Identification of a genetic locus involved in the biosynthesis of N-acetyl-D-mannosamine, a precursor of the (alpha 2--&gt;8)-linked polysialic acid capsule of serogroup B Neisseria meningitidis

Swartley, John S.; Stephens, David S.

doi:10.1128/jb.176.5.1530-1534.1994

Cited by 35 publications

(37 citation statements)

References 29 publications

(12 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The capsule-deficient mutant, M7, is unable to sialylate LOS (e.g., absent 4.8-kDa band in Fig. 2) since the Tn916 insertion interrupts synX, the first gene in the group B capsule biosynthesis operon required for the synthesis of CMP-N-acetylneuraminic acid (51). Since MAb 3F11 recognizes the unsialylated terminal galactose residue in the lacto-N-neotetraose structure (Gal␤134GlcNAc epitope [6]), parental NMB is MAb 3F11 positive, while mutant M7 is very positive in immunoblot screens.…”

Section: Resultsmentioning

confidence: 99%

Inner core biosynthesis of lipooligosaccharide (LOS) in Neisseria meningitidis serogroup B: identification and role in LOS assembly of the alpha1,2 N-acetylglucosamine transferase (RfaK)

et al. 1996

Self Cite

View full text Add to dashboard Cite

A lipooligosaccharide (LOS) mutant of Neisseria meningitidis serogroup B strain NMB (immunotype L3,7,9) was identified in a Tn916 (tetM) mutant bank by loss of reactivity with monoclonal antibody 3F11, which recognizes the terminal Gal␤134GlcNAc epitope in the lacto-N-neotetraose moiety of the wild-type LOS structure. The mutant, designated 559, was found to express a truncated LOS of 3.0 kDa. Southern and PCR analyses demonstrated that there was a single intact Tn916 insertion (class I) in the mutant 559 chromosome. Linkage of the LOS phenotype and the Tn916 insertion was confirmed by transformation of the wild-type parent. Nucleotide sequence analysis of the region surrounding the transposition site revealed a 1,065-bp open reading frame (ORF). A homology search of the GenBank/EMBL database revealed that the amino acid sequence of this ORF had 46.8% similarity and 21.2% identity with the ␣1,2 N-acetylglucosamine transferase (RfaK) from Salmonella typhimurium. Glycosyl composition and linkage analysis of the LOS produced by mutant 559 revealed that the lacto-N-neotetraose group which is attached to heptose I (HepI) and the N-acetylglucosamine and glucose residues that are attached to HepII in the inner core of the parental LOS were absent. These analyses also showed that the HepII residue in both the parent and the mutant LOS molecules was phosphorylated, presumably by a phosphoethanolamine substituent. The insertion of nonpolar and polar antibiotic resistance cartridges into the parental rfaK gene resulted in the expression of LOS with the same mobility as that produced by mutant 559. This result indicated that the inability to add the lacto-N-neotetraose group to the 559 LOS is not due to a polar effect on a gene(s) downstream of rfaK. Our data indicate that we have identified the meningococcal ␣1,2 N-acetylglucosamine transferase responsible for the addition of N-acetylglucosamine to HepII. We propose that the lack of ␣-chain extension from HepI in the LOS of mutant 559 may be due to structural constraints imposed by the incomplete biosynthesis of the LOS inner core.Lipooligosaccharide (LOS) is a major virulence factor of the obligate human pathogen Neisseria meningitidis (54, 55). Levels of LOS correlate with the release of inflammatory cytokines (interleukin 1, interleukin 6, and tumor necrosis factor alpha) important in the pathogenesis of meningococcemia and meningococcal meningitis (2). Direct cytotoxic effects of meningococcal LOS have also been observed in endothelial cell tissue models infected with N. meningitidis (7). The LOS structures of N. meningitidis have been well characterized both immunologically (53) and biochemically (9,17,23,31,35,57). Meningococcal LOS consists of a lipid A to which a conserved inner core of two heptoses and two 2-keto-3-deoxyoctulosonic acid (KDO) residues is attached. The sugar composition of the ␣-chain oligosaccharide attached to heptose I (HepI) of the inner core is variable (55) but in certain immunotypes is a perfect mimic of the sugars attached to the human sphi...

show abstract

Section: Resultsmentioning

confidence: 99%

Inner core biosynthesis of lipooligosaccharide (LOS) in Neisseria meningitidis serogroup B: identification and role in LOS assembly of the alpha1,2 N-acetylglucosamine transferase (RfaK)

et al. 1996

Self Cite

View full text Add to dashboard Cite

show abstract

“…We have previously reported the identification of mutants M7 and O8 (45) and partially characterized the genetic and biochemical defects in the M7 mutant (47). Both the M7 and O8 mutants contained truncated (class II) Tn916 insertions that interrupted the 1,134-bp synX gene (at nucleotide positions 710 and 412, respectively), the first gene in region A (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Meningococcal strains were grown on GC base agar (Difco) or in GC broth (38) at 37ЊC with 3.5% CO 2 . Minimal media with and without supplements were prepared as described previously (47). E. coli strains were grown on Luria-Bertani agar plates (Bethesda Research Laboratories) or in Luria-Bertani broth at 37ЊC.…”

Section: Methodsmentioning

confidence: 99%

“…Using a separate approach, Tn916 mutagenesis, we defined the first gene of the biosynthetic operon (synX), showed that this gene was either the N-acetyl-D-glucosamine-6-phosphate 2-epimerase or the N-acetyl-D-mannosamine-6-phosphate phosphatase, demonstrated that a mutation in synX produced a capsule-defective (Cap Ϫ ) phenotype, and clarified the nucleotide sequence of synB (45,47). Recently, Ganguli et al (18) reported the cloning and analysis of genes for group B polysialic acid biosynthesis in a third group B meningococcal strain and suggested, on the basis of amino acid homology with E. coli NeuB (1), that the protein encoded by synC (neuB) was the putative NANA synthetase.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Expression of sialic acid and polysialic acid in serogroup B Neisseria meningitidis: divergent transcription of biosynthesis and transport operons through a common promoter region

et al. 1996

Self Cite

View full text Add to dashboard Cite

show abstract

“…Conjugative transposons like Tn916 (9) are large elements that are self-transferable by conjugation to a variety of bacteria, where they integrate into the chromosome by transposition. When introduced into N. meningitidis, Tn916 (15) and a Tn916-like derivative (21) transposed into different sites, as evidenced by the identification of mutants presenting precise genetic defects (8,34,37). Conjugative transposons are, however, not practical for saturation mutagenesis of N. meningitidis for several reasons: (i) the frequency of transposition is low, and thus the number of mutants falls far short of the number statistically required for the mutagenesis of all N. meningitidis genes; (ii) the insertions are not perfectly stable (36); and (iii) transposition presents some site specificity.…”

mentioning

confidence: 99%

Mutagenesis of Neisseria meningitidis by In Vitro Transposition of Himar1 mariner

et al. 2000

View full text Add to dashboard Cite

Now that the meningococcal genome sequence has been completed, the lack of a suitable method for saturation mutagenesis remains a major obstacle to the unraveling of the pathogenic propensity of Neisseria meningitidis. Here, we demonstrate that in vitro Himar1 mariner transposition on chromosomal or PCRamplified meningococcal DNA, which is subsequently reintroduced into N. meningitidis by natural transformation, is an extremely efficient mutagenesis method. Southern blot analysis, sequencing the Himar1 insertion point in numerous transposition mutants, and a limited screening of the mutant libraries for clones impaired in maltose catabolism confirmed that Himar1 transposed randomly in N. meningitidis. Taken together, these data demonstrate that Himar1 in vitro transposition can lead to the exhaustive mutagenesis of N. meningitidis, allowing for the first time a genomic-scale mutational analysis of this important human pathogen.

show abstract

Identification of a genetic locus involved in the biosynthesis of N-acetyl-D-mannosamine, a precursor of the (alpha 2-->8)-linked polysialic acid capsule of serogroup B Neisseria meningitidis

Cited by 35 publications

References 29 publications

Inner core biosynthesis of lipooligosaccharide (LOS) in Neisseria meningitidis serogroup B: identification and role in LOS assembly of the alpha1,2 N-acetylglucosamine transferase (RfaK)

Inner core biosynthesis of lipooligosaccharide (LOS) in Neisseria meningitidis serogroup B: identification and role in LOS assembly of the alpha1,2 N-acetylglucosamine transferase (RfaK)

Expression of sialic acid and polysialic acid in serogroup B Neisseria meningitidis: divergent transcription of biosynthesis and transport operons through a common promoter region

Mutagenesis of Neisseria meningitidis by In Vitro Transposition of Himar1 mariner

Contact Info

Product

Resources

About