Identification, characterization, and variable expression of a naturally occurring inhibitor protein of IS1106 transposase in clinical isolates of Neisseria meningitidis

Abstract

Transposition plays a role in the epidemiology and pathogenesis of Neisseria meningitidis. Insertion sequences are involved in reversible capsulation and insertional inactivation of virulence genes encoding outer membrane proteins. In this study, we have investigated and identified one way in which transposon IS1106 controls its own activity. We have characterized a naturally occurring protein (Tip) that inhibits the transposase. The inhibitor protein is a truncated version of the IS1106 transposase lacking the NH(2)-terminal DNA binding sequence, and it regulates transposition by competing with the transposase for binding to the outside ends of IS1106, as shown by gel shift and in vitro transposition assays. IS1106Tip mRNA is variably expressed among serogroup B meningococcal clinical isolates, and it is absent in most collection strains belonging to hypervirulent lineages.

FIG. 1

Structure and nucleotide sequence of N. meningitidis IS1106. (A) Nucleotide sequence of IS1106 and deduced amino acid sequence of the putative transposase (IS1106T) gene. IS1106 has a length of 1,207 bp and is flanked by 19-bp-long IRs (IRL and IRR, arrows). Four-base-pair direct repeats (GGTC) border the ends of the IS. Amino acids corresponding to the IS1106T ORF are indicated in uppercase letters below the nucleotide sequence. Several control elements are also shown: (i) a14-bp-long perfect IR located 2 bp downstream from IRL (arrows), (ii) a repeated motif resembling part of the core sequence of the neisserial RS3 repeat (underlined sequences), and (iii) putative −10 and −35 promoter and Shine-Dalgarno (SD) sequences (asterisks). The nucleotide sequence downstream from the black arrowhead is also part of the rearranged element previously described (26). The sequence downstream from the open arrowhead is shared by IS1106Tip (Fig. 3B). (B) The deduced amino acid sequence of the putative IS1106T of N. meningitidis (N.m.) is aligned with the amino acid sequence of the 5A transposase protein of E. coli (E.c.) IS5. Plus signs indicate synonymous substitutions. The sequence downstream from the open arrowhead is shared by IS1106Tip (Fig. 3B). The overlined amino acids grouped in the N2, N3, and C1 regions are part of the DDE motif, the amino acid triad intimately involved in catalysis (32).

FIG. 2

Analysis of the IS1106-specific transcript. (A) Northern blot analysis. Total RNAs (10 μg) from carrier strains/isolates BF10, BF18, BF21, and BF8961 (lanes 1 to 4) and from invasive strains/isolates BL858, BS849, BL892, and BL947 (lanes 5 to-8) were hybridized to a 5′-end-labeled 1,126-bp DNA fragment spanning the entire coding region of IS1106T (Fig. 1A and Materials and Methods). Only the strand complementary to the RNA was labeled. Arrows indicate the IS1106-specific transcript and its approximate size deduced on the basis of the relative migration of the rRNAs. nt, nucleotides. (B) S1 nuclease mapping analysis. A 5′-end-labeled DNA fragment spanning the entire coding region of IS1106T (Fig. 1A) was used as a probe. The probe (lane 8) was hybridized to total RNA (10 μg) extracted from carrier strains/isolates BF10 and BF18 (lanes 3 and 4), from invasive strains/isolates BL858 and BS849 (panel B, lanes 5 and 6), or from yeast (lane 7). After treatment with S1 nuclease, the reaction products were resolved on a 6% polyacrylamide-urea denaturing gel. The sizes of the protected hybrids (arrows) were determined by running in parallel a sequencing reaction ladder (G and A, lanes 1 and 2) of the same DNA fragment used as a probe. The bar indicates the relative migration of the probe.

FIG. 3

Nucleotide sequence, deduced amino acid sequence, and structural features of the genomic region containing the transcriptionally active IS1106Tip gene. (A) Physical and genetic map of the genomic region containing the IS1106Tip gene. The IS1106Tip gene is located downstream of an IS1016-like element, which is preceded by the rho gene. The element is truncated at the 5′ end and is located immediately downstream from a neisserial SRE. Two copies of neisserial repetitive sequence RS3 are arranged in tandem downstream from the truncated element. The bent arrow indicates the transcription start point of the IS1106Tip mRNA. (B) Nucleotide and deduced amino acid sequences of the IS1106Tip gene. Arrows indicate IRs of the SRE (SRE-IRL and -IRR) and of IS1106 (IS1106-IRR). RS3 sequences are underlined. Bent arrows indicate the 5′ ends of the IS1106Tip mRNA. Asterisks mark the positions of a putative gearbox promoter sequence (5′-CACCAAGT-3′). Four nucleotide substitutions (indicated above the nucleotide sequence of BF18) were found in invasive strains/isolates BL859 and BL892 and map 12, 24, 82, and 86 nucleotides upstream of the transcription start site. Amino acid residues that are different from the deduced sequence of IS1106T (Fig. 1A) are underlined.

FIG. 4

Transcript mapping and translation of IS1106Tip. (A) S1 nuclease mapping of the putative start site(s) of the IS1106Tip transcript. Total RNAs (10 μg) from carrier strains/isolates BF10 and BF18 (lanes 5 and 6), from invasive strains/isolates BL858 and BS849 (lanes 3 and 4) or from yeast (lane 1) were hybridized to a 1,255-bp DNA fragment spanning the entire genomic region of IS1106Tip (Fig. 3 and Materials and Methods) that was labeled at the 5′ end of the strand complementary to the RNA (lane 2). The sizes of the protected hybrids (340 nucleotides [arrow] and 369 nucleotides [arrowhead]) were determined by running in parallel a sequencing reaction ladder (TCGA) of the same DNA fragment used as a probe. The bar indicates the relative migration of the probe. (B) In vitro transcription-translation of IS1106Tip gene. Plasmid pUH1I (lane 1) and derivative pUH1106Tip harboring the IS1106Tip gene (lane 2) were transcribed in vitro and translated by using an S30 extract derived from meningococcal strain BF18. The translation products were analyzed on an SDS–15% polyacrylamide gel. The bars on the left indicate vector-specific peptides. The arrow on the right marks the position of a specific translation product of about 16,000 Da produced by pUH1106Tip. The relative migration of molecular size markers is shown on the right.

FIG. 5

Dendrogram showing the genetic relatedness between meningococcal strains. The genetic distance between the meningococcal isolates was determined by comparing RFLP patterns in eight housekeeping genes and using collection strains as a reference as detailed in Materials and Methods.

FIG. 6

Southern blot analysis of IS1106 insertions in the genomes of meningococcal strains. (A) Physical and genetic map of an IS1106 element. The positions of the ClaI and HindIII sites are indicated. The dashed bar below the map corresponds to the 322-bp-long fragment used as a probe in the Southern blot experiment shown in panel B. (B) Total DNAs derived from the meningococcal strains indicated above the panels were digested with EcoRI and HindIII and hybridized to the 332-bp ³²P-labeled DNA fragment corresponding to the 5′-proximal one-third of the IS1106 sequence (A). The IS1106 sequence contains a unique HindIII site mapping downstream of the probe but does not contain any EcoRI site (A). Therefore, any band in the autoradiograph might be interpreted as an insertion of a single copy of either an intact or a truncated IS1106 element. The probe we used minimized the detection of rearranged elements because truncation of IS1106 occurred mostly at the 5′-proximal end (data not shown). The relative migrations of molecular size markers (sizes are in base pairs) are shown beside each panel. In addition to the names of the strains, their assignment to hypervirulent lineages is also indicated (lineage 3, ET-5, ET-37, IV-1, and other).

FIG. 7

DNA-protein interactions between the IS1106 ends and the transposase peptides. (A) Expression of recombinant histidine-tagged IS1106T and IS1106Tip in E. coli. S30 extracts derived from IPTG-induced or uninduced E. coli BL21 λDE3 cells harboring plasmid pET15b (lanes 1 and 2), pET1106Tip (lanes 3 and 4), or pET1106T (lanes 5 and 6) were analyzed by polyacrylamide gel electrophoresis. The arrows on the right indicate the relative migrations of recombinant IS1106T (Transposase) and IS1106Tip (Tip). The bars on the left indicate the positions of the molecular size markers that were run in parallel. (B) The DNA gel mobility shift assay was performed by incubating a 5′-end-labeled double-stranded oligonucleotide corresponding to the ends of IS1106 (lane 1) in the presence of partially purified, histidine-tagged IS1106T (lanes 2 to 4), histidine-tagged IS1106Tip (lanes 5 to 7), or a mixture of different amounts of both, as indicated (lanes 8 to 10). In lane 11, the probe was incubated with mock extract. Unlabeled competitor was added in 5- and 50-fold excesses over labeled DNA where indicated. The bars indicate specific DNA-protein complexes I, II, and III.