The Diadenylate Cyclase CdaA Is Critical for Borrelia turicatae Virulence and Physiology

Generation and complementation of the BtΔcdaA mutant.

c-di-AMP-dependent signaling is important for the virulence of several bacteria (44, 45, 54, 67–71). The chromosomally encoded protein BT0008, referred to as CdaA herein, is predicted to be the only DAC in B. turicatae, and homologs are encoded in all Borrelia spirochetes (33, 34, 64, 77, 78). B. turicatae CdaA is a 258-amino-acid, 29.02-kDa protein with a predicted pI of 7.74. CdaA is annotated as a TIGR00159 family protein, and the NCBI Conserved Domain Database identified amino acids 49 to 258 as belonging to the DisA_N superfamily (E value = 1.52e-83), both of which are consistent with CdaA homologs in other bacteria (82–84). Additionally, TMHMM-2.0 predicted CdaA to have three transmembrane domains (amino acids 15 to 32, 39 to 61, and 66 to 85) close to the N terminus (85, 86). The N terminus and C terminus would be located in the periplasm and cytoplasm, respectively, which is characteristic of CdaA proteins (34, 87). Finally, as is the case with other CdaA proteins, two probable coiled-coil domains were identified within the DisA_N superfamily domain (amino acids 109 to 149 and 213 to 251) by Waggawagga coiled-coil prediction analyses (34, 88). To determine the contribution of the B. turicatae cdaA homolog during mammalian infection, allelic exchange mutagenesis was used to replace an internal region of the open reading frame (ORF) in wild-type B. turicatae (BtWT) with an aacC1 gentamicin resistance cassette, generating BtΔcdaA (Fig. 1A). Genetic complementation of BtΔcdaA was achieved by inserting an aphI kanamycin resistance cassette and a copy of cdaA with the putative promoter region into the BtΔcdaA chromosome adjacent to the site of mutagenesis, creating the BtΔcdaA^C-cis strain (Fig. 1B). Genotypic confirmation of BtΔcdaA and BtΔcdaA^C-cis was performed using PCR specific for internal regions of cdaA, aacC1, and aphI genes (Fig. 1C). PCR to amplify a region of the flagellin (flaB) gene was also performed as an amplification control. PCR for cdaA only produced amplicons in BtWT and BtΔcdaA^C-cis. In addition, PCRs specific for the resistance markers failed to yield amplicons with BtWT, whereas BtΔcdaA and BtΔcdaA^C-cis were positive for the aacC1 gene and aphI gene, respectively. Finally, PCR for flaB resulted in amplicons of the expected size for all strains. Immunoblot analyses were then performed to assess CdaA production in BtWT, BtΔcdaA, and BtΔcdaA^C-cis (Fig. 1D). As expected, CdaA was detected in BtWT and BtΔcdaA^C-cis but undetectable in BtΔcdaA. Importantly, levels of FlaB, which was included as a loading control, were consistent across strains. In all, these results indicate successful mutation of cdaA and genetic complementation in BtΔcdaA and BtΔcdaA^C-cis, respectively.

The BtΔcdaA mutant is significantly attenuated in a murine needle-challenge model of RF.

Given the importance of c-di-AMP and DACs for virulence of other bacteria, we hypothesized that CdaA would be required for mammalian infection (44, 54). To test this hypothesis, a murine model of RF was used in which groups of four mice were intradermally needle inoculated with 10² BtWT, BtΔcdaA, or BtΔcdaA^C-cis bacteria (Fig. 2). On days 3 to 14 postinfection, blood samples were taken, and bacteremia was measured by quantitative PCR (qPCR). As expected, all four mice infected with BtWT and BtΔcdaA^C-cis experienced recurring bouts of spirochetemia, with the first peak occurring between days 4 to 6 and maximum bloodstream burden ranging from 10⁶ to 10⁸ bacteria/ml of blood (Fig. 2A and C). Conversely, mice infected with BtΔcdaA failed to reach detectable levels of spirochetemia by qPCR on any day postinfection (Fig. 2B).

Because the limit of detection (LOD) with our qPCR is 10⁴ bacteria/ml of blood, it was possible that BtΔcdaA bacteria are still infectious but failed to reach detectable levels (89). Therefore, we also attempted to culture bacteria out of the bloodstream of infected mice during this experiment, which is predicted to provide an LOD of 4 × 10² live bacteria/ml (see Materials and Methods). As expected, days when BtWT- and BtΔcdaA^C-cis-infected mice were positive by blood culture largely overlapped with days when the mice were positive by qPCR (data not shown). Alternatively, bacteria could not be cultured from the blood of BtΔcdaA-infected mice on any day postinfection (data not shown). As another metric to evaluate infection, mice were also screened for seroconversion at 14 days postinfection against BtWT whole-cell lysates; all BtWT- and BtΔcdaA^C-cis-infected mice seroconverted, but BtΔcdaA-infected mice did not (data not shown). This result suggested that BtΔcdaA failed to stimulate a strong humoral immune response because the mutant was cleared early during infection. In all, these murine infection studies support the hypothesis that CdaA is critical for B. turicatae mammalian infection.

The BtΔcdaA mutational strategy results in polar mutation effects.

Because the 3′ end of cdaA is located only 4 bp upstream of the coding region for bt0009 and cdaA is encoded on the same strand as the six genes that are immediately downstream, it was possible that our BtΔcdaA mutational approach may result in polar mutation effects. Quantitative reverse transcription-PCR (qRT-PCR) was used to measure expression of cdaA and adjacent genes in in vitro-cultured BtWT, BtΔcdaA, and BtΔcdaA^C-cis (Fig. 3). As expected, cdaA transcript was undetected in BtΔcdaA spirochetes, while expression was restored in the BtΔcdaA^C-cis strain, albeit at reduced levels relative to BtWT (0.55-fold change in expression). Expression of bt0007, the gene immediately upstream of bt0008, was unaffected in BtΔcdaA, but expression of the downstream genes bt0009 and bt0010 was reduced approximately 10-fold in the BtΔcdaA strain relative to BtWT. Importantly, this polar mutation effect was near-completely reversed upon complementation in the BtΔcdaA^C-cis strain (mean fold change in expression of 0.93 and 0.50 for bt0009 and bt0010, respectively, relative to BtWT). These results implied that the murine infection defect observed with BtΔcdaA could be due to either mutation of cdaA or to polar mutation effects associated with the mutational strategy. Therefore, additional experiments were required to determine if CdaA was essential for mammalian infection.

Addressing polar mutation effects associated with the BtΔcdaA mutant.

Because the BtΔcdaA mutational strategy resulted in polar mutation effects on genes downstream of bt0008 in the chromosome, we were unable to directly attribute the mammalian infection defect in BtΔcdaA to inactivation of cdaA. As complementation of BtΔcdaA in the BtΔcdaA^C-cis strain resulted in near-complete reversal of the polar mutational effect, we hypothesized that a similar strategy could be used to introduce a copy of cdaA with a point mutation rendering the encoded protein incapable of DAC activity into BtΔcdaA, thereby generating a cdaA mutant without the significant polar mutation effects. This mutant could subsequently be genetically complemented by replacing the point mutant cdaA allele with a wild-type copy to maintain similar expression of adjacent genes in mutant and complemented strains. Rosenburg et al. previously identified a conserved glycine residue in CdaA homologs that can be mutated to an alanine to abrogate DAC activity (90). Using an approach analogous to that employed for generation of BtΔcdaA^C-cis, the BtΔcdaA strain was transformed with a construct to insert the aphI kanamycin resistance cassette and a point mutant copy of cdaA, which contained a single-nucleotide change converting the glycine encoded at position 175 to an alanine (G175A), adjacent to the original mutagenesis site, creating BtΔcdaA^C-cis(G175A) (Fig. 4A). BtΔcdaA^C-cis(G175A) was then complemented by replacing the kanamycin resistance marker and G175A-mutated allele of cdaA with an aadA streptomycin resistance cassette and wild-type copy of cdaA, designated BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) (Fig. 4B). Consistent with the presence of cdaA alleles in BtΔcdaA^C-cis(G175A) and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT), both strains were positive by PCR for cdaA, while the BtΔcdaA mutant screened negative (Fig. 4C). As expected, BtΔcdaA^C-cis(G175A) and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) were positive for the kanamycin and streptomycin resistance markers, respectively, whereas BtΔcdaA, BtΔcdaA^C-cis(G175A), and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) were all PCR positive for the gentamicin marker. Additionally, PCR for flaB resulted in amplicons of the expected size in all B. turicatae strains. We further confirmed the presence and absence of the point mutation in cdaA in BtΔcdaA^C-cis(G175A) and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT), respectively, by PCR amplifying and Sanger sequencing an internal region of cdaA (data not shown). Finally, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analyses confirmed that BtΔcdaA^C-cis(G175A) failed to produce c-di-AMP. Alternatively, c-di-AMP was readily detectable in BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT), but an approximate 2-fold reduction in c-di-AMP levels was observed relative to BtWT (see Discussion) (Fig. 4D).

qRT-PCR analyses were performed to assess in vitro expression of genes adjacent to cdaA in BtΔcdaA^C-cis(G175A) and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) (Fig. 5). As expected, polar mutation effects observed in BtΔcdaA were near-completely reversed in BtΔcdaA^C-cis(G175A) and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT). Although modest reductions in expression were observed in bt0008, bt0009, and bt0010 (ranging from 0.54- to 0.85-fold relative to BtWT), levels of bt0007, cdaA, bt0009, and bt0010 transcripts were similar in BtΔcdaA^C-cis(G175A) and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT). These data imply that any phenotypic differences observed between these strains cannot be attributed to polar mutation effects. Therefore, BtWT, BtΔcdaA^C-cis(G175A), and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) were used in subsequent experiments to further characterize the impact of the cdaA mutation (see below).

BtΔcdaA^C-cis(G175A) is significantly attenuated in a murine needle-challenge model of RF.

To assess if the G175A point mutation in cdaA resulted in attenuated mammalian infection, groups of four mice were intradermally inoculated with 10² BtΔcdaA^C-cis(G175A) or BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) bacteria (Fig. 6). While BtΔcdaA^C-cis(G175A) spirochetes were not detected on any day postinfection in the bloodstream by qPCR (Fig. 6A), BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT)-infected mice reached levels of 10⁶ to 10⁸ bacteria/ml of blood during recurrent peaks (Fig. 6B), which is consistent with BtWT infection (Fig. 2A). Furthermore, bacteria could not be cultured from the blood of BtΔcdaA^C-cis(G175A)-infected mice, but BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) spirochetes were cultured from the blood on days postinfection when spirochetemia was detectable by qPCR (data not shown). Finally, all four BtΔcdaA^C-cis(G175A)-infected mice failed to seroconvert when BtWT lysates were probed with serum collected 14 days postinfection, indicating the bacteria were likely eliminated early during infection. Conversely, all four mice infected with the BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) strain seroconverted (data not shown). These results indicate that DAC activity of CdaA is crucial for B. turicatae mammalian infection and that polar mutation effects were not the sole reason for the attenuation seen with BtΔcdaA (Fig. 2).

cdaA mutants contain chromosomal single-nucleotide polymorphisms.

Because c-di-AMP signaling is essential for growth in many bacterial systems under standard culture conditions, it was possible that suppressor mutations were required to generate the BtΔcdaA mutant (34, 40, 56, 60–63). To test this, WGS was performed on two independently generated BtΔcdaA clones and the parental BtWT strain. These analyses identified SNPs in both mutants (Table 1). BtΔcdaA clone 1 had two missense mutations in the chromosomal genes bt0380 and bt0531, which encode an MgtE family Mg²⁺ transporter and N-acetylmuramoyl-l-alanine amidase, respectively. MgtE family transporters are widespread in eukaryotes and prokaryotes and primarily transport divalent cations, such as Mg²⁺ and Co²⁺ (91, 92). N-acetylmuramoyl-l-alanine amidases are ubiquitous bacterial proteins involved in turnover of the cell wall (93). In BtΔcdaA clone 2, four chromosomal SNPs were discovered. Missense mutations were identified in bt0219 and bt0747, which encode a ZIP family metal transporter and an oligopeptide transport system permease protein (OppB), respectively. ZIP family metal transporters are found in eukaryotes and prokaryotes, and they generally have broad specificity for divalent cations, including Zn²⁺, Fe²⁺, and Mn²⁺ (94, 95). OppB serves as a membrane permease that is part of the borrelial oligopeptide transport system (96). Additionally, nonsense mutations were found in bt0127 and bt0241B, which encode the 30S ribosomal protein S1 and a glycerophosphoryl diester phosphodiesterase (GlpQ), respectively. The 30S ribosomal protein S1 is a component of the prokaryotic 30S ribosomal subunit that participates in binding and unfolding of specific mRNA structures and allows for correct ribosomal positioning during translation initiation (97). GlpQ cleaves deacylated phospholipids to glycerol-3-phosphate, which can be used for phospholipid biosynthesis or be shunted into the glycolytic pathway via a dihydroxyacetone phosphate intermediate (98). In all, WGS of BtΔcdaA clones identified SNPs in proteins involved in membrane transport, metabolism, and translation, possibly representing compensatory suppressor mutations required for normal physiology during in vitro growth. Of note, BtΔcdaA clone 1 was used for initial infection experiments (Fig. 2), as well as for derivation of BtΔcdaA^C-cis(G175A) and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) clones used herein.

cdaA is required for normal growth and physiology in vitro.

The identification of SNPs in two independently generated BtΔcdaA clones could imply that suppressor mutations are required for in vitro viability in the absence of CdaA. To evaluate this possibility, a conditional mutational strategy was used (99, 100). A B. turicatae shuttle vector carrying a lacI cassette and cdaA under transcriptional control of a lac-inducible promoter was transformed into BtWT, generating BtiCdaA. BtiCdaA was then transformed with the ΔcdaA mutational construct and grown in the presence of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) to maintain cdaA expression until use of the conditional mutant in CdaA depletion experiments. Genotypic confirmation of the conditional mutant, designated BtiCdaA-ΔcdaA, was achieved by PCR to amplify a region flanking the site of mutation, as well as a PCR to amplify an internal region of the aacC1 gene (Fig. 7A). The PCR flanking the site of mutation (cdaA ext diag) revealed amplicons of the appropriate sizes in BtiCdaA and BtiCdaA-ΔcdaA, while the PCR for aacC1 only generated a product in the BtiCdaA-ΔcdaA strain, consistent with successful mutagenesis. PCRs for flaB resulted in amplicons of the appropriate size in BtiCdaA and BtiCdaA-ΔcdaA strains.

To determine whether depletion of CdaA resulted in an in vitro physiological defect, BtiCdaA-ΔcdaA was grown to late exponential phase, washed twice in media to remove IPTG, and then inoculated at an initial density of 10³ bacteria/ml of modified Barbour-Stoenner-Kelly (mBSK) medium with or without 1 mM IPTG. Growth was then quantified daily by dark-field microscopy (Fig. 7B). Interestingly, BtiCdaA-ΔcdaA grown in the absence of IPTG exhibited a growth defect, with significantly reduced densities on days 7 to 10 postinoculation relative to BtiCdaA-ΔcdaA cultured with IPTG. BtiCdaA-ΔcdaA cultured without IPTG also exhibited extensive membrane blebbing visible by bright-field microscopy (Fig. 7C and D). In fact, 53.5% of BtiCdaA-ΔcdaA spirochetes grown without IPTG had visual membrane blebbing, while only 10% of BtiCdaA-ΔcdaA bacteria grown with IPTG had membrane blebs. Finally, to confirm successful depletion of CdaA, immunoblot analyses were performed (Fig. 7E). As expected, CdaA was readily detectable in BtiCdaA-ΔcdaA cultured with IPTG, but the protein was undetectable in BtiCdaA-ΔcdaA grown without IPTG. Blots for FlaB were comparable regardless of IPTG treatment. Overall, these results indicated that conditional mutation of cdaA does not necessarily result in cell death in vitro, but CdaA is required for normal bacterial growth and physiology. Importantly, given that the two BtΔcdaA mutants used for WGS did not have an in vitro growth defect or any noticeable morphological changes by dark-field microscopy (data not shown), it is likely that the identified SNPs represent suppressor mutations which contribute to the c-di-AMP-responsive physiology of these clones in vitro.

Inactivation of cdaA does not impact bosR or rpoS expression or protein production.

In B. burgdorferi, the RpoS alternative sigma factor controls expression of several known virulence determinants and is essential for virulence (101–103). Borrelia oxidative stress regulator (BosR) in B. burgdorferi binds upstream of rpoS and serves as a transcriptional activator (104–107). Not surprisingly, BosR is also required for mammalian infection by B. burgdorferi (104, 106). Ye et al. demonstrated that conditional depletion of the c-di-AMP PDE, DhhP, in B. burgdorferi resulted in increased levels of c-di-AMP, as well as modestly reduced expression of bosR and significantly reduced expression of rpoS (64). BosR and RpoS protein levels were also markedly reduced, resulting in decreased production of virulence factors (64). This led us to hypothesize that mutation of cdaA could impact BosR and/or RpoS production in B. turicatae, possibly contributing to the observed infection defect. To evaluate this hypothesis, bosR and rpoS transcription (Fig. 8A) and protein production (Fig. 8B and C) were measured in in vitro cultured BtWT, BtΔcdaA^C-cis(G175A), and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT). Interestingly, there were only modest changes in expression of either regulator in BtΔcdaA^C-cis(G175A) or BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) relative to BtWT (1.12- and 0.81-fold change, respectively, for bosR; 0.94- and 1.03-fold change, respectively, for rpoS). Immunoblot analyses also revealed no substantial changes in BosR or RpoS production between BtWT, BtΔcdaA^C-cis(G175A), and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT), and levels of FlaB, which served as a loading control, were equivalent in the three strains. To confirm the specificity of the BosR and RpoS antiserum used in immunoblot analyses, a B. turicatae shuttle vector carrying a lacI cassette and either bosR or rpoS under transcriptional control of a lac-inducible promoter was transformed into BtWT, generating the strains BtiBosR and BtiRpoS, respectively. As expected, when BtiBosR and BtiRpoS were treated with 1 mM IPTG, increases in BosR and RpoS protein levels, respectively, were observed. In all, mutation of cdaA appeared to have no effect on bosR and rpoS transcription or protein production in vitro. This observation strongly suggests that c-di-AMP-mediated gene regulation differs between RF and LD spirochetes.

The cdaA mutant is sensitive to increased salt.

Considering the critical role of c-di-AMP signaling in osmoregulation in many bacterial systems, it is not surprising that mutation of c-di-AMP signaling pathway components results in altered susceptibility to salt treatment (42–44, 50, 58, 65, 108). To determine if mutation of cdaA in B. turicatae also results in altered resistance to salt treatment, the maximum concentrations of NaCl and KCl at which BtWT could grow was first determined using previously described MIC plating assays (109, 110). BtWT was able to grow in concentrations up to 50 mM for each salt (data not shown), so these concentrations were used in subsequent growth curve analyses with BtWT, BtΔcdaA^C-cis(G175A), and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) strains (Fig. 9). In standard mBSK media, BtΔcdaA^C-cis(G175A) and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) strains showed modestly increased growth rates relative to BtWT, with BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) having a significantly increased density on day 5 postinoculation (see Discussion) (Fig. 9A). With 50 mM NaCl supplementation, all strains grew slower than that seen in mBSK media (Fig. 9B). However, BtΔcdaA^C-cis(G175A) had a growth defect relative to BtWT, exhibiting significantly lower numbers of bacteria on 6 to 14 days postinoculation, and genetic complementation of BtΔcdaA^C-cis(G175A) failed to reverse this growth phenotype. Significant growth defects were also observed in BtΔcdaA^C-cis(G175A) and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) relative to BtWT when grown with 50 mM KCl; both strains had significantly reduced numbers of bacteria on days 6 to 7 postinoculation (Fig. 9C). The inability to complement the salt-dependent growth defect in BtΔcdaA^C-cis(G175A) suggests that (i) suppressor mutations, (ii) decreased c-di-AMP levels, or (iii) modestly reduced expression of genes downstream of cdaA in BtΔcdaA^C-cis(G175A) and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) strains could be responsible for the phenotype. Therefore, more evidence is required to elucidate a possible role for CdaA in survival under conditions of increased osmolarity (see Discussion).

Mutation of cdaA has no effect on growth under nutrient-limited conditions but results in a significant growth defect at decreased osmolarity.

Because c-di-AMP signaling and the bacterial stress response have been linked in S. aureus and L. monocytogenes (55–57), it was possible that CdaA may play a role in survival under nutrient-limited conditions. To assess this hypothesis, growth curve analyses were performed with BtWT, BtΔcdaA^C-cis(G175A), and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) strains in either standard mBSK media (Fig. 10A) or in media diluted 1:10 in 1× phosphate-buffered saline (PBS) to simulate nutrient-limited conditions (Fig. 10B) (111, 112). As expected, BtΔcdaA^C-cis(G175A) and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) strains exhibited an increased growth rate in standard mBSK relative to the BtWT strain (see Fig. 9A). In media diluted 1:10 in 1× PBS, however, BtΔcdaA^C-cis(G175A) failed to reach numbers detectable by dark-field microscopy. Alternatively, BtWT and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) strains were able to grow in the dilute media, indicating that CdaA is required for growth under these conditions.

It should be noted that the osmolarity of mBSK medium is 450 mOsm, and the osmolarity of 1× PBS is 300 mOsm (137 mM NaCl, 2.7 mM KCl, and 11.9 mM phosphates) (113). Therefore, dilution of the media with PBS alters both the nutrient composition and osmolarity of the media. Given the role of c-di-AMP signaling in osmoregulation in other bacteria, a possibility for the observed growth defect in media diluted 1:10 in 1× PBS could be the decrease in osmolarity (58). To address this alternative, growth curves were performed with BtWT, BtΔcdaA^C-cis(G175A), and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) in mBSK diluted 1:10 in 1.25× PBS (375 mOsm 171.25 mM NaCl, 3.38 mM KCl, and 14.88 mM phosphates) (Fig. 10C) and mBSK diluted 1:10 in 1.50× PBS (450 mOsm 205.5 mM NaCl, 4.05 mM KCl, and 17.85 mM phosphates) (Fig. 10D). BtWT and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) grew similarly in mBSK diluted 1:10 in 1.25× PBS, but BtΔcdaA^C-cis(G175A) exhibited a growth defect with significantly reduced densities on days 5 to 8 postinoculation. BtΔcdaA^C-cis(G175A) was not able to grow in mBSK diluted in 1× PBS, but detectable bacterial densities of BtΔcdaA^C-cis(G175A) (e.g., maximum of 10⁶ bacteria/ml) were observed in mBSK diluted in 1.25× PBS. Interestingly, in mBSK diluted 1:10 in 1.50× PBS, which is the same osmolarity as mBSK, growth was similar between all three strains. These data imply that, rather than being required to tolerate nutrient-limited conditions in vitro, CdaA is essential for growth at decreased osmolarity, suggesting a critical role for c-di-AMP in osmoregulation in B. turicatae.

The cdaA mutant requires pyruvate for growth in vitro.

c-di-AMP signaling is important for resistance to ROS in other bacterial systems (41, 51–53). Therefore, experiments were performed to assess if mutation of cdaA in B. turicatae led to altered susceptibility to oxidative stresses. Experiments to measure the sensitivity of Borrelia spirochetes to ROS are typically performed using media lacking pyruvate, as pyruvate can act as an ROS scavenger and decrease the assay sensitivity (89, 114, 115). When attempting to test the susceptibility of the cdaA mutant to oxidative agents, it was serendipitously found that BtΔcdaA^C-cis(G175A) failed to reach an adequate density in mBSK medium lacking pyruvate. Following this observation, growth curve analyses were performed to quantify differences in growth of BtWT, BtΔcdaA^C-cis(G175A), and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) in mBSK with or without pyruvate (Fig. 11). As expected, in the normal media formulation, BtΔcdaA^C-cis(G175A) and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) had modestly increased growth rates relative to the BtWT strain (Fig. 11A). However, BtΔcdaA^C-cis(G175A) exhibited a growth defect in mBSK lacking pyruvate, reaching significantly lower bacterial densities than BtWT on days 5 to 8 postinoculation (Fig. 11B). Importantly, genetic complementation in BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) reversed this phenotype. Therefore, while the contribution of cdaA to ROS resistance could not be determined using media lacking pyruvate (see Discussion), pyruvate was found to be critical for growth of the cdaA mutant in vitro. Given the critical role of pyruvate in bacterial metabolism, these results possibly link c-di-AMP signaling to an important function in central metabolism in B. turicatae.

Bacterial strains and culture conditions.

Bacterial strains used in this study are listed in Table 2. E. coli strain TOP10F′ (Life Technologies, Carlsbad, CA) was used for cloning and plasmid propagation, and E. coli strain C41(DE3) (Lucigen, Middleton, WI) was used for expression of recombinant protein. E. coli was cultured at 37°C in Luria-Bertani (LB) medium supplemented with 100 μg/ml ampicillin, 5 μg/ml gentamicin, 50 μg/ml kanamycin, 100 μg/ml spectinomycin, or 30 μg/ml chloramphenicol when appropriate. Low-passage B. turicatae strain 91E135 (Oz1), designated BtWT, was used in this study (149, 150). B. turicatae strains were passaged no more than twice beyond the original frozen stock. B. turicatae was cultured at 35°C with 3% CO₂ in modified Barbour-Stoenner-Kelly (mBSK) medium with 12% rabbit serum at pH 7.6 unless noted otherwise (151, 152). mBSK was supplemented with 40 μg/ml gentamicin, 150 μg/ml kanamycin, or 150 μg/ml streptomycin when appropriate.

Generation of constructs used in this study.

Plasmids and primers used in this study are detailed in Tables 2 and 3, respectively. PrimeStar Max DNA polymerase (TaKaRa Bio, Mountain View, CA) was used for PCR, and all amplicons were TA cloned into pGEM-T Easy (Promega Corp., Fitchburg, WI) and confirmed by Sanger sequencing. For expression of recombinant protein, the full bosR ORF (primers 5′ BtBosR ORF BamHI and 3′ BtBosR ORF SpeI), amino acids 120 to 131 of the rpoS ORF (primers 5′ BtRpoS-trun_BamHI and 3′ BtRpoS-trun_SpeI), and the cdaA ORF lacking the N-terminal region encoding the predicted signal sequence and transmembrane domains (amino acids 1 to 90; primers 5′ bt0008 w/o TM BamHI and 3′ bt0008 ORF BamHI) were amplified from genomic BtWT DNA (gDNA). The putative transmembrane domains of CdaA were predicted using TMHMM-2.0 (85, 86). cdaA, rpoS, and bosR fragments were excised with their respective restriction enzymes and ligated into linearized pProEX-HTb (Life Technologies) to generate pUAMS338, pUAMS233, and pUAMS159, respectively.

Allelic exchange mutagenesis was used for generation of the BtΔcdaA mutant and subsequent complementation. To make the ΔcdaA mutational construct, 5′ (primers 5′ F1 bt0008 KO and 3′ F1 bt0008 KO_AscI) and 3′ (primers 5′ F2 bt0008 KO_AscI and 3′ F2 bt0008 KO_BssHII) flanking regions were amplified from BtWT gDNA. These flanking regions were then ligated together with the B. turicatae-adapted gentamicin resistance cassette from pUAMS4 between them, generating the ΔcdaA construct pUAMS248B (89). In the final mutational construct, the aacC1 marker replaces a 419-bp internal region between nucleotides 86 and 504 of the bt0008 ORF. For complementation, a B. turicatae-adapted kanamycin resistance cassette was made by amplifying the putative promoter region for the flaB gene from BtWT gDNA (primers 5′ BtFlaB-aphI-BamHI and 3′ Kan ORF int -NdeI v.2) and the Tn903-derived aminoglycoside phosphotransferase ORF (aphI) from pBSV2 (primers 5′ Kan ORF int -NdeI v.2 and 3′ BtFlaB-aphI-BamHI) (153). These amplicons were then fused together by overlap extension PCR, and the resulting product was ligated into pGEM-T Easy, generating the plasmid pUAMS309. To create the ΔcdaA^C-cis complementation construct, a segment encoding the 5′ flanking region and disrupted cdaA ORF (primers 5′ F1 bt0008 KO and 5′ BtflgB-BamHI) was amplified from pUAMS248B, and a segment encoding the intact cdaA ORF with its adjacent upstream region, as well as a downstream segment to facilitate recombination (primers 5′ F2 bt0008 Comp-BamHI and 3′ F2 bt0008 Comp-BglII), was amplified from BtWT gDNA. These segments were then ligated together with the PflaB-aphI resistance cassette from pUAMS309 between them, yielding the final ΔcdaA^C-cis construct, pUAMS313B.

To complement the BtΔcdaA mutant with a mutated copy of cdaA containing a point mutation converting a glycine at position 175 to an alanine, an approach similar to the BtΔcdaA^C-cis complementation strategy was used. To introduce the G175A point mutation into cdaA, overlap extension PCR was performed with pUAMS313B serving as the template (primer pairs 5′ DAC/cis bt0008 and 3′ bt0008 G175A; 5′ bt0008 G175A and 3′ DAC/cis bt0008). The full-length product was then ligated into pUAMS313B, replacing the analogous region and yielding the ΔcdaA^C-cis(G175A) construct, pUAMS351B.

For complementation of BtΔcdaA^C-cis(G175A), a B. turicatae-adapted aadA streptomycin resistance cassette was first made by amplifying the putative promoter region for the flaB gene from BtWT gDNA (primers 5′ BtPflaB_BamHI and 3′ PflaB NdeI) and the aminoglycoside adenylyltransferase ORF (aadA) from pJD7 (primers 5′ BtflaB-aadA junc_NdeI and 3′ aadA ORF_BamHI) (154). The promoter and aadA ORF were fused by ligating the two amplicons together using the common NdeI restriction site. The final ligated PflaB-aadA cassette in pGEM-T Easy was designated pUAMS402. The PflaB-aadA cassette was subsequently ligated into pUAMS313B in place of the PflaB-aphI cassette using the BamHI sites, generating the final ΔcdaA^C-cis(G175A)::cdaA^C-cis(WT) construct pUAMS406B.

To generate the B. turicatae shuttle vector, pBtSV-JB, a region capable of autonomous replication in B. turicatae (BtOri), based on homology to B. burgdorferi and B. hermsii shuttle vectors, was assembled with an E. coli pUC/pMB1 ori and the aphI gene expressed from the B. turicatae flaB promoter (152, 153, 155). The pUC/pMB1 ori was amplified from the B. burgdorferi shuttle vector pJD44 (primers 5′ pJD44 ColE1 ori and 3′ pJD44 ColE1 ori). The BtOri region was then amplified from BtWT gDNA (primers 5′ UAMS BtOri BamHI and 3′ UAMS BtOri AscI). Next, the PflaB-aphI kanamycin resistance marker was amplified from pUAMS309 (primers 5′ UAMS-88 Kan BglII and 3′ UAMS-88 Kan AscI) and ligated into pJD44 to fuse the pJD44 multiple cloning site (MCS) to PflaB-aphI (pJD44::PflaB-aphI). pBtSV-JB was assembled by excising the MCS-PflaB-aphI fusion from the pJD44::PflaB-aphI intermediate with AscI and ligating with the BglII/AscI-digested ColE1 fragment and BamHI/AscI-digested BtOri region.

The lac-inducible expression constructs were generated in the backbone of pBtSV-JB (above). The region containing the MCS and lac-inducible promoter Bbluc+ of pJSB104 was moved into pBtSV-JB using BamHI and HindIII (154). For overproduction of LacI, the Borrelia codon-adapted BbLacI was expressed from the B. turicatae flagellar basal body rod protein (flgB) promoter. The flgB promoter was amplified from BtWT gDNA (primers 5′ Bt-PflgB/BamHI-HindIII and 3′ Bt-PflgB/BbLacI junc), and the BbLacI ORF was amplified from pJSB104 (primers 5′ Bt-PflgB/BbLacI junc and 3′ Bt-BbLacI/BglII). These amplicons were then fused together by overlap extension PCR, and the resulting product was ligated into pGEM-T Easy. For the construct used to overexpress rpoS and cdaA, the PflgB-BblacI fusion was excised with BamHI and BglII and then ligated into BglII-digested pBtSV-JB. For the construct used to overexpress bosR, the PflgB-BblacI fusion was excised with BamHI and BglII and then ligated into BamHI-digested pBtSV-JB. Next, the rpoS, cdaA, and bosR ORFs were amplified from BtWT gDNA with the following primer pairs: 5′ BtRpoS ORF-NdeI and 3′ BtRpoS ORF-HindIII, 5′ bt0008 ORF NdeI and 3′ bt0008 ORF HindIII, and 5′ BtBosR ORF NdeI and 3′ BtBosR ORF HindIII, respectively. These ORF regions were digested with NdeI and HindIII and ligated into the inducible expression constructs digested with the same enzymes to generate pUAMS267A (piRpoS), pUAMS346 (piCdaA), and pUAMS446B (piBosR), respectively.

Transformation of B. turicatae, clonal isolation, and genotypic confirmation.

Electroporation of B. turicatae was performed as previously described (156). Briefly, the mutagenesis construct, complementation construct, or shuttle vector of interest was electroporated into the appropriate strain. Following a 24-h recovery period in mBSK medium without selection, transformants were selected for antibiotic treatment. Clones were then isolated by serial dilution plating. All confirmatory PCRs herein were separated by electrophoresis in a 0.8% agarose gel and visualized by ethidium bromide staining. GeneRuler DNA Ladder mix (Thermo Fisher Scientific, Waltham, MA) served as the molecular weight standard. To confirm shuttle vector-transformed clones, plasmids were recovered by transformation into E. coli, analyzed by restriction digest, and Sanger sequenced (data not shown).

To confirm BtΔcdaA mutant and BtΔcdaA^C-cis clones, PCRs were performed to amplify a region within the replaced segment of cdaA (primers 5′ bt0008 int diag and 3′ bt0008 int diag), an internal segment of aacC1 (primers 5′ aacC1 diag and 3′ aacC1 diag), an internal region of aphI (primers 5′ aphI diag and 3′ aphI diag), and an internal segment of flaB as an amplification control (primers 5′ BtFlaB and 3′ BtFlaB). To confirm complementation of the BtΔcdaA strain with the mutated cdaA ORF [BtΔcdaA^C-cis(G175A)] and subsequent complementation with the wild-type copy of cdaA [BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT)], PCRs were conducted to amplify an internal segment of cdaA (see above), an internal segment of aphI (see above), an internal segment of aadA (primers 5′ aadA diag and 3′ aadA diag), and an internal segment of flaB (see above). A region of cdaA containing the point mutation was additionally amplified (primers 5′ bt0008 int diag and 3′ bt0008 ext diag), gel purified, and Sanger sequenced to confirm the G175A mutation.

The cdaA conditional mutant (BtiCdaA-ΔcdaA) was generated similar to previously described methods in B. burgdorferi (99, 100). BtWT was first transformed with pUAMS346 to generate BtiCdaA. A confirmed BtiCdaA clone was then transformed with the ΔcdaA mutational construct pUAMS248B, and bacteria were recovered after electroporation in the presence of 1 mM IPTG to maintain expression of cdaA in spirochetes that acquired the mutation. Following the 24-h recovery period, bacteria were passed into media containing kanamycin, gentamicin, and 1 mM IPTG to select for bacteria with the ΔcdaA mutation. BtiCdaA-ΔcdaA clones were then isolated using serial dilution plating (156). The conditional mutant was confirmed to harbor pUAMS346 by plasmid recovery in E. coli, and the mutation was confirmed by differential PCR using primers flanking the mutation (primers 5′ bt0008 ext diag and 3′ bt0008 ext diag), as well as PCRs for internal segments of the aacC1 marker (see above) and flaB gene (see above).

Expression and purification of recombinant protein.

Expression from pProEX-HTb generates recombinant protein with an N-terminal His₆ tag and tobacco etch virus (TEV) cleavage site. pUAMS338 was transformed into C41(DE3) E. coli, and expression of recombinant CdaA lacking the transmembrane domains was achieved by induction with 1 mM IPTG for 3 h at 37°C. For expression of recombinant truncated RpoS, pUAMS233 was also transformed into C41(DE3) E. coli, and expression was induced with 1 mM IPTG for 3 h at 37°C. For expression of recombinant BosR, pUAMS159 was cotransformed into C41(DE3) E. coli with pRARE (Novagen, Madison, WI), a plasmid isolated from RosettaBlue(DE3) cells that encodes tRNAs to express genes with rare E. coli codons (157). Expression of recombinant BosR was then achieved by induction with 1 mM IPTG for 3 h at 37°C. Recombinant CdaA, RpoS, and BosR were purified using HisPur nickel-nitrilotriacetic acid (Ni-NTA) resin (Thermo Fisher Scientific) under nonnative conditions. Briefly, cells were lysed using BugBuster protein extraction reagent (MilliporeSigma, Burlington, MA) in conjunction with Lysonase (MilliporeSigma) per the manufacturer’s instructions. Following cell lysis, soluble and insoluble fractions were separated by centrifugation at 24,000 × g for 15 min. Inclusion bodies were then solubilized in resin wash buffer (20 mM Tris, 20 mM NaCl, 5% glycerol, pH 7.5) supplemented with 0.3% N-lauryl-sarcosine. Ni-NTA resin was washed two times in this same buffer prior to combining with the solubilized inclusion body. Binding was then performed using an end-over-end rotator for >30 min. Following binding, the resin was washed in 10 bed volumes of buffer A (20 mM Tris, 20 mM NaCl, 20 mM imidazole, and 5% glycerol, pH 7.5) supplemented with 0.3% N-lauryl-sarcosine, followed by a wash in 10 resin bed volumes of buffer B (20 mM Tris, 1 M NaCl, and 5% glycerol, pH 7.5) supplemented with 0.3% N-lauryl-sarcosine. A final wash was then performed in 10 resin bed volumes of buffer A with 0.3% N-lauryl-sarcosine. Finally, the protein was eluted from the resin using buffer C (20 mM Tris, 200 mM NaCl, 250 mM imidazole, and 5% glycerol, pH 7.5) with 0.3% N-lauryl-sarcosine. Following purification, concentrations of purified recombinant proteins were calculated using the DC protein assay kit (Bio-Rad Laboratories, Hercules, CA).

Generation of CdaA-, RpoS-, and BosR-specific antiserum.

Rat immunizations were performed in accordance with the recommendations of the Public Health Science (PHS) Policy on Humane Care and Use of Laboratory Animals, the Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act, and the utilized protocol was approved by the University of Arkansas for Medical Sciences (UAMS) Institutional Animal Care and Use Committee (IACUC). Generation of CdaA-, RpoS-, and BosR-specific rat antiserum was performed as previously described (158). Briefly, 25 μg of recombinant protein in 200 μl of phosphate-buffered saline (PBS) was emulsified with 200 μl of complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO) and intraperitoneally injected into 3- to 4-week-old female Sprague-Dawley rats (Envigo, Indianapolis, IN, and Charles River Laboratories, Wilmington, MA). Rats were subsequently boosted twice at 4-week intervals by intraperitoneal injection of 25 μg of recombinant protein in 200 μl of PBS emulsified with 200 μl of incomplete Freund’s adjuvant (Sigma-Aldrich). Two weeks following the final boost, rats were euthanized, and serum was collected.

SDS-PAGE and immunoblotting.

Immunoblot analyses were performed as previously described (159). Briefly, whole-cell lysates were prepared from B. turicatae cultures grown to late exponential phase. A volume of lysate equivalent to 2 × 10⁷ spirochetes was then separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were subsequently probed with rat antiserum recognizing CdaA, BosR, or RpoS (see above). Horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as a secondary antibody. As a positive control, membranes were additionally probed for FlaB using chicken anti-B. burgdorferi FlaB IgY as the primary antibody and HRP-conjugated donkey anti-chicken IgY (Jackson ImmunoResearch Laboratories) as a secondary antibody (159). To assess seroconversion of mice from murine infection experiments (see below), BtWT lysates were probed with serum from infected or naive mice, and HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) served as the secondary antibody (data not shown). For all blots, colorimetric detection was achieved using 4-chloro-1-naphthol as a substrate, and Precision Plus Protein all blue prestained protein standard (Bio-Rad Laboratories) served as the molecular weight standard.

Murine infection experiments.

Murine infection experiments were performed in accordance with the recommendations of the PHS Policy on Humane Care and Use of Laboratory Animals, the Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act, and the utilized protocol was approved by the UAMS IACUC. Infections were performed as previously described (89). Briefly, 4- to 6-week-old female Swiss Webster mice (Charles River Laboratories) were intradermally/subcutaneously injected with 10² bacteria in the thoracic region. On days 3 to 14 postinfection, 2.5 μl of blood was taken by tail vein venipuncture, combined with 47.5 μl of SideStep lysis and stabilization buffer (Agilent Technologies, Santa Clara, CA), and stored at −80°C until qPCR analyses were performed to quantify bacteremia (see below). An additional 2.5 μl of blood was then collected and added to mBSK medium supplemented with Borrelia antibiotic mixture (BAM) (Monserate, San Diego, CA) to assess presence of live bacteria in the bloodstream. On day 14, mice were euthanized, blood was collected by brachial artery bleed, and serum was isolated for immunoblot analyses for seroconversion. Two weeks following collection of daily blood samples, cultures were assessed for presence of spirochetes; 10 fields of view were scanned by dark-field microscopy, and presence of one or more spirochetes was considered culture positive. Given the volume of blood taken during the murine infection experiments, the LOD for this analysis is equivalent to one spirochete/2.5 μl of blood (4 × 10² bacteria/ml). For use in qPCR standards, naive mice were euthanized, and blood was collected by brachial artery bleed and added to lysis and stabilization buffer at a blood-to-buffer ratio of 1:18. Blood/buffer mixtures from naive mice were stored at −80°C until standard preparation (see below).

qPCR for bacterial burdens.

qPCR analyses to detect spirochetemia in mice were performed as previously described (89). Briefly, in a 96-well real-time PCR plate, 3 μl of blood in lysis and stabilization buffer (see above) was added in a 20-μl reaction mixture containing 10 μl of SsoAdvanced universal probes supermix (Bio-Rad Laboratories) and final concentrations of 400 nM and 300 nM for primers (BtflaB F and BtflaB R) and probe (BtflaB-Probe), respectively. For qPCR standards, late-exponential BtWT cultures were pelleted, washed twice in PBS supplemented with 5 mM MgCl₂ (PBS-MgCl₂), and then resuspended in PBS-MgCl₂ and quantified by dark-field microscopy. This suspension was then used to make serial dilutions in PBS-MgCl₂ from 10⁴ to 10⁸ bacteria/ml. For use in the no-template control (NTC), nuclease-free water was diluted 10-fold in PBS-MgCl₂. These dilutions were then added to naive blood in lysis and stabilization buffer (see above) at a 1:19 ratio, and 3 μl of these preparations were added in a 20-μl reaction mixture, as described for murine blood samples above, to generate a standard curve. qPCRs for all samples and standards were performed in triplicate. The QuantStudio 6 Flex real-time PCR system (Thermo Fisher Scientific) was used for real-time PCR, and the reaction conditions consisted of an initial 2 min, 50°C hold followed by a 10 min, 95°C polymerase activation step. Forty cycles of DNA denaturation at 95°C for 15 s and primer annealing/DNA extension at 60°C for 60 s were then performed for DNA amplification. Data were then imported into GraphPad Prism version 8 (GraphPad Software, San Diego, CA) for analysis.

qRT-PCR analyses.

RNA extraction, cDNA synthesis, and qRT-PCR were performed as previously described (89, 154, 158). Briefly, B. turicatae cultures were grown to late exponential growth phase followed by addition of 10% RNA stop solution (154, 160). Bacteria were then collected by centrifugation and stored at −80°C until RNA isolation was performed. RNA was isolated by TRIzol extraction (Thermo Fisher Scientific), followed by purification with the RNeasy minikit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Treatment with RNase-free DNase I (Qiagen) was then performed to degrade possible contaminating DNA, and absence of B. turicatae gDNA was confirmed using PCR to amplify an internal segment of the flaB gene (see above). The iScript cDNA synthesis kit (Bio-Rad Laboratories) was then used to reverse transcribe purified RNA into cDNA via the manufacturer’s protocol. As a negative control, mock reactions were performed in the absence of reverse transcriptase. Successful cDNA synthesis was then confirmed by PCR amplification of an internal segment of the flaB gene (see above).

qRT-PCR was used to measure expression of bt0007 (primers S-bt0007_IDT-SYBR and AS-bt0007_IDT-SYBR), cdaA (primers S-bt0008_IDT-SYBR_V2 and AS-bt0008_IDT-SYBR_V2), bt0009 (primers S-bt0009_IDT-SYBR and AS-bt0009_IDT-SYBR), bt0010 (primers 2_bt0010_IDT-SYBR FWD and 2_bt0010_IDT-SYBR REV), bosR (primers bt0647 SYBR/IDT FWD Set 1 and bt0647 SYBR/IDT REV Set 1), rpoS (primers BtrpoS SYBR FWD and BtrpoS SYBR REV), and flaB (primers 5′ BtflaB SYBR/ABI and 3′ BtflaB SYBR/ABI). SYBR reactions were performed with SsoAdvanced universal SYBR green supermix (Bio-Rad Laboratories) per the manufacturer's instructions with 100 ng of cDNA serving as the template, and all reactions were performed in triplicate. The QuantStudio 6 Flex real-time PCR system was used for real-time PCR with reaction conditions consisting of an initial polymerase activation step at 95°C for 30 s and 40 cycles of DNA denaturation at 95°C for 10 s and primer annealing/DNA extension at 60°C for 30 s. Results were imported into GraphPad Prism version 8 for analysis.

Whole-genome sequencing and SNP analysis.

For WGS analyses, B. turicatae strains of interest were grown to late exponential growth phase, and DNA was purified using the DNeasy blood and tissue kit (Qiagen) via the manufacturer’s instructions. gDNA quality was then analyzed using the TapeStation 2200 system (Agilent Technologies), and only samples with DNA integrity numbers (DINs) above 8 were utilized. The Nextera XT DNA library preparation kit (Illumina Inc., San Diego, CA) was used for library preparation, and the MiSeq system (Illumina Inc.) was used for next-generation sequencing using 500-cycle, 2 × 250-bp chemistry. The paired-end FASTQ files generated were processed using fastp (v0.20.0) for quality filtering (Phred score ≥20), adapter trimming, and read correction (161). All sequencing data sets had a coverage of approximately ×400 to ×500 postfiltering and correction. Variant analysis was performed using an “all-in-one” variant calling pipeline, Snippy (v4.6.0), using default parameters (–mapqual 60, –basequal 13, –mincov 10, –minqual 100) with –cpus 30 (162). The updated, complete B. turicatae 91E135 genome generated by the Job Lopez laboratory at Baylor College of Medicine was used as the reference (J. E. Lopez and A. R. Kneubehl, unpublished data). BtWT was used to assess any differences between the B. turicatae 91E135 from our lab and what was sequenced by the Lopez laboratory. This analysis yielded only eight SNPs/variants between BtWT and the Lopez B. turicatae 91E135 genome. Variants were then assessed in BtΔcdaA clones 1 and 2 using the same reference genome. The variants reported herein were those found to be different from the BtWT variants, indicating differences between the mutant clones and their parental cell line compared to the reference genome. The variant analyses were performed on a System76 Thelio Massive Linux machine with an Intel Xeon Gold 6230 processor and 126 Gb ECC DDR4 2933 MHz RAM (System76, Denver, CO). All SNPs were confirmed by amplifying a segment containing the SNP, gel purifying the product, and Sanger sequencing. The following primer pairs were used for amplification of the regions containing the SNPs in bt0380, bt0531, bt0127, bt0219, bt0241B, and bt0747: 5′ BT0380 SNP Diag and 3′ BT0380 SNP Diag, 5′ BT0531 SNP Diag and 3′ BT0531 SNP Diag, 5′ BT0127 SNP Diag and 3′ BT0127 SNP Diag, 5′ BT0219 SNP Diag and 3′ BT0219 SNP Diag, 5′ BT0241B SNP Diag and 3′ BT0241B SNP Diag, and 5′ BT0747 SNP Diag and 3′ BT0747 SNP Diag, respectively.

Quantification of c-di-AMP.

For quantification of c-di-AMP, the B. turicatae strains of interest were grown to late exponential growth phase, and 10¹⁰ cells were pelleted and washed two times in filtered saline. Following the last wash, cells were resuspended in 50 μl of Milli-Q H₂O, heat inactivated for 10 min at 99°C, and stored at −80°C until c-di-AMP quantification was performed. Cell resuspensions were mixed with 25 μM heavy (C¹³N¹⁵) c-di-AMP, followed by 500 μl of methanol, and sonicated (20 s, 80% amplitude). Methanol was collected following centrifugation of cell lysates. The remaining cell lysates were then resuspended in 50 μl Milli-Q H₂O, mixed with 500 μl methanol, and sonicated (10 s, 80% amplitude) again. The second methanol fraction was pooled with the first following centrifugation. Fractions were dried in a speed vacuum concentrator, and resultant pellets were resuspended in 30 μl Milli-Q H₂O for mass spectrometry analysis. Quantification of c-di-AMP was based on the 659/136 mass transition (689/146 for the internal standard) as previously described (68).

Growth curve analyses.

For in vitro growth curve analyses of BtWT, BtΔcdaA^C-cis(G175A), and BtΔcdaA^C-cis(G175A)::cdaA^C-cis(WT), cultures were inoculated at a density of 10⁴ spirochetes/ml in normal mBSK medium or in the following conditions: (i) mBSK medium supplemented with 50 mM NaCl or 50 mM KCl; (ii) mBSK medium diluted 1:10 in 1× PBS, 1.25× PBS, or 1.50× PBS; or (iii) mBSK medium prepared without pyruvate. For in vitro growth curve analysis of BtiCdaA-ΔcdaA, an initial 1-ml starter culture was grown to late exponential growth phase in mBSK medium supplemented with 1 mM IPTG, followed by centrifugation at 5,283 × g for 5 min and resuspension in 1 ml mBSK without IPTG supplementation. This wash step was repeated twice to remove any remaining IPTG from the culture. Following the washes, BtiCdaA-ΔcdaA was inoculated into media with or without 1 mM IPTG at an initial density of 10³ bacteria/ml. On the indicated days postinoculation, bacteria were quantified by dark-field microscopy. Ten microscopic fields were counted for each biological replicate, and two biological replicates for each condition were analyzed.

Imaging of Borrelia spirochetes and quantification of membrane blebbing.

BtiCdaA-ΔcdaA was grown in the presence or absence of 1 mM IPTG to late exponential growth phase, centrifuged at 9,391 × g for 5 min, and resuspended in PBS-MgCl₂. This step was repeated one time, and the washed spirochetes in PBS-MgCl₂ were used for microscopy. To immobilize the spirochetes for visualization and imaging, bacterial resuspensions were spotted onto a 1% agarose pad on a microscope slide and covered with a coverslip (163). The Keyence BZ-X800 microscope (Keyence Corp., Itasca, IL) was used to visualize bacteria and capture bright-field images at ×60 magnification with oil immersion. Two biological replicates were analyzed, and 100 spirochetes from each replicate were assessed for membrane blebbing.

Statistical methods.

All statistical analyses were performed using GraphPad Prism version 8. Two-way analysis of variance (ANOVA) models with Dunnett’s test for multiple comparisons were used to compare growth of mutant and complemented strains to the BtWT parent. To compare growth of BtiCdaA-ΔcdaA grown with and without IPTG, a two-way ANOVA model with Bonferroni’s test for multiple comparisons was used. For comparison of numbers of bacteria exhibiting membrane blebbing in BtiCdaA-ΔcdaA grown with and without IPTG, an unpaired Student's t test was used. P values of 0<0.05 were considered statistically significant. For qRT-PCR analyses, the threshold cycle (2^−ΔΔCT) method was used to calculate fold change in gene expression relative to BtWT as previously described (89, 164).