Article Figures & Data
Figures
- Fig. 1.
The nar operon is strain specific. Southern blot of BamHI-digested total DNA of T. thermophilus HB8 (lane 1), T. thermophilus HB8narGH::kat (lane 2), T. thermophilus BPL7 (lane 3), T. thermophilus HB27 (lane 4), Thermus sp. strain ATCC 27737 (lane 5), and T. aquaticus YT1 (ATCC 25104) (lane 6). The nar operon was detected with 32P-probe A.
- Fig. 2.
Horizontal transfer of the nar operon. (A) Growth under anaerobic conditions of T. thermophilus HB8 (circles), T. thermophilus HB27Camr (diamonds), and the exconjugant T. thermophilusHB27Camr::nar (squares) in the presence (open symbols) or absence (closed symbols) of KNO3(20 mM). (B) PFGE of NdeI-digested chromosomal DNA fromT. thermophilusHB27Camr::nar (lane 1) and T. thermophilus HB27Camr (lane 2). (C) Southern blot of the PFGE sample shown in panel B labeled with probe A. Lanes are as in panel B. Numbers beside lanes in panel B are in kilobase pairs.
- Fig. 3.
Expression of the nar operon in T. thermophilus HB27Camr::nar. (A) Coomassie blue-stained SDS-PAGE sample showing proteins from the particulate fractions of T. thermophilus HB8 (lanes 2, 3, and 4), T. thermophilus HB27Camr (lane 5), andT. thermophilusHB27Camr::nar (lanes 6 and 7). Cells were grown for 24 h under microaerophilic (lanes 2, 3, 5, and 6) or anaerobic (lanes 4 and 7) conditions with nitrate (lanes 3 to 7). The sample in lane 2 was grown without nitrate. Lane 1 represents markers whose sizes (in thousands) are labeled at the left. (B) Parallel Western blot with antiserum against NarG. A 140-kDa protein which corresponds to NarG is indicated by an arrow. Lanes are as in panel A.
- Fig. 4.
Identification of oriV downstream of thenar operon. Restriction maps of the nar region (shaded bars) and of the plasmids used to localize a replicativeoriV. Genes narG, narH,narJ, and narI are labeled. The katgene used for their selection in Thermus strains is shown by black bars. The approximate position of the nar promoter (Pnar) is shown. Restriction enzyme abbreviations: E,EcoRI; K, KpnI; P, PstI; S,SalI; Sm, SmaI; X, XhoI.
- Fig. 5.
pNIT9kat replicates in Thermus strains. (A) Agarose gel electrophoresis of EcoRI-digested pNIT9kat purified from E. coli (lane 1) and EcoRI-digested plasmid fraction from T. thermophilus HB8 transformed with pNIT9kat (lane 2). (B) Parallel Southern blot labeled with probe B. Lanes are as in panel A. HindIII-digested DNAs from λ and φ29 phages were used as size markers (lane M).
- Fig. 6.
Sequence of the minimal oriV region. The 837-bp SmaI-XhoI region contains an origin for autonomous replication in nar-carrying Thermusstrains. The amino acid sequence shown below the DNA sequence corresponds to the C-terminal part of a protein homologous to the nitrite extrusion protein NarK. Inverted and direct repeats are labeled with arrows. Sites for restriction endonucleases SmaI andXhoI are also shown.
Tables
- Table 1.
Time-dependent horizontal transfera
Donor strain Level of KmrCamr colonies for a transfer time (min) of: 10 20 30 40 50 60 70 80 90 100 slrA::kat − + ++ ++ ++ ++ ++ ++ ++ ++ slpM::kat + ++ ++ +++ +++ +++ +++ +++ +++ +++ narGH::kat − − − − + ++ ++ ++ ++ ++ ↵a After the mating experiment (see Materials and Methods), we detected kanamycin- and chloramphenicol-resistant colonies at the following levels: −, none; +, less than 20; ++, 20 to 100; +++, more than 100.
- Table 2.
Identification of a replicative ori downstream of the nar operona
Host Level of Kmr colonies in the presence of plasmid: pMK18 pNIT9kat pNIT5kat1 pNIT5kat1XS pNIT5kat2 pNIT10kat HB8 +++ +++ ++++ ++++ ++ − HB27Camr::nar ++++ +++ ++++ ++++ ++ − HB27Camr ++++ − − − − − BPL7 ++ − − − − − E. coliTG1 ++++ +++ +++ +++ +++ ↵a Data represent the results of transformation of Thermus strains with plasmids purified from E. coli and the results of transformation of E. coli with the same plasmids isolated from Thermusstrains. pMK18 was used as a control for transformation efficiency. Number of colonies: −, none; ++, 50 to 100; +++, 100 to 500; ++++, >500.
