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. 2009 Jun;191(11):3482-91.
doi: 10.1128/JB.00365-09. Epub 2009 Apr 10.

Characterization of Streptococcus gordonii SecA2 as a paralogue of SecA

Barbara A Bensing  1 , Paul M Sullam
Affiliations

Characterization of Streptococcus gordonii SecA2 as a paralogue of SecA

Barbara A Bensing et al. J Bacteriol. 2009 Jun.

Abstract

The accessory Sec system of Streptococcus gordonii is essential for transport of the glycoprotein GspB to the bacterial cell surface. A key component of this dedicated transport system is SecA2. The SecA2 proteins of streptococci and staphylococci are paralogues of SecA and are presumed to have an analogous role in protein transport, but they may be specifically adapted for the transport of large, serine-rich glycoproteins. We used a combination of genetic and biochemical methods to assess whether the S. gordonii SecA2 functions similarly to SecA. Although mutational analyses demonstrated that conserved amino acids are essential for the function of SecA2, replacing such residues in one of two nucleotide binding folds had only minor effects on SecA2 function. SecA2-mediated transport is highly sensitive to azide, as is SecA-mediated transport. Comparison of the S. gordonii SecA and SecA2 proteins in vitro revealed that SecA2 can hydrolyze ATP at a rate similar to that of SecA and is comparably sensitive to azide but that the biochemical properties of these enzymes are subtly different. That is, SecA2 has a lower solubility in aqueous solutions and requires higher Mg(2+) concentrations for maximal activity. In spite of the high degree of similarity between the S. gordonii paralogues, analysis of SecA-SecA2 chimeras indicates that the domains are not readily interchangeable. This suggests that specific, unique contacts between SecA2 and other components of the accessory Sec system may preclude cross-functioning with the canonical Sec system.

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Figures

FIG. 1.
FIG. 1.
Comparison of the domain organization of SecAEc (ecSecA), SecASg (sgSecA), and SecA2Sg (sgSecA2). The domain boundaries of SecASg and SecA2Sg were approximated as determined by the Superfamily 1.69 structural comparison of proteins (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/hmm.html) and refined by comparing the primary sequence alignments with that of SecAEc (sequences were aligned with the Multalin algorithm [13]; see Fig. S1 in the supplemental material). CTD, extreme carboxy-terminal domain. HSD, HWD, IRA1, and CTD comprise the C domain.
FIG. 2.
FIG. 2.
The effects of replacement of select residues of SecA2 on the export of GspB736flag. (A) Alignment of the regions flanking the target residues (indicated in red). SecAEc, ecSecA; SecASg, sgSecA; SecA2Sg, sgSecA2. (B and C) Lanes correspond to the secA2 mutant strain PS1226 carrying the pMSP3535 vector only (lane 1) or the vector with wild-type secA2 (lane 2) or secA2 variants as indicated (lanes 3 to 8). (B) GspB736flag was detected by using an anti-FLAG monoclonal antibody. Lanes contain proteins corresponding to 25 μl of cultures grown for 6 h in the presence of nisin. Nearly identical results were seen even in the absence of nisin induction, indicating that very low levels of SecA2 are required for complementation (data not shown). M, medium fraction; P, protoplast fraction. (C) SecA2 variants were detected by using an anti-SecA2 polyclonal antiserum. Lanes contain protoplast proteins corresponding to 50 μl of cultures grown in the presence of nisin for 6 h.
FIG. 3.
FIG. 3.
The effect of sodium azide on SecASg- and SecA2Sg-dependent export. Lanes contain protein from 25 μl of spent medium of cultures grown at high density for 90 min. The secreted proteins were detected with an anti-FLAG monoclonal antibody. (A) SecASg-dependent export of GspB736flag variants. The upper panel shows export of a GspA-GspB736flag fusion protein from strain PS1025 (GspA is another cell wall protein of S. gordonii). The lower panel shows export of GspB736flag carrying a modified signal peptide (Δ8-68 G75L G79A G80C) from strain PS1139 (ΔsecA2 ΔgtfA). ss, signal sequence. (B) Export of GspB736flag from strains expressing variants of SecA2. wt, wild type.
FIG. 4.
FIG. 4.
Comparison of basal ATPase rates in vitro. (A) Effect of Mg2+ on the activity of the His6-tagged proteins. (B) Effect of azide on the activity of the His6-tagged proteins. (C) Effect of Mg2+ on the activity of the MalE fusion proteins. (D) Effect of azide on the activity of the MalE fusion proteins. 6His-SecA and 6His-SecA2 were purified in the presence of 0.1% CHAPS. The activity of 6His-SecA purified in the absence of CHAPS was not detectably different from that of 6His-SecA purified with CHAPS (data not shown). The enzymatic rates of ATP hydrolysis were determined after 30 min of incubation at 30°C. For determination of the effects of azide on ATP hydrolysis rates (B and D), the reaction mixtures included Mg2+ at concentrations that promoted maximal activity (0.1 mM Mg2+ for 6His-SecA and MalE-SecA, 0.5 mM for MalE-SecA2, and 2.5 mM for 6His-SecA2). Error bars show standard deviations. Pi, Pi.
FIG. 5.
FIG. 5.
Expression of the SecA-SecA2 chimeras in S. gordonii. Lanes contain protoplast proteins corresponding to 50 μl of cultures grown in the presence of nisin for 6 h. Chimeras were detected by using a polyclonal anti-SecA2 serum. The secA2 mutant strain PS1226 carried pMSP3535 with no insert (lane 1), wild-type secA2 (lane 2), or secA-secA2 constructs as indicated (lanes 3 to 6).
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References

    1. Bensing, B. A., B. W. Gibson, and P. M. Sullam. 2004. The Streptococcus gordonii platelet binding protein GspB undergoes glycosylation independently of export. J. Bacteriol. 186638-645. - PMC - PubMed
    1. Bensing, B. A., J. A. Lopez, and P. M. Sullam. 2004. The Streptococcus gordonii surface proteins GspB and Hsa mediate binding to sialylated carbohydrate epitopes on the platelet membrane glycoprotein Ibα. Infect. Immun. 726528-6537. - PMC - PubMed
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    1. Bensing, B. A., D. Takamatsu, and P. M. Sullam. 2005. Determinants of the streptococcal surface glycoprotein GspB that facilitate export by the accessory Sec system. Mol. Microbiol. 581468-1481. - PubMed

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