Identification of regions of the chromosome of Neisseria meningitidis and Neisseria gonorrhoeae which are specific to the pathogenic Neisseria species

Abstract

Neisseria meningitidis and Neisseria gonorrhoeae give rise to dramatically different diseases. Their interactions with the host, however, do share common characteristics: they are both human pathogens which do not survive in the environment and which colonize and invade mucosa at their port of entry. It is therefore likely that they have common properties that might not be found in nonpathogenic bacteria belonging to the same genetically related group, such as Neisseria lactamica. Their common properties may be determined by chromosomal regions found only in the pathogenic Neisseria species. To address this issue, we used a previously described technique (C. R. Tinsley and X. Nassif, Proc. Natl. Acad. Sci. USA 93:11109-11114, 1996) to identify sequences of DNA specific for pathogenic neisseriae and not found in N. lactamica. Sequences present in N. lactamica were physically subtracted from the N. meningitidis Z2491 sequence and also from the N. gonorrhoeae FA1090 sequence. The clones obtained from each subtraction were tested by Southern blotting for their reactivity with the three species, and only those which reacted with both N. meningitidis and N. gonorrhoeae (i.e., not specific to either one of the pathogens) were further investigated. In a first step, these clones were mapped onto the chromosomes of both N. meningitidis and N. gonorrhoeae. The majority of the clones were arranged in clusters extending up to 10 kb, suggesting the presence of chromosomal regions common to N. meningitidis and N. gonorrhoeae which distinguish these pathogens from the commensal N. lactamica. The sequences surrounding these clones were determined from the N. meningitidis genome-sequencing project. Several clones corresponded to previously described factors required for colonization and survival at the port of entry, such as immunoglobulin A protease and PilC. Others were homologous to virulence-associated proteins in other bacteria, demonstrating that the subtractive clones are capable of pinpointing chromosomal regions shared by N. meningitidis and N. gonorrhoeae which are involved in common aspects of the host interaction of both pathogens.

FIG. 1

Procedure for representational difference analysis. Sequences specific to the pathogen are represented in grey; those in common with N. lactamica are hatched. DNA from N. meningitidis or from N. gonorrhoeae was digested with frequently cutting restriction endonucleases and ligated to adapter pairs such that only the 5′ end of each DNA stand was covalently linked to the 24-bp adapter. On denaturing, mixing, and reannealing, only the (pathogen-specific) sequences with an adapter covalently linked were able to rehybridize with their complementary sequence. The fragments of randomly sheared N. lactamica chromosome are generally over 10 times as long as the restriction fragments from the pathogen and not only sequester all pathogen fragments having homologies in N. lactamica but also, in the large majority of cases, prevent the polymerase from synthesizing the complement of the adapter during the filling-in procedure. Hence, these common fragments are effectively prevented from being amplified, and only the pathogen-specific fragments possessing an adapter at each end can be exponentially amplified.

FIG. 2

Position of the pathogen-specific clones on the chromosomal map of N. meningitidis Z2491. Clones were mapped by Southern blotting and by comparison with the published partial genome sequence. Those derived from the N. gonorrhoeae-minus-N. lactamica subtraction are shown on the left (G-L libraries), and those from the N. meningitidis-minus-N. lactamica subtraction are shown on the right (M-L libraries). Clones from the two libraries derived from the same pathogen-specific region are marked with the same shading. Some clones were present in multiple copies (generally insertion sequences), and these are mapped only where they coincide with another identified locus.

FIG. 3

Comparison of the positions of the pathogen-specific clones on the chromosomes of N. gonorrhoeae FA1090 and N. meningitidis Z2491. The relative positions follow the lines of dislocation between the two chromosomes as previously described (11), with certain exceptions.

FIG. 4

Genetic arrangement of the regions surrounding pathogen-specific clones. Genes are shown as arrows, yellow for those with homologies to proteins in the databases and grey for ORFs without significant homology. Transposases are shown in red, and Correia sequences (marked C) are shown in blue. The positions of the subtractive clones are shown as orange bars below the bar representing the genes. Regions previously discovered as being N. meningitidis specific are shown in green. A scale (in kilobases) is shown above the sequences. (A) The pathogen-specific clones flank a region of low G+C content (46%) containing several ORFs with no homologies to previously described genes. Homologies of surrounding ORFs, at the amino acid level, are as follows: 1, SubI, E. coli; 2a, 2b, 2c, transposase, IS1106, N. meningitidis; 3, ORF B, IS150, E. coli; 4, integrase, phage φR73; 5, transposase IS1106, N. meningitidis; 6, transposase, Synechocystis sp. (accession no. BAA10234); 7, (3′ end) HI0270, H. influenzae; 8, (5′ end) GlcD, Synechocystis sp. (3′ end) and GlpC, Helicobacter pylori. (B) The pathogen-specific clones correspond to a region of particularly low G+C content (42%), containing ORFs with no homologies. Homologies of surrounding ORFs, at the amino acid level, are as follows: 1, NuoF, Rickettsia prowazekii; 2, NuoE, R. prowazekii; 3, NuoD, R. prowazekii; 4, NuoC, Rhodobacter capsulatus; 5, NuoB, Rickettsia prowazekii; 6, NuoA, Sinorhizobium meliloti; 7, transposase, IS1016, H. influenzae; 8, UvrD, E. coli; 9, HI1731, H. influenzae; 10, LamB homolog, H. influenzae; 11, BraB homolog, H. influenzae; 12; MTH939, Methanobacterium thermoautotrophicum; 13, transposase IS4351, N. meningitidis; 14, GlnE, E. coli; 15: PyrD, Salmonella typhimurium. (C) Homologies are as follows: 1, transposase IS4351, N. meningitidis; 2, ORF 288, Coxiella burnetii; 3, ORF 1244, Sphingomonas aromaticivorans; 4, YaeC, E. coli; 5, YaeE, E. coli; 6, ABC transporter (accession no. P30750), E. coli; 7, SLT70 transglycosylase (accession no. S56616), E. coli; 8, ribosomal protein S21, Burkholderia pseudomallei; 9, LporfX, Legionella pneumophila; 10, RegG, N. gonorrhoeae; 11, RegF, N. gonorrhoeae; 12, CadD, Staphylococcus aureus; 13, ribosomal protein L31, Haemophilus ducreyi; 14, putative acetyltransferase (accession no. CAA90593), Schizosaccharomyces pombe; 15, ResA, Bacillus subtilis; 16, YbaW, E. coli; 17, VacJ, Rickettsia prowazekii; 18, YrbC, E. coli; 19, HI1085, H. influenzae; 20, HI1086, H. influenzae; 21, HI1087, H. influenzae; 22, AldA, E. coli; 23, SsaI, Pasteurella haemolytica; 24, PabB, Helicobacter pylori; 25, OmpU, N. meningitidis; 26, HpuA, N. gonorrhoeae; 27, HpuB, N. meningitidis; 28, GroEL, N. gonorrhoeae; 29, GroES, N. gonorrhoeae; 30, transposase, IS1016, H. influenzae; 31, HI0736, H. influenzae; 32, LysA, Pseudomonas aeruginosa; 33, CyaY, E. coli; 34, HI1643, H. influenzae; 35, HI0931, H. influenzae; 36, YgaG, E. coli; 37, PolA, H. influenzae; 38, transposase IS1106, N. meningitidis; 39, Hap, H. influenzae; 40, ThdF, E. coli. (D) The subtractive clones flank the previously discovered N. meningitidis-specific region 2. Homologies are as follows: 1, SecB, E. coli; 2, RecG H. influenzae; 3, ArgC, Synechocystis sp.; 4, CvaA, plasmid ColV, E. coli; 5, CvaB, plasmid ColV, E. coli; 6, HI0276, H. influenzae; 7, YkvJ, Bacillus subtilis; 8, HI1190, H. influenzae; 9, HI1189, H. influenzae; 10, YcfO, 11, HI1586, H. influenzae; 12, MucD/HtrA serine protease homolog, Pseudomonas; 13, (5′ end) HI0489, H. influenzae, (3′ end) Nth endonuclease III, H. influenzae; 14, GluP, Brucella abortus; 15, NhaC, Bacillus firmus; 16, (5′ end) YbeY, E. coli, (3′ end) YbeX, E. coli; 17, HemC, Pseudomonas aeruginosa. (E) Homologies are as follows: 1, aq_1853, hypothetical protein, Aquifex aeolicus; 2, C09_orf404, Mycoplasma pneumoniae; 3, GepB, Dichelobacter nodosus; 4, GlrA, Actinobacillus actinomycetemcomitans; 5, RelA, Vibrio sp.; 6, putative transposase (accession no. AAD10186), Streptococcus pneumoniae; 7, TolC, E. coli; 8, HlyD, E. coli; 9, ORF C7, Ralstonia solanacearum/RstA1, CTX phage, Vibrio cholerae; 10, ORF1, TspB, N. meningitidis; 11, TspB, N. meningitidis; 12, MdaB, H. influenzae; 13, PntB, H. influenzae; 14, PntA, E. coli.