Phase-Variable Expression of an Operon Encoding Extracellular Alkaline Protease, a Serine Protease Homolog, and Lipase in Pseudomonas brassicacearum

ABSTRACT The rhizobacterium Pseudomonas brassicacearum forms phenotypic variants which do not show extracellular protease and lipase activity. The operon encoding these enzymes, a serine protease homolog, and a type I secretion machinery was characterized. Transcriptional lacZ gene fusions revealed that the expression of the operon is under the control of phase variation.

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Epigenetic Regulation of Virulence and Immunoevasion by Phase-Variable Restriction-Modification Systems in Bacterial Pathogens

Annual Review of Microbiology ◽

10.1146/annurev-micro-090817-062346 ◽

2020 ◽

Vol 74 (1) ◽

pp. 655-671

Author(s):

Kate L. Seib ◽

Yogitha N. Srikhanta ◽

John M. Atack ◽

Michael P. Jennings

Keyword(s):

Bacterial Pathogens ◽

Phase Variation ◽

Global Changes ◽

Type I ◽

Phase Variable ◽

Human Pathogens ◽

Variable Expression ◽

Host Immune Responses ◽

Restriction Modification ◽

Restriction Modification Systems

Human-adapted bacterial pathogens use a mechanism called phase variation to randomly switch the expression of individual genes to generate a phenotypically diverse population to adapt to challenges within and between human hosts. There are increasing reports of restriction-modification systems that exhibit phase-variable expression. The outcome of phase variation of these systems is global changes in DNA methylation. Analysis of phase-variable Type I and Type III restriction-modification systems in multiple human-adapted bacterial pathogens has demonstrated that global changes in methylation regulate the expression of multiple genes. These systems are called phasevarions (phase-variable regulons). Phasevarion switching alters virulence phenotypes and facilitates evasion of host immune responses. This review describes the characteristics of phasevarions and implications for pathogenesis and immune evasion. We present and discuss examples of phasevarion systems in the major human pathogens Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Helicobacter pylori, Moraxella catarrhalis, and Streptococcus pneumoniae.

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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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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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Coupled Phase-Variable Expression and Epitope Masking of Selective Surface Lipoproteins Increase Surface Phenotypic Diversity in Mycoplasma hominis

Infection and Immunity ◽

10.1128/iai.69.8.5177-5181.2001 ◽

2001 ◽

Vol 69 (8) ◽

pp. 5177-5181 ◽

Cited By ~ 18

Author(s):

Qijing Zhang ◽

Kim S. Wise

Keyword(s):

Membrane Protein ◽

High Frequency ◽

Phenotypic Diversity ◽

Phase Variation ◽

Mycoplasma Hominis ◽

Phase Variable ◽

Variable Expression ◽

Adaptive Capabilities ◽

Frequency Phase

ABSTRACT A new mechanism expanding mycoplasmal surface diversity is described. Exposure of surface epitopes on a constitutively expressed membrane protein (P56) of Mycoplasma hominis was subject to high-frequency phase variation due to phase-variable expression of the P120 antigen and its selective masking of P56 epitopes. Phase-variable masking may confer previously unrealized adaptive capabilities on mycoplasmas.

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Genotypic and Phenotypic Characterization of theO-Linked Protein Glycosylation System Reveals High Glycan Diversity in Paired Meningococcal Carriage Isolates

Journal of Bacteriology ◽

10.1128/jb.00794-17 ◽

2018 ◽

Vol 200 (16) ◽

Cited By ~ 8

Author(s):

Bente Børud ◽

Guro K. Bårnes ◽

Ola Brønstad Brynildsrud ◽

Elisabeth Fritzsønn ◽

Dominique A. Caugant

Keyword(s):

Neisseria Meningitidis ◽

Phase Variation ◽

Protein Glycosylation ◽

Phase Variable ◽

Upper Respiratory Tract ◽

Content Type ◽

Variable Expression ◽

Evolutionary Forces ◽

Strain Collection

ABSTRACTSpecies within the genusNeisseriadisplay significant glycan diversity associated with theO-linked protein glycosylation (pgl) systems due to phase variation and polymorphic genes and gene content. The aim of this study was to examine in detail thepglgenotype and glycosylation phenotype in meningococcal isolates and the changes occurring during short-term asymptomatic carriage. Paired meningococcal isolates derived from 50 asymptomatic meningococcal carriers, taken about 2 months apart, were analyzed with whole-genome sequencing. TheO-linked protein glycosylation genes were characterized in detail using the Genome Comparator tool at the https://pubmlst.org/ database. Immunoblotting with glycan-specific antibodies (Abs) was used to investigate the protein glycosylation phenotype. All majorpgllocus polymorphisms identified inNeisseria meningitidisto date were present in our isolate collection, with the variable presence ofpglGandpglH, both in combination with eitherpglBorpglB2. We identified significant changes and diversity in thepglgenotype and/or glycan phenotype in 96% of the paired isolates. There was also a high degree of glycan microheterogeneity, in which different variants of glycan structures were found at a given glycoprotein. The main mechanism responsible for the observed differences was phase-variable expression of the involved glycosyltransferases and theO-acetyltransferase. To our knowledge, this is the first characterization of thepglgenotype and glycosylation phenotype in a larger strain collection. This report thus provides important insight into glycan diversity inN. meningitidisand into the phase variability changes that influence the expressed glycoform repertoire during meningococcal carriage.IMPORTANCEBacterial meningitis is a serious global health problem, and one of the major causative organisms isNeisseria meningitidis, which is also a common commensal in the upper respiratory tract of healthy humans. In bacteria, numerous loci involved in biosynthesis of surface-exposed antigenic structures that are involved in the interaction between bacteria and host are frequently subjected to homologous recombination and phase variation. These mechanisms are well described inNeisseria, and phase variation provides the ability to change these structures reversibly in response to the environment. Protein glycosylation systems are becoming widely identified in bacteria, and yet little is known about the mechanisms and evolutionary forces influencing glycan composition during carriage and disease.

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Identification and Characterization ofpptA: a Gene Involved in the Phase-Variable Expression ofPhosphorylcholine on Pili of Neisseriameningitidis

Infection and Immunity ◽

10.1128/iai.71.12.6892-6898.2003 ◽

2003 ◽

Vol 71 (12) ◽

pp. 6892-6898 ◽

Cited By ~ 33

Author(s):

Matthew J. Warren ◽

Michael P. Jennings

Keyword(s):

High Frequency ◽

Phase Variation ◽

Open Reading Frames ◽

Phase Variable ◽

Dna Transformation ◽

Twitching Motility ◽

Variable Expression ◽

Factors Associated ◽

Identification And Characterization ◽

Reading Frames

ABSTRACT Pili of pathogenic Neisseria are major virulence factors associated with adhesion, cytotoxicity, twitching motility, autoaggregation, and DNA transformation. Pili are modified posttranslationally by the addition of phosphorylcholine. However, no genes involved in either the biosynthesis or the transfer of phosphorylcholine in Neisseria meningitidis have been identified. In this study, we identified five candidate open reading frames (ORFs) potentially involved in the biosynthesis or transfer of phosphorylcholine to pilin in N. meningitidis. Insertional mutants were constructed for each ORF in N. meningitidis strain C311#3 to determine their effect on phosphorylcholine expression. The effect of the mutant ORFs on the modification by phosphorylcholine was analyzed by Western analysis with phosphorylcholine-specific monoclonal antibody TEPC-15. Analysis of the mutants showed that ORF NMB0415, now defined as pptA (pilin phosphorylcholine transferase A), is involved in the addition of phosphorylcholine to pilin in N. meningitidis. Additionally, the phase variation (high frequency on-off switching of expression) of phosphorylcholine on pilin is due to changes in a homopolymeric guanosine tract in pptA.

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The Moraxella catarrhalis phase-variable DNA methyltransferase ModM3 is an epigenetic regulator that affects bacterial survival in an in vivo model of otitis media

BMC Microbiology ◽

10.1186/s12866-019-1660-y ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 3

Author(s):

Luke V. Blakeway ◽

Aimee Tan ◽

Joseph A. Jurcisek ◽

Lauren O. Bakaletz ◽

John M. Atack ◽

...

Keyword(s):

Otitis Media ◽

Middle Ear ◽

Moraxella Catarrhalis ◽

Nitrosative Stress ◽

Bacterial Load ◽

Phase Variation ◽

In Vivo Model ◽

Phase Variable ◽

Variable Expression

Abstract Background Moraxella catarrhalis is a leading cause of otitis media (OM) and chronic obstructive pulmonary disease (COPD). M. catarrhalis contains a Type III DNA adenine methyltransferase (ModM) that is phase-variably expressed (i.e., its expression is subject to random, reversible ON/OFF switching). ModM has six target recognition domain alleles (modM1–6), and we have previously shown that modM2 is the predominant allele, while modM3 is associated with OM. Phase-variable DNA methyltransferases mediate epigenetic regulation and modulate pathogenesis in several bacteria. ModM2 of M. catarrhalis regulates the expression of a phasevarion containing genes important for colonization and infection. Here we describe the phase-variable expression of modM3, the ModM3 methylation site and the suite of genes regulated within the ModM3 phasevarion. Results Phase-variable expression of modM3, mediated by variation in length of a 5′-(CAAC)n-3′ tetranucleotide repeat tract in the open reading frame was demonstrated in M. catarrhalis strain CCRI-195ME with GeneScan fragment length analysis and western immunoblot. We determined that ModM3 is an active N6-adenine methyltransferase that methylates the sequence 5′-ACm6ATC-3′. Methylation was detected at all 4446 5′-ACATC-3′ sites in the genome when ModM3 is expressed. RNASeq analysis identified 31 genes that are differentially expressed between modM3 ON and OFF variants, including five genes that are involved in the response to oxidative and nitrosative stress, with potential roles in biofilm formation and survival in anaerobic environments. An in vivo chinchilla (Chinchilla lanigera) model of otitis media demonstrated that transbullar challenge with the modM3 OFF variant resulted in an increased middle ear bacterial load compared to a modM3 ON variant. In addition, co-infection experiments with NTHi and M. catarrhalis modM3 ON or modM3 OFF variants revealed that phase variation of modM3 altered survival of NTHi in the middle ear during early and late stage infection. Conclusions Phase variation of ModM3 epigenetically regulates the expression of a phasevarion containing multiple genes that are potentially important in the progression of otitis media.

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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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GAA Trinucleotide Repeat Region Regulates M9/pMGA Gene Expression in Mycoplasma gallisepticum

Infection and Immunity ◽

10.1128/iai.68.2.871-876.2000 ◽

2000 ◽

Vol 68 (2) ◽

pp. 871-876 ◽

Cited By ~ 34

Author(s):

Li Liu ◽

Kevin Dybvig ◽

Victor S. Panangala ◽

Vicky L. van Santen ◽

Christopher T. French

Keyword(s):

Gene Expression ◽

Trinucleotide Repeat ◽

Mycoplasma Gallisepticum ◽

Avian Host ◽

Phase Variable ◽

Repeat Region ◽

Chromosomal Dna ◽

Promoter Regions ◽

Variable Expression ◽

Gaa Repeats

ABSTRACT Mycoplasma gallisepticum, the cause of chronic respiratory infections in the avian host, possesses a family of M9/pMGA genes encoding an adhesin(s) associated with hemagglutination. Nucleotide sequences of M9/pMGA gene family members indicate extensive sequence similarity in the promoter regions of both the transcribed and silent genes. The mechanism that regulates M9/pMGA gene expression is unknown, but studies have revealed an apparent correlation between gene expression and the number of tandem GAA repeat motifs located upstream of the putative promoter. In this study, transposon Tn4001was used as a vector with the Escherichia coli lacZ gene as the reporter system to examine the role of the GAA repeats in M9/pMGA gene expression in M. gallisepticum. A 336-bp M9 gene fragment (containing the GAA repeat region, the promoter, and the translation start codon) was amplified by PCR, ligated with alacZ gene from E. coli, and inserted into the Tn4001-containing plasmid pISM2062. This construct was transformed into M. gallisepticum PG31. Transformants were filter cloned on agar supplemented with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) to monitor lacZ gene expression on the basis of blue/white color selection. Several cycles of filter cloning resulted in cell lineages in which lacZ gene expression alternated between the On and Off states in successive generations of progeny clones. The promoter regions of the M9-lacZ hybrid genes of individual progeny clones were amplified by PCR and sequenced. The only differences between the promoter regions of the blue and white colonies were in the number of GAA repeats. Clones that expressedlacZ had exactly 12 tandem copies of the GAA repeat. Clones that did not express lacZ invariably had either more than 12 (14 to 16) or fewer than 12 (5 to 11) GAA repeats. Southern analysis of M. gallisepticum chromosomal DNA confirmed that the phase-variable expression of the lacZ reporter gene was not caused by Tn4001 transposition. These data strongly indicate that changes in the length of the GAA repeat region are responsible for regulating M9/pMGA gene expression.

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