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The haem-copper cytochrome oxidases are terminal catalysts of the respiratory chains in aerobic organisms. These integral membrane protein complexes catalyse the reduction of molecular oxygen to water and utilize the free energy of this reaction to generate a transmembrane proton gradient. Quinol oxidase complexes such as the Escherichia coli cytochrome bo belong to this superfamily. To elucidate the similarities as well as differences between ubiquinol and cytochrome c oxidases, we have analysed two-dimensional crystals of cytochrome bo by cryo-electron microscopy. The crystals diffract beyond 5 A. A projection map was calculated to a resolution of 6 A. All four subunits can be identified and single alpha-helices are resolved within the density for the protein complex. The comparison with the three-dimensional structure of cytochrome c oxidase shows the clear structural similarity within the common functional core surrounding the metal-binding sites in subunit I. It also indicates subtle differences which are due to the distinct subunit composition. This study can be extended to a three-dimensional structure analysis of the quinol oxidase complex by electron image processing of tilted crystals.

CyoA, an integral membrane protein, is a subunit of the Escherichia coli cytochrome o quinol oxidase complex. The C-terminal periplasmic domain of CyoA has been expressed in E. coli, purified and crystallized. Crystals were grown using ammonium sulphate as a precipitant. They have space group I222 or I2(1)2(1)2(1) and diffract X-rays to 2.3 A resolution.

The nucleotide sequence of the promoter distal region of the atp (or unc) operon of Escherichia coli has been determined. It encodes the gamma, beta and epsilon subunits of the ATP-synthase complex and includes a noncoding sequence in which transcription of the operon probably terminates. This work completes the nucleotide sequence of the operon which contains nine genes: eight encode structural proteins of the ATP-synthase complex; a ninth, the first in the operon, may be a pilot for assembly. The genes for the alpha and beta subunits have evolved from a common ancestor.

The nucleotide sequence is described of a region of the Escherichia coli chromosome extending from oriC to phoS that also includes the loci gid, unc and glmS. Taken with known sequences for asnA and phoS this completes the sequence of a segment of about 17 kilobases or 0.4 min of the E. coli genome. Sequences that are probably transcriptional promoters for unc and phoS can be detected and the identity of the unc promoter has been confirmed by experiments in vitro with RNA polymerase. Upstream of the promoter sequence is an extensive region that appears to be non-coding. Conserved sequences are found that may serve to concentrate RNA polymerase in the vicinity of the unc promoter. Hairpin loop structures resembling known rho-independent transcription termination signals are evident following the unc operon and glmS. The glmS gene encoding the amidotransferase, glucosamine synthetase, has been identified by homology with glutamine 5-phosphoribosylpyrophosphate amidotransferase.

An approach to sequencing proteins by the solid-phase method combined with isolation of proteins and polypeptides by gel electrophoresis is described. Mixtures of proteins or polypeptides resulting from digests are fractionated in the presence of dodecylsulphate in polyacrylamide gels. They are detected with Coomassie blue, eluted, selectively reacted with porous glass derivatives and sequenced in their amino-terminal regions with the aid of a new microsequencer. Alternatively they can be analysed or digested with enzymes and fingerprinted. It is a relatively rapid method of purifying proteins for sequence analysis which we have used to provide partial protein sequence data to complement DNA sequences. Nine genes, four from the unc operon of Escherichia coli encoding the alpha, beta, gamma and epsilon subunits of ATP synthase and five for capsid proteins of bacteriophage lambda, have been identified by this method.

The organization of genes encoding the blood group M-specific hemagglutinin (M-agglutinin) of Escherichia coli strain IH11165 was studied with a cloned 6.5-kb DNA segment. This DNA segment contains at least five genes which code for the polypeptides of 12.5, 30, 80, 18.5 and 21 kDa. The 30-, 80- and 21-kDa polypeptides are synthesized as precursors that are approximately 2 kDa larger. The 21-kDa polypeptide was identified as the M-agglutinin subunit by its reactivity with anti-M-agglutinin serum. Nucleotide sequence analysis of the corresponding gene showed that the M-agglutinin precursor had a 24-amino acid (aa) signal sequence, while the mature protein is 146 aa residues long. Although the organization of the M-agglutinin gene cluster resembles those of other E. coli adhesins, there is no significant sequence homology between the M-agglutinin subunit and the subunits of the other potentially related proteins in E. coli.