BACKGROUND
chlamydia pneumoniae is a gram negative, obligate intracellular pathogen that has been associated with community-acquired pneumonia  <cit> , atherosclerosis  <cit> , arthritis  <cit> , and alzheimer's disease  <cit> .
c. pneumoniae is characterized by a unique, biphasic life cycle beginning with an infectious, metabolically attenuated elementary body .
chlamydial invasion is initiated by attachment of the eb to the host eukaryotic cell membrane and recruitment of actin to the site of attachment.
this remodeling of the actin cytoskeleton is thought to be mediated by the type iii secretion  effector protein, the translocated actin recruitment protein , which facilitates chlamydial entry into the host cell  <cit> .
bacterial uptake involves modulation of the host mek-erk pathway and pi 3-kinase, possibly through the action of t3s effectors  <cit> .
once internalized, the remainder of the chlamydial life cycle takes place within a parasitophorous membrane-bound vesicle known as an inclusion, where ebs differentiate into the non-infectious, metabolically active form, termed a reticulate body .
within the inclusion, rbs acquire amino acids, nucleotides, lipids and cholesterol from the host cell, events possibly orchestrated via t3s across the inclusion membrane, while at the same time inhibiting apoptosis to ensure survival  <cit> .
golgi fragmentation appears to be a crucial step in intercepting host pathways to obtain these nutrients and compounds, as well as in the maturation of the chlamydiae sps.
within the inclusion  <cit> .
the rb interacts with the inclusion membrane until such time as inclusion membrane rb docking sites are no longer available and an unknown signal triggers detachment of the rb from the inclusion membrane followed by asynchronous differentiation into ebs  <cit> .
the newly formed ebs then exit the cell by either cellular lysis or a packaged release mechanism termed extrusion  <cit> .
c. pneumoniae encodes a full set of t3s genes scattered throughout the genome in several fragmented operons.
the t3s injectisome has a high amount of paralogy to the flagellar secretion system in structure and in function.
in the t3ss, cdsn is the atpase that aids in shuttling effectors through the needle, and is paralogous to flii  <cit> .
cdsl is orthologous to yscl and paralogous to flih.
in yersinia, yscl is the atpase tethering protein and functions to down-regulate enzymatic activity of yscn  <cit> .
copn, orthologous to yopn, is believed to function as a regulator of the system which plugs the injectisome pore prior to activation of t3s and is a known effector protein  <cit> .
cdsu, orthologous to yscu, plays an important role is substrate specificity and substrate switching from structural components to effector proteins upon host cell contact  <cit> .
recently, several reports have emerged characterizing protein interactions within the c. pneumoniae t3ss, describing novel protein complexes that form at the inner membrane.
johnson et al have shown that cdsd, a unique protein orthologous to yscd that contains two fork-head associated domains, interacts with the predicted c. pneumoniae atpase tethering protein, cdsl, and cdsq, a cytosolic component of the inner membrane that presumably forms the bulk of the t3s c-ring  <cit> .
stone et al extended these findings to show that cdsn, the atpase, is also involved in this complex as well as interacting with the proposed plug protein, copn  <cit> .
flagellar motility is an ancient, conserved mechanism that may have evolved from the same ancestor as t3s  <cit> .
this motility facilitates bacterial migration towards less hostile environments.
in non-motile bacteria, however, the presence of flagella would be evolutionarily redundant and energetically expensive, unless the proteins played a role in another mechanism involving bacterial replication or survival.
although c. pneumoniae is thought to be a non-motile bacteria, it has been shown to contain at least three orthologs of flagellar genes, namely flha, flif, and flii  <cit> .
microarray and proteomic experiments have suggested that these genes are expressed at mid-cycle  <cit> .
the proteins encoded by these genes are paralogs of the t3s proteins cdsv, cdsj and cdsn, respectively.
in motile bacteria, flha orthologs are integral membrane proteins required for flagellin export and swarming differentiation which interact with soluble components of the flagellar system  <cit> .
flif orthologs are integral membrane components that form the membrane and supramembrane  ring  <cit> .
flif forms a base for the other membrane components to form a molecular pore, through which components of the flagella that exist outside the cell membrane are exported.
the flagellar atpase, flii orthologs, provide energy for construction of the flagellum by aiding in export of flagellar proteins outside the bacterial cell where the proteins form molecular complexes  <cit> .
the presence of flii, flha and flif in c. pneumoniae is not sufficient to form a fully functional flagellar apparatus but they could potentially form a rudimentary base for a flagellum structure  <cit> .
in chlamydiae, the identity of other proteins  that play important roles in the flagellar apparatus is currently pending, but it is possible that the flagellar apparatus, if it exists, is a hybrid structure of c. pneumoniae t3s and flagellar proteins.
another possibility is that flagellar proteins are involved in t3s, aiding in secretion of effector proteins or structural components.
in pseudomonas, there is evidence to support that flagellar assembly actually antagonizes the t3ss, suggesting a negative cross-regulation of the two systems  <cit> .
no interaction between chlamydial t3s and flagellar components, however, has been reported to our knowledge.
the protein interactions that occur within the bacterial flagellar system have been characterized previously  <cit> .
genetic evidence, followed by direct biochemical assays, suggests an interaction of flha and flif  <cit> .
the c-terminal end of flha, which is predicted to be cytoplasmic, is known to interact with the soluble components of the flagellar system such as flii, flih and flij  <cit> .
flih acts as a negative regulator of the flagellar atpase, flii, and binds flii as a homodimer, forming a trimeric  <dig> complex  <cit> .
flij, a second soluble component which interacts with flha, acts as a general chaperone for the flagellar system to prevent premature aggregation of export substrates in the cytoplasm, and also interacts with the flih/flii complex  <cit> .
this complex of flii/flih/flij is believed to be crucial for selection of export substrates and construction of the flagellar apparatus, although the proton motive force could play a role in the actual secretion of flagellar proteins  <cit> .
in c. pneumoniae, flih and flij have not been annotated in the genome.
flii, the putative c. pneumoniae flagellar atpase ortholog, has significant amino acid similarity with both cdsn, the c. pneumoniae t3s atpase, and flii, the salmonella flagellar atpase, suggesting that it possesses enzymatic activity.
here we report an initial characterization of flii, the flagellar atpase, and show that it hydrolyzes atp at a rate similar to that of its t3s atpase paralog cdsn as well as orthologs in other bacteria  <cit> .
we have also characterized the protein-interactions occurring between flii, flif and flha, demonstrating a direct interaction of flii and flha, and flha and flif.
as well as interactions between the flagellar proteins, we have also characterized four novel interactions between the flagellar and t3s components.
the role of these interactions in the chlamydial replication cycle is discussed.
RESULTS
sequence analysis of flii, flha and flif
flii  is  <dig> amino acids in length with a predicted molecular mass of  <dig>  kda and a pi of  <dig> .
flii has significant sequence similarity with the β subunit of the f0f1-atpase and both its type iii secretion paralog, cdsn, and other flagellar atpases, although the sequence similarity is most striking in the atpase domain  <cit> .
flii has a non-conserved n-terminal region  which may be important for mediating protein-protein interactions, a catalytic domain , containing a conserved p loop, walker a and b domains, and a c-terminal non-conserved domain  of unknown function.
flii has a  <dig> percent sequence similarity to cdsn, the c. pneumoniae t3s atpase, and  <dig> percent similarity with flii from salmonella.
the active domain of flii has the most similarity to its paralogs and orthologs, while the n- and c-terminal regions have the lowest amount of similarity .
flha  is  <dig> amino acids in length with a predicted molecular mass of  <dig>  kda and a pi of  <dig> .
the flha paralog in c. pneumoniae is the t3s protein cdsv.
flha has seven predicted transmembrane regions in the n-terminal half of the protein , while the c-terminal half of the protein is predicted to be cytoplasmic .
flha from c. pneumoniae has  <dig> percent sequence orthology with flha from salmonella.
flif  is  <dig> amino acids in length with a predicted molecular mass of  <dig>  kda and a pi of  <dig> .
the flif paralog in c. pneumoniae is the t3s protein cdsj.
flif has two predicted tm regions, one located near the n-terminus and one located near the c-terminus.
flif from c. pneumoniae is only  <dig> percent similar to flif from salmonella.
expression and atpase activity of flii
flii has significant similarity with many characterized atpases, and this led us to explore the atpase activity of this protein.
purified gst tagged flii was tested for its ability to hydrolyze atp using the malachite green binding assay.
figure 2a shows that gst-flii was essentially free of contaminating proteins by sds-page and anti-gst western blot  or coomassie blue stain .
gst-flii hydrolyzed atp in a dose- and time-dependant manner at a rate of  <dig>  ± .
<dig> μmol min- <dig> mg- <dig> .
this level of activity is comparable to other flagellar atpases as well as the t3s paralog, cdsn  <cit> .
atpase activity of gst-flii peaked at 37°c, and at a ph of  <dig>  .
another gst-tagged protein, gst-copn, purified in the same manner as gst-flii had negligible atpase activity .
flha interacts with flif
flha is known to interact with the ms ring protein, flif, in other flagellar systems  <cit> .
we explored the interactions of these two proteins in c. pneumoniae.
two fragments of flif were cloned and expressed as his-tagged proteins.
his-flif1- <dig> lacked the distal c-terminal  <dig> amino acids while his-flif35- <dig> lacked the n-terminal  <dig> amino acids.
each fragment contained only one of the two predicted tm regions.
flif1- <dig> migrated with an apparent molecular weight of  <dig> kda, while his-flif35- <dig> migrated at  <dig> kda.
flha was cloned and expressed as a soluble fragment with either a gst or his tag.
flha308- <dig> encoded the c-terminal half of the protein, lacking the stretch of seven tm domains.
expression and detection of his-flha308- <dig> used as the bait protein in gst pull-down assays migrated at the expected molecular weight of  <dig> kda.
we used the bacterial-2-hybrid assay to test for interactions between flif and flha.
full length flha interacted significantly with full length flif, with a β-galactosidase activity of  <dig>  ±  <dig>  units of activity, as compared with a negative control value of  <dig>  ±  <dig>  units of activity .
we next used gst pull-downs to confirm the interactions found by the bacterial-2-hybrid system and to determine the exact regions of the proteins mediating these interactions .
all protein complexes were washed with either low or high salt buffers containing  <dig> % triton x- <dig> to dissociate spurious protein-protein interactions.
gst-flha308- <dig> co-purified with his-flif35- <dig> but not his-flif1- <dig>  suggesting that the c-terminus of flif  is required for interactions with the cytoplasmic portion of flha.
* flii, flif, flha, cdsl, copn, cpn <dig> and cpn <dig> were cloned into both the pt <dig> and pt <dig> vectors.
the bacterial-2-hybrid was performed in triplicate as described in the materials and methods section.
empty pt <dig> and pt <dig> vectors were used as a negative control while pt18-pknd + pt25-cdsd-fha- <dig> was used as a positive control.
the cut off for a positive interaction , is the mean of the negative control values  plus two standard deviations obtained from  <dig> assays.
flii interacts with flha
in orthologous systems, it has been shown that flha interacts with several soluble
components of the flagellar machinery, including the atpase, flii  <cit> .
therefore, we investigated the possibility of whether flha interacts with flii in c. pneumoniae.
the bacterial-2-hybrid system was initially used to screen for potential protein interactions.
flha interacted with flii, with β-galactosidase activity of  <dig>  ±  <dig>  units of activity as compared to the negative control with a value of  <dig>  ±  <dig>  units of activity .
to confirm these protein-protein interactions we used gst pull-down assays .
initially flii was cloned as three constructs, full length flii, a c-terminal truncation of flii  and a n-terminal truncation of flii .
these three constructs were tested for interaction with the his-flha308- <dig> construct.
full length gst-flii co-purified with his-flha308- <dig>  suggesting that the cytoplasmic fragment of flha contains the interactive domain.
to determine the region of flii that interacts with flha, we reacted the two truncation constructs of flii with the cytoplasmic domain of flha.
only flii1- <dig> was able to co-purify with flha, and not flii150- <dig>  suggesting that the flha binding domain resides in the n-terminal  <dig> amino acids of flii .
we next wanted to know if flii interacts with flif.
we therefore reacted gst-flii against the two flif constructs and found that there was no co-purification, suggesting that any interaction between flii and flif, if there is an association, would seem to be indirect and mediated through the action of flha or other intermediate proteins .
cpn <dig> interacts with flii and flha
cpn <dig> is a predicted  <dig> amino acid protein with a pi of  <dig>  and a molecular mass of  <dig>  kda.
the cpn <dig> orf is encoded directly upstream of flif and downstream of flii, the flagellar atpase.
based on its location relative to flii and flif, we hypothesized that it may interact with other flagellar components.
we used gst pull-down assays to explore this possibility.
initial gst pull-downs indicated that full length his-cpn <dig> interacts with gst-cpn <dig>  suggesting the presence of a dimerization domain .
to explore this observation we treated cpn <dig> with formaldehyde prior to page and observed the presence of a monomer and a dimer, migrating with apparent molecular weights of  <dig> kda and  <dig> kda .
we next explored the possible interaction of cpn <dig> with other flagellar proteins in c. pneumoniae.
using gst pull-downs, his-cpn <dig> co-purified with the full length gst-flii protein as well as the gst-flii1- <dig> protein, but not gst-flii150- <dig> .
this suggests that cpn <dig> binds to the n-terminus of flii.
gst pull-down assays showed an interaction between cpn <dig> and the flha308- <dig> protein, the cytoplasmic domain of flha .
cpn <dig> did not co-purify with either flif35- <dig> or flif1- <dig> , suggesting that cpn <dig> does not interact with flif.
flii and flha interact with t3s components
since chlamydia have no apparent flagella, we investigated whether the flagellar proteins flii, flha and flif interact with t3s components.
using bacterial-2-hybrid screening we found that flii and flha interacted with cdsl, the putative t3s atpase regulator and tethering protein, with a β-galactosidase activity of  <dig>  ±  <dig>  and  <dig> ±  <dig>  units of activity, respectively.
flii also interacted with copn, the putative t3s plug protein, with a β-galactosidase activity of  <dig>  ±  <dig>  units of activity.
we also found that flha interacted with the putative yscu ortholog, cdsu, with a β-galactosidase activity of  <dig>  ±  <dig>  units of activity, as well as cdsl, with a β-galactosidase activity of  <dig>  ±  <dig>  units of activity .
to corroborate these findings we utilized gst pull-down assays and showed that gst-flii interacted with cdsl and copn, but not cpn <dig> , and gst-flha co-purified with both cdsl and cdsu .
control gst coated beads did not co-purify with cdsl, copn or cdsu.
discussion
sequencing of several chlamydial genomes revealed a conserved set of flagellar orthologs, despite the fact that c. pneumoniae lack a flagellum and are considered non-motile bacteria  <cit> .
here we report an initial characterization of three annotated flagellar proteins of c. pneumoniae, flii, flha and flif, demonstrating atpase activity of flii and interactions between these flagellar orthologs.
we have demonstrated that flii hydrolyzes atp in a linear, time-and dose-dependant manner, with optimal activity at a ph of  <dig>  and a temperature of 37°c.
flii also interacts with the cytoplasmic domain of flha, while flha interacts with the c-terminal region of the flif protein.
no direct interaction of flii and flif was detected.
also, we have characterized an interaction of both flha and flii with cpn <dig>  a fourth unannotated protein.
we also show that flii interacts with cdsl and copn, two t3s components, while flha interacts with cdsl and a third t3s component, cdsu.
collectively, this data suggests that the flagellar proteins of c. pneumoniae interact in a specific way with t3s proteins and may play an important, as yet unidentified role in the chlamydial replication cycle.
flii hydrolyzes atp in a linear, time- and dose-dependant manner at a rate of  <dig>  ± .
<dig> μmol min- <dig> mg- <dig>  this rate is typical of other secretion atpases such as cdsn, escn, or flii from other bacterial species  <cit> .
the optimal ph for flii atpase activity is  <dig> , which is the same as that for other flagellar atpases  <cit> .
extreme low or high ph greatly reduced the activity, possibly due to protein denaturation.
also, the enzyme activity peaked at a temperature of 37°c and declined substantially beyond that.
although the formation of higher-order complexes was not explored here, other flagellar atpases are thought to form a hexameric complex  <cit> .
the presence of three flagellar genes in chlamydiae is intriguing since chlamydiae are thought to be non-motile and not to possess flagella.
flif, flha and flii alone do not contain all the necessary components for a functional flagella or secretion apparatus, however, a rudimentary basal body or pore complex could be formed by these three components.
it is known that the most rudimentary flagellar structure that can be assembled is the ms ring, which consists of only the flif protein  <cit> .
we have shown that these proteins interact with one another , most likely at the inner membrane of c. pneumoniae.
the interaction between flii and flha is mediated by the n-terminal  <dig> amino acids of flii and appears to be specific since it is not disrupted by high salt .
only the cytoplasmic domain of flha  was utilized in the gst pull-down, suggesting that any protein interactions that occur are within this region.
protein interaction studies with the full length flha protein are difficult due to the presence of seven transmembrane domains rendering full length flha insoluble and making this portion of the protein unable to bind to soluble flagellar components.
since flha is known to interact with soluble components of the flagellar apparatus in other bacteria, it is expected that the cytoplasmic domain mediates an interaction with flii  <cit> .
flif is known to form the ms ring in flagellated bacteria, and is one of the first components of the flagellar basal body to be incorporated into the membrane  <cit> .
we detected an interaction of the c-terminal  <dig> amino acids of flif with the cytoplasmic domain of flha.
these interactions were also stable in  <dig> mm nacl, suggesting that the interaction is specific.
we did not, however, detect any interaction between flii and flif, suggesting that any interaction between those two components may be mediated through the action of another protein, possibly flha
in c. pneumoniae, cpn <dig> is encoded directly downstream of the atpase, which led us to explore any interactions cpn <dig> may have with other flagellar proteins.
we initially found that cpn <dig> was able to interact with itself using a gst pull-down assay, suggesting that it could form dimers in solution.
to confirm this observation we utilized formaldehyde fixation followed by page analysis to visualize the formation of dimers.
using this method we saw that cpn <dig> migrated in two molecular forms, with sizes corresponding to both monomers and dimers.
we then explored possible interactions between cpn <dig> and the other flagellar proteins and detected interactions of cpn <dig> with both flii and flha, but not flif.
cpn <dig> bound to the n-terminal  <dig> amino acids of flii and the cytoplasmic region of flha.
the interaction of cpn <dig> with the cytoplasmic domain of flha was expected as flha is known to interact with soluble components of other flagellar systems  <cit> .
we considered the possibility that cpn <dig> may in fact be the flih ortholog in c. pneumoniae as cpn <dig> has minor sequence orthology to other flih proteins, but after further investigation we found that cpn <dig> did not appear to play a regulatory role with flii .
figure  <dig> summarizes the interactions between flii, flha and flif  and interactions between cpn <dig>  flha and flii .
bacterial type iii secretion  and flagellar secretion systems are structurally similar, and may have a common ancestry  <cit> .
although c. pneumoniae does not contain a full repertoire of flagellar genes, it does encode a complete t3s system which most likely consists of specific protein complexes located in the inner membrane  <cit> .
we have characterized an interaction of flii with cdsl, the t3s atpase tethering protein.
the c. pneumoniae flih ortholog has not yet been identified, and in the absence of flih, cdsl may play a regulatory role for both flii and cdsn.
flii also interacts with the copn, the t3s plug protein, suggesting that flii may be involved in either the secretion of effector proteins or regulation of the t3s system.
yscu orthologs have a flagellar paralog, flhb, and cpn <dig> is believed to be the c. pneumoniae yscu ortholog .
flhb is known to interact with flha, but in c. pneumoniae no flhb ortholog has been annotated.
we found that flha interacts with cdsu, suggesting integration of flha into the inner membrane, associating with t3s components.
CONCLUSIONS
based on all of our observations, it is possible that the flagellar proteins may interact with components of the t3s injectisome, forming a hybrid structure, thereby playing an ancillary or accessory role in secretion of type iii effectors across either the cytoplasmic or inclusion membrane.
another possibility is that there are other, as yet unannotated proteins that play a role in a putative flagellar system in c. pneumoniae.
for example, along with the flih/flii complex that is formed in other bacteria, another protein, flij, which is a general chaperone, is believed to be involved in this complex  <cit> .
flij has not been identified in c. pneumoniae.
in the absence of a genetic manipulation system for the chlamydiae, direct evidence for the role of these flagellar proteins remains elusive.
the fact that flii is enzymatically active and forms complexes in vitro with other flagellar proteins, all of which are present in all other chlamydiae sps.
studied to date, suggests that these proteins play an important role in chlamydial replication or survival.
further studies using heterologous systems and genetic complementation could help to decipher the exact role of these flagellar proteins in chlamydiae.

 chlamydia pneumoniae is a gram negative, obligate intracellular pathogen that has been associated with community-acquired pneumonia, atherosclerosis, arthritis, and alzheimer's disease. c. pneumoniae is characterized by a unique, biphasic life cycle beginning with an infectious, metabolically attenuated elementary body. chlamydial invasion is initiated by attachment of the eb to the host eukaryotic cell membrane and recruitment of actin to the site of attachment. this remodeling of the actin cytoskeleton is thought to be mediated by the type iii secretion effector protein, the translocated actin recruitment protein, which facilitates chlamydial entry into the host cell. bacterial uptake involves modulation of the host mek-erk pathway and pi 3-kinase, possibly through the action of t3s effectors. once internalized, the remainder of the chlamydial life cycle takes place within a parasitophorous membrane-bound vesicle known as an inclusion, where ebs differentiate into the non-infectious, metabolically active form, termed a reticulate body. within the inclusion, rbs acquire amino acids, nucleotides, lipids and cholesterol from the host cell, events possibly orchestrated via t3s across the inclusion membrane, while at the same time inhibiting apoptosis to ensure survival. golgi fragmentation appears to be a crucial step in intercepting host pathways to obtain these nutrients and compounds, as well as in the maturation of the chlamydiae sps. within the inclusion. the rb interacts with the inclusion membrane until such time as inclusion membrane rb docking sites are no longer available and an unknown signal triggers detachment of the rb from the inclusion membrane followed by asynchronous differentiation into ebs. the newly formed ebs then exit the cell by either cellular lysis or a packaged release mechanism termed extrusion. c. pneumoniae encodes a full set of t3s genes scattered throughout the genome in several fragmented operons. the t3s injectisome has a high amount of paralogy to the flagellar secretion system in structure and in function. in the t3ss, cdsn is the atpase that aids in shuttling effectors through the needle, and is paralogous to flii. cdsl is orthologous to yscl and paralogous to flih. in yersinia, yscl is the atpase tethering protein and functions to down-regulate enzymatic activity of yscn. copn, orthologous to yopn, is believed to function as a regulator of the system which plugs the injectisome pore prior to activation of t3s and is a known effector protein. cdsu, orthologous to yscu, plays an important role is substrate specificity and substrate switching from structural components to effector proteins upon host cell contact. recently, several reports have emerged characterizing protein interactions within the c. pneumoniae t3ss, describing novel protein complexes that form at the inner membrane. johnson et al have shown that cdsd, a unique protein orthologous to yscd that contains two fork-head associated domains, interacts with the predicted c. pneumoniae atpase tethering protein, cdsl, and cdsq, a cytosolic component of the inner membrane that presumably forms the bulk of the t3s c-ring. stone et al extended these findings to show that cdsn, the atpase, is also involved in this complex as well as interacting with the proposed plug protein, copn. flagellar motility is an ancient, conserved mechanism that may have evolved from the same ancestor as t3s. this motility facilitates bacterial migration towards less hostile environments. in non-motile bacteria, however, the presence of flagella would be evolutionarily redundant and energetically expensive, unless the proteins played a role in another mechanism involving bacterial replication or survival. although c. pneumoniae is thought to be a non-motile bacteria, it has been shown to contain at least three orthologs of flagellar genes, namely flha, flif, and flii. microarray and proteomic experiments have suggested that these genes are expressed at mid-cycle. the proteins encoded by these genes are paralogs of the t3s proteins cdsv, cdsj and cdsn, respectively. in motile bacteria, flha orthologs are integral membrane proteins required for flagellin export and swarming differentiation which interact with soluble components of the flagellar system. flif orthologs are integral membrane components that form the membrane and supramembrane ring. flif forms a base for the other membrane components to form a molecular pore, through which components of the flagella that exist outside the cell membrane are exported. the flagellar atpase, flii orthologs, provide energy for construction of the flagellum by aiding in export of flagellar proteins outside the bacterial cell where the proteins form molecular complexes. the presence of flii, flha and flif in c. pneumoniae is not sufficient to form a fully functional flagellar apparatus but they could potentially form a rudimentary base for a flagellum structure. in chlamydiae, the identity of other proteins that play important roles in the flagellar apparatus is currently pending, but it is possible that the flagellar apparatus, if it exists, is a hybrid structure of c. pneumoniae t3s and flagellar proteins. another possibility is that flagellar proteins are involved in t3s, aiding in secretion of effector proteins or structural components. in pseudomonas, there is evidence to support that flagellar assembly actually antagonizes the t3ss, suggesting a negative cross-regulation of the two systems. no interaction between chlamydial t3s and flagellar components, however, has been reported to our knowledge. the protein interactions that occur within the bacterial flagellar system have been characterized previously. genetic evidence, followed by direct biochemical assays, suggests an interaction of flha and flif. the c-terminal end of flha, which is predicted to be cytoplasmic, is known to interact with the soluble components of the flagellar system such as flii, flih and flij. flih acts as a negative regulator of the flagellar atpase, flii, and binds flii as a homodimer, forming a trimeric complex. flij, a second soluble component which interacts with flha, acts as a general chaperone for the flagellar system to prevent premature aggregation of export substrates in the cytoplasm, and also interacts with the flih/flii complex. this complex of flii/flih/flij is believed to be crucial for selection of export substrates and construction of the flagellar apparatus, although the proton motive force could play a role in the actual secretion of flagellar proteins. in c. pneumoniae, flih and flij have not been annotated in the genome. flii, the putative c. pneumoniae flagellar atpase ortholog, has significant amino acid similarity with both cdsn, the c. pneumoniae t3s atpase, and flii, the salmonella flagellar atpase, suggesting that it possesses enzymatic activity. here we report an initial characterization of flii, the flagellar atpase, and show that it hydrolyzes atp at a rate similar to that of its t3s atpase paralog cdsn as well as orthologs in other bacteria. we have also characterized the protein-interactions occurring between flii, flif and flha, demonstrating a direct interaction of flii and flha, and flha and flif. as well as interactions between the flagellar proteins, we have also characterized four novel interactions between the flagellar and t3s components. the role of these interactions in the chlamydial replication cycle is discussed.  sequence analysis of flii, flha and flif flii is amino acids in length with a predicted molecular mass of kda and a pi of. flii has significant sequence similarity with the β subunit of the f0f1-atpase and both its type iii secretion paralog, cdsn, and other flagellar atpases, although the sequence similarity is most striking in the atpase domain. flii has a non-conserved n-terminal region which may be important for mediating protein-protein interactions, a catalytic domain, containing a conserved p loop, walker a and b domains, and a c-terminal non-conserved domain of unknown function. flii has a percent sequence similarity to cdsn, the c. pneumoniae t3s atpase, and percent similarity with flii from salmonella. the active domain of flii has the most similarity to its paralogs and orthologs, while the n- and c-terminal regions have the lowest amount of similarity. flha is amino acids in length with a predicted molecular mass of kda and a pi of. the flha paralog in c. pneumoniae is the t3s protein cdsv. flha has seven predicted transmembrane regions in the n-terminal half of the protein, while the c-terminal half of the protein is predicted to be cytoplasmic. flha from c. pneumoniae has percent sequence orthology with flha from salmonella. flif is amino acids in length with a predicted molecular mass of kda and a pi of. the flif paralog in c. pneumoniae is the t3s protein cdsj. flif has two predicted tm regions, one located near the n-terminus and one located near the c-terminus. flif from c. pneumoniae is only percent similar to flif from salmonella. expression and atpase activity of flii flii has significant similarity with many characterized atpases, and this led us to explore the atpase activity of this protein. purified gst tagged flii was tested for its ability to hydrolyze atp using the malachite green binding assay. figure 2a shows that gst-flii was essentially free of contaminating proteins by sds-page and anti-gst western blot or coomassie blue stain. gst-flii hydrolyzed atp in a dose- and time-dependant manner at a rate of ±. μmol min- mg-. this level of activity is comparable to other flagellar atpases as well as the t3s paralog, cdsn. atpase activity of gst-flii peaked at 37°c, and at a ph of. another gst-tagged protein, gst-copn, purified in the same manner as gst-flii had negligible atpase activity. flha interacts with flif flha is known to interact with the ms ring protein, flif, in other flagellar systems. we explored the interactions of these two proteins in c. pneumoniae. two fragments of flif were cloned and expressed as his-tagged proteins. his-flif1- lacked the distal c-terminal amino acids while his-flif35- lacked the n-terminal amino acids. each fragment contained only one of the two predicted tm regions. flif1- migrated with an apparent molecular weight of kda, while his-flif35- migrated at kda. flha was cloned and expressed as a soluble fragment with either a gst or his tag. flha308- encoded the c-terminal half of the protein, lacking the stretch of seven tm domains. expression and detection of his-flha308- used as the bait protein in gst pull-down assays migrated at the expected molecular weight of kda. we used the bacterial-2-hybrid assay to test for interactions between flif and flha. full length flha interacted significantly with full length flif, with a β-galactosidase activity of ± units of activity, as compared with a negative control value of ± units of activity. we next used gst pull-downs to confirm the interactions found by the bacterial-2-hybrid system and to determine the exact regions of the proteins mediating these interactions. all protein complexes were washed with either low or high salt buffers containing % triton x- to dissociate spurious protein-protein interactions. gst-flha308- co-purified with his-flif35- but not his-flif1- suggesting that the c-terminus of flif is required for interactions with the cytoplasmic portion of flha. * flii, flif, flha, cdsl, copn, cpn and cpn were cloned into both the pt and pt vectors. the bacterial-2-hybrid was performed in triplicate as described in the materials and methods section. empty pt and pt vectors were used as a negative control while pt18-pknd + pt25-cdsd-fha- was used as a positive control. the cut off for a positive interaction, is the mean of the negative control values plus two standard deviations obtained from assays. flii interacts with flha in orthologous systems, it has been shown that flha interacts with several soluble components of the flagellar machinery, including the atpase, flii. therefore, we investigated the possibility of whether flha interacts with flii in c. pneumoniae. the bacterial-2-hybrid system was initially used to screen for potential protein interactions. flha interacted with flii, with β-galactosidase activity of ± units of activity as compared to the negative control with a value of ± units of activity. to confirm these protein-protein interactions we used gst pull-down assays. initially flii was cloned as three constructs, full length flii, a c-terminal truncation of flii and a n-terminal truncation of flii. these three constructs were tested for interaction with the his-flha308- construct. full length gst-flii co-purified with his-flha308- suggesting that the cytoplasmic fragment of flha contains the interactive domain. to determine the region of flii that interacts with flha, we reacted the two truncation constructs of flii with the cytoplasmic domain of flha. only flii1- was able to co-purify with flha, and not flii150- suggesting that the flha binding domain resides in the n-terminal amino acids of flii. we next wanted to know if flii interacts with flif. we therefore reacted gst-flii against the two flif constructs and found that there was no co-purification, suggesting that any interaction between flii and flif, if there is an association, would seem to be indirect and mediated through the action of flha or other intermediate proteins. cpn interacts with flii and flha cpn is a predicted amino acid protein with a pi of and a molecular mass of kda. the cpn orf is encoded directly upstream of flif and downstream of flii, the flagellar atpase. based on its location relative to flii and flif, we hypothesized that it may interact with other flagellar components. we used gst pull-down assays to explore this possibility. initial gst pull-downs indicated that full length his-cpn interacts with gst-cpn suggesting the presence of a dimerization domain. to explore this observation we treated cpn with formaldehyde prior to page and observed the presence of a monomer and a dimer, migrating with apparent molecular weights of kda and kda. we next explored the possible interaction of cpn with other flagellar proteins in c. pneumoniae. using gst pull-downs, his-cpn co-purified with the full length gst-flii protein as well as the gst-flii1- protein, but not gst-flii150-. this suggests that cpn binds to the n-terminus of flii. gst pull-down assays showed an interaction between cpn and the flha308- protein, the cytoplasmic domain of flha. cpn did not co-purify with either flif35- or flif1-, suggesting that cpn does not interact with flif. flii and flha interact with t3s components since chlamydia have no apparent flagella, we investigated whether the flagellar proteins flii, flha and flif interact with t3s components. using bacterial-2-hybrid screening we found that flii and flha interacted with cdsl, the putative t3s atpase regulator and tethering protein, with a β-galactosidase activity of ± and ± units of activity, respectively. flii also interacted with copn, the putative t3s plug protein, with a β-galactosidase activity of ± units of activity. we also found that flha interacted with the putative yscu ortholog, cdsu, with a β-galactosidase activity of ± units of activity, as well as cdsl, with a β-galactosidase activity of ± units of activity. to corroborate these findings we utilized gst pull-down assays and showed that gst-flii interacted with cdsl and copn, but not cpn, and gst-flha co-purified with both cdsl and cdsu. control gst coated beads did not co-purify with cdsl, copn or cdsu. discussion sequencing of several chlamydial genomes revealed a conserved set of flagellar orthologs, despite the fact that c. pneumoniae lack a flagellum and are considered non-motile bacteria. here we report an initial characterization of three annotated flagellar proteins of c. pneumoniae, flii, flha and flif, demonstrating atpase activity of flii and interactions between these flagellar orthologs. we have demonstrated that flii hydrolyzes atp in a linear, time-and dose-dependant manner, with optimal activity at a ph of and a temperature of 37°c. flii also interacts with the cytoplasmic domain of flha, while flha interacts with the c-terminal region of the flif protein. no direct interaction of flii and flif was detected. also, we have characterized an interaction of both flha and flii with cpn a fourth unannotated protein. we also show that flii interacts with cdsl and copn, two t3s components, while flha interacts with cdsl and a third t3s component, cdsu. collectively, this data suggests that the flagellar proteins of c. pneumoniae interact in a specific way with t3s proteins and may play an important, as yet unidentified role in the chlamydial replication cycle. flii hydrolyzes atp in a linear, time- and dose-dependant manner at a rate of ±. μmol min- mg- this rate is typical of other secretion atpases such as cdsn, escn, or flii from other bacterial species. the optimal ph for flii atpase activity is, which is the same as that for other flagellar atpases. extreme low or high ph greatly reduced the activity, possibly due to protein denaturation. also, the enzyme activity peaked at a temperature of 37°c and declined substantially beyond that. although the formation of higher-order complexes was not explored here, other flagellar atpases are thought to form a hexameric complex. the presence of three flagellar genes in chlamydiae is intriguing since chlamydiae are thought to be non-motile and not to possess flagella. flif, flha and flii alone do not contain all the necessary components for a functional flagella or secretion apparatus, however, a rudimentary basal body or pore complex could be formed by these three components. it is known that the most rudimentary flagellar structure that can be assembled is the ms ring, which consists of only the flif protein. we have shown that these proteins interact with one another, most likely at the inner membrane of c. pneumoniae. the interaction between flii and flha is mediated by the n-terminal amino acids of flii and appears to be specific since it is not disrupted by high salt. only the cytoplasmic domain of flha was utilized in the gst pull-down, suggesting that any protein interactions that occur are within this region. protein interaction studies with the full length flha protein are difficult due to the presence of seven transmembrane domains rendering full length flha insoluble and making this portion of the protein unable to bind to soluble flagellar components. since flha is known to interact with soluble components of the flagellar apparatus in other bacteria, it is expected that the cytoplasmic domain mediates an interaction with flii. flif is known to form the ms ring in flagellated bacteria, and is one of the first components of the flagellar basal body to be incorporated into the membrane. we detected an interaction of the c-terminal amino acids of flif with the cytoplasmic domain of flha. these interactions were also stable in mm nacl, suggesting that the interaction is specific. we did not, however, detect any interaction between flii and flif, suggesting that any interaction between those two components may be mediated through the action of another protein, possibly flha in c. pneumoniae, cpn is encoded directly downstream of the atpase, which led us to explore any interactions cpn may have with other flagellar proteins. we initially found that cpn was able to interact with itself using a gst pull-down assay, suggesting that it could form dimers in solution. to confirm this observation we utilized formaldehyde fixation followed by page analysis to visualize the formation of dimers. using this method we saw that cpn migrated in two molecular forms, with sizes corresponding to both monomers and dimers. we then explored possible interactions between cpn and the other flagellar proteins and detected interactions of cpn with both flii and flha, but not flif. cpn bound to the n-terminal amino acids of flii and the cytoplasmic region of flha. the interaction of cpn with the cytoplasmic domain of flha was expected as flha is known to interact with soluble components of other flagellar systems. we considered the possibility that cpn may in fact be the flih ortholog in c. pneumoniae as cpn has minor sequence orthology to other flih proteins, but after further investigation we found that cpn did not appear to play a regulatory role with flii. figure summarizes the interactions between flii, flha and flif and interactions between cpn flha and flii. bacterial type iii secretion and flagellar secretion systems are structurally similar, and may have a common ancestry. although c. pneumoniae does not contain a full repertoire of flagellar genes, it does encode a complete t3s system which most likely consists of specific protein complexes located in the inner membrane. we have characterized an interaction of flii with cdsl, the t3s atpase tethering protein. the c. pneumoniae flih ortholog has not yet been identified, and in the absence of flih, cdsl may play a regulatory role for both flii and cdsn. flii also interacts with the copn, the t3s plug protein, suggesting that flii may be involved in either the secretion of effector proteins or regulation of the t3s system. yscu orthologs have a flagellar paralog, flhb, and cpn is believed to be the c. pneumoniae yscu ortholog. flhb is known to interact with flha, but in c. pneumoniae no flhb ortholog has been annotated. we found that flha interacts with cdsu, suggesting integration of flha into the inner membrane, associating with t3s components.  based on all of our observations, it is possible that the flagellar proteins may interact with components of the t3s injectisome, forming a hybrid structure, thereby playing an ancillary or accessory role in secretion of type iii effectors across either the cytoplasmic or inclusion membrane. another possibility is that there are other, as yet unannotated proteins that play a role in a putative flagellar system in c. pneumoniae. for example, along with the flih/flii complex that is formed in other bacteria, another protein, flij, which is a general chaperone, is believed to be involved in this complex. flij has not been identified in c. pneumoniae. in the absence of a genetic manipulation system for the chlamydiae, direct evidence for the role of these flagellar proteins remains elusive. the fact that flii is enzymatically active and forms complexes in vitro with other flagellar proteins, all of which are present in all other chlamydiae sps. studied to date, suggests that these proteins play an important role in chlamydial replication or survival. further studies using heterologous systems and genetic complementation could help to decipher the exact role of these flagellar proteins in chlamydiae. 
the specific interaction of the four orthologous flagellar proteins in c. pneumoniae suggests that they interact in vivo and, taken together with their conservation across members of the chlamydiae sps., and their interaction with t3s components, suggests a role in bacterial replication and/or intracellular survival. flagellar secretion systems are utilized by a wide variety of bacteria to construct the flagellum, a conserved apparatus that allows for migration towards non-hostile, nutrient rich environments. using bacterial-2-hybrid and gst pull-down assays, the n-terminal domain of flii was shown to interact with the cytoplasmic domain of flha, but not with flif, and the cytoplasmic domain of flha was shown to interact with the c-terminus of flif. μmol min- mg- in a time- and dose-dependant manner. chlamydia pneumoniae is an obligate, intracellular pathogen whose genome contains at least three orthologs of flagellar proteins, namely flii, flha and flif, but the role of these proteins remains unknown. full length flii, and fragments of flha, flif, and flii, were cloned and expressed as either gst or his tagged proteins in e. coli. the gst-tagged full length flii protein was shown to possess atpase activity, hydrolyzing atp at a rate of ±. the absence of other flagellar orthologs led us to explore cross-reaction of flagellar proteins with type iii secretion proteins, and we found that flii interacted with cdsl and copn, while flha interacted with cdsl and cpn.
 flagellar secretion systems are utilized by a wide variety of bacteria to construct the flagellum, a conserved apparatus that allows for migration towards non-hostile, nutrient rich environments. chlamydia pneumoniae is an obligate, intracellular pathogen whose genome contains at least three orthologs of flagellar proteins, namely flii, flha and flif, but the role of these proteins remains unknown.  full length flii, and fragments of flha, flif, and flii, were cloned and expressed as either gst or his tagged proteins in e. coli. the gst-tagged full length flii protein was shown to possess atpase activity, hydrolyzing atp at a rate of ±. μmol min- mg- in a time- and dose-dependant manner. using bacterial-2-hybrid and gst pull-down assays, the n-terminal domain of flii was shown to interact with the cytoplasmic domain of flha, but not with flif, and the cytoplasmic domain of flha was shown to interact with the c-terminus of flif. the absence of other flagellar orthologs led us to explore cross-reaction of flagellar proteins with type iii secretion proteins, and we found that flii interacted with cdsl and copn, while flha interacted with cdsl and cpn.  the specific interaction of the four orthologous flagellar proteins in c. pneumoniae suggests that they interact in vivo and, taken together with their conservation across members of the chlamydiae sps., and their interaction with t3s components, suggests a role in bacterial replication and/or intracellular survival. 