Figure 1.
Diagrammatical representation of the mod genes of N. meningitidis and N. gonorrhoeae.
The methylase (mod) genes, restriction endonuclease (res) genes, and repeat regions that mediate phase variation are indicated. Also shown are the conserved, characteristic motifs found within type III R-M systems, which include in mod: the catalytic region (DPPY) and the AdoMet (methyl donor) binding pocket (FXGXG) [66],[67], and in res: the ATP binding motif (TGxGKT) the motif linked to ATP hydrolysis (DEAH), and the endonuclease domain [68]–[70]. The mod and res genes are colored to indicate differences in homology between both mod genes and both res genes, respectively. A variable region within mod (highlighted in stripes) contains the DNA recognition domain [14]. The percent distribution of the mod alleles in a N. meningitidis serogroup B collection and a N. gonorrhoeae clinical isolate collection is shown to the right of each gene. Strains and accession numbers that define the mod alleles are shown to the left. n indicates the number of repeats (refer to Table S1 and Table S2 for exact repeat numbers). A black circle on a line and black square on a line indicate the position of a frame shift mutation and large deletion, respectively (Table S1 and Table S2). †, some modB1 strains contain a premature stop codon after the DPP motif (Table S1). *, one N. gonorrhoeae strain does not have the modB gene. Others, minor/infrequent alleles.
Figure 2.
Phylogenetic tree inferred from aligned modA genes belonging to a collection of 107 N. meningitidis isolates.
More than 500 trees were generated using Clonalframe from which a 95% majority-rule consensus tree was derived and imported into MEGA version 4.0 for further interpretation. (A) Each modA gene was annotated according to clonal complex. (B) Each modA gene was annotated according to the serogroup of the corresponding isolate distinguishing modA12 genes belonging to capsule A meningococci.
Table 1.
Distribution of modA alleles in the N. meningitidis MLST strain collection by serogroup.
Figure 3.
Differences in Mod expression from alternate initiation codons.
A chromosomally located modA::lacZ reporter fusion in N. meningitidis strain MC58 was used to determine expression from all three possible reading frames generated by different repeat numbers. (A) Schematic diagram showing that translation of the mod gene could be initiated from one of three frames (Distal, Proximal, or Off) depending on the number of 5′-AGCC-3′ repeats. (B) Phenotypic differences of colonies from each reading frame as observed on brain–heart infusion (BHI) S-gal plates. The arrow shows a phase variant colony that has switched from Distal to OFF. (C) β-galactosidase assay showing quantitative differences in the level of mod gene expression between Distal, Proximal, and Off. A unit is defined as mg of O-nitrophenyl hydrolyzed per min–1. A Student's t-test confirmed a significant difference between expression of Distal and Proximal (p = 0.0024). A small difference was observed between Proximal and OFF (p = 0.0146), but this can be accounted for by phase variation in the population of the modA11 gene from OFF to ON.
Figure 4.
Analysis of wild-type MC58 modA11 ON, MC58 modA11 OFF, and MC58 modA11::kan for LbpA and LbpB expression.
(A) Quantitative RT-PCR of lbpA and lpbB expression. Relative gene expression of lbpA and lbpB is higher in the MC58 modA11::kan mutant compared to wild-type MC58 modA11 ON. (B) Effect of modA11 phase variation on expression of the lbpB gene. β-galactosidase assays showed a statistically significant difference in the level of lbpB::lacZ gene expression resulting from modA11 repeat tract changes. A 1.9-fold difference in expression was observed between modA11 ON and modA11 OFF. P-values were calculated using a Student's t-test (C). The LbpA specific monoclonal antibody 296-H1 was used to assess expression of LbpA. The positions of molecular weight standard proteins are shown on the right in kilo Daltons (kDa). The left panel shows coomasie-stained wild-type MC58 modA11 ON, phase-variant MC58 modA11 OFF, and MC58 modA11::kan whole cells to show equal loadings of cell extracts. The right panel shows the Western blot of wild-type MC58 modA11 ON, phase-variant MC58 modA11 OFF, and MC58 modA11::kan whole cells probed with a monoclonal LbpA specific antibody.
Table 2.
A selection of differentially expressed genes from microarray expression studies.
Figure 5.
Identification of the ModA13 recognition methylation target sequence.
(A) ApoI restriction digest of plasmid pCmGFP isolated from FA1090 modA13 ON and FA1090 modA13::kan cells. The modA13 ON lane shows the presence of a 722-bp fragment that results from lack of restriction at a single ApoI restriction site. In the modA13::kan lane, this fragment is cut into fragments of 527 and 195 bp. (B) The ApoI recognition sequence showing percentage inhibition of restriction by methylation of each adenosine as indicated by REBASE [27], and a schematic diagram of the 722 bp pair fragment showing the ApoI recognition site, overlapping with a putative ModA13 recognition site. The central panels show Southern blots of chromosomal DNA extracted from modA13 ON and modA13::kan FA1090 cells. (C) DNA digested with ApoI and probed with a PCR product containing an ApoI/AGAAA overlap showed inhibition of digestion in the modA13 ON lane compared to the modA13::kan lane. (D) DNA digested with AluI and probed with a PCR product containing an AluI/AGAAA overlap showing no difference in restriction between the modA13 methylated and unmethylated chromosomes. (E) DNA digested with RsaI and HindIII, and probed with a PCR product containing a HindIII/AGAAA overlap, showed restriction is inhibited in the modA13 ON lane as compared to the modA13::kan lane. Below each blot is the recognition site for each of the restriction enzymes used, and their known sensitivities to adenosine methylation as supplied by REBASE in the case of ApoI and HindIII [27]. Schematics of the probes used in each blot include the coordinates of the FA1090 genome to which the primers bind (see Table S7) and the overlap of the restriction enzyme recognition sequence with that of ModA13. (F) Chromosomal DNA extracted from N. gonorrhoeae strains FA1090 modA13 ON, modA13 OFF, modA13::kan, and 96D551 modA12 ON and modA12::kan cells, digested with ApoI and probed as in (C).
Figure 6.
Comparison of wild-type FA1090 modA13 ON and FA1090 modA13::kan mutant in an antimicrobial resistance assay.
(A) Wild-type FA1090 modA13 ON and FA1090 modA13::kan mutant cells were serially diluted and spotted onto GC plates containing increasing concentrations of Triton X-100 (x-axis) for determination of viable colony-forming units (y-axis). The white bars correspond to wild-type FA1090 modA13 ON, and the black bars correspond to FA1090 modA13::kan. A Student's t-test showed a significant difference between the two samples (P≤0.021) at each of the following concentrations of Triton X-100; 40 µg/ml, 50 µg/ml, 60 µg/ml, and 80 µg/ml. (B) Shows the ratio of FA1090 modA13 ON to FA1090 modA13 OFF at each of the following concentrations of Triton X-100: 0 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, and 80 µg/ml for FA1090 modA13 ON. †, a statistically significant difference was seen in the ON/OFF ratio between FA1090 modA13 ON 0 µg/ml Triton X-100 and the following FA1090 modA13 ON Triton X-100 concentrations: 40 µg/ml, 50 µg/ml, 60 µg/ml, indicating a selection to OFF organisms at these concentrations. N/D indicates not done. Calculations are shown in Table S8.
Figure 7.
Biofilm formation by N. gonorrhoeae strain O1G1370 modA13::kan, modA13 OFF, and O1G1370 modA13 ON.
(A) Confocal microscopy of the biofilm mass over 2 days of growth for (1) N. gonorrhoeae O1G1370 modA13 ON, (2) O1G1370 modA13::kan, and (3) O1G1370 modA13 OFF. These images are three-dimensional reconstructions of stacked z-series taken at 200× magnification, which were rendered by Volocity. These experiments were performed in quadruplicate on three different occasions, and representative images are shown. (B) Scanning electron microscopy of the surface of the biofilm mass over 2 days of growth on glass taken at 5,000× magnification. It can be noted that there are fewer cells in the O1G1370 modA13 ON biofilm than either the O1G1370 modA13::kan or O1G1370 modA13 OFF biofilms. (C) Scanning electron microscopy of the surface of the biofilm mass over 2 days growth on glass taken at 15,000× magnification. (D) Transmission electron microscopy of 70 nm thin-sections of the biofilm mass over 2 days of growth on glass taken at 10,000× magnification. (E) COMSTAT analysis of biomass, average, and maximum thickness of confocal z-series images of the O1G1370 modA13::kan, O1G1370 modA13 OFF, and O1G1370 modA13 ON biofilms grown for 2 days over glass, which are depicted in (A). COMSTAT was performed for all replicates, and results are as shown. Statistical significance was determined using a Student's t-test. There was no statistically significant difference between the biomass, average, or maximum thickness of the O1G1370 modA13::kan and O1G1370 modA13 OFF strains.
Figure 8.
N. gonorrhoeae O1G1370 association with, and intracellular survival within, primary human cervical epithelial (pex) cells.
Pex cells were challenged with N. gonorrhoeae strain O1G1370 as outlined in the text. Data shown represent the invasion index (left panel) or the survival index (right panel) following challenge of pex cells as outlined in the text. The invasion index represents the percentage of pex cell–associated gonococci that survive gentamicin treatment; whereas the survival index is the percentage of invasive gonococci that survive, intracellularly, within pex cells at 3 h post-invasion. There was no significant difference between the naturally occurring O1G1370 modA13 OFF isolate and the O1G1370 modA13::kan “knockout” strain in either the invasion (P = 0.091) or survival (P = 0.23) indices observed. A statistically significant difference (*) was obtained in the invasion (P = 0.046) and survival (P = 0.021) indices when comparing O1G1370 modA13 OFF to O1G1370 modA13 ON, and in the invasion (P = 0.019) and survival (P = 0.004) indices when comparing O1G1370 modA13::kan to O1G1370 modA13 ON. P-values were determined using a Student's t-test. (B) Shows the ratio of O1G1370 modA13 ON to O1G1370 modA13 OFF of the inoculum, and at the invasion and survival time points for O1G1370 modA13 ON and O1G1370 modA13 OFF. †, a statistically significant difference was seen in the ON/OFF ratio between the O1G1370 modA13 OFF inoculum and the O1G1370 modA13 OFF survival sample (P = 0.0234), indicating a selection for OFF organisms over the course of the 3-h assay (for full data, see Table S8).