Figure 1.
Schematic genetic maps of pDSK5191 and its derivative expression plasmids.
(A) Genetic map of the IncQ-group conjugation plasmid pDSK5191. Protein coding sequences are shown as block arrows. The pale gray rectangle represents the region of Ω-cassette in which T4-phage transcription and translation terminator sequences [23] are located at both ends. “Ori” represents the region containing the oriV and oriT sequences, which are derived from RSF1010 and are required for replication and mobilization of the plasmid, respectively [15]. (B) Expression constructs of the pDSK5191-derivative plasmids. Each construct was inserted into the unique EcoRI site of pDSK5191, as indicated by the arch-shaped arrow at the top of the panels. Protein-coding sequences are shown as block arrows. The pale gray rectangle represents the six-consecutive histidine-tag (6xhis) attached to the 5’ end of the pscA gene. “PpscA” and “ter” are a putative constitutive promoter and a ρ-independent transcription terminator, respectively, of the C. tepidum pscAB gene cluster.
Figure 2.
Restriction enzyme mappings of the plasmids in the C. tepidum and C. limnaeum transconjugants.
(A, B) Physical maps of the plasmids from the C. tepidum transconjugants. The maps were constructed with restriction enzymes EcoRI (A) and BglII (B). The restriction fragments were separated by agarose gel (1%) electrophoresis. The control plasmids pDSK5191 (lane 1), pDSK5191-cycA (lane 2), and pDSK5191-soxB (lane 3) were obtained from the donor S17-1 cultures. Lanes 4-11 are the plasmid samples of the C. tepidum cultures. The genotype of C. tepidum is indicated above each lane. The bars and numbers at the left side of the panel indicate mobility and size of the StyI digests of the λ-phage DNA. (C) Physical maps of the plasmids from C. limnaeum transconjugants. The maps were constructed with restriction enzymes EcoRI (lanes 1–3) and BamHI (lanes 4–6). Lanes 1 and 4 are pDSK5192 plasmids prepared from the donor S17-1 cultures. Lanes 2-3 and 5-6 are the plasmid samples of the C. limnaeum cultures. The genotype of C. limnaeum is indicated above each lane. The bars and numbers at the left side of the panel indicate mobility and sizes of the StyI digests of the λ-phage DNA, respectively.
Figure 3.
Growth curves of C. tepidum mutants after gene complementation experiments.
Growth curves of C. tepidum mutants used as host strains (A), transconjugant strains of ∆cycA mutant (B), and ∆soxB mutant (C). Each strain was grown in a liquid CL medium at 40°C (for details, see Materials and Methods), and its optical density (O.D.) was monitored at 660 nm. In the transconjugant cultures, 1 µg/mL of Em was added for the stable maintenance of plasmids. The average values and standard deviations, which were obtained from at least three independent experiments, were plotted.
Figure 4.
Absorption spectra of BChl a-associated proteins from various C. tepidum strains recovered by Ni2+-affinity purification.
Absorption spectra (traces 1-6) of BChl a-associated proteins, which were obtained from various C. tepidum strains with Ni2+-affinity chromatography purification procedure, were measured (see Materials and Methods). Each spectrum was normalized so that recovery rates per wet cell weights can be compared. In the inset, spectra of the eluates from the wild-type WT2321 strain (trace 1) and the 6HA-∆recA strain with pDSK5191-6xhis-pscAB (trace 5) were normalized at absorption peaks around 810 nm. The absorption band around 810 nm is contributed by antenna BChls a associated with FMO proteins and the RC complex as well, while the absorption shoulder around 840 nm is specific to a special pair of BChls a, P840, within the RC complex [3,13].