Franz Narberhaus

Negative regulation of bacterial heat shock genes

The expression of eubacterial heat shock genes is efficiently controlled at the transcriptional level by both positive and negative mechanisms. Positive control operates by the use of alternative sigma factors that target RNA polymerase to heat shock gene promoters. Alternatively, bacteria apply repressor‐dependent mechanisms, in which transcription of heat shock genes is initiated from a classical housekeeping promoter and cis‐acting DNA elements are used in concert with a cognate repressor protein to limit transcription under physiological conditions. Eight examples of negative regulation will be presented, among them the widespread CIRCE/HrcA system and the control by HspR in Streptomyces. Both mechanisms are designed to permit simple feedback control at the level of gene expression. Many bacteria have established sophisticated regulatory networks, often combining positive and negative mechanisms, in order to allow fine‐tuned heat shock gene expression in an environmentally responsive way.

Bacterial RNA thermometers: molecular zippers and switches

Bacteria use complex strategies to coordinate temperature-dependent gene expression. Many genes encoding heat shock proteins and virulence factors are regulated by temperature-sensing RNA sequences, known as RNA thermometers (RNATs), in their mRNAs. For these genes, the 5′ untranslated region of the mRNA folds into a structure that blocks ribosome access at low temperatures. Increasing the temperature gradually shifts the equilibrium between the closed and open conformations towards the open structure in a zipper-like manner, thereby increasing the efficiency of translation initiation. Here, we review the known molecular principles of RNAT action and the hierarchical RNAT cascade in Escherichia coli. We also discuss RNA-based thermosensors located upstream of cold shock and other genes, translation of which preferentially occurs at low temperatures and which thus operate through a different, more switch-like mechanism. Finally, we consider the potential biotechnological applications of natural and synthetic RNATs.

Molecular basis for temperature sensing by an RNA thermometer

Regulatory RNA elements, like riboswitches, respond to intracellular signals by three‐dimensional (3D) conformational changes. RNA thermometers employ a similar strategy to sense temperature changes in the cell and regulate the translational machinery. We present here the first 3D NMR structure of the functional domain of a highly conserved bacterial RNA thermometer containing the ribosome binding site that remains occluded at normal temperatures (30°C). We identified a region adjacent to the Shine–Dalgarno sequence that has a network of weak hydrogen bonds within the RNA helix. With the onset of heat shock at 42°C, destabilisation of the RNA structure initiates at this region and favours the release of the ribosome binding site and of the start codon. Deletion of a highly conserved G residue leads to the formation of a stable regular RNA helix that loses thermosensing ability. Our results indicate that RNA thermometers are able to sense temperature changes without the aid of accessory factors.

FourU: a novel type of RNA thermometer in Salmonella.

The translation of many heat shock and virulence genes is controlled by RNA thermometers. Usually, they are located in the 5′‐untranslated region (5′‐UTR) and block the Shine‐Dalgarno (SD) sequence by base pairing. Destabilization of the structure at elevated temperature permits ribosome binding and translation initiation. We have identified a new type of RNA thermometer in the 5′‐UTR of the Salmonella agsA gene, which codes for a small heat shock protein. Transcription of the agsA gene is controlled by the alternative sigma factor σ32. Additional translational control depends on a stretch of four uridines that pair with the SD sequence. Mutations in this region affect translation in vivo. Structure probing experiments demonstrate a temperature‐controlled opening of the SD region in vitro. Toeprinting (primer extension inhibition) shows that ribosome binding is dependent on high temperatures. Together with a postulated RNA thermometer upstream of the Yersinia pestis virulence gene lcrF (virF), the 5′‐UTR of Salmonella agsA might be the founding member of a new class of RNA thermometers that we propose to name ‘fourU’ thermometers.

Concerted Actions of a Thermo-labile Regulator and a Unique Intergenic RNA Thermosensor Control Yersinia Virulence

Expression of all Yersinia pathogenicity factors encoded on the virulence plasmid, including the yop effector and the ysc type III secretion genes, is controlled by the transcriptional activator LcrF in response to temperature. Here, we show that a protein- and RNA-dependent hierarchy of thermosensors induce LcrF synthesis at body temperature. Thermally regulated transcription of lcrF is modest and mediated by the thermo-sensitive modulator YmoA, which represses transcription from a single promoter located far upstream of the yscW-lcrF operon at moderate temperatures. The transcriptional response is complemented by a second layer of temperature-control induced by a unique cis-acting RNA element located within the intergenic region of the yscW-lcrF transcript. Structure probing demonstrated that this region forms a secondary structure composed of two stemloops at 25°C. The second hairpin sequesters the lcrF ribosomal binding site by a stretch of four uracils. Opening of this structure was favored at 37°C and permitted ribosome binding at host body temperature. Our study further provides experimental evidence for the biological relevance of an RNA thermometer in an animal model. Following oral infections in mice, we found that two different Y. pseudotuberculosis patient isolates expressing a stabilized thermometer variant were strongly reduced in their ability to disseminate into the Peyers patches, liver and spleen and have fully lost their lethality. Intriguingly, Yersinia strains with a destabilized version of the thermosensor were attenuated or exhibited a similar, but not a higher mortality. This illustrates that the RNA thermometer is the decisive control element providing just the appropriate amounts of LcrF protein for optimal infection efficiency.

Two different stator systems drive a single polar flagellum in Shewanella oneidensis MR-1.

The Gram‐negative metal ion‐reducing bacterium Shewanella oneidensis MR‐1 is motile by means of a single polar flagellum. We identified two potential stator systems, PomAB and MotAB, each individually sufficient as a force generator to drive flagellar rotation. Physiological studies indicate that PomAB is sodium‐dependent while MotAB is powered by the proton motive force. Flagellar function mainly depends on the PomAB stator; however, the presence of both stator systems under low‐sodium conditions results in a faster swimming phenotype. Based on stator homology analysis we speculate that MotAB has been acquired by lateral gene transfer as a consequence of adaptation to a low‐sodium environment. Expression analysis at the single cell level showed that both stator systems are expressed simultaneously. An active PomB–mCherry fusion protein effectively localized to the flagellated cell pole in 70–80% of the population independent of sodium concentrations. In contrast, polar localization of MotB–mCherry increased with decreasing sodium concentrations. In the absence of the Pom stator, MotB–mCherry localized to the flagellated cell pole independently of the sodium concentration but was rapidly displaced upon expression of PomAB. We propose that selection of the stator occurs at the level of protein localization in response to sodium concentrations.

Structure and function of the bacterial AAA protease FtsH

Proteolysis of regulatory proteins or key enzymes of biosynthetic pathways is a universal mechanism to rapidly adjust the cellular proteome to particular environmental needs. Among the five energy-dependent AAA(+) proteases in Escherichia coli, FtsH is the only essential protease. Moreover, FtsH is unique owing to its anchoring to the inner membrane. This review describes the structural and functional properties of FtsH. With regard to its role in cellular quality control and regulatory circuits, cytoplasmic and membrane substrates of the FtsH protease are depicted and mechanisms of FtsH-dependent proteolysis are discussed.

Chaperone activity and homo- and hetero-oligomer formation of bacterial small heat shock proteins.

Rhizobia are the only bacteria known to induce a multitude of small heat shock proteins (sHsps) upon temperature upshift. The sHsps of Bradyrhizobium japonicum fall into two different classes, class A and class B. Here, we studied the chaperone activity and oligomeric features of two representative members of each class. The purified sHsps were efficient chaperones, as demonstrated by their ability to prevent thermally induced aggregation of citrate synthase in vitro. Homo-oligomer formation of all four sHsps was demonstrated by gel filtration and by two independent co-purification approaches. Mixed oligomers were readily observed between members of the same class, even when these proteins originated from different species such as Escherichia coliand B. japonicum. The chaperone activity of purified hetero-oligomers was indistinguishable from the activity of homo-oligomers. Heteromeric complexes were never obtained between class A and class B sHsps, indicating that hetero-oligomer formation is restricted to sHsps of the same class.

Temperature-controlled structural alterations of an RNA thermometer.

Thermoresponsive structures in the 5′-untranslated region of mRNA are known to control translation of heat shock and virulence genes. Expression of many rhizobial heat shock genes is regulated by a conserved sequence element called ROSE for repression of heat shock gene expression. This cis-acting, untranslated mRNA is thought to prevent ribosome access at low temperature through an extended secondary structure, which partially melts when the temperature rises. We show here by a series of in vivo and in vitro approaches that ROSE is a sensitive thermometer responding in the physiologically relevant temperature range between 30 and 40 °C. Point mutations predicted to disrupt base pairing enhanced expression at 30 °C. Compensatory mutations restored repression, emphasizing the importance of secondary structures in the sensory RNA. Only moderate inducibility of a 5′-truncated ROSE variant suggests that interactions between individual stem loops coordinate temperature sensing. In the presence of a complementary oligonucleotide, the functionally important stem loop of ROSE was rendered susceptible to RNase H treatment at heat shock temperatures. Since major structural rearrangements were not observed during UV and CD spectroscopy, subtle structural changes involving the Shine-Dalgarno sequence are proposed to mediate translational control. Temperature perception by the sensory RNA is an ordered process that most likely occurs without the aid of accessory factors.

Direct observation of the temperature-induced melting process of the Salmonella fourU RNA thermometer at base-pair resolution

In prokaryotes, RNA thermometers regulate a number of heat shock and virulence genes. These temperature sensitive RNA elements are usually located in the 5′-untranslated regions of the regulated genes. They repress translation initiation by base pairing to the Shine–Dalgarno sequence at low temperatures. We investigated the thermodynamic stability of the temperature labile hairpin 2 of the Salmonella fourU RNA thermometer over a broad temperature range and determined free energy, enthalpy and entropy values for the base-pair opening of individual nucleobases by measuring the temperature dependence of the imino proton exchange rates via NMR spectroscopy. Exchange rates were analyzed for the wild-type (wt) RNA and the A8C mutant. The wt RNA was found to be stabilized by the extraordinarily stable G14–C25 base pair. The mismatch base pair in the wt RNA thermometer (A8–G31) is responsible for the smaller cooperativity of the unfolding transition in the wt RNA. Enthalpy and entropy values for the base-pair opening events exhibit linear correlation for both RNAs. The slopes of these correlations coincide with the melting points of the RNAs determined by CD spectroscopy. RNA unfolding occurs at a temperature where all nucleobases have equal thermodynamic stabilities. Our results are in agreement with a consecutive zipper-type unfolding mechanism in which the stacking interaction is responsible for the observed cooperativity. Furthermore, remote effects of the A8C mutation affecting the stability of nucleobase G14 could be identified. According to our analysis we deduce that this effect is most probably transduced via the hydration shell of the RNA.