DipA, a pore-forming protein in the outer membrane of Lyme disease spirochetes exhibits specificity for the permeation of dicarboxylates.

Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a single-channel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.

Oms38 is the first identified pore-forming protein in the outer membrane of relapsing fever spirochetes.

Relapsing fever is a worldwide, endemic disease caused by several spirochetal species belonging to the genus Borrelia. During the recurring fever peaks, borreliae proliferate remarkably quickly compared to the slow dissemination of Lyme disease Borrelia and therefore require efficient nutrient uptake from the blood of their hosts. This study describes the identification and characterization of the first relapsing fever porin, which is present in the outer membranes of B. duttonii, B. hermsii, B. recurrentis, and B. turicatae. The pore-forming protein was purified by hydroxyapatite chromatography and designated Oms38, for outer membrane-spanning protein of 38 kDa. Biophysical characterization of Oms38 was done by using the black lipid bilayer method, demonstrating that Oms38 forms small, water-filled channels of 80 pS in 1 M KCl that did not exhibit voltage-dependent closure. The Oms38 channel is slightly selective for anions and shows a ratio of permeability for cations over anions of 0.41 in KCl. Analysis of the deduced amino acid sequences demonstrated that Oms38 contains an N-terminal signal sequence which is processed under in vivo conditions. Oms38 is highly conserved within the four studied relapsing fever species, sharing an overall amino acid identity of 58% and with a strong indication for the presence of amphipathic beta-sheets.

An RND-type efflux system in Borrelia burgdorferi is involved in virulence and resistance to antimicrobial compounds.

Borrelia burgdorferi is remarkable for its ability to thrive in widely different environments due to its ability to infect various organisms. In comparison to enteric Gram-negative bacteria, these spirochetes have only a few transmembrane proteins some of which are thought to play a role in solute and nutrient uptake and excretion of toxic substances. Here, we have identified an outer membrane protein, BesC, which is part of a putative export system comprising the components BesA, BesB and BesC. We show that BesC, a TolC homolog, forms channels in planar lipid bilayers and is involved in antibiotic resistance. A besC knockout was unable to establish infection in mice, signifying the importance of this outer membrane channel in the mammalian host. The biophysical properties of BesC could be explained by a model based on the channel-tunnel structure. We have also generated a structural model of the efflux apparatus showing the putative spatial orientation of BesC with respect to the AcrAB homologs BesAB. We believe that our findings will be helpful in unraveling the pathogenic mechanisms of borreliae as well as in developing novel therapeutic agents aiming to block the function of this secretion apparatus.

Silver- and Coomassie-staining protocols: detection limits and compatibility with ESI MS.

Staining protocols for PAGE have to be sensitive and should not impair further MS analysis of selected samples. In this study, the MS compatibility of different silver- and Coomassie-staining protocols with a nano-LC-MS/MS system was systematically elucidated. Altogether, 13 different silver-staining, 1 imidazole-staining and 2 Coomassie-staining protocols were used and compared to each other for their achieved sequence coverage and their detection sensitivity. Three proteins were used as model proteins (bovine serum albumin, rabbit L-lactate dehydrogenase, bovine milk beta-lactoglobulin) in decreasing concentration (12 pmol down to 30 fmol) and different staining protocols were applied. The conclusion of this study is that two silver-staining protocols (Blum, H. et al.,. Electrophoresis 1987, 8, 93-99 and Shevchenko, A. et al.,. Anal. Chem. 1996, 68, 850-858) combine good sequence coverage and good sensitivity and are recommended for nano-LC-MS/MS analysis.

Ruthenium (II) tris-bathophenanthroline disulfonate is well suitable for Tris-Glycine PAGE but not for Bis-Tris gels.

Pre-cast bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) gels have proven to be very suitable for pre-fractionation for LC-MS/MS analysis due to high reliability and long stability. To visualize proteins within gels fluorescence dyes proved to be a good tradeoff between sensitivity and MS-compatibility. The custom-made ruthenium dye represents a low-cost alternative regarding fluorescence-based protein visualization with high sensitivity. We demonstrate, that this dye is incompatible with Bis-Tris gels, while using Tris-Glycine gels a competitive sensitivity to commercially available stains can be achieved.

Elimination of channel-forming activity by insertional inactivation of the p66 gene in Borrelia burgdorferi.

P66 is a chromosomally encoded 66-kDa integral outer membrane protein of the Lyme disease agent Borrelia burgdorferi exhibiting channel-forming activity. Herein, we inactivated and subsequently complemented the p66 gene in the B31-A (WT) strain. The P66 protein was also inactivated in two other channel-forming protein mutant strains, P13-18 (Deltap13) and Deltabba01, and then compared with the channel-forming activities of wild-type and various p66 mutant strains. We further investigated the ion-selectivity of native, purified P66. In conclusion, we show that the porin activity of P66 is eliminated by insertional inactivation and that this activity can be rescued by gene complementation.

The BBA01 protein, a member of paralog family 48 from Borrelia burgdorferi, is potentially interchangeable with the channel-forming protein P13.

The Borrelia burgdorferi genome exhibits redundancy, with many plasmid-carried genes belonging to paralogous gene families. It has been suggested that certain paralogs may be necessary in various environments and that they are differentially expressed in response to different conditions. The chromosomally located p13 gene which codes for a channel-forming protein belongs to paralog family 48, which consists of eight additional genes. Of the paralogous genes from family 48, the BBA01 gene has the highest homology to p13. Herein, we have inactivated the BBA01 gene in B. burgdorferi strain B31-A. This mutant shows no apparent phenotypic difference compared to the wild type. However, analysis of BBA01 in a C-terminal protease A (CtpA)-deficient background revealed that like P13, BBA01 is posttranslationally processed at its C terminus. Elevated BBA01 expression was obtained in strains with the BBA01 gene introduced on the shuttle vector compared to the wild-type strain. We could further demonstrate that BBA01 is a channel-forming protein with properties surprisingly similar to those of P13. The single-channel conductance, of about 3.5 nS, formed by BBA01 is comparable to that of P13, which together with the high degree of sequence similarity suggests that the two proteins may have similar and interchangeable functions. This is further strengthened by the up-regulation of the BBA01 protein and its possible localization in the outer membrane in a p13 knockout strain, thus suggesting that P13 can be replaced by BBA01.

Site-directed mutagenesis of the greasy slide aromatic residues within the LamB (maltoporin) channel of Escherichia coli: effect on ion and maltopentaose transport.

The 3D-structure of the maltooligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) is known from X-ray crystallography. The 3D structure suggests that a number of aromatic residues (Y6, Y41, W74, F229, W358 and W420) within the channel lumen are involved in carbohydrate and ion transport. All aromatic residues were replaced by alanine-scanning mutagenesis. Furthermore, LamB mutants were created in which two, three, four, five and all six aromatic residues were replaced to study their effects on ion and maltopentaose transport through LamB. The purified mutant proteins were reconstituted into lipid bilayer membranes and the single-channel conductance of the mutants was studied in conductance experiments. The results suggest that all aromatic residues provide some steric hindrance for ion transport through LamB. Highest impact is provided by Y6 and Y41 that are localized opposite Y118, which form the central constriction of the LamB channel. Stability constants for binding of maltopentaose to the mutant channels were measured using titration experiments with the carbohydrate. The mutation of one or several aromatic residue(s) led to a substantial decrease of the stability constant of binding. The highest effect was observed when all aromatic residues were replaced by alanine because no binding of maltopentaose could be detected in such a case. However, binding was again possible when Y118 was replaced by tryptophan. The carbohydrate-induced block of the channel function could be used also for the study of current noise through the different mutant LamB-channels. The analysis of the power density spectra of some of the mutants allowed the evaluation of the on-rate and off-rate constants (k1 and k(-1)) of carbohydrate binding to the binding site inside the channels. The results suggest that both on-rate and off-rate constants were affected by the mutations. For most mutants, k1 decreased and k(-1) increased. The possible influence of the aromatic residues of the greasy slide on carbohydrate and ion transport through LamB is discussed.