Microbial Primer: Phase variation - survival and adaptability by generation of a diverse population.

Phase variation is defined as the rapid and reversible switching of gene expression, and typically occurs in genes encoding surface features in small genome bacterial pathogens. Phase variation has evolved to provide an extra survival mechanism in bacteria that lack multiple 'sense-and-respond' gene regulation systems. Many bacterial pathogens also encode DNA methyltransferases that are phase-variable, controlling systems called 'phasevarions' (phase-variable regulons). This primer will summarize the current understanding of phase variation, describing the role of major phase-variable factors, and phasevarions, in bacterial pathobiology.

Quantitative method for measuring the proportion of bacterial cells expressing phase 1 or 2 of flagellin of Salmonella enterica serovar Typhimurium.

Salmonella enterica subsp. enterica is a major pathogen that causes zoonotic foodborne diseases worldwide. Some Salmonella serovars possess two antigenic phases for flagellin: phase 1 and 2. In Salmonella enterica serovar Typhimurium (S. Typhimurium), the flagellin is antigenically divided into "Hi" as phase 1 and "H1 or H2" as phase 2. Flagellin phase variation is regulated by inversion of hin gene. We focused on the inversion of hin and developed a real-time PCR system to quantitatively measure the proportion of bacterial cells expressing each phase of flagellin. In this study, we demonstrated that our newly developed real-time PCR system shows high quantitative accuracy and aligns with flagellin expression status. Furthermore, the newly developed real-time PCR system was applicable to various S. Typhimurium laboratory and field strains. This newly developed real-time PCR system has the potential to become a powerful tool for analyzing flagellin phase variation.

Novel manufacturing process of pneumococcal capsular polysaccharides using advanced sterilization methods.

Pneumococcal disease is caused by Streptococcus pneumoniae, including pneumonia, meningitis and sepsis. Capsular polysaccharides (CPSs) have been shown as effective antigens to stimulate protective immunity against pneumococcal disease. A major step in the production of pneumococcal vaccines is to prepare CPSs that meet strict quality standards in immunogenicity and safety. The major impurities come from bacterial proteins, nucleic acids and cell wall polysaccharides. Traditionally, the impurity level of refined CPSs is reduced by optimization of purification process. In this study, we investigated new aeration strategy and advanced sterilization methods by formaldehyde or ß-propiolactone (BPL) to increase the amount of soluble polysaccharide in fermentation supernatant and to prevent bacterial lysis during inactivation. Furthermore, we developed a simplified process for the CPS purification, which involves ultrafiltration and diafiltration, followed by acid and alcohol precipitation, and finally diafiltration and lyophilization to obtain pure polysaccharide. The CPSs prepared from formaldehyde and BPL sterilization contained significantly lower level of residual impurities compared to the refined CPSs obtained from traditional deoxycholate sterilization. Finally, we showed that this novel approach of CPS preparation can be scaled up for polysaccharide vaccine production.

Effect of capsular polysaccharide phase variation on biofilm formation, motility and gene expression in Vibrio vulnificus.

Vibrio vulnificus, a significant marine pathogen, undergoes opaque (Op)-translucent (Tr) colony switching based on whether capsular polysaccharide (CPS) is produced. CPS phase variation is sometime accompanied by genetic variation or down-regulation of particular genes, such as wzb. In addition, CPS prevents biofilm formation and is important to the virulence of V. vulnificus. However, the extent to which there is a difference in gene expression between Tr and Op colonies and the impact of CPS phase variation on other behaviors of V. vulnificus remain unknown. In this work, the data have shown that CPS phase variation of V. vulnificus is affected by incubation time. Tr and Op strains exhibited similar growth rates. However, Tr strains had enhanced biofilm formation capacities but reduced swimming motility compared to Op strains. The RNA-seq assay revealed 488 differentially expressed genes, with 214 downregulated and 274 upregulated genes, between Tr and Op colonies. Genes associated with Tad pili and CPS were downregulated, whereas those involved in flagellum were upregulated, in Tr colonies compared with Op colonies. In addition, 9 putative c-di-GMP metabolism-associated genes and 28 genes encoding putative regulators were significantly differentially expressed, suggesting that CPS phase variation is probably strictly regulated in V. vulnificus. Moreover, 8 genes encoding putative porins were also differentially expressed between the two phenotypic colonies, indicating that bacterial outer membrane was remodeled during CPS phase variation. In brief, this work highlighted the gene expression profiles associated with CPS phase variation, but more studies should be performed to disclose the intrinsic mechanisms in the future.

Phase variable colony variants are conserved across Gardnerella spp. and exhibit different virulence-associated phenotypes.

The Gardnerella genus, comprising at least 13 species, is associated with the polymicrobial disorder bacterial vaginosis (BV). However, the details of BV pathogenesis are poorly defined, and the contributions made by individual species, including Gardnerella spp., are largely unknown. We report here that colony phenotypes characterized by size (large and small) and opacity (opaque and translucent) are phase variable and are conserved among all tested Gardnerella strains, representing at least 10 different species. With the hypothesis that these different variants could be an important missing piece to the enigma of how BV develops in vivo, we characterized their phenotypic, proteomic, and genomic differences. Beyond increased colony size, large colony variants showed reduced vaginolysin secretion and faster growth rate relative to small colony variants. The ability to inhibit the growth of Neisseria gonorrhoeae and commensal Lactobacillus species varied by strain and, in some instances, differed between variants. Proteomics analyses indicated that 127-173 proteins were differentially expressed between variants. Proteins with increased expression in large variants of both strains were associated with amino acid and protein synthesis and protein folding, whereas those increased in small variants were related to nucleotide synthesis, phosphate transport, ABC transport, and glycogen breakdown. Furthermore, whole genome sequencing analyses revealed an abundance of genes associated with variable homopolymer tracts, implicating slipped strand mispairing in Gardnerella phase variation and illuminating the potential for previously unrecognized heterogeneity within clonal populations. Collectively, these results suggest that phase variants may be primed to serve different roles in BV pathogenesis.IMPORTANCEBacterial vaginosis is the most common gynecological disorder in women of childbearing age. Gardnerella species are crucial to the development of this dysbiosis, but the mechanisms involved in the infection are not understood. We discovered that Gardnerella species vary between two different forms, reflected in bacterial colony size. A slow-growing form makes large amounts of the toxin vaginolysin and is better able to survive in human cervix tissue. A fast-growing form is likely the one that proliferates to high numbers just prior to symptom onset and forms the biofilm that serves as a scaffold for multiple BV-associated anaerobic bacteria. Identification of the proteins that vary between different forms of the bacteria as well as those that vary randomly provides insight into the factors important for Gardnerella infection and immune avoidance.

Rapid evolutionary tuning of endospore quantity versus quality trade-off via a phase-variable contingency locus.

The UV resistance of bacterial endospores is an important quality supporting their survival in inhospitable environments and therefore constitutes an essential driver of the ecological success of spore-forming bacteria. Nevertheless, the variability and evolvability of this trait are poorly understood. In this study, directed evolution and genetics approaches revealed that the Bacillus cereus pdaA gene (encoding the endospore-specific peptidoglycan-N-acetylmuramic acid deacetylase) serves as a contingency locus in which the expansion and contraction of short tandem repeats can readily compromise (PdaAOFF) or restore (PdaAON) the pdaA open reading frame. Compared with B. cereus populations in the PdaAON state, populations in the PdaAOFF state produced a lower yield of viable endospores but endowed them with vastly increased UV resistance. Moreover, selection pressures based on either quantity (i.e., yield of viable endospores) or quality (i.e., UV resistance of viable endospores) aspects could readily shift populations between PdaAON and PdaAOFF states, respectively. Bioinformatic analysis also revealed that pdaA homologs within the Bacillus and Clostridium genera are often equipped with several short tandem repeat regions, suggesting a wider implementation of the pdaA-mediated phase variability in other sporeformers as well. These results for the first time reveal (1) pdaA as a phase-variable contingency locus in the adaptive evolution of endospore properties and (2) bet-hedging between what appears to be a quantity versus quality trade-off in endospore crops.

Helicobacter pylori glycan biosynthesis modulates host immune cell recognition and response.

Introduction: The pathogenic bacterium Helicobacter pylori has evolved glycan-mediated mechanisms to evade host immune defenses. This study tests the hypothesis that genetic disruption of H. pylori glycan biosynthesis alters immune recognition and response by human gastric epithelial cells and monocyte-derived dendritic cells. Methods: To test this hypothesis, human cell lines were challenged with wildtype H. pylori alongside an array of H. pylori glycosylation mutants. The relative levels of immune response were measured via immature dendritic cell maturation and cytokine secretion. Results: Our findings indicate that disruption of lipopolysaccharide biosynthesis diminishes gastric cytokine production, without disrupting dendritic cell recognition and activation. In contrast, variable immune responses were observed in protein glycosylation mutants which prompted us to test the hypothesis that phase variation plays a role in regulating bacterial cell surface glycosylation and subsequent immune recognition. Lewis antigen presentation does not correlate with extent of immune response, while the extent of lipopolysaccharide O-antigen elaboration does. Discussion: The outcomes of this study demonstrate that H. pylori glycans modulate the host immune response. This work provides a foundation to pursue immune-based tailoring of bacterial glycans towards modulating immunogenicity of microbial pathogens.

Type 1 fimbrial phase variation in multidrug-resistant asymptomatic uropathogenic Escherichia coli clinical isolates upon adherence to HTB-4 cells.

The adherence of bladder uroepithelial cells, subsequent expression, and regulation of type 1 fimbrial genes (key mediator of attachment) in clinical multidrug-resistant uropathogenic Escherichia coli (MDR-UPECs) isolated from individuals with asymptomatic bacteriuria (ABU) remain unexplored till date. Therefore, this study aimed to investigate the underlying molecular mechanisms associated with the adherence of clinical MDR-ABU-UPECs to human a uroepithelial cell line (HTB-4), both in the absence and presence of D-Mannose. These investigations focused on phase variation, expression, and regulation of type 1 fimbriae and were compared to a prototype ABU-strain (E. coli 83972) and symptomatic MDR-UPECs. Discordant to the ABU prototype strain, MDR-ABU-UPECs exhibited remarkable adhesive capacity that was significantly reduced after D-mannose exposure, fairly like the MDR symptomatic UPECs. The type 1 fimbrial phase variation, determined by the fim switch analysis, asserted the statistically significant incidence of "both OFF and ON" orientation among the adherent MDR-ABU-UPECs with a significant reduction in phase-ON colonies post-D-mannose exposure, akin to the symptomatic ones. This was indicative of an operative and alternating type 1 fimbrial phase switch. The q-PCR assay revealed a coordinated action of the regulatory factors; H-NS, IHF, and Lrp on the expression of FimB and FimE recombinases, which further controlled the function of fimH and fimA genes in ABU-UPECs, similar to symptomatic strains. Therefore, this study is the first of its kind to provide an insight into the regulatory crosstalk of different cellular factors guiding the adhesion of ABU-UPECs to the host. Additionally, it also advocated for the need to accurately characterize ABU-UPECs.

Mutation of wbtJ, a N-formyltransferase involved in O-antigen synthesis, results in biofilm formation, phase variation and attenuation in Francisella tularensis.

Two clinically important subspecies, Francisella tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) are responsible for most tularaemia cases, but these isolates typically form a weak biofilm under in vitro conditions. Phase variation of the F. tularensis lipopolysaccharide (LPS) has been reported in these subspecies, but the role of variation is unclear as LPS is crucial for virulence. We previously demonstrated that a subpopulation of LPS variants can constitutively form a robust biofilm in vitro, but it is unclear whether virulence was affected. In this study, we show that biofilm-forming variants of both fully virulent F. tularensis subspecies were highly attenuated in the murine tularaemia model by multiple challenge routes. Genomic sequencing was performed on these strains, which revealed that all biofilm-forming variants contained a lesion within the wbtJ gene, a formyltransferase involved in O-antigen synthesis. A ΔwbtJ deletion mutant recapitulated the biofilm, O-antigen and virulence phenotypes observed in natural variants and could be rescued through complementation with a functional wbtJ gene. Since the spontaneously derived biofilm-forming isolates in this study were a subpopulation of natural variants, reversion events to the wbtJ gene were detected that eliminated the phenotypes associated with biofilm variants and restored virulence. These results demonstrate a role for WbtJ in biofilm formation, LPS variation and virulence of F. tularensis.

Inflammation and bacteriophages affect DNA inversion states and functionality of the gut microbiota.

Reversible genomic DNA inversions control the expression of numerous gut bacterial molecules, but how this impacts disease remains uncertain. By analyzing metagenomic samples from inflammatory bowel disease (IBD) cohorts, we identified multiple invertible regions where a particular orientation correlated with disease. These include the promoter of polysaccharide A (PSA) of Bacteroides fragilis, which induces regulatory T cells (Tregs) and ameliorates experimental colitis. The PSA promoter was mostly oriented "OFF" in IBD patients, which correlated with increased B. fragilis-associated bacteriophages. Similarly, in mice colonized with a healthy human microbiota and B. fragilis, induction of colitis caused a decline of PSA in the "ON" orientation that reversed as inflammation resolved. Monocolonization of mice with B. fragilis revealed that bacteriophage infection increased the frequency of PSA in the "OFF" orientation, causing reduced PSA expression and decreased Treg cells. Altogether, we reveal dynamic bacterial phase variations driven by bacteriophages and host inflammation, signifying bacterial functional plasticity during disease.

Phage transcriptional regulator X (PtrX)-mediated augmentation of toxin production and virulence in Clostridioides difficile strain R20291.

Clostridioides difficile is a Gram-positive, anaerobic, and spore-forming bacterial member of the human gut microbiome. The primary virulence factors of C. difficile are toxin A and toxin B. These toxins damage the cell cytoskeleton and cause various diseases, from diarrhea to severe pseudomembranous colitis. Evidence suggests that bacteriophages can regulate the expression of the pathogenicity locus (PaLoc) genes of C. difficile. We previously demonstrated that the genome of the C. difficile RT027 strain NCKUH-21 contains a prophage-like DNA sequence, which was found to be markedly similar to that of the φCD38-2 phage. In the present study, we investigated the mechanisms underlying the φNCKUH-21-mediated regulation of the pathogenicity and the PaLoc genes expression in the lysogenized C. difficile strain R20291. The carriage of φNCKUH-21 in R20291 cells substantially enhanced toxin production, bacterial motility, biofilm formation, and spore germination in vitro. Subsequent mouse studies revealed that the lysogenized R20291 strain caused a more severe infection than the wild-type strain. We screened three φNCKUH-21 genes encoding DNA-binding proteins to check their effects on PaLoc genes expression. The overexpression of NCKUH-21_03890, annotated as a transcriptional regulator (phage transcriptional regulator X, PtrX), considerably enhanced toxin production, biofilm formation, and bacterial motility of R20291. Transcriptome analysis further confirmed that the overexpression of ptrX led to the upregulation of the expression of toxin genes, flagellar genes, and csrA. In the ptrX-overexpressing R20291 strain, PtrX influenced the expression of flagellar genes and the sigma factor gene sigD, possibly through an increased flagellar phase ON configuration ratio.

The Helicobacter pylori methylome is acid-responsive due to regulation by the two-component system ArsRS and the type I DNA methyltransferase HsdM1 (HP0463).

In addition to its role in genome protection, DNA methylation can regulate gene expression. In this study, we characterized the impact of acidity, phase variation, and the ArsRS TCS on the expression of the Type I m6A DNA methyltransferase HsdM1 (HP0463) of Helicobacter pylori 26695 and their subsequent effects on the methylome. Transcription of hsdM1 increases at least fourfold in the absence of the sensory histidine kinase ArsS, the major acid-sensing protein of H. pylori. hsdM1 exists in the phase-variable operon hsdR1-hsdM1. Phase-locking hsdR1 (HP0464), the restriction endonuclease gene, has significant impacts on the transcription of hsdM1. To determine the impacts of methyltransferase transcription patterns on the methylome, we conducted methylome sequencing on samples cultured at pH 7 or pH 5. We found differentially methylated motifs between these growth conditions and that deletions of arsS and/or hsdM1 interfere with the epigenetic acid response. Deletion of arsS leads to altered activity of HsdM1 and multiple other methyltransferases under both pH conditions indicating that the ArsRS TCS, in addition to direct effects on regulon transcription during acid acclimation, may also indirectly impact gene expression via regulation of the methylome. We determined the target motif of HsdM1 (HP0463) to be the complementary bipartite sequence pair 5'-TCAm6AVN6TGY-3' and 3'-AGTN6GAm6ACA-5'. This complex regulation of DNA methyltransferases, and thus differential methylation patterns, may have implications for the decades-long persistent infection by H. pylori. IMPORTANCE This study expands the possibilities for complex, epigenomic regulation in Helicobacter pylori. We demonstrate that the H. pylori methylome is plastic and acid sensitive via the two-component system ArsRS and the DNA methyltransferase HsdM1. The control of a methyltransferase by ArsRS may allow for a layered response to changing acidity. Likely, an early response whereby ArsR~P affects regulon expression, including the methyltransferase hsdM1. Then, a somewhat later effect as the altered methylome, due to altered HsdM1 expression, subsequently alters the expression of other genes involved in acclimation. The intermediate methylation of certain motifs supports the hypothesis that methyltransferases play a regulatory role. Untangling this additional web of regulation could play a key role in understanding H. pylori colonization and persistence.

The streptococcal phase-variable type I restriction modification system SsuCC20p dictates the methylome of Streptococcus suis impacting the transcriptome and virulence in a zebrafish larvae infection model.

IMPORTANCE: Phase variation allows a single strain to produce phenotypic diverse subpopulations. Phase-variable restriction modification (RM) systems are systems that allow for such phase variation via epigenetic regulation of gene expression levels. The phase-variable RM system SsuCC20p was found in multiple streptococcal species and was acquired by an emerging zoonotic lineage of Streptococcus suis. We show that the phase variability of SsuCC20p is dependent on a recombinase encoded within the SsuCC20p locus. We characterized the genome methylation profiles of the different phases of SsuCC20p and demonstrated the consequential impact on the transcriptome and virulence in a zebrafish infection model. Acquiring mobile genetic elements containing epigenetic regulatory systems, like phase-variable RM systems, enables bacterial pathogens to produce diverse phenotypic subpopulations that are better adapted to specific (host) environments encountered during infection.

Phasevarions in Haemophilus influenzae biogroup aegyptius control expression of multiple proteins.

IMPORTANCE: Haemophilus influenzae biogroup aegyptius is a human-adapted pathogen and the causative agent of Brazilian purpuric fever (BPF), an invasive disease with high mortality, that sporadically manifests in children previously suffering conjunctivitis. Phase variation is a rapid and reversible switching of gene expression found in many bacterial species, and typically associated with outer-membrane proteins. Phase variation of cytoplasmic DNA methyltransferases has been shown to play important roles in bacterial gene regulation and can act as epigenetic switches, regulating the expression of multiple genes as part of systems called phasevarions (phase-variable regulons). This study characterized two alleles of the ModA phasevarion present in H. influenzae biogroup aegyptius, ModA13, found in non-BPF causing strains and ModA16, unique to BPF causing isolates. Phase variation of ModA13 and ModA16 led to genome-wide changes to DNA methylation resulting in altered protein expression. These changes did not affect serum resistance in H. influenzae biogroup aegyptius strains.

The first genomic characterization of a stable, hemin-dependent small colony variant strain of Staphylococcus epidermidis isolated from a prosthetic-joint infection.

Phenotype switching from a wild type (WT) to a slow-growing subpopulation, referred to as small colony variants (SCVs), supports an infectious lifestyle of Staphylococcus epidermidis, the leading cause of medical device-related infections. Specific mechanisms underlying formation of SCVs and involved in the shaping of their pathogenic potential are of particular interest for stable strains as they have been only rarely cultured from clinical specimens. As the SCV phenotype stability implies the existence of genetic changes, the whole genome sequence of a stable, hemin-dependent S. epidermidis SCV strain (named 49SCV) involved in a late prosthetic joint infection was analyzed. The strain was isolated in a monoculture without a corresponding WT clone, therefore, its genome was compared against five reference S. epidermidis strains (ATCC12228, ATCC14990, NBRC113846, O47, and RP62A), both at the level of the genome structure and coding sequences. According to the Multilocus Sequence Typing analysis, the 49SCV strain represented the sequence type 2 (ST2) regarded as the most prominent infection-causing lineage with a worldwide dissemination. Genomic features unique to 49SCV included the absence of the Staphylococcal Cassette Chromosome (SCC), ~12 kb deletion with the loss of genes involved in the arginine deiminase pathway, and frameshift-generating mutations within the poly(A) and poly(T) homopolymeric tracts. Indels were identified in loci associated with adherence, metabolism, stress response, virulence, and cell wall synthesis. Of note, deletion in the poly(A) of the hemA gene has been considered a possible trigger factor for the phenotype transition and hemin auxotrophy in the strain. To our knowledge, the study represents the first genomic characterization of a clinical, stable and hemin-dependent S. epidermidis SCV strain. We propose that previously unreported indels in the homopolymeric tracts can constitute a background of the SCV phenotype due to a resulting truncation of the corresponding proteins and their possible biological dysfunction. Streamline of genetic content evidenced by the loss of the SCC and a large genomic deletion can represent a possible strategy associated both with the SCV phenotype and its adaptation to chronicity.

High-depth RNA-Seq data sets to investigate the differences in gene expression mediated by phasevarions in non-typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae (NTHi) is a major bacterial pathogen of the human airway. We report high-depth coverage RNA-Seq data from prototype NTHi strains 723 and R2866, encoding two of the most common phase-variable ModA alleles found in NTHi strains, ModA2 and ModA10, respectively.

Novel method for prediction of combinatorial phase-variable gene expression states.

Short sequence repeat mediated phase variation results in diverse phenotype presentation in many bacteria including Campylobacter and Neisseria species. Current methods for identifying the expression states of phase-variable genes involve taking a high number of single colonies. This approach is subject to bias, sampling effects and high workloads that reduce the ability to perform intermediary sampling. The use of high concentration colony sweeps provides a work around but reduces the resolution of combinatorial expression profiles (termed phasotypes). A parsimonious approach combining both single colony and sweep data was developed to overcome these limitations. The critical methodological advance is the use of an algorithm that utilises the experimental data from the two sample types and a parsimonious, iterative mathematical analysis that outputs the phasotype distribution with the highest likelihood of underpinning the experimental data sets. The advantages of this unified method are increased resolution and accuracy of gene expression state combinations as compared to conventional single colony sampling, reduced requirement for sampling large numbers of colonies leading to reduced costs, and a higher capacity for collecting samples and replicates.•Inputting of sweep and single colony data into an algorithm for a rapid determination of the combinatorial phase variation states (phasotypes) for repeat-mediated phase-variable bacterial genes•This method reduces the number of single colony samples required to produce accurate estimates of phasotypes•This method will reduce the costs of phasotype analyses and increase potential to analyse more time points or sample sites leading to an improved understanding of how phase variation contributes to bacterial host persistence and the ability to cause disease.

Phase variation as a major mechanism of adaptation in Mycobacterium tuberculosis complex.

Phase variation induced by insertions and deletions (INDELs) in genomic homopolymeric tracts (HT) can silence and regulate genes in pathogenic bacteria, but this process is not characterized in MTBC (Mycobacterium tuberculosis complex) adaptation. We leverage 31,428 diverse clinical isolates to identify genomic regions including phase-variants under positive selection. Of 87,651 INDEL events that emerge repeatedly across the phylogeny, 12.4% are phase-variants within HTs (0.02% of the genome by length). We estimated the in-vitro frameshift rate in a neutral HT at 100× the neutral substitution rate at [Formula: see text] frameshifts/HT/year. Using neutral evolution simulations, we identified 4,098 substitutions and 45 phase-variants to be putatively adaptive to MTBC (P < 0.002). We experimentally confirm that a putatively adaptive phase-variant alters the expression of espA, a critical mediator of ESX-1-dependent virulence. Our evidence supports the hypothesis that phase variation in the ESX-1 system of MTBC can act as a toggle between antigenicity and survival in the host.

A simple method for enrichment of phase I Coxiella burnetii.

Coxiella burnetii is the bacterial causative agent of the zoonosis Q fever. This bacterium undergoes lipopolysaccharide (LPS) phase transition similar to Enterobacteriaciae upon in vitro passage. Full-length, phase I C. burnetii LPS is a critical virulence factor and profoundly impacts vaccine-induced immunogenicity; thus, LPS phase is an important consideration in C. burnetii experimentation and Q fever vaccine design. Typically, phase I LPS-expressing organisms are obtained from the tissues of infected experimental animals. In this process, residual phase II LPS-expressing organisms are thought to be cleared by the host immune system. Here, we propose an efficient and non-animal-based method for the enrichment of C. burnetii phase I LPS-expressing bacteria in vitro. We utilize both Vero cell culture to selectively enrich solutions with phase I and intermediate phase LPS-expressing bacteria. This simple and quick method decreases reliance on experimental animals and is a sustainable solution for Q fever diagnostic and vaccine development hurdles.

Slipped-strand mispairing within a polycytidine tract in transcriptional regulator mga leads to M protein phase variation and Mga length polymorphism in Group A Streptococcus.

The M protein, a major virulence factor of Group A Streptococcus (GAS), is regulated by the multigene regulator Mga. An unexplained phenomena frequently occurring with in vitro genetic manipulation or culturing of M1T1 GAS strains is the loss of M protein production. This study was aimed at elucidating the basis for the loss of M protein production. The majority of M protein-negative (M-) variants had one C deletion at a tract of 8 cytidines starting at base 1,571 of the M1 mga gene, which is designated as c.1571C[8]. The C deletion led to a c.1571C[7] mga variant that has an open reading frame shift and encodes a Mga-M protein fusion protein. Transformation with a plasmid containing wild-type mga restored the production of the M protein in the c.1571C[7] mga variant. Isolates producing M protein (M+) were recovered following growth of the c.1571C[7] M protein-negative variant subcutaneously in mice. The majority of the recovered isolates with reestablished M protein production had reverted back from c.1571C[7] to c.1571C[8] tract and some M+ isolates lost another C in the c.1571C[7] tract, leading to a c.1571C[6] variant that encodes a functional Mga with 13 extra amino acid residues at the C-terminus compared with wild-type Mga. The nonfunctional c.1571C[7] and functional c.1571C[6] variants are present in M1, M12, M14, and M23 strains in NCBI genome databases, and a G-to-A nonsense mutation at base 1,657 of M12 c.1574C[7] mga leads to a functional c.1574C[7]/1657A mga variant and is common in clinical M12 isolates. The numbers of the C repeats in this polycytidine tract and the polymorphism at base 1,657 lead to polymorphism in the size of Mga among clinical isolates. These findings demonstrate the slipped-strand mispairing within the c.1574C[8] tract of mga as a reversible switch controlling M protein production phase variation in multiple GAS common M types.