The SecB chaperone is bifunctional in Serratia marcescens: SecB is involved in the Sec pathway and required for HasA secretion by the ABC transporter

Abstract

HasA is the secreted hemophore of the heme acquisition system (Has) of Serratia marcescens. It is secreted by a specific ABC transporter apparatus composed of three proteins: HasD, an inner membrane ABC protein; HasE, another inner membrane protein; and HasF, a TolC homolog. Except for HasF, the structural genes of the Has system are encoded by an iron-regulated operon. In previous studies, this secretion system has been reconstituted in Escherichia coli, where it requires the presence of the SecB chaperone, the Sec pathway-dedicated chaperone. We cloned and inactivated the secB gene from S. marcescens. We show that S. marcescens SecB is 93% identical to E. coli SecB and complements the secretion defects of a secB mutant of E. coli for both the Sec and ABC pathways of HasA secretion. In S. marcescens, SecB inactivation affects translocation by the Sec pathway and abolishes HasA secretion. This demonstrates that S. marcescens SecB is the genuine chaperone for HasA secretion in S. marcescens. These results also demonstrate that S. marcescens SecB is bifunctional, as it is involved in two separate secretion pathways. We investigated the effects of secB point mutations in the reconstituted HasA secretion pathway by comparing the translocation of a Sec substrate in various mutants. Two different patterns of SecB residue effects were observed, suggesting that SecB functions may differ for the Sec and ABC pathways.

FIG. 1.

Sequence comparison of SecB from various gram-negative bacteria. Sma, S. marcescens; Eco, E. coli; Sty, Salmonella enterica serovar Typhimurium; Ype, Yersinia pestis; Vch, Vibrio cholerae; Pmu, Pasteurella multocida; Hin, Haemophilus influenzae; Bsp, Buchnera sp.; Pfl, Pseudomonas fluorescens; Bap, Buchnera aphidicola; Pao, Pseudomonas aeruginosa; Xax, Xanthomonas axonopodis; Xca, Xanthomonas campestris; Xfa, Xylella fastidiosa; Nme, Neisseria meningitidis. Completely conserved residues are highlighted in black, and strongly conserved ones are in grey. Stars above the sequences point towards residues for which mutants have been obtained (see Fig. 6). The extreme N and C termini have been omitted from the alignments.

FIG. 2.

Complementation of an E. coli secB deletion mutant by SecB from S. marcescens (SecBSm). (A) Coomassie blue-stained gel of supernatants from MC4100(pSYCAC1 + pAM238) (lane 1), MC4100 ΔsecB(pSYCAC1 + pAM238) (lane 2), and MC4100 ΔsecB(pSYCAC1 + psecBSm/pAM238) (lane 3). In each case, the equivalent of 1 OD₆₀₀ unit was loaded on the gel. (B) immunodetection with anti-MBP antibody of whole-cell pellets after maltose induction of MC4100(pAM238) (lane 1), MC4100 ΔsecB(pAM238) (lane 2), and MC4100,ΔsecB (psecBSm/pAM238) (lane 3). The equivalent of 0.1 OD₆₀₀ unit was loaded on the gel. The star indicates a contaminating band. (C) Immunodetection of SecB with anti-E. coli SecB (SecBEc) antibodies. MC4100(pAM238) (lane 1), MC4100 ΔsecB(pAM238) (lane 2), and MC4100 ΔsecB (psecBSm/pAM238) (lane 3). The equivalent of 0.1 OD₆₀₀ unit was loaded on the gel. The star indicates a contaminating band.

FIG. 3.

Effect of secB deletion on pre-MBP processing in S. marcescens. (A) Immunodetection of SecB with anti-E. coli SecB antibodies in whole cells of E. coli MC4100, MC4100 ΔsecB, S. marcescens SM365, and SM365 ΔsecB::Kan. The equivalent of 0.1 OD₆₀₀ unit was loaded in each lane. (B) Immunodetection of MBP with anti-E. coli MBP antibodies in SM365 in the presence of 0.4% glucose or 0.4% maltose and in SM365 ΔsecB::Kan in the presence of 0.4% glucose or 0.4% maltose. (C) Pulse-chase analysis of pre-MBP processing in SM365 and SM365 ΔsecB::Kan after immunoprecipitation with anti-MBP antibodies. The lower two lanes correspond to a single pulse of 15 s.

FIG. 4.

Effect of secB deletion on HasA secretion in S. marcescens. (A) Immunodetection of HasA in the supernatant of several S. marcescens strains without (lanes 1, 3, 5, and 7) or with induction of the has system with 0.4 mM dipyridyl (lanes 2, 4, 6, and 8). The equivalent of 2 OD₆₀₀ units was loaded in each lane. Lanes 1 and 2, SM365 ΔsecB; lanes 3 and 4, SM365; lanes 5 and 6, SM365 ΔsecB(pAM238); lanes 7 and 8, SM365 ΔsecB(pSecBSm/pAM238). (B) Immunodetection of HasA in whole cells and supernatants from SM365 and SM365 ΔsecB harboring pSYC134/pAM without or with induction of the has operon with dipyridyl. Ct indicates SM365 not harboring pSYC134/pAM. (C) Pulse-chase analysis of HasA secretion in SM365 and SM365ΔsecB::Kan. Cells were labeled and HasA was immunoprecipitated in the various fractions (T, total; S, supernatant; C, cell pellet) at the times indicated (in minutes). (D) Immunodetection of PrtSM with anti-PrtSM antibody in supernatants of SM365 and SM365 ΔsecB. The equivalent of 2 OD₆₀₀ units was loaded in each lane.

FIG. 5.

Secretion of HasAΔ11-20 in various S. marcescens and E. coli strains. Immunodetection of HasAΔ11-20 in supernatants and whole cells of SM365, SM365 ΔsecB::Kan, and SM365UV3 harboring pHasAΔ11-20/pUC, in the absence or presence of 0.4 mM dipyridyl. The equivalent of 2 OD₆₀₀ units was loaded for the supernatants and of 0.2 OD₆₀₀ units for the whole-cell pellets. The lower part shows a Coomassie blue-stained gel of supernatants from E. coli MC4100(pSYC150 + pHasA/pUC), MC4100 ΔsecB(pSYC150 + pHasA/pUC), MC4100(pSYC150 + pHasAΔ11-20/pUC), and MC4100 ΔsecB(pSYC150 + pHasAΔ11-20/pUC).

FIG. 6.

Effect of secB point mutations on HasA secretion in the reconstituted system in E. coli. The name of the mutation and the residues affected are shown. (A) Coomassie blue-stained gel of supernatants from various secB mutants of MC4100 harboring pSYCAC1; the equivalent of 1 OD₆₀₀ unit was loaded in each lane. (B) Immunodetection of SecB in the corresponding whole-cell pellets.