The S (or RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli. While nearly absent in rapidly growing cells, S is strongly induced during entry into stationary phase and/or many other stress conditions and is essential for the expression of multiple stress resistances. Genome-wide expression profiling data presented here indicate that up to 10% of the E. coli genes are under direct or indirect control of S and that S should be considered a second vegetative sigma factor with a major impact not only on stress tolerance but on the entire cell physiology under nonoptimal growth conditions. This large data set allowed us to unequivocally identify a S consensus promoter in silico. Moreover, our results suggest that S -dependent genes represent a regulatory network with complex internal control (as exemplified by the acid resistance genes). This network also exhibits extensive regulatory overlaps with other global regulons (e.g., the cyclic AMP receptor protein regulon). In addition, the global regulatory protein Lrp was found to affect S and/or 70 selectivity of many promoters. These observations indicate that certain modules of the S -dependent general stress response can be temporarily recruited by stress-specific regulons, which are controlled by other stress-responsive regulators that act together with 70 RNA polymerase. Thus, not only the expression of genes within a regulatory network but also the architecture of the network itself can be subject to regulation.The general stress sigma factor S (or RpoS) is strongly induced when Escherichia coli cells are exposed to various stress conditions, which include starvation, hyperosmolarity, pH downshift, or nonoptimal high or low temperature (for a review of S regulation, see reference 24). By standard genetic and molecular biology methods, more than 80 S -controlled genes have been identified to date, indicating that S is the master regulator of a rather large regulon which represents the genetic basis of the E. coli general stress response (for summaries, see references 23 and 41).In their regulatory patterns, many S -controlled genes just follow the cellular S level; i.e., they are activated whenever S and therefore S -containing RNA polymerase (E S ) accumulate in the cell. Other S -dependent genes, however, exhibit highly specific regulation, with a narrow window of expression only under some sort of stress condition. The best-studied example of this type of S -controlled gene is the csiD gene, which is mainly induced by carbon starvation because the cyclic AMP (cAMP)-cAMP receptor protein (CRP) acts as an essential activator for S -containing RNA polymerase at the csiD promoter (21,46,49). Also, the leucine-responsive regulatory protein (Lrp) is involved in the regulation of certain S -dependent genes (9,13,33,64). These findings indicate that the S -containing RNA polymerase holoenzyme has the ability to cooperate with additional regulatory factors, just as the vegetative RNA polymerase containing 70 does. ...

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate a variety of substrates, ranging from ions to macromolecules, either out of or into the cytosol (hence defined as importers or exporters, respectively). It has been demonstrated that ABC exporters and importers function through a common mechanism involving conformational switches between inwardfacing and outward-facing states; however, the mechanism underlying their functions, particularly substrate recognition, remains elusive. Here we report the structures of an amino acid ABC importer Art(QN) 2 from Thermoanaerobacter tengcongensis composed of homodimers each of the transmembrane domain ArtQ and the nucleotide-binding domain ArtN, either in its apo form or in complex with substrates (Arg, His) and/or ATPs. The structures reveal that the straddling of the TMDs around the twofold axis forms a substrate translocation pathway across the membrane. Interestingly, each TMD has a negatively charged pocket that together create a negatively charged internal tunnel allowing amino acids carrying positively charged groups to pass through. Our structural and functional studies provide a better understanding of how ABC transporters select and translocate their substrates.ABC transporters | substrate selectivity | inward-facing state | two binding sites | type I importer

Previously, [1,3]dioxolo[4,5‐f][1,3]benzodioxole (DBD)‐based fluorophores used as highly sensitive fluorescence lifetime probes reporting on their microenvironmental polarity have been described. Now, a new generation of DBD dyes has been developed. Although they are still sensitive to polarity, in contrast to the former DBD dyes, they have extraordinary spectroscopic properties even in aqueous surroundings. They are characterized by long fluorescence lifetimes (10–20 ns), large Stokes shifts (≈100 nm), high photostabilities, and high quantum yields (>0.56). Here, the spectroscopic properties and synthesis of functionalized derivatives for labeling biological targets are described. Furthermore, thio‐reactive maleimido derivatives of both DBD generations show strong intramolecular fluorescence quenching. This mechanism has been investigated and is found to undergo a photoelectron transfer (PET) process. After reaction with a thiol group, this fluorescence quenching is prevented, indicating successful bonding. Being sensitive to their environmental polarity, these compounds have been used as powerful fluorescence lifetime probes for the investigation of conformational changes in the maltose ATP‐binding cassette transporter through fluorescence lifetime spectroscopy. The differing tendencies of the fluorescence lifetime change for both DBD dye generations promote their combination as a powerful toolkit for studying microenvironments in proteins.

Prokaryotic solute binding protein-dependent ATP-binding cassette import systems are divided into type I and type II and mechanistic differences in the transport process going along with this classification are under intensive investigation. Little is known about the conformational dynamics during the catalytic cycle especially concerning the transmembrane domains. The type I transporter for positively charged amino acids from Salmonella enterica serovar Typhimurium (LAO-HisQMP2) was studied by limited proteolysis in detergent solution in the absence and presence of co-factors including ATP, ADP, LAO/arginine, and Mg(2+) ions. Stable peptide fragments could be obtained and differentially susceptible cleavage sites were determined by mass spectrometry as Lys-258 in the nucleotide-binding subunit, HisP, and Arg-217/Arg-218 in the transmembrane subunit, HisQ. In contrast, transmembrane subunit HisM was gradually degraded but no stable fragment could be detected. HisP and HisQ were equally resistant under pre- and post-hydrolysis conditions in the presence of arginine-loaded solute-binding protein LAO and ATP/ADP. Some protection was also observed with LAO/arginine alone, thus reflecting binding to the transporter in the apo-state and transmembrane signaling. Comparable digestion patterns were obtained with the transporter reconstituted into proteoliposomes and nanodiscs. Fluorescence lifetime spectroscopy confirmed the change of HisQ(R218) to a more apolar microenvironment upon ATP binding and hydrolysis. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. Together, our data suggest similar conformational changes during the transport cycle as described for the maltose ABC transporter of Escherichia coli, despite distinct structural differences between both systems.

SummaryLon protease is a major protease in cellular protein quality control, but also plays an important regulatory role by degrading various naturally unstable regulators. Here, we traced additional such regulators by identifying regulons with co-ordinately altered expression in a lon mutant by genome-wide transcriptional profiling. Besides many members of the RcsA regulon (which validates our approach as RcsA is a known Lon substrate), many genes of the s S -dependent general stress response were upregulated in the lon mutant. However, the lon mutation did not affect s S levels nor s S activity in general, suggesting specific effects of Lon on secondary regulators involved in the control of subsets of s S -controlled genes. Lon-affected genes also included the major acid resistance genes (gadA, gadBC, gadE, hdeAB and hdeD), which led to the discovery that the essential acid resistance regulator GadE (whose expression is s S -controlled) is degraded in vivo in a Londependent manner. GadE proteolysis is constitutive as it was observed even under conditions that induce the system (i.e. at low pH or during entry into stationary phase). GadE degradation was found to rapidly terminate the acid resistance response upon shift back to neutral pH and to avoid overexpression of acid resistance genes in stationary phase.