Comparative Analysis of Streptococcus pneumoniae Type I Restriction-Modification Loci: Variation in hsdS Gene Target Recognition Domains

Streptococcus pneumoniae (pneumococcus) is a respiratory commensal pathogen that causes a range of infections, particularly in young children and the elderly. Pneumococci undergo spontaneous phase variation in colony opacity phenotype, in which DNA rearrangements within the Type I restriction-modification (R-M) system specificity gene hsdS can potentially generate up to six different hsdS alleles with differential DNA methylation activity, resulting in changes in gene expression. To gain a broader perspective of this system, we performed bioinformatic analyses of Type I R-M loci from 18 published pneumococcal genomes, and one R-M locus sequenced for this study, to compare genetic content, organization, and homology. All 19 loci encoded the genes hsdR, hsdM, hsdS, and at least one hsdS pseudogene, but differed in gene order, gene orientation, and hsdS target recognition domain (TRD) content. We determined the coding sequences of 87 hsdS TRDs and excluded seven from further analysis due to the presence of premature stop codons. Comparative analyses revealed that the TRD 1.1, 1.2, and 2.1 protein sequences had single amino acid substitutions, and TRD 2.2 and 2.3 each had seven differences. The results of this study indicate that variability exists among the gene content and arrangements within Type I R-M loci may provide an additional level of divergence between pneumococcal strains, such that phase variation-mediated control of virulence factors may vary significantly between individual strains. These findings are consistent with presently available transcript profile data.

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Functional Analysis of MmeI from Methanol Utilizer Methylophilus methylotrophus, a Subtype IIC Restriction-Modification Enzyme Related to Type I Enzymes

Applied and Environmental Microbiology ◽

10.1128/aem.01322-08 ◽

2008 ◽

Vol 75 (1) ◽

pp. 212-223 ◽

Cited By ~ 12

Author(s):

Joanna Nakonieczna ◽

Tadeusz Kaczorowski ◽

Agnieszka Obarska-Kosinska ◽

Janusz M. Bujnicki

Keyword(s):

Target Recognition ◽

Single Amino Acid ◽

Classical Type ◽

Type I ◽

Type Ii ◽

Terminal Portion ◽

Methylophilus Methylotrophus ◽

Putative Target ◽

Single Amino Acid Residue ◽

Restriction Modification

ABSTRACT MmeI from Methylophilus methylotrophus belongs to the type II restriction-modification enzymes. It recognizes an asymmetric DNA sequence, 5′-TCCRAC-3′ (R indicates G or A), and cuts both strands at fixed positions downstream of the specific site. This particular feature has been exploited in transcript profiling of complex genomes (using serial analysis of gene expression technology). We have shown previously that the endonucleolytic activity of MmeI is strongly dependent on the presence of S-adenosyl-l-methionine (J. Nakonieczna, J. W. Zmijewski, B. Banecki, and A. J. Podhajska, Mol. Biotechnol. 37:127-135, 2007), which puts MmeI in subtype IIG. The same cofactor is used by MmeI as a methyl group donor for modification of an adenine in the upper strand of the recognition site to N 6-methyladenine. Both enzymatic activities reside in a single polypeptide (919 amino acids [aa]), which puts MmeI also in subtype IIC of the restriction-modification systems. Based on a molecular model, generated with the use of bioinformatic tools and validated by site-directed mutagenesis, we were able to localize three functional domains in the structure of the MmeI enzyme: (i) the N-terminal portion containing the endonucleolytic domain with the catalytic Mg2+-binding motif D70-X9-EXK82, characteristic for the PD-(D/E)XK superfamily of nucleases; (ii) a central portion (aa 310 to 610) containing nine sequence motifs conserved among N 6-adenine γ-class DNA methyltransferases; (iii) the C-terminal portion (aa 610 to 919) containing a putative target recognition domain. Interestingly, all three domains showed highest similarity to the corresponding elements of type I enzymes rather than to classical type II enzymes. We have found that MmeI variants deficient in restriction activity (D70A, E80A, and K82A) can bind and methylate specific nucleotide sequence. This suggests that domains of MmeI responsible for DNA restriction and modification can act independently. Moreover, we have shown that a single amino acid residue substitution within the putative target recognition domain (S807A) resulted in a MmeI variant with a higher endonucleolytic activity than the wild-type enzyme.

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Genetic variation regulates the activation and specificity of Restriction-Modification systems in Neisseria gonorrhoeae

Scientific Reports ◽

10.1038/s41598-019-51102-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Leonor Sánchez-Busó ◽

Daniel Golparian ◽

Julian Parkhill ◽

Magnus Unemo ◽

Simon R. Harris

Keyword(s):

Neisseria Gonorrhoeae ◽

Single Molecule ◽

Regulation Of Gene Expression ◽

Phase Variation ◽

Type I ◽

Dam Methylase ◽

Different Strains ◽

Methylation Patterns ◽

Restriction Modification ◽

Restriction Modification Systems

Abstract Restriction-Modification systems (RMS) are one of the main mechanisms of defence against foreign DNA invasion and can have an important role in the regulation of gene expression. The obligate human pathogen Neisseria gonorrhoeae carries one of the highest loads of RMS in its genome; between 13 to 15 of the three main types. Previous work has described their organization in the reference genome FA1090 and has inferred the associated methylated motifs. Here, we studied the structure of RMS and target methylated motifs in 25 gonococcal strains sequenced with Single Molecule Real-Time (SMRT) technology, which provides data on DNA modification. The results showed a variable picture of active RMS in different strains, with phase variation switching the activity of Type III RMS, and both the activity and specificity of a Type I RMS. Interestingly, the Dam methylase was found in place of the NgoAXI endonuclease in two of the strains, despite being previously thought to be absent in the gonococcus. We also identified the real methylation target of NgoAXII as 5′-GCAGA-3′, different from that previously described. Results from this work give further insights into the diversity and dynamics of RMS and methylation patterns in N. gonorrhoeae.

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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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Synergistic Activity of Mobile Genetic Element Defences in Streptococcus pneumoniae

Genes ◽

10.3390/genes10090707 ◽

2019 ◽

Vol 10 (9) ◽

pp. 707 ◽

Cited By ~ 3

Author(s):

Min Jung Kwun ◽

Marco R. Oggioni ◽

Stephen D. Bentley ◽

Christophe Fraser ◽

Nicholas J. Croucher

Keyword(s):

Streptococcus Pneumoniae ◽

Mobile Genetic Element ◽

Horizontal Transmission ◽

Phase Variation ◽

Uninfected Cell ◽

Synergistic Activity ◽

Phase Variable ◽

Two Phase ◽

Strain Transformation ◽

Restriction Modification

A diverse set of mobile genetic elements (MGEs) transmit between Streptococcus pneumoniae cells, but many isolates remain uninfected. The best-characterised defences against horizontal transmission of MGEs are restriction-modification systems (RMSs), of which there are two phase-variable examples in S. pneumoniae. Additionally, the transformation machinery has been proposed to limit vertical transmission of chromosomally integrated MGEs. This work describes how these mechanisms can act in concert. Experimental data demonstrate RMS phase variation occurs at a sub-maximal rate. Simulations suggest this may be optimal if MGEs are sometimes vertically inherited, as it reduces the probability that an infected cell will switch between RMS variants while the MGE is invading the population, and thereby undermine the restriction barrier. Such vertically inherited MGEs can be deleted by transformation. The lack of between-strain transformation hotspots at known prophage att sites suggests transformation cannot remove an MGE from a strain in which it is fixed. However, simulations confirmed that transformation was nevertheless effective at preventing the spread of MGEs into a previously uninfected cell population, if a recombination barrier existed between co-colonising strains. Further simulations combining these effects of phase variable RMSs and transformation found they synergistically inhibited MGEs spreading, through limiting both vertical and horizontal transmission.

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Deletion of the Zinc Transporter Lipoprotein AdcAII Causes Hyperencapsulation of Streptococcus pneumoniae Associated with Distinct Alleles of the Type I Restriction-Modification System

mBio ◽

10.1128/mbio.00445-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 1

Author(s):

Claire Durmort ◽

Giuseppe Ercoli ◽

Elisa Ramos-Sevillano ◽

Suneeta Chimalapati ◽

Richard D. Haigh ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Mouse Models ◽

Zinc Uptake ◽

Invasive Infection ◽

Type I ◽

Modification System ◽

Content Type ◽

Restriction Modification System ◽

Capsule Thickness ◽

Restriction Modification

ABSTRACT The capsule is the dominant Streptococcus pneumoniae virulence factor, yet how variation in capsule thickness is regulated is poorly understood. Here, we describe an unexpected relationship between mutation of adcAII, which encodes a zinc uptake lipoprotein, and capsule thickness. Partial deletion of adcAII in three of five capsular serotypes frequently resulted in a mucoid phenotype that biochemical analysis and electron microscopy of the D39 adcAII mutants confirmed was caused by markedly increased capsule thickness. Compared to D39, the hyperencapsulated ΔadcAII mutant strain was more resistant to complement-mediated neutrophil killing and was hypervirulent in mouse models of invasive infection. Transcriptome analysis of D39 and the ΔadcAII mutant identified major differences in transcription of the Sp_0505-0508 locus, which encodes an SpnD39III (ST5556II) type I restriction-modification system and allelic variation of which correlates with capsule thickness. A PCR assay demonstrated close linkage of the SpnD39IIIC and F alleles with the hyperencapsulated ΔadcAII strains. However, transformation of ΔadcAII with fixed SpnD39III alleles associated with normal capsule thickness did not revert the hyperencapsulated phenotype. Half of hyperencapsulated ΔadcAII strains contained the same single nucleotide polymorphism in the capsule locus gene cps2E, which is required for the initiation of capsule synthesis. These results provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identified an unexpected linkage between capsule thickness and mutation of ΔadcAII. Further investigation will be needed to characterize how mutation of adcAII affects SpnD39III (ST5556II) allele dominance and results in the hyperencapsulated phenotype. IMPORTANCE The Streptococcus pneumoniae capsule affects multiple interactions with the host including contributing to colonization and immune evasion. During infection, the capsule thickness varies, but the mechanisms regulating this are poorly understood. We have identified an unsuspected relationship between mutation of adcAII, a gene that encodes a zinc uptake lipoprotein, and capsule thickness. Mutation of adcAII resulted in a striking hyperencapsulated phenotype, increased resistance to complement-mediated neutrophil killing, and increased S. pneumoniae virulence in mouse models of infection. Transcriptome and PCR analysis linked the hyperencapsulated phenotype of the ΔadcAII strain to specific alleles of the SpnD39III (ST5556II) type I restriction-modification system, a system which has previously been shown to affect capsule thickness. Our data provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identify an unexpected link between capsule thickness and ΔadcAII, further investigation of which could further characterize mechanisms of capsule regulation.

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Genetic variation regulates the activation and specificity of Restriction-Modification systems in Neisseria gonorrhoeae

10.1101/630434 ◽

2019 ◽

Author(s):

Leonor Sánchez-Busó ◽

Daniel Golparian ◽

Julian Parkhill ◽

Magnus Unemo ◽

Simon R. Harris

Keyword(s):

Neisseria Gonorrhoeae ◽

Single Molecule ◽

Regulation Of Gene Expression ◽

Phase Variation ◽

Type I ◽

Dam Methylase ◽

Different Strains ◽

Methylation Patterns ◽

Restriction Modification ◽

Restriction Modification Systems

ABSTRACTRestriction-Modification systems (RMS) are one of the main mechanisms of defence against foreign DNA invasion and can have an important role in the regulation of gene expression. The obligate human pathogen Neisseria gonorrhoeae carries one of the highest loads of RMS in its genome; between 13 to 15 of the three main types. Previous work has described their organization in the reference genome FA1090 and has experimentally inferred the associated methylated motifs. Here, we studied the structure of RMS and target methylated motifs in 25 gonococcal strains sequenced with Single Molecule Real-Time (SMRT) technology, which provides data on DNA modification. The results showed a variable picture of active RMS in different strains, with phase variation switching the activity of Type III RMS, and both the activity and specificity of a Type I RMS. Interestingly, the Dam methylase was found in place of the NgoAXI endonuclease in two of the strains, despite being previously thought to be absent in the gonococcus. We also identified the real methylation target of NgoAX as 5’-GCAGA-3’, different from that previously described. Results from this work give further insights into the diversity and dynamics of RMS and methylation patterns in N. gonorrhoeae.

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Methylation Warfare: Interaction of Pneumococcal Bacteriophages with Their Host

Journal of Bacteriology ◽

10.1128/jb.00370-19 ◽

2019 ◽

Vol 201 (19) ◽

Cited By ~ 9

Author(s):

Leonardo Furi ◽

Liam A. Crawford ◽

Guillermo Rangel-Pineros ◽

Ana S. Manso ◽

Megan De Ste Croix ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Lytic Replication ◽

Host Cells ◽

Transcriptional Analysis ◽

Type I ◽

Phase Variable ◽

Abortive Infection ◽

Content Type ◽

Variable Type ◽

Restriction Modification

ABSTRACTVirus-host interactions are regulated by complex coevolutionary dynamics. InStreptococcus pneumoniae, phase-variable type I restriction-modification (R-M) systems are part of the core genome. We hypothesized that the ability of the R-M systems to switch between six target DNA specificities also has a key role in preventing the spread of bacteriophages. Using the streptococcal temperate bacteriophage SpSL1, we show that the variants of both the SpnIII and SpnIV R-M systems are able to restrict invading bacteriophage with an efficiency approximately proportional to the number of target sites in the bacteriophage genome. In addition to restriction of lytic replication, SpnIII also led to abortive infection in the majority of host cells. During lytic infection, transcriptional analysis found evidence of phage-host interaction through the strong upregulation of thenrdRnucleotide biosynthesis regulon. During lysogeny, the phage had less of an effect on host gene regulation. This research demonstrates a novel combined bacteriophage restriction and abortive infection mechanism, highlighting the importance that the phase-variable type I R-M systems have in the multifunctional defense against bacteriophage infection in the respiratory pathogenS. pneumoniae.IMPORTANCEWith antimicrobial drug resistance becoming an increasing burden on human health, much attention has been focused on the potential use of bacteriophages and their enzymes as therapeutics. However, the investigations into the physiology of the complex interactions of bacteriophages with their hosts have attracted far less attention, in comparison. This work describes the molecular characterization of the infectious cycle of a bacteriophage in the important human pathogenStreptococcus pneumoniaeand explores the intricate relationship between phase-variable host defense mechanisms and the virus. This is the first report showing how a phase-variable type I restriction-modification system is involved in bacteriophage restriction while it also provides an additional level of infection control through abortive infection.

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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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Recombination of the Phase-VariablespnIIILocus Is Independent of All Known Pneumococcal Site-Specific Recombinases

Journal of Bacteriology ◽

10.1128/jb.00233-19 ◽

2019 ◽

Vol 201 (15) ◽

Cited By ~ 11

Author(s):

M. De Ste Croix ◽

K. Y. Chen ◽

I. Vacca ◽

A. S. Manso ◽

C. Johnston ◽

...

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Streptococcus Pneumoniae ◽

Colony Morphology ◽

Type I ◽

Site Specific ◽

Content Type ◽

Sequence Types ◽

Restriction Modification ◽

Genetic Rearrangements

ABSTRACTStreptococcus pneumoniaeis one of the world’s leading bacterial pathogens, causing pneumonia, septicemia, and meningitis. In recent years, it has been shown that genetic rearrangements in a type I restriction-modification system (SpnIII) can impact colony morphology and gene expression. By generating a large panel of mutant strains, we have confirmed a previously reported result that the CreX (also known as IvrR and PsrA) recombinase found within the locus is not essential forhsdSinversions. In addition, mutants of homologous recombination pathways also undergohsdSinversions. In this work, we have shown that these genetic rearrangements, which result in different patterns of genome methylation, occur across a wide variety of serotypes and sequence types, including two strains (a 19F and a 6B strain) naturally lacking CreX. Our gene expression analysis, by transcriptome sequencing (RNAseq), confirms that the level ofcreXexpression is impacted by these genomic rearrangements. In addition, we have shown that the frequency ofhsdSrecombination is temperature dependent. Most importantly, we have demonstrated that the other known pneumococcal site-specific recombinases XerD, XerS, and SPD_0921 are not involved inspnIIIrecombination, suggesting that a currently unknown mechanism is responsible for the recombination of these phase-variable type I systems.IMPORTANCEStreptococcus pneumoniaeis a leading cause of pneumonia, septicemia, and meningitis. The discovery that genetic rearrangements in a type I restriction-modification locus can impact gene regulation and colony morphology led to a new understanding of how this pathogen switches from harmless colonizer to invasive pathogen. These rearrangements, which alter the DNA specificity of the type I restriction-modification enzyme, occur across many different pneumococcal serotypes and sequence types and in the absence of all known pneumococcal site-specific recombinases. This finding suggests that this is a truly global mechanism of pneumococcal gene regulation and the need for further investigation of mechanisms of site-specific recombination.

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Type I Interferons Regulate the Magnitude and Functionality of Mouse Polyomavirus-Specific CD8 T Cells in a Virus Strain-Dependent Manner

Journal of Virology ◽

10.1128/jvi.00199-16 ◽

2016 ◽

Vol 90 (10) ◽

pp. 5187-5199 ◽

Cited By ~ 7

Author(s):

Qingsong Qin ◽

Shwetank ◽

Elizabeth L. Frost ◽

Saumya Maru ◽

Aron E. Lukacher

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cell Response ◽

Cd8 T Cell ◽

T Cell Response ◽

Single Amino Acid ◽

Type I ◽

Type I Ifns ◽

Vp1 Capsid Protein

ABSTRACTMouse polyomavirus (MPyV) is a ubiquitous persistent natural mouse pathogen. A glutamic acid (E)-to-glycine (G) difference at position 91 of the VP1 capsid protein shifts the profile of tumors induced by MPyV from an epithelial to a mesenchymal cell origin. Here we asked if this tropism difference affects the MPyV-specific CD8 T cell response, which controls MPyV infection and tumorigenesis. Infection by the laboratory MPyV strain RA (VP1-91G) or a strain A2 mutant with an E-to-G substitution at VP1 residue 91 [A2(91G)] generated a markedly smaller virus-specific CD8 T cell response than that induced by A2(VP1-91E) infection. Mutant A2(91G)-infected mice showed a higher frequency of memory precursor (CD127hiKLRG1lo) CD8 T cells and a higher recall response than those of A2-infected mice. Using T cell receptor (TCR)-transgenic CD8 T cells and immunization with peptide-pulsed dendritic cells, we found that early bystander inflammation associated with A2 infection contributed to recruitment of the larger MPyV-specific CD8 T cell response. Beta interferon (IFN-β) transcripts were induced early during A2 or A2(91G) infections. IFN-β inhibited replication of A2 and A2(91G)in vitro. Using mice lacking IFN-αβ receptors (IFNAR−/−), we showed that type I IFNs played a role in controlling MPyV replicationin vivobut differentially affected the magnitude and functionality of virus-specific CD8 T cells recruited by A2 and A2(91G) viral infections. These data indicate that type I IFNs are involved in protection against MPyV infection and that their effect on the antiviral CD8 T cell response depends on capsid-mediated tropism properties of the MPyV strain.IMPORTANCEIsolates of the human polyomavirus JC virus from patients with the frequently fatal demyelinating brain disease progressive multifocal leukoencephalopathy (PML) carry single amino acid substitutions in the domain of the VP1 capsid protein that binds the sialic acid moiety of glycoprotein/glycolipid receptors on host cells. These VP1 mutations may alter neural cell tropism or enable escape from neutralizing antibodies. Changes in host cell tropism can affect recruitment of virus-specific CD8 T cells. Using mouse polyomavirus, we demonstrate that a single amino acid difference in VP1 known to shift viral tropism profoundly affects the quantity and quality of the anti-polyomavirus CD8 T cell response and its differentiation into memory cells. These findings raise the possibility that CD8 T cell responses to infections by human polyomaviruses may be influenced by VP1 mutations involving domains that engage host cell receptors.

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