Restriction–Modification Systems as a Barrier for Genetic Manipulation of Staphylococcus aureus

2014 ◽  
pp. 9-23 ◽  
Author(s):  
Marat R. Sadykov
Keyword(s):  
Staphylococcus Aureus ◽  
Genetic Manipulation ◽  
Restriction Modification ◽  
Restriction Modification Systems

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

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.

scholarly journals 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

2006 ◽  
Vol 188 (15) ◽  
pp. 5578-5585 ◽  
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.

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

2009 ◽  
Vol 75 (10) ◽  
pp. 3034-3038 ◽  
Author(s):  
Helena Veiga ◽  
Mariana G. Pinho
Keyword(s):  
Staphylococcus Aureus ◽  
Genetic Manipulation ◽  
Type I ◽  
Modification System ◽  
Single Strain ◽  
Pathogenic Organism ◽  
Restriction Modification System ◽  
High Transformation ◽  
Foreign Dna ◽  
Restriction Modification

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.

Cloning and sequence comparison ofAvaI andBsoBI restriction-modification systems

1996 ◽  
Vol 252 (6) ◽  
pp. 695-699 ◽  
Author(s):  
H. Ruan ◽  
K. D. Lunnen ◽  
M. E. Scott ◽  
L. S. Moran ◽  
B. E. Slatko ◽  
...  
Keyword(s):  
Sequence Comparison ◽  
Restriction Modification ◽  
Restriction Modification Systems

scholarly journals Reconstruction ofmreBExpression in Staphylococcus aureus via a Collection of New Integrative Plasmids

2014 ◽  
Vol 80 (13) ◽  
pp. 3868-3878 ◽  
Author(s):  
Ana Yepes ◽  
Gudrun Koch ◽  
Andrea Waldvogel ◽  
Juan-Carlos Garcia-Betancur ◽  
Daniel Lopez
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Biology ◽  
Genetic Manipulation ◽  
Protein Localization ◽  
Antibiotic Resistance Genes ◽  
Wall Material ◽  
Content Type ◽  
Genetic Manipulations ◽  
Discrete Structures

ABSTRACTProtein localization has been traditionally explored in unicellular organisms, whose ease of genetic manipulation facilitates molecular characterization. The two rod-shaped bacterial modelsEscherichia coliandBacillus subtilishave been prominently used for this purpose and have displaced other bacteria whose challenges for genetic manipulation have complicated any study of cell biology. Among these bacteria is the spherical pathogenic bacteriumStaphylococcus aureus. In this report, we present a new molecular toolbox that facilitates gene deletion in staphylococci in a 1-step recombination process and additional vectors that facilitate the insertion of diverse reporter fusions into newly identified neutral loci of theS. aureuschromosome. Insertion of the reporters does not add any antibiotic resistance genes to the chromosomes of the resultant strains, thereby making them amenable for further genetic manipulations. We used this toolbox to reconstitute the expression ofmreBinS. aureus, a gene that encodes an actin-like cytoskeletal protein which is absent in coccal cells and is presumably lost during the course of speciation. We observed that inS. aureus, MreB is organized in discrete structures in association with the membrane, leading to an unusual redistribution of the cell wall material. The production of MreB also caused cell enlargement, but it did not revert staphylococcal shape. We present interactions of MreB with key staphylococcal cell wall-related proteins. This work facilitates the useS. aureusas a model system in exploring diverse aspects of cellular microbiology.

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