Phase-variable Expression Research Articles

Inversion of Correia repeat enclosed elements in Neisseria gonorrhoeae.

Neisseria gonorrhoeae is capable of causing gonorrhoea and more complex diseases in the human host. Within the gonococcal genome are over 100 copies of the insertion sequence-like Correia repeat enclosed element (CREE), which has been predicted to be mobile within the neisserial genomes. Although there is evidence of ancestral movement of these elements, no previous study has provided evidence for current mobilization. CREE has the ability to alter gene expression and regulation in many ways: by insertional mutagenesis, by introducing promoter elements, by generating mRNA processing sites and by association with non-coding RNAs. Previous studies have compared the genomic locations of CREEs in the Neisseria spp., demonstrating that otherwise identical regions have either the element or the target TA insertion site. In this study, we report for the first time, to our knowledge, movement of CREEs, through inversion of the element at its chromosomal location. Analysis of Ion Torrent generated genome sequence data from N. gonorrhoeae strain NCCP11945 passaged for 8 weeks in the laboratory under standard conditions and stress conditions revealed a total of 37 inversions: 24 were exclusively seen in the stressed sample, 7 were seen in the control sample and the remaining 3 were seen in both samples. These inversions have the capability to alter gene expression in N. gonorrhoeae through the previously determined activities of the sequence features of these elements, potentially resulting in reversible phase-variable gene expression.

Phase variable DNA repeats in Neisseria gonorrhoeae influence transcription, translation, and protein sequence variation.

There are many types of repeated DNA sequences in the genomes of the species of the genus Neisseria, from homopolymeric tracts to tandem repeats of hundreds of bases. Some of these have roles in the phase-variable expression of genes. When a repeat mediates phase variation, reversible switching between tract lengths occurs, which in the species of the genus Neisseria most often causes the gene to switch between on and off states through frame shifting of the open reading frame. Changes in repeat tract lengths may also influence the strength of transcription from a promoter. For phenotypes that can be readily observed, such as expression of the surface-expressed Opa proteins or pili, verification that repeats are mediating phase variation is relatively straightforward. For other genes, particularly those where the function has not been identified, gathering evidence of repeat tract changes can be more difficult. Here we present analysis of the repetitive sequences that could mediate phase variation in the Neisseria gonorrhoeae strain NCCP11945 genome sequence and compare these results with other gonococcal genome sequences. Evidence is presented for an updated phase-variable gene repertoire in this species, including a class of phase variation that causes amino acid changes at the C-terminus of the protein, not previously described in N. gonorrhoeae.

A biphasic epigenetic switch controls immunoevasion, virulence and niche adaptation in non-typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae contains an N6-adenine DNA-methyltransferase (ModA) that is subject to phase-variable expression (random ON/OFF switching). Five modA alleles, modA2, modA4, modA5, modA9 and modA10, account for over two-thirds of clinical otitis media isolates surveyed. Here, we use single molecule, real-time (SMRT) methylome analysis to identify the DNA-recognition motifs for all five of these modA alleles. Phase variation of these alleles regulates multiple proteins including vaccine candidates, and key virulence phenotypes such as antibiotic resistance (modA2, modA5, modA10), biofilm formation (modA2) and immunoevasion (modA4). Analyses of a modA2 strain in the chinchilla model of otitis media show a clear selection for ON switching of modA2 in the middle ear. Our results indicate that a biphasic epigenetic switch can control bacterial virulence, immunoevasion and niche adaptation in an animal model system.

Selection and Counterselection of Hia Expression Reveals a Key Role for Phase-Variable Expression of Hia in Infection Caused by Nontypeable Haemophilus influenzae.

Hia is a major adhesin of nontypeable Haemophilus influenzae (NTHi) and has long been investigated as a vaccine candidate. Here we show that Hia phase variation is controlled by changes in the length of a polythymidine tract located in the hia promoter. Studies of an invasive clinical isolate (strain R2866) show that strains expressing high Hia levels are more efficiently killed by opsonophagocytosis. An opsonophagocytic assay was used to select for a subpopulation of variants that expressed a low level of Hia, which facilitated their escape from killing by anti-Hia antisera. Conversely, a subpopulation of variants expressing a high level of Hia was selected for during passaging through Chang cells. In both cases, phase variation of Hia expression corresponded directly with discrete modal changes in polythymidine tract length. In the chinchilla model of NTHi infection, we observed consistent selection for high Hia expression upon nasopharyngeal colonization, confirming the key role of phase-variable expression of Hia within a specific niche in vivo.

Phase-variable expression of lptA modulates the resistance of Neisseria gonorrhoeae to cationic antimicrobial peptides.

Phosphoethanolamine (PEA) decoration of lipid A produced by Neisseria gonorrhoeae has been linked to bacterial resistance to cationic antimicrobial peptides/proteins (CAMPs) and in vivo fitness during experimental infection. We now report that the lptA gene, which encodes the PEA transferase responsible for this decoration, is in an operon and that high-frequency mutation in a polynucleotide repeat within lptA can influence gonococcal resistance to CAMPs.

Prevalence and Phase Variable Expression Status of Two Autotransporters, NalP and MspA, in Carriage and Disease Isolates of Neisseria meningitidis

Neisseria meningitidis is a human nasopharyngeal commensal capable of causing life-threatening septicemia and meningitis. Many meningococcal surface structures, including the autotransporter proteins NalP and MspA, are subject to phase variation (PV) due to the presence of homopolymeric tracts within their coding sequences. The functions of MspA are unknown. NalP proteolytically cleaves several surface-located virulence factors including the 4CMenB antigen NhbA. Therefore, NalP is a phase-variable regulator of the meningococcal outer membrane and secretome whose expression may reduce isolate susceptibility to 4CMenB-induced immune responses. To improve our understanding of the contributions of MspA and NalP to meningococcal-host interactions, their distribution and phase-variable expression status was studied in epidemiologically relevant samples, including 127 carriage and 514 invasive isolates representative of multiple clonal complexes and serogroups. Prevalence estimates of >98% and >88% were obtained for mspA and nalP, respectively, with no significant differences in their frequencies in disease versus carriage isolates. 16% of serogroup B (MenB) invasive isolates, predominately from clonal complexes ST-269 and ST-461, lacked nalP. Deletion of nalP often resulted from recombination events between flanking repetitive elements. PolyC tract lengths ranged from 6–15 bp in nalP and 6–14 bp in mspA. In an examination of PV status, 58.8% of carriage, and 40.1% of invasive nalP-positive MenB isolates were nalP phase ON. The frequency of this phenotype was not significantly different in serogroup Y (MenY) carriage strains, but was significantly higher in invasive MenY strains (86.3%; p<0.0001). Approximately 90% of MenB carriage and invasive isolates were mspA phase ON; significantly more than MenY carriage (32.7%) or invasive (13.7%) isolates. This differential expression resulted from different mode mspA tract lengths between the serogroups. Our data indicates a differential requirement for NalP and MspA expression in MenB and MenY strains and is a step towards understanding the contributions of phase-variable loci to meningococcal biology.

Analysis of Nontypeable Haemophilus influenzae Phase-Variable Genes During Experimental Human Nasopharyngeal Colonization

Studies of nontypeable Haemophilus influenzae (NTHi) have demonstrated that a number of genes associated with infectivity have long repeat regions associated with phase variation in expression of the respective gene. The purpose of this study was to determine the genes that underwent phase variation during a 6-day period of experimental human nasopharyngeal colonization. Strain NTHi 2019Str(R)1 was used to colonize the nasopharynx of human subjects in a study of experimental colonization. Thirteen phase-variable genes were analyzed in NTHi 2019Str(R)1. Samples of NTHi 2019Str(R)1 were cultured from subjects during the 6-day colonization period. We used capillary electrophoresis and Roche 454 pyrosequencing to determine the number of repeats in each gene from each sample. A significant number of samples switched licA and igaB from phase off in the inoculated strain to phase on during the 4-day period of observation. lex2A also showed variability as compared to baseline, but the differences were not significant. The remaining genes showed no evidence of phase variation. Our studies suggest that the phase-on genotypes of licA and igaB are important for early human nasopharynx colonization. lex2A showed a trend from phase off to phase on, suggesting a potentially important role in the colonization process.

Identification of the lic1ABCD operon that controls the phase-variable expression of phosphorylcholine on lipopolysaccharide from Avibacterium paragallinarum

Phosphorylcholine (ChoP) is an important virulence factor found on the surface of many mucosal pathogens and its expression enhances bacterial colonization on mucous membranes and reduces susceptibility to antimicrobial peptides. Whole-genome sequencing analyses showed that Avibacterium paragallinarum contained an operon with strong sequence similarity to the lic1ABCD operon from Haemophilus influenzae and the pcgDABC operon from Pasteurella multocida; both operons are involved in metabolism and addition of ChoP on bacterial lipopolysaccharide (LPS). Western immunoblot analysis with ChoP-specific monoclonal antibody showed that ChoP is present on LPS of Av. paragallinarum and the expression of ChoP is controlled by phase variation mediated by the number of 5′-CAAT-3′ tetranucleotide tandem repeats within the coding region of the lic1A gene. The number of tetranucleotide repeats varied widely among strains, and variation in the number of repeats was observed following in vivo passage but not in vitro passage. Antimicrobial susceptibility assays showed that ChoP expression decreased susceptibility of Av. paragallinarum to chicken antimicrobial peptide fowlicidin-1. This is the first report showing that ChoP is present on LPS from Av. paragallinarum and that Av. paragallinarum contains a phase-variable gene. These results could be valuable for understanding the mechanism of pathogenicity of Av. paragallinarum.

Population Genomic Scan for Candidate Signatures of Balancing Selection to Guide Antigen Characterization in Malaria Parasites

Acquired immunity in vertebrates maintains polymorphisms in endemic pathogens, leading to identifiable signatures of balancing selection. To comprehensively survey for genes under such selection in the human malaria parasite Plasmodium falciparum, we generated paired-end short-read sequences of parasites in clinical isolates from an endemic Gambian population, which were mapped to the 3D7 strain reference genome to yield high-quality genome-wide coding sequence data for 65 isolates. A minority of genes did not map reliably, including the hypervariable var, rifin, and stevor families, but 5,056 genes (90.9% of all in the genome) had >70% sequence coverage with minimum read depth of 5 for at least 50 isolates, of which 2,853 genes contained 3 or more single nucleotide polymorphisms (SNPs) for analysis of polymorphic site frequency spectra. Against an overall background of negatively skewed frequencies, as expected from historical population expansion combined with purifying selection, the outlying minority of genes with signatures indicating exceptionally intermediate frequencies were identified. Comparing genes with different stage-specificity, such signatures were most common in those with peak expression at the merozoite stage that invades erythrocytes. Members of clag, PfMC-2TM, surfin, and msp3-like gene families were highly represented, the strongest signature being in the msp3-like gene PF10_0355. Analysis of msp3-like transcripts in 45 clinical and 11 laboratory adapted isolates grown to merozoite-containing schizont stages revealed surprisingly low expression of PF10_0355. In diverse clonal parasite lines the protein product was expressed in a minority of mature schizonts (<1% in most lines and ∼10% in clone HB3), and eight sub-clones of HB3 cultured separately had an intermediate spectrum of positive frequencies (0.9 to 7.5%), indicating phase variable expression of this polymorphic antigen. This and other identified targets of balancing selection are now prioritized for functional study.

Phase variable genes of Campylobacter jejuni exhibit high mutation rates and specific mutational patterns but mutability is not the major determinant of population structure during host colonization

Phase variation of surface structures occurs in diverse bacterial species due to stochastic, high frequency, reversible mutations. Multiple genes of Campylobacter jejuni are subject to phase variable gene expression due to mutations in polyC/G tracts. A modal length of nine repeats was detected for polyC/G tracts within C. jejuni genomes. Switching rates for these tracts were measured using chromosomally-located reporter constructs and high rates were observed for cj1139 (G8) and cj0031 (G9). Alteration of the cj1139 tract from G8 to G11 increased mutability 10-fold and changed the mutational pattern from predominantly insertions to mainly deletions. Using a multiplex PCR, major changes were detected in ‘on/off’ status for some phase variable genes during passage of C. jejuni in chickens. Utilization of observed switching rates in a stochastic, theoretical model of phase variation demonstrated links between mutability and genetic diversity but could not replicate observed population diversity. We propose that modal repeat numbers have evolved in C. jejuni genomes due to molecular drivers associated with the mutational patterns of these polyC/G repeats, rather than by selection for particular switching rates, and that factors other than mutational drift are responsible for generating genetic diversity during host colonization by this bacterial pathogen.

Promoter Orientation of Prokaryotic Phase-variable Genes by PCR.

One major mechanism of phase variable gene expression in prokaryotes is through inversion of the promoter element for a gene or operon. This protocol describes how to detect the promoter orientation of a phase-variable gene by PCR. This protocol, including primer design, is specific to detection of the promoter orientations of hyxR, a LuxR-like response regulator in Extraintestinal Pathogenic Escherichia coli (ExPEC) isolates (Bateman and Seed, 2012); however, this protocol can be generalized to other organisms and genes to discriminate prokaryotic promoter inversions by PCR through size discrimination of the amplification products. Expression of hyxR is regulated through bidirectional phase inversion of the upstream promoter region mediated by a member of the family of site-specific tyrosine recombinases called Fim-like recombinases. The recombinases recognize inverted DNA repeat sequences flanking the promoter and produce a genomic rearrangement, orientating the promoter in favor or disfavor of gene expression.

Phasevarion Mediated Epigenetic Gene Regulation in Helicobacter pylori

Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae and pathogenic Neisseria, the random switching of the modA gene, associated with a phase-variable type III restriction modification (R-M) system, controls expression of a phase-variable regulon of genes (a “phasevarion”), via differential methylation of the genome in the modA ON and OFF states. Phase-variable type III R-M systems are also found in Helicobacter pylori, suggesting that phasevarions may also exist in this key human pathogen. Phylogenetic studies on the phase-variable type III modH gene revealed that there are 17 distinct alleles in H. pylori, which differ only in their DNA recognition domain. One of the most commonly found alleles was modH5 (16% of isolates). Microarray analysis comparing the wild-type P12modH5 ON strain to a P12ΔmodH5 mutant revealed that six genes were either up- or down-regulated, and some were virulence-associated. These included flaA, which encodes a flagella protein important in motility and hopG, an outer membrane protein essential for colonization and associated with gastric cancer. This study provides the first evidence of this epigenetic mechanism of gene expression in H. pylori. Characterisation of H. pylori modH phasevarions to define stable immunological targets will be essential for vaccine development and may also contribute to understanding H. pylori pathogenesis.

In the NadR Regulon, Adhesins and Diverse Meningococcal Functions Are Regulated in Response to Signals in Human Saliva

The Neisseria meningitidis regulator NadR was shown to repress expression of the NadA adhesin and play a major role in NadA phase-variable expression. In this study, we identified through microarray analysis over 30 genes coregulated with nadA in the NadR mutant and defined members of the NadR regulon through in vitro DNA-binding assays. Two distinct types of promoter architectures (I and II) were identified for NadR targets, differing in both the number and position of NadR-binding sites. All NadR-regulated genes investigated were found to respond to 4-hydroxyphenylacetic acid (4HPA), a small molecule secreted in human saliva, which was previously demonstrated to induce nadA expression by alleviating NadR-dependent repression. Interestingly, two types of NadR 4HPA responsive activities were found on different NadR targets corresponding to the two types of genes identified by different promoter architectures: while NadA and the majority of NadR targets (type I) are induced, only the MafA adhesins (type II) are corepressed in response to the same 4HPA signal. This alternate behavior of NadR was confirmed in a panel of strains in response to 4HPA and after incubation in saliva. The in vitro NadR binding activity at type I and type II promoter regions is differentially affected by 4HPA, suggesting that the nature of the NadR binding sites may define the regulation to which they will be subjected. We conclude that NadR coordinates a broad transcriptional response to signals present in human saliva, mimicked in vitro by 4HPA, enabling the meningococcus to adapt to the relevant host niche.

The CgrA and CgrC Proteins Form a Complex That Positively Regulates cupA Fimbrial Gene Expression in Pseudomonas aeruginosa

The CgrA and CgrC proteins of Pseudomonas aeruginosa are coregulators that are required for the phase-variable expression of the cupA fimbrial genes. Neither CgrA nor CgrC resembles a classical transcription regulator, and precisely how these proteins exert their regulatory effects on cupA gene expression is poorly understood. Here, we show that CgrA and CgrC interact with one another directly. We identify a mutant of CgrC that is specifically defective for interaction with CgrA and demonstrate that this mutant cannot restore the phase-variable expression of the cupA fimbrial genes to cells of a cgrC mutant strain. Using this mutant, we also show that CgrC associates with the cupA promoter regardless of whether or not it interacts with CgrA. Our findings establish that interaction between CgrA and CgrC is required for the phase-variable expression of the cupA fimbrial genes and suggest that CgrC exerts its regulatory effects directly at the cupA promoter, possibly by recruiting CgrA. Because the regions of CgrA and CgrC that we have identified as interacting with one another are highly conserved among orthologs, our findings raise the possibility that CgrA- and CgrC-related regulators present in other bacteria function coordinately through a direct protein-protein interaction.

Decreased virulence of a uropathogenic Escherichia coli pst mutant is attributed to the repression of Type 1 fimbriae

Extra-intestinal pathogenic E. coli cause urinary tract infections (UTIs), newborn meningitis, abdominal sepsis and septicemia. UTIs affect millions of women annually, and result in significant health care costs and morbidity worldwide. Uropathogenic E. coli (UPEC) is the predominant urinary tract pathogen, causing up to 85% of UTIs. Despite appropriate therapy, recurrent episode of UTI are common and bacterial strains are increasingly more resistant to many currently used antimicrobial agents. The pstSCAB-phoU operon encodes the phosphate specific transport system (Pst) and belongs to the Pho regulon, which is regulated by the two-component regulatory system (TCRS) PhoBR. Inactivation of the Pst system in E. coli and other bacteria leads to constitutive activation of the Pho regulon, perturbations in cellular adaptation, and decreased virulence. The role of the Pst system in uropathogenic E. coli (UPEC) was assessed by deleting the pstSCA genes in UPEC strain CFT073. In competitive (co-challenge) and single-strain infections, the pst mutant was attenuated for colonization of both the bladder and kidneys of CBA/J mice and was impaired for production of Type 1 fimbriae. Type 1 fimbriae are essential for UPEC virulence and their phase-variable expression is positively and negatively regulated by FimB and FimE, respectively. In vitro, in LB broth and human urine, repression of the fim structural gene fimA in the pst mutant correlated with increased orientation of the fim promoter in the OFF-position. In vivo, down-regulation of fimA in CFT073 Δpst correlated with the up-regulation of fimE. To confirm the specific role of repression of fim expression by the pst mutant during UTI, fim phase locked-ON pst derivatives of the pst mutant and WT CFT073 strains were constructed. Compared to the pst mutant, the fim phase locked-ON pst derivative demonstrated a significant gain in colonization of the bladder, that was similar to that of CFT073 WT and CFT073 fim locked-ON strains. As Type 1 fimbriae are important for UPEC virulence, by promoting adhesion, our results suggest that the reduced bladder colonization by the pst mutant during UTI is predominantly attributed to down-regulation of these fimbriae. Since the Pho regulon is controlled by the TCRS PhoBR, molecules inducing the expression of the Pho regulon through inactivation of Pst or activation of PhoBR could potentially impair UPEC virulence by inhibiting colonization and the infection cycle, which is dependent on expression of type 1 fimbrial adhesins.

Sticky and Sweet: The Role of Post-Translational Modifications on Neisserial Pili

Sticky and Sweet: The Role of Post-Translational Modifications on Neisserial Pili

Legionella pneumophila carrying the virulence-associated lipopolysaccharide epitope possesses two functionally different LPS components

Phase-variable expression of Legionella pneumophila lipopolysaccharide (LPS) has not been described in detail for strains possessing the virulence-associated epitope recognized by the monoclonal antibody (mAb) 3/1 of the Dresden Panel. About 75 % of cases of community-acquired legionellosis are caused by mAb 3/1-positive strains. In this study, the LPS architecture of the mAb 3/1-positive Corby strain was investigated during its life cycle in broth culture and inside monocytic host cells. During the exponential growth phase in broth, the highly acetylated and therefore strongly hydrophobic mAb 3/1 epitope is expressed continuously, but only 3 % of the bacteria can be detected using mAb 59/1, which recognizes a short-chain variant of the Legionella LPS that is less hydrophobic due to missing acetylations of the O-chain. The percentage of mAb 59/1-positive legionellae increases up to 34 % in the post-exponential growth phase. LPS shed in broth during the exponential phase is mAb 59/1-negative, and mAb 3/1-positive components do not possess short-chain molecules. The LPS pattern expressed and shed inside U937 cells and A/J mouse macrophages points to the same regulatory mechanisms. During the so-called 'pregnant pause', the period for establishment of the replicative phagosomes, the mAb 3/1-positive LPS is shed into the phagosome and seems to pass through the phagosomal membrane, while mAb 59/1-positive LPS is detectable only on the bacterial surface. After egress of the legionellae into the cytoplasm followed by host cell lysis, individual bacteria are mAb 3/1-positive and mAb 59/1-negative. Intracellularly formed Legionella clusters consist of surface-located mAb 3/1-positive bacteria, which are predominantly mAb 59/1-negative. They surround less hydrophobic and therefore closely packed mAb 59/1-positive bacteria. Based on the different degrees of hydrophobicity, bacteria are able to support the expression of two functionally different LPS architectures, namely more hydrophobic LPS for surviving in aerosols and more hydrophilic LPS for close-packing of legionellae inside clusters.

Opacity proteins increase Neisseria gonorrhoeae fitness in the female genital tract due to a factor under ovarian control.

The neisserial opacity (Opa) proteins are a family of antigenically distinct outer membrane proteins that undergo phase-variable expression. Opa(+) variants of Neisseria gonorrhoeae strain FA1090 are selected in a cyclical pattern from the lower genital tract of estradiol-treated mice. Here we show that cyclical recovery of Opa(+) gonococci does not occur in ovariectomized mice; therefore, the reproductive cycle plays a role in the selection kinetics in vivo. As predicted by the selection pattern shown by wild-type gonococci, we demonstrated that a constitutive Opa-expressing strain was more fit than an Opa-deficient mutant in the early and late phases of infection. We found no evidence that Opa-mediated colonization selects for Opa(+) variants during murine infection based on adherence assays with cultured murine epithelial cells. We also tested the hypothesis that complement selects for Opa protein expression during infection. Although some Opa(+) variants of a serum-sensitive derivative of strain FA1090 were more resistant to the bactericidal activity of normal human serum, selection for Opa expression was not abrogated in C3-depleted mice. Finally, as previously reported, Opa(+) gonococci were more sensitive to serine proteases. Thus, proteases or protease inhibitors may contribute to the observed in vivo selection pattern. We concluded that Opa proteins promote persistence of N. gonorrhoeae in the female genital tract and that opa gene phase variation allows gonococci to evade or capitalize upon unidentified host factors of the mammalian reproductive cycle. This work revealed an intimate interaction between pathogen and host and provides evidence that hormonally related factors shape bacterial adaptation.

Simple sequence repeats in Helicobacter canadensis and their role in phase variable expression and C-terminal sequence switching

BackgroundHelicobacter canadensis is an emerging human pathogen and zoonotic agent. The genome of H. canadensis was sequenced previously and determined to contain 29 annotated coding regions associated with homopolymeric tracts.ResultsTwenty-one of the repeat-associated coding regions were determined to be potentially transcriptionally or translationally phase variable. In each case the homopolymeric tract was within the predicted promoter region or at the 5' end of the coding region, respectively. However, eight coding sequences were identified with simple sequence repeats toward the 3' end of the open reading frame. In these cases, the repeat tract would be too far into the coding region to be mediating translational phase variation. All of the 29 coding region-associated homopolymeric tracts display variability in tract length in the sequencing read data.ConclusionsTwenty-nine coding regions have been identified in the genome sequence of Helicobacter canadensis strain NCTC13241 that show variations in homopolymeric tract length in the bacterial population, indicative of phase variation. Five of these are potentially associated with promoter regions, which would lead to transcriptional phase variation. Translational phase variation usually switches expression of a gene ON and OFF due to the repeat region being located sufficiently close to the initiation codon for the resulting frame-shift to lead to a premature termination codon and stop the translation of the protein. Sixteen of the 29 coding regions have homopolymeric tracts characteristic of translational phase variation. For eight coding sequences with repeats located later in the reading frame, changes in the repeat tract length would alter the protein sequence at the C-terminus but not stop the expression of the protein. This mechanism of C-terminal phase variation has implications for stochastic switching of protein sequence in bacterial species that already undergo transcriptional and translational phase variation.

A Novel Phase Variation Mechanism in the Meningococcus Driven by a Ligand-Responsive Repressor and Differential Spacing of Distal Promoter Elements

Phase variable expression, mediated by high frequency reversible changes in the length of simple sequence repeats, facilitates adaptation of bacterial populations to changing environments and is frequently important in bacterial virulence. Here we elucidate a novel phase variable mechanism for NadA, an adhesin and invasin of Neisseria meningitidis. The NadR repressor protein binds to operators flanking the phase variable tract and contributes to the differential expression levels of phase variant promoters with different numbers of repeats likely due to different spacing between operators. We show that IHF binds between these operators, and may permit looping of the promoter, allowing interaction of NadR at operators located distally or overlapping the promoter. The 4-hydroxyphenylacetic acid, a metabolite of aromatic amino acid catabolism that is secreted in saliva, induces NadA expression by inhibiting the DNA binding activity of the repressor. When induced, only minor differences are evident between NadR-independent transcription levels of promoter phase variants and are likely due to differential RNA polymerase contacts leading to altered promoter activity. Our results suggest that NadA expression is under both stochastic and tight environmental-sensing regulatory control, both mediated by the NadR repressor, and may be induced during colonization of the oropharynx where it plays a major role in the successful adhesion and invasion of the mucosa. Hence, simple sequence repeats in promoter regions may be a strategy used by host-adapted bacterial pathogens to randomly switch between expression states that may nonetheless still be induced by appropriate niche-specific signals.