BACKGROUND
bacterial cell division occurs in the middle of the dividing cell mediated by protein complex division machinery called divisome.
ftsz  is the principal protein of the divisome in escherichia coli, and assembles into the z ring at the cell division site; it is bound to the inner membrane and is stabilized by ftsa and zipa
<cit> .
this event is necessary for the sequential addition of at least twelve other membrane proteins responsible for peptidoglycan assembly and membrane constriction
<cit> .
inactivation of the corresponding genes blocks the cell division and give rise to long multinucleated cells that fail to propagate and eventually die
<cit> .
the z-ring and its dynamics during the cell process have been observed by fluorescence microscopy in living cells
<cit>  and in liposomes
<cit> .
although, the organization of ftsz in the z-ring is not known, based on the in vitro studies it is believed that the basic units to form the ring are the protofilaments.
in order to explain the z-ring dynamics at the molecular level, two theoretical models have been proposed.
one model involves the hydrolysis of gtp to generate the force for invagination of the plasma membrane
<cit>  and the other model proposes a microphase transition of the ftsz filaments that is energized by lateral interactions between filaments during the constriction of the plasma membrane
<cit> .
in both models, the lateral interactions between the protofilaments are crucial for the membrane constriction.
however, the role of lateral interactions has been questioned because the interaction entropy was not considered in the formation of the filaments
<cit> .
therefore, the question regarding the role of lateral interactions still remains unclear.
mutations on the lateral interface of ftsz induce the formation of an aberrant z-ring in e. coli cells and cell division can be compromised
<cit> .
apparently, the lateral interactions to form the z-ring are also important in b. subtilis <cit> .
recently, the z-ring was observed in vivo at a higher resolution.
the authors proposed that ftsz polymerizes into short filaments that assemble into loose bundles mediated by longitudinal and lateral interactions to finally locate as a z-ring at the division site
<cit> .
these observations suggest that lateral interactions between ftsz protofilaments could be important for in vivo formation of the functional z ring.
however, the elements that play a key role in the z-ring formation remain unknown.
in vitro, lateral interactions of ftsz stabilize sheets and rings
<cit>  and zipa, mentioned earlier as a membrane anchor protein, stimulates the formation of ftsz sheets
<cit> .
moreover, ftsz also polymerizes into sheets and tubules in the presence of cations such as calcium or deae-dextran
<cit> , or by the ftsz-interacting protein sepf
<cit> .
the sheets and tubules are stabilized by lateral interactions similar to those found in tubulin polymers
<cit> .
moreover, arc-like filaments that could interact laterally have been observed by electron cryotomography at the constriction site
<cit> , but the importance of these lateral interactions has remained controversial
<cit> .
ca2+-induced ftsz sheets are composed of protofilaments that are aligned in parallel and antiparallel way, while in zn2+-induced tubulin sheets are arranged only in an antiparallel manner.
thus, from a low-resolution sheet structure of methanococcus jannaschii ftsz, it has been proposed that two protofilaments interact through the same lateral face, in a parallel form, to produce double-stranded filaments called thick filaments, which subsequently form sheets through antiparallel interactions
<cit> .
e. coli ftsz also polymerases into thick filaments
<cit> , indicating that these filaments can be the basic units to form sheets.
phylogenetic and structural studies show that ftsz is the most-conserved cell division protein and it shares structural and functional features with eukaryotic tubulins
<cit> .
the heterodimer αβ-tubulin polymerizes into microtubules and two types of interactions are involved between the heterodimer subunits: the longitudinal interactions between heterodimers to form the protofilaments, and the lateral interactions between  <dig> or  <dig> parallel protofilaments to organize into the microtubule.
the lateral interactions result from the contacts between α- and β-tubulin subunits of neighboring protofilaments and are mediated by central structural elements, such as the interaction of the m loop of one subunit with the h <dig> helix of the neighboring subunit
<cit> .
a newly discovered bacterial tubulin, btuba/b, also contains the h <dig> helix and m loop, however it is believed that the last one is a reminiscent structure from eukaryotic tubulin
<cit> .
interestingly, the h <dig> helix is highly conserved among ftszs while the m loop is absent.
therefore, we believe that h <dig> helix of ftsz can participate in the lateral interactions in a similar manner to its related tubulins.
the aim of this work was to investigate the role of the h <dig> helix in lateral interactions of ftsz from e. coli.
we mutated the highly conserved glutamate  <dig> and arginine  <dig> residues to glutamine by site-directed mutagenesis and evaluated the effect of these mutations on the viability of the cells, the cell morphology and localization of ftsz, as well as the gtpase activity and the ability to polymerize in vitro, in the presence and in the absence of calcium and zipa.
RESULTS
design of ftsz and ftsz mutants
we choose as candidates for mutation two conserved residues located in the lateral face of ftsz, specifically in the bend of h <dig> helix.
this was based on the similarities in the crystal structures between ftsz and tubulin and the degree of conservation of amino acids among ftsz sequences.
the three dimensional structure of an e. coli ftsz modela  was compared with the structure of the αβ-tubulin heterodimer.
the helix h <dig> of tubulin, located on the lateral face and known for the role in the lateral interactions, is also present in ftsz.
the helix h <dig> of ftsz is located in the left lateral face
<cit> .
helix h <dig> is present in most of the ftsz sequences from gram positive and negative bacteria, mitochondria and chloroplasts.
within the h <dig> helix, glutamate at position  <dig> is 100% conserved and the positive charge of arginine at  <dig> is highly conserved  among ftsz sequences from  <dig> different organisms .
interestingly, the two residues are located in the bend of the helix h <dig> and are exposed to the surface of the left face.
hence, we hypothesized that these residues, e <dig> and r <dig>  participate in the interactions of ftsz to form filaments, tubules or sheets.
to this end, the bend in h <dig> could be necessary to maintain both the topography of the monomer for the interactions and the gtpase activity in the polymer.
in microtubules, the lateral interactions are principally mediated between the helix h <dig> from a protofilament and the m loop of the adjacent protofilament
<cit> .
the m loop is even present in bacterial tubulin a/b, though smaller than its counterpart tubulin, and is also believed to participate in the lateral interactions to form a bacterial microtubules  composed by five protofilaments
<cit> .
however, ftsz lacks the m loop hence, the lateral interactions are expected to be weaker between ftsz protofilaments
<cit> , hence an effect on longitudinal interactions should affect both the length and the gtpase activity of the polymers.
viability of e. coli vip <dig> cells in the presence of ftsz and ftsz
to evaluate the functionality of the ftsz mutants in vivo, the e83q, and r85q proteins were expressed independently and the cell viability was determined in a strain containing ftsz thermonull mutant, vip2/plar <dig>  the parental strain, vip2/plar <dig> has been successfully employed to demonstrate that ftsz is essential for cell division and survival in e. coli <cit> .
for the purpose of our study we modified the parental strain to create a de <dig> lysogen of vip <dig>  that contains a copy of t <dig> rna polymerase gene under the control of placuv <dig>  so the expression of a target protein is inducible by iptg addition.
thus, if vip2/plar <dig> is transformed with a second plasmid that contains the ftsz mutants , the transformed cells will contain a ftsz mutant allele from pmfv-derived plasmid under the control of a t <dig> promoter, a wild-type allele encoded on plasmid plar <dig>  and a genomic null allele.
in summary, at the permissive temperature , vip2/plar9/pmfv- will express both ftsz alleles , whereas at the restrictive temperature  plar <dig> cannot replicate, so the ftsz expression originates from the pmfv-derivatives.
the production of ftsz ceases completely after 2– <dig> h of shifting the temperature, once plar <dig> is segregated by lack of replication.
in this regard, this system is different from those utilizing a ftsz mutant nonfunctional at 42°c, because in this case after shifting to the non-permissive temperature ftsz will be nonfunctional almost instantly.
to simplify the nomenclature we will refer henceforth to the strain vip <dig>  containing plar <dig>  as vip <dig>
vip5/pmfv-wt cell grown at 42°c  and at 30°c show similar cfu/ml in the absence of iptg, indicating that the basal non-induced expression level from pmfv-wt  was enough to complement the lack of ftsz from plar <dig> at 42°c .
hence, in order to avoid an excessive expression of ftsz, iptg was not added in all our experiments.
as expected, the survival of vip <dig> at 42°c  decreases about four orders of magnitude.
the small survival fraction observed in vip <dig> is explained as due to the reversion frequency of the chromosomal mutation
<cit> .
figure
<dig> shows that the viability of cells expressing ecftsz mutants e83q and r85q at the restrictive temperature diminished about  <dig>  and  <dig> orders of magnitude, respectively, compared to the non-restrictive temperature.
these values indicate that the cells could not support normal cell division but at the same time the ecftsz mutants did not completely lose functionality in vivo.
hence, the survival assay was useful to characterize the functionality of ecftsz mutants in vivo and the quantification of the viability of the cells expressing ecftsz mutants was used as an indication of the efficiency in the cell division.
consequently, the efficiency of the different forms of ecftsz in terms of cell viability can be interpreted as: wild type > e83q > r85q.
cell morphology and in situ localization of ftsz mutants using immunofluorescence microscopy
the ftsz ring is formed in the middle of the cell and is attached to the cytoplasmic membrane through zipa
<cit> .
in order to evaluate the effect of these mutations on cell morphology and ftsz localization, vip <dig> cells in exponential phase of growth carrying plasmids with ftsz, ftsz, and ftsz mutants were incubated in the absence of iptg at 30°c  and at 42°c.
figure
<dig> shows that, in the negative control at 42°c  the cell division is blocked, and ftsz localization is dispersed along the very long filamentous cells , while at 30°c the cells are normal.
cells carrying the plasmid ftsz at 42°c  reverts the cell filamentation, and at 30°c  as expected the morphology and ftsz localization is normal.
the co-expression at 30°c of ftsz from plar <dig> with the mutants ftsz  and ftsz  also display a normal phenotype.
however, the expression of the ftsz mutants at 42°c ) have an effect on cell division, producing filamentous cells .
interestingly, ftsz still localizes as discrete bands along the filamentous cells, probably at the potential cell division sites .
in contrast, the expression of ftsz produced shorter filamentous cells with a disperse localization of the protein along the filaments, and no clear bands were observed .
the same experiments were performed with iptg  <dig>  mm and similar results were obtained .
in order to keep the cells in exponential phase of growth for a longer time, the morphology experiments were repeated starting the culture for the temperature shift at an od <dig> of  <dig> .
after  <dig> h of incubation no difference in the cell morphology of the mutants respect to the experiments presented in figure
<dig> was observed.
localization of the ftsz mutants in the single cell population  that appear at early stationary phase  shows that ftsz is located with greater intensity at the middle of the cell but presents some degree of dispersion, while ftsz is rather distributed in a gradient shape from the mid-cell toward the cell poles.
the zipa distribution was similar to that observed for ftsz mutants , which suggests that the mutations in ftsz do not affect its interaction with zipa.
binding experiments between ftsz  and zipa inserted into proteoliposomes confirmed this hypothesis .
in summary, the residues glutamate  <dig> and arginine  <dig> located in bend of helix h <dig> on the left face of ftsz plays a role in the formation of the z-ring  and consequently zipa presents an altered localization in cells expressing the ftsz mutants .
structural characterization of r85q and e83q
in order to evaluate the impact of the point mutations e83q and r85q on the protein structure, we performed circular dichroism on purified ftsz wt, r85q and e83q.
all three purified proteins showed the expected molecular mass and had a very similar secondary structure content ; therefore, presumably the mutations did not cause significant changes in the tertiary structure of ftsz.
gtpase activity of e83q and r85q
in order to understand the possible effects of the ftsz mutations on the formation of the z ring, we purified the mutant proteins and measured their abilities to hydrolyze gtp.
the wild-type ftsz and e83q exhibited a constant initial velocity of gtp hydrolysis for at least  <dig> minutes at a protein concentration of  <dig>  μm .
by contrast, r85q showed a lag time  of  <dig>  minutes and a reduced gtpase activity.
we next compared the gtp hydrolysis efficiency by calculating the apparent catalytic constants .
wild-type ftsz, e83q and r85q hydrolyzed gtp with apparent catalytic constants of  <dig>  ±  <dig>  min- <dig>   <dig>  ±  <dig>  min- <dig> and  <dig>  ±  <dig>  min- <dig> respectively.
r85q was further characterized by measuring the dependence of gtp hydrolysis on protein concentration .
the progress curves show a sigmoidal behaviour, i.e., the lag time was dependent on the protein concentration .
this lag time should disappear at the extrapolated concentration of  <dig> μm.
the similarity of the progress curves at  <dig>  and  <dig>  μm of protein is probably due to gtp becoming limiting.
these results suggest that the r85q mutation does not directly alter the rate of gtp hydrolysis, but rather affects ftsz polymerization, which is known to induce gtpase activity.
therefore, the effect is reversed when the concentration of r85q is increased to stimulate polymerization.
the gtpase activity of ftsz wt and e83q is not affected by zipa, which is in agreement with previous study
<cit> .
interestingly, zipa increased around twofold the gtpase activity of r85q , suggesting that zipa stimulates the longitudinal interactions of r85q protofilaments during polymerization, similar to the effect of maps on microtubules
<cit> .
polymerization of ftsz mutants with and without zipa
in vitro, ftsz polymerizes into protofilaments in the presence of gtp
<cit> , and these protofilaments associate to form bundles in the presence of zipa
<cit> .
single protofilaments are monomers that interact longitudinally whereas double protofilaments  consist of two parallel protofilaments interacting laterally
<cit> .
antiparallel lateral interactions of these double filaments induce the formation of sheets or bundles
<cit> .
in order to evaluate the effect of the mutations on the polymerization capability, we followed the polymerization of ftsz mutants by 90° light scattering at  <dig> nm and the polymers morphology by electron microscopy.
the polymerization reaction was started with the addition of the protein, and the reaction was followed for one hour for wild type and e83q, while r85q was followed for almost three hours due to the long lag period observed for the gtpase activity .
the mutant r85q apparently did not form long filaments at the protein concentration used for ftsz wild type and e83q , producing a small increment in turbidity that was stimulated around two-fold by zipa additional file
1: figure s3a), whereas ftsz wild type and e83q showed a fast polymerization kinetics that could not be measured.
e83q formed polymers that remained in solution around three times longer than those formed by ftsz wild type, with a rate of depolymerization  <dig>  times slower.
this increase in the time in which the filaments remain polymerized can be explained by the small apparent catalytic constant for the gtpase activity.
the time traces of light scattering  indicate that the polymerization and the subsequent depolymerization of ftsz are altered by the mutations on the arginine  <dig> and glutamate  <dig>  additional file
1: figure s <dig> shows that calcium increased the ligth scattering, and for ftsz wild type and e83q this was due to the formation of bundles, as shown in additional file
1: figure s <dig>  on the other hand, r85q presented a smaller increase in light scattering and no formation of bundles was observed.
<dig>  after a stable base line of the buffer containing 1mm gtp, with or without zipa, ftsz was added to initiate the polymerization reaction.
the change in light scattering at  <dig> nm was recorded during the polymerization reaction and the difference between the base line and the maximum value reached after ftsz addition is shown in the bar graph.
electron microscopy of negatively stained polymers of wild type ftsz, e83q and r85q polymerized as in .
the samples for ftsz wt, e83q and r85q were taken at  <dig> min,  <dig> min and 2h  <dig> min, respectively .
electron micrographs were taken at a magnification of  <dig>  for wild type ftsz and e83q, and of  <dig>  for r85q.
scale bar,  <dig> nm.
histogram of ftsz polymers width distribution shows that the mutations e83q and r85q decrease the number of protofilaments per polymer.
at least  <dig> polymers were measured for each case using imagej.
figure
5a shows the maximum light scattering value reached by ftsz in  <dig> mm gtp in the presence and absence of zipa.
the mutants presented a decrease in the polymerization levels of ~20% and ~75% for e83q and r85q, respectively.
in the presence of zipa, the maximum light scattering value of ftsz wild type increased slightly, , while for both mutants the increment was significantly larger: 90% and 150% for e83q and r85q, respectively .
in the absence of zipa, ftsz wild type polymerized into long and mostly straight filaments of  <dig> to  <dig> nm width  suggesting that the main population of these filaments consists of  <dig> assembled protofilaments of  <dig> nm width for each protofilament .
e83q polymerized into curved filaments with a width of  <dig> to  <dig> nm .
the greatest effect was caused by the mutation r85q, in which the polymers were mainly short and straight, and occasionally, spirals probably composed of single or double protofilaments were found on the grids .
zipa induced bundling of wt and e83q, which is in agreement with previous studies
<cit> ; however, we did not observe a clear bundling effect in the r85q mutant, even given the increase in light scattering.
the light scattering contribution of zipa was subtracted, therefore we can rule out the possibility that the increase in the light scattering of r85q is due to zipa.
further characterization of r85q will be done in a future study.
all the results together indicate that arginine  <dig> and glutamate  <dig> participate in the longitudinal and in the lateral interactions, shown by their reduced gtpase activity and the effect on polymerization, respectively.
interestingly, the addition of a stabilizer of the lateral interactions, such as zipa to r85q, did not induce bundles  but stimulated the gtpase activity , indicating that protofilaments are induced.
on the other hand, in the case of e83q, even though the protofilament lateral interactions were diminished  and zipa induced bundles that were thinner than those with ftsz wild type, the gtpase activity was not greatly compromised.
therefore, we believe that the ability of ftsz to form bundles, presumably through lateral interactions, is important to form the z-ring in the cell, and consequently for cell division, a conclusion consistent with our immunofluorescence experiments  and cell viability results .
discussion
the aim of this work was to characterize the role of two conserved amino acid residues, e <dig> and r <dig>  located in the bend of helix h <dig> at the lateral face of ftsz .
our results show that these amino acids are relevant for cell division, ftsz polymerization, gtpase activity and stability of the polymers.
the properties of these ftsz mutants were evaluated through their kinetics and critical concentrations for polymerization and gtpase activities in the absence and presence of zipa and calcium.
the ftsz mutant proteins had the same secondary structure as the wild type form  indicating an intact global fold.
interestingly, although the mutations are located far from the gtpase catalytic site  both mutant proteins hydrolyzed gtp with less efficiency than wild type in the following order: wt > e83q > r85q.
compared to the wild type ftsz, filaments with mutant proteins were thinner with altered curvatures, indicating that the mutations affect lateral interactions between ftsz protofilaments.
zipa, which in wild type ftsz is known to induce bundling without affecting gtpase activity, induced thinner bundles in e83q, while in the case of r85q was unable to cause bundling, suggesting that both glutamate  <dig> and arginine  <dig> are important for lateral interactions between protofilaments.
r85q filaments were short and abnormally bent, indicating that the mutation also affects the geometry of the longitudinal interactions.
the formation of short filaments is consistent with the ability of r85q to hydrolyze gtp, which requires longitudinal interaction between two ftsz molecules
<cit> .
also the neighbor mutation d86k, which induces twined filaments, shows a reduction in gtpase activity further suggesting a role of this region in longitudinal interactions
<cit> .
the long lag period of the progress curve of r85q gtpase activity was suppressed by increasing the protein concentration, confirming that this activity was directly related to polymerization.
this result could be explained by a reduction of the nucleation free energy over the free energy for the elongation process.
the bend in h <dig> is likely caused by the negatively-charged amino acids ded located just before the bend which destabilize the helix by negative charge repulsion.
at the other end of the h <dig> bend, r <dig> is followed by d86; therefore, the positive charge of r <dig> could destabilize the positive dipole of the helix in this region.
thus, altering the charges in this region could induce a conformational change, explaining the effects of the r85q, e83q and d86k
<cit>  mutations on the functionality of ftsz.
based on our results we hypothesize that, directly or via a subtle conformational change, the charges of amino acids located in the bend of the h <dig> helix are involved in longitudinal and lateral interactions.
we infer that the negative charge of glutamate  <dig> weakens these interactions while the positive charge of arginine  <dig> stabilizes them, probably regulating the dynamics of the z-ring.
thus, cells carrying ftsz or ftsz were affected in cell division and showed reduced survival and cell filamentation in agreement with previously described lateral-interaction-ftsz mutants that could not complement the temperature sensitive ftsz <dig> <cit> .
we have quantified survival rates at the restrictive temperature  as a means to evaluate the effect of ftsz mutations on cell division, similar to studies on the effects of proteins expression from unstable plasmids
<cit> .
the e83q and r85q mutations diminished the apparent catalytic constant of gtpase activity by 60% and 95%, respectively.
presumably, the positive charge of r <dig> is involved in longitudinal and lateral interactions between protofilaments explaining the inhibitory effect of the r85q mutation on gtpase activity and polymerization when compared to the e83q or d86k mutants
<cit> .
both, longitudinal and lateral interactions are important in models for the conversion of gtp hydrolysis into mechanical energy during z-ring constriction
<cit> .
the observed contraction of tubular liposomes in the presence of gtp and a chimeric, membrane-attached ftsz indicates that polymerization of ftsz is sufficient to generate a mechanical force to bend the membrane.
our results indicate that h <dig> bend mutations affecting longitudinal and lateral interactions in vitro reduce cell viability in vivo while they cause cell filamentation.
cell filamentation and the immunofluorescence analysis  together with the polymerization results support a model of a z-ring composed of several polymers that are recruited to the septation site by lateral interactions, as has been found in vivo <cit> .
athe mutations are positioned on the surface and their location on the structure are shown in figure
<dig>  left: the left lateral surface; right: the right lateral surface.
aa, nº = amino acid number.
bthe % viability was determined from data of figure
<dig>  n = no growth of ftsz <dig> at the restrictive temperature.
cgtpase activity was measured with  <dig> μm of protein in all cases.
db, bundles; s, short filaments; nd, not determined.
the shape of the polymers was determined by electron microscopy .
CONCLUSIONS
this study describes the role of the positively charged arginine  <dig> and the negatively charged glutamate  <dig> in longitudinal and lateral interactions to form ftsz protofilaments and two-dimensional polymers of protofilaments, respectively.
we also show that gtpase activity could be fine-tuned by the net charge of the h <dig> helix bend.
finally, our results suggest that the conserved amino acids e83q and r85q located in the bend of h <dig> are involved in the formation of a functional z-ring.

 bacterial cell division occurs in the middle of the dividing cell mediated by protein complex division machinery called divisome. ftsz is the principal protein of the divisome in escherichia coli, and assembles into the z ring at the cell division site; it is bound to the inner membrane and is stabilized by ftsa and zipa. this event is necessary for the sequential addition of at least twelve other membrane proteins responsible for peptidoglycan assembly and membrane constriction. inactivation of the corresponding genes blocks the cell division and give rise to long multinucleated cells that fail to propagate and eventually die. the z-ring and its dynamics during the cell process have been observed by fluorescence microscopy in living cells and in liposomes. although, the organization of ftsz in the z-ring is not known, based on the in vitro studies it is believed that the basic units to form the ring are the protofilaments. in order to explain the z-ring dynamics at the molecular level, two theoretical models have been proposed. one model involves the hydrolysis of gtp to generate the force for invagination of the plasma membrane and the other model proposes a microphase transition of the ftsz filaments that is energized by lateral interactions between filaments during the constriction of the plasma membrane. in both models, the lateral interactions between the protofilaments are crucial for the membrane constriction. however, the role of lateral interactions has been questioned because the interaction entropy was not considered in the formation of the filaments. therefore, the question regarding the role of lateral interactions still remains unclear. mutations on the lateral interface of ftsz induce the formation of an aberrant z-ring in e. coli cells and cell division can be compromised. apparently, the lateral interactions to form the z-ring are also important in b. subtilis. recently, the z-ring was observed in vivo at a higher resolution. the authors proposed that ftsz polymerizes into short filaments that assemble into loose bundles mediated by longitudinal and lateral interactions to finally locate as a z-ring at the division site. these observations suggest that lateral interactions between ftsz protofilaments could be important for in vivo formation of the functional z ring. however, the elements that play a key role in the z-ring formation remain unknown. in vitro, lateral interactions of ftsz stabilize sheets and rings and zipa, mentioned earlier as a membrane anchor protein, stimulates the formation of ftsz sheets. moreover, ftsz also polymerizes into sheets and tubules in the presence of cations such as calcium or deae-dextran, or by the ftsz-interacting protein sepf. the sheets and tubules are stabilized by lateral interactions similar to those found in tubulin polymers. moreover, arc-like filaments that could interact laterally have been observed by electron cryotomography at the constriction site, but the importance of these lateral interactions has remained controversial. ca2+-induced ftsz sheets are composed of protofilaments that are aligned in parallel and antiparallel way, while in zn2+-induced tubulin sheets are arranged only in an antiparallel manner. thus, from a low-resolution sheet structure of methanococcus jannaschii ftsz, it has been proposed that two protofilaments interact through the same lateral face, in a parallel form, to produce double-stranded filaments called thick filaments, which subsequently form sheets through antiparallel interactions. e. coli ftsz also polymerases into thick filaments, indicating that these filaments can be the basic units to form sheets. phylogenetic and structural studies show that ftsz is the most-conserved cell division protein and it shares structural and functional features with eukaryotic tubulins. the heterodimer αβ-tubulin polymerizes into microtubules and two types of interactions are involved between the heterodimer subunits: the longitudinal interactions between heterodimers to form the protofilaments, and the lateral interactions between or parallel protofilaments to organize into the microtubule. the lateral interactions result from the contacts between α- and β-tubulin subunits of neighboring protofilaments and are mediated by central structural elements, such as the interaction of the m loop of one subunit with the h helix of the neighboring subunit. a newly discovered bacterial tubulin, btuba/b, also contains the h helix and m loop, however it is believed that the last one is a reminiscent structure from eukaryotic tubulin. interestingly, the h helix is highly conserved among ftszs while the m loop is absent. therefore, we believe that h helix of ftsz can participate in the lateral interactions in a similar manner to its related tubulins. the aim of this work was to investigate the role of the h helix in lateral interactions of ftsz from e. coli. we mutated the highly conserved glutamate and arginine residues to glutamine by site-directed mutagenesis and evaluated the effect of these mutations on the viability of the cells, the cell morphology and localization of ftsz, as well as the gtpase activity and the ability to polymerize in vitro, in the presence and in the absence of calcium and zipa.  design of ftsz and ftsz mutants we choose as candidates for mutation two conserved residues located in the lateral face of ftsz, specifically in the bend of h helix. this was based on the similarities in the crystal structures between ftsz and tubulin and the degree of conservation of amino acids among ftsz sequences. the three dimensional structure of an e. coli ftsz modela was compared with the structure of the αβ-tubulin heterodimer. the helix h of tubulin, located on the lateral face and known for the role in the lateral interactions, is also present in ftsz. the helix h of ftsz is located in the left lateral face. helix h is present in most of the ftsz sequences from gram positive and negative bacteria, mitochondria and chloroplasts. within the h helix, glutamate at position is 100% conserved and the positive charge of arginine at is highly conserved among ftsz sequences from different organisms. interestingly, the two residues are located in the bend of the helix h and are exposed to the surface of the left face. hence, we hypothesized that these residues, e and r participate in the interactions of ftsz to form filaments, tubules or sheets. to this end, the bend in h could be necessary to maintain both the topography of the monomer for the interactions and the gtpase activity in the polymer. in microtubules, the lateral interactions are principally mediated between the helix h from a protofilament and the m loop of the adjacent protofilament. the m loop is even present in bacterial tubulin a/b, though smaller than its counterpart tubulin, and is also believed to participate in the lateral interactions to form a bacterial microtubules composed by five protofilaments. however, ftsz lacks the m loop hence, the lateral interactions are expected to be weaker between ftsz protofilaments, hence an effect on longitudinal interactions should affect both the length and the gtpase activity of the polymers. viability of e. coli vip cells in the presence of ftsz and ftsz to evaluate the functionality of the ftsz mutants in vivo, the e83q, and r85q proteins were expressed independently and the cell viability was determined in a strain containing ftsz thermonull mutant, vip2/plar the parental strain, vip2/plar has been successfully employed to demonstrate that ftsz is essential for cell division and survival in e. coli. for the purpose of our study we modified the parental strain to create a de lysogen of vip that contains a copy of t rna polymerase gene under the control of placuv so the expression of a target protein is inducible by iptg addition. thus, if vip2/plar is transformed with a second plasmid that contains the ftsz mutants, the transformed cells will contain a ftsz mutant allele from pmfv-derived plasmid under the control of a t promoter, a wild-type allele encoded on plasmid plar and a genomic null allele. in summary, at the permissive temperature, vip2/plar9/pmfv- will express both ftsz alleles, whereas at the restrictive temperature plar cannot replicate, so the ftsz expression originates from the pmfv-derivatives. the production of ftsz ceases completely after 2– h of shifting the temperature, once plar is segregated by lack of replication. in this regard, this system is different from those utilizing a ftsz mutant nonfunctional at 42°c, because in this case after shifting to the non-permissive temperature ftsz will be nonfunctional almost instantly. to simplify the nomenclature we will refer henceforth to the strain vip containing plar as vip vip5/pmfv-wt cell grown at 42°c and at 30°c show similar cfu/ml in the absence of iptg, indicating that the basal non-induced expression level from pmfv-wt was enough to complement the lack of ftsz from plar at 42°c. hence, in order to avoid an excessive expression of ftsz, iptg was not added in all our experiments. as expected, the survival of vip at 42°c decreases about four orders of magnitude. the small survival fraction observed in vip is explained as due to the reversion frequency of the chromosomal mutation. figure  shows that the viability of cells expressing ecftsz mutants e83q and r85q at the restrictive temperature diminished about and orders of magnitude, respectively, compared to the non-restrictive temperature. these values indicate that the cells could not support normal cell division but at the same time the ecftsz mutants did not completely lose functionality in vivo. hence, the survival assay was useful to characterize the functionality of ecftsz mutants in vivo and the quantification of the viability of the cells expressing ecftsz mutants was used as an indication of the efficiency in the cell division. consequently, the efficiency of the different forms of ecftsz in terms of cell viability can be interpreted as: wild type > e83q > r85q. cell morphology and in situ localization of ftsz mutants using immunofluorescence microscopy the ftsz ring is formed in the middle of the cell and is attached to the cytoplasmic membrane through zipa. in order to evaluate the effect of these mutations on cell morphology and ftsz localization, vip cells in exponential phase of growth carrying plasmids with ftsz, ftsz, and ftsz mutants were incubated in the absence of iptg at 30°c and at 42°c. figure  shows that, in the negative control at 42°c the cell division is blocked, and ftsz localization is dispersed along the very long filamentous cells, while at 30°c the cells are normal. cells carrying the plasmid ftsz at 42°c reverts the cell filamentation, and at 30°c as expected the morphology and ftsz localization is normal. the co-expression at 30°c of ftsz from plar with the mutants ftsz and ftsz also display a normal phenotype. however, the expression of the ftsz mutants at 42°c ) have an effect on cell division, producing filamentous cells. interestingly, ftsz still localizes as discrete bands along the filamentous cells, probably at the potential cell division sites. in contrast, the expression of ftsz produced shorter filamentous cells with a disperse localization of the protein along the filaments, and no clear bands were observed. the same experiments were performed with iptg mm and similar results were obtained. in order to keep the cells in exponential phase of growth for a longer time, the morphology experiments were repeated starting the culture for the temperature shift at an od of. after h of incubation no difference in the cell morphology of the mutants respect to the experiments presented in figure  was observed. localization of the ftsz mutants in the single cell population that appear at early stationary phase shows that ftsz is located with greater intensity at the middle of the cell but presents some degree of dispersion, while ftsz is rather distributed in a gradient shape from the mid-cell toward the cell poles. the zipa distribution was similar to that observed for ftsz mutants, which suggests that the mutations in ftsz do not affect its interaction with zipa. binding experiments between ftsz and zipa inserted into proteoliposomes confirmed this hypothesis. in summary, the residues glutamate and arginine located in bend of helix h on the left face of ftsz plays a role in the formation of the z-ring and consequently zipa presents an altered localization in cells expressing the ftsz mutants. structural characterization of r85q and e83q in order to evaluate the impact of the point mutations e83q and r85q on the protein structure, we performed circular dichroism on purified ftsz wt, r85q and e83q. all three purified proteins showed the expected molecular mass and had a very similar secondary structure content; therefore, presumably the mutations did not cause significant changes in the tertiary structure of ftsz. gtpase activity of e83q and r85q in order to understand the possible effects of the ftsz mutations on the formation of the z ring, we purified the mutant proteins and measured their abilities to hydrolyze gtp. the wild-type ftsz and e83q exhibited a constant initial velocity of gtp hydrolysis for at least minutes at a protein concentration of μm. by contrast, r85q showed a lag time of minutes and a reduced gtpase activity. we next compared the gtp hydrolysis efficiency by calculating the apparent catalytic constants. wild-type ftsz, e83q and r85q hydrolyzed gtp with apparent catalytic constants of ± min- ± min- and ± min- respectively. r85q was further characterized by measuring the dependence of gtp hydrolysis on protein concentration. the progress curves show a sigmoidal behaviour, i.e., the lag time was dependent on the protein concentration. this lag time should disappear at the extrapolated concentration of μm. the similarity of the progress curves at and μm of protein is probably due to gtp becoming limiting. these results suggest that the r85q mutation does not directly alter the rate of gtp hydrolysis, but rather affects ftsz polymerization, which is known to induce gtpase activity. therefore, the effect is reversed when the concentration of r85q is increased to stimulate polymerization. the gtpase activity of ftsz wt and e83q is not affected by zipa, which is in agreement with previous study. interestingly, zipa increased around twofold the gtpase activity of r85q, suggesting that zipa stimulates the longitudinal interactions of r85q protofilaments during polymerization, similar to the effect of maps on microtubules. polymerization of ftsz mutants with and without zipa in vitro, ftsz polymerizes into protofilaments in the presence of gtp, and these protofilaments associate to form bundles in the presence of zipa. single protofilaments are monomers that interact longitudinally whereas double protofilaments consist of two parallel protofilaments interacting laterally. antiparallel lateral interactions of these double filaments induce the formation of sheets or bundles. in order to evaluate the effect of the mutations on the polymerization capability, we followed the polymerization of ftsz mutants by 90° light scattering at nm and the polymers morphology by electron microscopy. the polymerization reaction was started with the addition of the protein, and the reaction was followed for one hour for wild type and e83q, while r85q was followed for almost three hours due to the long lag period observed for the gtpase activity. the mutant r85q apparently did not form long filaments at the protein concentration used for ftsz wild type and e83q, producing a small increment in turbidity that was stimulated around two-fold by zipa additional file 1: figure s3a), whereas ftsz wild type and e83q showed a fast polymerization kinetics that could not be measured. e83q formed polymers that remained in solution around three times longer than those formed by ftsz wild type, with a rate of depolymerization times slower. this increase in the time in which the filaments remain polymerized can be explained by the small apparent catalytic constant for the gtpase activity. the time traces of light scattering indicate that the polymerization and the subsequent depolymerization of ftsz are altered by the mutations on the arginine and glutamate additional file 1: figure s shows that calcium increased the ligth scattering, and for ftsz wild type and e83q this was due to the formation of bundles, as shown in additional file 1: figure s on the other hand, r85q presented a smaller increase in light scattering and no formation of bundles was observed. after a stable base line of the buffer containing 1mm gtp, with or without zipa, ftsz was added to initiate the polymerization reaction. the change in light scattering at nm was recorded during the polymerization reaction and the difference between the base line and the maximum value reached after ftsz addition is shown in the bar graph. electron microscopy of negatively stained polymers of wild type ftsz, e83q and r85q polymerized as in. the samples for ftsz wt, e83q and r85q were taken at min, min and 2h min, respectively. electron micrographs were taken at a magnification of for wild type ftsz and e83q, and of for r85q. scale bar, nm. histogram of ftsz polymers width distribution shows that the mutations e83q and r85q decrease the number of protofilaments per polymer. at least polymers were measured for each case using imagej. figure  5a shows the maximum light scattering value reached by ftsz in mm gtp in the presence and absence of zipa. the mutants presented a decrease in the polymerization levels of ~20% and ~75% for e83q and r85q, respectively. in the presence of zipa, the maximum light scattering value of ftsz wild type increased slightly,, while for both mutants the increment was significantly larger: 90% and 150% for e83q and r85q, respectively. in the absence of zipa, ftsz wild type polymerized into long and mostly straight filaments of to nm width suggesting that the main population of these filaments consists of assembled protofilaments of nm width for each protofilament. e83q polymerized into curved filaments with a width of to nm. the greatest effect was caused by the mutation r85q, in which the polymers were mainly short and straight, and occasionally, spirals probably composed of single or double protofilaments were found on the grids. zipa induced bundling of wt and e83q, which is in agreement with previous studies; however, we did not observe a clear bundling effect in the r85q mutant, even given the increase in light scattering. the light scattering contribution of zipa was subtracted, therefore we can rule out the possibility that the increase in the light scattering of r85q is due to zipa. further characterization of r85q will be done in a future study. all the results together indicate that arginine and glutamate participate in the longitudinal and in the lateral interactions, shown by their reduced gtpase activity and the effect on polymerization, respectively. interestingly, the addition of a stabilizer of the lateral interactions, such as zipa to r85q, did not induce bundles but stimulated the gtpase activity, indicating that protofilaments are induced. on the other hand, in the case of e83q, even though the protofilament lateral interactions were diminished and zipa induced bundles that were thinner than those with ftsz wild type, the gtpase activity was not greatly compromised. therefore, we believe that the ability of ftsz to form bundles, presumably through lateral interactions, is important to form the z-ring in the cell, and consequently for cell division, a conclusion consistent with our immunofluorescence experiments and cell viability results. discussion the aim of this work was to characterize the role of two conserved amino acid residues, e and r located in the bend of helix h at the lateral face of ftsz. our results show that these amino acids are relevant for cell division, ftsz polymerization, gtpase activity and stability of the polymers. the properties of these ftsz mutants were evaluated through their kinetics and critical concentrations for polymerization and gtpase activities in the absence and presence of zipa and calcium. the ftsz mutant proteins had the same secondary structure as the wild type form indicating an intact global fold. interestingly, although the mutations are located far from the gtpase catalytic site both mutant proteins hydrolyzed gtp with less efficiency than wild type in the following order: wt > e83q > r85q. compared to the wild type ftsz, filaments with mutant proteins were thinner with altered curvatures, indicating that the mutations affect lateral interactions between ftsz protofilaments. zipa, which in wild type ftsz is known to induce bundling without affecting gtpase activity, induced thinner bundles in e83q, while in the case of r85q was unable to cause bundling, suggesting that both glutamate and arginine are important for lateral interactions between protofilaments. r85q filaments were short and abnormally bent, indicating that the mutation also affects the geometry of the longitudinal interactions. the formation of short filaments is consistent with the ability of r85q to hydrolyze gtp, which requires longitudinal interaction between two ftsz molecules. also the neighbor mutation d86k, which induces twined filaments, shows a reduction in gtpase activity further suggesting a role of this region in longitudinal interactions. the long lag period of the progress curve of r85q gtpase activity was suppressed by increasing the protein concentration, confirming that this activity was directly related to polymerization. this result could be explained by a reduction of the nucleation free energy over the free energy for the elongation process. the bend in h is likely caused by the negatively-charged amino acids ded located just before the bend which destabilize the helix by negative charge repulsion. at the other end of the h bend, r is followed by d86; therefore, the positive charge of r could destabilize the positive dipole of the helix in this region. thus, altering the charges in this region could induce a conformational change, explaining the effects of the r85q, e83q and d86k mutations on the functionality of ftsz. based on our results we hypothesize that, directly or via a subtle conformational change, the charges of amino acids located in the bend of the h helix are involved in longitudinal and lateral interactions. we infer that the negative charge of glutamate weakens these interactions while the positive charge of arginine stabilizes them, probably regulating the dynamics of the z-ring. thus, cells carrying ftsz or ftsz were affected in cell division and showed reduced survival and cell filamentation in agreement with previously described lateral-interaction-ftsz mutants that could not complement the temperature sensitive ftsz. we have quantified survival rates at the restrictive temperature as a means to evaluate the effect of ftsz mutations on cell division, similar to studies on the effects of proteins expression from unstable plasmids. the e83q and r85q mutations diminished the apparent catalytic constant of gtpase activity by 60% and 95%, respectively. presumably, the positive charge of r is involved in longitudinal and lateral interactions between protofilaments explaining the inhibitory effect of the r85q mutation on gtpase activity and polymerization when compared to the e83q or d86k mutants. both, longitudinal and lateral interactions are important in models for the conversion of gtp hydrolysis into mechanical energy during z-ring constriction. the observed contraction of tubular liposomes in the presence of gtp and a chimeric, membrane-attached ftsz indicates that polymerization of ftsz is sufficient to generate a mechanical force to bend the membrane. our results indicate that h bend mutations affecting longitudinal and lateral interactions in vitro reduce cell viability in vivo while they cause cell filamentation. cell filamentation and the immunofluorescence analysis together with the polymerization results support a model of a z-ring composed of several polymers that are recruited to the septation site by lateral interactions, as has been found in vivo. athe mutations are positioned on the surface and their location on the structure are shown in figure  left: the left lateral surface; right: the right lateral surface. aa, nº = amino acid number. bthe % viability was determined from data of figure  n = no growth of ftsz at the restrictive temperature. cgtpase activity was measured with μm of protein in all cases. db, bundles; s, short filaments; nd, not determined. the shape of the polymers was determined by electron microscopy.  this study describes the role of the positively charged arginine and the negatively charged glutamate in longitudinal and lateral interactions to form ftsz protofilaments and two-dimensional polymers of protofilaments, respectively. we also show that gtpase activity could be fine-tuned by the net charge of the h helix bend. finally, our results suggest that the conserved amino acids e83q and r85q located in the bend of h are involved in the formation of a functional z-ring. 
in vitro, both, r85q and e83q reduced the rate of ftsz polymerization and gtpase activity. the expression of the mutant proteins diminished cell viability as follows: wild type > e83q > r85q. in this study, we demonstrate that the highly conserved negative charge of glutamate and the positive charge of arginine located in the helix h bend of ftsz are required for in vitro ftsz lateral and longitudinal interactions, respectively and for in vivo cell division. in vitro, ftsz polymerizes and induces gtpase activity through longitudinal interactions to form the protofilaments, whilst lateral interactions result within formation of bundles. the interactions that participate in the protofilaments are similar to its eukaryotic homologue tubulin and are well characterized; however, lateral interactions between the inter protofilaments are less defined. ftsz forms double protofilaments in vitro, though the key elements on the interface of the inter-protofilaments remain unclear as well as the structures involved in the lateral interactions in vivo and in vitro. in the presence of zipa, r85q gtpase activity increased two fold, but no bundles were formed suggesting that lateral interactions were affected. we found that glutamate and arginine located in the bend of helix h at the lateral face are required for the protofilament lateral interaction and also affects the inter-protofilament lateral interactions that ultimately play a role in the functional localization of the ftsz ring at the cell division site. r85q protein polymerized into shorter filaments compared to wt and e83q, with a gtpase lag period that was inversely proportional to the protein concentration.
 ftsz is an essential cell division protein, which localizes at the middle of the bacterial cell to mediate cytokinesis. in vitro, ftsz polymerizes and induces gtpase activity through longitudinal interactions to form the protofilaments, whilst lateral interactions result within formation of bundles. the interactions that participate in the protofilaments are similar to its eukaryotic homologue tubulin and are well characterized; however, lateral interactions between the inter protofilaments are less defined. ftsz forms double protofilaments in vitro, though the key elements on the interface of the inter-protofilaments remain unclear as well as the structures involved in the lateral interactions in vivo and in vitro. in this study, we demonstrate that the highly conserved negative charge of glutamate and the positive charge of arginine located in the helix h bend of ftsz are required for in vitro ftsz lateral and longitudinal interactions, respectively and for in vivo cell division.  the effect of mutation on the widely conserved glutamate- and arginine- residues located in the helix h was evaluated by in vitro and in situ experiments. the morphology of the cells expressing escherichia coli ftsz mutant at 42°c formed filamented cells while those expressing ftsz formed shorter filamented cells. in situ immunofluorescence experiments showed that the ftsz mutant formed rings within the filamented cells whereas those formed by the ftsz mutant were less defined. the expression of the mutant proteins diminished cell viability as follows: wild type > e83q > r85q. in vitro, both, r85q and e83q reduced the rate of ftsz polymerization and gtpase activity. r85q protein polymerized into shorter filaments compared to wt and e83q, with a gtpase lag period that was inversely proportional to the protein concentration. in the presence of zipa, r85q gtpase activity increased two fold, but no bundles were formed suggesting that lateral interactions were affected.  we found that glutamate and arginine located in the bend of helix h at the lateral face are required for the protofilament lateral interaction and also affects the inter-protofilament lateral interactions that ultimately play a role in the functional localization of the ftsz ring at the cell division site. bacterial divisionftsz polymerizationlongitudinal/lateral interactionszipa 