Ti plasmid-encoded octopine and nopaline catabolism in Agrobacterium: specificities of the LysR-type regulators OccR and NocR, and protein-induced DNA bending

The occ and noc regions in octopine and nopaline Ti plasmids, respectively, are responsible for the catabolism of octopine and nopaline in Agrobacterium. The functions are activated in the presence of the opines by OccR and NocR, two related regulatory proteins, and the promoters contain common sequence motifs. We have investigated heterologous interactions between the regulators and the promoters. Previous experiments using all possible heterologous combinations of opines, regulators, and promoters in vivo had demonstrated that only the combination of nopalme, NocR, and the occ promoter led to limited promoter activation. We now show that OccR and NocR bind to the heterologous promoters in vitro and in vivo. The weak or non-existent promoter activation actually observed could be explained by the assumption that OccR and NocR use different activation mechanisms; we investigated protein-induced DNA bending because of reports that the two regulators differ in this respect. Analysis with a bending vector showed that both OccR and NocR induced a DNA bend that is relaxed in the presence of the respective opine. The data suggest that subtle differences in regulator/promoter interactions are responsible for the inactivity of the heterologous combinations. Investigations with a chimeric NocR/OccR protein indicated that it induced a DNA bend in both promoters. No opine-induced relaxation was detectable with the hybrid, and the inducible promoter was not activated. These findings suggest that bend relaxation may be an integral part of promoter activation.     

Ti plasmid-encoded octopine and nopaline catabolism in Agrobacterium: specificities of the LysR-type regulators OccR and NocR,and protein-induced DNA bending

The occ and noc regions in octopine and nopaline Ti plasmids, respectively, are responsible for the catabolism of octopine and nopaline in Agrobacterium. The functions are activated in the presence of the opines by OccR and NocR, two related regulatory proteins, and the promoters contain common sequence motifs. We have investigated heterologous interactions between the regulators and the promoters. Previous experiments using all possible heterologous combinations of opines, regulators, and promoters in vivo had demonstrated that only the combination of nopalme, NocR, and the occ promoter led to limited promoter activation. We now show that OccR and NocR bind to the heterologous promoters in vitro and in vivo. The weak or non-existent promoter activation actually observed could be explained by the assumption that OccR and NocR use different activation mechanisms; we investigated protein-induced DNA bending because of reports that the two regulators differ in this respect. Analysis with a bending vector showed that both OccR and NocR induced a DNA bend that is relaxed in the presence of the respective opine. The data suggest that subtle differences in regulator/promoter interactions are responsible for the inactivity of the heterologous combinations. Investigations with a chimeric NocR/OccR protein indicated that it induced a DNA bend in both promoters. No opine-induced relaxation was detectable with the hybrid, and the inducible promoter was not activated. These findings suggest that bend relaxation may be an integral part of promoter activation.     

Opine-regulated promoters and LysR-type regulators in the nopaline (noc) and octopine (occ) catabolic regions of Ti plasmids of Agrobacterium tumefaciens.

Essential steps in the uptake and catabolism of the plant tumor metabolites nopaline and octopine in Agrobacterium spp. are performed by proteins encoded in the nopaline catabolic (noc) and octopine catabolic (occ) regions of Ti plasmids. We investigated the opine activation of the genes by using (i) promoter studies of Agrobacterium spp. and (ii) analysis of the promoter interaction with the regulatory proteins NocR (noc) and OccR (occ). The noc region contained two nopaline-induced promoters (Pi1[noc] and Pi2[noc]) and one autogenously regulated promoter (Pr [control of NocR expression]). Pi2 and Pr overlapped and were divergently oriented (Pi2 [noc]). DNA binding studies and DNase I footprints indicated that NocR bound specifically to single binding sites in Pi1[noc] and Pi2/Pr[noc] and that Pi2 and Pr were regulated from the same binding site. The binding was independent of the inducer nopaline, and nopaline caused small changes in the footprint. The promoters in the noc and occ regions shared sequence motif and contained the sequence T-N11-A, which is characteristic for LysR-type-regulated promoters. The occ region contained one octopine-induced and one autogenously regulated promoter (Pi/Pr[occ]) in the same arrangement as Pi2/Pr[noc] in the noc region. Promoter deletions indicated that sequences flanking the OccR binding site determined the extent of induction, although they did not bind OccR. The promoter bound OccR in the absence and presence of octopine. The opine caused a change in the mobility of the DNA-protein complex with the complete promoter. The resected fragments did not reveal this opine-induced shift, and it was also not detectable with the DNA-NocR complexes with the two promoters of the noc region.     

Repression and catabolite gene activation in the araBAD operon.

Catabolite gene activation of the araBAD operon was examined by using catabolite gene activator protein (CAP) site deletion mutants. A high-affinity CAP-binding site between the divergently orientated araBAD and araC operons has been previously identified by DNase I footprinting techniques. Subsequent experiments disagreed as to whether this site is directly involved in stimulating araBAD expression. In this paper, we present data showing that deletions generated by in vitro mutagenesis of the CAP site led to a five- to sixfold reduction in single-copy araBAD promoter activity in vivo. We concluded that catabolite gene activation of araBAD involves this CAP site. The hypothesis that CAP stimulates the araBAD promoter primarily by relieving repression was then tested. The upstream operator araO2 was required for repression, but we observed that the magnitude of CAP stimulation was unaffected by the presence or absence of araO2. We concluded that CAP plays no role in relieving repression. Other experiments showed that when CAP binds it induces a bend in the ara DNA; similar bending has been reported upon CAP binding to lac DNA. This conformational change in the DNA may be essential to the mechanism of CAP activation.     

A fluorescence stopped-flow kinetic study of the conformational activation of alpha-chymotrypsin and several mutants

The kinetic activation parameters (activation free energy, activation free enthalpy, and activation free entropy change) of the conformational change of alpha-chymotrypsin from an inactive to the active conformation were determined after a pH jump from pH 11.0 to pH 6.8 by the fluorescence stopped-flow method. The conformational change was followed by measuring changes in the protein fluorescence. For the bovine wild-type protein, the same kinetic parameters are obtained as in the study of proflavin binding. Several mutants were made with the goal to accelerate or decelerate this conformational transition. The inspiration for the choice of the mutants came from a previous modelling study done on the bovine wild-type chymotrypsin. The results of the fluorescence stopped flow experiments show that several mutants behaved as was expected based on the information provided by the modeling study on the wild-type variant. For some mutants our assumptions were not correct, and therefore additional modeling studies of the activation pathways of these mutant proteins are necessary to be able to explain the observed kinetic behavior.     

Enhancement of the Potential To Utilize Octopine in the Nonfluorescent Pseudomonas sp. Strain 92

The nonfluorescent Pseudomonas sp. strain 92 requires the presence of a supplementary carbon source for growth on octopine, whereas the spontaneous mutant RB100 has acquired the capacity to utilize this opine as the sole carbon and nitrogen source. Insertional mutagenesis of RB100 with transposon Tn5 generated mutants which were unable to grow on octopine and others which grew slowly on this substrate. Both types of mutants yielded revertants that had regained the ability to utilize octopine. Some of the revertants had lost the transposon, whereas in others the transposon was retained but with rearrangements of the insertion site. Genes of octopine catabolism from strain 92 were cloned on a cosmid vector to generate pK3. The clone pK3 conferred the ability to utilize octopine as the sole carbon and nitrogen source on the host Pseudomonas putida KT2440. Although they conferred an equivalent growth phenotype, the mutant genes carried by RB100 and the cloned genes on pK3 differed in their regulation. Utilization of [¹⁴C]octopine was inducible by octopine in RB100 and was constitutive in KT2440(pK3).