Fig. 1. Southern blot analysis of ClaI-digested chromosomal DNA from different strains belonging to the genus Neisseria. Hybridization was performed under conditions of moderate stringency (see Materials and methods). The probe used in (A) was a PCR-generated fragment amplified between oligonucleotides C1-152 and C1-8 on the pilC1 promoter and corresponding to the CREN of pilC1. Neisseria meningitidis (Nm) strains were LNP6505 (1), LNP6548 (2), LNP8013 (3), LNP10824 (4), LNP12681 (5), LNP12787 (6), LNP12886 (7), LNP12963 (8), LNP12970 (9), LNP13150 (10), LNP13407 (11), LNP13473 (12). Neisseria gonorrhoeae (Ng) strain was MS11 (13). Commensal Neisseria were: N.mucosa strain LNP405 (14), N.lactamica strain LNP415 (15), N.flavescens strain LNP414 (16), N.cinerea strain LNP415 (17), N.polysaccharia strain LNP462 (18), N.sicca strain LNP3265 (19). The probe used in (B) was an internal fragment of crgA generated by PCR using oligonucleotides 98-4 and 98-7. Nm strains were LNP10824 (1), LNP7381 (2), LNP6505 (3), LNP12873 (4), LNP12792 (5), LNP13146 (6), LNP12870 (7), LNP8013 (8), LNP13083 (9) and LNP13145 (10). These strains belong to different genetic lineages (Guibourdenche et al., 1997). The probe used in (C) was an internal fragment of crgA generated by PCR using oligonucleotides 98-4 and 98-7. Nm strain LNP8013 (1), Moraxella catarrhalis (Mc) LNP417 (2), N.gonorrhoeae strains MS11, LNP403 and LNP6911 (lanes 3–5); commensal Neisseria were: N.lactamica strain LNP415 (6), N.polysaccharia strain LNP462 (7), N.cinerea strain LNP415 (8), N.perflava strain LNP407 (9). All these strains were reported previously (Taha and Marchal, 1990; Guibourdenche et al., 1997).

Fig. 2. (A) Schematic representation of the crgA locus in Nm (Clone 12). Recombinant plasmid pTC1-202 was constructed from the original cosmid pNM5-266 as described in the text. Thick black arrows indicate the organization of ORFs. Recombinant plasmid pTC1-208 was constructed by subcloning the fragment SmaI–MluI of pTC1-202 into pUC18 vector. crgA was then disrupted by the insertion of a cassette encoding an antibiotic resistance (kanamycin or spectinomycin) into the PstI site. Recombinant plasmid pAD5 contains the crgA–lacZ–aph-3′ transcriptional fusion (see Materials and methods for detailed description). Small arrows indicate oligonucleotides used in this study, white boxes correspond to DNA of vectors. The figure is not drawn to scale. (B) Alignment of the nucleotide sequence of the CREN of pilC1 and crgA. The TSPs (P2 for crgA and PC1.3 for pilC1), the beginnings of the ORFs and the Shine–Dalgarno sequence (SD) are indicated. The CREN of both genes is in a box. The GG-N8-(A/G)C motif is in bold. The sequences of pilC1 and crgA that bind CrgA are upstream of the boxed region and the T-N11-A motifs are underlined.

Fig. 3. Nuclease S1 and reverse transcriptase promoter mapping of the TSPs of crgA. (A) S1 mapping was performed using an end-labelled fragment obtained by PCR between oligonucleotides 98-2 and 98-3. Total RNA was extracted from Clone 12 grown on GCB medium. (1) Total RNA from Clone 12 was incubated with the PCR fragment in the presence of S1 enzyme; (2) the PCR fragment was incubated in the presence of S1 enzyme but no RNA was added; (3) total RNA from Clone 12 was incubated with the PCR fragment but no S1 enzyme was added. Arrowheads indicate the TSPs P1 and P2. DNA sequence of the coding strand is shown with P1 and P2 indicated. (B) Reverse transcriptase mapping experiments were performed using total RNA extracted (1) from Hec-1-B epithelial cells, (2) from Hec-1-B cells infected with Clone 12 for 1 h, and (3) from Clone 12 grown alone in cell culture medium. The arrowhead indicates the TSP P2.

Fig. 4. Adhesion level of Clone 12 and its derivatives on Hec-1-B epithelial cells. Infection was performed as described in Materials and methods. At 1, 4 and 9 h post-infection, cells were lifted off the plates and the adhesion level was measured.

Fig. 5. EM micrographs after conA/peroxidase staining of Hec-1-B epithelial cells infected for 9 h by Clone 12 (wild-type). (A) Numerous bacteria are in close contact with the cell on the right (arrowheads). The cell on the left is devoid of bacteria. Note that the conA/peroxidase reaction preferentially delimited microvilli on this cell. (B) A higher magnification from (A) (framed). Arrowheads indicate bacteria intimately adhered to the cell surface.

Fig. 6. EM micrographs after conA/peroxidase staining Hec-1-B epithelial cells infected for 9 h by strain NM98-3 (crgA mutant). (A) Bacteria are spread on the cell surface, but are not involved in intimate adhesion. Few pili (small arrows) are present. (B) One diplococcus is seen in close proxmity to the cell membrane but is still separated from the cell surface by a space in which the product of the conA/peroxidase reaction is present (arrow).

Fig. 7. Colony blotting to monitor capsule level. Adhesion of Clone 12 (wild-type) and strain NM98-3 was performed as described in Materials and methods. After bacterial counting, serial dilutions of whole bacteria were spotted onto a nitrocellulose membrane. Equal amounts of bacteria were spotted, 2 × 10⁷ (1), 4 × 10⁶ (2) and 8 × 10⁵ (3). The membrane was immunoblotted using a rabbit anti-serogroup C serum and revealed using the ECL-kit (Amersham). A strain of serogroup B (LNP17114) and epithelial cells alone were also tested as controls.

Fig. 8. Gel retardation analysis with purified CrgA. The promoter regions used were those of crgA, pilC1 and pilT. These regions were amplified by PCR using oligonucleotides 98-2/98-3 and 98-1/98-3 (crgA), C1-354/C1-8 and C1-128/C1-8 (pilC1) and 99-17 and 99-18 (pilT). PCR fragments were end labelled using T4 polynucleotide kinase and [γ-³²P]ATP (3000 Ci/mmol; Amersham). For each sample, 25 ng of labelled fragment were incubated with increasing amounts of purified CrgA (top) as described in Materials and methods. Heterologous DNA (500 ng) was present in all cases. Competition experiments with unlabelled homologous DNA (100 ng) were also performed for crgA and pilC1.

Fig. 9. RT–PCR analysis of the expression of crgA and porA genes. Oligonucleotides used were 98-4 and 98-7 (crgA) and porA0 and porA101 (porA). The bacteria tested are indicated above each lane. Size markers are indicated on the left. RT, reverse transcriptase.