TABLE 1.
Bacterial strains, plasmids, and oligonucleotides used in this study
| Bacterial strain, plasmid, or oligonucleotidea | Description (relevant genotype or phenotype) or sequence (5′ to 3′)b | Source, reference, or RE sitec |
|---|---|---|
| Escherichia coli strains | ||
| DH10B (K-12 strain) | dam + dcm + ΔhsdRMS endA1 recA1 | Invitrogen |
| BW25113 (K-12 strain) | dam + dcm + hsdMS+ hsdR514 | 15 |
| IBEC55 | Δdcm ΔhsdMS in the BW25113 background; Dam methylation only | 15 |
| IBEC56 | Δdam ΔhsdMS in the BW25113 background; Dcm methylation only | 15 |
| IBEC57 | Δdam Δdcm in the BW25113 background; Hsd methylation only | 15 |
| IBEC58 | Δdam Δdcm ΔhsdMS in the BW25113 background; no methylation | 15 |
| DC10B | Δdcm in the DH10B background; Dam methylation only | This study |
| BL21(DE3) (B strain) | F– ompT hsdSB (rB– mB–) gal dcm (DE3); IPTG-inducible T7 RNA polymerase | Novagen |
| Staphylococcus strains | ||
| Newman | ST8; CC8 isolated in 1952 human clinical MSSA; genome sequenced | 4 |
| Newman ΔhsdR | Newman with a deletion of Sae0139 | This study |
| Newman ΔsauUSI | Newman with a deletion of Sae2386 | This study |
| Newman ΔhsdR ΔsauUSI | Newman with a deletion of both Sae0139 and Sae2386 | This study |
| Newman ΔhsdR sauUSIECORV | Restoration of Sae2386 in the ΔhsdR ΔsauUSI background | This study |
| Newman Δnudix | Newman with a deletion of Sae2385 | This study |
| NRS384 | USA300-14 clone obtained from NARSA | NARSA collection |
| NRS384 hsdRINT | Targetron insertion at nucleotide 735 of hsdR | This study |
| NRS384 sauUSIINT | Targetron insertion at nucleotide 739 of sauUSI | This study |
| NRS384 sauUSIINT hsdRINT | Targetron insertion in hsdR made in the NRS384sauUSIINT background | This study |
| NRS384 sauUSIECORV hsdRINT | Restoration of the sauUSI mutation with a silent EcoRV site in the double insertion mutant background | This study |
| RN4220 | ST8; CC8; chemically mutagenized derivative of 8325-4, transformable with E. coli DNA; premature stop codon in both hsdR and sauUSI | 11 |
| RN4220 sauUSI+ | Nonsense mutation in sauUSI corrected to wild type with 8325-4 sequence | This study |
| Cowan | ST30; CC30 MSSA;high-level protein A producer; ATCC 12598 | 25 |
| Cowan ΔsauUSI | Deletion of sauUSI | This study |
| N315 | ST5 CC5 MSSA; genome sequenced | 29 |
| LAC | ST8 CC8 CA-MRSA; USA300 | 6 |
| MRSA252 | ST36 CC30 MRSA; genome sequenced | 36 |
| Oxford 13 | ST22 CC22 | 37 |
| Oxford 19 | ST10 CC16 | 37 |
| Oxford 71 | ST1 CC1 | 37 |
| Oxford 159 | ST25 CC25 | 37 |
| Oxford 207 | ST15 CC15 | 37 |
| Oxford 233 | ST45 CC45 | 37 |
| Oxford 560 | ST121 CC51 | 37 |
| Oxford 3177 | ST97 CC16 | 37 |
| RP62a | Methicillin-resistant, biofilm-forming Staphylococcus epidermidis isolate; genome sequenced | 24 |
| RP62a ∆mcrR | Deletion of Serp2052; able to accept DNA at a low frequency from wild-type E. coli | This study |
| Enterococcus faecalis strains | ||
| OG1RF | Rifampin- and fusic acid-resistant E. faecalis clone derived for OG1 | 38 |
| JH2-2 | Rifampin- and fusic acid-resistant E. faecalis clone derived for JH2 | 39 |
| V583 | Vancomycin-resistant clinical isolate of E. faecalis | 40 |
| Plasmids | ||
| pNL9164 | Temperature-sensitive targetron plasmid for S. aureus pT181 replicon; Ampr Eryr | Sigma |
| pNL9164(hsdR) | pNL9164 retargeted for hsdR of NRS384 | This study |
| pNL9164(sauUSI) | pNL9164 retargeted for sauUSI of NRS384 | This study |
| pKD4 | Plasmid for amplification of frt-kan-frt for E. coli gene deletion; Ampr Kanr | 26 |
| pKD46 | E. coli temperature-sensitive plasmid containing λ red recombinase genes under the control of an arabinose-inducible promoter; Ampr | 26 |
| pCP20 | E. coli temperature-sensitive plasmid containing flp required for antibiotic marker excision; Ampr Cmr | 10 |
| pIMC | Site-specific integrating vector; p15A low-copy-number origin of replication; RP4 conjugative origin of transfer and Phelp-driven chloramphenicol resistance marker; pBluescript MCS; Cmr | 34 |
| pKOR1 | Temperature-sensitive shuttle vector for allelic exchange in S. aureus; Ampr Cmr | 19 |
| pVE6007 | pWV01ts-derived plasmid that cannot replicate in E. coli; Cmr | 21 |
| pIMC5 | Temperature-sensitive Gram-positive replicon from pVE6007 with an E. coli replicon; MCS and antibiotic resistance from pIMC; Cmr (IM46/IM47/IM48/IM49) | This study |
| pIMAY | pIMC5 with tetracycline; inducible secY antisense from pKOR1; Cmr (IM72/IM73) | This study |
| pIMAYΔhsdR | A deletion encompassing the entire hsdR gene (between the ATG and TAA codons); amplified from Newman (IM93/IM3/IM4/IM94) | This study |
| pIMAYΔsauUSI(CC8) | A deletion encompassing the entire sauUSI gene (between the ATG and TAA codons); amplified from Newman (IM89/IM90/IM91/IM92) | This study |
| pIMAYΔsauUSI(CC30) | A deletion encompassing the entire sauUSI gene (between the ATG and TAA codons); amplified from Cowan (IM89/IM90/IM91/IM150) | This study |
| pIMAYΔmcrR(S.epi) | A deletion encompassing the entire mcrR gene (between the ATG and TAA codons); amplified from RP62a (IM216/IM217/IM218/IM219) | This study |
| pIMAY sauUSIEcoRV | A silent EcoRV site was introduced into the middle of the sauUSI gene (with DNA flanking for gene restoration in the ΔsauUSI mutant) (IM89/IM350/IM351/IM92) | This study |
| pIMAY(RN4220sauUSI+) | A 1-kb fragment amplified from Newman surrounding the premature stop codon in RN4220 sauUSI (IM108/IM109) | This study |
| pIMAYΔnudix | A deletion encompassing the entire putative nudix gene (between the ATG and TAA codons); amplified from Newman (IM222/IM223/IM224/IM225) | This study |
| pET21d+ | C-terminal hexahistadine tagging vector; Ampr | Novagen |
| pET21d+sauUSI | The entire sauUSI gene amplified from Newman and fused to a C-terminal His tag (IM196/IM197) | This study |
| Oligonucleotides | ||
| IM46 (pVE6007 F) | ATATGCATGCGTT TTAGCGTTTATTTC GTTTAGTTATCGG | SphI |
| IM47 (pVE6007 R) | GTATTGCTATTAATC GCAACATCAAACC | |
| IM48 (pIMC F) | GATGTTGCGATTAATAGC AATACATTCTATAATAGA AGGTATGGAGGATG | |
| IM49 (pIMC R) | AGATCTCCTCTCGC CTGTCCCCTCAGTTC AGTAATTTCC | BglII |
| IM72 (anti secY F) | ATATAGATCTTGATC TAATGATTCAAACCCTTGTG | BglII |
| IM73 (anti secY R) | ATATGCATGCTGAAG TTACCATCACGGAAAAAGG | SphI |
| IM93 (ΔhsdR-AFwd) | ATATGGTACCGTGGC CACACATTACAGTATTCCC | KpnI |
| IM2 (ΔhsdR-B) | CATTCATATCCCCTT CCATACACTTTCTATTGC | |
| IM3 (ΔhsdR-C) | TATGGAAGGGGATATGA ATGTAATGATTCAGCCCC CTCGCTAGATTAGTG | |
| IM94 (ΔhsdR-DRev) | ATATGAGCTCATTCAT CTTTGTATTCTTTCATGTTTCC | SacI |
| IM5 (hsdR-outF) | AGTCATAGTGAATTGCA GTCAATTGC | |
| IM6 (hsdR-outR) | ATATAACAAGAACTTA ATTTCAGCCG | |
| IM89 (ΔsauUSI-AFwd) | ATATGGTACCGTGTAT GAAAATGCATGGAGTAGAGC | KpnI |
| IM90 (ΔsauUSI-B) | CATATTATCCCTCAGT CATAATTTTATTAACG | |
| IM91 (ΔsauUSI-C) |
CGTTAATAAAATTATG
ACTGAGGGATAATATGT
AATGTAAACCGAAAAATG
AATGTTAGTAAAG |
|
| IM92 (ΔsauUSI-DRev) | ATATGAGCTCCCAA TCCTCTGGATTCCAT ATTCTTTCC | SacI |
| IM150 (CC30 sauUSI-DRev) | ATATGAGCTCAAA CTCTTCGTCACGAAATCCTTCC | SacI |
| IM110 (sauUSI-OUT F) | ACAGCCCCAAGACA ATACTTTTCAC | |
| IM111 (sauUSI-OUTR) | ATACAGGACCAATCC TCTGGATTCC | |
| IM108 (RNsauUSIcomp)F | ATATGGTACCGTGCATT AGATGTTAGAGAAGTAAACC | KpnI |
| IM109 (RNsauUSIcomp)R | ATATGAGCTCATTTAATG ATACTGCATCCAATGAATTG | SacI |
| IM350 (384sauUSIcomp)B | GATATCACTTTCTAATG CTGCTTGTAACC | EcoRV |
| IM351 (384sauUSIcomp)C | ACAAGCAGCATTAGAAA GTGATATCTTATGTCCATT TCATTATTTTGGTGTG | EcoRV |
| IM196 (His-SauUSI F) | ATATCCATGGGTAGATTA CTAAATGATTTCAATC | NcoI |
| IM197 (His-SauUSI R) | ATATCTCGAGATTTGTTA GATAACGATATATATCATCTC | XhoI |
| IM216 (Se ΔmcrR-AFwd) | ATATGTCGACTCTAATAT ATTAAGTATGTAAACCACG | SalI |
| IM217 (Se ΔmcrR-B) | CAATCTAATTCTCCTCTATTATACG | |
| IM218 (Se ΔmcrR-C) | GTATAATAGAGGAGAATTA GATTGTAATTACTTATACTA AATTATTATTTATTG | |
| IM219 (Se ΔmcrR-DRev) | ATATGAATTCTGAATCACA GATCAAAAATGAAGACC | EcoRI |
| IM220 (Se mcrR-OUTF) | GAATTGAAAATTTTAGG TATTCAGATGG | |
| IM221 (Se mcrR-OUTR) | AAACCTTTAATAATTA TCAAGACAGC | |
| IM222 (Δnudix-AFwd) | ATATGGTACCACCTTCACC AAGACCGAATTTTCC | KpnI |
| IM223 (Δnudix-B) | CATAAGACTCACCCTTCA ATTTAAAATC | |
| IM224 (Δnudix-C) | TTAAATTGAAGGGTGAGTC TTATGTAATATGAGTAGATT ACTAAATGATTTC | |
| IM225 (Δnudix-DRev) | ATATGAGCTCATAGTA GACAGTAAAACATTATGC | SacI |
| IM226 (nudix OUT F) | TTTAAATAACGCGCTAAACCTAATGC | |
| IM227 (nudix OUT R) | CACTATCAACTAAATCGCCATTTTTC | |
| IM261 (Ec Δdcm F) |
TGTAATTATGTTAACCTG
TCGGCCATCTCAGATGGC
CGGTGAAATCTATGGTG
TAGGCTGGAGCTGCTTC |
|
| IM262 (Ec Δdcm R) |
TTGTGCCTCTTGCTGACG
CAACGCCACCGCCTGTTTG
ATTTTTGGCTCAAGGTCCAT
ATGAATATCCTCCTTAG |
|
| IM251 (Ec Δdcm OUTF) | AGAAGAGACGCGTCGCCTGCTCC | |
| IM252 (Ec Δdcm OUTR) | TACTGGTCACGTTGGGAAAATATCTC | |
| IMS80 (hsdR IBS) | AAAAAAGCTTATAATTA TCCTTACTTCTCCCGCAT GTGCGCCCAGATAGGGTG | HindIII |
| IMS81 (hsdR EBS1d) |
CAGATTGTACAAATGTGGTG
ATAACAGATAAGTCCCGCAT
ACTAACTTACCTTTCTTTGT |
BsrGI |
| IMS82 (hsdR EBS2) | TGAACGCAAGTTTCTAATT TCGGTTAGAAGTCGATAG AGGAAAGTGTCT | |
| IMS83 (hsdR OUT F) | AGTATACGACTTACCTCAA | |
| IMS84 (hsdR OUT R) | TCAGTTGTTTCTGCCACG | |
| IMS85 (sauUSI IBS) | AAAAAAGCTTATAATTATC CTTAAAAGACAAGGCGGT GCGCCCAGATAGGGTG | HindIII |
| IMS86 (sauUSI EBS1d) |
CAGATTGTACAAATGTGGTG
ATAACAGATAAGTCAAGGCG
TTTAACTTACCTTTCTTTGT |
BsrGI |
| IMS87 (sauUSI EBS2) | TGAACGCAAGTTTCTAATTTC GGGTTTCTTTCCGATAGAGG AAAGTGTCT | |
| IMS90 (sauUSI OUT F) | ATGAGTAGATTACTAAATG | |
| IMS91 (sauUSI OUT R) | CGTTACTACGTTTGAACC | |
| IMSuni | CGAAATTAGAAACTTGCGTTCAGTAAAC | |
| IM151 (pIMAY MCS F) | TACATGTCAAGAATAAACTGCCAAAGC | |
| IM152 (pIMAY MCS R) | AATACCTGTGACGGAAGATCACTTCG | |
a
S. aureus and S. epidermidis gene designations are taken from http://kegg.jp. For oligonucleotides, anti secY stands for antisense secY RNA.
b
The description (relevant genotype, phenotype, or other characteristic) is shown for bacterial strains and plasmids. MSSA, methicillin-sensitive S. aureus.
The primers used in the construction of recombinant plasmids are shown in parentheses at the end of the entry. The sequences for primers are shown. Restriction sites are indicated by underlining. Regions of homology for SOE PCR with the B primer are shown in italic type, and regions of homology for recombineering in E. coli are shown in bold type.
c
The source or reference is shown for bacterial strains and plasmids. The restriction enzyme (RE) site is shown for oligonucleotides.