Two additional components of the accessory sec system mediating export of the Streptococcus gordonii platelet-binding protein GspB

Abstract

The gspB-secY2A2 locus of Streptococcus gordonii strain M99 encodes the platelet-binding glycoprotein GspB, along with proteins that mediate its glycosylation and export. We have identified two additional components of the accessory Sec system (Asp4 and Asp5) encoded just downstream of gtfB in the gspB-secY2A2 locus. These proteins are required for GspB export and for normal levels of platelet binding by M99. Asp4 and Asp5 may be functional homologues of SecE and SecG, respectively.

FIG. 1.

Genetic map of the gspB-secY2A2 locus in M99 and in the derived mutants. The nucleotide sequence between orf6 and orf7 is also shown. The dotted arrows above the sequence represent an inverted repeat. The bold sequences represent stop codons for orf6 and orf7. Striped arrows, spectinomycin resistance gene (spc); gray arrows, gspB; black arrows, target genes for gene replacement; and open arrows, the other genes in this locus.

FIG. 2.

Alignments of the deduced amino acid sequences of Orf5 and Orf6 of S. gordonii strain M99 with SecE and SecG of other gram-positive species. (A) Alignment of Orf5 with SecE of B. subtilis 168 (accession number, CAB11876) and with SecE of S. gordonii Challis. (B) Alignment of Orf6 with SecG of B. subtilis 168 (CAB15368) and with SecG of S. gordonii Challis. The SecE and SecG sequences of B. subtilis 168 were obtained from GenBank. The SecE and SecG sequences of S. gordonii Challis were retrieved from the genome sequence data for this strain (recently available from TIGR at http://www.tigr.org) by performing a BLAST search with the SecE (AAK76075) and SecG (AAK75095) sequences of S. pneumoniae TIGR4. The sequences were aligned using ClustalW (http://www.ddbj.nig.ac.jp/search/clustalw-e.html). Identities and similarities between proteins were calculated on the basis of the alignments. Dashes indicate gaps in the aligned sequences. Bars and colons between two sequences indicate identical amino acid residues and conservative changes, respectively. Underlined sequences represent TMS predicted by using TMpred (http://www.ch.embnet.org/software/TMPRED_form.html). S.g., S. gordonii; B.s., B. subtilis.

FIG. 3.

Effect of orf5, orf6, and orf7 disruption on the export and expression of GspB. (A) GspB expression and export by PS436 (gspB::pM995′Bint), M99, PS851 (Δorf5::spc), PS842 (Δorf6::spc), and PS887 (Δorf7::spc). (B and C) Effect of proteinase K treatment of M99, PS851, and PS842. Bacterial cells suspended in DPBS were incubated for 1 h at 37°C with (+) or without (−) proteinase K. Cells were recovered by washing, and protoplasts were generated as described in the text. Proteins were separated by electrophoresis through a 3 to 8% polyacrylamide gradient gel and then subjected to Western blot analysis using a polyclonal anti-GspB serum. All proteins shown in this figure migrated above the highest standard (250 kDa). Each lane contains cell wall proteins extracted from bacteria in 75 μl of a broth culture or protoplast proteins extracted from bacteria in 50 μl of a broth culture. CW, cell wall proteins; P, protoplast proteins.

FIG. 4.

Complementation analysis of the orf5 and orf6 mutant strains (PS851 and PS842, respectively). Protein expression was induced with nisin, as described in the text. Extracted cell wall proteins were separated by electrophoresis through a 3 to 8% polyacrylamide gradient gel and then subjected to Western blot analysis using the anti-GspB serum. Each lane contains proteins extracted from 200 μl of broth culture.

FIG. 5.

Platelet binding by M99 and the derivative strains. (A) Platelet binding by strains M99, PS851 (Δorf5::spc), PS842 (Δorf6::spc), and PS436 (gspB::pM995′Bint). Data were collected from two experiments (n = 8) with platelets from two different human donors. For each strain, binding is expressed as a percentage of the binding levels achieved by the parent strain M99 (mean ± standard deviation). Differences in platelet binding were compared by the unpaired t test with the Welch modification. Asterisks indicate values that are significantly different (P < 0.0001) from that for M99. (B) Platelet binding by strains M99 (pMSP3545), PS851 (pMSP3545), PS851 (pORF5C), PS842 (pMSP3545), and PS842 (pORF6C). Data were collected from two experiments (n = 12) with platelets from two different human donors. For each strain, binding is expressed as a percentage of the binding levels achieved by M99 (pMSP3545) (mean ± standard deviation).