He S. enterica Castanospermine serovar cerro 87 dptBCDE genes in E. coli BW25113. A. Orthologous DNA phosphorothioation gene clusters from S. lividans (dndABCDE) and S. enterica (dptBCDE). The cysteine desulfurase gene dndA of S. lividans is required for DNA phosphorothioation. The S. enterica dptBCDE gene cluster lacks a dndA ortholog. The dndA function may be performed by an unknown, unlinked gene in S. enterica and also in E. coli expressing dptBCDE. B. The three cysteine desulfurases in the E. coli genome. C. E. coli 301353-96-8 cost BW25113 DNA becomes phosphorothioated when expressing dptBCDE of S. enterica. Ethidium bromide-stained agarose gels containing total genomic DNA, separated in Tris-acetate EDTA (TAE) buffer. TAE (top panel), 23727046 untreated samples; PAA (bottom panel), identical DNA samples after incubation in TAE containing 1 per-acetic acid (PAA). Lane 1, E. coli BW25113 (wild-type, not S-modified); lane 2, S. enterica serovar 87 (wild-type, containing phosphorothioate DNA); lane 3, E. coli BW25113 expressing the S. enterica serovar cerro 87 dptBCDE gene cluster. The fluorescent smear in lanes 2 and 3 of the lower gel indicates that the DNA was phosphorothioate modified. doi:10.1371/journal.pone.0051265.gTable 1. Strains that are used in this study.STRAINS Salmonella enterica Cerro 87 E. coli DH10B E. coli BW25113 BL21(DE3)pLysS JW2514-4 JW1670-1 JW2781-1 JW2513-1 JW3955-2 JW3956-1 JW2512-1 JW2508-1 JW0810-2 JW3779-3 JW3435-1 JW0413-1 AXH034 E. coli XL1-Blue MR E. coli XL1-Blue MRF9 KanCHARACTERISTICS Strain containing naturally S-modified DNA, source of the dptB-E gene cluster Non-restricting host strain for gene cloning acIq rrnBT14 DlacZWJ16 hsdR514 DaraBADAH33 DrhaBADLD78, strain used for creating gene knockouts Lacks lon and ompT proteases Cmlr E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DiscS776::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DsufS755::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DcsdA738::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DiscU775::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, rph-1, D(rhaD-rhaB)568, DthiS762::kan, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, rph-1, D(rhaD-rhaB)568, DthiF763::kan, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DiscA774::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DiscX770::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DmoeB726::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, rph-1, DcyaY752::kan, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DyhhP(tusA)725::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), DthiI780::kan, l-, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DiscS1191::kan, rph-1, D(rhaD-rhaB)568, hsdR514 Host strain for propagating pBT and pTRG recombinants D(mcrA)183 D(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96; relA1 lac Derivative of XL1-Blue MR. Reporter strain for two-hybird test using pBT and pTRG derivatives D(mcrA)183 D(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F9 proAB lacIqZDM15 Tn5 (Kanr)]REFERENCE [6] [7] [12] Nova.He S. enterica serovar cerro 87 dptBCDE genes in E. coli BW25113. A. Orthologous DNA phosphorothioation gene clusters from S. lividans (dndABCDE) and S. enterica (dptBCDE). The cysteine desulfurase gene dndA of S. lividans is required for DNA phosphorothioation. The S. enterica dptBCDE gene cluster lacks a dndA ortholog. The dndA function may be performed by an unknown, unlinked gene in S. enterica and also in E. coli expressing dptBCDE. B. The three cysteine desulfurases in the E. coli genome. C. E. coli BW25113 DNA becomes phosphorothioated when expressing dptBCDE of S. enterica. Ethidium bromide-stained agarose gels containing total genomic DNA, separated in Tris-acetate EDTA (TAE) buffer. TAE (top panel), 23727046 untreated samples; PAA (bottom panel), identical DNA samples after incubation in TAE containing 1 per-acetic acid (PAA). Lane 1, E. coli BW25113 (wild-type, not S-modified); lane 2, S. enterica serovar 87 (wild-type, containing phosphorothioate DNA); lane 3, E. coli BW25113 expressing the S. enterica serovar cerro 87 dptBCDE gene cluster. The fluorescent smear in lanes 2 and 3 of the lower gel indicates that the DNA was phosphorothioate modified. doi:10.1371/journal.pone.0051265.gTable 1. Strains that are used in this study.STRAINS Salmonella enterica Cerro 87 E. coli DH10B E. coli BW25113 BL21(DE3)pLysS JW2514-4 JW1670-1 JW2781-1 JW2513-1 JW3955-2 JW3956-1 JW2512-1 JW2508-1 JW0810-2 JW3779-3 JW3435-1 JW0413-1 AXH034 E. coli XL1-Blue MR E. coli XL1-Blue MRF9 KanCHARACTERISTICS Strain containing naturally S-modified DNA, source of the dptB-E gene cluster Non-restricting host strain for gene cloning acIq rrnBT14 DlacZWJ16 hsdR514 DaraBADAH33 DrhaBADLD78, strain used for creating gene knockouts Lacks lon and ompT proteases Cmlr E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DiscS776::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DsufS755::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DcsdA738::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DiscU775::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, rph-1, D(rhaD-rhaB)568, DthiS762::kan, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, rph-1, D(rhaD-rhaB)568, DthiF763::kan, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DiscA774::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DiscX770::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DmoeB726::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, rph-1, DcyaY752::kan, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DyhhP(tusA)725::kan, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), DthiI780::kan, l-, rph-1, D(rhaD-rhaB)568, hsdR514 E.coli F-, D(araD-araB)567, DlacZ4787(::rrnB-3), lambda2, DiscS1191::kan, rph-1, D(rhaD-rhaB)568, hsdR514 Host strain for propagating pBT and pTRG recombinants D(mcrA)183 D(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96; relA1 lac Derivative of XL1-Blue MR. Reporter strain for two-hybird test using pBT and pTRG derivatives D(mcrA)183 D(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F9 proAB lacIqZDM15 Tn5 (Kanr)]REFERENCE [6] [7] [12] Nova.
