An Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus is negatively autoregulated.

The archaeal transcriptional initiation machinery closely resembles core elements of the eukaryal polymerase II system. However, apart from the established basal archaeal transcription system, little is known about the modulation of gene expression in archaea. At present, no obvious eukaryal-like transcriptional regulators have been identified in archaea. Instead, we have previously isolated an archaeal gene, the Pyrococcus furiosus lrpA, that potentially encodes a bacterial-like transcriptional regulator. In the present study, we have for the first time addressed the actual involvement of an archaeal Lrp homologue in transcription modulation. For that purpose, we have produced LrpA in Escherichia coli. In a cell-free P. furiosus transcription system we used wild-type and mutated lrpA promoter fragments to demonstrate that the purified LrpA negatively regulates its own transcription. In addition, gel retardation analyses revealed a single protein-DNA complex, in which LrpA appeared to be present in (at least) a tetrameric conformation. The location of the LrpA binding site was further identified by DNaseI and hydroxyl radical footprinting, indicating that LrpA binds to a 46-base pair sequence that overlaps the transcriptional start site of its own promoter. The molecular basis of the transcription inhibition by LrpA is discussed.

Structural and functional analysis of the phosphoenolpyruvate carboxylase gene from the purple nonsulfur bacterium Rhodopseudomonas palustris No. 7.

The ppc gene, encoding phosphoenolpyruvate carboxylase (PEPC), from Rhodopseudomonas palustris No. 7 was cloned and sequenced. Primer extension analysis identified a transcriptional start site 42 bp upstream of the ppc initiation codon. An R. palustris No. 7 PEPC-deficient strain showed a slower doubling time compared with the wild-type strain either anaerobically in the light or aerobically in the dark, when pyruvate was used as a carbon source.

The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene.

In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIA(Glc), a component of the phosphotransferase system for glucose transport. In strains deficient in EnzymeIIA(Glc), cAMP levels are very low. Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIA(Glc). In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIA(Glc) were obtained. The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end. Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIA(Glc). In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished. This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose. Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain.

Role of the phosphotransferase system in Escherichia coli strains deficient in hexose phosphate transport.

In Escherichia coli, glucose 6-phosphate is transported via the Uhp system which is inducible by glucose 6-phosphate. We showed that, in a uhp-deficient strain, glucose 6-phosphate was dephosphorylated in the periplasm and that the resulting glucose was subsequently transported into the cells via the phosphotransferase system. The uptake of glucose generated from glucose 6-phosphate allowed the bacteria to produce an increased level of cAMP compared to cells grown on non-limiting concentrations of glucose.

Structural and functional relationships between Pasteurella multocida and enterobacterial adenylate cyclases.

The Pasteurella multocida adenylate cyclase gene has been cloned and expressed in Escherichia coli. The primary structure of the protein (838 amino acids) deduced from the corresponding nucleotide sequence was compared with that of E. coli. The two enzymes have similar molecular sizes and, based on sequence conservation at the protein level, are likely to be organized in two functional domains: the amino-terminal catalytic domain and the carboxy-terminal regulatory domain. It was shown that P. multocida adenylate cyclase synthesizes increased levels of cyclic AMP in E. coli strains deficient in the catabolite gene activator protein compared with wild-type strains. This increase does not occur in strains deficient in both the catabolite gene activator protein and enzyme III-glucose, indicating that a protein similar to E. coli enzyme III-glucose is involved in the regulation of P. multocida adenylate cyclase. It also indicates that the underlying process leading to enterobacterial adenylate cyclase activation has been conserved through evolution.