Mercury resistance is encoded by transferable giant linear plasmids in two chesapeake bay Streptomyces strains

Abstract

The Streptomyces strains CHR3 and CHR28, isolated from the Baltimore Inner Harbor, contained two and one, respectively, giant linear plasmids which carry terminally bound proteins. The plasmids pRJ3L (322 kb), from CHR3, and pRJ28 (330 kb), from CHR28, carry genes homologous to the previously characterized chromosomal Streptomyces lividans 66 operon encoding resistance against mercuric compounds. Both plasmids are transmissible (without any detectable rearrangement) to the chloramphenicol-resistant S. lividans TK24 strain lacking plasmids and carrying a chromosomal deletion of the mer operon. S. lividans TK24 conjugants harboring pRJ3L or pRJ28 exhibited profiles of mercury resistance to mercuric compounds similar to those of Streptomyces strains CHR3 and CHR28.

FIG. 1

(A) PFGE and (B) Southern hybridization with probe MER-A and MER-B analysis of Streptomyces strains CHR3 and CHR28. Lanes 1 and 6, λ concatamer ladder; lane 2, strain CHR3; lane 2, strain CHR28; lane 3, S. lividans 66; lane 4, S. lividans TK24. Pulse time was 30 s for 24 h (6 V/cm at 14°C).

FIG. 2

(A) PFGE of DNA plugs of strains CHR3 and CHR28. Lanes 1 and 4, S. cerevisiae chromosomes; lane 2, CHR3 with proteinase K treatment; lane 3, CHR3 without proteinase K treatment; lane 5, CHR28 with proteinase K treatment; lane 6, CHR3 without proteinase K treatment. Pulse time was 30 s. (B) SDS-PFGE of strains CHR3 and CHR28. Lane 1, λ concatamer ladder; lane 2, CHR3 with proteinase K treatment; lane 3, CHR3 without proteinase K treatment; lane 4, CHR28 with proteinase K treatment; lane 5, CHR28 without proteinase K treatment. Pulse time was 30 s for 24 h (6 V / cm at 14°C). (C) Retardation gel assay. Plasmid bands from samples treated (+) or untreated (−) with proteinase K were excised from an SDS-PFGE gel and digested with restriction enzyme XbaI. Lane 1, λ DNA digested with HindIII; lane 2, λ concatamer ladder; lane 3, pRJ28 from sample prepared without proteinase K treatment digested with XbaI; lane 4, pRJ28 from sample prepared with proteinase K treatment digested with XbaI. Ramping time was 12 to 15 s for 20 h (6 V/cm at 14°C).

FIG. 3

Restriction map of pRJ3L and pRJ28. X, XbaI; H, HindIII. The relative locations of the mercuric resistance genes are indicated by the solid black bar. The terminal proteins are indicated by a gray circle.

FIG. 4

Analysis of conjugants. (A) PFGE and (B) Southern hybridization with probe MER-A and MER-B of parents and conjugants of strains CHR3 and CHR28. Lanes 1 and 9, λ concatamer ladder; lanes 2 and 3, conjugants of strain CHR3; lane 4, parent strain CHR3; lane 5, recipient strain S. lividans TK24; lane 6, parent strain CHR28; lanes 7 and 8, conjugants of strain CHR28. Pulse time was 30 s for 24 h (6 V / cm at 14°C). (C) PFGE analysis of total DNA digested with restriction enzyme DraI. Lane 1, Saccharomyces cerevisiae chromosomes; lane 2 and 3: conjugants of strain CHR3; lane 4, parent strain CHR3; lane 5, recipient strain S. lividans TK24; lane 6, parent strain CHR28; lanes 7 and 8, conjugants of strain CHR28. The arrow indicates the linear uncut plasmid. Pulse time ramping was 60 to 180 s for 26 h (6 V / cm at 14°C). (D) Restriction digest of plasmids from parents and conjugants. Lanes 1 and 10, λ concatamer ladder; lane 2, pRJ28T digested with XbaI; lane 3, pRJ28T digested with HindIII; lane 4, pRJ28 digested with XbaI; lane 5, pRJ28 digested with HindIII; lane 6, pRJ3LT digested with XbaI; lane 7, pRJ3LT digested with HindIII; lane 8, pRJ3L digested with XbaI; lane 9, pRJ3L digested with HindIII. Ramping time was 8 to 14 s for 20 h (6 V/cm at 14°C).

FIG. 5

Mercury resistance profiles to mercuric chloride (A) and phenylmercuric acetate (B). Streptomyces strains CHR3 (•) and CHR28 (■) and conjugants CHR3T (○) and CHR28T (□) are as marked. The control strains were S. lividans 66 (▴) and S. lividans TK24 (▵). The horizontal line indicates the inhibition zone diameter used as a resistance criterion.