Sodium/proton antiporters are important membrane proteins that export sodium in exchange with protons for internal pH homeostasis and play roles in energy metabolism, nutrient acquisition, and more [35]. These transporter proteins can be found in a wide variety of eukaryotic and prokaryotic cells [35, 36, 49]. Loss of such proteins can have significant consequences to the cell thus making these proteins interesting drug target candidates. In this study, growth, biofilm formation, swarming motility, and resistance to weak acids under different environmental conditions were examined using transposon insertion mutants in P. aeruginosa. The primary transporters NQR and Complex I were studied, as well as the secondary antiporters Mrp, NhaB, and NhaP.
The loss of the NQR did not affect growth under challenging sodium and pH conditions, which confirms a recent study by Raba et al. [37] who suggested that in contrast to NQR in other species, such as Vibrio [3, 23, 27, 51], Pa-NQR is a proton pump with no affinity for sodium. In addition to that, our results suggest that loss of NQR does not affect pH homeostasis, biofilm formation, or swarming motility, which might be due to complementary proton pumping activity of Complex I. This hypothesis should be tested with a double deletion mutant of nqr and nuo in the future.
NhaB is not essential in V. cholerae as it has a supporting function and is, when lost, easily replaced by other sodium transporters, such as NhaA or NhaD, which are missing in P. aeruginosa [21]. Based on our results Pa-NhaB is unlikely a major contributor to sodium transport as its mutation did not interfere with growth, biofilm formation, or swarming motility at any of the tested conditions. In addition, it had been reported that when Pa-NhaB was expressed in everted membrane vesicles at pH 8.0 it showed a higher affinity to lithium than sodium [28]. This could suggest that Pa-NhaB has only a supportive function in sodium transport. To further investigate this theory, a triple deletion mutant strain lacking NhaA, NhaP, and Mrp should be constructed as these proteins can likely complement each other’s sodium transport in single mutants.
Pa-NhaP has also only been assayed when expressed in everted membrane vesicles of the antiporter-deficient E. coli strain KNabc, where Pa-NhaP was found to be a major sodium but not lithium transporter [28]. While we did not evaluate NhaP in regards to lithium, we tested two independent P. aeruginosa NhaP mutants in different pH and sodium conditions and did not observe an increase of sodium sensitivity compared to the wild-type strain. Also unaffected were biofilm formation and swarming motility. This lack of effect is likely due to a concerted effort of other sodium pumps (such as Mrp) in challenging environmental conditions and should be further investigated in the future.
When the MrpA subunit was mutated, neither growth nor biofilm formation or swarming was affected at pH 6.5, but significant changes occurred at pH 7.5 and particularly at pH 8.5. At pH 7.5 and early log phase, sodium concentrations of ≥ 200 mM led to a prolonged lag phase, while at pH 8.5, ≥ 200 mM NaCl resulted in a growth arrest from which the strain did not recover. An involvement of the Pa-mrp gene cluster (previously also designated sha [26];) as a major contributor to sodium transport at alkaline pH was previously demonstrated by the quenching method using everted membrane vesicles of the sodium sensitive E. coli TO114 which lacks three major Na+/H+ antiporters [41] and by disruption of PA01-shaA [26]. In our experiments, the Pa-Mrp mutant experienced a severely prolonged lag phase when exposed to high concentrations of sodium (400 and 500 mM NaCl) at pH 7.5 and a complete growth retardation for at least 24 h at pH 8.5 when ≥ 200 mM NaCl was added, but showed successful formation of biofilms under those conditions. This could be explained by altered physiological characteristics often found when planktonic bacteria become sessile, such as a vastly altered gene expression profiles and slowed growth rate intended to make the sessile form more resilient to adverse environmental conditions (suggested by [11]). When biofilm formation is high, motility/swarming activity is usually low; this phenomena is a basic function in bacterial physiology and is antagonistically regulated [5, 48].
Swarming is the rapid multicellular movement of bacteria across an > 0.3% agar surface along a nutrient gradient generated by the bacteria, and the movement is powered by a rotating flagella [24]. While in some bacteria, like V. cholerae, flagellar rotation is powered by sodium; this does not seem to be the case in P. aeruginosa [5, 12], which has a polar flagella driven by a dual set of motor proteins, MotA/B/Y and MotC/D [12]. Different to many other bacteria, swarming in P. aeruginosa seems to be more complex and in some capacity also supported by Type IV pili [25, 34]. Swarming is often defined as the surface-motility used to translocate cells to a more favorable environment [12]. It can therefore be assumed that lack of swarming in unfavorable environmental conditions might result in heightened biofilm formation; however, from our results we conclude that swarming, or lack-thereof, is a poor predictor of biofilm production in P. aeruginosa. Regardless of the generally favorable effects of sodium addition to growth of our tested strains at pH 7.5 and 8.5 (Fig. 2), the Complex I mutant showed significantly reduced swarming at pH 7.5 and no added sodium (Fig. 4b; p < 0.0001), where we would likely expect higher swarming to get the cells into a more sodium-rich environment. A possible explanation is that this transporter is a major contributor to the protein motive force and thus provides the power necessary for flagella rotation. However, generally, this mutation did not alter any other phenotypes tested in this study, suggesting that other proteins collaborate with Complex I to enable normal phenotypes. In contrast at pH 8.5, the Complex I mutant revealed an increased swarming phenotype at the unfavorable sodium-depleted condition and reduced swarming in sodium-rich conditions, which supports the translocation to more favorable conditions idea (Fig. 4c; p < 0.0001). Clearly, future studies are required to better understand these phenotypes.
The Mrp mutant showed significantly reduced swarming motility compared to the wild-type strain at pH 7.5 and 400 mM added sodium (Fig. 4b; p < 0.0001). However, at these environmental conditions, the strain’s growth was also significantly slowed and reduced compared to the wild-type strain (Fig. 2e), confirming Mrp’s involvement in sodium transport. However, this reduced growth could also explain the quantitatively reduced, sodium-independent swarming motility (Fig. 4b). Overall, the Mrp protein becomes most relevant when sodium levels in the environment are elevated (≥ 200 mM) and pH is 7.5, and it becomes essential at pH 8.5 in the presence sodium. In addition at those conditions (≥ pH 7.5 and ≥ 200 mM NaCl), lack of a functional Mrp protein complex resulted in higher biofilm formation, even though at pH 8.5 no growth and thus no swarming motility were evident. In summary, Mrp is clearly important to P. aeruginosa’s survival in challenging environments.
Mrp and Complex I seem to have some evolutionary relationship based on the homology between MrpC and NuoK [31], MrpA and NuoL, and MrpD and NuoMN [33]. Interestingly, the NuoL subunit has been suggested to transport sodium in E. coli [17, 22, 43]. While not reported as statistically significant in this work due to our stringent p value threshold and the randomness of the occurrences, a few differences between the wild-type and the NuoB mutant might be notable, for example at pH 8.5, the growth of the NuoB mutant was slightly reduced when 500 mM NaCl was added (p = 0.0165), or slightly elevated Biofilm Indexes were seen at pH 6.5 and 100 mM NaCl (p = 0.0246), as well as at pH 7.5 and 500 mM NaCl (p = 0.0323).