Complete Bypass of Restriction Systems for Major Staphylococcus aureus Lineages - PubMed (original) (raw)

Complete Bypass of Restriction Systems for Major Staphylococcus aureus Lineages

Ian R Monk et al. mBio. 2015.

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Abstract

Staphylococcus aureus is a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into clinical isolates of S. aureus. Here, we describe the construction and characterization of the IMXXB series of Escherichia coli strains that mimic the type I adenine methylation profiles of S. aureus clonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of major S. aureus lineages.

Importance: The genetic manipulation of clinical S. aureus isolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset of S. aureus strains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite of E. coli strains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer into S. aureus. The IMXXB series of E. coli strains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation of S. aureus.

Copyright © 2015 Monk et al.

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Figures

FIG 1

FIG 1

SMRT sequencing for the identification of adenine methylation in S. aureus and engineered E. coli. (A) The target recognition motifs (TRMs) for the type I HsdMS systems encoded by each S. aureus strain were identified, and the positions of methylated adenine residues on the chromosome plotted with Circos (34). Each adenine methylation on the chromosome is represented by a line whose length corresponds with the interpulse duration of the read. (B and C) PacBio reads were analyzed by the SMRT suite pipeline version 2.2.0/motif finder 1.3.1 to identify conserved adenine-methylated residues (in boldface) and the TRMs for the HsdS alleles of each S. aureus (B) and IMXXB E. coli (C) strain.1, Mean modification QV is defined as the quality value of the base calls within the motif.2, Mean motif coverage is defined as the average depth of read coverage within a motif.3, GATC methylation encoded by dam in the E. coli strain is not present in S. aureus.

FIG 2

FIG 2

Transformation of NRS384 with HsdMS-methylated plasmid. Three different CC8 HsdMS combinations were expressed from an IPTG-inducible plasmid (pET21) in E. coli BL21 (including the hybrid _hsdM2_-hsdS1). The coextracted shuttle vector pIMK7 (5 µg total plasmid DNA) was transformed into CA-MRSA strain NRS384. pIMK7 isolated from NRS384 was included as the maximal transformation of fully modified plasmid. The number of TRMs on pIMK7 for CC8 strains are indicated next to the plasmid name. The transformation efficiencies are expressed as the mean numbers of all transformants obtained in each experiment ± standard deviations (error bars) from three replicates. The graph shows data representative of the data from three independent experiments.

FIG 3

FIG 3

Construction and characterization of IMXXB E. coli strains. (A) Schematic of the construction of an E. coli strain expressing CC-specific hsdMS alleles from strong promoters at neutral locations in the DC10B (DH10B Δ_dcm_) chromosome. (B to D) Transformation profiles of S. aureus strains (grey bars) and their isogenic hsdR mutants (defective in type I restriction) (white bars) with plasmid pRAB11 (5 µg) or pIMAY-Z (2.5 µg) isolated from DC10B or the respective IMXXB strain of E. coli. The number of TRMs on either plasmid for the CC of the strain is denoted next to the plasmid name. NT, no transformants were detected. The transformation efficiencies are expressed as the mean numbers of all transformants obtained in each experiment ± standard deviations (error bars) from three replicates. The graph shows data representative of the data from three independent experiments.

FIG 4

FIG 4

Effect of plasmid concentration for S. aureus electroporation. Different concentrations of plasmid DNA (pRAB11 at 0.1, 0.5, 1, 2.5, 5, and 10 µg) were isolated from E. coli strains IM01B, IM08B, and IM30B for transformation into S. aureus MW2, NRS384, and MRSA252, respectively. A dose-dependent response was observed for up to 5 µg of plasmid with pRAB11. The transformation efficiencies are expressed as the mean numbers of all transformants obtained in each experiment ± standard deviations (error bars) from three replicates. The graph shows representative data from one experiment.

FIG 5

FIG 5

Determination of the TRM for each TRD. Clustal Omega alignment of HsdS proteins from CC1, CC5, CC8, CC30, and ST93. The protein sequences of the HsdS variants were aligned with Clustal Omega, and TRDs that match exactly are color coded in the same color.

FIG 6

FIG 6

Transformation of JKD6159 and type I restriction mutants with pRAB11. Plasmid pRAB11 was isolated from E. coli strains DC10B and IM93B and S. aureus strain JKD6159 for transformation into JKD6159 and the respective type I-deficient mutants. pRAB11 contains 4 TRM sites in ST93-1, 2 in ST93-2, and 2 in ST93-3. The transformation efficiencies were expressed as the mean numbers of all transformants obtained in each experiment ± standard deviations (error bars) from three replicates. The graph shows representative data from one experiment. NT, no transformants were isolated.

FIG 7

FIG 7

Transformation of S. aureus NRS384 and NRS384 ΔhsdR with plasmid isolated from IMXXB E. coli. Plasmid pRAB11 or pIMAY-Z was isolated from E. coli strains IM01B, IM08B, and IM30B for transformation into a CC8 host (NRS384) and the respective hsdR mutant. The transformation efficiencies are expressed as the mean numbers of all transformants obtained in each experiment ± standard deviations (error bars) from three replicates. The graph shows representative data from one experiment.

FIG 8

FIG 8

Transformation of additional representative isolates from CC1, CC8/ST239, and CC30 with plasmid isolated from IMXXB strains. Plasmid pRAB11 (5 µg) was isolated from either E. coli strain DC10B or the compatible IMXXB strain for transformation into additional representative S. aureus isolates of CC1, CC8/ST239, and CC30. The transformation efficiencies are expressed as the mean numbers of all transformants obtained in each experiment ± standard deviations (error bars) from three replicates. The graph shows representative data from one experiment.

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