Inactivation of the SauI Type I Restriction-Modification System Is Not Sufficient To Generate Staphylococcus aureus Strains Capable of Efficiently Accepting Foreign DNA

ABSTRACT Genetic manipulation of Staphylococcus aureus is limited by the availability of only a single strain, RN4220, that is capable of easily accepting foreign DNA. Inactivation of the hsdR gene of the SauI type I restriction-modification system was shown previously to be responsible for the high transformation efficiency of RN4220 (D. E. Waldron and J. A. Lindsay, J Bacteriol. 188:5578-5585, 2006). However, deletion of this gene in three different S. aureus strains was not sufficient to make them readily transformable, which would be remarkably useful for genetic studies of this pathogenic organism. These results indicate that another unknown factor(s) is required for the transformable phenotype in S. aureus.

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Sau1: a Novel Lineage-Specific Type I Restriction-Modification System That Blocks Horizontal Gene Transfer into Staphylococcus aureus and between S. aureus Isolates of Different Lineages

Journal of Bacteriology ◽

10.1128/jb.00418-06 ◽

2006 ◽

Vol 188 (15) ◽

pp. 5578-5585 ◽

Cited By ~ 191

Author(s):

Denise E. Waldron ◽

Jodi A. Lindsay

Keyword(s):

Staphylococcus Aureus ◽

Resistance Genes ◽

Genetic Manipulation ◽

Type I ◽

Sequence Specificity ◽

Modification System ◽

Major Mechanism ◽

Restriction Modification System ◽

Resistance Transfer ◽

Restriction Modification

ABSTRACT The Sau1 type I restriction-modification system is found on the chromosome of all nine sequenced strains of Staphylococcus aureus and includes a single hsdR (restriction) gene and two copies of hsdM (modification) and hsdS (sequence specificity) genes. The strain S. aureus RN4220 is a vital intermediate for laboratory S. aureus manipulation, as it can accept plasmid DNA from Escherichia coli. We show that it carries a mutation in the sau1hsdR gene and that complementation restored a nontransformable phenotype. Sau1 was also responsible for reduced conjugative transfer from enterococci, a model of vancomycin resistance transfer. This may explain why only four vancomycin-resistant S. aureus strains have been identified despite substantial selective pressure in the clinical setting. Using a multistrain S. aureus microarray, we show that the two copies of sequence specificity genes (sau1hsdS1 and sau1hsdS2) vary substantially between isolates and that the variation corresponds to the 10 dominant S. aureus lineages. Thus, RN4220 complemented with sau1hsdR was resistant to bacteriophage lysis but only if the phage was grown on S. aureus of a different lineage. Similarly, it could be transduced with DNA from its own lineage but not with the phage grown on different S. aureus lineages. Therefore, we propose that Sau1 is the major mechanism for blocking transfer of resistance genes and other mobile genetic elements into S. aureus isolates from other species, as well as for controlling the spread of resistance genes between isolates of different S. aureus lineages. Blocking Sau1 should also allow genetic manipulation of clinical strains of S. aureus.

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Unstable chromosome rearrangements in Staphylococcus aureus cause phenotype switching associated with persistent infections

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904861116 ◽

2019 ◽

Vol 116 (40) ◽

pp. 20135-20140 ◽

Cited By ~ 17

Author(s):

Romain Guérillot ◽

Xenia Kostoulias ◽

Liam Donovan ◽

Lucy Li ◽

Glen P. Carter ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Genetic Basis ◽

Host Cells ◽

Type I ◽

Modification System ◽

Persistent Infections ◽

Restriction Modification System ◽

Long Read ◽

Small Colony ◽

Restriction Modification

Staphylococcus aureus small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically important phenomenon is poorly understood, hampered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rifampicin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of S. aureus linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the S. aureus chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type–like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete S. aureus genomes showed strong signatures of recombination between hsdMS genes, suggesting that analogous CI has repeatedly occurred during S. aureus evolution. Using qPCR and long-read amplicon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major S. aureus lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in S. aureus associated with SCV generation and persistent infections.

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Improved transformation efficiency of group A Streptococcus by inactivation of a type I restriction modification system

PLoS ONE ◽

10.1371/journal.pone.0248201 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0248201

Author(s):

Meredith B. Finn ◽

Kathryn M. Ramsey ◽

Hunter J. Tolliver ◽

Simon L. Dove ◽

Michael R. Wessels

Keyword(s):

Genetic Transformation ◽

Parent Strain ◽

Transformation Efficiency ◽

Genetic Manipulation ◽

Spontaneous Mutation ◽

Type I ◽

Modification System ◽

Restriction Modification System ◽

Group A ◽

Restriction Modification

Streptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal glomerulonephritis. Genetic manipulation of this important pathogen is complicated by resistance of the organism to genetic transformation. Very low transformation efficiency is attributed to recognition and degradation of introduced foreign DNA by a type I restriction-modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten GAS strains that had been previously transformed with an unrelated plasmid revealed that six of the ten harbored a spontaneous mutation in hsdR, S, or M. The mutations were all different, and at least five of the six were predicted to result in loss of function of the respective hsd gene product. The unexpected occurrence of such mutations in previously transformed isolates suggested that the process of transformation selects for spontaneous inactivating mutations in the Hsd system. We investigated the possibility of exploiting the increased transformability of hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate representative of the globally dominant M1T1 clonal group. Mutant strain 854ΔhsdM exhibited a 5-fold increase in electrotransformation efficiency compared to the wild type parent strain and no obvious change in growth or off-target gene expression. We conclude that genetic transformation of GAS selects for spontaneous mutants in the hsdRSM restriction modification system. We propose that use of a defined hsdM mutant as a parent strain for genetic manipulation of GAS will enhance transformation efficiency and reduce the likelihood of selecting spontaneous hsd mutants with uncharacterized genotypes.

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Faculty Opinions recommendation of Sau1: a novel lineage-specific type I restriction-modification system that blocks horizontal gene transfer into Staphylococcus aureus and between S. aureus isolates of different lineages.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1104205.560206 ◽

2008 ◽

Author(s):

Edward Feil

Keyword(s):

Staphylococcus Aureus ◽

Gene Transfer ◽

Horizontal Gene Transfer ◽

Type I ◽

Modification System ◽

Restriction Modification System ◽

Restriction Modification

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Functional genomics reveals extensive diversity in Staphylococcus epidermidis restriction modification systems compared to Staphylococcus aureus

10.1101/644856 ◽

2019 ◽

Author(s):

Jean YH Lee ◽

Glen P Carter ◽

Sacha J Pidot ◽

Romain Guérillot ◽

Torsten Seemann ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Staphylococcus Epidermidis ◽

Genetic Manipulation ◽

Gene Arrangement ◽

Type I ◽

Efficient Approach ◽

Molecular Studies ◽

Foreign Dna ◽

Clinically Significant ◽

Restriction Modification

AbstractStaphylococcus epidermidis is a significant opportunistic pathogen of humans. Molecular studies in this species have been hampered by the presence of restriction-modification (RM) systems that limit introduction of foreign DNA. Here we establish the complete genomes and methylomes for seven clinically significant, genetically diverse S. epidermidis isolates and perform the first systematic genomic analyses of the type I RM systems within both S. epidermidis and Staphylococcus aureus. Our analyses revealed marked differences in the gene arrangement, chromosomal location and movement of type I RM systems between the two species. Unlike S. aureus, S. epidermidis type I RM systems demonstrate extensive diversity even within a single genetic lineage. This is contrary to current assumptions and has important implications for approaching the genetic manipulation of S. epidermidis. Using Escherichia coli plasmid artificial modification (PAM) to express S. epidermidis hsdMS, we readily overcame restriction barriers in S. epidermidis, and achieved transformation efficiencies equivalent to those of modification deficient mutants. With these functional experiments we demonstrate how genomic data can be used to predict both the functionality of type I RM systems and the potential for a strain to be transformation proficient. We outline an efficient approach for the genetic manipulation of S. epidermidis from diverse genetic backgrounds, including those that have hitherto been intractable. Additionally, we identified S. epidermidis BPH0736, a naturally restriction defective, clinically significant, multidrug-resistant ST2 isolate as an ideal candidate for molecular studies.ImportanceStaphylococcus epidermidis is a major cause of hospital-acquired infections, especially those related to implanted medical devices. Understanding how S. epidermidis causes disease and devising ways to combat these infections has been hindered by an inability to genetically manipulate “hospital-adapted” strains that cause clinical disease. Here we provide the first comprehensive analyses of the mechanisms whereby S. epidermidis resists the uptake of foreign DNA and demonstrate that these are distinct from those described for S. aureus. Until now it had been assumed that these are the same. Using these insights, we demonstrate an efficient approach for the genetic manipulation of S. epidermidis to enable the study of clinically relevant isolates for the first time.

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Development of a Markerless Genetic Exchange System for Desulfovibrio vulgaris Hildenborough and Its Use in Generating a Strain with Increased Transformation Efficiency

Applied and Environmental Microbiology ◽

10.1128/aem.01839-09 ◽

2009 ◽

Vol 75 (24) ◽

pp. 7682-7691 ◽

Cited By ~ 56

Author(s):

Kimberly L. Keller ◽

Kelly S. Bender ◽

Judy D. Wall

Keyword(s):

Transformation Efficiency ◽

Genetic Manipulation ◽

Kanamycin Resistance ◽

Deletion Strain ◽

Desulfovibrio Vulgaris ◽

Type I ◽

Wild Type ◽

Modification System ◽

Single Strain ◽

Restriction Modification System

ABSTRACT In recent years, the genetic manipulation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has seen enormous progress. In spite of this progress, the current marker exchange deletion method does not allow for easy selection of multiple sequential gene deletions in a single strain because of the limited number of selectable markers available in D. vulgaris. To broaden the repertoire of genetic tools for manipulation, an in-frame, markerless deletion system has been developed. The counterselectable marker that makes this deletion system possible is the pyrimidine salvage enzyme, uracil phosphoribosyltransferase, encoded by upp. In wild-type D. vulgaris, growth was shown to be inhibited by the toxic pyrimidine analog 5-fluorouracil (5-FU), whereas a mutant bearing a deletion of the upp gene was resistant to 5-FU. When a plasmid containing the wild-type upp gene expressed constitutively from the aph(3′)-II promoter (promoter for the kanamycin resistance gene in Tn5) was introduced into the upp deletion strain, sensitivity to 5-FU was restored. This observation allowed us to develop a two-step integration and excision strategy for the deletion of genes of interest. Since this in-frame deletion strategy does not retain an antibiotic cassette, multiple deletions can be generated in a single strain without the accumulation of genes conferring antibiotic resistances. We used this strategy to generate a deletion strain lacking the endonuclease (hsdR, DVU1703) of a type I restriction-modification system that we designated JW7035. The transformation efficiency of the JW7035 strain was found to be 100 to 1,000 times greater than that of the wild-type strain when stable plasmids were introduced via electroporation.

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Improved transformation efficiency of group A Streptococcus by inactivation of a type I restriction modification system

10.1101/2021.02.23.432441 ◽

2021 ◽

Author(s):

Meredith B. Finn ◽

Kathryn M. Ramsey ◽

Simon L. Dove ◽

Michael R. Wessels

Keyword(s):

Genetic Transformation ◽

Parent Strain ◽

Transformation Efficiency ◽

Genetic Manipulation ◽

Spontaneous Mutation ◽

Type I ◽

Modification System ◽

Restriction Modification System ◽

Group A ◽

Restriction Modification

AbstractStreptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal glomerulonephritis. Genetic manipulation of this important pathogen is complicated by resistance of the organism to genetic transformation. Very low transformation efficiency is attributed to recognition and degradation of introduced foreign DNA by a type I restriction-modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten GAS strains that had been previously transformed with an unrelated plasmid revealed that six of the ten harbored a spontaneous mutation in hsdR, S, or M. The mutations were all different, and at least five of the six were predicted to result in loss of function of the respective hsd gene product. The unexpected occurrence of such mutations in previously transformed isolates suggested that the process of transformation selects for spontaneous inactivating mutations in the Hsd system. We investigated the possibility of exploiting the increased transformability of hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate representative of the globally dominant M1T1 clonal group. Mutant strain 854ΔhsdM exhibited a 5-fold increase in transformation efficiency compared to the wild type parent strain and no obvious change in growth or off-target gene expression. We conclude that genetic transformation of GAS selects for spontaneous mutants the hsdRSM restriction modification system. We propose that use of a defined hsdM mutant as a parent strain for genetic manipulation of GAS will enhance transformation efficiency and reduce the likelihood of selecting spontaneous hsd mutants with uncharacterized genotypes.

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Biological characteristics of a type I restriction-modification system in Staphylococcus aureus.

Journal of Bacteriology ◽

10.1128/jb.133.3.1144-1149.1978 ◽

1978 ◽

Vol 133 (3) ◽

pp. 1144-1149 ◽

Cited By ~ 8

Author(s):

J E Sjöström ◽

S Löfdahl ◽

L Philipson

Keyword(s):

Staphylococcus Aureus ◽

Biological Characteristics ◽

Type I ◽

Modification System ◽

Restriction Modification System ◽

Restriction Modification

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Complete Genome Sequence of Brevibacterium linens SMQ-1335

Genome Announcements ◽

10.1128/genomea.01242-16 ◽

2016 ◽

Vol 4 (6) ◽

Cited By ~ 1

Author(s):

Alessandra G. de Melo ◽

Simon J. Labrie ◽

Jeannot Dumaresq ◽

Richard J. Roberts ◽

Denise M. Tremblay ◽

...

Keyword(s):

Complete Genome Sequence ◽

Open Reading Frames ◽

Type I ◽

Industrial Strain ◽

Modification System ◽

Brevibacterium Linens ◽

Restriction Modification System ◽

New Type ◽

Restriction Modification ◽

Reading Frames

Brevibacterium linens is one of the main bacteria found in the smear of surface-ripened cheeses. The genome of the industrial strain SMQ-1335 was sequenced using PacBio. It has 4,209,935 bp, a 62.6% G+C content, 3,848 open reading frames, and 61 structural RNAs. A new type I restriction-modification system was identified.

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