Deletions of Tn916‐like transposons are implicated in tetM‐mediated resistance in pathogenic Neisseria

Abstract

Summary Using the tetM‐containing conjugative transposon Tn916 as a mutagenesis tool, we identified two distinct classes of transposon insertions in the meningococcal chromosome. Class I Insertions have an intact copy of Tn916 that appears to have transposed by a novel recombinational mechanism, similar to the transposition of conjugative transposons in Gram‐positive bacteria. Class II insertions were characterized by deletions of Tn916 but preservation of the tetM determinant. In addition, we identified Class II Tn916‐like insertions in the naturally occurring 25.2 MDa tetM‐containing plasmids of both Neisseria meningitidis and Neisseria gonorrhoeae. The truncated Tn916 like insertions appeared to be present in the same site in these two plasmids; however, the deletions of the transposon were different. Plasmid sequence adjacent to the truncated transposon in the 25.2 MDa plasmids was found in a tetracycline‐sensitive N. gonorrhoeae 24.5 MDa conjugative plasmid. These data suggest that the 25.2 MDa plasmids are the result of one or a series of Class II Tn916‐like insertions into 24.5 MDa conjugative plasmids. Class II insertions of Tn916‐like transposons are implicated in the dissemination of tetM resistance in pathogenic Neisseria.

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

“…We have previously described the creation, by Tn916 insertion, of a mutant library of the serogroup B meningococcal strain NMB (45) and characterized two distinct classes of Tn916 insertions (class I intact or class II truncated) in this library (46). Transformants of the Tn916 library were screened by colony immunoblots with group B polysialic acid-specific monoclonal antibody 5C1-3 for mutants that were capsule defective (Cap Ϫ ).…”

“…We used the single-specific-primer PCR technique described by Shyamala and Ames (43) as described previously (46).…”

“…Standard PCRs were performed as described previously (46). PCR products were visualized by 1.2% agarose gel electrophoresis and UV detection after ethidium bromide staining.…”

Mutagenesis of Neisseria meningitidis by In Vitro Transposition of Himar1 mariner

“…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. Some of these drawbacks are absent in shuttle mutagenesis, where cloned neisserial DNA is mutated by transposition within E. coli and subsequently transferred into Neisseria, where it inactivates the corresponding genes via allelic exchange (33).…”

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

“…pJS1934 was sequenced with the Sequenase ds-DNA sequencing kit (United States Biochemical, Cleveland, Ohio). The junctions between the ends of the truncated transposon and the flanking meningococcal chromosomal sequence were then identified by comparison with known Tn916 sequences (19).…”