Multiple phase-variable type III R-M systems in pathogenic Neisseria

To investigate whether the type III R-M systems of the pathogenic Neisseria behave as a phasevarion [15], we first carried out a phylogenetic analysis of mod genes associated with type III R-M systems of N. meningitidis and N. gonorrhoeae. A comparison of the available genome sequences revealed that each strain contains two distinct phase variable mod genes, which we define as modA and modB, that share only 37% similarity to each other along the full length of the Mod deduced amino acid sequence. Both genes contain tracts of simple tandem repeats, 5′-AGCC-3′ (modA) and 5′-CCCAA-3′ (modB), that mediate phase variation of mod gene expression (Figure 1). ModA is highly homologous (>90% identity along the length of the Mod deduced amino acid sequence excluding the variable DNA recognition domain) to the mod gene of H. influenzae strain Rd (HI1058/56) [4],[15]. Differences in the modA DNA recognition domain [14] (Figure 1) have previously been observed in H. influenzae [16] with 17 distinct mod alleles defined in this organism (modA1–17; [17]). The Neisseria modA alleles present in the genome strains surveyed have the designations modA11, 12 and 13 and share >94% similarity to each other along the length of the Mod deduced amino acid sequence, excluding the variable DNA recognition domain. Our recent work also shows that the modA gene of H. influenzae and Neisseria are essentially the same gene with evidence of horizontal transfer of this gene in both directions between these organisms [17]. Unlike modA, the modB gene appears to be specific to Neisseria species. Two distinct modB alleles, modB1 and 2, distinguished by differences in their DNA recognition domain, were also observed (Figure 1). ModB1 and 2 share >95% similarity to each other along the length of the Mod deduced amino acid sequence, excluding the variable DNA recognition domain (which shares <33% identity).

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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.

https://doi.org/10.1371/journal.ppat.1000400.g001

To investigate whether additional alleles of modA and modB are present in these organisms, and to look at the distribution of mod alleles and their repeat sequence type and number, sequence analysis of a large, genetically diverse set of N. meningitidis and N. gonorrhoeae isolates was performed. This analysis revealed that all strains examined contained both modA and modB genes. Sequencing of the repeat region of the mod alleles revealed that the repeat numbers vary in length between different strains, resulting in the mod genes being in-frame (ON) or out-of-frame (OFF) for expression, consistent with phase variation of the mod genes (Table S1, Table S2). The N. gonorrhoeae strains contained either the modA12 or modA13 allele, and only the modB1 allele. One strain was found not to have a modB gene (Figure 1, Table S2).

A complete analysis of modA allele distribution was conducted in N. meningitidis, which has a well characterized population structure defined by multi locus sequence typing (MLST; [18]). The complete 107 strain MLST modA survey revealed that the majority of N. meningitidis strains had either the modA11 or modA12 allele, with modA15 found in two strains and modA4 and modA18 found in one isolate each (Figure 1, Figure 2A and 2B, Table S1). The most notable associations were in capsule type, where 100% of serogroup A strains and 92% of serogroup C strains contained the modA12 allele (Table 1, Figure 2B). Some association with clonal complex was also observed, with meningococci belonging to the ST-32 clonal complex predominantly harbouring the modA11 allele. Further clustering could be seen among ST-41/44 and ST-8 clonal complexes. Unlike N. gonorrhoeae, which contained only one type of modB allele, modB1, N. meningitidis strains contained either modB1 or modB2. There were seven strains, all from the ST-32 group, which contained point mutations in modB1 suggesting the gene is inactive in these strains (Figure 1, Table S1).

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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.

https://doi.org/10.1371/journal.ppat.1000400.g002

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Table 1. Distribution of modA alleles in the N. meningitidis MLST strain collection by serogroup.

https://doi.org/10.1371/journal.ppat.1000400.t001

ModA expression and phase variation

The modA genes of N. meningitidis and N. gonorrhoeae have two alternate initiation codons (Distal ATG and Proximal ATG) that are predicted to code for proteins of either 589 aa/640 aa or 707 aa/758 aa, depending on the number of tetranucleotide repeats that are present (Figure 3A). As this study is focused on modA phase variation and expression, a clear understanding of the relationship between tetranucleotide repeat number and modA expression was established. ModA expression was examined in the three frames; Distal, Proximal and OFF (which has no candidate ATG and a stop codon immediately after the 5′-AGCC-3′ repeats), by constructing a modA::lacZ reporter fusion in the N. meningitidis strain MC58 chromosome (Figure 3A and Figure S1). Maximal expression was found to be from the Distal ATG only and unlike H. influenzae strain Rd [15], minimal expression was observed from the Proximal ATG and the OFF frame (Figure 3B and 3C).

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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.

https://doi.org/10.1371/journal.ppat.1000400.g003

Natural modA ON and OFF colonies of N. meningitidis strain MC58 and N. gonorrhoeae strain FA1090 were required for microarray analysis and biological characterization experiments. N. meningitidis strain MC58 has 21 5′-AGCC-3′ repeats resulting in the modA11 gene being out-of frame (OFF) from the Distal ATG. Single colonies of MC58 were picked and screened by PCR and sequencing to find modA11 in-frame (ON) with the Distal ATG (see Figure 3). Similarly, single colonies of N. gonorrhoeae strain FA1090 were picked and screened by PCR and sequencing to find modA13 in-frame and also out-of-frame with the Distal ATG. During this process, ModA13 repeat tracts ranging from 13 (ON) to 26 (OFF) and also 37 (ON) were observed, demonstrating phase variation of N. gonorrhoeae strain FA1090 modA13 (results not shown).

Analysis of differentially expressed genes in N. meningitidis modA11 and modA12 phasevarions

Having established the relationship between modA repeats and modA expression (see Figure 3), we were in a position to conduct studies to determine whether phase variation of the various modA alleles in pathogenic Neisseria resulted in changes in gene expression. These studies were initiated with N. meningitidis strain MC58 modA11 gene, where the modA11 gene was inactivated by insertion of a kan^r cassette to make the mutant strain MC58 modA11::kan (Figure S2). Wild-type MC58 modA11 ON and MC58 modA11::kan were compared by microarray analysis using N. gonorrhoeae/meningitidis genome arrays (Materials and Methods). Initially, microarray analysis was performed using RNA isolated from wild-type MC58 modA11 ON and MC58 modA11::kan strains grown under standard culture conditions. However, these studies revealed no statistically significant difference in gene expression. Experiments were then performed in which N. meningitidis were cultured under iron-limiting conditions to more closely reflect in vivo conditions. Using this more physiologically relevant culture condition, many genes were found to have an expression ratio of 1.5-fold and over, with 162 genes up-regulated in MC58 modA11::kan relative to wild-type and 123 genes down-regulated, confirming modA11 phase variation has an influence on gene expression (Table 2, Table S3). Five of these genes encode surface exposed proteins, including NMB1540 (lbpA) and NMB1541 (lbpB), encoding LbpA and LbpB respectively, which are part of the lactoferrin receptor that allows acquisition and binding of iron from lactoferrin containing compounds. LbpA is the TonB-dependent integral outer membrane lactoferrin receptor and iron transport channel. LbpB is an accessory lipoprotein anchored to the outer membrane that contributes to lactoferrin binding/use [19],[20]. The lactoferrin receptor is a potential vaccine candidate in N. meningitidis [21]. Quantitative real time PCR (RT-PCR) was performed to confirm that the lbpA and lbpB genes were expressed at a higher level in MC58 modA11::kan compared to the MC58 modA11 ON parent strain (see Figure 4A). Altered expression was further confirmed by an lbpB::lacZ fusion (Figure S3) located on the chromosome of each strain, which showed ∼2-fold higher expression in the modA11::kan mutant strain compared to the modA11 ON parent strain (see Figure 4B). Consistent with this, western blot analysis confirmed the effect of modA11 phase variation on expression of LbpA, with an apparent reduction in expression when modA11 is ON (Figure 4C). The same effect is seen when comparing a wild-type modA11 ON strain to either a modA11::kan mutant or a natural phase variant in which the modA11 gene had switched OFF due to an alteration in the 5′-AGCC-3′ repeat tract (from 22 to 21 AGCC repeats; see Figure 4C), confirming the regulation of LbpA by ModA11 is not related to the use of a kan^r inactivated modA11 gene.

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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.

https://doi.org/10.1371/journal.ppat.1000400.g004

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Table 2. A selection of differentially expressed genes from microarray expression studies.

https://doi.org/10.1371/journal.ppat.1000400.t002

A similar microarray study was conducted using a N. meningitidis modA12 clinical isolate, B6116/77. To determine whether phase variable expression of the N. meningitidis strain B6116/77 modA12 gene also led to alteration in global gene expression, the modA12 gene was inactivated by insertion of a kan^r cassette to make the mutant strain B6116/77 modA12::kan (Figure S2). Wild-type B6116/77 modA12 ON and B6116/77 modA12::kan were compared by microarray analysis using N. gonorrhoeae/meningitidis genome arrays. Experiments were performed under the same conditions as described above. Twenty six genes were found to have an expression ratio of 1.5-fold and over, with 14 genes up-regulated in B6116/77modA12::kan relative to wild-type and 12 genes down-regulated, confirming modA12 phase variation has an influence on gene expression (Table 2, Table S4). The set of genes differentially expressed in the modA12 mutant were different to the modA11 set of genes. This is consistent with the differences in the DNA recognition domain between modA11 and modA12, and confirms that these distinct alleles control different phasevarions in N. meningitidis.

Analysis of differentially expressed genes in N. gonorrhoeae modA13

An additional phasevarion study was conducted in N. gonorrhoeae. In this case, a modA13 knockout mutant was constructed by interrupting the modA13 gene with a kan^r cassette (Figure S2). Comparison of the phenotype of the FA1090 modA13::kan mutant strain with wild-type FA1090 modA13 ON formed the basis of expression and phenotypic studies.

Global gene expression was compared between wild-type FA1090 modA13 ON and FA1090 modA13::kan under iron-limiting conditions. 34 genes were up-regulated in FA1090 modA13::kan relative to wild-type, and 20 genes were down-regulated (Table 2, Table S5). Five of the differentially regulated genes have obvious roles in virulence; four in oxidative stress and one in antimicrobial resistance. NGO0929 (metF) and NGO0928 (metE) are part of the MetFE operon, which plays a role in the methylation of homocysteine, the final step of methionine biosynthesis, and is involved in defence against oxidative stress [22]. NGO0554 encodes a gonococcal-specific hypothetical protein that is shown to protect against damage caused by high levels of H₂O₂ [23]. NGO0650 (recN) encodes the DNA repair protein RecN. The gonococcal RecN protein is demonstrated to be involved in DNA repair and DNA transformation [24] and plays an important role in H₂O₂ damage protection as well as resistance to killing by polymorphonuclear leukocytes [25]. NGO1368 (mtrF) encodes the inner membrane protein, MtrF, and has been shown to have a role in antimicrobial resistance [26].

Our phylogenetic analysis revealed that N. gonorrhoeae strains have one of two distinct modA alleles (modA12 or 13; see Figure 1), indicating that different phasevarions may exist within N. gonorrhoeae and that strains with the same mod allele may regulate similar sets of genes. To determine if a strain with the same DNA recognition domain as FA1090 (modA13 allele) would result in the same set of genes being regulated, we chose a N. gonorrhoeae clinical isolate, strain O1G1370, from a representative set of N. gonorrhoeae strains (Table S2) that also contains a modA13 allele. A modA13::kan knockout mutant was made using the same approach as described for FA1090 (Materials and Methods). Quantitative RT-PCR on the metF, mtrF and NGO1581 genes confirmed that metF and mtrF, which are up-regulated in expression in the FA1090 modA13::kan mutant, are also up-regulated in the O1G1370 modA13::kan mutant (Table 2). NGO1581, which was down-regulated in the FA1090 modA13::kan mutant, is also down-regulated in the O1G1370 modA13::kan mutant (Table 2). Furthermore, similar results were seen with quantitative RT-PCR on the same set of genes when comparing a wild-type FA1090 modA13 ON strain to a natural phase variant of FA1090 in which the FA1090 modA13 gene had switched OFF due to an alteration in the 5′-AGCC-3′ repeat tract (from 37 to 26 AGCC repeats) (Table 2), confirming the regulation of this set of genes by ModA13 is not related to the use of a kan^r inactivated modA13 allele.

When we conducted microarray analysis of a N. gonorrhoeae modA12 strain 96D551, comparison of 96D551 modA12 ON and 96D551 modA12::kan (OFF) strains revealed a distinct set of genes being regulated compared to the modA13 data above (Table 1, Table S6). These results are consistent with the differences in the DNA recognition domain between modA13 and modA12 (see below), and supports the idea that these distinct alleles control different phasevarions in N. gonorrhoeae. However, unlike the modA11, modA13 and modA12 (N. meningitidis) expression studies described above, the 96D551 modA12 ON and 96D551 modA12::kan (OFF) strains showed a significant difference in growth rate for the cultures used to make RNA (Figure S4D). We cannot rule out the possibility that these differential growth rates may have influenced the gene expression data in Table 1 and Table S6.

N. gonorrhoeae strain FA1090 ModA13 recognition site is 5′-AGAAA-3′

In all cases, phase variation or mutagenesis of modA of pathogenic Neisseria results in altered gene expression, defining these systems as functional phasevarions. In order to determine whether the observed changes in modA expression correspond to global changes in DNA methylation, thereby indicating this as the likely mechanism of gene control, it was necessary to identify one or more of the modA target sites. In addition to confirming global changes in methylation, target site identification would also facilitate future studies on the molecular mechanisms operating at individual promoters within the phasevarion. In order to identify methylation target sites, a strategy based on inhibition of DNA restriction was used. In initial studies, plasmid pCmGFP was isolated from N. gonorrhoeae or N. meningitidis modA11, modA12 or modA13 ON strains and their corresponding modA::kan mutants, and digested with a range of restriction enzymes known to be inhibited by methylation of an adenine within their recognition sequence (see Materials and Methods). Differences in digestion patterns between plasmid extracted from modA ON cells (ModA methylated DNA) and modA::kan cells (DNA not methylated by ModA) would indicate an overlap of the respective ModA methylated target and the restriction enzyme used. No such fortuitous inhibition pairs were seen with modA11 or modA12 strains, but were with modA13. Figure 5A shows an obvious difference in the restriction pattern of plasmid extracted from modA13 ON and modA13::kan cells, indicating overlap between the ModA13 site and ApoI. The recognition sequence of ApoI is 5′-RAATTY-3′. The specific ApoI site displaying inhibition (5′-AAATTC-3′) was not unique on the plasmid. Comparison between the inhibited ApoI site and other ApoI sites whose sequence was also 5′-AAATTC-3′ revealed that the overlap with ModA13 must be 5′ to the ApoI site. As seen in Figure 5B, methylation of the different adenines in the ApoI recognition site inhibits digestion to varying degree. Since over-digestion did not result in the 722 bp band being cleaved into the two smaller fragments, it can be assumed that the first adenine of the ApoI sequence was not the methylation target, as methylation of this adenine would result in only 10% inhibition of restriction [27], an effect which could potentially be overcome by over-digestion. Therefore, depending on which of the other two adenines was methylated, the ModA13 recognition sequence must be found within 5′-CAGAAA-3′.

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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).

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To confirm which adenine is the ModA13 methylation target, and to further specify the ModA13 recognition sequence, overlaps of the putative ModA13 recognition sequence and ApoI restriction sites were identified on the FA1090 chromosome. Chromosomal DNA was extracted from FA1090 modA13 ON and FA1090 modA13::kan cells, digested with ApoI and examined by Southern blot. Inhibition of ApoI restriction at the internal ApoI/ModA13 overlap results in the presence of one large band at 3408 bp, while the unmethylated DNA (modA13::kan) is cleaved at this site by ApoI into two smaller bands of 2500 and 908 bp (Figure 5C). This confirms that ApoI restriction can be inhibited in DNA methylated by ModA13, as shown by the plasmid digest (Figure 5A). Similar studies were done with three other ApoI restriction sites in the genome that overlap ModA13 with 5′-TAGAAA-3′, 5′-GAGAAA-3′ or 5′-AAGAAA-3′. In each of these cases, ApoI restriction of ModA13 methylated DNA was inhibited compared with DNA extracted from modA13::kan cells (data not shown). This confirms the ModA13 recognition sequence to be 5′-AGAAA-3′.

To identify which of the two potential adenines in 5′-AGAAA-3′ is methylated, ModA13 recognition sequences in the FA1090 chromosome were identified which overlapped with restriction enzymes other than ApoI and were known to be inhibited by methylation of adenines. Two of these enzymes were HindIII (5′-AAGCTT-3′) and AluI (5′-AGCT-3′). Although the recognition sequence of HindIII contains two adenines (Figure 5E), both these adenines are part of the ModA13 recognition sequence. Methylation of the adenine in the AluI recognition sequence is known to result in complete inhibition [28]. When chromosomal DNA digested with AluI is probed using a PCR product containing an AluI/ModA13 overlap (Figure 5D) no difference in restriction is seen between the modA13 ON and modA13::kan lanes, indicating that the common adenine in this overlap is not methylated by ModA13. This suggests that ModA13 methylates AGAAA on the second most 3′ adenine of recognition site 5′-AGAAA-3. Information on the sensitivity of HindIII to hemimethylation is only known for the 5′ adenine of the HindIII recognition site. Hemimethylation of this adenine results in a 95% inhibition of restriction [27]. Using a random site in the FA1090 genome where the overlap between the ModA13 target site and HindIII resulted in the 5′ adenine of HindIII site corresponding to the second last adenine of the AGAAA (see Figure 5E) we were able to determine whether this was the residue methylated by ModA13. The results shown in Figure 5E confirm the expected restriction inhibition phenotype [27] allowing us to conclude that the ModA13 methylation site is AGAA^mA, with the methyl group being added to the third adenine in the sequence.

Having established the ModA13 target sequence, we tested DNA derived from a modA12 strain and confirmed that the ModA12 target site was distinct as there is no difference between modA12 ON and modA12::kan (OFF) DNA in a ModA13/ApoI inhibition assay (Figure 5F). Analysis of the FA1090 genome has revealed a total of 5135 ModA13 target sites.

The role of the N. gonorrhoeae modA13 phasevarion in model systems

To determine whether the phasevarion mediated changes in gene expression correspond to altered phenotypes in model systems, we chose to focus on the modA13 allele of N. gonorrhoeae. Several model systems were available from our previous studies on oxidative stress, biofilm formation and bacterial - host cell interactions [29]–[32]. Furthermore, strains FA1090 and O1G1370 provided an opportunity to test the reproducibility of key phenotypes in two independent modA13 strains.

Phasevarion switching alters resistance to an antimicrobial agent

Previous studies using N. gonorrhoeae strain FA19 demonstrate that mtrF is required for induction of high-level antimicrobial resistance to Triton X-100 by gonococci [33]. Our data show that MtrF expression is up-regulated in the modA13 mutant relative to the wild-type under iron-limiting conditions. To test whether differences in antimicrobial-resistance could be observed between wild-type FA1090 modA13 ON and the FA1090 modA13::kan mutant, an antimicrobial-resistance assay was performed using increasing Triton X-100 concentrations (Figure 6A). The FA1090 modA13::kan mutant was found to be more resistant than wild-type FA1090 modA13 ON, consistent with the higher level of expression of MtrF in this modA13 OFF strain. As modA13 ON is free to phase vary to OFF, and OFF cells appear to be fitter in this assay, the status of modA13 expression was monitored by PCR with fluorescent primers across the repeat region to determine whether ON to OFF phase variants had been selected in the survivor colonies at various Triton X-100 concentrations. This analysis revealed that the FA1090 modA13 ON culture plated on zero Triton X-100 remained ON, with only 11.21% OFF cells. However, cells plated on increasing Triton X-100 concentrations changed to 46.99% OFF, 80.29% OFF and 80.15% OFF over the course of the assay for 40, 50 and 60 µg/ml Triton X-100, respectively (Figure 6B).

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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.

https://doi.org/10.1371/journal.ppat.1000400.g006

Phasevarion switching alters efficiency of biofilm formation in modA13 strains

A number of studies have shown that N. gonorrhoeae can form a biofilm in a continuous-flow chamber and over primary human genital tract epithelial cells in culture [30],[31]. Biofilms provide a number of advantages in survival of the bacteria. It is suggested that biofilm formation by N. gonorrhoeae may contribute to its ability to persist in an asymptomatic state in the female genital tract [34]. In addition, bacteria within biofilms show increased resistance to antimicrobial agents [35],[36] and links between biofilm formation and oxidative stress defenses have been observed in N. gonorrhoeae [30].

The ability of N. gonorrhoeae O1G1370 modA13 ON, O1G1370 modA13 OFF and O1G1370 modA13::kan (OFF) to form a biofilm was evaluated after two days of growth under continuous flow conditions. Three-dimensional images of these biofilms were created in Volocity (Materials and Methods). These images show that O1G1370 modA13::kan and modA13 OFF form a thick and dense biofilm, while O1G1370 modA13 ON forms an extremely weak biofilm with a few sparse patches of cells scattered across the surface of attachment (Figure 7A). The O1G1370 modA13 ON strain also formed biofilms with lower maximum thicknesses than the O1G1370 modA13::kan and O1G1370 modA13 OFF strains. (Figure 7D). Scanning electron microscopy of the surface of the biofilm taken at 5,000× magnification shows that there are gaps between clusters of biofilm in the O1G1370 modA13 ON strain, unlike the O1G1370 modA13 OFF and O1G1370 modA13::kan strain biofilms, where there are no areas where the glass surface of attachment is visible. There are also large areas where no biofilm is present in the O1G1370 modA13 ON samples (Figure 7B). Scanning electron microscopy taken at 15,000× magnification shows that O1G1370 modA13::kan and O1G1370 modA13 OFF form biofilms that are tightly enmeshed in an extracellular material that obscures the structure of individual cells, while cells in the modA13 ON biofilm are clearly distinguishable (Figure 7C). Transmission electron microscopy shows that O1G1370 modA13::kan forms a biofilm where individual cells are shedding copious amounts of membrane, as seen in the numerous enclosed membrane blebs on the surface of the cells, while there is no evidence of blebbing in the O1G1370 modA13 ON biofilm. Cells in the O1G1370 modA13 OFF biofilm also appear to be blebbing, like those in O1G1370 modA13::kan biofilm, as numerous blebs can be seen forming on the surface of the O1G1370 modA13 OFF strain. These electron micrographs suggest that the extracellular matrices of the O1G1370 modA13::kan and O1G1370 modA13 OFF biofilms may be at least partially composed of fused membrane blebs (Figure 7C). COMSTAT [37] was used to quantitatively assess the biomass, and average and maximum thickness of confocal z-series photomicrographs taken for each flow chamber. COMSTAT analysis showed that the O1G1370 modA13::kan and O1G1370 modA13 OFF strains form significantly thicker biofilms with significantly more biomass than the O1G1370 modA13 ON strain. Specifically, O1G1370 modA13 ON had 3.5% of the biomass and 4.2% of the thickness of the O1G1370 modA13::kan mutant on average and 4.7% of the biomass and 5.2% of the thickness of the O1G1370 modA13 OFF (Figure 7E). Similar results were observed using N. gonorrhoeae strains FA1090 modA13 ON and FA1090 modA13::kan (Figure S5).

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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.

https://doi.org/10.1371/journal.ppat.1000400.g007

ModA13 phase variation results in the altered fitness of N. gonorrhoeae strain O1G1370 to survive within primary human cervical epithelial cells

The use of primary human cervical epithelial (pex) cells as a model system of gonococcal cervicitis is well established and has been used in a number of studies, such as the examination of the role of oxidative stress regulators in host-pathogen interactions [29],[32]. To determine the biological significance of O1G1370 modA13 expression using this pex cell culture model, we performed quantitative association, invasion, and survival assays using O1G1370 modA13 ON, O1G1370 modA13 OFF, and O1G1370 modA13::kan mutant gonococci (Figure 8). These data revealed that there was no significant (P≥0.2338) difference in the ability of the O1G1370 modA13 OFF and O1G1370 modA13::kan strains to adhere to, invade, or survive within pex cells. This confirmed that the modA13::kan knockout allele behaves in the same way as a natural phase variant modA13 OFF strain. In contrast, behavior of the O1G1370 modA13 ON strain was significantly (P≤0.001) different from that obtained with the use of either the O1G1370 modA13 OFF or O1G1370 modA13::kan strains in parallel assays. In this regard, a modA13 ON phenotype resulted in the increased ability of gonococci to associate with pex cells, whereas a modA13 OFF configuration augmented the ability of gonococci to invade (Figure 8A, invasion index) and survive within pex cells following invasion (Figure 8A, survival index). These data suggest a possible role for Mod-dependent phase variation in promoting the adaptive changes required for gonococci to switch from an extracellular to an intracellular existence. This idea is supported by our observation of selection for a switch from ON to OFF in the O1G1370 modA13 ON strain. Fragment analysis confirmed that the O1G1370 modA13 ON inoculum, which contains only 5.86% OFF, changes to ∼49.84% OFF by the time the 3 hour intracellular survival sample was taken (Figure 8B, Table S9). An independent N. gonorrhoeae modA13 strain, 1291, displayed the same intracellular survival and modA13 switching phenotype (Figure S6).

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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).

https://doi.org/10.1371/journal.ppat.1000400.g008

Bacterial strains and growth conditions

N. meningitidis and N. gonorrhoeae strains were grown at 37°C with 5% CO₂ in either GC broth or GC agar with IsoVitaleX (Becton Dickinson). E. coli strains DH5α and JM109 (Promega) were used to propagate plasmids and were grown at 37°C in Luria-Bertani (LB) broth supplemented with either ampicillin (100 µg/ml) or kanamycin (100 µg/ml).

DNA manipulation and analysis

All enzymes were sourced from New England Biolabs. Sequencing was performed on PCR products using QiaQuick PCR purification kit (Qiagen) and Big-Dye (Perkin Elmer) sequencing kits. Data was analysed using MacVector v9.0 (Accelrys).

Mod allele specific PCR

PCR products specific for the DNA recognition domain and repeat regions of modA and modB were generated using the primers listed in Table S7. N. meningitidis isolates [58] and N. gonorrhoeae DGI and MI clinical isolates [59] were used as templates. The reaction was performed in 50 µl using 1× Taq buffer, 1.5 mM MgCl₂, and 1 unit of Taq DNA polymerase (Promega) with the following cycling conditions for the DNA recognition domain: 30 cycles of 94°C for 30 sec, 57°C for 30 sec, 72°C for 2 min and 1 cycle of 72°C for 7 min with 5 µM of the primer pair ModADRDF and ModADRDR for modA or ModBDRDF and ModBDRDR for modB. For modA, a 597 bp region containing the DNA recognition domain (393 bp downstream of ModADRDF and 101 bp upstream from ModADRDR) was compared to the genome strains to determine the modA allele group. For modB a 537 bp region containing the DNA recognition domain (461 bp downstream from ModBDRDF and 285 bp upstream from ModBDRDR) was compared to the genome strains to determine the modB allele group. The following cycling conditions were used for the repeat region: 30 cycles of 94°C for 30 sec, 57°C for 30 sec, 72°C for 30 sec and 1 cycle of 72°C for 7 min with 5 µM of the primer pair ModAF and ModAREPEATR or ModBREPEATF and ModBREPEATR. PCR products were cleaned using the QIAquick PCR Purification Kit (Qiagen).

Res specific PCR

PCR products specific for the res gene were generated using the primers listed in Table S7. N. meningitidis isolates [58] and N. gonorrhoeae DGI and MI clinical isolates [59] were used as templates. The reaction was performed in 50 µl using 1× Taq buffer, 1.5 mM MgCl₂, and 1 unit of Taq DNA polymerase (Promega) with the following cycling conditions for the DRD: 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 3 min and 1 cycle of 72°C for 7 min with 5 µM of the primer pair ResF and ResEDR2. PCR products were sequenced using ResF, ResR, ResEDF2 and ResEDR2.

Nucleotide sequence manipulation and analysis

The modA nucleotide sequences were assembled using the Staden sequence analysis package [60] and all sequences aligned manually in the Seqlab alignment program (Genetics Computer Group, Madison, Wis.). Phylogenetic analysis was undertaken using the software package ClonalFrame version 1.1, which is a statistical model-based method initially described for inferring bacterial clonal relationships using multilocus sequence data [61]. Inference is performed in a Bayesian framework and a neutral coalescent model is assumed based on the hypothesis that the bacteria in the sample come from a constant-sized population in which each bacterium is equally likely to reproduce, irrespective of its previous history. The key assumption of ClonalFrame is that recombination events introduce a constant rate of substitutions to a contiguous region of sequence with the end result that a clonal frame can be inferred. In the present study, over 50,000 iterations were performed with every hundredth tree sampled after which, a 95% majority-rule consensus tree was derived. ClonalFrame is available at available at http://bacteria.stats.ox.ac.uk. The modA gene is composed of relatively conserved N and C-terminal regions with the DNA recognition domain in between. Consequently, sequence input into ClonalFrame was undertaken by firstly adding the N-terminal region starting at bp 359 in the modA gene belonging to the reference N. meningitidis isolate MC58, followed by the DNA recognition domain occurring from 416 to 795 bp and ending with the C-terminal region 796 to 1242 bp. Annotation was then undertaken by importing the tree into the Molecular Evolutionary Genetics Analysis software package (MEGA ver 4.0) [62].

Plasmid pCmGFP (Source M A Apicella) was extracted from N. meningitidis strain C311. Primers used to sequence this plasmid are listed in Table S7. Sequencing reactions were prepared using the plasmid as template and Big-Dye sequencing kit (Perkin-Elmer). Samples were analysed using a 3130×l Capillary Electrophoresis Genetic Analyser (Applied Biosystems International). Data were analysed and plasmid map constructed using MacVector (version 9.0). The plasmid sequence is deposited in GeneBank under accession number FJ172221).

Identification of ModA13 modification site by inhibition of restriction assays

Construction of a translation fusion between the modA gene and lacZ gene and insertion into N. meningitidis strain MC58

A modA::lacZ fusion was constructed in N. meningitidis MC58. The gene fusion was initially constructed in E. coli with subsequent transformation into the N. meningitidis chromosome. In the fusion construct, the codons for LacZ are in the same translational frame as ModA resulting in an in-frame Mod-LacZ fusion protein. A 4 kb fragment of a promoterless lacZ::kan fragment was amplified by PCR using the primer pair LacZStyI1 and KanStyI. The plasmid pBluescriptlacZ::kan was used as template. Following digestion with StyI, the 4.0 kb lacZ::kan fragment was then ligated into the XbaI site of pGEMmodA. The ligation mixture was transformed into E. coli JM109 and transformants were selected on LB agar plates supplemented with kanamycin (50 µg/ml; Sigma). The orientation and sequence of the insert were checked and found to be correct. The resulting construct was named pGEMmodA::lacZ::kan. This plasmid was linearized with SacII and used to transform competent N. meningitidis. The MC58modA::lacZ::kan transformants were streaked on BHI plates containing Levinthal supplement and X-gal (40 µg/ml).

Construction of knockout mutants of the modA11, modA12, and modA13 gene and insertion into N. meningitidis strain MC58, N. meningitidis strain B6116/77, N. gonorrhoeae strain FA1090, clinically isolated N. gonorrhoeae strains O1G1370 and 96D551

The modA open reading frame (ORF) was amplified using PCR with primers ModAF and ModAR (see Figure S1). N. meningitidis strain MC58 was used as template. The PCR product was cloned into vector pGEM-Teasy (Promega) and named pGEMmodA. The pGEMmodA construct was digested with XbaI and blunt ended using Klenow Polymerase (New England Biolabs). The Tn903 kan resistance gene from the pUC4K vector (Pharmacia) was excised using HincII and inserted into the blunt XbaI site. Previous work has demonstrated that the pUC4Kan kanamycin cassette has no promoter or terminator that is active in Neisseria and will neither affect transcription nor have a polar effect on expression of adjacent genes [29]. The resulting plasmid, pGEMmodA::kan was linearized by digestion with SphI and used to transform competent N. meningitidis strains MC58 and B6116/77 or N. gonorrhoeae strains FA1090, O1G1370 or 96D551. MC58 modA11::kan, B6116/77 modA12::kan, FA1090 modA13::kan O1G1370 modA13::kan and 96D551 modA12::kan transformants were selected on BHI plates containing Levinthal supplement and 100 µg/ml kanamycin. Transformants were confirmed by PCR and sequence analysis using primers ModAF2 and kanamycin specific primers. RNA midi-preps of both the wild-type (MC58 modA11 ON, B6116/77 modA12 ON, FA1090 modA13 ON and O1G1370 modA13 ON) and mutant (MC58 modA11::kan, B6116/77 modA12::kan, FA1090 modA13::kan and O1G1370 modA13::kan) were made using the RNeasy Midiprep kit (Qiagen). Wild-type colonies, from which RNA was isolated for microarray analysis, were sequenced using primers ModAF and ModAREPEATR to check that the mod repeat region was in-frame.

RNA extraction

Triplicate cultures of N. meningitidis strain MC58 modA11 ON and the MC58 modA11::kan mutant, N. meningitidis strain B6116/77 modA12 ON and the B6116/77 modA12::kan mutant or N. gonorrhoeae strain FA1090 modA13 ON and the FA1090 modA13::kan mutant, O1G1370 modA13 ON and the O1G1370 modA13::kan and 96D551 modA12 ON and 96D551 modA12::kan mutant were grown to exponential phase (optical density at 600 nm = 0.5 to 0.6) with 30 µM desferal (Sigma) in GC broth prior to RNA extraction. Growth rates of strain pairs used to make RNA for microarray comparison were determined (Figure S4) and were found to be equivalent ensuring that the samples taken were in the same growth phase. Only 96D551 modA12 ON and 96D551 modA12::kan (OFF) strains showed a significant difference in growth rate (see Figure S4). Culture media for RNA preps was free of antibiotics as once the modA::kan mutation is transferred to the chromosome by double crossover we observed that it is stable without selection. Approximately 100 µg of total RNA was prepared from each sample using the RNeasy Maxi Kit according to the manufacturer's instructions (Qiagen). The triplicate samples were pooled and the integrity and concentration of RNA was determined via micro-fluidic analysis on a bio-analyser (Agilent Technologies).

Microarray analysis

All microarray analysis was performed on N. gonorrhoeae/meningitidis genome arrays (TIGR; http://pfgrc.tigr.org/). Each microarray consists of 6,389 70mer oligonucleotides representing open reading frames (ORFs) from N. gonorrhoeae strains FA1090 and ATCC 700825 (reference strain), as well as N. meningitidis strains Z2491 (serogroup A) and MC58 (serogroup B). Methods and analysis were as previously described [29]. All primary data was imported into an in-house installation of the comprehensive microarray relational database, BASE (accessible at: http://kidney.scgap.org/base login: Nmmod, password: Nmmod, login: NmmodA12, password: NmmodA12, login: NgmodA12, password: NgmodA12 or login: Ngmod, password: Ngmod).

Quantitative real-time PCR

Oligonucleotides (Table S7) were designed using Primer Express 1.0 software (ABI Prism; PE Biosystems) and are named according to the ORF being amplified. All real-time PCR reactions were performed in a 25 µl mixture containing 1/5 volume of cDNA preparation (5 µl), 10XSYBR Green buffer (PE Applied Biosystems) and 2 µM of each primer. We used 16S RNA as the standard control in each quantitative PCR. Amplification and detection of specific products were performed with the ABI Prism 7700 sequence-detection system (PE Applied Biosystems) with the following cycle profile: 95°C for 10 min, followed by 45 cycles of 95°C for 15 sec and 60°C for 1 min. Data was analysed with ABI prism 7700 (version 1.7) analysis software. Relative gene expression between the MC58 modA11::kan mutant and wild-type MC58 modA11 ON, N. meningitidis strain B6116/77 modA12 ON and the B6116/77modA12::kan mutant or the FA1090 modA13::kan mutant and wild-type FA1090 modA13 ON was determined using the 2^ΔΔCT relative quantification method.

Semi-quantitative RT-PCR

Total RNA was isolated using the RNeasy kit (Qiagen). The equivalent of 1 µg of the total RNA preparation was treated with RQ1 RNase-free DNase (Promega). RT-PCR was performed using the TaqMan RT-PCR kit (PE Applied Biosystems) as recommended by the manufacturer. PCR was carried out in 50 µl using 1× Taq buffer, 1.5 mM MgCl₂, and 1 unit of Taq DNA polymerase (Promega) and cDNA amplified using gene specific primers designed for Real-time PCR (Table S7) with the following cycling conditions: 30 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 30 s and 1 cycle of 72°C for 7 min. 16S rRNA internal standards for comparison were used with amplification resulting in a 200 bp RT-PCR product. PCR products (20 µl) were run on a 3% agarose gel.

Growth studies comparing wild-type mod ON to the modA::kan mutants

Growth experiments were carried out in GC medium supplemented with IsoVitaleX, at 37°C with 5% CO2, under iron-limiting conditions (30 µM desferal). Triplicate cultures of the strain pairs being compared were adjusted to an identical initial OD₆₀₀. One milliliter of culture was removed at fixed times to measure the OD₆₀₀.

Analysis of LbpA expression

Wild-type MC58 modA11 ON, wild-type MC58 modA11 OFF and MC58 modA11::kan mutant bacterial cells were grown under iron-limiting conditions to an optical density at 600 nm = 0.55–0.6. Cells were spun down at 5000 rpm for 5 min and then washed once in PBS, pH 7.2. Cells were then re-suspended in PBS to an optical density at 600 nm = 2.5 and separation was carried out on a 4–12% Nu-PAGE Novex Bis-Tris gel (Invitrogen) according to the manufacturer's instructions. The Nu-PAGE semi-dry system was used to transfer protein from gel to nitrocellulose membrane (0.22 µM pore, Bio-Rad), as recommended by Invitrogen. Immunoblotting of membranes was carried out in a 1∶2000 dilution of LbpA specific monoclonal 269-H1 [19] in 5% skimmed milk powder in TBS. Bands were visualized following incubation in 1∶5000 dilution of alkaline phosphatase-conjugated anti-mouse IgG secondary antibody (Sigma).

Construction of a translation fusion between the lbpB gene and lacZ gene and insertion into N. meningitidis strain MC58

An lbpB::lacZ fusion was constructed in N. meningitidis strain MC58. The gene fusion was initially constructed in E. coli with subsequent transformation into the N. meningitidis chromosome. In the fusion construct, the codons for LacZ are in the same translational frame as lbpB resulting in an in-frame LbpB-LacZ fusion protein. A 1.7 kb DNA fragment was amplified by PCR using the primer pair LbpBF and LbpBR. MC58 was used as the template. The reaction was performed in 50 µl using 1× Taq buffer, 1.5 mM MgCl₂, and 1 unit of Taq DNA polymerase (Promega) with the following cycling conditions: 30 cycles of 94°C for 30 sec, 57°C for 30 sec, 72°C for 1 min and 1 cycle of 72°C for 7 min. The fragment was then cloned into vector pGEM-Teasy (Promega). A 4 kb fragment of a promoterless lacZ::kan fragment was amplified by PCR using the primer pair LacZStyI+1 and KanStyI. The plasmid pBluescriptlacZ::kan (M. Dieckelman, personal communication) was used as template. Following digestion with StyI, the 4.0 kb lacZ::kan fragment was blunted using Klenow Polymerase and then inserted into the EcoRV site of the lbpB construct. The ligation mixture was transformed into E. coli JM109 and transformants were selected on LB agar plates supplemented with kanamycin (50 µg/ml). The orientation and sequence of the insert were checked and found to be correct. The resulting construct was named pGEMlbpB::lacZ::kan. This plasmid was linearized with NcoI and used to transform competent N. meningitidis strain MC58 with a naturally derived number of mod ON and OFF repeats. The MC58lbpB::lacZ::kan mod ON and MC58lbpB::lacZ::kan mod OFF transformants were streaked on BHI plates containing Levinthal supplement and X-gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside; 40 µg/ml).

β-galactosidase assay

MC58lbpB::lacZ::kan modA11 OFF and MC58lbpB::lacZ::kan modA11 ON strains were grown on GC plates with 15 µM desferal at 37°C over night. The next day triplicate cultures of iron-starved strains were grown to exponential phase (optical density at 600 nm = 0.55 to 0.6) with 30 µM desferal in GC broth. Cells were spun down at 15,000×g for 10 min, resuspended in PBS and lysed by repeated freeze-thaw cycles. The cells debris was spun down at 15,000×g for 5 min. The amount of protein was calculated by using the BCA protein assay reagent kit (Pierce). The amount of β-galactosidase in the cell extracts was measured in Miller units, in triplicate, as described [64]. Miller units were calculated as follows: Units (1000×A₄₂₀)/(t×v×C), where t is the time of the assay (in mins), v is the volume of cell extract used in the assay, and C is the total protein concentration (in µg/ml).

Antimicrobial resistance assay

The antimicrobial resistance assay was adapted from a method described by Dougherty et al. [65]. In brief, N. gonorrhoeae FA1090 wild-type modA13 ON and FA1090 modA13::kan mutant colonies were re-suspended in PBS to a density of 10⁶ colony forming units (CFUs), and 5 µl of serial ten-fold dilutions were spotted in triplicate onto GC agar plates containing 15 µM desferal, supplemented with IsoVitaleX and increasing concentrations of Triton X-100 (40, 50, 60, and 80 µg/ml). The plates were then incubated at 37°C under 5% CO₂ for 24 h. Colony counts were used to compare wild-type FA1090 modA13 ON to the FA1090 modA13::kan mutant by plating each dilution in triplicate. The experiment was repeated on three separate occasions. The ratio of FA1090 modA13 ON to FA1090 modA13 OFF at the following concentrations of Triton X-100 (40, 50 and 60 ug/ml) was calculated as follows. Colonies were taken from the triplicate samples of the original inoculum and each of the increasing concentrations of Triton X-100 from FA1090 modA13 ON and used as PCR template. The percentage of modA13 ON and modA13 OFF from the starting inoculum and the three different Triton X-100 concentrations was verified via fragment analysis [17] using primers ModAF6Fam and ModAREPEATR (Table S7). A Student's t-test was used to determine the statistical significance between the percentage of modA13 ON and modA13 OFF from the original inoculum of modA13 ON and the percentage of modA13 ON and modA13 OFF from the three different Triton X-100 of modA13 ON.

Biofilm formation by N. gonorrhoeae

For examination of biofilm formation via confocal microscopy, the N. gonorrhoeae FA1090 modA13::kan and wild-type FA1090 modA13 ON strains and N. gonorrhoeae strains O1G1370 modA13::kan, modA13 OFF and modA13 ON were transformed with a plasmid encoding a green fluorescent protein, pCmGFP. Formation and analysis of biofilms was as described previously, except the cells were grown under the same iron-limiting conditions as for the microarray analysis [29]. Colonies used to inoculate cultures for biofilm assays were assessed for morphology to ensure equivalent level of piliation. Biofilms images are three-dimensional reconstructions of stacked z-series taken at 200× magnification, which were rendered by Volocity.

Electron microscopy

Biofilms of N. gonorrhoeae strain FA1090 modA13::kan and modA13 ON and N. gonorrhoeae strains O1G1370 modA13::kan, modA13 OFF and modA13 ON were grown at in glass flow chambers at 37°C and a flow rate of 180 µl/min in 1∶10 GC broth diluted in PBS with 10 ml/L IsoVitaleX, 3 µM desferal, and 100 µM sodium nitrite. The modA13 status of the starting inoculum was verified via fragment analysis [17] using primers ModAF6Fam and ModAREPEATR. After 48 hours of growth, biofilms were prepared for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) as follows. Glass coverslips, which served as the surface of attachment for biofilm, were removed from the chambers and fixed in 1% osmium perfluorocarbon for 1 h. The coverslips were then gently rinsed for 15 min with pure perfluorocarbon three times. To avoid destruction of the biofilm, rinse solution was gently added to coverslips in a 100 mm Petri dish, allowed to incubate at room temperature for 15 min, then the rinse was aspirated and another rinse was applied. The samples were then dehydrated with 100% ethanol by performing another three 15 min rinses. At this point, the coverslips were cut in half and one half was processed for SEM, while the other half was processed for TEM. SEM samples were transitioned into HMDS for two 15 min washes and then allowed to air dry. SEM samples were then sputter-coated and viewed with the Hitachi S-4800 SEM. TEM samples were infiltrated with a 50% Eponate-12 resin (epon) in ethanol for 1 h. The coverslips were then inverted and imbedded in 100% epon at 42°C overnight. Thin-sections (70 nm) were prepared on an ultramicrotome, mounted on a grid, and then stained with uranyl acetate and lead citrate. TEM samples were viewed with the JEOL 1230 TEM.

Primary, human, cervical epithelial cell culture and infection studies

Surgical cervical biopsies were used to seed primary cervical epithelial (pex) cell cultures and were procured and maintained as described previously [32]. Quantitative association, invasion, and survival assays were performed as previously described using a multiplicity of infection of 100 [32] with modification as follows. Our previous studies demonstrate that pex cells produce a full alternative pathway of complement, and that iC3b serves as a critical opsonin for CR3-mediated gonococcus adherence to and invasion of these cells. Thereby, antibiotic-free medium was harvested from uninfected pex cell monolayers and treated overnight with 30 µM desferal (Sigma). Our previous (unpublished) studies have revealed that N. gonorrhoeae strain FA1090 uniquely becomes cytotoxic to human, primary cervical and male urethral epithelial cells within 2 to 3 hours post-challenge, which prohibits their confident use in gentamicin survival assays for time periods totaling greater than 90 min. Therefore, N. gonorrhoeae strains O1G1370 modA13 ON, O1G1370 modA13 OFF, and the O1G1370 modA13::kan mutant, and 1291 modA13 ON, 1291 modA13 OFF, and 1291modA13::kan were selected to elucidate the role of mod-dependent phase variation during pex cell challenge. Complement-containing, iron-depleted, primed medium was inoculated with 5×10⁶ gonococci per ml. Colonies used to inoculate cultures for these assays were assessed for morphology to ensure equivalent level of piliation. Bacterial cultures were incubated (37°C, with shaking) for 2 h, after which the optical density of the gonococcal cultures was adjusted to 10⁷ gonococci per ml and directly used to challenge (new) pex cell monolayers. Pex cell infections were then allowed to progress at 37°C, 5% CO₂. Association (90 min infection), invasion (90 min infection plus a 30 min incubation in 100 µg/ml gentamicin), and survival (90 min infection, 30 min gentamicin treatment, plus a 3 h incubation in antibiotic-free medium) assays were performed using a modified gentamicin-resistance assay as described previously [32].Serial dilutions of the cervical cell lysates were plated to determine CFUs. The percent association, invasion, and survival were determined as functions of the original inoculum. From these data the invasion and survival indices were determine as follows: Invasion index, percent invasion/percent association; Survival index, percent survival/percent invasion. P-values were determined for the actual data points using a Kruskal-Wallis non-parametric analysis of variance. A Student's t-test was used to determine the statistical significance of the invasion and survival indices. The ratio of modA13 ON and modA13 OFF within the O1G1370 modA13 ON and O1G1370 modA13 OFF original inoculum, association, invasion, and survival time points were determined as follows. Samples were taken from the original inoculum, association, invasion, and survival time points from three independent assays and chromosomal DNA extracted. The percentage of modA13 ON and modA13 OFF from the starting inoculum was verified via fragment analysis [17] using primers ModAF6Fam and ModAREPEATR (Table S7). A Student's t-test was used to determine the statistical significance between the percentage of modA13 ON and modA13 OFF from the original inoculum of O1G1370 modA13 ON and the percentage of modA13 ON and modA13 OFF from the invasion time point and survival time point of O1G1370 modA13 ON. Similarly, a Student's t-test was used to determine statistical significance between the percentage of modA13 ON and modA13 OFF from the original inoculum of O1G1370 modA13 OFF and the percentage of modA13 ON and modA13 OFF from the invasion time point and survival time point of O1G1370 modA13 OFF.

Production of anti-Mod antisera

The mod gene was amplified from H. influenzae strain Rd chromosomal DNA using primers listed in Table S7. The NcoI restriction site at the 5′ end and the BamHI site at the 3′ end of the mod gene were introduced. The resulting PCR fragment was subsequently digested with NcoI and BamHI and cloned into the digested pET16b expression vector (Novagen & EMD, San Diego, CA, USA) carrying the same enzyme cutting sites, leading to the construct, pET16b::mod. The sequence of the insert was confirmed and then used for generating the recombinant Mod protein with the (His)₁₀-tag (MGHHHHHHHHHH) attached at the N-terminal end. For generating the recombinant Mod protein, the construct, pET16b::mod, was transformed into E. coli strain BL21(DE3) and the cells were grown in LB broth at 20°C. Induction of the expression was initiated by adding IPTG to the final concentration of 0.1 mM and then incubated at 10°C for 3 days. After harvesting the bacteria by centrifuging at 6,000 rpm for 30 min at 4°C, the bacterial pellet was lysed with the lysis buffer (25 mM Tris-HCl, 300 mM KCl, 5 mM imidazole, pH 7.5) plus protease inhibitor, Complete cocktail EDTA-free (Roche, Switzerland). Soluble proteins were obtained from the supernatant by centrifuging at 20,000 rpm for 20 min at 4°C to remove the cell debris and precipitates. The Mod protein was purified using the Ni²⁺-nitilotriacetic acid (Ni-NTA) column (Amersham Biosciences, Piscataway, NJ, USA) with an elution gradient from 25–500 mM imidazole in the buffer solution (25 mM Tris-HCl, 300 mM KCl, pH 7.5). The purity of the eluted protein was examined by SDS–PAGE analysis and the concentration determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). The pure fractions were collected and transferred to 25 mM Tris, pH 7.5 by the HiPrep 26/10 Desalting column (Amersham Biosciences, USA) and store at −80°C.

Rabbits (New Zealand White strain, weighing 3–3.5 kg, were immunized by intrasplenic injection with the soluble recombinant Mod protein at 300 µg per immunization. The antigen was administered together with an equal amount of Gold TiterMax adjuvant (CytRx, Norcross, GA, USA). The rabbit antisera were collected from weeks 4∼9 and the titers of rabbit sera from weeks 4∼6 were analyzed using Western blot assays. Antiserum of week 6 had a high titer of 5,000,000 against 1 µg of the Mod protein. The antisera recognized a single band in wild type modA ON H. influenzae strain RD and N. meningitidis strain MC58, but not in their corresponding modA::kan mutants (not shown). For the subsequent Western blot experiments, 1/1,000 dilution of the antiserum of week 6 was used.