Figure 1.
Heterologous expression of the 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), 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.
Table 1.
Strains that are used in this study.
Table 2.
Plasmids that are used in this study.
Table 3.
Primers that are used in this study.
Figure 2.
E.coli iscS is required for DNA phosphorothioation.
Ethidium bromide-stained agarose gels containing E. coli total genomic DNA, separated in Tris-acetate EDTA (TAE) buffer. Top gel (TAE), untreated samples; bottom gel (PAA), identical DNA samples after incubation in TAE containing 1% per-acetic acid (PAA). A fluorescent smear in the lower gel indicates that the DNA was S-modified. Lanes 1–8, Dnd (DNA degradation) phenotypes of E. coli cysteine desulfurase deletion mutants (ΔiscS, ΔsufS, ΔcsdA) containing the S. enterica dptBCDE gene cluster cloned on pJTU3510 (lane 1–8); lanes 9–12, trans complementation of the chromosomal ΔiscS mutation by pJTU3619 containing mutant derivatives of iscS (lanes 9–12). E. coli hosts: wt, wild type. The mutations ΔiscS, ΔsufS and ΔcsdA are in the E. coli chromosome. pJTU3510: −, no plasmid; +, pJTU3510 expressing dptBCDE. pJTU3619 (compatible with pJTU3510) containing the following genes: S+, wild-type E. coli iscS; 111, 170, 328, mutant iscS genes containing the aa changes Cys111Ala, Cys170Ala or Cys328Ala, respectively. −, no plasmid. TAE, gel running buffer; PAA, TAE containing per-acetic acid.
Figure 3.
IscS might participate DNA phosphorothioation directly.
Ethidium bromide-stained agarose gels. TAE (top gel), samples run in normal TAE buffer; PAA (bottom gel), samples run in TAE containing PAA. Expression of S. enterica dptB-E resulted in DNA S-modification and a fluorescent smear in all samples, except for E. coli ΔiscS. IscS was therefore the only gene that was required for DNA S-modification among the tested deletions.
Figure 4.
Protein interactions between IscS and Dpt proteins.
A.The bar graph shows protein interactions that enable the E. coli cells to survive on medium containing 3AT (3-amino-1,2,4-triazole). F, pBT-LGF2; P, pTRG-Gal11P; S, pBT-IscS; B, pTRG-DptB; C, pTRG-DptC; D, pTRG-DptD; E, pTRG-DptE; G, pTRG only. F and P were co-expressed as positive control; S and G were co-expressed as negative control. E. coli can grow on 3-AT selective screening medium only when there is a binding interaction between the fusion proteins expressed from the bait and target plasmids. B. Dual selection plate containing 3-amino-1,2,4-triazole and streptomycin. F+P, LGF2+GallP (growth, positive control); S+B, IscS+DptB (no growth, no interaction); S+C, IscS+DptC (growth indicating protein interaction); S+D, IscS+DptD (no growth, no interaction); S+E, IscS+DptE (growth indicating protein interaction); S+G, IscS+pTRG (no growth, negative control). C. Interactions between IscS and DptC as well as IscS and DptE confirmed by pull-down experiments. Left panel: IscS (N terminus Strep tagged) extraction was mixed with GSTDptC or GSTDptE extraction and then purified by Streptactin affinity purification. Western blot was done using antibody against GST. Right panel: the mixture was purified by GST affinity purification. Western blotting was done using antibody against StreptagII.