BACKGROUND
the closely related pathogens neisseria gonorrhoeae and neisseria meningitidis colonize human mucosal epithelia, however, at different sites in the body.
neisserial motility is enabled by type iv pili , which are long and dynamic filaments expressed by a phylogenetically diverse set of bacterial species, such as pseudomonas aeruginosa, vibrio cholerae, legionella pneumophila, moraxella bovis, escherichia coli and myxococcus xanthus .
in addition to mediating what is termed twitching motility, tfp are also involved in a multitude of other functions, including attachment to host cells, microcolony and biofilm formation and dna uptake  <cit> .
tfp can extend and retract via the assembly and disassembly of pilin subunits, called pile, from an inner membrane protein pool  <cit> .
the pile of pathogenic neisseria is divided into two classes: class i and class ii pilin.
the latter is only present in a subset of n. meningitidis strains.
in contrast to class i pilin, class ii pilin very rarely undergo antigenic variation and is shorter due to a deletion in the hypervariable region  <cit> .
the assembled filaments can extend up to several micrometers from the bacterial surface in contrast to the bacterial diameter, which is approximately 1 μm.
on the other hand, the pilus cross-sectional diameter is only 6–8 nm  <cit> , far below the diffraction limit of visible light, which makes it impossible to observe single tfp using bright field microscopy.
tfp biogenesis and extension in neisseria spp.
depends on a core set of 12– <dig> highly conserved proteins  <cit> , whereas the retraction of tfp is powered by the atpase pilt  <cit> .
based on x-ray crystallography data, the functional unit of pilt is proposed to be a hexamer  <cit> .
in rod-shaped bacteria, such as p. aeruginosa and m. xanthus, pili are present at one pole although the atpases necessary for assembly and retraction are distributed to both poles  <cit> .
in pathogenic neisseria, pili extend in all directions  <cit>  and a study on pilt localization in n. gonorrhoeae indicated that it is found in the cytoplasm and associated with the inner membrane  <cit> .
of the two pathogenic neisseria species, mainly n. gonorrhoeae has been used to study twitching motility.
optical tweezers experiments have demonstrated that a single gonococcal pilus is able to retract with peak forces up to  <dig> pn, and bundles of pili can exert even stronger forces  <cit> .
the average pilus retraction rate in n. gonorrhoeae is  <dig>  ±  <dig>  μm/s at forces below  <dig> pn  <cit> .
this rate may vary depending on multiple factors including the pilt concentration, the expression of the pilt paralog pilt <dig>  the external force applied to the pilus and the oxygen concentration  <cit> .
movement of n. gonorrhoeae during long time scales is consistent with a random walk while persistent movement in one direction can be observed on shorter time scales .
increasing the number of pili in n. gonorrhoeae increases the persistence time  <cit> .
in this work, we have investigated motility in n. meningitidis and n. gonorrhoeae, including the influence of pile sequence, pilin class and pilt expression.
since it is important to understand the relation between pilus filaments and bacterial motility, we also monitored the speed and the number of visible filaments during bacterial motility using a combination of phase contrast and total internal reflection fluorescence  microscopy.
we observe differences between gonococci and meningococci in their motility characteristics that are not related to pilt expression.
pile sequence or pilin class.
furthermore, we demonstrate that pilt expression is influenced by a species-specific single nucleotide variation in the pilt promoter.
RESULTS
n. gonorrhoeae and n. meningitidis exhibit different speed during twitching motility
to characterize motility in pathogenic neisseria, we studied twitching motility using live-cell phase contrast imaging and automated particle tracking of moving bacteria.
the bacteria were allowed to adhere to poly-d-lysine-coated glass and observed over a period of 60 s, with  <dig> images acquired per second.
to determine the level of background noise in our system, a nonpiliated mutant derived from n. gonorrhoeae strain ms <dig> was included in the motility analysis and its speed was measured to  <dig>  μm/s .
the tracking data revealed significant differences in the mean speed between the n. gonorrhoeae and n. meningitidis strains tested .
the average speed of n. gonorrhoeae strains ms <dig> and fa <dig> ranged between  <dig> - <dig>  μm/s, whereas n. meningitidis strains had a speed of  <dig> - <dig>  μm/s which included strains of serogroup b , c  and w .
pile sequence variation in neisseria affects the level of host cell adhesion and to determine whether it may also have an impact on motility, we tracked isogenic clones of ms <dig> with different pile sequences  <cit> .
variants 3: <dig>  5: <dig> and 8: <dig> carry two amino acid substitutions in the variable mini-cassette   <dig> .
further, variants 3: <dig> and 8: <dig> both contain a lysine to glutamate substitution in mc <dig> and variant 8: <dig> has another five substitutions in mc <dig>  ms <dig> 6: <dig> has several amino acid changes in mc <dig>   <dig>   <dig> and  <dig>  different from the other three ms <dig> pile sequence variants.
in addition, the variants 3: <dig>  5: <dig> and 6: <dig> have a larger substitution in the mc <dig> sequence.
the hypervariable mc <dig> is unaltered in all pile variants.
piliation level is similar between variants but approximately 50% lower in comparison to the parental ms <dig> strain  <cit> .
tracking analysis showed that there was no difference in speed between the pile sequence variants and the parental ms <dig> strain .
in order to determine whether the pile class affected motility speed, the fam <dig> native pile gene encoding a class ii pilin was exchanged with the pile sequence obtained from the pile expression locus in the gonococcal strain fa <dig>  which is similar to class i pilins of n. meningitidis.
tracking analysis demonstrated that the pile swap mutant of fam <dig>  although expressing slightly less pile and pili , moved with the same speed as the wild-type , indicating that pilin of gc most likely is not in itself causing the lower speed of n. gonorrhoeae.
the surface coating poly-d-lysine is positively charged at neutral ph.
to confirm the difference in speed between meningococci and gonococci on a biologically relevant surface material with a neutral net charge at ph 7– <dig>  we assessed twitching motility on collagen.
as shown in figure 1b, n. gonorrhoeae and n. meningitidis display a similar difference in speed on a collagen-coated surface.
in conclusion, these data suggest that n. meningitidis moves faster than n. gonorrhoeae on both poly-d-lysine- and collagen-coated glass and that bacterial speed is independent of pile sequence variation.figure  <dig> average motility speed of neisseria strains on a solid surface.
n. gonorrhoeae strains fa <dig> and ms <dig>  a nonpiliated ms <dig> and four clones of ms <dig> with different pile sequences .
n. meningitidis strains fam <dig> , c <dig> , c <dig> , jb <dig> , c <dig>  and the pile sequence swap mutant in fam <dig>  the bacteria were allowed to adhere to poly-d-lysine-coated glass for 60 min at 37°c in 5% co2-saturated gc broth.
attempts to track strains of serogroup a failed, as these strains did not adhere to the poly-d-lysine-coated surface.
motility speed of n. gonorrhoeae fa <dig> and n. meningitidis fam <dig> on collagen-coated glass.
the bacteria were allowed to adhere to the glass for 60 min prior to tracking.
motile bacteria were studied using live-cell imaging and particle tracking.
the data are presented as the average values of at least  <dig> tracks acquired in at least three independent experiments.
the error bars indicate the standard error of the mean.
significant differences  between gonococcal and meningococcal strains are marked with an asterisk *.
expression of pilt and pilf are equal but higher in n. gonorrhoeae
the atpases driving pili extension and retraction are pilf and pilt, respectively.
pilt-mediated retraction is specifically essential for functional tfp and twitching motility  <cit> .
a previous study demonstrated a higher expression of pilf in gonococci in comparison to meningococci  <cit> .
we could confirm higher levels of pilf mrna in fa <dig> than in fam <dig> .
because pilf expression differs between the species, we chose to also quantify pilt mrna and protein levels by using quantitative real-time pcr and a two-color western blot assay with an in-house generated polyclonal pilt antibody raised against pilt from n. gonorrhoeae.
indeed, fa <dig> expressed higher relative levels of pilt mrna than fam <dig>  and pilt expression matched that of pilf in both species .
the western blot, with elongation factor tu  as loading control, showed that n. gonorrhoeae fa <dig> expressed nearly twice as much pilt protein as n. meningitidis fam <dig> .
because both the pilt and ef-tu amino acid sequences of n. gonorrhoeae and n. meningitidis are identical, the antibodies should react equally well in both species.
in conclusion, n. gonorrhoeae expresses more pilf and pilt in comparison to n. meningitidis and because the atpase expression levels are matched it suggests that the pilt expression is not a factor underlying the difference in speed between pathogenic neisseria.table  <dig>
pilt
versus
pilf
mrna expression
strain
reference gene
pilt
/reference gene
pilf
/reference gene
16s rrna
rplp
rpod
16s rrna
rplp
rpod
a single base pair variation alters pilt expression
to determine the reason behind the different pilt expression in pathogenic neisseria, we analyzed the pilt promoter.
an alignment of the pilt promoter from representative strains is shown in figure 2c and all available sequenced strains are shown in additional file 2: figure s <dig>  while close to identical, the pilt promoter in a majority of n. meningitidis strains contained the − <dig> promoter element 5’tacaat while the pilt promoter of all n. gonorrhoeae strains contained the canonical σ <dig> - <dig> sequence 5’tataat.
an additional c/t polymorphism was found downstream of the transcriptional start site .
to experimentally investigate the effect of the - <dig> promoter sequence on pilt expression, we switched the pilt promoter between n. meningitidis and n. gonorrhoeae.
we then assessed the role of the atpase ratio in neisseria motility.
promoter mutagenesis in the − <dig> sequence  in both species demonstrated that the 5’tacaat sequence confers lower pilt mrna  and protein levels  without changing pilf mrna expression .
neither the motility speed or the level of piliation, quantified in the fam <dig> mutants by using a whole cell elisa and transmission electron microscopy , differed between the promoter variants .
in conclusion, the 5'tataat sequence specifically conserved in the gonococcal pilt promoter results in higher expression of pilt.
however, the change in pilt/pilf ratio applied in this work does not significantly affect motility speed.figure  <dig> analysis of pilt expression in pilt promoter mutants.
the pilt promoter constructs used to transform n. gonorrhoeae n <dig> and n. meningitidis fam <dig> carried either the 5’tataat or the 5’tacaat sequence with a chloramphenicol resistance cassette  oriented on the opposite strand and in the opposite direction of the pilt gene.
pilt mrna transcription level in n <dig>  and fam <dig> , as quantified using qpcr.
mrna expression was normalized to three different reference genes  and compared to the wt level.
the experiment was performed two to three times.
the bars show the mean ± standard deviation.
pilt protein quantification of whole cell lysates from n <dig>  and fam <dig>  by western blot.
the relative pilt/ef-tu band intensities were averaged over two to four separate blots and normalized against wt strains.
the error bars represent the standard deviation.
visualizing type iv pili in motile n. gonorrhoeae and n. meningitidis
to determine whether the observed difference in motility speed between n. gonorrhoeae and n. meningitidis could be linked to the distribution or number of pili, we stained bacteria and pili with an nhs-ester-based fluorescent dye and visualized pili using live-cell tirf microscopy.
the stain did not significantly affect speed .
tirf microscopy provides a highly specific excitation of fluorophores up to ~100 nm from the glass surface.
when using this illumination system, the background fluorescence is drastically reduced and the relatively weak fluorescence of stained pili becomes visible.
hence, it was possible to monitor pili in neisseria during bacterial crawling, similar to previous visualization of tfp in pseudomonas  <cit> .
the time for tirf imaging was limited to 10– <dig> seconds due to fading of the fluorescent stain.
it is important to note that this technique did not permit the distinction between a single pilus and a bundle of pili.
therefore, observed pili will collectively be referred to as visible filaments.
we monitored bacterial crawling on a poly-d-lysine-coated glass surface and captured more than  <dig> bacterial tracks of n. gonorrhoeae fa <dig> and ms <dig> and n. meningitidis fam <dig> motility.
in general, the number of visible filaments varied over time during the movement of a bacterium.
filaments were observed in all directions around the bacterium, which is in accordance with previous data  <cit> .
figure 4a shows representative images of motile, fluorescently stained n. gonorrhoeae and n. meningitidis.
to quantify the relative frequency of visible filaments during motility, a manual single-blinded frame-by-frame counting was performed on the collected time-lapse movies .
when moving on a solid surface, n. gonorrhoeae most frequently displayed one filament, while n. meningitidis most often exhibited three filaments.
similar results as seen with tirf were obtained after quantification of single pili and pili bundles in tem  although only a trend and not a significant difference could be detected.
comparison of pilus bundle size showed that the majority of bundles observed in both meningococci and gonococci contained two or three pili, corresponding to a bundle width of 10–25 nm .
the relative amount of pili that were present as single filaments versus as bundles was slightly higher in gonococcal strains than in fam <dig> .
figure 6a shows an example of tfp-mediated motility involving primarily a single filament, as exemplified by n. gonorrhoeae strain fa <dig> where the mean track speed was  <dig>  μm/s and the distance travelled being  <dig>  μm over the  <dig>  s shown.
the corresponding track of movement is presented in figure 6c with dots indicating the position and the colors showing the speed in each frame.
figure 6b exemplifies bacterial movement by n. meningitidis strain fam <dig> where the mean track speed was  <dig>  μm/s and the distance travelled being  <dig>  μm over the  <dig>  s shown.
the full time-lapse videos are shown in video s <dig> and s <dig> .
the track in figure 6d indicates that meningococcal movement involving the apparent retraction of several filaments simultaneously results in more frequent changes in direction.
still, a graph relating the number of filaments to the motility speed only depicted a weak correlation and n. gonorrhoeae with the same number of filaments as meningococci in tirf movies still moved at a lower speed .
to summarize, the results suggest that there is no correlation between the difference in number of filaments and motility speed in n. meningitidis and n. gonorrhoeae.
nevertheless, n. meningitidis fam <dig> displayed multiple filaments more frequently than n. gonorrhoeae, which may influence changes in motility direction.figure  <dig> visualization of tfp during movement using tirf microscopy.
live bacteria were stained with the dylight™ <dig> nhs-ester prior to experiments.
representative pictures of stained n. gonorrhoeae and n. meningitidis crawling on a poly-d-lysine-coated surface.
the scale bar corresponds to 5 μm.
percent of observations with x number of visible filaments during movement on a solid surface.
more than  <dig>  individual frames from the time-lapse movies were analyzed for each species, and the number of visible filaments was counted in each frame.
discussion
tfp-mediated motility is accomplished via repeated steps of pilus assembly and pilt-mediated pilus retraction.
in this work, we aimed to further characterize and quantify pilus dynamics and twitching motility in n. meningitidis and n. gonorrhoeae.
bacterial tracking demonstrated that the speed of n. meningitidis strains was significantly higher than that of n. gonorrhoeae strains .
live visualization of the filaments visible during twitching motility indicated that n. meningitidis fam <dig> displays more pili than n. gonorrhoeae fa <dig> and ms <dig> .
however, the tirf data did not reveal a clear correlation between the higher number of visible pili in n. meningitidis fam <dig> and the higher motility speed .
by using an nhs-ester-based fluorescent dye, we were able to observe tfp during live neisserial motility.
newly synthesized unlabeled pili or pili bundles may have contributed to motility.
however, we could also detect the elongation of labeled filaments , suggesting the possibility that labeled pile subunits are recycled in neisseria, analogous to previous reports in p. aeruginosa  <cit> .
tirf time-lapse experiments do not reveal whether a filament pulls, pushes, or is relaxed, but the observed straight shape of filaments is consistent with stretched filaments due to pulling.
the reported narrow and solvent-inaccessible channel at the center as well as the flexibility of pili suggests a relatively small effect of tfp pushing on motility  <cit> .
the tirf technique enables observation of labeled pili and bundles within 100 nm of the surface and filaments attaching at a large angle would be more difficult to image.
however, given the fact that one pilus is 6–8 nm in diameter and the most frequent bundles are up to 24 nm in diameter, there is a large margin that should permit the observation of most filaments.
the presence of fewer visible filaments on moving n. gonorrhoeae in comparison to n. meningitidis suggests a difference in tfp-dynamics between these two species.
for n. meningitidis, the frames with the highest speed most often displayed several spread filaments, rather than several filaments pointing in the same direction .
the most clear trend from quantification of pili bundles in tem indicate that n. meningitidis strain fam <dig> more frequently display two to four bundles in comparison to n. gonorrhoeae .
few published articles have explored the influence of pili bundles on motility.
a recently published model suggested that persistence is largely dependent on the re-elongation of pili from stable tfp membrane complexes and enhanced by small bundles containing two to three pili  <cit> .
the formation of tfp bundles is influenced by pile sequence variation  <cit> , increases with higher pile expression  <cit>  and is reduced in n. gonorrhoeae when grown in protein rich medium  <cit> .
bundles could in theory influence motility since they support stronger and more sustained retraction in comparison to single pili  <cit> .
however, at this point our data does not clearly show a correlation between pili bundles and enhanced motility speed.
meningococci express a polysaccharide capsule, which n. gonorrhoeae lacks.
unfortunately, an unencapsulated meningococcal mutant did not move on the tested surfaces, which made it impossible to compare this mutant with the wild-type strain.
quantification of the speed of serogroup a strains was not possible because these strains did not bind to the surface.
analysis of pilt promoters and the mutagenesis experiment in pathogenic neisseria strains revealed that a conserved difference in the σ <dig> - <dig> element between meningococci and gonococci  is linked to a lower pilt expression in meningococci .
the − <dig> promoter sequence 5’tacaat has previously been linked to suboptimal transcription in helicobacter pylori  <cit> .
the mechanism behind the difference in pilf expression remains an open question despite a previous attempt to link expression levels to a repetitive dna sequence present in the meningococcal but absent from the gonococcal pilf promoter  <cit> .
the relative expression of pilt and pilf in pathogenic neisseria are matched, suggesting that this balance is important for tfp dynamics.
although the change in pilt expression and atpase ratio through promoter mutagenesis reported here did not alter bacterial speed, a more considerable reduction in pilt expression has previously been demonstrated to influence tfp dynamics in ms <dig>  <cit> .
the amino acid sequence of pilt is identical between gonococci and meningococci, excluding intrinsic pilt differences as the source of motility variation.
because the pilt paralog pilt <dig> has been shown to increase pilus retraction in gonococci  <cit> , we cannot exclude the possibility that differences in pilt <dig> expression between meningococci and gonococci might influence their relative speed.
optical tweezers experiments investigating n. meningitidis pilus retraction speed are lacking but the highly conserved tfp biogenesis machinery and the similar pilus retraction force and speed in the more distantly related m. xanthus  <cit>  support similar pilus retraction rates in both pathogenic neisseria species.
it is intriguing that all sequenced n. meningitidis strains have a suboptimal pilt promoter in comparison to n. gonorrhoeae.
at this point, we can only speculate on the underlying reasons.
one theory could be that events altering the expression of certain tfp genes  may have disrupted the balance of tfp biogenesis and function, driving the entire system back to homeostasis.
another hypothesis could be that the higher expression of atpases is linked to optimal colonization of the gonococcal niche.
the origin and further implications of this divergence remain to be investigated.
CONCLUSIONS
in summary, due to a point variation in the pilt − <dig> promoter element, gonococci expressed a higher level of pilt in comparison to meningococci which is matched by a higher pilf expression.
furthermore, we observed distinct motility characteristics for n. gonorrhoeae and n. meningitidis, with respect to both the mean speed and the number of visible filaments during motility.
however, the difference in motility speed between n. gonorrhoeae and n. meningitidis does not appear to be correlated with the difference in number of visible filaments or pile sequence.
nevertheless, our data suggests a difference in tfp-dynamics between these two species.

 the closely related pathogens neisseria gonorrhoeae and neisseria meningitidis colonize human mucosal epithelia, however, at different sites in the body. neisserial motility is enabled by type iv pili, which are long and dynamic filaments expressed by a phylogenetically diverse set of bacterial species, such as pseudomonas aeruginosa, vibrio cholerae, legionella pneumophila, moraxella bovis, escherichia coli and myxococcus xanthus. in addition to mediating what is termed twitching motility, tfp are also involved in a multitude of other functions, including attachment to host cells, microcolony and biofilm formation and dna uptake. tfp can extend and retract via the assembly and disassembly of pilin subunits, called pile, from an inner membrane protein pool. the pile of pathogenic neisseria is divided into two classes: class i and class ii pilin. the latter is only present in a subset of n. meningitidis strains. in contrast to class i pilin, class ii pilin very rarely undergo antigenic variation and is shorter due to a deletion in the hypervariable region. the assembled filaments can extend up to several micrometers from the bacterial surface in contrast to the bacterial diameter, which is approximately 1 μm. on the other hand, the pilus cross-sectional diameter is only 6–8 nm, far below the diffraction limit of visible light, which makes it impossible to observe single tfp using bright field microscopy. tfp biogenesis and extension in neisseria spp. depends on a core set of 12– highly conserved proteins, whereas the retraction of tfp is powered by the atpase pilt. based on x-ray crystallography data, the functional unit of pilt is proposed to be a hexamer. in rod-shaped bacteria, such as p. aeruginosa and m. xanthus, pili are present at one pole although the atpases necessary for assembly and retraction are distributed to both poles. in pathogenic neisseria, pili extend in all directions and a study on pilt localization in n. gonorrhoeae indicated that it is found in the cytoplasm and associated with the inner membrane. of the two pathogenic neisseria species, mainly n. gonorrhoeae has been used to study twitching motility. optical tweezers experiments have demonstrated that a single gonococcal pilus is able to retract with peak forces up to pn, and bundles of pili can exert even stronger forces. the average pilus retraction rate in n. gonorrhoeae is  ±   μm/s at forces below pn. this rate may vary depending on multiple factors including the pilt concentration, the expression of the pilt paralog pilt the external force applied to the pilus and the oxygen concentration. movement of n. gonorrhoeae during long time scales is consistent with a random walk while persistent movement in one direction can be observed on shorter time scales. increasing the number of pili in n. gonorrhoeae increases the persistence time. in this work, we have investigated motility in n. meningitidis and n. gonorrhoeae, including the influence of pile sequence, pilin class and pilt expression. since it is important to understand the relation between pilus filaments and bacterial motility, we also monitored the speed and the number of visible filaments during bacterial motility using a combination of phase contrast and total internal reflection fluorescence microscopy. we observe differences between gonococci and meningococci in their motility characteristics that are not related to pilt expression. pile sequence or pilin class. furthermore, we demonstrate that pilt expression is influenced by a species-specific single nucleotide variation in the pilt promoter.  n. gonorrhoeae and n. meningitidis exhibit different speed during twitching motility to characterize motility in pathogenic neisseria, we studied twitching motility using live-cell phase contrast imaging and automated particle tracking of moving bacteria. the bacteria were allowed to adhere to poly-d-lysine-coated glass and observed over a period of 60 s, with images acquired per second. to determine the level of background noise in our system, a nonpiliated mutant derived from n. gonorrhoeae strain ms was included in the motility analysis and its speed was measured to  μm/s. the tracking data revealed significant differences in the mean speed between the n. gonorrhoeae and n. meningitidis strains tested. the average speed of n. gonorrhoeae strains ms and fa ranged between -  μm/s, whereas n. meningitidis strains had a speed of -  μm/s which included strains of serogroup b, c and w. pile sequence variation in neisseria affects the level of host cell adhesion and to determine whether it may also have an impact on motility, we tracked isogenic clones of ms with different pile sequences. variants 3: 5: and 8: carry two amino acid substitutions in the variable mini-cassette. further, variants 3: and 8: both contain a lysine to glutamate substitution in mc and variant 8: has another five substitutions in mc ms 6: has several amino acid changes in mc and different from the other three ms pile sequence variants. in addition, the variants 3: 5: and 6: have a larger substitution in the mc sequence. the hypervariable mc is unaltered in all pile variants. piliation level is similar between variants but approximately 50% lower in comparison to the parental ms strain. tracking analysis showed that there was no difference in speed between the pile sequence variants and the parental ms strain. in order to determine whether the pile class affected motility speed, the fam native pile gene encoding a class ii pilin was exchanged with the pile sequence obtained from the pile expression locus in the gonococcal strain fa which is similar to class i pilins of n. meningitidis. tracking analysis demonstrated that the pile swap mutant of fam although expressing slightly less pile and pili, moved with the same speed as the wild-type, indicating that pilin of gc most likely is not in itself causing the lower speed of n. gonorrhoeae. the surface coating poly-d-lysine is positively charged at neutral ph. to confirm the difference in speed between meningococci and gonococci on a biologically relevant surface material with a neutral net charge at ph 7– we assessed twitching motility on collagen. as shown in figure 1b, n. gonorrhoeae and n. meningitidis display a similar difference in speed on a collagen-coated surface. in conclusion, these data suggest that n. meningitidis moves faster than n. gonorrhoeae on both poly-d-lysine- and collagen-coated glass and that bacterial speed is independent of pile sequence variation.figure average motility speed of neisseria strains on a solid surface. n. gonorrhoeae strains fa and ms a nonpiliated ms and four clones of ms with different pile sequences. n. meningitidis strains fam, c, c, jb, c and the pile sequence swap mutant in fam the bacteria were allowed to adhere to poly-d-lysine-coated glass for 60 min at 37°c in 5% co2-saturated gc broth. attempts to track strains of serogroup a failed, as these strains did not adhere to the poly-d-lysine-coated surface. motility speed of n. gonorrhoeae fa and n. meningitidis fam on collagen-coated glass. the bacteria were allowed to adhere to the glass for 60 min prior to tracking. motile bacteria were studied using live-cell imaging and particle tracking. the data are presented as the average values of at least tracks acquired in at least three independent experiments. the error bars indicate the standard error of the mean. significant differences between gonococcal and meningococcal strains are marked with an asterisk *. expression of pilt and pilf are equal but higher in n. gonorrhoeae the atpases driving pili extension and retraction are pilf and pilt, respectively. pilt-mediated retraction is specifically essential for functional tfp and twitching motility. a previous study demonstrated a higher expression of pilf in gonococci in comparison to meningococci. we could confirm higher levels of pilf mrna in fa than in fam. because pilf expression differs between the species, we chose to also quantify pilt mrna and protein levels by using quantitative real-time pcr and a two-color western blot assay with an in-house generated polyclonal pilt antibody raised against pilt from n. gonorrhoeae. indeed, fa expressed higher relative levels of pilt mrna than fam and pilt expression matched that of pilf in both species. the western blot, with elongation factor tu as loading control, showed that n. gonorrhoeae fa expressed nearly twice as much pilt protein as n. meningitidis fam. because both the pilt and ef-tu amino acid sequences of n. gonorrhoeae and n. meningitidis are identical, the antibodies should react equally well in both species. in conclusion, n. gonorrhoeae expresses more pilf and pilt in comparison to n. meningitidis and because the atpase expression levels are matched it suggests that the pilt expression is not a factor underlying the difference in speed between pathogenic neisseria.table pilt versus pilf mrna expression strain reference gene pilt /reference gene pilf /reference gene 16s rrna rplp rpod 16s rrna rplp rpod a single base pair variation alters pilt expression to determine the reason behind the different pilt expression in pathogenic neisseria, we analyzed the pilt promoter. an alignment of the pilt promoter from representative strains is shown in figure 2c and all available sequenced strains are shown in additional file 2: figure s while close to identical, the pilt promoter in a majority of n. meningitidis strains contained the − promoter element 5’tacaat while the pilt promoter of all n. gonorrhoeae strains contained the canonical σ - sequence 5’tataat. an additional c/t polymorphism was found downstream of the transcriptional start site. to experimentally investigate the effect of the - promoter sequence on pilt expression, we switched the pilt promoter between n. meningitidis and n. gonorrhoeae. we then assessed the role of the atpase ratio in neisseria motility. promoter mutagenesis in the − sequence in both species demonstrated that the 5’tacaat sequence confers lower pilt mrna and protein levels without changing pilf mrna expression. neither the motility speed or the level of piliation, quantified in the fam mutants by using a whole cell elisa and transmission electron microscopy, differed between the promoter variants. in conclusion, the 5'tataat sequence specifically conserved in the gonococcal pilt promoter results in higher expression of pilt. however, the change in pilt/pilf ratio applied in this work does not significantly affect motility speed.figure analysis of pilt expression in pilt promoter mutants. the pilt promoter constructs used to transform n. gonorrhoeae n and n. meningitidis fam carried either the 5’tataat or the 5’tacaat sequence with a chloramphenicol resistance cassette oriented on the opposite strand and in the opposite direction of the pilt gene. pilt mrna transcription level in n and fam, as quantified using qpcr. mrna expression was normalized to three different reference genes and compared to the wt level. the experiment was performed two to three times. the bars show the mean ± standard deviation. pilt protein quantification of whole cell lysates from n and fam by western blot. the relative pilt/ef-tu band intensities were averaged over two to four separate blots and normalized against wt strains. the error bars represent the standard deviation. visualizing type iv pili in motile n. gonorrhoeae and n. meningitidis to determine whether the observed difference in motility speed between n. gonorrhoeae and n. meningitidis could be linked to the distribution or number of pili, we stained bacteria and pili with an nhs-ester-based fluorescent dye and visualized pili using live-cell tirf microscopy. the stain did not significantly affect speed. tirf microscopy provides a highly specific excitation of fluorophores up to ~100 nm from the glass surface. when using this illumination system, the background fluorescence is drastically reduced and the relatively weak fluorescence of stained pili becomes visible. hence, it was possible to monitor pili in neisseria during bacterial crawling, similar to previous visualization of tfp in pseudomonas. the time for tirf imaging was limited to 10– seconds due to fading of the fluorescent stain. it is important to note that this technique did not permit the distinction between a single pilus and a bundle of pili. therefore, observed pili will collectively be referred to as visible filaments. we monitored bacterial crawling on a poly-d-lysine-coated glass surface and captured more than bacterial tracks of n. gonorrhoeae fa and ms and n. meningitidis fam motility. in general, the number of visible filaments varied over time during the movement of a bacterium. filaments were observed in all directions around the bacterium, which is in accordance with previous data. figure 4a shows representative images of motile, fluorescently stained n. gonorrhoeae and n. meningitidis. to quantify the relative frequency of visible filaments during motility, a manual single-blinded frame-by-frame counting was performed on the collected time-lapse movies. when moving on a solid surface, n. gonorrhoeae most frequently displayed one filament, while n. meningitidis most often exhibited three filaments. similar results as seen with tirf were obtained after quantification of single pili and pili bundles in tem although only a trend and not a significant difference could be detected. comparison of pilus bundle size showed that the majority of bundles observed in both meningococci and gonococci contained two or three pili, corresponding to a bundle width of 10–25 nm. the relative amount of pili that were present as single filaments versus as bundles was slightly higher in gonococcal strains than in fam. figure 6a shows an example of tfp-mediated motility involving primarily a single filament, as exemplified by n. gonorrhoeae strain fa where the mean track speed was  μm/s and the distance travelled being  μm over the  s shown. the corresponding track of movement is presented in figure 6c with dots indicating the position and the colors showing the speed in each frame. figure 6b exemplifies bacterial movement by n. meningitidis strain fam where the mean track speed was  μm/s and the distance travelled being  μm over the  s shown. the full time-lapse videos are shown in video s and s. the track in figure 6d indicates that meningococcal movement involving the apparent retraction of several filaments simultaneously results in more frequent changes in direction. still, a graph relating the number of filaments to the motility speed only depicted a weak correlation and n. gonorrhoeae with the same number of filaments as meningococci in tirf movies still moved at a lower speed. to summarize, the results suggest that there is no correlation between the difference in number of filaments and motility speed in n. meningitidis and n. gonorrhoeae. nevertheless, n. meningitidis fam displayed multiple filaments more frequently than n. gonorrhoeae, which may influence changes in motility direction.figure visualization of tfp during movement using tirf microscopy. live bacteria were stained with the dylight™ nhs-ester prior to experiments. representative pictures of stained n. gonorrhoeae and n. meningitidis crawling on a poly-d-lysine-coated surface. the scale bar corresponds to 5 μm. percent of observations with x number of visible filaments during movement on a solid surface. more than individual frames from the time-lapse movies were analyzed for each species, and the number of visible filaments was counted in each frame. discussion tfp-mediated motility is accomplished via repeated steps of pilus assembly and pilt-mediated pilus retraction. in this work, we aimed to further characterize and quantify pilus dynamics and twitching motility in n. meningitidis and n. gonorrhoeae. bacterial tracking demonstrated that the speed of n. meningitidis strains was significantly higher than that of n. gonorrhoeae strains. live visualization of the filaments visible during twitching motility indicated that n. meningitidis fam displays more pili than n. gonorrhoeae fa and ms. however, the tirf data did not reveal a clear correlation between the higher number of visible pili in n. meningitidis fam and the higher motility speed. by using an nhs-ester-based fluorescent dye, we were able to observe tfp during live neisserial motility. newly synthesized unlabeled pili or pili bundles may have contributed to motility. however, we could also detect the elongation of labeled filaments, suggesting the possibility that labeled pile subunits are recycled in neisseria, analogous to previous reports in p. aeruginosa. tirf time-lapse experiments do not reveal whether a filament pulls, pushes, or is relaxed, but the observed straight shape of filaments is consistent with stretched filaments due to pulling. the reported narrow and solvent-inaccessible channel at the center as well as the flexibility of pili suggests a relatively small effect of tfp pushing on motility. the tirf technique enables observation of labeled pili and bundles within 100 nm of the surface and filaments attaching at a large angle would be more difficult to image. however, given the fact that one pilus is 6–8 nm in diameter and the most frequent bundles are up to 24 nm in diameter, there is a large margin that should permit the observation of most filaments. the presence of fewer visible filaments on moving n. gonorrhoeae in comparison to n. meningitidis suggests a difference in tfp-dynamics between these two species. for n. meningitidis, the frames with the highest speed most often displayed several spread filaments, rather than several filaments pointing in the same direction. the most clear trend from quantification of pili bundles in tem indicate that n. meningitidis strain fam more frequently display two to four bundles in comparison to n. gonorrhoeae. few published articles have explored the influence of pili bundles on motility. a recently published model suggested that persistence is largely dependent on the re-elongation of pili from stable tfp membrane complexes and enhanced by small bundles containing two to three pili. the formation of tfp bundles is influenced by pile sequence variation, increases with higher pile expression and is reduced in n. gonorrhoeae when grown in protein rich medium. bundles could in theory influence motility since they support stronger and more sustained retraction in comparison to single pili. however, at this point our data does not clearly show a correlation between pili bundles and enhanced motility speed. meningococci express a polysaccharide capsule, which n. gonorrhoeae lacks. unfortunately, an unencapsulated meningococcal mutant did not move on the tested surfaces, which made it impossible to compare this mutant with the wild-type strain. quantification of the speed of serogroup a strains was not possible because these strains did not bind to the surface. analysis of pilt promoters and the mutagenesis experiment in pathogenic neisseria strains revealed that a conserved difference in the σ - element between meningococci and gonococci is linked to a lower pilt expression in meningococci. the − promoter sequence 5’tacaat has previously been linked to suboptimal transcription in helicobacter pylori. the mechanism behind the difference in pilf expression remains an open question despite a previous attempt to link expression levels to a repetitive dna sequence present in the meningococcal but absent from the gonococcal pilf promoter. the relative expression of pilt and pilf in pathogenic neisseria are matched, suggesting that this balance is important for tfp dynamics. although the change in pilt expression and atpase ratio through promoter mutagenesis reported here did not alter bacterial speed, a more considerable reduction in pilt expression has previously been demonstrated to influence tfp dynamics in ms. the amino acid sequence of pilt is identical between gonococci and meningococci, excluding intrinsic pilt differences as the source of motility variation. because the pilt paralog pilt has been shown to increase pilus retraction in gonococci, we cannot exclude the possibility that differences in pilt expression between meningococci and gonococci might influence their relative speed. optical tweezers experiments investigating n. meningitidis pilus retraction speed are lacking but the highly conserved tfp biogenesis machinery and the similar pilus retraction force and speed in the more distantly related m. xanthus support similar pilus retraction rates in both pathogenic neisseria species. it is intriguing that all sequenced n. meningitidis strains have a suboptimal pilt promoter in comparison to n. gonorrhoeae. at this point, we can only speculate on the underlying reasons. one theory could be that events altering the expression of certain tfp genes may have disrupted the balance of tfp biogenesis and function, driving the entire system back to homeostasis. another hypothesis could be that the higher expression of atpases is linked to optimal colonization of the gonococcal niche. the origin and further implications of this divergence remain to be investigated.  in summary, due to a point variation in the pilt − promoter element, gonococci expressed a higher level of pilt in comparison to meningococci which is matched by a higher pilf expression. furthermore, we observed distinct motility characteristics for n. gonorrhoeae and n. meningitidis, with respect to both the mean speed and the number of visible filaments during motility. however, the difference in motility speed between n. gonorrhoeae and n. meningitidis does not appear to be correlated with the difference in number of visible filaments or pile sequence. nevertheless, our data suggests a difference in tfp-dynamics between these two species. 
neisseria motility has mainly been studied in n. gonorrhoeae whereas the motility of n. meningitidis has not yet been characterized. live-cell imaging using total internal reflection fluorescence microscopy demonstrated that n. gonorrhoeae more often moved with fewer visible retracting filaments when compared to n. meningitidis. we observed that n. meningitidis moved over surfaces at an approximate speed of  μm/s, whereas n. gonorrhoeae moved with a lower speed. the type iv pili of pathogenic neisseria are essential for twitching motility. neisseriatype iv pilipilttwitching motilityissue-copyright-statement© the author 2015 nevertheless, motile gonococci that had the same number of filaments as n. meningitidis still moved with a lower speed. by tracking mutants with altered pilt expression or pile sequence, we concluded that the difference in motility speed was independent of both. these data reveal differences in both speed and piliation between the pathogenic neisseria species during twitching motility, suggesting a difference in tfp-dynamics. correspondingly, meningococci also displayed a higher level of piliation in transmission electron microscopy. in this work, we analyzed bacterial motility and monitored tfp retraction using live-cell imaging of freely moving bacteria. an alignment of the meningococcal and gonococcal pilt promoters revealed a conserved single base pair variation in the − promoter element that influence pilt expression.
 the type iv pili of pathogenic neisseria are essential for twitching motility. tfp retraction, which is dependent on the atpase pilt, generates the forces that move bacteria over surfaces. neisseria motility has mainly been studied in n. gonorrhoeae whereas the motility of n. meningitidis has not yet been characterized.  in this work, we analyzed bacterial motility and monitored tfp retraction using live-cell imaging of freely moving bacteria. we observed that n. meningitidis moved over surfaces at an approximate speed of  μm/s, whereas n. gonorrhoeae moved with a lower speed. an alignment of the meningococcal and gonococcal pilt promoters revealed a conserved single base pair variation in the − promoter element that influence pilt expression. by tracking mutants with altered pilt expression or pile sequence, we concluded that the difference in motility speed was independent of both. live-cell imaging using total internal reflection fluorescence microscopy demonstrated that n. gonorrhoeae more often moved with fewer visible retracting filaments when compared to n. meningitidis. correspondingly, meningococci also displayed a higher level of piliation in transmission electron microscopy. nevertheless, motile gonococci that had the same number of filaments as n. meningitidis still moved with a lower speed.  these data reveal differences in both speed and piliation between the pathogenic neisseria species during twitching motility, suggesting a difference in tfp-dynamics. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords neisseriatype iv pilipilttwitching motilityissue-copyright-statement© the author 2015 