Borrelia burgdorferi locus BB0795 encodes a BamA orthologue required for growth and efficient localization of outer membrane proteins

Abstract

The outer membrane (OM) of the pathogenic diderm spirochete, Borrelia burgdorferi, contains integral beta-barrel outer membrane proteins (OMPs) in addition to its numerous outer surface lipoproteins. Very few OMPs have been identified in B. burgdorferi, and the protein machinery required for OMP assembly and OM localization is currently unknown. Essential OM BamA proteins have recently been characterized in Gram-negative bacteria that are central components of an OM beta-barrel assembly machine and are required for proper localization and insertion of bacterial OMPs. In the present study, we characterized a putative B. burgdorferi BamA orthologue encoded by open reading frame bb0795. Structural model predictions and cellular localization data indicate that the B. burgdorferi BB0795 protein contains an N-terminal periplasmic domain and a C-terminal, surface-exposed beta-barrel domain. Additionally, assays with an IPTG-regulatable bb0795 mutant revealed that BB0795 is required for B. burgdorferi growth. Furthermore, depletion of BB0795 results in decreased amounts of detectable OMPs in the B. burgdorferi OM. Interestingly, a decrease in the levels of surface-exposed lipoproteins was also observed in the mutant OMs. Collectively, our structural, cellular localization and functional data are consistent with the characteristics of other BamA proteins, indicating that BB0795 is a B. burgdorferi BamA orthologue.

Fig. 1

Domain organization and predicted structural features of BB0795. A. Schematic diagram of the BB0795 protein. The N-terminal region is composed of a putative signal peptide (amino acids 1–27; indicated by a black box) followed by five putative POTRA domains, indicated as P1–P5 (amino acids 28–433). The BB0795 C-terminal region (amino acids 434–821) is composed of a putative β-barrel domain. B. Comparison of the B. burgdorferi putative POTRA domains P1–P5 with E. coli POTRA domain P2. The canonical β1-α1-α2-β2-β3 POTRA domain secondary structure is predicted to be conserved between all of the putative BB0795 POTRA domains and the known E. coli BamA POTRA domain. Secondary structure predictions were performed as indicated in the Experimental procedures. Regions predicted to be in β-sheet are indicated in blue and regions predicted to be in α-helical conformation are shown in red. At bottom is the consensus secondary structure regions identified in the comparison. C. Predicted topology of the BB0795 β-barrel domain. Amino acids 434–821 of the BB0795 protein (corresponding to the C-terminal putative β-barrel domain) were subjected to the PRED-TMBB β-barrel prediction programme (http://bioinformatics.biol.uoa.gr/PRED-TMBB) (Bagos et al., 2004a,b). Topology results from the Viterbi, N-Best and Posterior decoding algorithms used by PRED-TMBB are indicated as #1, #2 and #3 respectively. Predicted transmembrane regions (red), extracellular domains (blue) and periplasmic domains (green) are indicated for each topology prediction algorithm.

Fig. 2

BB0795 structural model and predicted membrane topology. A. X-ray crystal structure co-ordinates from E. coli BamA POTRA domains 1–4 (pdb co-ordinates: 2qdf) were used to model the structure of the putative BB0795 POTRA domains 1–4. The known E. coli BamA POTRA crystal structure (left) and the predicted structure of POTRA domains 1–4 from BB0795 (right) are displayed in ribbon model format. B. The ribbon models for the known E. coli structure and the predicted BB0795 POTRA domains were overlaid to demonstrate structural similarity. C. Predicted topology of the BB0795 protein. The BB0795 P1–P4 model and a structural model of POTRA domain P5, predicted by the Robetta programme (see Experimental procedures), were combined with the BB0795 β-barrel prediction to create the topographical model displayed. The BB0795 protein is predicted to contain five soluble POTRA domains and an 18-stranded outer membrane β-barrel domain (indicated by anti-parallel arrows) with eight periplasmic and nine surface-exposed loops.

Fig. 3

BB0795 is a surface-exposed OMP. A. BB0795 is amphipathic and partitions into the detergent-enriched phase after Triton X-114 phase partitioning. Whole-cell lysates of B. burgdorferi B31cF were subjected to Triton X-114 phase partitioning, and equal amounts of the detergent-enriched and aqueous phases were subjected to immunoblot analysis with BB0795 antisera. Molecular weight standards, in kDa, are indicated at left. To ensure proper phase separation, a known detergent phase protein and a soluble aqueous phase protein, OspA and BBA57, respectively, were included as controls. B. BB0795 contains surface-exposed epitopes. Whole-cell lysates of B. burgdorferi B31cF cells overexpressing BB0795 were either mock-treated (−) or proteinase K-treated (+) before being immunoblotted with rat anti-β-barrel domain antibodies (left panel), rat anti-POTRA domain antibodies (middle panel) or rabbit anti-FlaB antibodies (right panel). The asterisk in the middle panel indicates the liberated ~47 kDa periplasmic POTRA domain that is protected from protease treatment. An identical membrane transfer was immunoblotted with rabbit anti-FlaB antibodies to ensure equal loading of the mock-treated and proteinase K-treated whole-cell lysates. Molecular weight standards, in kDa, are indicated at left. C. BB0795 is localized to the B. burgdorferi OM. Strains B. burgdorferi B31cF (cF) and the BB0795 overexpressing strain (795-cF) were separated into OM and PC fractions. Whole-cell equivalents of each fraction were subjected to immunoblot analysis with rat anti-BB0795 antibodies. To determine the effect of BB0795 overexpression on the OM levels of other surface-localized proteins, the OM and PC samples were also probed with antisera to various known integral OMPs (p66, BesC and BB0405) and to the surface-exposed lipoprotein CspA. As a control for the fractionation procedure, antisera to the inner membrane-anchored OppAIV lipoprotein was also included to ensure OM fractions were not contaminated with PC components.

Fig. 4

Construction of an IPTG-regulated bb0795 gene in B. burgdorferi B31-A3-LK. A. The native bb0795 promotor was replaced with the IPTG-regulatable promotor, flacp, in the B. burgdorferi B31-A3-LK strain. The pTLflacp-795 construct was composed of a streptomycin resistance cassette fused to flacp and flanked by homologous DNA from bb0794 and bb0795. The construct was inserted upstream of the bb0795 start codon by homologous recombination. B. Insertion of the streptomycin resistance cassette fused to the flacp promoter upstream of bb0795 was verified by PCR using primers BB0794 (KpnI) F and BB0795 (784–762) R (arrowheads). A 1.5 kb amplicon was amplified from the wild-type B. burgdorferi B31-A3-LK strain (lane B31-A3-LK), as expected. Amplification of the mutant flacp-795-LK DNA produced a 3 kb amplicon, consistent with insertion of the streptomycin-flacp insert between the bb0794 and bb0795 genes. A PCR reaction with no template DNA was also included as a negative control. Molecular weight standards, in kb, are indicated at left.

Fig. 5

BB0795 is an essential protein in B. burgdorferi. A. IPTG is required for growth of the B. burgdorferi flacp-795-LK strain. flacp-795-LK cultures were seeded at 3000 organisms per ml and incubated in media containing 0 mM ( ), 0.05 mM ( ) or 1 mM ( ) IPTG in triplicate. The parental B31-A3-LK strain ( ) was seeded in IPTG-deplete media. Spirochetes were enumerated daily by dark-field microscopy for 13 days post inoculation. B. IPTG dose-dependent expression of BB0795 in B. burgdorferi strain flacp-795-LK. Whole-cell lysates of B. burgdorferi flacp-795-LK cultivated in media containing 0.05 mM or 1 mM IPTG were subjected to immunoblot analysis using rat anti-BB0795 antibodies. Lysates were also probed with a LacI monoclonal antibody and FlaB antisera to ensure equivalent amounts of the LacI repressor protein and equivalent amounts of whole-cell lysates were loaded, respectively. Immunoblots of whole-cell lysates of the parental B31-A3-LK strain (grown in IPTG-deplete media) are shown for comparison.

Fig. 6

BB0795 depletion inhibits proper localization of known B. burgdorferi outer membrane proteins and lipoproteins into the OM. OM and PC fractions were isolated from B. burgdorferi flacp-795-LK cultures grown in either 0.05 mM or 1.0 mM IPTG, and from B31-A3-LK cultures grown in IPTG-deplete media. Equivalent amounts of OMs (left panels) and PCs (right panels) from each culture condition were subjected to immunoblot analysis with rat anti-BB0795 antibodies, and with antisera specific to known integral OMPs p66, BesC and BB0405, as well as to the surface lipoproteins CspA and OspA. As a control for the fractionation procedure, antisera to the inner membrane-anchored OppAIV lipoprotein was also included to ensure OM fractions were not contaminated with PC components.