Bacillus cereus efflux protein BC3310 - A multidrug transporter of the unknown major facilitator family, UMF-2

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Abstract

Phylogenetic classification divides the major facilitator superfamily (MFS) into 82 families, including 25 families that are comprised of transporters with no characterized functions. This study describes functional data for BC3310 from Bacillus cereus ATCC 14579, a member of the "unknown major facilitator family-2" (UMF-2). BC3310 was shown to be a multidrug efflux pump conferring resistance to ethidium bromide, SDS and silver nitrate when heterologously expressed in Escherichia coli DH5a ΔacrAB. A conserved aspartate residue (D105) in putative transmembrane helix 4 was identified, which was essential for the energy dependent ethidium bromide efflux by BC3310. Transport proteins of the MFS comprise specific sequence motifs. Sequence analysis of UMF-2 proteins revealed that they carry a variant of the MFS motif A, which may be used as a marker to distinguish easily between this family and other MFS proteins. Genes orthologous to bc3310 are highly conserved within the B. cereus group of organisms and thus belong to the core genome, suggesting an important conserved functional role in the normal physiology of these bacteria.

Figures

  • TABLE 1 | Primers used in this study.
  • FIGURE 1 | Dendrogram comparing BC3310 from Bacillus cereus ATCC 14579 with orthologous proteins and other multidrug transporters from the DHA1 and DHA3 families. BC3310 from B. cereus ATCC 14579 (UniProt accession number: Q81B77; bold font) and orthologous proteins from B. cereus ATCC 10987 (Q734U9), B. cereus ATCC 10876 (C2N377), B. anthracis str. Ames (Q81N75), B. cereus ssp. cytotoxis (A7GQF7), B. weihenstephanensis (A9VLS6), B. mycoides (C3AET4), B. pseudomycoides (C3BM93), Geobacillus sp. Y4.1MC1 (E3IFM2), Halobacillus halophilus (I0JJA0), B. subtilis (O34929), Listeria innocua (Q92AX8), Listeria monocytogenes (S5JWD1), Geobacillus kaustophilus (Q5L2X3), Lysinibacillus sphaericus (B1HUQ2), Exiguobacterium sibiricum (B1YK36), Anoxybacillus flavithermus (B7GFW5), M. caseolyticus (B9E839), Brevibacillus brevis (C0ZL32), Escherichia coli (P21503), and DHA1 proteins from Lactococcus lactis (Q48658), B. subtilis (Q797E3, O34546, P39843, P33449), Pseudomonas aeruginosa (P32482) and DHA3 proteins from Streptococcus pyrogenes (P95827), B. subtilis (P39642, O31600, P42112), B. clausii (Q5WAS7), Pseudomonas syringae (Q887F7), Clostridium perfringens (Q46305) and the sugar transporter AraE from B. subtilis (P96710) as an outgroup were used to build the tree. Posterior probability values are shown at each node and the bar represents the expected number of amino acid substitutions per site. The seven protein sequences marked with ∗ were aligned in Figure 5.
  • TABLE 2 | Minimal inhibition concentration (MIC) of E. coli DH5α acrAB expressing BC3310 (pbc3310) compared to empty vector control (pTTQ18) and Bacillus cereus ATCC 14579 bc3310 ( bc3310) compared to B. cereus ATCC 14579 (wild type).
  • FIGURE 2 | Purification of BC3310-RGSHis6 . BC3310 was expressed with RGS(His)6-tag in E. coli and inner and outer membranes were separated. BC3310-RGSHis6 was solubilized with DDM and purified by immobilized metal affinity chromatography (IMAC). The SDS-PAGE was loaded as follows and stained with Coomassie Blue: (1) molecular weight marker; (2) inner membrane fraction; (3) solubilized protein; (4) unsolubilized protein; (5) flow-through after IMAC binding; (6) eluted protein. (7) Western blot detection of eluted BC3310 protein after affinity chromatography using an antibody to the RGSHis6 epitope.
  • FIGURE 4 | Uncoupler-sensitive efflux of ethidium associated with expression of BC3310. Accumulation of ethidium bromide after 30 min was measured in IPTG-induced E. coli DH5α acrAB host cells either expressing bc3310 (pBC3310, dark gray) or not using an empty vector control (pTTQ18, light gray) without (-CCCP) and with addition of CCCP (+ CCCP). Values are means of four independent experiments and error bars indicate standard deviations, ∗p < 0.01, unpaired Student’s t-test.
  • FIGURE 3 | Circular dichroism analysis of purified BC3310-RGSHis6 protein. The analysis was performed at 1 nm intervals over 270–190 nm with a scan rate of 10 nm min−1. The spectrum represents an averaged accumulation of 20 scans, from which the buffer contribution was subtracted.
  • FIGURE 5 | Multiple sequence alignment of BC3310 and its homologs. BC3310 from B. cereus ATCC 14579 (UniProt accession number: Q81B77) was aligned using MUSCLE with orthologs from Brevibacillus brevis (C0ZL32), Exiguobacterium sibiricum (B1YK36), Listeria innocua (Q92AX8), B. subtilis (O34929), Geobacillus kaustophilus (Q5L2X3) and Halobacillus halophilus (I0JJA0). Shading corresponds to >80% (dark blue), >60% (blue), >40% (light blue), and ≤40% (white) amino acid identity, respectively. The conserved acidic residue in transmembrane region 4 is displayed in red. Transmembrane regions have gray bars under and conserved MFS motifs are depicted above the sequence.
  • FIGURE 6 | Western blot detection of the BC3310 protein and its mutants in Escherichia coli. E. coli DH5α acrAB was transformed with empty pTTQ18 vector plasmid or pTTQ18 with the indicated insert; wild type BC3310 with his-tag and mutants D105N, D105A, and D105E under an IPTG inducible promoter. The cells were grown and prepared for SDS-PAGE as described in Section “Materials and Methods”. Numbers indicate: (1) empty pTTQ18 vector; (2) BC3310 wild type; (3) D105N mutant; (4) D105A mutant; (5) molecular weight marker; (6) D105E mutant. BC3310 and its mutants migrate at ∼30 kDa.

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Kroeger, J. K., Hassan, K., Vörös, A., Simm, R., Saidijam, M., Bettaney, K. E., … Kolstø, A. B. (2015). Bacillus cereus efflux protein BC3310 - A multidrug transporter of the unknown major facilitator family, UMF-2. Frontiers in Microbiology, 6(OCT). https://doi.org/10.3389/fmicb.2015.01063

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