E, reminiscent of the oxygen response of E. coli [45]. Furthermore, pilus retractions were not detected after depletion of PMF via CCCP, indicating that depletion of PMF induces the switch to the non-motile state. Thus, speed switching in M. xanthus may be triggered by a so far undiscovered environmental input. If reduction of cellular ATP levels and hence the occupation of the hexameric MedChemExpress IQ 1 retraction ATPase PilT with ATPs would be exclusively responsible for speed switching, we expect that M. xanthus and N. gonorrhoeae responded in a similar way to cellular energy depletion. Recently, Sun et al. have shown that treatment of M. xanthus with nigericin does not affect T4Pdependent motility [46] in contrast to our results with N. gonorrhoeae. We conclude, therefore, that although the mechanical characteristics of T4P dynamics are wellconserved between bacterial species with different lifestyles, they have evolved to respond to different regulatory inputs. In contrast to M. xanthus, which is an obligate aerobe, oxygen-dependent speed switching may have evolved in N. gonorrhoeae due to its oxygen-limited habitat in the human urogenital mucosa. In this scenario, N. gonorrhoeae switches to the low speed mode, which serves as a “power saving mode”, to save ATP for processes of vital importance when PMF is reduced and ATP is limited. This behavior could prevent energy depletion without needing to switch off T4P dynamics completely.Contribution of the components of PMFAs it was expected from experiments in B. subtilis, Escherichia coli, or Helicbacter pylori [37?9], we have shown that pHin, pH as well as are functions of pHex in N. gonorrhoeae. Consequently, pH decreased with increasing pHex, whereas increased to stabilize the PMF at changing pHex. We conclude therefore, that our qualitative behavior for pH as well as agree well with the literature values, even though pH homeostasis appears to be relatively poor. Please note that comparison of absolute bioenergetic parameters between different studies is quite challenging, since they strongly depend on experimental conditions such as surface charge [40].The role of the denitrification pathway in speed switchingIt has been shown that N. gonorrhoeae can grow under anaerobic conditions in the presence of nitrite, using a truncated denitrification pathway. Anaerobiosis plays an important physiological role, since biofilm formation is accompanied by a transition to anaerobic conditions followed by anaerobic growth [28,41,42]. Therefore we addressed the question whether denitrification in the presence of nitrite can affect oxygen-dependent speed switching. Twitching motility assays revealed that the time point of global switching was unchanged. This was expected because denitrification must be induced by the oxygen-sensing transcription factor FNR [26] [29]. We did not observe a global switching back to the high speed mode even 60 min after the first global switching event, indicating that the denitrification pathway does not show a major influence on oxygen-dependent speed switching. Interestingly, both Entified S192, located on the flexible loop in the binding cleft reductases of the truncated denitrification pathway, AniA and NorB, do not support PMF generation [43,44], indicating that PMF is significantly lower under exclusive nitrite respiration. Therefore, we hypothesize that the high speed mode, which is very likely coupled to the PMF by a so far unknown mechanism, cannot be driven by denitrification exclusively. We would like to point out, however, that we d.E, reminiscent of the oxygen response of E. coli [45]. Furthermore, pilus retractions were not detected after depletion of PMF via CCCP, indicating that depletion of PMF induces the switch to the non-motile state. Thus, speed switching in M. xanthus may be triggered by a so far undiscovered environmental input. If reduction of cellular ATP levels and hence the occupation of the hexameric retraction ATPase PilT with ATPs would be exclusively responsible for speed switching, we expect that M. xanthus and N. gonorrhoeae responded in a similar way to cellular energy depletion. Recently, Sun et al. have shown that treatment of M. xanthus with nigericin does not affect T4Pdependent motility [46] in contrast to our results with N. gonorrhoeae. We conclude, therefore, that although the mechanical characteristics of T4P dynamics are wellconserved between bacterial species with different lifestyles, they have evolved to respond to different regulatory inputs. In contrast to M. xanthus, which is an obligate aerobe, oxygen-dependent speed switching may have evolved in N. gonorrhoeae due to its oxygen-limited habitat in the human urogenital mucosa. In this scenario, N. gonorrhoeae switches to the low speed mode, which serves as a “power saving mode”, to save ATP for processes of vital importance when PMF is reduced and ATP is limited. This behavior could prevent energy depletion without needing to switch off T4P dynamics completely.Contribution of the components of PMFAs it was expected from experiments in B. subtilis, Escherichia coli, or Helicbacter pylori [37?9], we have shown that pHin, pH as well as are functions of pHex in N. gonorrhoeae. Consequently, pH decreased with increasing pHex, whereas increased to stabilize the PMF at changing pHex. We conclude therefore, that our qualitative behavior for pH as well as agree well with the literature values, even though pH homeostasis appears to be relatively poor. Please note that comparison of absolute bioenergetic parameters between different studies is quite challenging, since they strongly depend on experimental conditions such as surface charge [40].The role of the denitrification pathway in speed switchingIt has been shown that N. gonorrhoeae can grow under anaerobic conditions in the presence of nitrite, using a truncated denitrification pathway. Anaerobiosis plays an important physiological role, since biofilm formation is accompanied by a transition to anaerobic conditions followed by anaerobic growth [28,41,42]. Therefore we addressed the question whether denitrification in the presence of nitrite can affect oxygen-dependent speed switching. Twitching motility assays revealed that the time point of global switching was unchanged. This was expected because denitrification must be induced by the oxygen-sensing transcription factor FNR [26] [29]. We did not observe a global switching back to the high speed mode even 60 min after the first global switching event, indicating that the denitrification pathway does not show a major influence on oxygen-dependent speed switching. Interestingly, both reductases of the truncated denitrification pathway, AniA and NorB, do not support PMF generation [43,44], indicating that PMF is significantly lower under exclusive nitrite respiration. Therefore, we hypothesize that the high speed mode, which is very likely coupled to the PMF by a so far unknown mechanism, cannot be driven by denitrification exclusively. We would like to point out, however, that we d.
