the cre expressing plasmid, were verified by PCR using primers internal to the dacA gene and external to the dacA-locus.

Figure 1. Cre-lox Deletion of dacA and ΔdacA Suppressor Mutations.

Figure 1

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(A) Schematic representation of the L. monocytogenes strain used to delete dacA. The ΔdacA mutants were recovered after cre was induced during infection of BMDMs from a Ts plasmid that could be cured from ΔdacA mutants.

(B) BHI broth growth curve of wild-type, floxed-dacA (dacAfl), and ΔdacA mutants (for genotypes see Table S1). Data are representative of three independent experiments.

(C) Sensitivity to the toxic tripeptide bialaphos as measured by disk diffusion. Data are mean ± SEM of three independent experiments. p-oppB represents oppB complemented under its native promoters. The dotted line represents the L.o.D. and *p ≤ 0.01 by two-tailed Student’s t test as compared to wild-type.

(D) Frequency of opp and relA suppressor mutations within a collection of 284 ΔdacA mutants.

(E) Depiction of RelA protein with conserved hydrolase, synthase, ThrRS-GTPase-SpoT (TGS), and Aspartokinase-Chorismate-mutase-TyrA (ACT) domains. Annotation of specific amino acid changes as a result of suppressor mutations: R295S was the first identified in ΔdacA.3 and R216P was identified twice.

Five mutants, numbered ΔdacA.1–ΔdacA.5, were chosen for initial characterization. As expected, these mutants grew poorly in brain-heart infusion (BHI) broth, a rich medium commonly used for cultivating L. monocytogenes (Figure 1B). We hypothesized that dacA was essential but that these ΔdacA strains contained suppressor mutations that bypassed the essential functions of c-di-AMP. Genome sequencing of strains ΔdacA.1–ΔdacA.5 and the parent dacAfl confirmed that dacA was absent and revealed two groups of mutations not found in the parent strain (Table S1). Four strains contained mutations in the 5-gene operon oppABCDF encoding a previously identified oligopeptide permease (Opp) (Borezee et al., 2000). These four mutants displayed decreased sensitivity to killing by the toxic tri-peptide bialaphos that is transported exclusively by the Opp (Figure 1C) (Borezee et al., 2000). The only strain without an opp mutation (ΔdacA.3) encoded a point mutation (R295S) in the synthase domain of the previously identified bi-functional guanosine penta- and tetraphosphate ((p)ppGpp) synthase/hydrolase relA (Bennett et al., 2007; Taylor et al., 2002) and remained bialaphos sensitive (Figures 1C and 1E). These data indicated that the opp nucleotide changes were loss-of-function mutations, consistent with the disruptive nature of these polymorphisms (frameshifts and a premature stop codon) and that the relA mutation in the ΔdacA.3 strain did not affect Opp activity.

To further investigate dacA essentiality, an additional 284 ΔdacA mutants were selected for characterization. All of the ΔdacA strains isolated encoded mutations. Of these mutants, 94.37% were resistant to bialaphos, suggesting mutations in opp genes, and 1.76% harbored relA mutations as determined by Sanger sequencing of the synthase domain of the relA gene (Figure 1D). The additional relA alleles identified are depicted in Figure 1E. Genome sequencing of the remaining 11 mutants, ΔdacA.6–ΔdacA.16, that were sensitive to bialaphos and did not harbor relA synthase domain mutations revealed that each strain contained more than one mutation (Table S1). These additional mutations often recurred in the same genes (three of which share cystathionine-β-synthase [CBS] domains), appeared in genes encoding identified c-di-AMP binding proteins (Sureka et al., 2014) and included opp and relA mutations that escaped detection (Table S1). Characterization of opp and other suppressor mutations will be the subject of future studies.

Development of a dacA Essentiality Assay

The above results suggested that dacA was essential and that each of the ΔdacA mutants had accumulated one or more suppressor mutations. To expand upon these studies, we developed a rapid essentiality assay based on the method of co-transduction of linked genetic markers used to show gene essentiality in Escherichia coli (De Las Peñas et al., 1997). Two donor L. monocytogenes strains were constructed, dacA and ΔdacA using Cre-lox, in which the dacA locus was marked by a kana- mycin resistance gene (kanR) 3′ to the dacA locus. A himar1 transposon (ermR) encoding an erythromycin resistance gene was present 16.6 kb 5′ of the dacA locus (Figure 2A). The assay was performed by (1) lysogenizing recipient L. monocytogenes strains with phage derived from either of the two drug-resistant donor strains, (2) selecting for transduction on erythromycin, and (3) analyzing genetic linkage by scoring transductants for kanamycin resistance (Figure 2A). In a wild-type recipient, the dacA-kanR allele displayed approximately 35% linkage with the ermR gene, while co-transduction of the ΔdacA-kanR allele was below the limit of detection (L.o.D.) (Figure 2B). This linkage disequilibrium was ameliorated in recipients merodiploid for dacA (WT p-dacA), indicating the difference in linkage between dacA-kanR and ΔdacA-kanR alleles was specific to deletion of the dacA gene (Figure 2B). Moreover, the genetic linkage analysis produced similar results when performed with an alternative himar1 transposon 10 kb 5′ of the dacA locus, demonstrating that the location of the himar1 transposon had no effect on the results (Figure S1).

Figure 2. dacA Essentiality Assay.

Figure 2

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(A) Schematic of dacA essentiality assay depicting erythromycin resistance genes (ermR), kana-mycin resistance genes (kanR) 3′ of dacA and ΔdacA, erythromycin (Erm)/kanamycin (Kan)-containing medium agar, and resistant trans-ductants (ermR/kanR). See text for description.

(B) Genetic linkage of dacA or ΔdacA with co-transduced antibiotic resistance marker in wild-type or mutants merodiploid for dacA (p-dacA).

(C) dacA essentiality assay of ΔdacA suppressor mutants, ΔdacA.3 (encoding a relAR295S mutation) was not determined due to an inability to obtain ermR transductants in this background. The relAR295S mutation was interrogated instead by reconstructing the mutation in a wild-type background. Dotted line indicates L.o.D., and all data are mean ± SEM of at least three independent experiments. See also Figure S1.

The ΔdacA phage lysate used for the essentiality assay was derived from a strain that presumably harbors suppressor mutations as a result of the Cre-lox-mediated deletion of dacA. Given the results of the linkage experiments, these suppressor mutations were not linked to the dacA locus. To further assess the impact of suppressor mutations, ΔdacA.1–5 mutants generated through Cre-lox recombination were subjected to the identical linkage analysis described above (Figure 2A). Linkage was unaffected in ΔdacA.1, 2, 4, and 5, verifying the existence of suppressor mutations and that the dacA gene was no longer essential in these strains (Figure 2C). Linkage analysis was not determined for ΔdacA.3 (encoding a relAR295S mutation) owing to an inability to obtain erythromycin-resistant transductants in this background for an unknown reason. This limitation was overcome by reconstructing the relAR295S mutation in a wild-type background. dacA was also not essential in this background, confirming the relAR295S mutation suppresses dacA essentiality (Figure 2C). These data were consistent with dacA being essential to wild-type L. monocytogenes and established an assay whereby comparison of dacA and ΔdacA genetic linkage is a measure of dacA essentiality.

Suppressor Mutations in relA Decreased (p)ppGpp Accumulation in Response to Starvation

We chose to characterize the suppressor mutations in relA because these mutations appeared sufficient to ablate dacA essentiality and because of the previously documented nucleotide cross-talk between c-di-AMP and (p)ppGpp (Corrigan et al., 2015; Rao et al., 2010; Sureka et al., 2014). The DdacA suppressor mutations in relA clustered within or near the synthase domain of the RelA protein (Figure 1E). RelA (encoded by a homolog of the E. coli relA/spoT gene) synthesizes (p)ppGpp in response to starvation during the “stringent response” by transferring two phosphates from ATP to either GTP or GDP to produce pppGpp or ppGpp, respectively (collectively referred to as (p)ppGpp). In Firmicutes, RelA is also a hydrolase that degrades (p)ppGpp when nutrients are abundant (Mechold et al., 1996). The hydrolase and synthase enzymatic activities can be separated by point mutations in their respective domains (Hogg et al., 2004). The impact of the relAR295S suppressor mutation was interrogated by reconstructing the mutation in the chromosome of wild-type L. monocytogenes and measuring 32P-labeled intracellular nucleotides by thin-layer chromatography (TLC). These experiments were performed in low-phosphate defined medium supplemented with tryptone, which mimicked rich medium and stimulated uptake of added 32P (Taylor et al., 2002). Amino acid starvation was simulated using serine hydroxamate (SHX) and (p)ppGpp was quantified as a proportion of (p)ppGpp + GTP levels. Control experiments demonstrated that wild-type E. coli (CF1943) accumulated (p)ppGpp in response to starvation while E. coli carrying a disrupted relA gene (CF1944) did not (Figures 3A and 3B). Wild-type L. monocytogenes also accumulated (p)ppGpp in response to starvation; however, mutants expressing relAR295S did not (Figures 3A and 3C), supporting the supposition that this mutation disrupted RelA synthase activity.

Figure 3. ΔdacA Suppressor Mutations in relA Affect Starvation-Induced (p)ppGpp.

Figure 3

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TLC of 32P-labeled intracellular nucleotides from bacterial mutants. Bacteria were grown in low-phosphate defined medium plus tryptone, and where indicated, starvation was induced using SHX. Wild-type E. coli (CF1943) and relA251::kan E. coli (CF1944) are included as controls; all other strain are L. monocytogenes.

(A–C) A representative TLC is shown (A), and quantification of the ratio (pppGpp+ppGpp)/ (pppGpp+ppGpp+GTP) as a percent of wild-type is shown for E. coli (B) and L. monocytogenes (C). All data are representative of n = 10 independent experiments, graphed data are mean ± SEM of pooled data, *p ≤ 0.05 by two-tailed Students t test, and ns denotes not significant (p > 0.05).

In related Firmicutes, two proteins (RelP and RelQ) in addition to RelA are capable of synthesizing (p)ppGpp, although RelA is the only synthase predicted to respond to starvation (Nanamiya et al., 2008). The other two small alarmone synthases, identified here as relP (lmo0802) and relQ (lmo0967) based on their homology to B. subtilis, S. aureus, and Streptococcus mutans (Geiger et al., 2014; Lemos et al., 2007; Nanamiya et al., 2008), were likely responsible for the basal levels of (p)ppGpp observed in the untreated condition (Figures 3A and 3C). RelA is unique because it is the only identified (p)ppGpp hydrolase in L. monocytogenes. The hydrolase function of RelA was revealed by increased (p)ppGpp levels in a ΔrelA mutant as compared to wild-type (Figures 3A and 3C). The difference between the levels of (p)ppGpp in the untreated ΔrelA and relAR295S strains was therefore due to the functional hydrolase component of the Re-lAR295S protein, which can degrade (p)ppGpp synthesized by RelP and RelQ. These data demonstrated that the suppressor mutation in relA encoded a hydrolase-only form of the protein.

Accumulation of (p)ppGpp Is Toxic to ΔdacA Mutants

In Firmicutes, (p)ppGpp inhibits DNA primase and enzymes that catalyze GTP synthesis (Kriel et al., 2012; Wang et al., 2007). The net effect of increased (p)ppGpp is both a transcriptional and translational response that results in a decreased growth rate (Dalebroux and Swanson, 2012). We hypothesized that the relAR295S mutation suppressed dacA essentiality by decreasing (p)ppGpp that may have accumulated as a consequence of deletion of dacA. We tested the first part of this hypothesis by measuring (p)ppGpp under non-starvation conditions in a dacA conditional depletion strain (cΔdacA), which expressed dacA under the control of an IPTG inducible promoter (Witte et al., 2013). In comparison to wild-type, conditional depletion of dacA led to an increase in (p)ppGpp levels in non-starvation conditions (Figures 4A and 4B).

Figure 4. (p)ppGpp Accumulates during Depletion of c-di-AMP Leading to dacA Essentiality and Decreased Growth Rate.

Figure 4

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(A and B) TLC analysis of 32P-labeled intracellular nucleotides from bacteria grown in low-phosphate defined medium plus tryptone without starvation. Chromosomal dacA was deleted in a strain harboring an IPTG-inducible dacA gene to construct a conditional dacA depletion strain (cΔdacA) (Witte et al., 2013). A representative TLC is shown (A), and quantification of the ratio (pppGpp + ppGpp)/(pppGpp + ppGpp + GTP) is shown as a percent of wild-type (B). Data are representative of n = 11 independent experiments, graphed data are mean ± SEM of pooled data, *p ≤ 0.05 by two-tailed Students t test, and ns denotes not significant.

(C) dacA essentiality assay. Complemented genes indicated by (p-) were introduced at a neutral site using their native promoter. Dotted line indicates L.o.D.; data are mean ± SEM of at least three independent experiments.

(D and E) BHI broth growth curves, with or without (+/−) IPTG in (E). Data are representative of three independent experiments.

To further evaluate the role of increased (p)ppGpp in dacA essentiality, we constructed a L. monocytogenes strain lacking (p) ppGpp by sequentially deleting the relP, relQ, and relA genes (ΔrelAPQ) and subjected this strain to the dacA essentiality assay in BHI (Figure 2A). The dacA gene was no longer essential in the ΔrelAPQ background (Figure 4C). Complementation of ΔrelAPQ with any of the three (p)ppGpp synthases using their native promoters restored the essentiality of the dacA gene (Figure 4C). These data indicated that dacA was essential due to accumulation of the nucleotide (p)ppGpp rather than an interaction with any single (p)ppGpp synthase. Additionally, relA was sufficient to render dacA essential, which suggested that although RelA is a bifunctional synthase/hydrolase, in the absence of c-di-AMP, RelA functioned as a synthase.

The dacA gene was not essential in a ΔrelAPQ background, although the ΔdacAΔrelAPQ mutant grew slowly compared to wild-type (Figure 4D). These data established arole for (p)ppGpp in dacA essentiality. Additionally, we hypothesized that the accumulation of (p)ppGpp observed after depletion of dacA (Figures 4A and 4B) might be partially responsible for the growth defect of the cΔdacA strain (Witte et al., 2013). This hypothesis was tested by measuring growth rate of a conditional dacA depletion strain constructed in a wild-type or ΔrelAPQ background (cΔdacAΔrelAPQ) in BHI. In the presence of IPTG, the conditional dacA depletion strains cΔdacAΔrelAPQ and cΔdacA grew similarly to wild-type. In the absence of IPTG (when dacA is depleted), the cΔdacAΔrelAPQ strain displayed an increased growth rate compared to the cΔdacA strain (Figure 4E). These data are consistent with a role for c-di-AMP in maintaining low (p)ppGpp levels that are otherwise detrimental for growth.

A Screen for Mutations that Rescue the Virulence Defect of a ΔrelAPQ Mutant Reveals a Critical Role for CodY

We next sought to understand the function(s) of (p)ppGpp in L. monocytogenes. relA mutants are attenuated for pathogenesis (Bennett et al., 2007), although the role of (p)ppGpp in infection is still unclear, since our data indicated that relA mutants have elevated levels of (p)ppGpp (Figures 3A and 3C). The ΔrelAPQ mutant grew similarly to wild-type in rich medium, despite lacking all sources of (p)ppGpp (Figure 4D); however, it was severely attenuated in a plaque assay, an in vitro infection model that serves as a surrogate for virulence (Figure 5A). In this assay, confluent mammalian fibroblasts are infected with L. monocytogenes, and intracellular growth and cell-to-cell spread of the bacteria produce a zone of clearance (plaque) that is quantifiable and high-throughput (Sun et al., 1990). Small plaques often correlate to virulence defects in vivo, and the ΔrelAPQ strain produced small plaques that were 30% the area of wild-type plaques (Figure 5A).

Figure 5. A Screen for Suppressor Mutations of the ΔrelAPQ Virulence Defect Reveals a Critical Role for Inactivation of CodY.

Figure 5

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(A) A murine fibroblast (L2 cell) monolayer was infected with L. monocytogenes mutants from Table 1, producing plaques. The reduced plaque area correlates with reduction in virulence. Data are mean ± SEM of pooled data from least three independent experiments; the dotted line represents ΔrelAPQ plague area for reference; P preceding a gene name indicates the transposon is in the promoter; *p ≤ 0.05 by one-way ANOVA compared with ΔrelAPQ.

(B) Recovered CFU at 48 hr post-infection from CD-1 mice intravenously infected via tail vein with 105 CFU of each indicated strain. Data are pooled results from at least two independent experiments, bars indicate median value, p-relA represents relA complemented under its native promoter, and *p ≤ 0.05 using two-tailed Student’s t test.

These results suggested that (p)ppGpp was necessary for a productive infection. We speculated that the contribution of (p) ppGpp to virulence and toxicity to ΔdacA mutants were related and performed a transposon mutagenesis screen for mutations that rescued the small plaque phenotype of the ΔrelAPQ mutant. We identified 98 mutants from over 10,000-screened that displayed increased plaque size. DNA sequencing of the region adjacent to the transposon insertions and phage transduction led to the identification of 14 genes, that when disrupted, significantly increased the ΔrelAPQ plaque size (Figure 5A; Table 1).

Table 1.

Transposon Mutations that Suppress the ΔrelAPQ Plaque Defect

Plaque Area (%WT ± SEM)
Gene Namea Annotationb himar1 Locationc ΔrelAPQd ΔrelAPQ codY::spcd wild-typed
codY (lmo1280) GTP-responsive transcriptional regulator LMRG 00730::119 84 ± 2.3 N/A 89 ± 1.2
PspoVG (Plmo0196) Promoter of genes similar to spoVG LMRG 02618::-144 72 ± 1.6 89 ± 1.6 105 ± 1.6
guaB2 (lmo2758) Inosine-5’-monophosphate (IMP) dehydrogenase LMRG 01938::220 71 ± 1.0 77 ± 1.0 93 ± 1.4
lmo0808 Spermidine/putrescine ABC transporter LMRG_02789::1593 49 ± 0.9 83 ± 2.3 108 ± 1.6
lmo0948 GntR family transcriptional regulator LMRG _02047::494 45 ± 1.5 84 ± 1.2 111 ± 2.4
lmo0006/7 Between DNA Gyrase subunits B/A LMRG_02435::-26 44 ± 1.2 70 ± 1.1 99 ± 1.3
lmo0218 S1 RNA binding domain protein similar to yabR LMRG 02640::121 43 ± 1.2 72 ± 2.6 102 ± 2.7
lmo0767 Sugar ABC transporter permease LMRG 00455::222 42 ± 1.4 80 ± 0.9 100 ± 1.3
lmo1884 Xanthine uptake transporter similar to pbuX LMRG 01031::319 41 ± 0.7 77 ± 1.5 107 ± 2.9
Plmo0974 Promoter of D-alanine-poly(phosphoribitol) ligase
(dlt) operon		 LMRG _02073::-223 41 ± 1.1 77 ± 1.6 100 ± 1.9
purr (lmo0192) Purine associated transcriptional repressor LMRG 02614::471 39 ± 1.8 70 ± 1.5 93 ± 1.6
anrB (lmo2115) FtsX family ABC transporter permease associated
with nisin resistance		 LMRG_01269::1842 39 ± 1.1 80 ± 1.9 104 ± 2.4
rsbX
(lmo0896)		 Negative regulator of sigma-B (serine
phosphatase)		 LMRG _02320::2103 38 ± 1.5 59 ± 0.9 72 ± 1.2
lmo1843 RluA family 23S pseudouridylate synthase LMRG _00990::876 37 ± 0.9 80 ±1.1 96 ± 2.0
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a

Annotated using EGD-e ordered loci and previously published name where appropriate. P indicates the transposon location is within a predicted promoter of the annotated gene.

b

Gene similarity based on Bacillus subtilis genome annotation.

c

Sequence-mapped transposon insertion site (10403S ordered genetic locus::nucleotides 3′ of ORF start codon).

d

Genetic background of the transposon mutant; data represent the mean ± SEM for at least three independent experiments; bold-face numbers indicate plaque area was significantly different from background strain (p ≤ 0.05 by one-way ANOVA, Tukey test).

Transposon insertions in codY produced the most significant increase in the ΔrelAPQ plaque size (Figure 5A; Table 1). CodY is activated by high GTP levels and branch-chain amino acids (BCAAs) to promote DNA binding that transcriptionally represses a large regulon of genes, but also is capable of transcriptional activation of a few genes (Geiger and Wolz, 2014). In B. subtilis, (p)ppGpp inhibits GTP synthesis at multiple enzymatic steps and the subsequent decrease in GTP leads to CodY deactivation (Kriel et al., 2012). The ΔrelAPQ mutant is incapable of modulating GTP levels via (p)ppGpp, and thus CodY remains constitutively activated. In L. monocytogenes, codY mutations likely rescue the plaque defect of the ΔrelAPQ mutant by phenocopying (p)ppGpp-dependent inhibition of GTP synthesis that takes place in wild-type bacteria and demonstrates a critical role for CodY deactivation during infection. Other identified suppressor mutations (such as guaB2, lmo1884, and purR) in purine nucleotide synthesis/acquisition might have affected CodY activity by modulating intracellular GTP levels. We confirmed that these mutations were epistatic to mutations in codY by constructing a marked deletion in codY (codY::spc) and transducing the isolated transposons into the ΔrelAPQ codY::spc background (Table 1). Only mutations in the promoter of spoVG and lmo0948 further increased the ΔrelAPQ codY::spc plaque size. However, lmo0948::himar1 also increased the plaque area of wild-type and thus was likely not specific to (p)ppGpp (Table 1). Although spoVG is regulated by (p)ppGpp in B. subtilis (Tagami et al., 2012) and has been identified with divergent phenotypes in multiple organisms (Jutras et al., 2013; Matsuno and Sonenshein, 1999; Meier et al., 2007), it is unclear how this mutation contributed to the virulence of the ΔrelAPQ mutant.

In a mouse model of infection, the ΔrelAPQ mutant was approximately 100-fold less virulent compared to wild-type or the ΔrelAPQ strain complemented with relA under its native promoter (Figure 5B). The codY::spc mutation suppressed the virulence defect of the ΔrelAPQ strain to the level of a codY::spc mutation alone, approximately 10-fold less virulent than wild-type (Figure 5B). The virulence defect of the codY::spc mutant is consistent with previous reports demonstrating a virulence defect for a codY mutant (Lobel et al., 2014; 2012), and mutations in codY suppress the virulence defect of other pathogenic Firmicutes with decreased (p)ppGpp (Geiger and Wolz, 2014). Our data suggested that the principle role of (p)ppGpp during infection was the inhibition of GTP synthesis leading to inactivation of CodY.

(p)ppGpp-Dependent Inactivation of CodY Is Necessary for the Essentiality of dacA

We speculated that the function of (p)ppGpp during infection overlaps with the role of (p)ppGpp in dacA essentiality. Accordingly, dacA may not be essential in the ΔrelAPQ mutant, because in the absence of (p)ppGpp, GTP remains elevated, and CodY is highly active. We examined the role of CodY in the essentiality of dacA by comparing the ΔrelAPQ and ΔrelAPQ codY::spc mutants in the genetic assay for dacA essentiality (Figure 2A). While dacA was not essential in the ΔrelAPQ mutant, addition of a codY mutation returned dacA to its original essential phenotype (Figure 6A). Addition of the spoVG mutation to the ΔrelAPQ mutant strain did not alter dacA essentiality (Figure 6A). These results suggested that among the diverse functions of (p)ppGpp, inactivation of GTP synthesis and thus inactivation of CodY was selectively toxic to ΔdacA mutants. Further, these findings imply that elements of the CodY regulon, which are necessary for infection, may be toxic in the absence of c-di-AMP.

Figure 6. dacA Is Essential in Rich Medium Due to CodY Inactivation but dacA Is Not Essential in Minimal Medium.

Figure 6

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(A and B) dacA essentiality assay.

(B) The commonly used rich medium is BHI, and minimal medium for L. monocytogenes is detailed in the Supplemental Experimental Procedures. Dotted line indicates L.o.D.; data are mean ± SEM of at least three independent experiments.

dacA Is Not Essential in Minimal Medium

We hypothesized that dacA might no longer be essential in growth conditions that favored inactivation of CodY. The best example of such a condition is in minimal medium, where a B. subtilis strain unable to produce (p)ppGpp cannot grow without a codY mutation (Kriel et al., 2012; 2014). Similarly in L. monocytogenes, the ΔrelAPQ mutant does not grow on minimal medium (data not shown), prompting us to examine these growth conditions. Unlike rich medium, dacA was no longer essential in a defined minimal medium (Figure 6B) (Phan-Thanh and Gormon, 1997). Remarkably, in-frame ΔdacA deletions were readily obtainable by allelic exchange when bacteria were cultivated in minimal medium. Genome sequencing of ΔdacA mutants constructed on minimal medium using a marked dacA deletion confirmed the absence of suppressor mutations. These data suggested a model in which c-di-AMP is essential for growth in rich medium, because in the absence of c-di-AMP, (p)ppGpp accumulated and indirectly inactivated CodY, which facilitated transcriptional changes selectively toxic to ΔdacA mutants. This work identified that mutations that decreased (p) ppGpp or replacement with a medium favoring CodY inactivation were sufficient to reverse the essentiality of dacA.

DISCUSSION

There is an emerging consensus that c-di-AMP is an essential molecule in Firmicutes (Corrigan and Gründling, 2013). However, here we report the isolation of L. monocytogenes dacA deletion mutants that lack c-di-AMP. As conventional approaches of isolating mutations were unsuccessful, a strain was constructed in which loxP sites were introduced into the L. monocytogenes chromosome flanking the dacA gene, and Cre recombinase was expressed from the PrfA-regulated actA promoter that is induced in host cells (Reniere et al., 2015). Upon infection of macrophages, Cre was expressed leading to deletion of dacA, thereby providing an unbiased selection for L. monocytogenes mutants able to grow in the absence of the c-di-AMP. All DdacA mutants isolated from the infected cells contained one or more suppressor mutations that promoted bacterial growth in rich medium. Mutations in the synthase domain of the bi-functional RelA/SpoT homologrelA that retained (p)ppGpp hydrolase activity or deletion of all three L. monocytogenes (p)ppGpp synthase genes (ΔrelAPQ) suppressed dacA essentiality. Although the growth defect of the ΔdacAΔrelAPQ strain implies additional roles for c-di-AMP in bacterial physiology, these data suggested that ΔdacA mutants failed to grow in rich medium and in cells because (p)ppGpp levels were elevated, a condition known to inhibit bacterial growth (Dalebroux and Swanson, 2012).

We reasoned that there might be an overlap between the (p) ppGpp-regulated genes required for pathogenesis and those that suppressed dacA essentiality. Therefore, we screened for transposon mutations that suppressed the plaque defect of a strain lacking (p)ppGpp (ΔrelAPQ), which was nearly 100-fold less virulent in mice. Fourteen loci were identified and transposon mutations disrupting codY produced the largest plaque. Mutations in codY restored the virulence of L. monocytogenes strains lacking (p)ppGpp to that of the codY mutant alone. CodY is a GTP-responsive transcriptional regulator whose function is inextricably linked with (p)ppGpp levels (Geiger and Wolz, 2014). During exponential growth in rich medium, CodY is GTP bound and represses dozens of biosynthetic operons (Sonenshein, 2007). CodY also enhances transcription of genes involved in GTP synthesis, most notably guaB, causing a feedforward regulatory loop that maintains CodY activation (Bennett et al., 2007). During starvation, (p)ppGpp interrupts this feed-forward loop through allosteric inhibition of GTP-synthesis enzymes, thereby allowing for the expression of many biosynthetic operons (Kriel et al., 2012). Other mutations that suppressed virulence defects of the ΔrelAPQ mutant, such as guaB2, lmo1884, and purR, were epistatic to codY because they likely recapitulate the role of (p)ppGpp, decreasing GTP abundance and therefore the activity of CodY. Most importantly however, the codY mutation restored the essentiality of dacA in a ΔrelAPQ mutant. Therefore, c-di-AMP essentiality is likely caused by one or more CodY-regulated genes that are inappropriately expressed in rich medium due to elevated levels of (p)ppGpp. If this were correct, one would predict that dacA might not be essential in conditions favoring expression of CodY-regulated genes, such as minimal medium where CodY-repressed genes are essential for growth (Kriel et al., 2014). Indeed, we were able to construct supressorless ΔdacA mutants in minimal medium using conventional methods of allelic exchange.

It is not clear why c-di-AMP is essential in rich but not minimal media. However, c-di-AMP is an allosteric inhibitor of L. monocytogenes PycA, an enzyme that catalyzes the conversion of pyruvate to oxaloacetate and entry of carbon into the TCA cycle and is essential for growth in minimal medium (Schär et al., 2010; Sureka et al., 2014). Depletion of c-di-AMP leads to overactivity of PycA and thus increased levels of TCA cycle intermediates, such as glutamate/glutamine, primarily because L. monocytogenes has an incomplete TCA cycle and lacks α-ketoglutarate dehydrogenase. Consequently, mutations in citrate synthase (citZ), the first step of the TCA cycle, relieve the buildup of glutamate/glutamine and suppress the virulence defect of a conditional dacA depletion strain (Sureka et al., 2014). Interestingly, among our ΔdacA suppressor mutants that contained multiple mutations, we identified missense and promoter mutations in pycA and a premature stop codon in citZ, suggesting that mutations which lower potentially toxic concentrations of glutamate/glutamine counter dacA essentiality. Although it is not clear how increased glutamate/glutamine levels might result in toxicity, it may be noteworthy that in E. coli, glutamate functions as the principle counterion to K+ (McLaggan et al., 1990; 1994), which is an indispensible cation for balancing osmotic stress in bacteria (Epstein, 2003). In S. aureus, three of the four identified c-di-AMP-binding proteins modulate intracellular potassium levels (Corrigan et al., 2013), and in a diverse set of organisms, the c-di-AMP-binding ydaO-yuaA riboswitch regulates potassium transporters and osmoprotection genes (Nelson et al., 2013). We hypothesize that bacteria lacking c-di-AMP accumulate both K+ and glutamate and are unable to regulate a subsequent lethal change in internal osmotic pressure. In support of this, we identified mutations in the glycine betaine/proline osmoprotection transporter in some of our ΔdacA suppressors mutants that contained multiple mutations, although we were unable to rescue ΔdacA essentiality by adding osmoprotectants or altering salt concentrations (data not shown). In minimal medium, dacA may not be essential, because glutamate is required for synthesis of many additional metabolites made under nutrient stress (Sonenshein, 2007). In addition, CodY overactivation (in the absence of (p)ppGpp) could remedy dacA essentiality by repressing glutamate synthase or altering L. monocytogenes metabolism to provide decreased PycA precursors (Brinsmade et al., 2014; Sonenshein, 2007).

The results of this study suggest that there is a signaling loop between (p)ppGpp and c-di-AMP, which is not surprising since c-di-AMP-specific phosphodiesterases are inhibited by (p)ppGpp (Corrigan et al., 2015; Huynh et al., 2015; Rao et al., 2010). What is surprising is that deletion of c-di-AMP phosphodiesterases pgpH and gdpP in L. monocytogenes and S. aureus, respectively, led to increased (p)ppGpp during stress despite containing elevated levels of c-di-AMP (Corrigan et al., 2015; Liu et al., 2006), the opposite of the phenotype predicted by work presented here. We hypothesize that both high and low c-di-AMP can contribute to increased (p)ppGpp by altering central metabolism and amino acid biosynthesis, specifically the levels of the BCAAs valine, leucine, and isoleucine. (p)ppGpp production is stimulated by low BCAA levels, which are uniquely poised as sensors of nutrient stress because they require precursors from carbon, nitrogen, and sulfur metabolism for their synthesis (Somerville and Proctor, 2009). Valine and leucine are derived from pyruvate, whereas isoleucine is derived from oxaloacetate (Sonenshein, 2007). In L. monocytogenes, c-di-AMP may affect the abundance of these precursors by regulating PycA activity (Schär et al., 2010; Sureka et al., 2014). When over-active (low c-di-AMP), PycA activity leads to pyruvate depletion and potentially low levels of valine and leucine. When underactive (high c-di-AMP), the bacterium is depleted of oxaloacetate. This hypothesis is consistent with the demonstrated toxicity of excess c-di-AMP (Huynh et al., 2015; Mehne et al., 2013) and places c-di-AMP as a key regulator of metabolic homeostasis.

Both c-di-AMP and (p)ppGpp contribute to bacterial stress responses; for example, in related Firmicutes, mutations in relP and relQ increase antibiotic sensitivity while mutations in c-di-AMP-dependent phosphodiesterases lead to increased antibiotic resistance (Abranches et al., 2009; Corrigan and Gründling, 2013; Geiger et al., 2014). Importantly, there are fundamental differences between (p)ppGpp and c-di-AMP: whereas the former is only made during acute stress, the latter appears to be present during all growth conditions (Corrigan and Gründling, 2013). Even in minimal medium, where c-di-AMP is not essential, DdacA mutants grew slowly compared to wild-type (data not shown). Another fundamental difference is that c-di-AMP is secreted by L. monocytogenes during growth in media and in cells, perhaps altering intracellular nucleotide concentrations or regulating extracellular processes (Woodward et al., 2010). There is also evidence that Chlamydia trachomatis and Mycobacterium tuberculosis are capable of secreting c-di-AMP during infection (Barker et al., 2013; Yang et al., 2014). Collectively, these properties make c-di-AMP an ideal pathogen-associated molecular pattern (PAMP); i.e., it is highly expressed, conserved, essential for virulence, and secreted, thereby triggering STING a central hub of host innate immunity (Danilchanka and Mekalanos, 2013; Vance et al., 2009).

EXPERIMENTAL PROCEDURES

Generation of ΔdacA Suppressor Mutants by Cre-lox

The dacAfl PactA-cre strain was grown at 30°C overnight without agitation, and bone-marrow-derived macrophages (BMDMs) were infected as previously described using gentamicin to kill extracellular bacteria (Witte et al., 2013). After infection, bacteria were grown intracellularly for 4 hr to allow for adequate cytosolic access and actA induction. Infected BMDMs were then washed three times with sterile PBS, lysed with 0.1% NP-40, and plated on media agar at 37°C, curing the cre-containing plasmid. ΔdacA mutants were verified by PCR using primers internal to dacA and primers external to the dacA locus. In some cases, BMDM lysates were initially plated at 30°C on selective BHI-agar to enrich for bacteria that retained the PactA-cre plasmid, prior to plasmid-curing at 37°C. For generating 284 additional ΔdacA suppressor mutants, 24 independent infections with dacAfl PactA-cre were used, dacA deletion was confirmed by PCR, bacteria were grown in minimal medium with bialaphos to analyze Opp activity, the synthase domain of relA was sequenced with primers relA-syn-F/R, and Opp activity was reanalyzed by disk diffusion.

dacA Essentiality Assay

The dacA essentiality assay was performed by adapting previously described methods (De Las Peñas et al., 1997). Three transducing lysates were constructed from dacAfl-kanR lmo2103/2104::himar1 (dacA lysate) and three transducing lysates were constructed from ΔdacA-kanR lmo2103/2104:: himar1 (ΔdacA lysate) produced by Cre-lox deletion of dacA. dacA essentiality in a recipient strain was analyzed by transducing with each of the six lysates and selecting for erythromycin-resistant (ermR) transductants on the indicated media. At 48 hr, 50 transductants were patched from each transduction onto appropriate media agar containing either erythromycin or kanamycin. The proportion of kanamycin resistant colonies is a measure of genetic linkage, and one out of 50 colonies defined the L.o.D. at 2%. As a control, the essentiality assay was also performed with a lmo2110::himar1 transposon insertion instead of lmo2103/2104::himar1. The mean genetic linkage from the three transducing lysates per genotype constituted one experiment; data represent the mean ± SEM for at least three independent experiments. For wild-type merodiploid for dacA, an IPTG-inducible dacA was introduced into wild-type, and the experiment was performed in the presence of IPTG.

Virulence Analysis

In vivo virulence analysis was performed as previously described (Reniere et al., 2015) with the following changes: female, 8- to 12-week-old CD-1 mice (Charles River) were injected via tail-vein with 200 µl of PBS containing 105 CFU of L. monocytogenes. Mice were euthanized 48hr post-infection, liver and spleen removed, organs homogenized in filter-sterilized 0.1% NP40, and the CFU of the liver and spleen enumerated by plating serial dilutions on LB-Agar containing streptomycin. Statistical significance was determined by a two-tailed heteroscedastic Student’s t test. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All protocols were reviewed and approved by the Animal Care and Use Committee at the University of California, Berkeley (MAUP# R235–0815B).

For detailed explanations of bacterial strains and culture conditions, DNA manipulations and strain construction, genome sequencing, disk diffusions, phage transduction, (p)ppGpp quantification, plaque assay, ΔrelAPQ virulence suppressor screen, a table of L. monocytogenes strains, a table of plasmids and E. coli strains, and a table of oligonucleotides used in this study, please see Supplemental Experimental Procedures.

Supplementary Material

Suppl 1
Suppl 2

Highlights.

  • c-di-AMP is predicted to be essential in many pathogens and is thus an ideal PAMP

  • ΔdacA mutants lacking c-di-AMP contain suppressor mutations within a (p)ppGpp synthase

  • Decreased c-di-AMP increases (p)ppGpp, which inhibits growth via inactivation of CodY

  • In minimal medium, which favors CodY inactivation, dacA is no longer essential

ACKNOWLEDGMENTS

The authors would like to thank Joshua Woodward (University of Washington) for helpful advice, Peter Lauer and Bill Hanson (Aduro BioTech) for sharing genetic tools, Michael Cashel (National Institutes of Health) for generously providing E. coli strains, Michelle Reniere for helpful discussions and critical reading of the manuscript, and Regina Matthew for technical assistance. This work used the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH S10 Instrumentation Grants S10RR029668 and S10RR027303. This work was supported by National Institutes of Health grants 1P01 AI063302 and 1R01 AI27655 to D.A.P.; A.T.W. is supported by the NSF GRFP DGE 1106400 and the UC Berkeley Center for Emerging and Neglected Diseases Irving H. Wiesenfeld Graduate Fellowship. D.A.P. has a consulting relationship with and a financial interest in Aduro BioTech, Inc., and both he and the company stand to benefit from the commercialization of the results of this research.

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information includes one figure, one table, and Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j.chom.2015.05.006

AUTHOR CONTRIBUTIONS

A.T.W. and A.J.P. performed experiments; A.T.W. and D.A.P. designed the study and wrote the manuscript; all authors participated in discussing results and commented on the manuscript.

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