Transcription regulation of the nir gene cluster encoding nitrite reductase of Paracoccus denitrificans involves NNR and NirI, a novel type of membrane protein.

The nirIX gene cluster of Paracoccus denitrificans is located between the nir and nor gene clusters encoding nitrite and nitric oxide reductases respectively. The NirI sequence corresponds to that of a membrane-bound protein with six transmembrane helices, a large periplasmic domain and cysteine-rich cytoplasmic domains that resemble the binding sites of [4Fe-4S] clusters in many ferredoxin-like proteins. NirX is soluble and apparently located in the periplasm, as judged by the predicted signal sequence. NirI and NirX are homologues of NosR and NosX, proteins involved in regulation of the expression of the nos gene cluster encoding nitrous oxide reductase in Pseudomonas stutzeri and Sinorhizobium meliloti. Analysis of a NirI-deficient mutant strain revealed that NirI is involved in transcription activation of the nir gene cluster in response to oxygen limitation and the presence of N-oxides. The NirX-deficient mutant transiently accumulated nitrite in the growth medium, but it had a final growth yield similar to that of the wild type. Transcription of the nirIX gene cluster itself was controlled by NNR, a member of the family of FNR-like transcriptional activators. An NNR binding sequence is located in the middle of the intergenic region between the nirI and nirS genes with its centre located at position -41.5 relative to the transcription start sites of both genes. Attempts to complement the NirI mutation via cloning of the nirIX gene cluster on a broad-host-range vector were unsuccessful, the ability to express nitrite reductase being restored only when the nirIX gene cluster was reintegrated into the chromosome of the NirI-deficient mutant via homologous recombination in such a way that the wild-type nirI gene was present directly upstream of the nir operon.

Cloning and nucleotide sequence determination of twelve mutant dnaA genes of Escherichia coli.

Plasmids carrying different regions of the wild-type dnaA gene were used for marker rescue analysis of the temperature sensitivity of twelve strains carrying dnaA mutations. The different dnaA(Ts) mutations could be unambiguously located within specific regions of the dnaA gene. The mutant dnaA genes were cloned on pBR322-derived plasmids and on nucleotide sequencing by dideoxy chain termination the respective mutations were determined using M13 clones carrying the relevant parts of the mutant dnaA gene. Several of the mutant dnaA genes were found to have two mutations. The dnaA5, dnaA46, dnaA601, dnaA602, dnaA604, and dnaA606 genes all had identical mutations corresponding to an amino acid change from alanine to valine at amino acid 184 in the DnaA protein, close to the proposed ATP binding site, but all carried one further mutation giving rise to an amino acid substitution. The dnaA508 gene also had two mutations, whereas dnaA167, dnaA203, dnaA204, dnaA205, and dnaA211 each had only one. The pairs dnaA601/602, dnaA604/606, and dnaA203/204 were each found to have identical mutations. Plasmids carrying the different dnaA mutant genes intact were introduced into the respective dnaA mutant strains. Surprisingly, these homopolyploid mutant strains were found to be temperature resistant in most cases, indicating that a high intracellular concentration of the mutant DnaA protein can compensate for the decreased activity of the protein.

Titration of DnaA protein by oriC DnaA-boxes increases dnaA gene expression in Escherichia coli.

Binding of the DnaA protein to its binding sites, the DnaA-boxes (TTATCCACA), was measured by a simple physiological approach. The presence of extra DnaA-boxes in growing cells leads to a derepression of dnaA gene expression, measured as beta-galactosidase activity of a dnaA-lacZ fusion polypeptide. Different DnaA-boxes caused different degrees of derepression indicating that the DnaA protein requires sequences in addition to the DnaA-box for efficient binding. The DnaA-boxes in oriC might act cooperatively in binding of the DnaA protein. The derepressed levels of DnaA protein obtained in a strain carrying an oriC+-pBR322 chimera were very high and sufficient to activate oriC on the chimeric plasmid, which was maintained at a copy number more than three times that of pBR322.