Anoxygenic photosynthetic growth of Rhodobacter sphaeroides, a member of the ␣ subclass of the class Proteobacteria, requires the response regulator PrrA. PrrA and the sensor kinase PrrB are part of a twocomponent signaling pathway that influences a wide range of processes under oxygen-limited conditions. In this work we characterized the pathway of transcription activation by PrrB and PrrA by purifying these proteins, analyzing them in vitro, and characterizing a mutant PrrA protein in vivo and in vitro. When purified, a soluble transmitter domain of PrrB (cPrrB) could autophosphorylate, rapidly transfer phosphate to PrrA, and stimulate dephosphorylation of phospho-PrrA. Unphosphorylated PrrA activated transcription from a target cytochrome c 2 gene (cycA) promoter, P2, which contained sequences from ؊73 to ؉22 relative to the transcription initiation site. However, phosphorylation of PrrA increased its activity since activation of cycA P2 was enhanced up to 15-fold by treatment with the low-molecular-weight phosphodonor acetyl phosphate. A mutant PrrA protein containing a single amino acid substitution in the presumed phosphoacceptor site (PrrA-D63A) was not phosphorylated in vitro but also was not able to stimulate cycA P2 transcription. PrrA-D63A also had no apparent in vivo activity, demonstrating that aspartate 63 is necessary both for the function of PrrA and for its phosphorylation-dependent activation. The cellular level of wild-type PrrA was negatively autoregulated so that less PrrA was present in the absence of oxygen, conditions in which the activities of many PrrA target genes increase. PrrA-D63A failed to repress expression of the prrA gene under anaerobic conditions, suggesting that this single amino acid change also eliminated PrrA function in vivo.

The existence of a global gene regulatory system in the hyperthermophilic archaeon Sulfolobus solfataricus is described. The system is responsive to carbon source quality and acts at the level of transcription to coordinate synthesis of three physically unlinked glycosyl hydrolases implicated in carbohydrate utilization. The specific activities of three enzymes, an α-glucosidase (malA), a β-glycosidase (lacS), and an α-amylase, were reduced 4-, 20-, and 10-fold, respectively, in response to the addition of supplementary carbon sources to a minimal sucrose medium. Western blot analysis using anti-α-glucosidase and anti-β-glycosidase antibodies indicated that reduced enzyme activities resulted exclusively from decreased enzyme levels. Northern blot analysis of malA and lacSmRNAs revealed that changes in enzyme abundance arose primarily from reductions in transcript concentrations. Culture conditions precipitating rapid changes in lacS gene expression were established to determine the response time of the regulatory system in vivo. Full induction occurred within a single generation whereas full repression occurred more slowly, requiring nearly 38 generations. SincelacS mRNA abundance changed much more rapidly in response to a nutrient down shift than to a nutrient up shift, transcript synthesis rather than degradation likely plays a role in the regulatory response.

The hyperthermophilic archaeon Sulfolobus solfataricus employs a catabolite repression-like regulatory system to control enzymes involved in carbon and energy metabolism. To better understand the basis of this system, spontaneous glycosyl hydrolase mutants were isolated using a genetic screen for mutations, which reduced expression of the lacS gene. The specific activities of three glycosyl hydrolases, including an α-glucosidase (malA), a β-glycosidase (lacS), and the major secreted α-amylase, were measured in the mutant strains using enzyme activity assays, Western blot analysis, and Northern blot analysis. On the basis of these results the mutants were divided into two classes. Group I mutants exhibited a pleiotropic defect in glycosyl hydrolase expression, while a single group II mutant was altered only in lacS expression. PCR, Southern blot analysis, comparative heterologous expression in Escherichia coli, and DNA sequence analysis excluded cis-acting mutations as the explanation for reduced lacS expression in group I mutants. In contrast lacS and flanking sequences were deleted in the group II mutant. Revertants were isolated from group I mutants using a lacS-specific screen and selection. These revertants were pleiotropic and restored glycosyl hydrolase activity either partially or completely to wild-type levels as indicated by enzyme assays and Western blots. The lacS mutation in the group II mutant, however, was nonrevertible. The existence of group I mutants and their revertants reveals the presence of a trans-acting transcriptional regulatory system for glycosyl hydrolase expression.