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 PF-3084014 manufacturer 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 BB0795, 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, BB0324 and BB0028.
We also demonstrate that depletion of BamA, using an IPTG-regulated B. burgdorferi mutant, results in loss of BB0324-BB0028 interactions, suggesting selleck inhibitor that the lipoproteins do not associate without the presence of BamA. Additionally, we determined that both BB0324 and BB0028 are OM-anchored, and are localized to the inner leaflet of the OM. While HSP990 nmr sequence analysis strongly suggests that BB0324 is a BamD ortholog containing TPR domains similar to those predicted for the N. meningitidis and E. coli BamD lipoproteins , BB0028 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-A3 , strain B31-A3-LK , and Galeterone strain flacp-795-LK  were cultivated at 34°C in Barbour-Stoenner-Kelly (BSK-II) liquid medium  containing 6% heat-inactivated rabbit serum (complete BSK-II). The B31-A3 strain was supplemented with kanamycin (200 μg/mL), and the B31-A3-LK strain was supplemented with kanamycin and gentamicin (40 μg/mL). Strain flacp-795-LK was supplemented with 100 μg/mL streptomycin (selection for the flacp regulatable promoter), in addition to kanamycin and gentamycin. Strain flacp-795-LK was also cultivated in 0.05 mM or 1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG), as indicated. Isolation of B. burgdorferi outer membrane vesicles and protoplasmic cylinders For Blue Native PAGE (BN-PAGE) and cellular localization assays, B.