BACKGROUND
borrelia burgdorferi, the spirochetal agent of lyme disease, possesses a dual-membraned  architecture, which is composed of a peptidoglycan layer associated with the inner membrane  and an outer membrane   <cit> .
in gram-negative bacteria, cytoplasmic precursor outer membrane proteins  are synthesized with an amino-terminal signal peptide sequence, which typically targets a protein for sec-mediated translocation.
after the precursor omp crosses the im through the secyeg translocase, the signal peptide is cleaved by signal peptidase i, and the mature protein is subsequently released into the periplasmic space  <cit> .
once in the periplasm, the unfolded omp is bound by chaperones that help direct the omp to the om for proper folding and membrane insertion  <cit> .
until recently, these latter steps of periplasmic omp trafficking and om assembly have remained largely uncharacterized.
in  <dig>  however, tommassen and coworkers identified an essential β-barrel omp whose function is dedicated to the proper om-assembly of most known omps  <cit> .
this protein, now known as bama  <cit> , is evolutionarily well-conserved since putative orthologs can be found in all known diderm bacteria, as well as in dual-membraned eukaryotic organelles, such as mitochondria and chloroplasts  <cit> .
the functional importance of bama was illustrated when researchers discovered that bama was essential for the viability of both n. meningitidis and e. coli, and that its depletion resulted in dramatically decreased levels of properly-inserted omps in the om of both organisms  <cit> .
in e. coli, combined genetic and biochemical studies have now revealed that bama exists in a multiprotein om complex, termed the beta-barrel assembly machine   <cit> .
this complex is composed of the om-imbedded bama protein and four om-anchored accessory lipoproteins, termed bamb, bamc, bamd, and bame   <cit> .
more recent studies have revealed that all of the bam components are important at some level for omp assembly and/or for the stability of the bam complex.
the bamb lipoprotein interacts directly with bama within the complex, and this association is independent of the other bam lipoproteins  <cit> .
bamb is thought to be an important scaffolding protein for the bam complex, and although bamb deletion mutants are viable, they have reduced levels of various omps  <cit> .
bamc- and bame-null strains have relatively mild omp assembly defects; however, they both show moderate om permeability defects, and biochemical studies show that their presence in the complex is important for the bama-bamd interaction  <cit> .
the bamd protein, however, is essential for cell viability, and depletion of bamd causes a phenotype similar to that observed in bama mutants  <cit> .
additionally, bamd is the most evolutionarily conserved lipoprotein in the bam complex.
like bama, bamd orthologs are predicted to be present in all diderm bacteria  <cit> , and they are proposed to contain conserved tetratricopeptide repeat  domains which have been shown to function in protein-protein interactions  <cit> .
bam complexes have now been characterized from other gram-negative bacteria, such as n. meningitidis and caulobacter crescentus  <cit> .
in n. meningitidis, co-immunoprecipitation studies showed that, along with bama and bamd, the bam complex contains bamc and bame, as well as an additional protein component, rmpm  <cit> .
a bamb homolog, however, was not identified in n. meningitidis.
the bam complex in c. crescentus was recently reported to contain all of the known bam lipoproteins except bamc, but includes an additional lipoprotein termed pal, which contains an ompa-type peptidoglycan binding domain that is similar to rmpm  <cit> .
these studies suggest that bacterial bam complexes likely contain not only conserved orthologs and proteins with conserved structural motifs, such as bamd, but also non-conserved proteins which may provide specific requirements for omp assembly in a particular species of bacteria.
in b. burgdorferi, the only member of the bam complex identified to date is bb <dig>  which we previously determined to be a structural and functional b. burgdorferi bama ortholog  <cit> .
in the present study, we examined whether b. burgdorferi bama, like other known bama proteins, exists as a member of a multiprotein om complex.
we report that native b. burgdorferi bama forms high molecular-weight om complexes and that bama co-immunoprecipitates specifically with two putative b. burgdorferi lipoproteins, bb <dig> and bb <dig>  we also demonstrate that depletion of bama, using an iptg-regulated b. burgdorferi mutant, results in loss of bb0324-bb <dig> interactions, suggesting that the lipoproteins do not associate without the presence of bama.
additionally, we determined that both bb <dig> and bb <dig> are om-anchored, and are localized to the inner leaflet of the om.
while sequence analysis strongly suggests that bb <dig> is a bamd ortholog containing tpr domains similar to those predicted for the n. meningitidis and e. coli bamd lipoproteins  <cit> , bb <dig> did not have significant sequence homology to any other known bam components.
the combined results suggest that b. burgdorferi contains fewer proteins in its bam complex, which is likely reflective of its distinct evolutionary phylogeny and unique om ultrastructure.
methods
bacterial strains and growth conditions
borrelia burgdorferi strain b31-mi, strain b31-a <dig>  <cit> , strain b31-a3-lk  <cit> , and strain flacp-795-lk  <cit>  were cultivated at 34°c in barbour-stoenner-kelly  liquid medium  <cit>  containing 6% heat-inactivated rabbit serum .
the b31-a <dig> strain was supplemented with kanamycin , and the b31-a3-lk strain was supplemented with kanamycin and gentamicin .
strain flacp-795-lk was supplemented with  <dig> μg/ml streptomycin , in addition to kanamycin and gentamycin.
strain flacp-795-lk was also cultivated in  <dig>  mm or  <dig>  mm isopropyl-β-d-thiogalactopyranoside , as indicated.
isolation of b. burgdorferi outer membrane vesicles and protoplasmic cylinders
for blue native page  and cellular localization assays, b. burgdorferi strain b31-a <dig> outer membranes  and protoplasmic cylinders  were isolated by discontinuous sucrose density gradient centrifugation followed by a continuous sucrose density gradient centrifugation protocol as previously described  <cit> .
blue native page  analysis
b. burgdorferi strain b31-a <dig> om complexes were analyzed by bn-page under native conditions as described  <cit> .
briefly, the isolated om preparations were resuspended in  <dig>  m aminocaproic acid,  <dig> mm bis-tris  and β-dodecyl maltoside  .
the protein solution was incubated for  <dig> min on ice and centrifuged at  <dig>  × g for  <dig> min, and the resulting supernatant was separated using a 5-14% gradient polyacrylamide gel at 4°c.
the protein migration pattern in the bn gel was analyzed visually, or electrophoretically transferred to nitrocellulose for anti-bama immunoblot analysis, as described below.
sds-page and immunoblot analyses
for denaturing page and immunoblots, protein samples were prepared and separated by sds-page, followed by electrophoretic transfer to nitrocellulose membranes, as described previously  <cit> .
for flab immunoblots, membranes were probed with a 1: <dig>  dilution of rabbit anti-flab antisera  <cit> , followed by incubation with a 1: <dig>  dilution of horseradish peroxidase -conjugated goat anti-rabbit secondary antibodies .
subsequent chromogenic development was performed using 4-chloronapthol and hydrogen peroxide.
for all other immunoblots, enhanced chemiluminescence  was used, as described by kenedy et al.
<cit> .
after primary antibody incubation , membranes were incubated in a 1: <dig>  dilution of goat anti-rat , goat anti-rabbit , or goat anti-mouse  secondary antibodies.
washed membranes were subsequently developed using supersignal west pico ecl reagent according to manufacturer's instructions .
sequence analyses and alignments
the n. meningitidis bamd  protein sequence was used to search the b. burgdorferi b <dig> peptide database using the j. craig venter comprehensive microbial resource blast server .
bb <dig> and bb <dig> hydrophilicity analyses were performed using macvector version  <dig>  sequence analysis software  according to the method of kyte and doolittle  <cit> , and prediction of putative signal peptides and the canonical lipoprotein signal peptidase ii cleavage sites was performed using the signalp  <dig>  server  <cit>  and the lipop  <dig>  server  <cit> , respectively.
bb <dig> tetratricopeptide repeat  domains were predicted using tprpred  and by comparison with the original published tpr consensus sequence  <cit> .
the predicted tpr-containing regions from nm-bamd, e. coli bamd, and bb <dig>  were aligned using the macvector version  <dig>  multiple sequence alignment program , followed by manual editing using the jalview  <dig> multiple alignment editor  <cit> .
generation and purification of recombinant proteins
to generate bb <dig>  bb <dig>  and bb <dig> recombinant proteins, dna sequences corresponding to each full-length mature protein lacking the putative signal peptide were pcr-amplified from b <dig> genomic dna.
primers used for amplification of the bb <dig> dna region are as follows : 5'-gcgggatccttaacaaaagaaactccttatgg-3' , and 5'-ttttttattattttctattttatttaata-3' .
primers used for amplification of the bb <dig> dna region are as follows: 5'-gcgggatccgctaatcttgatcaaataaaaaatc-3'  and 5'-gcggaatccttaagggtttttattgtccttttc-3' .
primers used for amplification of the bb <dig> dna region are as follows: 5'-aagaattctcaagcgaatccatattttcac-3' , and 5'-aactcgagttattctttagttaattttctgttttcca-3' .
the bb <dig>  bb <dig>  and bb <dig> amplicons were ligated into the topo-ta pbad/thio vector , the pgex- <dig> t- <dig> vector , and the pgex-6p <dig> vector , respectively.
the resulting constructs were transformed into electrocompetent e. coli dh5α cells, and prior to protein purification, selected transformants were verified to contain the correct insert sequence by restriction digest and by nucleotide sequence analysis.
for protein purification, recombinant bb <dig> was purified as a thioredoxin fusion using a solubilization protocol described previously  <cit> .
recombinant bb <dig> and bb <dig> were purified as glutathione-s-transferase  fusion proteins and cleaved free of the gst moiety using procedures described previously  <cit> .
antibodies
antibodies to the bb <dig> and bb <dig> recombinant proteins were generated in rats as previously described  <cit> .
rabbit anti-bb <dig> antibodies were described elsewhere  <cit> .
rat anti-bama  antibodies were generated previously  <cit> , and mouse anti-lp <dig>  antibodies were also generated as described previously  <cit> .
mouse anti-oppaiv antibodies were generously provided by drs.
justin radolf and melissa caimano, university of connecticut health center, farmington, ct. rabbit anti-flab, rat anti-thio, rat anti-ospa, and rat anti- <dig> antibodies were generated as previously described  <cit> .
all animal procedures were approved by the oklahoma university health sciences center institutional animal care and use committee .
cell lysate preparation and co-immunoprecipitation
for each co-ip sample, cell lysates were prepared by using mid-log phase cultures  of b. burgdorferi strain b31-mi, b31-a3-lk, or flacp-795-lk .
cells were centrifuged at  <dig>  × g for  <dig> min and subsequently washed four times in pbs .
prior to cell lysis for co-ip, washed cells  from each culture condition were subjected to anti-bama immunoblot analysis to verify the regulatable bama phenotype.
for co-ip experiments, cell pellets were solubilized and lysed by resuspension in 1× bugbuster reagent .
the solubilized cell solution was supplemented with  <dig> μl lysonase bioprocessing reagent  and  <dig> μl of protease inhibitor cocktail  per co-ip sample, and the mixture was subsequently rocked at room temperature  for  <dig> min.
finally, the cell debris was pelleted at  <dig>  × g for  <dig> min at 4°c, and the supernatant  was used for the co-ip experiments.
co-ips were performed using the sigma protein g immunoprecipitation kit according to manufacturer's instructions, with the following modifications: 1) the 1× and  <dig> × ip buffers were supplemented with  <dig> % triton x- <dig>  and 2) prior to immunoprecipitation, the lysates were pre-cleared overnight to reduce background binding.
after immunoprecipitation, bound proteins were eluted in  <dig> μl final sample buffer , subjected to sds-page, and analyzed by silver stain according to the procedure of morrissey  <cit> , or by immunoblot, as described above.
for protein identification, excised sds-page gel bands were submitted to the molecular biology-proteomics facility  for tryptic digestion and hplc-ms/ms analysis, followed by mascot database search for protein identification.
triton x- <dig>  phase partitioning
to determine whether bb <dig> and bb <dig> have the amphipathic properties of typical lipid-modified proteins, b. burgdorferi strain b31-mi cells  were harvested and phase-partitioned as described previously  <cit> .
proteinase k  surface accessibility
to determine whether bb <dig> and bb <dig> contain surface-exposed regions, pk experiments were performed as previously described  <cit> .
briefly, spirochetes  were harvested at  <dig>  × g, washed four times in 1× pbs , and the washed cells were either mock-treated or pk-treated ; sigma chemical co.) for one hour at rt.
after addition of pmsf , samples were prepared for sds-page and immunoblot analysis, as described above.
to verify that bb <dig> and bb <dig> were not resistant to pk activity, cell membranes were disrupted as previously described  <cit> .
cells  were pelleted at  <dig>  × g, washed, and incubated for  <dig> m in  <dig> μl pk lysis buffer containing  <dig> mm tris,  <dig> % triton x- <dig>   <dig> %, β-mercaptoethanol, and  <dig> μg of lysozyme.
samples were then incubated in the absence or presence of pk.
the reaction was stopped with pmsf and prepared for immunoblot as indicated above.
RESULTS
b. burgdorferi bama forms multi-protein complexes in the om
previously, we performed a structural and functional characterization of the om-localized b. burgdorferi bama protein  <cit> .
since other bama orthologs are known to exist in a hetero-oligomeric protein complex  <cit> , we wanted to determine if native b. burgdorferi bama could be detected in high molecular weight om complexes.
to perform this assay, we isolated om vesicles from b. burgdorferi strain b31-a <dig> and subjected the om sample to one-dimensional blue native -page, followed by anti-bama immunoblot analysis.
results from the immunoblot showed multiple protein bands between the  <dig> and  <dig>  kda mw markers , with two prominent bands that resolved at approximately  <dig> kda and  <dig>  kda .
in addition, samples from the om fraction and from the protoplasmic cylinder  fraction were separated by denaturing sds-page and immunoblotted against the periplasmic flab protein to verify om purity .
these results demonstrate that native b. burgdorferi bama is present in multiple high molecular weight om complexes, which may indicate that bama associates with other om-localized proteins or protein complexes.
in silico analysis of b. burgdorferi bam orthologs
to identify possible components of the b. burgdorferi bam complex, our initial approach was to search the b. burgdorferi protein database for putative orthologs of the e. coli bam lipoproteins, bamb, bamc, bamd, and bame  <cit> .
although protein blast  searches using each of the bam proteins provided no significant sequence matches, blastp searches using each of the n. meningitidis bam lipoproteins as a search query yielded one b. burgdorferi protein.
this protein, encoded by open reading frame  bb <dig>  has significant similarity  to the n. meningitidis bamd lipoprotein.
bb <dig> is a 119-residue polypeptide of unknown function that is predicted to contain an n-terminal signal peptide with a signal peptidase ii lipoprotein modification and processing site as determined by a combination of hydrophilicity, signalp  <dig> , and lipop  <dig>  computer analyses as described in methods.
the identification of a canonical lipoprotein processing and modification site strongly suggested that bb <dig> is the b. burgdorferi lipoprotein bamd ortholog.
comparative sequence analyses indicate that bb <dig> aligns with the n-terminus of n. meningitidis bamd, such that almost the entire bb <dig> amino acid sequence aligns with the first  <dig> residues of the 267-residue n. meningitidis bamd protein .
importantly, this region of n. meningitidis bamd is predicted to contain two conserved tpr sequences, which are also predicted to exist in bb <dig> .
the tpr sequence is a degenerate 34-residue consensus sequence that forms a helix-turn-helix secondary structure element  <cit> , and such motifs are known to be involved in protein-protein interactions  <cit> .
only a few positions within the consensus tpr sequence are highly conserved , and therefore individual tprs can vary substantially at the primary sequence level.
e. coli bamd is also predicted to contain n-terminal tpr sequences that can be aligned with those of bb <dig> and n. meningitidis bamd .
the combined results from the protein blast searches and the sequence alignment analyses further support the contention that bb <dig> is a b. burgdorferi bamd ortholog.
b. burgdorferi bama forms a complex with bb <dig> and bb0028
to identify additional bam accessory proteins, we next performed anti-bama co-immunoprecipation  experiments.
since our bama antisera was generated against recombinant bama proteins with a 5' thioredoxin fusion , we utilized anti-thioredoxin  antisera as our negative control antibody for the co-ip assays.
when we analyzed the immunoprecipitated proteins by coomassie stain, we detected an ~ <dig> kda band that was prominent in the bama-precipitated samples as compared with the anti-thio control .
to determine the identity of the protein contained within the  <dig> kda band identified, this region  were subsequently excised, trypsin-digested, and subjected to lc-ms/ms analysis.
after mascot database search, the unknown protein from the bama co-ip was identified as a 349-residue polypeptide encoded by the b. burgdorferi orf bb <dig>  this protein was not identified in the band extracted from the thio co-ip elution, suggesting that it co-immunoprecipitated specifically with bama.
similar to bb <dig>  computer analyses of the bb <dig> protein indicated that it contains a signal peptide with a consensus signal peptidase ii lipoprotein modification and processing site, suggesting that bb <dig> is also a b. burgdorferi lipoprotein.
interestingly, blastp analyses failed to identify any bb <dig> conserved domains or any significant protein matches outside of the borrelia genus.
to determine if bb <dig>  and bb <dig> are bam accessory proteins that specifically associate with bama, we performed anti-bama, anti-bb <dig>  and anti-bb <dig> immunoprecipitation experiments .
the immunoprecipitation assays were then subjected to immunoblot analysis with specific antibodies to bama, bb <dig>  and bb <dig> .
as shown in figure  <dig>  b. burgdorferi bama co-immunoprecipitated bb <dig> and bb <dig>  additionally, bb <dig> antibodies co-immunoprecipitated bama and bb <dig> while bb <dig> antibodies co-immunoprecipitated bama and bb <dig> .
however, none of the three proteins were detected in the thio co-immunoprecipitation experiment control sample .
additionally, when immunoprecipitated proteins from all experiments were probed with antibodies to lp <dig> , which is a lipoprotein known to be localized to the inner leaflet of the borrelial om  <cit> , there was no detectable co-immunoprecipitation of lp <dig>  .
the lp <dig>  immunoblot data demonstrated that an unrelated lipoprotein was not immunoprecipitated, which helped to confirm the specificity of the immunoprecipitation assays.
the combined data indicate that bama physically associates with bb <dig> and bb <dig>
bama expression is required for interaction with bb <dig> and bb0028
although the above co-immunoprecipitation data indicated that bb <dig> and bb <dig> specifically interact with bama, it was still unclear if bb <dig> and bb <dig> interacted with each other.
we therefore wanted to determine if native bb <dig> and bb <dig> form their own complexes in b. burgdorferi, or if they interact only in the presence of bama as constituents of the larger bam complex.
to examine this issue, we utilized the regulatable b. burgdorferi strain  that was engineered to express an iptg-inducible chromosomal bama gene.
we previously illustrated that in low concentrations of iptg , total cellular levels of bama protein were dramatically reduced, and as a result, b. burgdorferi om preparations contained reduced levels of omps  <cit> .
by performing immunoprecipitation experiments with flacp-795-lk cultivated in a low concentration  or high concentration  of iptg, we were able to observe the effects of bama depletion on the bama-bb0324-bb <dig> interactions.
as shown by immunoblot analysis, bama depletion resulted in less bb <dig> being immunoprecipitated by bb <dig> antibodies as compared to the parental b31-lk strain .
similarly, bama depletion also resulted in less bb <dig> being immunoprecipitated by bb <dig> antibodies as compared to the parental b31-lk strain .
however, it should be noted that there was no detectable difference in the levels of bb <dig> or bb <dig> expression after bama depletion .
these data indicate that the loss of bama did not affect the amount of bb <dig> or bb <dig> protein being expressed in the flacp-795-lk or parental lk strains.
consistent with this observation, whole-cell lysates  from the flacp-795-lk strain grown in  <dig>  mm or  <dig>  mm iptg and the parental lk strain contained similar levels of bb <dig> and bb <dig> as shown in figure 5c.
the combined data revealed that bama depletion does not affect expression of bb <dig> or bb <dig>  but instead causes a decrease in the amount of bb <dig> that is immunoprecipitated with bb <dig>  and also causes a decrease in the amount of bb <dig> that is immunoprecipitated by bb <dig>  thus, the bb <dig> and bb <dig> interactions with bama appear to be severely affected by the loss of bama expression, which also indicates that they require bama in order to efficiently form the larger bam complex.
bb <dig> and bb <dig> are outer membrane-associated subsurface proteins
currently, all known accessory proteins of e. coli bam complex, besides bama, are lipoproteins anchored to the inner leaflet of the om  <cit> .
therefore, we next examined whether both bb <dig> and bb <dig> are localized to the periplasmic leaflet of the om.
to begin our cellular localization assays, we first performed triton x- <dig>  phase partitioning studies with b. burgdorferi cells to determine if bb <dig> and bb <dig> are amphiphilic.
as shown in figure 6a, both bb <dig> and bb <dig> partitioned exclusively into the detergent-enriched fraction, which is characteristic of amphiphilic proteins.
additionally, a known membrane-anchored lipoprotein  and a soluble protein  were used as detergent phase and aqueous phase controls, respectively.
we next examined the cellular location of bb <dig> and bb <dig> to confirm their presence in the om.
as shown in figure 6b, bb <dig> and bb <dig> were detected in the isolated oms of b. burgdorferi, demonstrating that both proteins are localized to the om.
cell fractions were also probed with antibodies to the om-localized bama and lp <dig>  proteins, as well as to the im-anchored oppaiv lipoprotein, to verify om specificity and purity.
to determine if bb <dig> or bb <dig> are anchored to the periplasmic leaflet of the om, we next incubated whole b. burgdorferi cells in the presence or absence of proteinase k .
these experiments revealed that there was no difference between mock- or pk-treated samples when probed with anti-bb <dig> or anti-bb <dig>  indicating neither protein is surface-exposed .
as controls for pk activity and om integrity, lysates from the mock- and pk-treated cells were also probed with antibodies against the surface-localized bb <dig> protein  <cit>  and the periplasmic flab protein, respectively.
importantly, when cell membranes were disrupted prior to pk treatment, bb <dig> and bb <dig> were both degraded in the presence of pk  confirming that these proteins are subsurface proteins and not intrinsically resistant to pk activity.
discussion
current working model for the b. burgdorferi bam complex
the bacterial beta-barrel assembly machine, or bam, is a multiprotein om complex that is composed of the essential integral omp bama, as well as a number of conserved and nonconserved accessory lipoproteins that are anchored to the inner leaflet of the om  <cit> .
to date, few bam complexes have been studied, and since only those from proteobacteria have been characterized, it is yet to be determined what elements of various bam complexes are conserved between different bacterial groups.
in this study we report that the diderm spirochete, b. burgdorferi, also contains an om-localized bam complex, which is composed of bama and at least two accessory lipoproteins, bb <dig> and bb <dig>  additionally, co-immunoprecipitation experiments using a bama regulatable b. burgdorferi mutant strain indicated that bama is required for efficient association of bb <dig> and bb <dig>  further cellular localization assays indicated that both bb <dig> and bb <dig> are om anchored subsurface lipoproteins, although only bb <dig> is predicted to be an ortholog to a currently identified bam accessory lipoprotein .
as determined from our initial immunoprecipitation experiments with b. burgdorferi strain b31-mi, the bb <dig> and bb <dig> proteins associate specifically with bama as a heterooligomeric om protein complex .
additional data from the bama regulatable mutant provided further insight into the bama-bb0324-bb <dig> interactions.
when the bama iptg-regulatable strain was cultivated in decreasing concentrations of iptg  it was immediately apparent that the bama and bb0324/bb <dig> associations were dramatically affected as compared to the parental, wildtype strain b31-lk .
although these data are insufficient to provide conclusions on the detailed organization of the bam complex, it is apparent that bb <dig> and bb <dig> do not efficiently co-immunoprecipitate each other when bama is depleted.
these data suggest that bb <dig> and bb <dig> do not readily associate in b. burgdorferi without the presence of bama, and that they likely come together only to form the functional bam complex.
however, the molecular architecture of the b. burgdorferi bam complex is still unknown, and it is unclear what specific interactions create the bama-bb0324-bb <dig> complex.
in our model, bb <dig> and bb <dig> may associate indirectly through individual direct contacts with bama.
alternatively, bb <dig> and bb <dig> may bind directly with each other, where only one of them binds bama.
further experiments using b. burgdorferi bb <dig> and bb <dig> partial and/or full deletion mutants  should help to clarify the molecular architecture and binding partners within the bam complex.
potential interactions between the periplasmic bama polypeptide transport-associated  domains and the bb <dig> and bb <dig> accessory lipoproteins
the periplasmic n-terminus of bama contains five repeating potra  domains, which have been designated p1-p <dig>  <cit> , that are responsible for the bama-accessory lipoprotein interactions  <cit> .
it has been shown in e. coli that deleting any of the potra domains other than p <dig> results in disruption of accessory lipoprotein interactions  <cit> .
similar to the e. coli bam accessory lipoproteins, it is likely that bb <dig> and bb <dig> also associate with bama through potra domain contacts.
future co-immunoprecipitation experiments with different b. burgdorferi bama potra domain mutants as well as bb <dig>  and/or bb <dig> mutants will help clarify exactly which potra domains are needed for bb <dig> and bb <dig> accessory protein binding.
bb <dig> is a putative bamd ortholog with a truncated c-terminus
blastp searches and sequence analyses indicate that the bb <dig> protein is a putative b. burgdorferi bamd ortholog.
bamd is predicted to be ubiquitous in diderm bacteria  <cit> , and it appears to be both essential for cell survival and central to the function of the bam complex, as demonstrated in e. coli and in n. meningitidis  <cit> .
it is predicted that all bamd orthologs possess n-terminal tpr domains  <cit> , and in e. coli and n. meningitidis, bamd appears to contain two .
although such structural features are still predicted for e. coli and n. meningitidis, a recently-determined crystal structure from the rhodothermus marinus bamd confirms the presence of tpr domains within this protein  <cit> .
although tprs form a characteristic helix-loop-helix structure, their propensity for sequence variation is likely a reason that we were initially unable to identify a bamd ortholog in b. burgdorferi, even though bb <dig> contains consensus tpr sequences  <cit> .
in addition, bb <dig> is considerably smaller than the bamd proteins currently identified in other bacteria.
the putative borrelial bamd lipoprotein has a predicted mw of ~ <dig> kda, which is less than half the size of proteobacterial bamd proteins from e. coli, n. meningitidis, and c. crescentus.
interestingly, it has been proposed that the tpr domain region fulfills the major functional requirements for bamd , and that the tprs may be the only essential feature of the bamd proteins  <cit> .
this idea has been discussed in previous reports, and it originates from the discovery of a viable transposon mutant of the neisseria gonorrhoeae bamd protein, also known as coml  <cit> .
as noted by volokhina et al., this truncated mutant contains only  <dig> amino acids of the mature 267-residue protein, indicating that the coml n-terminus, which comprises the tpr motifs, is sufficient for viability  <cit> .
although viable, the coml mutant displayed reduced colony size and was deficient in transformation competency  <cit> .
similarly, an e. coli transposon mutant lacking only the c-terminal  <dig> amino acids of the 245-residue bamd protein was also viable but had greatly reduced bamd function  <cit> .
these phenotypic characteristics suggest that the bamd c-terminus, although nonessential, fulfills some functional requirement for neisseria and for e. coli  that is either unnecessary for b. burgdorferi, or is provided by a different protein.
interestingly, it has been shown that the c-terminus of the e. coli bamd binds bamc and bame, and is therefore important for the stability of this part of the bam complex  <cit> .
thus, a truncated b. burgdorferi bamd may simply be the result of this organism having no requirement for an extended c-terminal region to interact with additional accessory lipoproteins such as bamc or bame, since we were not able to identify other accessory lipoproteins in b. burgdorferi.
CONCLUSIONS
in the current study, we have identified two accessory components of the b. burgdorferi bam complex.
based on the knowledge gained from studying other proteobacterial organisms, it is possible that b. burgdorferi contains one or more other bam accessory lipoprotein components in addition to bb <dig> and bb <dig> that are still unidentified.
as indicated by bn-page in figure 1a, multiple high molecular weight  complexes containing bama are present between approximately  <dig> kda and over  <dig>  kda.
these data accommodate the possibility that additional protein species may be co-migrating with bama, especially since the smallest of the two most prominent bands, which migrates at ~ <dig> kda, has an approximate mw that is larger than the expected mw of bama, bb <dig>  and bb <dig> combined .
alternatively, these large protein complexes may contain multiple copies of the same protein, such as multiple bb <dig> molecules, and/or be homo-oligomers of the entire bam complex.
it should be noted, however, that b. burgdorferi contains a relatively small number of integral omps  compared to e. coli  <cit> ; hence, it may require a less complicated bam complex system for omp assembly.
indeed, silhavy and coworkers proposed that the major function of the nonessential e. coli bamb, bamc, and bame lipoproteins is most likely to increase efficiency of omp assembly, or to stabilize the complex, since individual mutants were viable and showed relatively mild assembly defects  <cit> .
it is, therefore, possible that an om with a more limited omp repertoire, such as that of b. burgdorferi, does not necessitate additional bam complex members to provide the essential functions for complete om biogenesis.
in this regard, it is tempting to speculate that the b. burgdorferi bam constituents identified here constitute a "minimal" bacterial bam complex, which can now be further studied as a model system to not only further our understanding of b. burgdorferi om biogenesis, but also to contribute to our current knowledge of bacterial om biogenesis in general.
authors' contributions
tl carried out the experiments for figures  <dig>   <dig>   <dig>   <dig> and 6a-c and drafted the initial manuscript.
mk participated in the design of the studies and performed experiments for 6d and provided intellectual input and editing assistance for the manuscript.
xy and up provided the data for figure  <dig>  da conceived of the study, participated in its design and coordination, and helped to draft and edit the manuscript.
all authors read and approved the final manuscript.

 borrelia burgdorferi, the spirochetal agent of lyme disease, possesses a dual-membraned architecture, which is composed of a peptidoglycan layer associated with the inner membrane and an outer membrane. in gram-negative bacteria, cytoplasmic precursor outer membrane proteins are synthesized with an amino-terminal signal peptide sequence, which typically targets a protein for sec-mediated translocation. after the precursor omp crosses the im through the secyeg translocase, the signal peptide is cleaved by signal peptidase i, and the mature protein is subsequently released into the periplasmic space. once in the periplasm, the unfolded omp is bound by chaperones that help direct the omp to the om for proper folding and membrane insertion. until recently, these latter steps of periplasmic omp trafficking and om assembly have remained largely uncharacterized. in however, tommassen and coworkers identified an essential β-barrel omp whose function is dedicated to the proper om-assembly of most known omps. this protein, now known as bama, is evolutionarily well-conserved since putative orthologs can be found in all known diderm bacteria, as well as in dual-membraned eukaryotic organelles, such as mitochondria and chloroplasts. the functional importance of bama was illustrated when researchers discovered that bama was essential for the viability of both n. meningitidis and e. coli, and that its depletion resulted in dramatically decreased levels of properly-inserted omps in the om of both organisms. in e. coli, combined genetic and biochemical studies have now revealed that bama exists in a multiprotein om complex, termed the beta-barrel assembly machine. this complex is composed of the om-imbedded bama protein and four om-anchored accessory lipoproteins, termed bamb, bamc, bamd, and bame. more recent studies have revealed that all of the bam components are important at some level for omp assembly and/or for the stability of the bam complex. the bamb lipoprotein interacts directly with bama within the complex, and this association is independent of the other bam lipoproteins. bamb is thought to be an important scaffolding protein for the bam complex, and although bamb deletion mutants are viable, they have reduced levels of various omps. bamc- and bame-null strains have relatively mild omp assembly defects; however, they both show moderate om permeability defects, and biochemical studies show that their presence in the complex is important for the bama-bamd interaction. the bamd protein, however, is essential for cell viability, and depletion of bamd causes a phenotype similar to that observed in bama mutants. additionally, bamd is the most evolutionarily conserved lipoprotein in the bam complex. like bama, bamd orthologs are predicted to be present in all diderm bacteria, and they are proposed to contain conserved tetratricopeptide repeat domains which have been shown to function in protein-protein interactions. bam complexes have now been characterized from other gram-negative bacteria, such as n. meningitidis and caulobacter crescentus. in n. meningitidis, co-immunoprecipitation studies showed that, along with bama and bamd, the bam complex contains bamc and bame, as well as an additional protein component, rmpm. a bamb homolog, however, was not identified in n. meningitidis. the bam complex in c. crescentus was recently reported to contain all of the known bam lipoproteins except bamc, but includes an additional lipoprotein termed pal, which contains an ompa-type peptidoglycan binding domain that is similar to rmpm. these studies suggest that bacterial bam complexes likely contain not only conserved orthologs and proteins with conserved structural motifs, such as bamd, but also non-conserved proteins which may provide specific requirements for omp assembly in a particular species of bacteria. in b. burgdorferi, the only member of the bam complex identified to date is bb which we previously determined to be a structural and functional b. burgdorferi bama ortholog. in the present study, we examined whether b. burgdorferi bama, like other known bama proteins, exists as a member of a multiprotein om complex. we report that native b. burgdorferi bama forms high molecular-weight om complexes and that bama co-immunoprecipitates specifically with two putative b. burgdorferi lipoproteins, bb and bb we also demonstrate that depletion of bama, using an iptg-regulated b. burgdorferi mutant, results in loss of bb0324-bb interactions, suggesting that the lipoproteins do not associate without the presence of bama. additionally, we determined that both bb and bb are om-anchored, and are localized to the inner leaflet of the om. while sequence analysis strongly suggests that bb is a bamd ortholog containing tpr domains similar to those predicted for the n. meningitidis and e. coli bamd lipoproteins, bb did not have significant sequence homology to any other known bam components. the combined results suggest that b. burgdorferi contains fewer proteins in its bam complex, which is likely reflective of its distinct evolutionary phylogeny and unique om ultrastructure. methods bacterial strains and growth conditions borrelia burgdorferi strain b31-mi, strain b31-a, strain b31-a3-lk, and strain flacp-795-lk were cultivated at 34°c in barbour-stoenner-kelly liquid medium containing 6% heat-inactivated rabbit serum. the b31-a strain was supplemented with kanamycin, and the b31-a3-lk strain was supplemented with kanamycin and gentamicin. strain flacp-795-lk was supplemented with μg/ml streptomycin, in addition to kanamycin and gentamycin. strain flacp-795-lk was also cultivated in mm or mm isopropyl-β-d-thiogalactopyranoside, as indicated. isolation of b. burgdorferi outer membrane vesicles and protoplasmic cylinders for blue native page and cellular localization assays, b. burgdorferi strain b31-a outer membranes and protoplasmic cylinders were isolated by discontinuous sucrose density gradient centrifugation followed by a continuous sucrose density gradient centrifugation protocol as previously described. blue native page analysis b. burgdorferi strain b31-a om complexes were analyzed by bn-page under native conditions as described. briefly, the isolated om preparations were resuspended in m aminocaproic acid, mm bis-tris and β-dodecyl maltoside. the protein solution was incubated for min on ice and centrifuged at × g for min, and the resulting supernatant was separated using a 5-14% gradient polyacrylamide gel at 4°c. the protein migration pattern in the bn gel was analyzed visually, or electrophoretically transferred to nitrocellulose for anti-bama immunoblot analysis, as described below. sds-page and immunoblot analyses for denaturing page and immunoblots, protein samples were prepared and separated by sds-page, followed by electrophoretic transfer to nitrocellulose membranes, as described previously. for flab immunoblots, membranes were probed with a 1: dilution of rabbit anti-flab antisera, followed by incubation with a 1: dilution of horseradish peroxidase -conjugated goat anti-rabbit secondary antibodies. subsequent chromogenic development was performed using 4-chloronapthol and hydrogen peroxide. for all other immunoblots, enhanced chemiluminescence was used, as described by kenedy et al.. after primary antibody incubation, membranes were incubated in a 1: dilution of goat anti-rat, goat anti-rabbit, or goat anti-mouse secondary antibodies. washed membranes were subsequently developed using supersignal west pico ecl reagent according to manufacturer's instructions. sequence analyses and alignments the n. meningitidis bamd protein sequence was used to search the b. burgdorferi b peptide database using the j. craig venter comprehensive microbial resource blast server. bb and bb hydrophilicity analyses were performed using macvector version sequence analysis software according to the method of kyte and doolittle, and prediction of putative signal peptides and the canonical lipoprotein signal peptidase ii cleavage sites was performed using the signalp server and the lipop server, respectively. bb tetratricopeptide repeat domains were predicted using tprpred and by comparison with the original published tpr consensus sequence. the predicted tpr-containing regions from nm-bamd, e. coli bamd, and bb were aligned using the macvector version multiple sequence alignment program, followed by manual editing using the jalview multiple alignment editor. generation and purification of recombinant proteins to generate bb bb and bb recombinant proteins, dna sequences corresponding to each full-length mature protein lacking the putative signal peptide were pcr-amplified from b genomic dna. primers used for amplification of the bb dna region are as follows: 5'-gcgggatccttaacaaaagaaactccttatgg-3', and 5'-ttttttattattttctattttatttaata-3'. primers used for amplification of the bb dna region are as follows: 5'-gcgggatccgctaatcttgatcaaataaaaaatc-3' and 5'-gcggaatccttaagggtttttattgtccttttc-3'. primers used for amplification of the bb dna region are as follows: 5'-aagaattctcaagcgaatccatattttcac-3', and 5'-aactcgagttattctttagttaattttctgttttcca-3'. the bb bb and bb amplicons were ligated into the topo-ta pbad/thio vector, the pgex- t- vector, and the pgex-6p vector, respectively. the resulting constructs were transformed into electrocompetent e. coli dh5α cells, and prior to protein purification, selected transformants were verified to contain the correct insert sequence by restriction digest and by nucleotide sequence analysis. for protein purification, recombinant bb was purified as a thioredoxin fusion using a solubilization protocol described previously. recombinant bb and bb were purified as glutathione-s-transferase fusion proteins and cleaved free of the gst moiety using procedures described previously. antibodies antibodies to the bb and bb recombinant proteins were generated in rats as previously described. rabbit anti-bb antibodies were described elsewhere. rat anti-bama antibodies were generated previously, and mouse anti-lp antibodies were also generated as described previously. mouse anti-oppaiv antibodies were generously provided by drs. justin radolf and melissa caimano, university of connecticut health center, farmington, ct. rabbit anti-flab, rat anti-thio, rat anti-ospa, and rat anti- antibodies were generated as previously described. all animal procedures were approved by the oklahoma university health sciences center institutional animal care and use committee. cell lysate preparation and co-immunoprecipitation for each co-ip sample, cell lysates were prepared by using mid-log phase cultures of b. burgdorferi strain b31-mi, b31-a3-lk, or flacp-795-lk. cells were centrifuged at × g for min and subsequently washed four times in pbs. prior to cell lysis for co-ip, washed cells from each culture condition were subjected to anti-bama immunoblot analysis to verify the regulatable bama phenotype. for co-ip experiments, cell pellets were solubilized and lysed by resuspension in 1× bugbuster reagent. the solubilized cell solution was supplemented with μl lysonase bioprocessing reagent and μl of protease inhibitor cocktail per co-ip sample, and the mixture was subsequently rocked at room temperature for min. finally, the cell debris was pelleted at × g for min at 4°c, and the supernatant was used for the co-ip experiments. co-ips were performed using the sigma protein g immunoprecipitation kit according to manufacturer's instructions, with the following modifications: 1) the 1× and × ip buffers were supplemented with % triton x- and 2) prior to immunoprecipitation, the lysates were pre-cleared overnight to reduce background binding. after immunoprecipitation, bound proteins were eluted in μl final sample buffer, subjected to sds-page, and analyzed by silver stain according to the procedure of morrissey, or by immunoblot, as described above. for protein identification, excised sds-page gel bands were submitted to the molecular biology-proteomics facility for tryptic digestion and hplc-ms/ms analysis, followed by mascot database search for protein identification. triton x- phase partitioning to determine whether bb and bb have the amphipathic properties of typical lipid-modified proteins, b. burgdorferi strain b31-mi cells were harvested and phase-partitioned as described previously. proteinase k surface accessibility to determine whether bb and bb contain surface-exposed regions, pk experiments were performed as previously described. briefly, spirochetes were harvested at × g, washed four times in 1× pbs, and the washed cells were either mock-treated or pk-treated; sigma chemical co.) for one hour at rt. after addition of pmsf, samples were prepared for sds-page and immunoblot analysis, as described above. to verify that bb and bb were not resistant to pk activity, cell membranes were disrupted as previously described. cells were pelleted at × g, washed, and incubated for m in μl pk lysis buffer containing mm tris, % triton x- %, β-mercaptoethanol, and μg of lysozyme. samples were then incubated in the absence or presence of pk. the reaction was stopped with pmsf and prepared for immunoblot as indicated above.  b. burgdorferi bama forms multi-protein complexes in the om previously, we performed a structural and functional characterization of the om-localized b. burgdorferi bama protein. since other bama orthologs are known to exist in a hetero-oligomeric protein complex, we wanted to determine if native b. burgdorferi bama could be detected in high molecular weight om complexes. to perform this assay, we isolated om vesicles from b. burgdorferi strain b31-a and subjected the om sample to one-dimensional blue native -page, followed by anti-bama immunoblot analysis. results from the immunoblot showed multiple protein bands between the and kda mw markers, with two prominent bands that resolved at approximately kda and kda. in addition, samples from the om fraction and from the protoplasmic cylinder fraction were separated by denaturing sds-page and immunoblotted against the periplasmic flab protein to verify om purity. these results demonstrate that native b. burgdorferi bama is present in multiple high molecular weight om complexes, which may indicate that bama associates with other om-localized proteins or protein complexes. in silico analysis of b. burgdorferi bam orthologs to identify possible components of the b. burgdorferi bam complex, our initial approach was to search the b. burgdorferi protein database for putative orthologs of the e. coli bam lipoproteins, bamb, bamc, bamd, and bame. although protein blast searches using each of the bam proteins provided no significant sequence matches, blastp searches using each of the n. meningitidis bam lipoproteins as a search query yielded one b. burgdorferi protein. this protein, encoded by open reading frame bb has significant similarity to the n. meningitidis bamd lipoprotein. bb is a 119-residue polypeptide of unknown function that is predicted to contain an n-terminal signal peptide with a signal peptidase ii lipoprotein modification and processing site as determined by a combination of hydrophilicity, signalp, and lipop computer analyses as described in methods. the identification of a canonical lipoprotein processing and modification site strongly suggested that bb is the b. burgdorferi lipoprotein bamd ortholog. comparative sequence analyses indicate that bb aligns with the n-terminus of n. meningitidis bamd, such that almost the entire bb amino acid sequence aligns with the first residues of the 267-residue n. meningitidis bamd protein. importantly, this region of n. meningitidis bamd is predicted to contain two conserved tpr sequences, which are also predicted to exist in bb. the tpr sequence is a degenerate 34-residue consensus sequence that forms a helix-turn-helix secondary structure element, and such motifs are known to be involved in protein-protein interactions. only a few positions within the consensus tpr sequence are highly conserved, and therefore individual tprs can vary substantially at the primary sequence level. e. coli bamd is also predicted to contain n-terminal tpr sequences that can be aligned with those of bb and n. meningitidis bamd. the combined results from the protein blast searches and the sequence alignment analyses further support the contention that bb is a b. burgdorferi bamd ortholog. b. burgdorferi bama forms a complex with bb and bb0028 to identify additional bam accessory proteins, we next performed anti-bama co-immunoprecipation experiments. since our bama antisera was generated against recombinant bama proteins with a 5' thioredoxin fusion, we utilized anti-thioredoxin antisera as our negative control antibody for the co-ip assays. when we analyzed the immunoprecipitated proteins by coomassie stain, we detected an ~ kda band that was prominent in the bama-precipitated samples as compared with the anti-thio control. to determine the identity of the protein contained within the kda band identified, this region were subsequently excised, trypsin-digested, and subjected to lc-ms/ms analysis. after mascot database search, the unknown protein from the bama co-ip was identified as a 349-residue polypeptide encoded by the b. burgdorferi orf bb this protein was not identified in the band extracted from the thio co-ip elution, suggesting that it co-immunoprecipitated specifically with bama. similar to bb computer analyses of the bb protein indicated that it contains a signal peptide with a consensus signal peptidase ii lipoprotein modification and processing site, suggesting that bb is also a b. burgdorferi lipoprotein. interestingly, blastp analyses failed to identify any bb conserved domains or any significant protein matches outside of the borrelia genus. to determine if bb and bb are bam accessory proteins that specifically associate with bama, we performed anti-bama, anti-bb and anti-bb immunoprecipitation experiments. the immunoprecipitation assays were then subjected to immunoblot analysis with specific antibodies to bama, bb and bb. as shown in figure b. burgdorferi bama co-immunoprecipitated bb and bb additionally, bb antibodies co-immunoprecipitated bama and bb while bb antibodies co-immunoprecipitated bama and bb. however, none of the three proteins were detected in the thio co-immunoprecipitation experiment control sample. additionally, when immunoprecipitated proteins from all experiments were probed with antibodies to lp, which is a lipoprotein known to be localized to the inner leaflet of the borrelial om, there was no detectable co-immunoprecipitation of lp. the lp immunoblot data demonstrated that an unrelated lipoprotein was not immunoprecipitated, which helped to confirm the specificity of the immunoprecipitation assays. the combined data indicate that bama physically associates with bb and bb bama expression is required for interaction with bb and bb0028 although the above co-immunoprecipitation data indicated that bb and bb specifically interact with bama, it was still unclear if bb and bb interacted with each other. we therefore wanted to determine if native bb and bb form their own complexes in b. burgdorferi, or if they interact only in the presence of bama as constituents of the larger bam complex. to examine this issue, we utilized the regulatable b. burgdorferi strain that was engineered to express an iptg-inducible chromosomal bama gene. we previously illustrated that in low concentrations of iptg, total cellular levels of bama protein were dramatically reduced, and as a result, b. burgdorferi om preparations contained reduced levels of omps. by performing immunoprecipitation experiments with flacp-795-lk cultivated in a low concentration or high concentration of iptg, we were able to observe the effects of bama depletion on the bama-bb0324-bb interactions. as shown by immunoblot analysis, bama depletion resulted in less bb being immunoprecipitated by bb antibodies as compared to the parental b31-lk strain. similarly, bama depletion also resulted in less bb being immunoprecipitated by bb antibodies as compared to the parental b31-lk strain. however, it should be noted that there was no detectable difference in the levels of bb or bb expression after bama depletion. these data indicate that the loss of bama did not affect the amount of bb or bb protein being expressed in the flacp-795-lk or parental lk strains. consistent with this observation, whole-cell lysates from the flacp-795-lk strain grown in mm or mm iptg and the parental lk strain contained similar levels of bb and bb as shown in figure 5c. the combined data revealed that bama depletion does not affect expression of bb or bb but instead causes a decrease in the amount of bb that is immunoprecipitated with bb and also causes a decrease in the amount of bb that is immunoprecipitated by bb thus, the bb and bb interactions with bama appear to be severely affected by the loss of bama expression, which also indicates that they require bama in order to efficiently form the larger bam complex. bb and bb are outer membrane-associated subsurface proteins currently, all known accessory proteins of e. coli bam complex, besides bama, are lipoproteins anchored to the inner leaflet of the om. therefore, we next examined whether both bb and bb are localized to the periplasmic leaflet of the om. to begin our cellular localization assays, we first performed triton x- phase partitioning studies with b. burgdorferi cells to determine if bb and bb are amphiphilic. as shown in figure 6a, both bb and bb partitioned exclusively into the detergent-enriched fraction, which is characteristic of amphiphilic proteins. additionally, a known membrane-anchored lipoprotein and a soluble protein were used as detergent phase and aqueous phase controls, respectively. we next examined the cellular location of bb and bb to confirm their presence in the om. as shown in figure 6b, bb and bb were detected in the isolated oms of b. burgdorferi, demonstrating that both proteins are localized to the om. cell fractions were also probed with antibodies to the om-localized bama and lp proteins, as well as to the im-anchored oppaiv lipoprotein, to verify om specificity and purity. to determine if bb or bb are anchored to the periplasmic leaflet of the om, we next incubated whole b. burgdorferi cells in the presence or absence of proteinase k. these experiments revealed that there was no difference between mock- or pk-treated samples when probed with anti-bb or anti-bb indicating neither protein is surface-exposed. as controls for pk activity and om integrity, lysates from the mock- and pk-treated cells were also probed with antibodies against the surface-localized bb protein and the periplasmic flab protein, respectively. importantly, when cell membranes were disrupted prior to pk treatment, bb and bb were both degraded in the presence of pk confirming that these proteins are subsurface proteins and not intrinsically resistant to pk activity. discussion current working model for the b. burgdorferi bam complex the bacterial beta-barrel assembly machine, or bam, is a multiprotein om complex that is composed of the essential integral omp bama, as well as a number of conserved and nonconserved accessory lipoproteins that are anchored to the inner leaflet of the om. to date, few bam complexes have been studied, and since only those from proteobacteria have been characterized, it is yet to be determined what elements of various bam complexes are conserved between different bacterial groups. in this study we report that the diderm spirochete, b. burgdorferi, also contains an om-localized bam complex, which is composed of bama and at least two accessory lipoproteins, bb and bb additionally, co-immunoprecipitation experiments using a bama regulatable b. burgdorferi mutant strain indicated that bama is required for efficient association of bb and bb further cellular localization assays indicated that both bb and bb are om anchored subsurface lipoproteins, although only bb is predicted to be an ortholog to a currently identified bam accessory lipoprotein. as determined from our initial immunoprecipitation experiments with b. burgdorferi strain b31-mi, the bb and bb proteins associate specifically with bama as a heterooligomeric om protein complex. additional data from the bama regulatable mutant provided further insight into the bama-bb0324-bb interactions. when the bama iptg-regulatable strain was cultivated in decreasing concentrations of iptg it was immediately apparent that the bama and bb0324/bb associations were dramatically affected as compared to the parental, wildtype strain b31-lk. although these data are insufficient to provide conclusions on the detailed organization of the bam complex, it is apparent that bb and bb do not efficiently co-immunoprecipitate each other when bama is depleted. these data suggest that bb and bb do not readily associate in b. burgdorferi without the presence of bama, and that they likely come together only to form the functional bam complex. however, the molecular architecture of the b. burgdorferi bam complex is still unknown, and it is unclear what specific interactions create the bama-bb0324-bb complex. in our model, bb and bb may associate indirectly through individual direct contacts with bama. alternatively, bb and bb may bind directly with each other, where only one of them binds bama. further experiments using b. burgdorferi bb and bb partial and/or full deletion mutants should help to clarify the molecular architecture and binding partners within the bam complex. potential interactions between the periplasmic bama polypeptide transport-associated domains and the bb and bb accessory lipoproteins the periplasmic n-terminus of bama contains five repeating potra domains, which have been designated p1-p, that are responsible for the bama-accessory lipoprotein interactions. it has been shown in e. coli that deleting any of the potra domains other than p results in disruption of accessory lipoprotein interactions. similar to the e. coli bam accessory lipoproteins, it is likely that bb and bb also associate with bama through potra domain contacts. future co-immunoprecipitation experiments with different b. burgdorferi bama potra domain mutants as well as bb and/or bb mutants will help clarify exactly which potra domains are needed for bb and bb accessory protein binding. bb is a putative bamd ortholog with a truncated c-terminus blastp searches and sequence analyses indicate that the bb protein is a putative b. burgdorferi bamd ortholog. bamd is predicted to be ubiquitous in diderm bacteria, and it appears to be both essential for cell survival and central to the function of the bam complex, as demonstrated in e. coli and in n. meningitidis. it is predicted that all bamd orthologs possess n-terminal tpr domains, and in e. coli and n. meningitidis, bamd appears to contain two. although such structural features are still predicted for e. coli and n. meningitidis, a recently-determined crystal structure from the rhodothermus marinus bamd confirms the presence of tpr domains within this protein. although tprs form a characteristic helix-loop-helix structure, their propensity for sequence variation is likely a reason that we were initially unable to identify a bamd ortholog in b. burgdorferi, even though bb contains consensus tpr sequences. in addition, bb is considerably smaller than the bamd proteins currently identified in other bacteria. the putative borrelial bamd lipoprotein has a predicted mw of ~ kda, which is less than half the size of proteobacterial bamd proteins from e. coli, n. meningitidis, and c. crescentus. interestingly, it has been proposed that the tpr domain region fulfills the major functional requirements for bamd, and that the tprs may be the only essential feature of the bamd proteins. this idea has been discussed in previous reports, and it originates from the discovery of a viable transposon mutant of the neisseria gonorrhoeae bamd protein, also known as coml. as noted by volokhina et al., this truncated mutant contains only amino acids of the mature 267-residue protein, indicating that the coml n-terminus, which comprises the tpr motifs, is sufficient for viability. although viable, the coml mutant displayed reduced colony size and was deficient in transformation competency. similarly, an e. coli transposon mutant lacking only the c-terminal amino acids of the 245-residue bamd protein was also viable but had greatly reduced bamd function. these phenotypic characteristics suggest that the bamd c-terminus, although nonessential, fulfills some functional requirement for neisseria and for e. coli that is either unnecessary for b. burgdorferi, or is provided by a different protein. interestingly, it has been shown that the c-terminus of the e. coli bamd binds bamc and bame, and is therefore important for the stability of this part of the bam complex. thus, a truncated b. burgdorferi bamd may simply be the result of this organism having no requirement for an extended c-terminal region to interact with additional accessory lipoproteins such as bamc or bame, since we were not able to identify other accessory lipoproteins in b. burgdorferi.  in the current study, we have identified two accessory components of the b. burgdorferi bam complex. based on the knowledge gained from studying other proteobacterial organisms, it is possible that b. burgdorferi contains one or more other bam accessory lipoprotein components in addition to bb and bb that are still unidentified. as indicated by bn-page in figure 1a, multiple high molecular weight complexes containing bama are present between approximately kda and over kda. these data accommodate the possibility that additional protein species may be co-migrating with bama, especially since the smallest of the two most prominent bands, which migrates at ~ kda, has an approximate mw that is larger than the expected mw of bama, bb and bb combined. alternatively, these large protein complexes may contain multiple copies of the same protein, such as multiple bb molecules, and/or be homo-oligomers of the entire bam complex. it should be noted, however, that b. burgdorferi contains a relatively small number of integral omps compared to e. coli; hence, it may require a less complicated bam complex system for omp assembly. indeed, silhavy and coworkers proposed that the major function of the nonessential e. coli bamb, bamc, and bame lipoproteins is most likely to increase efficiency of omp assembly, or to stabilize the complex, since individual mutants were viable and showed relatively mild assembly defects. it is, therefore, possible that an om with a more limited omp repertoire, such as that of b. burgdorferi, does not necessitate additional bam complex members to provide the essential functions for complete om biogenesis. in this regard, it is tempting to speculate that the b. burgdorferi bam constituents identified here constitute a "minimal" bacterial bam complex, which can now be further studied as a model system to not only further our understanding of b. burgdorferi om biogenesis, but also to contribute to our current knowledge of bacterial om biogenesis in general. authors' contributions tl carried out the experiments for figures and 6a-c and drafted the initial manuscript. mk participated in the design of the studies and performed experiments for 6d and provided intellectual input and editing assistance for the manuscript. xy and up provided the data for figure da conceived of the study, participated in its design and coordination, and helped to draft and edit the manuscript. all authors read and approved the final manuscript. 
cellular localization assays indicate that bb and bb are om-associated subsurface lipoproteins, and in silico analyses indicate that bb is a putative bamd ortholog. although the mechanism of omp biogenesis is still not well-understood, recent studies have indicated that a heterooligomeric om protein complex, known as bam is required for proper assembly of omps into the bacterial om. similar to gram-negative bacteria, the outer membrane of the pathogenic spirochete, borrelia burgdorferi, contains integral om-spanning proteins, as well as membrane-anchored lipoproteins. in the current study, we report on the identification of two additional protein components of the b. burgdorferi bam complex, which were identified as putative lipoproteins encoded by orfs bb and bb biochemical assays with a bama-depleted b. burgdorferi strain indicate that bb and bb do not readily interact with the bam complex without the presence of bama, suggesting that the individual b. burgdorferi bam components may associate only when forming a functional bam complex. we previously identified and characterized the essential β-barrel omp component of this complex in b. burgdorferi, which we determined to be a functional bama ortholog. the combined data suggest that the bam complex of b. burgdorferi contains unique protein constituents which differ from those found in other proteobacterial bam complexes. the novel findings now allow for the b. burgdorferi bam complex to be further studied as a model system to better our understanding of spirochetal om biogenesis in general.
 similar to gram-negative bacteria, the outer membrane of the pathogenic spirochete, borrelia burgdorferi, contains integral om-spanning proteins, as well as membrane-anchored lipoproteins. although the mechanism of omp biogenesis is still not well-understood, recent studies have indicated that a heterooligomeric om protein complex, known as bam is required for proper assembly of omps into the bacterial om. we previously identified and characterized the essential β-barrel omp component of this complex in b. burgdorferi, which we determined to be a functional bama ortholog.  in the current study, we report on the identification of two additional protein components of the b. burgdorferi bam complex, which were identified as putative lipoproteins encoded by orfs bb and bb biochemical assays with a bama-depleted b. burgdorferi strain indicate that bb and bb do not readily interact with the bam complex without the presence of bama, suggesting that the individual b. burgdorferi bam components may associate only when forming a functional bam complex. cellular localization assays indicate that bb and bb are om-associated subsurface lipoproteins, and in silico analyses indicate that bb is a putative bamd ortholog.  the combined data suggest that the bam complex of b. burgdorferi contains unique protein constituents which differ from those found in other proteobacterial bam complexes. the novel findings now allow for the b. burgdorferi bam complex to be further studied as a model system to better our understanding of spirochetal om biogenesis in general. 