The ATP synthase atpHAGDC (F1) operon from Rhodobacter capsulatus

Abstract

The atpHAGDC operon of Rhodobacter capsulatus, containing the five genes coding for the F1 sector of the ATP synthase, has been cloned and sequenced. The promoter region has been defined by primer extension analysis. It was not possible to obtain viable cells carrying atp deletions in the R. capsulatus chromosome, indicating that genes coding for ATP synthase are essential, at least under the growth conditions tested. We were able to circumvent this problem by combining gene transfer agent transduction with conjugation. This method represents an easy way to construct strains carrying mutations in indispensable genes.

FIG. 1

(A) Determination of transcription initiation. The sequencing ladder, to the left of the lane containing the primer extension product, shows the actual sequence of the template strand; for the sake of clarity, however, the letters at the top of the lanes have been changed to correspond to the sense strand, and the sequence should be read from top to bottom. The corresponding sequence, on the right, should also be read from top to bottom, as indicated by the arrow. The boldface A corresponds to the 5′ end of the mRNA. (B) For the promoter region, the sequence includes 40 bases upstream of the start of transcription, which is marked by +1. The −10 and −35 elements are identified by their distance from the +1 position. For the terminator, the TGA at the 5′ of the sequence corresponds to the stop codon of the last gene of the operon (atpC).

FIG. 2

Alignment of the amino acid sequences of β (A), γ (B), and ɛ (C) subunits of Rhodobacter capsulatus (rc), E. coli (ec), and bovine heart mitochondria (bt). Asterisks indicate identical residues, dots indicate conservative substitutions, E’s and double underlines indicate β strands, and H’s and black boxes indicate α helices. The secondary structures of β and γ subunits have been obtained from the atomic coordinates of bovine heart mitochondria F₁ (1), and the secondary structural elements of the ɛ subunit have been obtained from nuclear magnetic resonance models of E. coli (33).

FIG. 2

Alignment of the amino acid sequences of β (A), γ (B), and ɛ (C) subunits of Rhodobacter capsulatus (rc), E. coli (ec), and bovine heart mitochondria (bt). Asterisks indicate identical residues, dots indicate conservative substitutions, E’s and double underlines indicate β strands, and H’s and black boxes indicate α helices. The secondary structures of β and γ subunits have been obtained from the atomic coordinates of bovine heart mitochondria F₁ (1), and the secondary structural elements of the ɛ subunit have been obtained from nuclear magnetic resonance models of E. coli (33).

FIG. 2

Alignment of the amino acid sequences of β (A), γ (B), and ɛ (C) subunits of Rhodobacter capsulatus (rc), E. coli (ec), and bovine heart mitochondria (bt). Asterisks indicate identical residues, dots indicate conservative substitutions, E’s and double underlines indicate β strands, and H’s and black boxes indicate α helices. The secondary structures of β and γ subunits have been obtained from the atomic coordinates of bovine heart mitochondria F₁ (1), and the secondary structural elements of the ɛ subunit have been obtained from nuclear magnetic resonance models of E. coli (33).

FIG. 3

(A) Restriction maps of plasmids pRCA51, pRCA107, and pRCA108. Thick lines represent Rhodobacter capsulatus DNA. Gray bars indicate atp genes, and open bars represent ORFs. Solid bars in pRCA107 and pRCA108 are kanamycin resistance cassettes inserted at the positions shown. Restriction site abbreviations: B, BamHI; X, XhoI; N, NotI; E, EcoRI; Bg, BglII; EV, EcoRV. Vectors are not shown. (B) Hybridization pattern of Rhodobacter capsulatus total DNA digested with EcoRI. Lanes: 1 and 4, wild-type B100; 2 and 5, strain RCAK1; 3 and 6, deletion mutant RCAK4. Hybridization was done with a γ-subunit probe (lanes 1 to 3) and then with a kanamycin resistance probe (lanes 4 to 6). The two panels are pictures of the same filter hybridized with the first probe, stripped, and rehybridized with the second probe.