Viability of a capsule- and lipopolysaccharide-deficient mutant of Neisseria meningitidis

Abstract

Neisseria meningitidis is the only lipopolysaccharide (LPS)-producing gram-negative bacterial species shown to be viable also without LPS. It was thought that the presence of capsular polysaccharide is necessary for this unusual feature. However, we show now that no part of the capsule gene cluster is required for maintaining LPS deficiency in N. meningitidis.

FIG. 1.

Analysis of unencapsulated neisserial lpxA mutants. (A) PCR analysis using primers LpxA-For and LpxA-Rev (Table 1) annealing at the 5′ and 3′ ends of the lpxA gene, respectively. Lane 3 shows the product obtained when chromosomal DNA from strain HB-1 was used as template. The PCR products obtained with the DNA from strains HB-1-1 and HB-1-2 (lanes 1 and 2, respectively) were identical in size to that obtained with plasmid pLAK33, containing the lpxA::kan allele (13), as the template (lane 4). (B) Silver-stained Tricine-SDS-PAGE gel containing, in each lane, equal amounts (as inferred from optical density measurements) of proteinase K-treated whole-cell lysates. L3 and L8 represent two wild-type LPS immunotypes of strain H44/76 that can arise through phase variation (9). Note that the LPS of strain HB-1 shows a higher electrophoretic mobility. In lanes 4 and 5, the lpxA mutants HB-1-1 and HB-1-2 were analyzed. Samples and gels were processed as described previously (3). (C) Colony immunoblot probed with capsule-specific monoclonal antibody 735 (Dade-Behring) (8) followed by alkaline-phosphatase-conjugated goat anti-mouse immunoglobulin G antibodies. Approximately 10 colonies of the indicated strains were mixed and streaked twice on nitrocellulose. A strong reaction is seen only in the case of the capsule-producing wild-type strain H44/76.

FIG. 2.

Neisserial capsule locus organizations. (A) The capsule locus organization in strain MC58 was compiled from the published genome sequence (14) and that in strain B1940 from reference and from B1940 capsule locus sequences deposited in GenBank (accession numbers Z13995, L09188, and L09189). The organization in pMF121 was assembled after sequencing of the regions indicated by dashed lines. Sizes of the regions, indicated by capital letters, and genes are not drawn to scale. Arrows indicate annealing sites for the primers listed in Table 1. Solid lines between B1940 and pMF121 indicate the homologous regions where crossover could take place to disrupt the chromosomal locus. Regions A, B, and C contain genes involved in capsule biogenesis. Region D contains genes involved in LPS biosynthesis, while region D′ is a duplication of region D with a truncation of the galE gene. Region E comprises a single open reading frame encoding a putative DNA-binding protein, and region MT contains methyltransferase genes (18). The B1940 capsule locus contains an additional open reading frame with homology to xcbA, a gene involved in serogroup X capsule biosynthesis (16). The NMB numbers refer to open reading frames as annotated in the MC58 genome sequence (14). Vector and pIM13 indicate sequences of the cloning vectors of the B1940 capsule locus and the erythromycin resistance cassette, respectively. c, LipA-For; d, LipB-Rev; e, CtrA-For; f, CtrD-Rev; g, SiaC-For; h, SiaD-Rev; i, LipA-Rev; j, Region-D-For; k, MT-Rev; l, Region-E-For; m, Ery-Rev. (B) Analysis of capsule locus organizations. Chromosomal DNA from strains MC58 (lane 1), B1940 (lane 2), and H44/76 (lane 3) was used in PCRs with primers i and j (Fig. 2A). (C) Plasmid pMF121 (lanes a) and chromosomal DNA from strains H44/76 (lanes b) and HB-1 (lanes c) were used in a series of PCRs indicated at the top of the gel. Primers (Table 1; Fig. 2A) used were as follows: for PCR 1, c and d; for PCR 2, e and f; for PCR 3, g and h; for PCR 4, m and f; and for PCR 5, k and l. The left lane shows size markers.

FIG. 3.

Phenotype of an H44/76 lpxA mutant (H44/76 lpxA cps) after capsule locus deletion. (A) Colony immunoblot probed with anti-capsule monoclonal antibody 735 as described in the legend to Fig. 1C. Panel 1, H44/76 lpxA cps; panel 2, H44/76 lpxA. (B) Silver-stained Tricine-SDS-PAGE gel containing, in each lane, equal amounts (as inferred from optical density measurements) of proteinase K-treated whole-cell lysates. Lane wt, H44/76 wild type; lane 1, H44/76 lpxA cps; lane 2, H44/76 lpxA.