The Metallophore Staphylopine Enables Staphylococcus aureus To Compete with the Host for Zinc and Overcome Nutritional Immunity

ABSTRACT During infection, the host sequesters essential nutrients, such as zinc, to combat invading microbes. Despite the ability of the immune effector protein calprotectin to bind zinc with subpicomolar affinity, Staphylococcus aureus is able to successfully compete with the host for zinc. However, the zinc importers expressed by S. aureus remain unknown. Our investigations have revealed that S. aureus possesses two importers, AdcABC and CntABCDF, which are induced in response to zinc limitation. While AdcABC is similar to known zinc importers in other bacteria, CntABCDF has not previously been associated with zinc acquisition. Concurrent loss of the two systems severely impairs the ability of S. aureus to obtain zinc and grow in zinc-limited environments. Further investigations revealed that the Cnt system is responsible for the ability of S. aureus to compete with calprotectin for zinc in culture and contributes to acquisition of zinc during infection. The cnt locus also enables S. aureus to produce the broad-spectrum metallophore staphylopine. Similarly to the Cnt transporter, loss of staphylopine severely impairs the ability of S. aureus to resist host-imposed zinc starvation, both in culture and during infection. Further investigations revealed that together staphylopine and the Cnt importer function analogously to siderophore-based iron acquisition systems in order to facilitate zinc acquisition by S. aureus. Analogous systems are found in a broad range of Gram-positive and Gram-negative bacterial pathogens, suggesting that this new type of zinc importer broadly contributes to the ability of bacteria to cause infection.

ability of A. baumannii, S. Typhimurium, and P. aeruginosa to resist CP-imposed Zn limitation (17,23,24,35). The ability of S. aureus to compete with CP for Zn is presumably mediated by the expression of high-affinity Zn importers. However, the identity of the Zn importers expressed by S. aureus remains unknown.
Given the ability of S. aureus to compete with the host for Zn, we sought to elucidate the identity of the staphylococcal Zn importers. We show that S. aureus obtains Zn using an AdcABC permease and an additional importer, CntABCDF, which has not previously been associated with Zn uptake. Analysis of CntABCDF, a member of the Opp/NikA family of ABC transporters, shows that it functions in conjunction with the metallophore staphylopine (StP). Further analyses of this system showed that while StP can bind a broad range of first-row transition metals, S. aureus employs it as a zincophore. Analysis of the respective roles of the two Zn acquisition systems in overcoming nutritional immunity revealed that the Cnt-StP system, but not AdcABC, is responsible for the ability of S. aureus to compete with CP for Zn and serves as the major uptake pathway used by the bacterium to obtain this metal during infection. Further analysis revealed that similar zincophore systems are present in a diverse collection of pathogens. Collectively, these findings significantly expand our understanding of how bacteria obtain this essential nutrient and compete with the host for Zn during infection.

RESULTS
S. aureus utilizes an uncharacterized import pathway to obtain zinc. Examination of the staphylococcal genome identified a single putative Zn-associated ABC permease (NWMN_2306, 1458, and 1459). The transporter was designated AdcABC due to its similarity to other Gram-positive Zn-specific ABC permeases. Bioinformatic analysis of AdcA (NWMN_2306), the metal ion-recruiting component of the permease, revealed that it is comprised of two domains. The N-terminal domain (amino acids 1 to 322) belonged to the cluster A-I subgroup of solute binding proteins (SBPs), which have specific roles in Mn, Fe, and Zn recruitment (36). The C-terminal domain (amino acids 323 to 531) showed high similarity to ZinT, a periplasmic metallochaperone present in some Gram-negative bacteria (37). This SBP composition is unique to the Adc permeases of Gram-positive bacteria such as S. pneumoniae (38). The ABC transporter AdcBC is comprised of a transmembrane component, AdcB (NWMN_1458), and a nucleotide-binding domain, AdcC (NWMN_1459). Unusually, the gene encoding S. aureus AdcA was not clustered with the ABC transporter.
Initially, we examined the role of the putative AdcABC permease in Zn acquisition by assessing its transcriptional response to transition metal limitation. Transcription of adcA was increased in Zn-depleted medium (Fig. 1A) and in a strain in which the Zn-responsive repressor Zur had been deleted (Fig. 1B) (39). In contrast, no change in adcA transcription was observed in Mn-or Fe-depleted medium (Fig. 1A) or in strains that lacked the Mn-responsive regulator MntR or the Fe-responsive regulator Fur (Fig. 1B) (40,41). Taken together, the S. aureus AdcABC system shows structural and transcriptional features consistent with a bacterial Zn importer (17,(42)(43)(44). We then investigated the phenotypic impact of metal restriction on an S. aureus strain that lacks AdcA (the ⌬adcA mutant). Limiting Mn and Fe availability had no impact on the growth of the ⌬adcA mutant (Fig. 1C). Surprisingly, Zn-depleted medium also had no effect on the growth of the ⌬adcA mutant despite the apparent lack of other Zn import pathways in the staphylococcal genome ( Fig. 1C and S1A). Collectively, these findings suggested that in addition to the AdcABC permease, S. aureus possesses an import pathway for Zn that is distinct from previously described mechanisms for obtaining this metal.
A new class of zinc importers facilitates zinc uptake by S. aureus. In Zn-limited medium, in addition to AdcABC, S. aureus expresses the Opp/NikA family transporter CntABCDF (45). However, in these studies loss of CntA did not result in a Zn uptake defect. Loss of CntA did reduce the ability of S. aureus to transport Co and Ni. These results led to the suggestion that Co and Ni are the physiological substrates of the system (45,46). Building on our observation that the AdcABC permease is not the sole Zn import pathway, we reevaluated the CntABCDF permease and its potential contribution to Zn import. Consistent with prior results (45), the cnt locus was induced in response to Zn limitation (Fig. 1A). Notably, addition of Co and Ni had no impact on cnt transcription ( Fig. S1B and C). Removal of Fe or Mn from the growth medium also resulted in a modest increase in expression (Fig. 1A). When Zn, Mn, and Fe were omitted from the medium, expression of the system increased by 21-fold, compared to 4.6-fold in medium that lacked only Zn (Fig. 1A). To explore the regulation of this system further, cnt transcription in strains lacking Zur, Fur, and MntR was assessed. Loss of Zur and Fur, but not MntR, resulted in increased expression of the cnt locus (Fig. 1B). Together, these results show that while the expression of Cnt is responsive to the We then evaluated the contribution of the Cnt system to Zn acquisition. To accomplish this goal, we generated a strain lacking CntA (the ΔcntA mutant). We observed that, consistent with prior studies (45,46), there was no discernible growth defect of the ΔcntA mutant in Zn-depleted medium ( Fig. 1C and S1A). Given the potential for overlapping function with the AdcABC system, we assessed the growth of a strain lacking both potential Zn import pathways (the ΔadcA ΔcntA mutant). Growth of the double mutant in medium lacking Zn, Fe, and Mn was severely compromised ( Fig. 1C and S1C). Supplementation with either Mn, Fe, Ni, or Co failed to reverse the growth defect of the ΔadcA ΔcntA mutant ( Fig. 1C and S1D). Notably, addition of either Mn or Fe further reduced the growth of the ΔadcA ΔcntA mutant. In contrast, supplementation with Zn restored the growth of the ΔadcA ΔcntA mutant to wild-type levels. Ectopic expression of either adcA or cntA also reversed the growth defect of the ΔadcA ΔcntA mutant in Zn-depleted medium ( Fig. 1D and E, and Fig. S1E and F). We also observed that in metal-replete medium, loss of AdcA resulted in increased expression of the cnt locus while loss of CntA did not result in increased expression of adcA ( Fig. S1G and H). This result suggests that AdcA is the first system used by S. aureus to obtain Zn, with the Cnt system being induced when the bacterium cannot meet the cellular demand for this metal using the Adc system. Collectively, these results show that both AdcA and CntA contribute to the ability of S. aureus to grow in Zn-limited environments.
Whole-cell metal accumulation was then assessed to directly determine the contribution of Adc and Cnt pathways to S. aureus metal uptake. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of wild-type S. aureus and ΔadcA, ΔcntA, and ΔadcA ΔcntA mutants revealed a significant decrease in Zn accumulation by ΔadcA and ΔadcA ΔcntA strains ( Fig. 2A). A modest reduction in Mn accumulation was also observed in the double mutant (Fig. 2B). However, this small reduction is unlikely to be contributing to the observed growth phenotype of the double mutant as the addition of Mn further exacerbated the growth defect of the ΔadcA ΔcntA strain (Fig. 1C). No change in the accumulation of Fe, Co, Ni, or Cu was observed with any of the mutant strains ( Fig. 2C to F). Collectively, these results directly show that AdcABC and CntAB-CDF contribute to S. aureus Zn import. Further, CntABCDF, which has not been previously associated with Zn acquisition, does not functionally contribute to the uptake of metal ions other than Zn in S. aureus.
Zinc acquisition by the Cnt transporter facilitates competition with calprotectin. We then sought to delineate the relative contribution of the two systems to resisting CP-imposed Zn starvation. Initially, we assessed how CP influenced transcription of the two Zn uptake systems. In response to CP, both systems were significantly upregulated (Fig. 3A). To assess the relative contributions of each system to Zn acquisition in the presence of CP, we then evaluated the growth of ΔadcA, ΔcntA, and ΔadcA ΔcntA mutants in the presence of CP. Consistent with our earlier observations, the ΔadcA ΔcntA mutant showed greater sensitivity to CP than did wild-type S. aureus at all concentrations tested (Fig. 3B). Loss of CntA diminished the ability of S. aureus to grow relative to the wild type when CP concentrations exceeded 120 g/ml. In contrast, the ΔadcA mutant was no more sensitive to CP than the wild type (Fig. 3B). Ectopic expression of cntABCDF in ΔcntA and ΔadcA ΔcntA mutants restored wild-type growth in the presence of CP (Fig. 3C). Plasmid-mediated expression of adcA in the ΔadcA ΔcntA mutants permitted growth similar to that of the ΔcntA mutant (Fig. 3D). Additionally, loss of any of the genes associated with the Cnt importer in the methicillin-resistant strain USA300 (JE2) increased the sensitivity of S. aureus to CP (Fig. S2A). To determine if the Cnt system was important for resisting host-imposed Mn and/or Zn starvation, we used CP variants that lack either the Zn site (ΔZn, which binds both Mn and Zn) or the Mn/Zn site (ΔMn/Zn, which binds only Zn) (20). Expression of both the adcA and the cnt loci was induced in the presence of either CP site mutant (Fig. 3E). Both ΔcntA and ΔadcA ΔcntA mutants were sensitive to both the ΔZn and the ΔMn/Zn CP binding site mutants, as both can bind Zn (Fig. 3F), indicating that the increased sensitivity of these strains is due to a reduced ability to compete for Zn. Unexpectedly, we observed that the ΔadcA mutant is more sensitive than wild-type S. aureus to the ΔMn/Zn site mutant. As the ΔadcA mutant is not more sensitive to wild-type CP or ΔZn CP, this result suggests that the Cnt system is a less effective Zn importer when other metals, which could block Zn transport, are freely available, as would occur in the presence of the ΔMn/Zn site mutant, which lacks the ability to restrict Mn availability (Fig. 3F). In summary, these data demonstrate that the Cnt system contributes more to resisting CP-mediated Zn restriction than AdcABC.
Staphylopine functions as a zincophore enabling S. aureus to compete with CP for Zn. Distinct from the direct metal ion recruitment mechanisms of the cluster A-I SBPs, the cluster C SBPs, employed by Opp/NikA ABC permeases, bind metal chelates in a process analogous to siderophore-iron acquisition systems. The cnt locus also encodes CntKLM and CntE, which produce and secrete, respectively, the broadspectrum metallophore staphylopine (StP). Extracellular StP can then be imported by the CntABCDF transporter (46). Loss of StP, similar to that of CntA (Fig. 1C) (46), does not inhibit the growth of S. aureus in medium that has had the metal content reduced using conventional approaches (Fig. 4A) (46). Our results suggest that the presence of the Adc permease may have obscured a role for StP in Zn uptake. However, the promiscuity of Opp/NikA-type transporters (47-49) raises the possibility that StP may Grim et al.
® not be the metallophore involved in Zn acquisition. We sought to address this by evaluating the ability of an ΔadcA ΔcntKLM mutant to grow in Zn-depleted medium. Similarly to the ΔadcA ΔcntA strain, this mutant was severely attenuated for growth in Zn-restricted medium and profoundly more sensitive to CP ( Fig. 4A and B). The addition of Zn, but not Fe or Mn, reversed the growth defect of the ΔadcA ΔcntKLM mutant (Fig. 4A). The growth defect of the ΔadcA ΔcntKLM mutant was also reversed by ectopic expression of adcA or cntKLM (Fig. S3). Similarly to the ΔcntA mutant, the ΔcntKLM mutant was also more sensitive to CP (Fig. 4B). This sensitivity was abolished by expression of cntKLM from a plasmid in the ΔcntKLM mutant (Fig. S3D). Similar results were also observed with USA300 (JE2) (Fig. S2B). The ΔcntKLM and ΔadcA ΔcntKLM mutants were also more sensitive than wild-type S. aureus to both CP variants (ΔZn CP Staphylopine-Mediated Zinc Acquisition ® and ΔMn/Zn CP), indicating that the observed growth defect is attributable to impaired Zn acquisition (Fig. 4C). Collectively, these results show that StP contributes to the ability of S. aureus to overcome host-imposed Zn starvation.
Next, we evaluated if StP is promoting growth in Zn-restricted environments by functioning as a zincophore. If StP is a zincophore, it should function in trans and be dependent on the ABC permease CntABCDF. To evaluate this possibility, supernatants harvested from Zn-starved wild-type S. aureus and ΔcntA and ΔcntKLM mutants were tested for their ability to rescue the growth of various staphylococcal mutants. The supernatants harvested from wild-type S. aureus and the ΔcntA mutant rescued the growth of both ΔcntKLM and ⌬adcA ΔcntKLM strains but not strains lacking the SBP CntA ( Fig. 4D and S4). These data indicate that CntABCDF and StP function together to promote resistance to CP-mediated Zn starvation. Supernatant harvested from a strain deficient in StP synthesis, the ΔcntKLM mutant, was unable to rescue the growth of any strain tested (Fig. 4D and S4). Collectively, these results demonstrate that StP functions as a zincophore, enhancing the ability of S. aureus to compete with CP for Zn.
The Cnt-StP system is the dominant Zn importer utilized during systemic infection. To evaluate the contributions of AdcABC, CntABCDF, and StP to staphylococcal infection, a systemic retroorbital infection model was used. For these studies, C57BL/6 mice were infected with either wild-type S. aureus or the ΔadcA, ΔcntA, ΔcntKLM, ΔadcA ΔcntA, or ΔadcA ΔcntKLM mutant (Fig. 5A to D). Mice infected with the double mutants lost significantly less weight than those infected with wild-type S. aureus or the single mutants (Fig. 5A). Consistent with the CP growth assays, mice infected with the ΔadcA strain had bacterial burdens comparable to those infected with the wild type. Compared to the wild type, the ΔcntA mutant showed a significant reduction in bacterial burden in the heart, while the ΔcntKLM mutant had a reduced burden in the liver. Both ΔadcA ΔcntA and ΔadcA ΔcntKLM mutants had reduced bacterial burdens in the liver, heart, and kidneys ( Fig. 5B to D). Together, these results indicate that while both Zn import pathways can contribute to the ability of S. aureus to cause disease, the Cnt-StP system is sufficient to facilitate Zn acquisition during infection.
Staphylopine analogs are widely distributed in bacteria. Having established a role for StP in the pathogenesis of S. aureus, we then investigated the distribution of the StP synthesis locus using genome neighborhood network (GNN) analysis. StP is produced by CntK, a histidine racemase; CntL, an enzyme with low similarity to nicotianamine synthase; and CntM, which attaches a pyruvate moiety to the StP precursor. CntM, the only member of a Pfam/InterPro protein family, was used to anchor the analysis ( Fig. 6 and Table S1). The CntM InterPro family (IPR016935, 184 nonredundant, nonobsolete, queriable members) contains~90 unique species. Using this data set, 92% analysis of the latter group revealed that 70% of the sequences shared a high degree of similarity (50% sequence identity or greater) with CntL from S. aureus. Surprisingly, CntK, CntL homologs, and the methyltransferase_31 Pfam family are largely restricted to the Firmicutes. Predicted importer and efflux system were associated with all of the synthesis clusters. While there was variability in the importer associated with the synthesis loci, the putative efflux pump belonged to either the major facilitator superfamily (MFS) or the EamA family of transporters. Notably, the MFS-containing loci were primarily associated with CntABCDF importer homologs and divergent staphylopine synthesis machinery, while the EamA loci were associated with a variety of potential importers but contained conserved core synthesis machinery. Additionally, for at least 20 of the genomes analyzed, additional enzymes were encoded proximal to cntLM that may result in the production of modified StP-like molecules. Collectively, these observations suggest that a diverse collection of StP analogs is present in a variety of Gram-positive and Gram-negative organisms.

DISCUSSION
During infection, nutritional immunity severely restricts the bioavailability of the essential nutrient Zn (8,12). Despite this challenge, successful pathogens, such as S. aureus, remain capable of causing devastating disease. The success of S. aureus and other invaders is mediated by an ability to compete with the host for Zn (13,20). Our  Table S1 in the supplemental material for a complete list of the loci identified. Abbreviations: NBD, nucleotide-binding domain; TMD, transmembrane domain; DH, dehydrogenase; NAS, nicotianamine synthase.

Grim et al.
® work shows that S. aureus possesses two distinct types of ABC permeases, AdcABC and CntABCDF, involved in Zn acquisition. AdcABC is homologous to ABC permeases associated with direct recruitment of Zn. CntABCDF belongs to the NikA/Opp family of ABC permeases, which have not previously been associated with Zn acquisition. Our work revealed that CntABCDF functions in conjunction with the recently identified broad-spectrum metallophore StP to specifically promote Zn acquisition. These results indicate that, although StP can bind a variety of metals in vitro, it functions as a staphylococcal zincophore. Collectively, our findings conclusively establish the existence of a new class of bacterial Zn ABC importers.
Many bacteria, including S. aureus, contain an array of distinct Fe and Mn acquisition systems. Due to their overlapping functions, the disruption of multiple metal transporters is frequently required to observe a phenotype (12,(50)(51)(52)(53). The presence of multiple Zn uptake pathways in bacteria is also well established, but typically these transporters all belong to the Znu/Adc ABC permease family (54,55). Loss of CntA or StP does not impair the ability of S. aureus to grow or to obtain Zn in medium rendered Zn depleted using conventional approaches. Despite the transcriptional responsiveness of the cnt locus to Zn abundance, this led to the conclusion that the Cnt-StP system was not a Zn importer (45,46). At the time that the studies were conducted, there was a paucity of data on the Zn acquisition systems possessed by S. aureus. Identification of the AdcABC Zn importer in S. aureus suggests that the prior lack of a Zn-associated phenotype in CntA and StP single mutants is due to overlapping functions. The observations that strains lacking both the Cnt-StP system and Adc permease have major growth defects in Zn-limited medium and selectively fail to accumulate Zn demonstrate that these systems serve as the major Zn importers of the pathogen. Consequently, this work defines the Cnt-StP system as the founding member of a new class of Zn importers and expands the use of bacterially produced metallophores beyond Fe.
StP is a broad-spectrum metallophore, and transport assays following growth in Zn-depleted medium have demonstrated that the Cnt-StP system can import Cu, Co, and Ni (45,46). This raises the possibility that the system could contribute to the ability of S. aureus to obtain these metals. However, metal content analyses revealed only defects in Zn accumulation. Additionally, high-affinity metal importers are typically regulated by the cellular abundance of their cognate metal (11,43,56). Our work and that of others (45) have shown that Co and Ni abundance does not influence the expression of the cnt locus. Furthermore, S. aureus possess two bona fide Ni transporters, the loss of which does reduce accumulation of Ni (57). With respect to Cu accumulation, due to its potent toxicity, even low levels of this metal are sufficient to induce the expression of dedicated efflux pumps. As such, it is unlikely that S. aureus actively accumulates this metal (58). Taken together, the balance of evidence indicates that Co, Ni, and Cu are not physiological substrates of the Cnt-StP system. Distinctly from Co and Ni, our work and that of others (59) suggest that the Cnt-StP system is modestly responsive to Mn and Fe. However, these metals exert an influence on transcription only in the absence of Zn. This suggests that Zn abundance is the principal regulatory factor controlling expression of the system. Further supporting a role in Zn transport is the observation that Mn and Fe supplementation suppresses growth of the ΔadcA ΔcntA mutant. Collectively, these data indicate that the physiological role of the Cnt-StP system is as a Zn acquisition pathway.
StP synthesis loci are present in numerous pathogens, including multiple staphylococcal, Yersinia, and Pseudomonas species, suggesting that StP analogs may play an important role in the pathogenesis of several microbes. Intriguingly, while all of the putative synthesis loci contain genes encoding CntM and CntL, 45% lacked a gene encoding a CntK homolog, suggesting that both D-and L-isomers of StP are produced, depending on the species. Additional genes that appear to encode small-moleculemodifying enzymes were associated with some of the StP synthesis loci. These observations suggest that bacteria produce an array of diverse metal chelators that are related to but are distinct from StP. This inference is supported by the observation that importers that are not homologous to the CntABCDF permease are associated with StP loci in other bacteria. Microbes are known to steal siderophores produced by other organisms; thus, the production of StP variants may serve as a mechanism to prevent their use by other microorganisms (32,34,(60)(61)(62). It is also tempting to speculate that this diversity may serve as a mechanism to prevent the host from binding the zincophore produced by a pathogen, akin to the production of modified siderophores that evade binding by the host immune effector lipocalin (63)(64)(65).
In S. Typhimurium and A. baumannii, loss of the AdcABC family importer severely impairs their ability to compete with CP for Zn and cause infection (17,23). Loss of AdcABC permeases also impairs the ability of Vibrio cholerae, Streptococcus pneumoniae, Listeria monocytogenes, and other pathogens to cause infection (38,55,66,67). In P. aeruginosa, loss of the AdcA homolog ZnuA modestly diminishes the ability of the bacterium to grow in the presence of CP and in Zn-limited medium (24,68). Differing from these pathogens, loss of AdcABC alone does not diminish the ability of S. aureus to grow in the presence of CP or cause disease. However, the Cnt-StP system is critical to the ability of S. aureus to resist CP-imposed Zn starvation. In combination with the virulence defects associated with ΔcntKLM, ΔcntA, and ΔcntE ( Fig. 5A to D) (59), these results indicate that this zincophore-based importer is the main system used by S. aureus to compete with the host for Zn during infection. Unfortunately, mice lacking CP, in the context of a S. aureus infection, do not have defects in Zn sequestration, preventing this idea from being directly tested as has been done for the staphylococcal Mn transporters (8,12). Further supporting this supposition is the observation that the virulence defects of strains lacking CntKLM or CntA are exacerbated by concurrent loss of AdcA. Similarly to S. aureus, Yersinia pestis lacking the Adc permease does not have a virulence defect (43). This observation is potentially explained by the presence of an StP synthesis locus in Y. pestis, which is Zur regulated (69). The presence of an StP analog could also explain the modest phenotypes of P. aeruginosa strains lacking the Znu system. While the P. aeruginosa StP analog is reported to be a siderophore, it is regulated by Zur, which strongly suggests a role in Zn acquisition (68,70). These observations suggest that StP analogs and their cognate transporters likely contribute to the ability of multiple pathogens to compete with the host for Zn.
The identification of the Zn acquisition systems employed by S. aureus offers new opportunities to disrupt the ability of pathogens to compete with the host for Zn. The widespread prevalence of the StP synthesis machinery in both Gram-positive and Gram-negative pathogens suggests that information gained by studying these systems will provide critical insight into how numerous pathogens circumvent nutritional immunity.

MATERIALS AND METHODS
Ethics statement. All animal experiments were approved by the University of Illinois at Urbana-Champaign Institutional Animal Care and Use Committee (IACUC license number 15059) and performed according to NIH guidelines, the Animal Welfare Act, and U.S. federal law.
Bacterial strains. For routine overnight cultures, S. aureus strains were inoculated into 5 ml of tryptic soy broth (TSB) in 15-ml conical tubes and grown at 37°C on a roller drum. To preculture bacteria in limited-metal environments, overnight growth was performed in 5 ml of Chelex-treated RPMI medium (NRPMI) supplemented with 1% Casamino Acids, 1 mM MgCl 2 , 100 M CaCl 2 , and 1 M FeCl 2 in 15-ml conical tubes and bacteria were grown at 37°C on a roller drum. S. aureus Newman and derivatives were used for all experiments, unless otherwise noted. The ΔcntA and ΔcntKLM mutants were generated by amplifying the 5= and 3= flanking regions of the genes using the indicated primers (see Table S2 in the supplemental material). These fragments were then cloned into pKOR1, and the deletions were generated using allelic replacement, as previously described (71). The adcA::erm mutant was generated by phage transducing the allele from USA300 (JE2) adcA::erm into Newman via ⌽85 phage. For complementation constructs, the cntA, adcA, and cntKLM coding sequences were amplified using the indicated primers (Table S2). The cntA and cntKLM coding sequences were cloned into pRMC2, which contains an anhydrotetracycline-inducible promoter (72). The adcA coding sequence was cloned into pOS1 under the control of the lgt promoter. For the fluorescent reporters, the promoters of the cnt operon and adcA were cloned into the yellow fluorescent protein (YFP)-containing vector pAH5 (73). All constructs were verified by sequencing, and all mutants were confirmed to be hemolytic. See Tables S3 and S4 for a full list of the strains and plasmids used in this study, respectively.
BLAST search was performed separately for CntL and CntK (http://www.uniprot.org/blast/; generated using default parameters and BLOSUM62 matrix), and the resulting list was searched against the complete list of genome proximal sequences returned in the IPR016935 GNN. Cytoscape v3.2.0 (78) was used for visualization and analysis of the SSN and GNN.
Quantification and statistical analysis. All statistical analyses were performed using GraphPad Prism version 6. The specific statistical test used is indicated in each figure legend.