A Copper-Responsive Two-Component System Governs Lipoprotein Remodeling in Listeria monocytogenes

ABSTRACT

Bacterial lipoproteins are membrane-associated proteins with a characteristic acylated N-terminal cysteine residue anchoring C-terminal globular domains to the membrane surface. While all lipoproteins are modified with acyl chains, the number, length, and position can vary depending on host. The acylation pattern also alters ligand recognition by the Toll-like receptor 2 (TLR2) protein family, a signaling system that is central to bacterial surveillance and innate immunity. In select Listeria monocytogenes isolates carrying certain plasmids, copper exposure converts the lipoprotein chemotype into a weak TLR2 ligand through expression of the enzyme lipoprotein intramolecular acyltransferase (Lit). In this study, we identify the response regulator (CopR) from a heavy metal-sensing two-component system as the transcription factor that integrates external copper levels with lipoprotein structural modifications. We show that phosphorylated CopR controls the expression of three distinct transcripts within the plasmid cassette encoding Lit2, prolipoprotein diacylglyceryl transferase (Lgt2), putative copper resistance determinants, and itself (the CopRS two-component system). CopR recognizes a direct repeat half-site consensus motif (TCTACACA) separated by 3 bp that overlaps the −35 promoter element. Target gene expression and lipoprotein conversion were not observed in the absence of the response regulator, indicating that CopR phosphorylation is the dominant mechanism of regulation.

IMPORTANCE Copper is a frontline antimicrobial used to limit bacterial growth in multiple settings. Here, we demonstrate how the response regulator CopR from a plasmid-borne two-component system in the opportunistic pathogen L. monocytogenes directly induces lipoprotein remodeling in tandem with copper resistance genes due to extracellular copper stress. Activation of CopR by phosphorylation converts the lipoprotein chemotype from a high- to low-immunostimulatory TLR2 ligand. The two-component system-mediated coregulation of copper resistance determinants, in tandem with lipoprotein biosynthesis demonstrated here in L. monocytogenes, may be a common feature of transmissible copper resistance cassettes found in other Firmicutes.

INTRODUCTION

Bacterial lipoproteins are globular proteins maintained at the membrane surface through an acylated N-terminal cysteine residue (1–4). The initial steps in lipoprotein biosynthesis are highly conserved across all bacteria. Prolipoproteins are translated with a characteristic N-terminal export signal peptide sequence preceding a signature lipobox motif containing an invariant cysteine residue. Once exported, the cysteine side chain is modified with diacyl-glycerol acquired from a phospholipid donor by prolipoprotein diacylglyceryl transferase (Lgt). The signal sequence is removed by lipoprotein signal peptidase (Lsp) to produce diacylated lipoprotein (DA-LP), which can be further modified by additional bacterial enzymes in a species-specific fashion (5, 6). In Firmicutes, a Staphylococcus aureus two-gene system named LnsAB adds a third acyl chain to the α-amino group of cysteine to make triacyl-lipoprotein (TA-LP) (7). In Enterococcus faecalis, lipoprotein intramolecular acyltransferase (Lit) makes lyso-form lipoprotein from a DA-LP precursor (8). Lit catalyzes an internal transacylation whereby the ester-linked sn-2 glycerol acyl chain migrates to form an amide bond at the α-amino group of cysteine (9). The recently reported crystal structure of Lit from Bacillus cereus supports a mechanistically unique intramolecular acyl transfer involving an eight-atom cyclic intermediate (10). Enzymes responsible for synthesizing other chemotypes, including lipoprotein N-acetylation systems in S. carnosus (11) and B. subtilis (12), await identification and characterization.

The reason for such lipoprotein chemotype structural diversity observed among Firmicutes is not fully understood. Lipoproteins are ligands for the Toll-like receptor 2 (TLR2) protein family, an integral component of innate immunity responsible for detecting bacteria (13, 14). Changes in lipoprotein structure can alter TLR2 signaling outcomes. DA-LP to lyso-form lipoprotein conversion upon Lit expression decreases TLR2 activation by 100- to 1,000-fold in E. faecalis (15), while switching from DA-LP to TA-LP in S. aureus mutes TRL2 signaling by ~10-fold (7). Evidence for non-TLR2-related selective pressures comes from the occurrence of lit gene paralogs (lit2) on plasmids from certain Listeria monocytogenes isolates (15). Multiple genes throughout these plasmids and flanking lit2 encode proteins with sequence similarity to heavy metal resistance determinants, including to copper (16). Copper usage as a general antimicrobial agent is ubiquitous in the agriculture (17–19), food processing (20, 21), water treatment (22), and health care (23–25) sectors. The antimicrobial properties stem in part from redox cycling and the resulting oxidative cellular damage (26), as well as mis-metallation and inhibition of essential cellular processes, including iron-sulfur cluster assembly (27). Remnants of insertion sequence transposases flank both ends of the lit2-copper resistance encoding cassette on plasmids in certain L. monocytogenes isolates, suggesting potential for mobility and horizontal gene transfer events. Indeed, other genera, including Enterococcus sp. isolated from pig farms have acquired the cassette either as part of a larger plasmid or integrated into the chromosome (28). Understanding how the lit2-copper resistance cassette senses copper, governs lipoprotein chemotype conversion, and attenuates TLR2 detection is therefore of interest.

We previously showed that supplementing growth media with copper increases plasmid-borne lit2 and lgt2 transcript levels in L. monocytogenes (15). Whether induction was triggered by a general stress response resulting from copper intoxication or if copper itself was directly sensed was not addressed. Herein, we identify and characterize a response regulator (RR) from a two-component (CopRS) system coresident on the same heavy metal resistance plasmid as the lit2-cassette that controls expression from three distinct promoters. We define the DNA binding motif and demonstrate an essential role for phosphorylated CopR RR in lipoprotein chemotype conversion. In the companion article (29), we show extracellular cuprous ion specifically activates the sensor histidine kinase CopS. We also identify the extracellular electron transport (EET) system as the major source of extracellular cuprous ion ligands that induce the CopS under oxygen-limiting conditions.

RESULTS

Three distinct copper-inducible transcripts are controlled by the two-component system response regulator CopR.

The lit2 gene is encoded within a 10.7-kb cassette as part of a larger 52-kb plasmid carried by select L. monocytogenes isolates (Fig. 1A). Within the cassette itself, bioinformatic analysis of protein groups genes into two functionally related sets involved in either lipoprotein biosynthesis (lit2 and a putative prolipoprotein diacylglyceryl transferase [lgt2]) or copper trafficking (cop and cue genes) (Fig. 1A). To begin to understand how lipoprotein remodeling is coregulated with respect to copper, we first experimentally determined the 5′ transcriptional start sites (TSS) by 5′-RACE (rapid amplification of cDNA ends) mapping (Fig. 1B). Three 5′ ends located within intergenic gaps 24 to 32 bp upstream of putative protein initiation codons were enriched in cultures grown with copper. Two TSS are proximal to each other but located on opposite strands (P_lgt2 and P_lit2) while the third TSS is upstream of a putative copper oxidase (P_cueO). Initiation at all three TSS with termination at the respective computationally predicted Rho-independent terminators would yield transcripts consistent with lengths previously estimated by Northern blotting (15). While ideally spaced −10 promoter elements could be readily identified for all putative TSSs, candidate −35 elements are positioned farther upstream (20, 19, and 22 bp from the end of −10) than the 17 bp generally considered optimal for typical Gram-positive promoters (30). To confirm promoter activity, β-galactosidase gene expression reporter probes were constructed with each of the putative promoter elements. Copper increased β-galactosidase expression in all three cases, indicating the mapped TSSs marked functional promoters (Fig. 1C). There was only basal expression in the absence of copper in comparison to the promoterless control construct, consistent with tight regulation.

FIG 1.

The copper-responsive lipoprotein remodeling operon in L. monocytogenes CFSAN023459 is induced by the two-component response regulator CopR at three separate promoters. (A) The 10.7-kb DNA segment from plasmid pCFSAN023459_02 (52687 bp, accession number CP014254) is flanked by insertion sequence/transposon remnants (KO07_15320, IS6 family on the left and KO07_15680, ISL3 family on the right) and an uncharacterized type II restriction enzyme (Type II RE, KO07_15325). Putative transcriptional units are assigned based on 5′-RACE (rapid amplification of cDNA ends) mapping of transcriptional start sites (TSS; arrows) and Rho-independent terminator locations (Ω) predicted using ARNold (83). Genes encoding proteins with similarity to enzymes involved in lipoprotein modifications (shaded) and copper resistance (hatched) are indicated. A second CopY ortholog cotranscribed with CtpA and with a CopY DNA binding box on the antisense strand is located ~5 kb downstream from P_cueO transcriptional terminator (not shown, see Discussion). (B) The DNA sequences for each promoter, along with transcript length (parenthesis), is shown. Bases in common with Gram-positive bacterial consensus sequences for −10 (TGNTATAAT from references 30, 84), −35 (TTGACA from reference 30), and Shine-Dalgarno (SD) (GGAGG from reference 85) elements are indicated (capital letters). Experimentally determined 5′ TSS (+1 base in bold) and potential start codons (capital letters) of Lit2, Lgt2, and CueO are shown. The center of the CopR binding motif as shown in Fig. 5 is highlighted in gray. (C) Gene expression reporter constructs were used to measure induction of β-galactosidase by each putative promoter (P_lit2, P_lgt2, P_lgt2, or P_con which lacked any insert) in response to copper in the Wt background. Statistical significances were calculated by using Student’s t tests: *, P < 0.05; ***, P < 0.001; ns = not significant. (D) Levels of lit2 containing transcripts were probed by Northern blotting in Wt, ΔcopY, ΔcopR, and ΔcopR plasmid back complemented (pcopR) strain backgrounds. A single ~1.4 kb band induced by 1 mM copper was only detected in CopR⁺ strain backgrounds. (E) Promoter activity was measured using β-galactosidase reporter assay as in panel C but using a ΔcopR strain background.

We next sought to determine which protein regulated expression at P_lit2. Within the cassette, there are two candidate open reading frames (ORFs) in CopY (KO07_15350) and CopR (KO07_15365) with similarity to characterized copper-responsive transcriptional regulators (Fig. 1A). CopY is a well-studied cytosolic copper sensor containing an N-terminal helix-wing-helix DNA binding domain and a C-terminal metal binding domain that is common to many Firmicutes (31–33). Copper binding decreases CopY affinity for cognate operator DNA, leading to the upregulation of copper resistance determinants (34). As copY is cotranscribed with lit2 and a heavy metal domain containing copper chaperone/efflux pump fragment, we initially hypothesized CopY governed lipoprotein remodeling in response to cytosolic copper. Deletion of copY, however, did not impart constitutive lit2 expression or alter copper induction as would have been expected for a repressor (Fig. 1D). We next turned our attention to the adjacent P_cueO-directed transcript (CueOCopRS) encoding a putative multicopper oxidase CueO (KO07_15360) and a two-component system CopRS (KO07_15365 and 15370). The membrane-bound signal transduction component CopS has an extracytoplasmic N-terminal domain with similarity to other heavy metal sensor kinases involved in copper sensing (35–42), while the cytoplasmic C-terminal domain looks to be a typical ATP-dependent histidine kinase. We thus deleted the putative cognate response regulator copR and remeasured β-galactosidase expression (Fig. 1E). All three promoters exhibited low basal activity even when copper was included in the media, indicating that CopR coregulates both lipoprotein remodeling genes (lit2/lgt2) as well as copper resistance from three distinct promoters.

CopR is required for lyso-LP detection by matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Lit2 catalyzes an intramolecular transfer requiring no ATP or additional substrates beyond DA-LP (9). Partial LP chemotype conversion has thus been observed in wild-type L. monocytogenes even when synthetic minimal media were not supplemented with copper (15). Basal lit2 transcription due to weak secondary CopR-independent promoters with activities below the detection limits of Northern blotting and β-galactosidase assays was probed by direct characterization of LP acylation states using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) tandem mass spectrometry (MS/MS) (Fig. 2). Consistent with earlier reports (15), we observed a mixed population of DA-LP and lyso-LP forms for the LP KO07_11695 (a putative peptide ABC transporter substrate-binding protein) when wild type (Wt) was grown without copper (Fig. 2C) that was converted to all lyso-LP upon copper addition to the growth medium (Fig. 2D). DA-LP chemotype was retained in the ΔcopR strain regardless of copper supplementation to the media (Fig. 2E and F). Copper-induced chemotype conversion from DA-LP to lyso-LP could be restored by back complementation of CopR (Fig. S1 in the supplemental material), indicating Lit2 expression is entirely dependent on the response regulator CopR.

FIG 2.

Lipoprotein modification by Lit2 is dependent on CopR. MALDI-TOF tandem MS/MS spectra of N-terminal lipopeptides produced by trypsin treatment of the KO07_11695 lipoprotein (putative peptide ABC transporter substrate-binding protein) isolated from either Wt or ΔcopR L. monocytogenes strains grown in the presence or absence of 1 mM CuCl₂. (A and B) Sodiated primary ions (1,283 m/z for both diacyl- [A] and lyso- [B] LP isomers) were fragmented to distinguish between isomeric LP chemotypes by assigning the corresponding dehydro-alanyl diagnostic peptide fragment ions m/z 676 (free α-amino terminus, DA-LP parent ion) and m/z 900 (N-acylated terminus, lyso-LP parent ion), as has been described (79). (C and D) The wild-type strain produces a mixture of DA-LP and lyso-LP in media only (C) that is converted to all lyso-LP by copper (D). (E and F) In the ΔcopR strain, only DA-LP is detected even in the presence of copper. Plasmid back complementation of ΔcopR restored lipoprotein N-acylation (Fig. S1 in the supplemental material).

Phosphorylation of CopR increases binding to lit2/lgt2 and cueO promoter DNA fragments.

CopR belongs to the OmpR/PhoB-family of two-component system response regulators (43–45), with a seemingly typical N-terminal RR domain followed by a DNA binding winged helix-turn-helix domain. All key OmpR/PhoB-family RR residues (46), including the aspartate phosphoacceptor residue, are conserved in the CopR response receiver domain. To determine how phosphorylation impacts DNA operator recognition, recombinant CopR was purified from E. coli and subjected to in vitro phosphorylation reactions (Fig. 3). We tested low-molecular-weight phosphoryl donors to generate phosphorylation of CopR (CopR~P) as has been described for other regulators (47). While acetyl phosphate was ineffective (Fig. 3A), the phosphoramidate (PA) donor approached complete CopR phosphorylation as judged by Phos-tag acrylamide gel electrophoresis separation (48). We next tested whether CopR~P bound the three promoter elements using two equally sized DNA fragments, one containing the divergent P_lgt2/lit2 promoters and the other P_cueO. Electrophoretic mobility shift assays (EMSA) demonstrated high-molecular-weight complexes for both DNA binding probes that were absent from control promoter DNA (Fig. 3B). Of note, the P_lgt2/lit2 promoter complex migrated at a higher molecular weight, suggesting higher CopR~P binding stoichiometry in comparison to P_cueO.

FIG 3.

Chemically phosphorylated CopR specifically binds lit2/lgt2 and cueO promoter DNA fragments. (A) Recombinant CopR (100 μM) was incubated for 1 h at room temperature in either buffer alone (−, lane 1) or with acetyl phosphate (50 mM AcP, lane 2) or with phosphoramidate (50 mM PA, lane 3) before separation using a 12% Zn²⁺-Phos-tag acrylamide gel to selectively slow migration of phosphorylated protein. Protein was stained using Coomassie blue dye. (B) Recombinant CopR that had been previously phosphorylated with PA (CopR~P) was incubated with dually labeled 5′-FAM DNA fragments encoding P_lit2/lgt2 (made with primers TM1703-1704, 136 bp long), P_cueO (made with primers TM1857-1858, 138 bp long), or P_pen (made with primers TM1859-1860, 137 bp long). Free and protein-bound DNA was separated on an 8% polyacrylamide gel, and DNA was imaged by fluorescence.

We next quantified the binding affinity of CopR and CopR~P for P_lgt2/lit2 and P_cueO. While unphosphorylated CopR had an apparent K_d of 3.8 ± 1.7 μM for P_lgt2/lit2 DNA probe, binding affinity increased for CopR~P (K_d 0.53 ± 0.14 μM, Fig. 4). Formation of multiple high molecular species prevented quantitative estimation of P_cueO affinity, but the relative DNA binding affinity was likewise enhanced by CopR phosphorylation (Fig. S2). The data, in tandem with promoter analyses, support the identification of P_lgt2/lit2 and P_cueO as CopR-responsive promoters.

FIG 4.

Phosphorylation of CopR enhances affinity for P_lit2/lgt2 DNA. (A) Increasing concentrations of CopR (0 to 20 μM) were incubated with 5′ FAM P_lit2/lgt2 DNA promoter fragment (made with primers TM1703-1704, 136 bp long) and protein bound-DNA was separated from free DNA on an 8% polyacrylamide gel. Gels were imaged for FAM fluorescence (top), and signals were quantified using Bio-Rad Image Lab 5.0 software. The bound DNA fraction was calculated and fitted with the Hill equation, from which apparent disassociation (K_d) binding constants curves were derived. (B) As in panel A, except freshly phosphorylated CopR (CopR~P, 0 to 2 μM) was utilized. One representative of three separate experiments is shown. Complementary experiments using P_cueO DNA promoter fragments are in Fig. S2 in the supplemental material.

CopR ~P binds an 8-bp-long TCTACACA half-site direct repeat motif overlapping the −35 site.

We employed DNase I footprinting of 5′-end-labeled DNA binding probes corresponding to P_lgt2/lit2 and P_cueO to establish the CopR~P binding motif (Fig. 5). Comparison of electropherograms produced by capillary electrophoresis of DNA fragments specifically protected by CopR~P nominated two regions in P_lgt2/lit2 (Fig. 5A) and a single region in P_cueO (Fig. 5B) as putative binding sites. All three protected DNA segments were located upstream of TSSs, were approximately 24-bp long, and spanned a region from midspacer to the beginning of each of the −35 sites. Sequence alignment revealed a common core footprint centered at the −30/31 bp position (with respect to the TSS) that was flanked by two 8-bp-long imperfect direct repeat elements separated by 3-bp (Fig. 5C). In each of the three separate promoter elements, at least 12 of 16 positions (with no less than 5 of 8 in a given half site, 41 of 48 total possible bp) match a half site consensus sequence of TCTACACA (Fig. 5D). While insight is limited by the overall numbers of CopR~P-responsive promoters available to analyze, the 3-bp spacer separating half sites is enriched in AT-bp (9 of 9 total possible bp).

FIG 5.

DNase I footprinting protection assay of promoter DNA fragments by CopR~P. (A) P_lit2/lgt2 DNA probe containing a single 5′-FAM fluorescently labeled end (made with primers TM1949-1950, 267 bp long) was incubated without (red trace) or with (blue trace) CopR~P before partial digestion with DNase I. Fragments were separated by capillary electrophoresis, and electropherograms aligned with di-deoxynucleotide terminator sequencing control reaction electropherograms to assign bases peaks as described previously (81, 82). Areas specifically protected from DNase I digestion are indicated by black bars. Numbers below traces indicate relative bp position with respect to the TSS for each promoter. TSS sites and promoter strand orientation are indicated with arrows. (B) As in panel A, except 5′-FAM-labeled P_cueO DNA probe was utilized (made with primers TM1951-1952, 272 bp long). (C) The DNA regions protected from DNase I digestion by CopR~P for each promoter were aligned, resulting in the identification of two 8-bp-long imperfect direct repeats (underlined) separated by3-bp and centered at the −31/30 position (yellow highlighted). (D) Weblogo (86) alignment of all six putative consensus half sites involved in CopR~P binding. Base pairs matching the consensus sequence are capitalized in Fig. 5C.

We next tested whether the consensus half-site TCTACACA operator identified above was important for CopR~P binding. The footprinting analysis indicated DNase I access to CopR~P protected DNA in between the divergently orientated P_lgt2 and P_lit2 binding probe (Fig. 5A), so we were able to design three separate sets of DNA binding probes (P_lit2, P_lgt2, and P_cueO) wherein each half site or both half-site motifs were randomized (Fig. 6A). In all cases, the first 6-bp from each half site was replaced with the random sequence GACGTG to maintain relative spacing. The isolated P_lit2 DNA probe was recognized by CopR~P, confirming the two divergently transcribed promoters can independently be recognized (Fig. 6B). Replacing either the up or down half site decreased the relative amount of DNA-CopR~P complex observed by EMSA, while no shift could be seen when both half sites were replaced. A similar pattern was observed for the P_lgt2 and P_cueO DNA probe set (Fig. 6C and D). For all three promoter sets and particularly for the P_cueO probes, some high-molecular-weight complex formed when only a single half site was removed. This suggests a single half site can weakly bind CopR~P in vitro or that the randomized half site retained partial function. In either case, DNA binding probes with a single intact half site clearly had lower affinity compared to the corresponding full CopR~R binding operator core defined by the consensus sequence TCTACACANNNTCTACACA.

FIG 6.

Both half-site direct repeat motifs are important for CopR~P binding. (A) DNA binding probes with 5′-FAM end labels were generated to span from the P_lit2, P_lgt2, and P_cueO binding motifs to past the respective TSS. Probes encoded either the wild-type CopR~P binding sequence (Native), had the first 6-bp of each half site identified in Fig. 5C individually replaced with GACGTG (Up or Down), or had both repeats replaced (both). The native 5-bp sequence located between repeats was retained in all probes. (B) DNA migration shift either without (−) or with CopR~P addition was assessed by EMSA as in Fig. 3B using the P_lit2 DNA binding probe set. (C) As in panel B, except P_lgt2 binding probes were tested. (D) As in panel B, except P_cueO binding probes were tested.

DISCUSSION

Bacterial cells utilize two mechanistically distinct strategies to respond to copper depending on cellular location (49, 50). One-component sensors have metal-sensing domains coupled with DNA binding domains to directly alter transcriptional output, while two-component systems use an integral membrane protein to bind extracellular copper and then relay that signal to a RR transcription factor through activation by phosphorylation. In the case of the plasmid-borne copper resistance operon from L. monocytogenes studied here (Fig. 1), phosphorylation of the two-component system RR CopR regulates genes required for lipoprotein remodeling, copper resistance, as well as its own expression. We had initially presumed that CopY, a cytoplasmic sensor, would regulate lit2 expression since it is cotranscribed (Fig. 1A). Deletion of CopY (KO07_15350), however, did not exert any effect on basal expression or copper induction (Fig. 1D). Closer inspection of the amino acid sequence suggests this may be a nonfunctional ORF (referred to as CopY’ from here forward), as the signature cysteine-rich CxC motif (31, 51) at the extreme C terminus is conspicuously absent from the metal binding domain. Second, CopY family repressors typically bind a highly conserved operator DNA sequence (called the CopY box) that has been well characterized in other Firmicutes (32, 33, 52). The CopY box motif is absent in the P_lit2, P_lgt2, and P_cueO promoters, although a complete CopY box does appear ~5 kb past the end of the P_cueO transcript on the same plasmid preceding a second CopY ortholog (KO07_15415). KO07_15415 has an intact CXC motif and thus appears to be functional. Studies on a closely related plasmid in L. monocytogenes strain DRDC8 suggested the functional CopY ortholog located outside the locus studied here regulates a cotranscribed copper translocating P-type ATPase (CtpA) that is important for copper trafficking and virulence (16, 53, 54). Multiple other heavy resistance determinants and cognate regulators are coresident on these plasmids, and L. monocytogenes also encodes a chromosomally encoded CsoR-family cytosolic copper-sensing repressor (55). We only observed cis-regulation of P_lit2, P_lgt2, and P_cueO promoters by the CopR RR, as ΔcopR remained uninduced even with copper supplementation (Fig. 1D and E). All three regulatory networks (CopRS and CopY from plasmid along with CsoR from chromosome) thus appear to operate as isolated regulons sampling a common pool of copper distributed either inside (CopY and CsoR) or outside (CopRS) of the cell. In contrast, cross-recognition by chromosomal and plasmid-borne copper resistance two-component systems determinants in Pseudomonas syringae (38, 56, 57) and E. coli has been documented (40). Bacteria acquire and integrate various copper trafficking elements to achieve unique copper homeostasis network architectures to suit particular needs (49). The high number of insertion sequence elements and transposons on pCFSAN023459_02 indicates multiple independent acquisition events of copper resistance elements. To prevent subsequent regulatory cross-talk and isolate the extracellular copper-sensing CopRS regulon from cytoplasmic CopY, loss of function in the cis-encoded CopY’ (KO07_15350) would have been necessary and may explain the lack of a phenotype observed here.

The consensus binding sequence proposed for CopR~P contains two imperfect direct repeat half sites separated by three bases (TCTACACANNNTCTACACA; Fig. 5 and 6). The centers of each half site are spaced by 11 bp on the center, corresponding to the pitch of a B-type DNA helix (10.5 to 11 bp). Since the DNA binding domains of OmpR/PhoB-family RRs generally bind as head-to-tail homodimers (43, 58), both CopR~P subunits would be predicted to bind to the same face and recruit RNA polymerase (RNAP) holoenzyme to the opposite side of the helix through protein-protein interactions. This binding mode for transcriptional activators has been characterized for other class II type RR within the OmpR/PhoB-family having binding sites that overlap the −35 element (59–62). The CopR~P imperfect direct repeat binding motif is centered at −30/31 from the TSS in all three promoters (Fig. 1B). In this regard, the binding site is similar to that of two well-characterized RR, PhoB (63, 64), and PmrA (65–67). All have a downstream half site within the −35/10 spacer and an upstream site that at least partially overlaps with where a typical −35 element would be located. The CopR~P half site motif defined here, however, shares 4 of 6 bp with the consensus −35 element (TTGACA) sequence in two reading frames (Fig. 5). The CopR upstream binding half sites are suboptimally spaced from strong −10 elements, being located 19, 20, and 22 bp away. This likely prevents efficient RNAP holoenzyme recruitment in the absence of CopR~P. Many MerR-family transcriptional activators, including the copper-responsive cytoplasmic sensor CueR (68, 69), overcome suboptimal spacing by introducing torsional stress and bending to reshape the core promoter and help facilitate productive interactions between −35 elements and RNAP holoenzyme. Whether the candidate −35 elements (labeled in Fig. 1B) directly contribute to RNAP recruitment or transcriptional activation is driven solely by canonical CopR~P transactivation loop interactions with the σ⁷⁰ RNAP subunit akin to those in model class II OmpR-family activators is, at present, unknown.

The binding site motif of CopR from L. monocytogenes is unique from other characterized OmpR/PhoB-family copper-sensing RR two-component systems. The RR from P. syringae (CopR [57]) and E. coli (PcoR [40] and CusR [39]) recognize an inverted repeat (cop-box) situated immediately adjacent or upstream of the −35 position. In the cyanobacterium Synechocystis sp. PCC 6803, a TTTCAT direct repeat separated by 5 bp and flanking the −35 region was proposed as the CopR binding site (35). The direct repeat TGAAGATTTnnTGAAGATTT in Corynebacterium glutamicum was identified by EMSA (41). Once more, none of these two-component systems are known to directly coregulate lipoprotein biosynthesis genes as observed here (Fig. 1C to E). Intriguingly, the plasmid-borne two-component system LcoRS (43% and 28% identical amino acid sequence to CopRS in L. monocytogenes pCFSAN023459_02) was partially characterized from Lactococcus lactis (70–72). LcoRS was shown to be responsive to copper and control expression of a neighboring operon encoding a multicopper oxidase (CueO; 29% identical) and Lgt (42% identical) homolog. Although the CopR binding site in L. lactis was not determined, a putative motif centered at −34 from the mapped TSS motif matches 13 of 16 bp to the consensus determined here can be identified (Fig. S3A).

The modest overall sequence similarity between genes on both the L. lactis and L. monocytogenes cassettes, when considered in tandem with differences in gene content and orientation, does not support a common origin for both cassettes (Fig. S3B). Rather, it appears that two distinct gene assembly trajectories aided by mobile genetic element activity have brought lipoprotein biosynthesis under the control of copper-sensing two-component systems in separate Firmicute lineages. At present, the functional link driving coregulation in Firmicutes is not completely understood. Copper ions have been proposed to bind at the N terminus of lipoproteins, leading to an increase in oxidative damage by redox cycling and toxic cytoplasmic uptake (15). Nuclear magnetic resonance experiments support specific N-terminal copper binding by free α-amino DA-LP chemotypes in vitro (10). Evidence for copper-lipoprotein interactions also comes from studies in E. coli, where copper induces a cell envelope stress response that surveils for defective lipoprotein trafficking (73). Enhanced lipoprotein biosynthesis flux, and in particular N-terminal acylation by Lit2 to remove a ligand for metal coordination in lyso-LP, may thus help cells deal with excess copper. What form of extracellular copper triggers lipoprotein remodeling through binding to the sensor histidine kinase CopS in L. monocytogenes is an outstanding question. In the companion article (29), we demonstrate a central role of EET in generating cuprous ion for CopRS signaling and lipoprotein remodeling.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

Strains were routinely grown in either Luria broth (LB; E. coli) or tryptic soy broth (TSB; L. monocytogenes) at 37°C with shaking unless otherwise noted. Carbenicillin (carb, 100 μg/mL), kanamycin (kan; 50 μg/mL), or chloramphenicol (chl; 20 μg/mL) were used for plasmid selection in E. coli, while chl was used at either 10 μg/mL (for pKFC plasmid) or 5 μg/mL (for integrated pPL2 or pKFC) in L. monocytogenes. LB agar supplemented with polymyxin (5 μg/mL) was utilized to counterselect E. coli donor strains used in conjugation. Strains and plasmids used in this study are listed in Table 1.

TABLE 1.

Bacterial strains and plasmids used in this study

Plasmid or strain	Relevant genotype/phenotypea	Source
pKFC	E. coli-Gram-positive shuttle vector with temp-sensitive replicon; carbR chlR	74
pPL2	E. coli-L. monocytogenes shuttle integration vector; chlR	76
pTXM1070	pET22(b+) modified with N-terminal 6×His TEV site and NheI/HindIII cloning sites; carbR	Lab stock
pTXM1113	pTXM1070 N-terminal 6×His TEV site copR of L. monocytogenes; carbR	This study
pZL207	E. coli lacZ reporter gene translated from the ribosome binding site and leader peptide of B. subtilis SpoVG; carbR	87
pCN59	E. coli-S. aureus shuttle vector containing transcriptional terminator (TT) of blaZ; carbR ermR	75
pTXM1525	pPL2-Ppen SAOUHSC_00822	7
pTXM1071	pKFC-copY deletion vector	This study
pTXM1113	pTXM1070 copR	This study
pTXM1122	pKFC-copR deletion vector	This study
pTXM1188	pPL2-Ppen copR	This study
pTXM1190	pPL2-TTblaZ-KpnI-lacZ	This study
pTXM1191	pPL2-TTblaZ Plit2-lacZ	This study
pTXM1192	pPL2-TTblaZ Plgt2-lacZ	This study
pTXM1193	pPL2-TTblaZ PcueO-lacZ	This study
L. monocytogenes
Wild type (Wt)	Listeria monocytogenes CFSAN023459 with endogenous plasmid pCFSAN023459_02	Dwayne Roberson, FDA
KA1064	Wt ΔcopY using pTXM1071	This study
TXM1157	Wt ΔcopR using pTXM1122	This study
TXM1195	TXM1157 attB::pTXM1188	This study
TXM1199	Wt attB::pTXM1191	This study
TXM1200	Wt attB::pTXM1192	This study
TXM1201	Wt attB::pTXM1193	This study
TXM1203	Wt attB::pTXM1190	This study
TXM1211	TXM1157 attB::pTXM1191	This study
TXM1212	TXM1157 attB::pTXM1192	This study
TXM1213	TXM1157 attB::pTXM1193	This study
TXM1215	TXM1157 attB::pTXM1190	This study
Escherichia coli
TXM1152	E. coli HST04 F-, endA1, supE44, thi-1, recA1, relA1, gyrA96, phoA, Φ80d lacZΔ M15, Δ(lacZYA-argF) U169, Δ(mrr-hsdRMS-mcrBC), ΔmcrA, λ-; pRK2013 kanR	Lab stock

^achl, Chloramphenicol; carb, carbenicillin; kan, kanamycin; TT, transcriptional terminator; TEV, tobacco etch virus.

Construction of plasmids, deletion strains, and β-galactosidase gene expression reporter constructs in L. monocytogenes.

The shuttle vector pKFC with a temperature-sensitive Gram-positive origin of replication was used for generating unmarked in-frame gene deletions (74). DNA homology arms (1 kb in length) flanking the gene targeted were generated by PCR using P1-P2 and P3-P4 primers (all primers used in this study are listed in Table S1) and assembled with EcoRI/HindIII cut pKFC vector using In-Fusion assembly seamless cloning (TaKaRa Bio). Plasmids were introduced into L. monocytogenes by electroporation and plasmid transformants selected under permissive growth conditions (5 μg/mL chl at 30°C). Cointegrants were isolated by plating at 40°C, and plasmids were outcrossed by successive passaging at room temperature in the absence of antibiotic. All deletion alleles were verified by colony PCR using up- and downcheck primers. To construct the β-galactosidase reporter strains, a strong transcriptional terminator (blaZ TT from pCN59 [75]) was amplified by PCR (primers TM1196-1808) and assembled upstream of an E. coli lacZ gene cassette containing the strong Gram-positive ribosomal binding site of Bacillus subtilis SpoVG (amplified from pZL207 using primers TM1176-1809) through three-piece assembly using BamHI/KpnI digested L. monocytogenes integration vector pPL2 (76). The resulting plasmid (pTXM1190) has a single KpnI site, into which PCR amplicons of putative promoter element were inserted (made with primer pairs forward/reverse primer pairs TM1845-1850). The 185-bp promoter fragment for P_lit2 and P_lgt2 was identical (stretching from +40 to P_lgt2 TSS to +46 of P_lit2 TSS) but cloned in opposite orientation, and P_cueO was 198 bp long (stretching from −157 to +40 of candidate TSS). Plasmids (pTXM1190-1193) were introduced into L. monocytogenes-recipient strains by conjugation using as donor E. coli Stellar cells (TaKaRa Bio) carrying the pRK2013 RP4-based conjugation plasmid (77). The pPL2-based copR complementation vector (pTXM1188) was constructed by amplifying the constitutive P_pen promoter (from pTXM1525 using primers TM1817-KK892) and copR gene (primers TM1818-1819) and assembling into BamHI/KpnI digested pPL2. The recombinant CopR expression plasmid pTXM1113 was built by amplifying copR (primers TM1753-1754) and assembling into NheI/HindIII digested pTXM1070, which is a pET22(b+) expression vector (Millipore Sigma) modified with an N-terminal 6×His tag followed by a TEV protease cut site.

Total RNA isolation, 5′ transcriptional start site mapping, and Northern blot analysis.

Total RNA was isolated from bacteria in the exponential growth phase that had been grown in TSB media with or without 1 mM CuSO₄ as has been described previously (15). Transcription start sites were identified using the 5/3′-RACE kit Second Generation (Roche) with lit2, lgt2, and cueO SP1/SP2 oligonucleotides (Table S1) according to manufacturer’s directions. Transcripts containing lit2 were detected by Northern blotting using biotin-labeled antisense RNA probe with chemiluminescence detection (15).

β-Galactosidase assay.

Dilutions of overnight L. monocytogenes cultures (TXM1199-1201, 1203 and TXM1211-1213, 1215) carrying chromosomally integrated promoter-lacZ reporter cassettes were diluted 1:100 vol/vol in 3 mL of TSB with either 0 or 1 mM CuCl₂. At mid-log growth, cells were pelleted (3,000 × g, 5 min), washed with 1 mL of Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, and 50 mM β-mercaptoethanol), and frozen at −20°C. Cell pellets were resuspended in 500 μL of Z buffer with 10 μg/mL of mutanolysin (Sigma). Cells were mixed with an equal volume of 0.1 μM zirconium glass beads and broken (3 cycles × 20 s each at 7,000 rpm on Roche MagNaLyzer). After a slow speed spin to remove beads (1,000 × g, 2 min), 200 μL of supernatant was transferred to a 96-well microplate and mixed with 40 μL of 2-nitrophenyl β-d-galactopyranoside (4 mg/mL) in 0.1 M sodium phosphate buffer, pH 7.0. Total protein concentrations in supernatants were checked using the Bradford assay to confirm equal lysis between samples and adjusted by dilution when necessary. Reaction mixtures were incubated for 3 h at room temperature, after which 100 μL of 1 M Na₂CO₃ was added to quench reactions. Absorbance was read at 420 nm and Miller units calculated to quantify LacZ-specific activity (78).

MALDI-TOF mass spectrometry analysis and MS/MS.

KO07_11695 lipoprotein was prepared for mass spectrometry as previously described (8, 79). Briefly, the Triton X-114 phase partitioning method was used to extract lipoproteins, which were then separated with a Tris-glycine 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. Bands corresponding to KO07_11695 were cut and trypsinized overnight. Samples were eluted from the membranes and mixed with α-cyano-4-hydroxycinnamic acid matrix. Analysis was performed on an Ultraflextreme (Bruker Daltonics) MALDI-TOF mass spectrometer in positive reflector mode and MS-MS spectra were acquired in Lift mode.

Protein purification.

E. coli BL21(DE3) cells carrying pTXM1113 were grown in 1 L of LB at 37°C until optical density at 600 nm reached 0.5. The culture was chilled to 16°C, induced with 1 mM isopropyl β-D-1-thiogalactopyranoside, and grown at 16°C overnight with shaking (250 rpm). Cells were collected by centrifugation (4,000 × g for 10 min), and pellets were stored frozen at −20°C. Cells were resuspended in column buffer (20 mM Tris pH 8, 300 mM NaCl, 5 mM imidazole, and 1 mM DTT), lysed with a French pressure cell (3 passes at 14000 lb/in²), and clarified by centrifugation (20,000 × g 30 min). Recombinant 6×His-CopR was then purified from the ensuing supernatant using HisPur cobalt resin (ThermoFisher Scientific) according to the manufacturer’s instructions. The 6×His tag was cleaved by incubation with TurboTEV Protease (1:200 6×His-CopR; BioVision) at 4°C overnight in dialysis buffer (5 mM DTT, 10% glycerol, 300 mM NaCl and 50 mM sodium phosphate, pH 7.4). Dialysate was then passed through HisPur cobalt resin to remove the cleaved His tag fragment. Cleaved CopR was further purified using a Superdex 10/300 gel filtration column (GE Healthcare) preequilibrated with buffer (20 mM sodium phosphate pH 7.4, 150 mM NaCl, 0.1 mM DTT, and 10% glycerol). Fractions containing purified CopR were pooled, concentrated, and supplemented with glycerol to a 20% final concentration before being flash frozen in liquid nitrogen and stored at −80°C.

Recombinant CopR protein phosphorylation.

Recombinant CopR (100 μM) was incubated for 1 to 2 h at room temperature in either buffer alone (50 mM Tris pH 8.0, 50 mM NaCl and 20 mM MgCl₂) or with 50 mM acetyl phosphate (AcP, Sigma) or with 50 mM phosphoramidate (PA; 50 mM). The ammonium salt form of PA was synthesized from orthophosphate and ethyl isocyanate according to published protocols (80). Phosphorylation reaction progress was monitored by PAGE using a 12% Zn²⁺-Phos-tag acrylamide gel matrix (NARD Institute, Ltd.). Total protein was stained using GelCode Blue Safe Protein Stain (ThermoScientific).

Electrophoretic mobility shift assays.

For gel shift assays, different concentrations of CopR or CopR~P (freshly prepared CopR phosphorylated for 2 h at room temperature with PA as detailed above) were incubated with 40 ng of fluorescently labeled promoter DNA fragments for 30 min at room temperature in binding buffer [25 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM MgCl₂, 0.1 mg/mL BSA, 0.8 mU/mL Poly(dI-dC), and 10% glycerol]. DNA binding probes encoding P_lit2/lgt2 (made with primers TM1703-1704, 136 bp long), P_cueO (made with primers TM1857-1858, 138 bp long), or P_pen (made with primers TM1859-1860, 137 bp long) were 5′-fluorescein (FAM) labeled at both ends by PCR using chemically modified primers (Table S1). For the DNA binding probe with 6-bp randomized half sites, probes were FAM labeled at a single 5′ end incorporated with a common primer for each set of promoter elements. Paired primers were then used to introduce a randomized sequence (GACGTG) in place of endogenous binding motifs within the putative CopR operator. Probes corresponding to the wild-type sequence (Native), with a randomized upstream motif (Up), with a randomized downstream motif (Down), or with both motifs replaced (Both) were thus generated. The native 5-bp sequence intervening between the up and down repeats was retained in all probes. Binding probes for the native P_lit2 promoter region were made using either primers TM1973-1949 (Native, 156 bp long for all P_lit2 probes), primers TM1974-1949 (Up), TM1975-1949 (Down), or primers TM1974-1998 to replace both motifs with GACGTG (Both). For P_lgt2 operator probes (174 bp long for all), DNA with an endogenous binding motif (Native, made using TM1976-2045), an upstream randomized motif (Up, made using primers TM1977-2045), a randomized downstream motif (Down, made using primers TM1978-2045), or both motifs randomized (Both, made using primers TM1999-2045) was likewise made by PCR. A set of P_cueO operator DNA probes (167 bp long) was made with the same strategy using either primers TM1979-1951 (Native), TM1980-1951 (Up), TM1981-1951 (Down), or primers TM2000-1951 (Both). All PCR-generated DNA binding probes were purified by TAE-agarose gel electrophoresis (2% agarose) to remove mis-amplification products and unincorporated primers. Protein-bound DNA was separated from free DNA on an 8% polyacrylamide minigel run at 130 V for 90 min at 4°C. Gels were imaged and bands quantified using Bio-Rad Image Lab 5.0 software. Data were plotted in GraphPad Prism, and binding curves were fitted with the Hill equation to determine apparent disassociation constants (K_d).

Fluorescent capillary electrophoresis DNase I footprinting.

Capillary electrophoresis (CE) DNase I footprinting reactions were performed according to published protocols (81, 82). DNA operator probes containing a single 5′-FAM fluorescent label were generated by PCR (primers TM1949-1950 for 267-bp long P_lit2/lgt2 and TM1951-1952 for 272-bp long P_cueO) and purified by 2% agarose gel electrophoresis. CopR was first phosphorylated with PA as described above and then 10 μM CopR-P was incubated with 40 ng of fluorescent DNA probe in 25 μL of reaction buffer (25 mM Tris pH 8.0, 25 mM NaCl, 2 mM MgCl_2, 0.1 mg/mL BSA, and 5% glycerol). After a 30-min incubation at room temperature, 3 μL of 10× DNase I reaction buffer (NEB) was followed by 2 μL of a DNase I solution. The optimal concentration of DNase I was determined to be 0.06 units per reaction. Digestions were allowed to proceed for 3 min at room temperature before being quenched with 25 μL of 0.5 M EDTA. Control reactions were likewise performed in parallel except CopR~P was withheld. DNA fragments were purified using Monarch PCR & DNA cleanup kit (NEB) and separated by CE (Penn State Genomics Core Facility, University Park, PA). Footprinting electropherograms were aligned with di-deoxynucleotide terminator sequencing control reaction electropherograms (Thermo Sequenase Dye Primer Manual Cycle Sequencing kit; USB) generated using FAM-labeled oligonucleotides as sequencing primers (primers TM1949 or TM1950) and unlabeled P_lit2/lgt2 or P_cueO DNA as the template. Data were analyzed using Peak Scanner software (ThermoFisher Scientific), and base pair positions were assigned as has been described previously (81, 82).