Daniela Fischer

The response regulator RssB controls stability of the sigma(S) subunit of RNA polymerase in Escherichia coli.

The rpoS‐encoded sigma(S) subunit of RNA polymerase is a central regulator in a regulatory network that governs the expression of many stationary phase‐induced and osmotically regulated genes in Escherichia coli. sigma(S) is itself induced under these conditions due to an increase in rpoS transcription (only in rich media) and rpoS translation as well as a stabilization of sigma(S) protein which in growing cells is subject to rapid turnover. We demonstrate here that a response regulator, RssB, plays a crucial role in the control of the cellular sigma(S) content. rssB null mutants exhibit nearly constitutively high levels of sigma(S) and are impaired in the post‐transcriptional growth phase‐related and osmotic regulation of sigma(S). Whereas rpoS translational control is not affected, sigma(S) is stable in rssB mutants, indicating that RssB is essential for sigma(S) turnover. RssB contains a unique C‐terminal output domain and is the first known response regulator involved in the control of protein turnover.

Identification and molecular analysis of glgS, a novel growth‐phase‐regulated and rpoS‐dependent gene involved in glycogen synthesis in Escherichia coli

The putative stationary‐phase sigma factor (σS) encoded by rpoS is essential for giycogen synthesis, but is not required for the transcription of glgC and glgA, which encode ADP‐glucose‐pyrophosphorylase and glycogen synthase, respectively. Using a mini‐Mu random chromosomal library and a screen for glycogen overproduction, we identified a novel gene (glgS) involved in glycogen synthesis. glgS maps at 66.6 min (3247 kb) on the chromosome and constitutes a mono‐cistronic operon. It encodes a hydrophilic and highly charged small protein, with a molecular weight of 7886, which is strongly expressed in minicells. Experiments with single‐copy chromosomal glgS:: lacZ gene fusions indicated that glgS expression is controlled by σS as well as by cAMP. Two transcriptional start sites were mapped in the upstream regulatory region of glgS. The glgS p1 transcript was absent in a cya mutant, whereas an rpoS mutant did not synthesize the glgS p2 transcript. Although glycogen synthesis is strongly stimulated by overproduction of GlgS and is inhibited by a glgS null mutation, glgS does not affect the expression of the glgCAP operon. Its potential role in the metabolic control of glycogen synthesis is discussed. Also, evidence is presented to show that the amount of glycogen accumulated in vivo in early stationary‐phase cells is mainly determined by σS‐controlled gene expression and allosteric activation of GlgC, whereas the absolute levels of expression of glgCAP as well as the intracellular concentration of cAMP are of minor importance.

Regulation of RssB‐dependent proteolysis in Escherichia coli: a role for acetyl phosphate in a response regulator‐controlled process

σS (RpoS) is a highly unstable global regulatory protein in Escherichia coli, whose degradation is inhibited by various stress signals, such as carbon starvation, high osmolarity and heat shock. As a consequence, these stresses result in the induction of σS‐regulated stress‐protective proteins. The two‐component‐type response regulator, RssB, is essential for the rapid proteolysis of σS and is probably involved in the transduction of some of these stress signals. Acetyl phosphate can be used as a phosphodonor for the phosphorylation of various response regulators in vitro and, in the absence of the cognate sensor kinases, acetyl phosphate can also modulate the activities of several response regulators in vivo. Here, we demonstrate increased in vivo half‐lives of σS and the RpoS742::LacZ hybrid protein (also a substrate for RssB‐dependent proteolysis) in acetyl phosphate‐free (pta–ackA) deletion mutants, even though no sensor kinase was eliminated. The in vivo data indicate that acetyl phosphate acts through the response regulator, RssB. In vitro, efficient phosphotransfer from radiolabelled acetyl phosphate to the Asp‐58 residue of RssB (the expected site of phosphorylation in the RssB receiver domain) was observed. Via such phosphorylation, acetyl phosphate may thus modulate RssB activity even in an otherwise wild‐type background. While acetyl phosphate is not essential for the transduction of specific environmental stress signals, it could play the role of a modulator of RssB‐dependent proteolysis that responds to the metabolic status of the cells reflected in the highly variable cellular acetyl phosphate concentration.

Occurrence of glucosylsucrose [α‐D‐glucopyranosyl‐ (1→2)‐α‐D‐glucopyranosyl‐(1→2)‐β‐D‐fructofuranoside] and glucosylated homologues in cyanobacteria

Little is known about the structure and function of oligosaccharides in cyanobacteria. In this study, a new class of saccharides from Nostoc was identified by MS and NMR techniques, consisting of α‐d‐glucopyranosyl‐(1→2)‐[α‐d‐glucopyranosyl‐(1→2)]n‐β‐d‐fructofuranosides ranging from the trisaccharide (n = 1) to decasaccharide (n = 8). In Nostoc ellipsosporum the cell content of saccharides increased 10–20‐fold after heat stress (1 day, 40 °C) or during prolonged cultivation. Under these conditions the abundance of homologues of higher molecular mass (> pentasaccharide) increased and finally exceeded that of homologues of lower molecular mass including sucrose. Total intracellular content of the saccharides after heat stress was 5–10 mg·(g dry weight)−1 corresponding to intracellular concentrations of 0.25–0.5% (w/v). A possible role of the oligosaccharides identified is in the protection of enzymes against heat inactivation. Whereas amylase from Nostoc was only weakly protected by the decasaccharide, α‐amylase from porcine pancreas was more efficiently stabilized by the octasaccharide and decasaccharide. Evidence is presented for the widespread occurrence of the newly identified saccharides in cyanobacteria. The results are discussed including previous reports on cyanobacterial oligosaccharides and with respect to possible functions of these compounds in the living cell.Little is known about the structure and function of oligosaccharides in cyanobacteria. In this study, a new class of saccharides from Nostoc was identified by MS and NMR techniques, consisting of alpha-D-glucopyranosyl-(1-->2)-[alpha-D-glucopyranosyl-(1-->2)]n-beta-D-fructofuranosides ranging from the trisaccharide (n = 1) to decasaccharide (n = 8). In Nostoc ellipsosporum the cell content of saccharides increased 10-20-fold after heat stress (1 day, 40 degrees C) or during prolonged cultivation. Under these conditions the abundance of homologues of higher molecular mass (> pentasaccharide) increased and finally exceeded that of homologues of lower molecular mass including sucrose. Total intracellular content of the saccharides after heat stress was 5-10 mg x (g dry weight)(-1) corresponding to intracellular concentrations of 0.25-0.5% (w/v). A possible role of the oligosaccharides identified is in the protection of enzymes against heat inactivation. Whereas amylase from Nostoc was only weakly protected by the decasaccharide, alpha-amylase from porcine pancreas was more efficiently stabilized by the octasaccharide and decasaccharide. Evidence is presented for the widespread occurrence of the newly identified saccharides in cyanobacteria. The results are discussed including previous reports on cyanobacterial oligosaccharides and with respect to possible functions of these compounds in the living cell.