Phase-variable bacterial loci: how bacteria gamble to maximise fitness in changing environments

Abstract Phase-variation of genes is defined as the rapid and reversible switching of expression — either ON-OFF switching or the expression of multiple allelic variants. Switching of expression can be achieved by a number of different mechanisms. Phase-variable genes typically encode bacterial surface structures, such as adhesins, pili, and lipooligosaccharide, and provide an extra contingency strategy in small-genome pathogens that may lack the plethora of ‘sense-and-respond’ gene regulation systems found in other organisms. Many bacterial pathogens also encode phase-variable DNA methyltransferases that control the expression of multiple genes in systems called phasevarions (phase-variable regulons). The presence of phase-variable genes allows a population of bacteria to generate a number of phenotypic variants, some of which may be better suited to either colonising certain host niches, surviving a particular environmental condition and/or evading an immune response. The presence of phase-variable genes complicates the determination of an organism's stably expressed antigenic repertoire; many phase-variable genes are highly immunogenic, and so would be ideal vaccine candidates, but unstable expression due to phase-variation may allow vaccine escape. This review will summarise our current understanding of phase-variable genes that switch expression by a variety of mechanisms, and describe their role in disease and pathobiology.

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Stabilizing Genetically Unstable Simple Sequence Repeats in the Campylobacter jejuni Genome by Multiplex Genome Editing: a Reliable Approach for Delineating Multiple Phase-Variable Genes

mBio ◽

10.1128/mbio.01401-21 ◽

2021 ◽

Author(s):

Shouji Yamamoto ◽

Sunao Iyoda ◽

Makoto Ohnishi

Keyword(s):

Campylobacter Jejuni ◽

Simple Sequence Repeat ◽

Phase Variation ◽

Developed Countries ◽

Phase Variable ◽

Phenotypic Stability ◽

Content Type ◽

Multiple Phase ◽

Simple Sequence ◽

Variable Genes

Campylobacter jejuni is the leading bacterial cause of foodborne gastroenteritis in developed countries and occasionally progresses to the autoimmune disease Guillain-Barré syndrome. A relatively large number of hypermutable simple sequence repeat (SSR) tracts in the C. jejuni genome markedly decreases its phenotypic stability through reversible changes in the ON or OFF expression states of the genes in which they reside, a phenomenon called phase variation.

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Systematic Analysis of REBASE Identifies Numerous Type I Restriction-Modification Systems with Duplicated, Distinct hsdS Specificity Genes That Can Switch System Specificity by Recombination

mSystems ◽

10.1128/msystems.00497-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

John M. Atack ◽

Chengying Guo ◽

Thomas Litfin ◽

Long Yang ◽

Patrick J. Blackall ◽

...

Keyword(s):

Vaccine Development ◽

Phase Variation ◽

Dna Methyltransferases ◽

Inverted Repeats ◽

Global Changes ◽

Type I ◽

Phase Variable ◽

Global Methylation ◽

Content Type ◽

Restriction Modification

ABSTRACT N6-Adenine DNA methyltransferases associated with some Type I and Type III restriction-modification (R-M) systems are able to undergo phase variation, randomly switching expression ON or OFF by varying the length of locus-encoded simple sequence repeats (SSRs). This variation of methyltransferase expression results in genome-wide methylation differences and global changes in gene expression. These epigenetic regulatory systems are called phasevarions, phase-variable regulons, and are widespread in bacteria. A distinct switching system has also been described in Type I R-M systems, based on recombination-driven changes in hsdS genes, which dictate the DNA target site. In order to determine the prevalence of recombination-driven phasevarions, we generated a program called RecombinationRepeatSearch to interrogate REBASE and identify the presence and number of inverted repeats of hsdS downstream of Type I R-M loci. We report that 3.9% of Type I R-M systems have duplicated variable hsdS genes containing inverted repeats capable of phase variation. We report the presence of these systems in the major pathogens Enterococcus faecalis and Listeria monocytogenes, which could have important implications for pathogenesis and vaccine development. These data suggest that in addition to SSR-driven phasevarions, many bacteria have independently evolved phase-variable Type I R-M systems via recombination between multiple, variable hsdS genes. IMPORTANCE Many bacterial species contain DNA methyltransferases that have random on/off switching of expression. These systems, called phasevarions (phase-variable regulons), control the expression of multiple genes by global methylation changes. In every previously characterized phasevarion, genes involved in pathobiology, antibiotic resistance, and potential vaccine candidates are randomly varied in their expression, commensurate with methyltransferase switching. Our systematic study to determine the extent of phasevarions controlled by invertible Type I R-M systems will provide valuable information for understanding how bacteria regulate genes and is key to the study of physiology, virulence, and vaccine development; therefore, it is critical to identify and characterize phase-variable methyltransferases controlling phasevarions.

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Phase Variation During Host Colonization and Invasion by Campylobacter jejuni and Other Campylobacter Species

Frontiers in Microbiology ◽

10.3389/fmicb.2021.705139 ◽

2021 ◽

Vol 12 ◽

Author(s):

Caroline Cayrou ◽

Natalie A. Barratt ◽

Julian M. Ketley ◽

Christopher D. Bayliss

Keyword(s):

Bacterial Species ◽

Foodborne Pathogen ◽

Phase Variation ◽

Phase Variable ◽

Bacterial Survival ◽

Zoonotic Infections ◽

Infection Models ◽

The Status ◽

Host Tissues ◽

Variable Genes

Phase variation (PV) is a phenomenon common to a variety of bacterial species for niche adaption and survival in challenging environments. Among Campylobacter species, PV depends on the presence of intergenic and intragenic hypermutable G/C homopolymeric tracts. The presence of phase-variable genes is of especial interest for species that cause foodborne or zoonotic infections in humans. PV influences the formation and the structure of the lipooligosaccharide, flagella, and capsule in Campylobacter species. PV of components of these molecules is potentially important during invasion of host tissues, spread within hosts and transmission between hosts. Motility is a critical phenotype that is potentially modulated by PV. Variation in the status of the phase-variable genes has been observed to occur during colonization in chickens and mouse infection models. Interestingly, PV is also involved in bacterial survival of attack by bacteriophages even during chicken colonization. This review aims to explore and discuss observations of PV during model and natural infections by Campylobacter species and how PV may affect strategies for fighting infections by this foodborne pathogen.

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Mecanismos moleculares de adaptación del patógeno respiratorio Haemophilus influenzae y desarrollo de nuevos antimicrobianos

10.48035/tesis/2454/41079 ◽

2020 ◽

Author(s):

◽

Ariadna Fernández Calvet

Keyword(s):

Haemophilus Influenzae ◽

Molecular Mechanisms ◽

Therapeutic Potential ◽

Phase Variation ◽

Opportunistic Pathogen ◽

Phase Variable ◽

Nontypeable Haemophilus Influenzae ◽

Molecular Systems ◽

Bacterial Surface ◽

Human Airways

This PhD Thesis work tackled three aspects of the interaction between the colonizing opportunistic pathogen nontypeable Haemophilus influenzae (NTHi) and the human airways, by considering the concepts of phase-variable regulation of pathoadaptive traits (Chapter 1), the importance of molecular systems involved in maintaining the bacterial surface integrity (Chapter 2), and the therapeutic potential of xenohormetic molecules (Chapter 3). Altogether, this work contributes expanding our understanding on molecular mechanisms of NTHi pathoadaptation regulated by phase variation, provides evidence for VacJ/MlaA as a key bacterial factor modulating NTHi survival at the human airway upon exposure to hydrophobic molecules, and highlights the therapeutic potential of xenohormetic molecules against NTHi infection.

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Stabilizing genetically unstable simple sequence repeats in the Campylobacter jejuni genome by multiplex genome editing: a reliable approach for delineating multiple phase-variable genes

10.1101/2021.05.14.444138 ◽

2021 ◽

Author(s):

Shouji Yamamoto ◽

Sunao Iyoda ◽

Makoto Ohnishi

Keyword(s):

Genome Editing ◽

Campylobacter Jejuni ◽

Simple Sequence Repeats ◽

Antibiotic Resistance Gene ◽

Phase Variation ◽

Phase Variable ◽

Dna Fragments ◽

Variable Expression ◽

Simple Sequence ◽

Variable Genes

Hypermutability of simple sequence repeats (SSR) through DNA slippage is a major mechanism of phase variation in Campylobacter jejuni . The presence of multiple SSR-mediated phase-variable genes encoding enzymes that modify surface structures, including capsular polysaccharide (CPS) and lipooligosaccharide (LOS), generates high levels of structural variants within bacterial populations, thereby promoting adaptation to selective pressures in host environments. Therefore, the phenotypic diversity generated by phase variation can limit the reproducibility of results with C. jejuni ; therefore, researchers need to genetically control the mutability of multiple SSRs. Here, we show that natural “cotransformation” is an effective method for C. jejuni genome editing. Cotransformation is a trait of naturally competent bacteria that causes uptake and integration of multiple different DNA fragments, which has been recently adapted to multiplex genome editing by natural transformation (MuGENT), a method for introducing multiple scarless mutations into the genomes of these bacteria. We found that the cotransformation frequencies of antibiotic resistance gene-marked DNA fragments and unmarked DNA fragments reached ~40% in C. jejuni . To examine the feasibility of MuGENT in C. jejuni , we “locked” either different polyG SSR tracts in strain NCTC11168 (which are located in the biosynthetic CPS and LOS gene clusters) into either the ON or OFF configurations by interrupting the continuous runs of G residues without changing the encoded amino acids. This approach, termed “MuGENT-SSR,” enabled the generation of all eight edits within 2 weeks and the identification of a phase-locked strain with a highly stable type of Penner serotyping, a CPS-based serotyping scheme. Furthermore, extensive genome editing of this strain by MuGENT-SSR identified a phase-variable gene that determines the Penner serotype of NCTC11168. Thus, MuGENT-SSR provides a platform for genetic and phenotypic engineering of genetically unstable C. jejuni , making it a reliable approach for elucidating the mechanisms underlying phase-variable expression of specific phenotypes.

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Identification and Functional Mapping of the Mycoplasma fermentans P29 Adhesin

Infection and Immunity ◽

10.1128/iai.70.9.4925-4935.2002 ◽

2002 ◽

Vol 70 (9) ◽

pp. 4925-4935 ◽

Cited By ~ 24

Author(s):

Spencer A. Leigh ◽

Kim S. Wise

Keyword(s):

Disulfide Bond ◽

Hela Cells ◽

Mammalian Cells ◽

Flow Cytometric Analysis ◽

Phase Variation ◽

Surface Proteins ◽

Mycoplasma Species ◽

Phase Variable ◽

Binding Region ◽

Mycoplasma Fermentans

ABSTRACT Initial adherence interactions between mycoplasmas and mammalian cells are important for host colonization and may contribute to subsequent pathogenic processes. Despite significant progress toward understanding the role of specialized, complex tip structures in the adherence of some mycoplasmas, particularly those that infect humans, less is known about adhesins through which other mycoplasmas of this host bind to diverse cell types, even though simpler surface components are likely to be involved. We show by flow cytometric analysis that a soluble recombinant fusion protein (FP29), representing the abundant P29 surface lipoprotein of Mycoplasma fermentans, binds human HeLa cells and inhibits M. fermentans binding to these cells, in both a quantitative and a saturable manner, whereas analogous fusion proteins representing other mycoplasma surface proteins did not. Constructs representing nested N- or C-terminal truncations of FP29 allowed initial mapping of this specific adherence function to a central region of the P29 sequence containing a 36-amino-acid disulfide loop. A derivative of FP29 containing a mutation converting one participating Cys to Ser, precluding intrachain disulfide bond formation, retained full activity. Together these results suggest that the direct interaction of M. fermentans with a ligand on the HeLa cell surface involves a limited segment of the P29 surface lipoprotein and requires neither the disulfide bond nor the contribution of adjacent portions of the protein. Earlier results indicating phase-variable display of monoclonal antibody surface epitopes on P29, now recognized to be outside this ligand binding region, raise the possibility that variation of mycoplasma surface architecture might alter the presentation of the binding region and the adherence phenotype. Preliminary results further indicated that FP29 could inhibit binding to HeLa cells by Mycoplasma hominis, a distinct human mycoplasma species displaying the phase-variable adhesin Vaa, but not that by Mycoplasma capricolum, an organism infecting caprine species. This result raises the additional, testable possibility that a common host cell ligand for two human mycoplasma species may be recognized through structurally dissimilar adhesins that undergo phase variation by two distinct mechanisms, governing protein expression (Vaa) or surface masking (P29).

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Elucidation of the Monoclonal Antibody 5G8-Reactive, Virulence-Associated Lipopolysaccharide Epitope of Haemophilus influenzae and Its Role in Bacterial Resistance to Complement-Mediated Killing

Infection and Immunity ◽

10.1128/iai.73.4.2213-2221.2005 ◽

2005 ◽

Vol 73 (4) ◽

pp. 2213-2221 ◽

Cited By ~ 11

Author(s):

Ruth Griffin ◽

Andrew D. Cox ◽

Katherine Makepeace ◽

James C. Richards ◽

E. Richard Moxon ◽

...

Keyword(s):

Monoclonal Antibody ◽

Haemophilus Influenzae ◽

Bacterial Resistance ◽

Inner Core ◽

Phase Variation ◽

Phase Variable ◽

Type B ◽

Variable Expression ◽

Virulent Type ◽

Unknown Structure

ABSTRACT The phase-variable locus lex2 is required for expression of a Haemophilus influenzae lipopolysaccharide (LPS) epitope of previously unknown structure. This epitope, which is reactive with monoclonal antibody (MAb) 5G8, has been associated with virulence of type b strains. When strain RM118 (from the same source as strain Rd), in which the lex2 locus and MAb 5G8 reactivity are absent, was transformed with lex2 DNA, transformants that were reactive with MAb 5G8 were obtained. Surprisingly, the 5G8 reactivity of these transformants was phase variable, although the lex2 locus lacked tetrameric repeats and was constitutively expressed. This phase variation was shown to be the result of phase-variable expression of phosphorylcholine (PCho) such that MAb 5G8 reacted only in the absence of PCho. Structural analysis showed that, compared to RM118, the lex2 transformant had acquired a tetrasaccharide, Gal-α1,4-Gal-β1,4-Glc-β1,4-Glc-β1,4, linked to the proximal heptose (HepI). A terminal GalNAc was detected in a minority of glycoforms. LPS derived from a mutant of RM7004, a virulent type b strain which naturally expresses lex2 and has LPS containing the same tetrasaccharide linked to HepI as the sole oligosaccharide extension from the inner core, confirmed that GalNAc is not a part of the MAb 5G8-reactive epitope. Thus, MAb 5G8 specifically binds to the structure Gal-α1,4-Gal-β1,4-Glc-β1,4-Glc-β attached via a 1,4 linkage to HepI of H. influenzae LPS, and we show that the ability to synthesize this novel tetrasaccharide was associated with enhanced bacterial resistance to complement-mediated killing.

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Analysis of Invasive NontypeableHaemophilus influenzaeIsolates Reveals Selection for the Expression State of Particular Phase-Variable Lipooligosaccharide Biosynthetic Genes

Infection and Immunity ◽

10.1128/iai.00093-19 ◽

2019 ◽

Vol 87 (5) ◽

Cited By ~ 6

Author(s):

Zachary N. Phillips ◽

Charles Brizuela ◽

Amy V. Jennison ◽

Megan Staples ◽

Keith Grimwood ◽

...

Keyword(s):

Gene Expression ◽

Phase Variation ◽

Phase Variable ◽

Chronic Infections ◽

Biosynthetic Genes ◽

Content Type ◽

Invasive Infections ◽

Acetyl Group ◽

Overt Disease ◽

Expression Status

ABSTRACTNontypeableHaemophilus influenzae(NTHi) is a major human pathogen, responsible for several acute and chronic infections of the respiratory tract. The incidence of invasive infections caused by NTHi is increasing worldwide. NTHi is able to colonize the nasopharynx asymptomatically, and the exact change(s) responsible for transition from benign carriage to overt disease is not understood. We have previously reported that phase variation (the rapid and reversible ON-OFF switching of gene expression) of particular lipooligosaccharide (LOS) glycosyltransferases occurs during transition from colonizing the nasopharynx to invading the middle ear. Variation in the structure of the LOS is dependent on the ON/OFF expression status of each of the glycosyltransferases responsible for LOS biosynthesis. In this study, we surveyed a collection of invasive NTHi isolates for ON/OFF expression status of seven phase-variable LOS glycosyltransferases. We report that the expression state of the LOS biosynthetic genesoafAON andlic2AOFF shows a correlation with invasive NTHi isolates. We hypothesize that these gene expression changes contribute to the invasive potential of NTHi. OafA expression, which is responsible for the addition of anO-acetyl group onto the LOS, has been shown to impart a phenotype of increased serum resistance and may serve as a marker for invasive NTHi.

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