Regulation of adenylate cyclase synthesis in Escherichia coli: nucleotide sequence of the control region.

The regulatory region of the cya gene from Escherichia coli has been characterized by nucleotide sequence analysis and genetic approaches. Two promoters, P1 and P2, organized in that order with respect to the beginning of the cya open reading frame, were identified. Using cya-lac operon and protein fusions, it was possible to show that both promoters are active in vivo. P1 activity seemed sensitive to catabolite repression whereas activity of the stronger promoter, P2, did not respond to inhibition by glucose. No effect of cAMP or its receptor, catabolite activator protein (CAP), could be found although the DNA sequence reveals a consensus CAP site downstream of P2. The 548 nucleotides situated at the 3' end of the sequence carry an open reading frame which can tentatively be assigned to the beginning of adenylate cyclase. Among noteworthy features of the corresponding sequence are an UUG codon as the putative start site of cyclase, and a long hydrophobic stretch of amino acids resembling leader peptides in secreted or membrane proteins.     

A short N-proximal region of prochymosin inhibits the secretion of hybrid proteins from Escherichia coli

A gene encoding bovine prochymosin (PC) was fused to the coding sequence (phoA) for the Escherichia coli alkaline phosphatase (AP) signal peptide and expressed in E. coli under the control of the phoA promoter. Upon induction, an AP-PC fusion protein was produced which was neither processed nor exported into the periplasm. We investigated this lack of secretion by constructing a series of gene fusions in which different regions of the PC gene were inserted between the coding regions of the AP leader and mature protein. Analysis of the cellular location of the proteins encoded by these fusions revealed that a region of PC (between amino acids 6 and 29) prevented processing and secretion of an AP-PC fusion when inserted near to the AP signal peptide. In contrast, when this 'blocking sequence' was inserted elsewhere in AP the hybrid proteins were efficiently processed and translocation was initiated.     

NMR study of the structural changes induced in the E. coli lac promoter by the specific binding of the CAP protein.

We have studied the binding of the CAP protein to an 18 base pair lac promoter sequence comprising the core of the CAP recognition sequence. Specific binding of this sequence was established by competition binding assays and comparison of the relative affinities of a number of lac promoter, lac operator, and unspecific sequences of different lengths. The effect of the binding of CAP to the 18 base pair promoter sequence and, for comparison, to an 18 base pair symmetric operator and an oligonucleotide of unrelated sequence have been studied by 1H NMR. Binding of CAP does not bring about any changes in the chemical shift values of the imino proton resonances of the DNA, but causes the selective line broadening of two of the resonances. The comparison of these data with results of gel retardation assays published previously (1) allows the identification and localization of a kink induced in the DNA by the CAP binding to its specific site on the lac promoter.     

Evidence that modulation requires sequences downstream of the promoters of two vir-repressed genes of Bordetella pertussis.

Gene expression in Bordetella pertussis is altered by environmental signals in a process called antigenic modulation. In the presence of modulating signals, expression of several known virulence factors and outer membrane proteins is coordinately reduced. From a bank of TnphoA fusions, we have identified five genes whose expression profiles are reciprocal of those of the major virulence determinants; that is, alkaline phosphatase activity is maximal during growth in the presence of the modulators nicotinic acid and MgSO4 (S. Knapp and J. J. Mekalanos, J. Bacteriol. 170:5059-5066, 1988). We have called these loci vir-repressed genes (vrg). Two of these gene fusions (vrg-6 and vrg-18) have been cloned in Escherichia coli, returned on low-copy-number plasmids to several strains of B. pertussis, and found to be regulated similarly to the fusions harbored on the chromosome. Deletions of the two vrg promoters were constructed and returned to B. pertussis. Regulation was maintained even when all but 24 nucleotides upstream of the vrg-18 initiation codon and 60 nucleotides upstream of the vrg-6 initiation codon were deleted, suggesting that cis-acting regulatory elements of these genes lie very near or within the coding region. We observed a 21-base palindromic sequence overlapping an 8-base direct repeat within the signal sequence coding region of vrg-6; insertion of a 6-bp linker in this region abolished regulation. These repetitive sequences are also at the site of greatest primary sequence identify between vrg-6 and vrg-18 and correspond to the signal sequence coding region. We propose models that involve recognition of this region by a vir-regulated gene product.     

Partitioning of plasmid R1. Structural and functional analysis of the parA locus

The stability locus, parA+, of plasmid R1 is shown to be localized within a 1500 base-pair region of DNA on the largest EcoRI restriction fragment of plasmid R1. The nucleotide sequence of the region revealed the presence of two open reading frames, one of 320 codons, and another of 60 codons. The larger open reading frame encodes a polypeptide of 36,000 Mr. Deletions covering the promoter distal end of the 36,000 Mr reading frame give rise to synthesis of large amounts of truncated protein. Construction of promoter fusions between the parA+ promoter and the lacZ gene showed that the parA+ region encodes a factor that negatively regulates the expression of the 36,000 Mr protein. The locus exerting parA+-associated incompatibility, denoted incA+, was mapped to a 60 base-pair region covering the parA+ promoter. Most likely, this region is involved both in the negative regulation of the parA+ operon and in the parA+-associated incompatibility. Two explanations are suggested to explain this possible dual function of the parA+ promoter region. The parA+ region was cloned into an unstably inherited (par-) derivative of a mini-F derivative. The low copy number plasmid mini-F devoid of its own partition genes was stabilized more than 100-fold by carrying the parA+ genes. This observation is in accordance with the proposal that the parA+ locus specifies the true partition function of plasmid R1.