Enterococcus faecalis is a gram-positive facultative anaerobe and opportunistic pathogen routinely identified among diverse microorganisms in the animal gastrointestinal tract (GIT), ranging from mammals to insects (Martin and Mundt, 1972; Krawczyk et al., 2021). Studies have demonstrated E. faecalis to be capable of withstanding a wide range of pH, temperatures, and salinity, highlighting its inherent resilience, and contributing to its persistence in diverse hosts and environments.

The adaptability of E. faecalis has made it a useful model for elucidating the mechanisms underlying host-microbe interactions in non-mammalian systems, such as lepidopterans, where the dynamic GIT environment presents unique physiological challenges for transient, resident, and invasive pathogens. Species in the order Lepidoptera, which include caterpillars and moths, have been used extensively to characterize the toxicity of insecticidal proteins produced by Bacillus thuringiensis (Bt) (Schnepf et al., 1998; Jurat-Fuentes et al., 2011; Kerns et al., 2023). However, the holometabolous life cycle and proportionately large GIT, genetic tractability, and microbiome plasticity have contributed in recent decades to their emergence as useful models to characterize host-microbe interactions.

The lepidopteran midgut presents a unique anatomical and physiological environmental niche including high luminal alkalinity, elevated potassium concentration, reduced oxygen availability, short retention times, and the presence of a peritrophic matrix that protects the epithelium from mechanical damage, pathogens, and toxins (Dow, 1992; Lehane, 1997; Johnson and Barbehenn, 2000; Brinkmann and Tebbe, 2007). While the broad-range utility of lepidopteran systems for the investigation of host-microbe interactions has increased, studies on the molecular mechanisms underlying persistence in the GIT have not. Moreover, these studies have relied on destructive sampling approaches which prevent longitudinal tracking of persistence dynamics in individuals over time (Tang et al., 2004; Holt et al., 2015; Teh et al., 2016; Mazumdar et al., 2021; Zhang et al., 2022). Given the combined alkalinity and potassium levels in the GIT of the lepidopteran species, it is plausible that proteins involved in osmoregulation and ionic homeostasis contribute to bacterial persistence in this environment.

Initially described in Enterococcus hirae strain ATCC 9790, the ntpJ gene is located at 3′ end of the vacuolar V₀V₁ type Na⁺-ATPase (ntp) operon. This operon comprises 11 open reading frames (ORFs), including ntpF, -I, -K, -E, -C, -G, -A, -B, -D, -H and -J, and has homologs in the Cyanobacterium Synechocystis sp. PCC 6803 as well as many clinical E. faecalis isolates. Our BLASTp and tBLASTn queries of the E. faecalis OG1RF genome (GenBank: CP002621.1) with accession numbers corresponding to each of the ntp genes in E. hirae indicate that 10 of the 11 genes described by Murata et al. are present, with ntpJ located ∼1.025 Mbp away from the remaining gene cluster (Murata et al., 1996). These findings suggest ntpJ is independently transcribed from the other ntp operon genes and are consistent with a report which showed the variation of potassium transporter architecture across the Enterococcus genus was strain specific (Acciarri et al., 2023). The ntpJ gene encodes a 49-kDa integral membrane protein with sequence similarity to K⁺-transporters in Escherichia coli (TrkH/TrkG) and Saccharomyces cerevisiae (Trk1/Trk2), both of which mediate K⁺ uptake (Takase et al., 1994; Schlosser et al., 1995; Murata et al., 1996; Petrezselyova et al., 2011). Northern blot analysis of total RNA extracted from E. hirae revealed that elevated intracellular Na⁺ and/or alkaline pH induce polycistronic expression of ntpJ along with the other ntp operon genes (Takase et al., 1994; Murata et al., 1996). NtpJ encodes KtrB, the translocating subunit of the high-affinity K⁺ uptake systems in E. hirae (KtrII) and E. faecalis (KtrAB). The E. faecalis cytoplasmic nucleotide-sensitive regulator, KtrA, binds to ATP which is followed by a conformational change that allows it to form a KtrAB complex under osmotic stress and alkalinity. Under alkaline conditions, which diminish proton motive force (PMF) and membrane potential (Δψ), the Na⁺-ATPase extrudes Na⁺ to generate a sodium motive force (SMF). This SMF is subsequently utilized by KtrB to drive K⁺/Na⁺ symport (Murata et al., 1996; Kawano et al., 2000, 2001; Berry et al., 2003; Matsuda et al., 2004; Acciarri et al., 2023).

In this report, we characterized the role of KtrB during E. faecalis adaptation to alkaline stress and persistence in the H. zea GIT. We hypothesize that KtrB facilitates ionic homeostasis, which in turn contributes to survival and persistence in the H. zea GIT. To test this hypothesis, we assessed the impact of transposon mutagenesis of ntpJ on bacterial growth kinetics in vitro, biofilm formation, surface adhesion, and persistence using a non-destructive frass-based H. zea sampling model.

In this study, we demonstrate that KtrB–the integral membrane component of the two-component KtrAB Na⁺/K⁺ symporter encoded by ntpJ–contributes to E. faecalis adaptation and persistence in the alkaline environment of lepidopteran GIT. The data suggest that KtrB is essential for the planktonic progression into mid-log phase growth and biofilm formation in vitro but is not required for adhesion to semi-solid substrates. The ntpJ transposon mutant (OG1RF ntpJ-Tn), which lacks a functional KtrB, exhibits a persistence defect in the H. zea GIT after 48 h. We hypothesize that KtrB facilitates E. faecalis growth under alkaline conditions by maintaining osmotic balance during the initial stages post-ingestion.

The E. faecalis OG1RF genome encodes four primary K⁺ transport systems, including two regulator of conductance of K⁺ (RCK)-gated channels (KtrAB and KtrAD) and two K⁺/H⁺ symporters (Kup and KimA). A comparative analysis of the E. faecalis OG1RF genome (454 pyrosequencing, Solexa, Sanger) by Bourgogne et al., 2008, BLAST (BLASTp, and tBLASTn) against E. faecalis V583 kdpA-E protein sequences, and KEGG/Orthology analyses confirmed the absence of the kdpFABC operon and its regulatory system, kdpDE (Bourgogne et al., 2008).

To test the functional role of KtrB (ntpJ) during growth in vitro under neutral conditions, OG1RFS wild type and OG1RF ntpJ-Tn were inoculated into standard YPD (pH 6.8-7) and monitored spectrophotometrically. As shown in Figure 1A and Figure 2A, OG1RF ntpJ-Tn exhibited a significant delay in the progression to mid-log phase relative to the wild-type strain, suggesting the combined K⁺ influx through KtrD, Kup, and KimA was insufficient to compensate for the loss of KtrB and restore wild-type growth kinetics and supporting a role for KtrB as the primary K⁺ transporter under K⁺-limiting conditions. These results are consistent with Acciarri et al., 2023 (Figure 2) in which an E. faecalis JH2-2 derivative (ΔkupΔktrA) experienced a significant growth defect relative to the wild type in mLBG at neutral pH, and align with previous reports detailing that KtrB provides the highest-affinity for K⁺ influx under neutral conditions, followed by Kup and KimA which provide progressively lower-affinity backup K⁺ influx (Kawano et al., 2000; Petrezselyova et al., 2011; Gries et al., 2013; Acciarri et al., 2023).

Under alkaline conditions, Enterococcus hirae and E. faecalis compensate for the loss of proton motive force (PMF) by activating the V₀V₁ type Na⁺-ATPase (V-ATPase) which utilizes the energy from ATP hydrolysis to pump Na⁺ to the extracellular environment. The Na⁺ gradient (sodium motive force, SMF) drives K⁺ influx via KtrB restoring pH and osmotic balance (Kobayashi et al., 1986; Ikegami et al., 1999; Krulwich et al., 2011). To test the functional role of KtrB under alkaline-adjusted conditions, OG1RFS wild type and OG1RF ntpJ-Tn were inoculated into YPD adjusted to a pH of 10 with either 3.6 M KOH or 5 M NaOH and monitored spectrophotometrically. We hypothesized that collapse of PMF would impair H⁺-coupled symport of K⁺ through Kup and KimA. Simultaneously, elevation of the extracellular [K⁺] from KOH dissociation would place the burden of K⁺ influx on KtrD, potentially causing OG1RF ntpJ-Tn growth defects. As shown in Figure 1B, OG1RF ntpJ-Tn did not exhibit a growth delay in the progression to mid-log phase relative to the wild-type strain, suggesting the elevated extracellular [K⁺] from KOH dissociation allowed KtrD and, potentially, Kup and KimA to compensate for the loss of KtrB and restore wild-type growth kinetics despite the collapse of PMF. These results are consistent with findings of Acciarri et al., 2023 (Figure 2C), which demonstrated K⁺ supplementation of mLBG at pH 9 partially restored the growth defects of KtrB- and KtrD-deficient E. faecalis JH2-2 derivatives. Combined, these data substantiate the compensatory roles of alternative K⁺ transporters in the absence of SMF-driven KtrB-mediated K⁺ influx. We extended our analysis of the role of KtrB under alkaline conditions to include growth in YPD adjusted to a pH of 10 using NaOH. Under these conditions, the K⁺ concentration remained at baseline (see Figure 1A), while we propose the addition of NaOH collapsed the PMF and simultaneously elevated the extracellular [Na⁺]. Interestingly, Figure 1C shows that OG1RF ntpJ-Tn exhibited no significant growth defect relative to the wild-type, suggesting the hyperpolarized membrane potential (ΔΨ) driven by the V-ATPase in tandem with Na⁺ from NaOH dissociation was sufficient to create an electrical gradient that could drive K⁺ influx through KtrD, Kup and KimA under K⁺-limited conditions. Consistent with our observation and subsequent proposal that hyperpolarized ΔΨ contributed to the growth of OG1RF ntpJ-Tn in NaOH-adjusted YPD, Acciarri et al., 2025 (Figure 5) demonstrated that E. faecalis JH2-2 double mutants (ΔktrAΔkimA → Kup⁺ and ΔktrAΔkup → KimA⁺) can sustain growth in a modified sodium Spizizen medium at near neutral pH (7.0–8.0) in which the pH difference across the plasma membrane is minimized and ΔΨ serves as the primary driver of Kup- and KimA-mediated K⁺/H⁺ symport. In alkaline-adjusted YPD, both E. faecalis strains showed similar progression to the mid-log phase, despite exhibiting differential sensitivity to ionic stress evidenced by the effect sizes. The wild-type strain was moderately sensitive to both ions, whereas the ntpJ-Tn mutant was more sensitive to Na⁺ than K⁺ (Figure 2). While these data revealed a non-significant delay in the progression to the mid-log phase by the ntpJ-Tn mutant in alkaline-adjusted media supplemented with K⁺ or Na⁺, the competition with other enteric microorganisms for nutrients and the peristaltic activity of the host GIT would potentially amplify this phenotype. Moreover, the elevated differential sensitivity of the ntpJ-Tn mutant suggests that, in addition to Na⁺-dependent K⁺ uptake, KtrB may play a broader role in ionic stress adaptation. The lack of transcriptional terminators in EfaMarTn would allow the native ntpJ promoter to drive read-through into the napA open reading frame, reducing the likelihood of a polar effect. Furthermore, studies on the E. faecalis strain ATCC 33186 transcriptional response to alkaline conditions (pH 10) showed that napA expression is downregulated ∼1.64-fold compared to controls (pH 7.4) (Ran et al., 2015). In combination, the transcriptional read-through and alkaline-induced repression of napA suggest NapA did not contribute to these phenotypes.

The ntpJ-Tn mutant showed significantly impaired early-stage biofilm formation relative to the wild-type strain under all conditions tested. In standard YPD, the density of biofilms formed by the wild-type strain was significantly larger than those formed by the ntpJ-Tn mutant at 24 h and 48 h, supported by increasing effect sizes. This biofilm defect was consistent but diminished under the alkaline-adjusted conditions (Figure 3). Physiologically, attempts to maintain K⁺ and Na⁺ homeostasis may underlie this observation. As mentioned earlier, the compensatory pumping of Na⁺ by the V₀V₁ type Na⁺-ATPase to establish SMF under alkaline conditions would reduce the availability of ATP for cell replication–a phenotype that would be exacerbated in the ntpJ-Tn mutant leading to reduced biofilm density (Kobayashi et al., 1986; Ikegami et al., 1999). This agrees with the results of the planktonic growth experiments in which the ntpJ-Tn mutant showed a growth delay in standard YPD relative to the wild-type strain and suggests that an indirect consequence of reduced planktonic fitness is impaired early-stage biofilm formation. Several previous studies including those in Mycobacterium tuberculosis, Staphylococcus aureus, and Pseudomonas aeruginosa have adopted mariner transposon-based approaches for genome-wide mutagenesis and the subsequent identification of genes driving phenotypes of interest (Sassetti et al., 2001; Gallagher et al., 2011; Fey et al., 2013). We tested the effects of high pH (pH 10) in combination with elevated [K⁺] or [Na⁺] on the growth dynamics of an E. faecalis ntpJ-Tn, allowing us to isolate the phenotypic consequences of KtrB disruption under pH and ionic stress.

It was demonstrated in a previous study that Saccharomyces cerevisiae biofilm mats exhibit spatially distinct regions, a non-adherent rim, and an adherent hub, influenced by adhesin expression, glucose concentration, and pH gradients. Specifically, Flo11p was shown to be essential for both biofilm formation and adhesion, while glucose concentration and pH were higher in the rim relative to the hub (Reynolds et al., 2008). We adapted the yeast overlay adhesion assay described in the study to further compare the E. faecalis OG1RFS WT and OG1RF ntpJ-Tn biofilms grown on standard and alkaline-adjusted low-density agar. Our results indicate that environmental pH and the chemical properties of the base used to adjust pH influence biofilm morphology and surface adhesion. Both strains formed weakly adherent biofilms on standard YPD supporting a uniform distribution of metabolically active cells in the rim and hub regions (Figure 4). In contrast, biofilms grown on KOH-adjusted YPD agar showed stronger adherence in the hub relative to the rim, suggesting that nutrient depletion in the hub promoted stronger surface attachment. Most notably, both strains showed the strongest uniform adhesion on NaOH-adjusted YPD agar, suggesting that Na⁺ ions may alter nutrient utilization in a manner that enhances attachment or promotes stronger surface attachment directly, the latter of which is consistent with a previous report demonstrating that environmental factors, such as bile salts and ionic composition, influence E. faecalis surface charge heterogeneity enhancing adhesion independently of known adhesive proteins (van Merode et al., 2006) (Figure 6).

In studies of continuous flow cultures, Rolf Freter demonstrated that early-stage adaptation to environmental stress and efficient acquisition of limiting nutrients are critical for establishing stable populations in the mucosal layer of the mammalian GIT (Freter, 1981, 1988; Freter et al., 1983). Relative to mammalian model systems, the GIT of lepidopteran larvae presents a stringently alkaline environment that poses significant physiological challenges to transient and stably colonized microorganisms. The alkalinity of the H. zea midgut, with high luminal pH and K⁺, is known to disrupt proton motive force and challenge bacterial ionic homeostasis (Kakinuma, 1987; Dow and Harvey, 1988; Dow, 1992; Fallingborg, 1999). In the H. zea bioassays, we demonstrate that KtrB promotes E. faecalis persistence in the alkaline GIT of H. zea. While frass production by all larvae did not differ and the wild-type and mutant strains were initially recovered at similar levels 24 h post-ingestion, the ntpJ-Tn mutant was undetectable by 48 h, indicating a significant persistence defect. Dow and Harvey (1988) used flame photometry to estimate the total and active [K+] in the Manduca sexta (tobacco hornworm) midgut to be 204 ± 6 mM and 83 ± 1, respectively (Dow and Harvey, 1988). To date, there have been no direct measurements of the [K⁺] concentration in the H. zea GIT. However, given the similarity in the functional mechanisms used by goblet cells to maintain alkaline pH in the lepidopteran midgut, it is appropriate to use as a proxy the experimentally determined concentrations in M. sexta.

While the in vitro experiments suggest that the elevated [K⁺] in the GIT would allow the E. faecalis OG1RF ntpJ-Tn mutant to compensate for PMF collapse, the rapid turnover of intestinal contents further exploits the loss of KtrB and explains the observed persistence defect. Furthermore, we would expect the elevated [K⁺] in the H. zea midgut lumen to fully saturate all four K⁺ transporters (e.g., KtrD, KtrB, Kup, and KimA). Because the midgut is also extremely alkaline, the collapsed PMF would significantly diminish the symporter activities of Kup and KimA even with an abundant supply of K⁺. Under the same conditions, the transport activities of KtrB and KtrD would depend on whether the epithelial cells are able to maintain a negative potential in the cytoplasm, which would draw in K⁺. The collapsed PMF and subsequent reduced Kup- and KimA-mediated H⁺/K⁺ symport, the downregulated expression of kimA, and the decreased electrogenic import of H⁺ and K⁺ by KtrD force E. faecalis to rely primarily on KtrB under alkaline conditions (Acciarri et al., 2025). The loss of KtrB, consequently, leads to significant persistence defects in the H. zea GIT. These findings support the hypothesis that the KtrB, the integral membrane component of the KtrAB potassium uptake system, plays a significant role in maintaining osmotic balance and pH under these conditions. That the ntpJ-Tn mutant did not persist beyond 24 h suggests that K⁺ translocation by KtrB is essential for maintaining optimal ionic balance. Furthermore, while both strains showed increased surface attachment to low-density agar, the persistence bioassay results suggest that amplification of the planktonic growth differences in vivo limits the density of the planktonic population that may enter adherent biofilms (Figures 6, 7). This observation also agrees with previous work using this non-destructive model, suggesting that multiple genetic signaling pathways mediate survival and persistence in lepidopteran GIT (Jackson et al., 2024).

In conclusion, these data demonstrate that KtrB-mediated K⁺ and Na⁺ transport plays a significant role in the adaptation of E. faecalis to alkaline stress and its ability to withstand the dynamic and inherently alkaline H. zea GIT. Loss of KtrB impaired early-stage planktonic growth, biofilm formation in vitro, and persistence in vivo. While transport by KtrB was not essential to the surface adhesion of biofilms on low-density agar, its role during ionic homeostasis supported E. faecalis persistence in the H. zea GIT. These results emphasize the importance of K⁺ and Na⁺ transport systems during bacterial persistence in alkaline environments and highlight the efficacy of the non-destructive model for functionally assessing bacterial circuitry underscoring GIT persistence.