Figure 1.
Genomic organization of the scpB-lmb intergenic region.
Dark grey box represents the noncoding spacer region. Primer set scpB3382/rlmb51 was used to determine the structure of the scpB-lmb intergenic region. The presence of the insertion sequence IS1548 and of the group II intron GBSi1 in the spacer was then confirmed using primer sets scpB3382/r1S1548, and scpB3382/rGBSi143.
Table 1.
Distribution of 109 GBS strains according to the structure of scpB-lmb intergenic region and according to clonal complexes as determined by MLST.
Table 2.
Mobile genetic elements (MGEs) in scpB-lmb intergenic region of CC17, CC19, CC23, and CC10 GBS strains from infected and colonized. patients.
Figure 2.
Properties of 26 GBS isolates in relation to the scpB-lmb intergenic region structure (8 strains without MGE, 9 strains with GBSi1 and 9 strains with IS1548).
(A) Relative transcription levels of scpB (filled boxes) and lmb (open boxes) genes. The amount of transcripts of each gene was normalized to the amount of gyrA transcripts and expressed relative to the level of transcription in L19 GBS isolate that has no MGE. Boxes are means and bars are standard deviation of the means of the relative level of transcription in each group of strains. ** The mean transcription level of lmb gene was significantly higher in the group of strains with IS1548 than in the other two groups of strains (P<0.001). (B) Binding ability of GBS isolates to immobilized human fibronectin (filled boxes) and to immobilized human laminin (open boxes). Boxes are means and bars are standard deviation of the means of the fibronectin and laminin binding percentages in each group of strains. ** The mean binding ability to laminin was significantly higher in the group of strains with IS1548 than in the other two groups of strains (P≤0.001).
Table 3.
lmb gene expression in 14 strains from two phylogenetic lineages in relation to the presence of the IS1548 sequence upstream lmb gene.
Figure 3.
Expression of Lmb protein on GBS cells in relation to the scpB-lmb intergenic region structure.
Lmb protein was quantified in an ELISA-type assay. GBS cells (8 strains without MGE, 9 strains with GBSi1, 9 strains with IS1548), were immobilized in microtiter wells and incubated with rabbit anti-Lmb antibodies. Binding of antibodies to the Lmb protein was quantified by addition of peroxidase-conjugated goat anti-rabbit IgG, and subsequent addition of OPD peroxidase substrate. Boxes are means and bars are standard deviation of the means of the absorbance at 492 nm (A492) per adhering bacteria in each group of strains. ** The amount of Lmb protein was significantly higher in the group of strains with IS1548 as compared with the other two groups of strains (P<0.001).
Figure 4.
Properties of L29 wild type GBS strain and isogenic IS1548 deletion mutant.
Relative transcription levels of lmb gene, binding ability of GBS cells to immobilized human laminin, and expression of Lmb protein on GBS cells as determined by ELISA, in an isogenic mutant deleted for IS1548 sequence between scpB and lmb genes (L29ΔIS1548) (striped boxes) as compared to the parent strain (L29 WT) (open boxes). Each experiment was performed at least three times. Boxes are means and bars are standard deviation of the means.
Figure 5.
Detection of Lmb protein by Western blot in L29 wild type GBS strain and isogenic IS1548 deletion mutants.
10 µg of total bacterial cell proteins isolated from strains 1: NEM316 (no MGE), 2: ΔscpB-lmb NEM316 mutant, 3: L29 wild type (IS1548), 4 to 7: four independent L29ΔIS1548 mutant strains were separated by SDS PAGE and transferred to an Immobilon P polyvinylidene difluoride membrane. The blots were probed with rabbit anti-Lmb antibodies that were detected with a horseradish peroxidase labeled secondary anti-rabbit antibody that was then visualized by the ECL system (Amersham Biosciences). Molecular sizes in kDa are depicted at the left.
Table 4.
Oligonucleotide primers.