Article Figures & Data
Figures
- FIG. 1.
stp and stk are cotranscribed. (A) Schematic of overlapping pattern of SA1062 (stp) and SA1063 (stk) and primer annealing sites. The four-nucleotide overlap is in bold. The stop codon for SA1062 is underlined. Primer binding sites are indicated by arrows. (B) Results of PCRs using cDNA, gDNA, or cDNA reaction mixtures lacking RT as templates. Lanes 1, 4, and 7 were performed using primer set 44/107; lanes 2, 5, and 8 utilized 108/109; and lanes 3, 6, and 9 utilized 110/46, annealing on flanking or overlapping regions of stp/stk as indicated in panel A.
- FIG. 2.
Autoradiographs of in vitro kinase reactions. (A) Purified rSTK was incubated in kinase buffer with [γ-32P]ATP and Mn2+ alone and in the presence of MBP and rSTP as indicated. Proteins were resolved by SDS-PAGE and visualized by autoradiography. Shown are representative autoradiographs. (B) Increasing amounts of purified rSTK were added to separate kinase reaction mixtures to observe the dose-dependent effect. One microgram of rSTK was used for the time course and cation dependency experiments, where minutes and cations are shown above the autoradiographs. (C) Thin-layer chromatography results showing phosphoamino acid standards stained with ninhydrin (left) and autophosphorylated rSTK hydrolysis products revealed by autoradiography (right). The first and second dimensions are indicated by arrows.
- FIG. 3.
Confirmation of mutant strains. (A) PCR analysis of gDNA isolated from the indicated strains with primers specific for stp and stk. (B) Western blot assays of total cell lysates probed separately with (top) anti-rSTP and (bottom) anti-rSTK production sera (right) and corresponding prebleed (PB, left) sera. Lanes: 1, N315; 2, N315ΔSTP; 3, N315ΔSTK; 4, N315ΔSTP/STK; 5, N315pCN40tet; 6, N315ΔSTPΩSTP; 7, N315ΔSTKΩSTK; 8, N315ΔSTP/STKΩSTP/STK. Arrows indicate the locations of STK and STP within the lysates.
- FIG. 4.
Analysis of cell morphology by TEM. Shown are representative fields of TSB-grown stationary-phase wild-type (control) and mutant S. aureus strains, as indicated. White arrows indicate abnormal septation; black arrows indicate evidence of apparent wall or membrane shedding. Magnifications: A, ×30,500; B, ×120,000.
- FIG. 5.
Lysostaphin sensitivity assays. Results indicate the susceptibilities of (A) N315; (B) N315ΔSTP, (C) N315ΔSTK, and (D) N315ΔSTP/STK; and their appropriate complemented strains (triangles) to lysostaphin. Autolysis (no lysostaphin) control results are shown as closed squares. Data shown are the averages of three independent experiments. Error bars indicate standard deviations. Data are presented as percentages of the original culture turbidity. OD620, optical density at 620 nm.
Tables
- TABLE 1.
Plasmids and strains used in this study
Plasmid or strain Description Source or reference Plasmids pET14B N-terminally His-tagged expression vector Novagen pET14B-STP pET14B containing entire SA1062 gene This study pET14B-STK pET14B containing entire SA1063 gene This study pMAD E. coli-S. aureus shuttle vector for gene inactivation 2 pDC123 Source of chloramphenicol acetyltransferase resistance gene (chl) 8 pMADΔSTKchl pMAD containing up- and downstream regions of SA1063 flanking chl This study pKOR1 E. coli-S. aureus shuttle vector with ccdB selection and lambda recombination capabilities 4 pKOR1ΔSTP pKOR1 containing up- and downstream regions of SA1062 This study pKOR1ΔSTP/STK pKOR1 containing regions upstream of SA1062 and downstream of SA1063 This study pCN36 Source of tet(M) gene for tetracycline resistance 9 pCN40 E. coli-S. aureus shuttle vector 9 pCN40tet pCN40 with ermC gene replaced with tet(M) from pCN36 This study pCN40tet-STP pCN40tet containing entire SA1062 gene and putative promoter upstream This study pCN40tet-STK pCN40tet containing entire SA1063 gene and putative promoter upstream of SA1062 This study pCN40tet-STPSTK pCN40tet containing overlapping SA1062 and SA1063 genes and putative promoter upstream of SA1062 This study E. coli strains XL-1-Blue endA1 gyrA96(Nalr) thi-1 recA1 relA1 lac glnV44 [F′::Tn10 proAB+lacIq Δ(lacZ)M15] hsdR17(rK− mK+) Stratagene DH5α Subcloning efficiency F− φ80dlacZΔM15 λ(lacZYA-argF)U169 recA1 endA1 hsdR17 (rK− mK+) phoA supE44 λ−thi-1 gyrA96 relA1 Invitrogen BL21(DE3)pLysS F−ompT hsdSB(rB− mB−) gal dcm(DE3)pLysS (Camr) Invitrogen S. aureus strains RN4220 Restriction-deficient derivative of NCTC 8325-4 24 N315 Methicillin-resistant S. aureus parent strain 26 N315ΔSTP N315 lacking SA1062 This study N315ΔSTK N315 lacking SA1063 This study N315ΔSTP/STK N315 lacking both SA1062 and SA1063 This study N315pCN40tet N315 containing pCN40tet (vector-only control) This study N315ΔSTPΩSTP Complemented N315ΔSTP strain containing pCN40tet-STP This study N315ΔSTKΩSTK Complemented N315ΔSTK strain containing pCN40tet-STK This study N315ΔSTP/STKΩSTP/STK Complemented N315ΔSTP/STK strain containing pCN40tet-STPSTK This study - TABLE 2.
Primers used in this study
Use and name Sequence (5′ → 3′)a Restriction site Cotranscription assay #44 GCCATGTTAACTTAATTCCTGTCAACC #107 CGCAACCAATTTTCAGCTTGATG #108 ACAGATAAACGTGTGAGTCCAGA #109 CTTCTTCATCAACATCGATCATACTTAC #110 GAATCGGTAGATGTACCATACACTG #46 TCGACCTGTCTTCAAAATGGCA Recombinant proteins STP-F CTACGGCCATATGCTAGAGGCACAATTTTTTACTGATACTGGAC NdeI STP-R CCGCGGATCCTCATACTTTATCACCTTCAATAGCCG BamHI STK-F CTACGGCCATATGATAGGTAAAATAATAAATGAACG NdeI STK-R CCGCGGATCCTTAAACATCATCATAGCTGACTTCTTTTTC BamHI Deletion strains AF GCGGATCCCATAAAGCAGGAGAAGTTGCAAG BamHI AR CTAGCTAGCTCATACTTTATCACCTTCAATAGC NheI BF CTAGCTAGCAATTGAAGTAAATGTACCGAGG NheI BR TGCCATGGCACTTACCGACACTGATTGACCAC NcoI catF CGGCTAGCCGTTGACTTTTAAAAAAGGATTGATTC NheI catR CGGCTAGCCTTATAAAAGCCAGTCATTAGGC NheI #33 GGGGACCACTTTGTACAAGAAAGCTGGGTGAAACAGATTTTTGAATGGTTATATCAA #34 GTTTCACCGCGGGTTTCTACTTGTCGTTCCTTTGC SacII #58 GTTTCACCGCGGAGTATGATAGGTAAAATAATAAATG SacII #59 GGGGACAAGTTTGTACAAAAAAGCAGGCTCTGGCTTTTGGAATTGCTGATGATGAG #35 GTTTCACCGCGGATAATTGAAGTAAATGTACCGAG SacII #36 GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATATCGATTTAATTCAAGAA Complementation #73 CGCGGATCCCTTGTGGTCAATTAAGAGCA BamHI #65 CCGGAATTCTCATACTTTATCACCTTCAATAG EcoRI #66 CCGGAATTCTTAAATATCATCATAGCTGACTTC EcoRI #87 CTACGGCCATATGTTGTCTTTACCTCGTTTCTACTTGTCG NdeI #88 CTACGGCCATATGATGATAGGTAAAATAATAAATGAACG NdeI ↵ a Restriction sites are in bold, and att recombination sites are in italics.
- TABLE 3.
MICs of antibiotics tested
Antibiotic(s) MIC(s) (μg/ml) for strain: N315 N315ΔSTP N315ΔSTK N315ΔSTKΩSTK N315ΔSTP/STK N315ΔSTP/ STKΩSTP/STK Most affected Cefazolin >16 >16 >16 ≤8 Cefotaxime >256 >256 43 >256a 12 >256 Ceftriaxone >32 32 ≤8 >32 ≤8 >32 Ertapenem >32 >32 18 >32 0.6 >32 Imipenem >8 8 8 ≤4 Unaffected Amoxicillin-clavulanate >4/2 >4/2 >4/2 >4/2 Ampicillin-sulbactam ≤8/4 ≤8/4 ≤8/4 ≤8/4 Ampicillin >8 >8 >8 >8 Clindamycin >4 >4 >4 >4 Erythromycin >4 >4 >4 >4 Gentamicin ≤4 ≤4 ≤4 ≤4 Levofloxacin ≤2 ≤2 ≤2 ≤2 Linezolid 2 2 2 2 Moxifloxacin ≤2 ≤2 ≤2 ≤2 Nitrofurantoin ≤32 ≤32 ≤32 ≤32 Oxacillin >2 >2 >2 >2 Penicillin >8 >8 >8 >8 Rifampin ≤1 ≤1 ≤1 ≤1 Synercid ≤0.5 ≤0.5 ≤0.5 ≤0.5 Tetracycline ≤4 ≤4 ≤4 ≤4 Trimethoprim-sulfamethoxazole ≤0.5/9.5 ≤0.5/9.5 ≤0.5/9.5 ≤0.5/9.5 ↵ a The complementation MICs shown are averages of three independent experiments with Etest strips. The antibiotic gradients on the Etest strips were 0.016 to 256 μg/ml (cefotaxime and ceftriaxone) and 0.002 to 32 μg/ml (ertapenem).
