Over-expression of stress protein-encoding genes helps Clostridium acetobutylicum to rapidly adapt to butanol stress.

The toxicity of n-butanol in microbial fermentations limits its formation. The stress response of Clostridium acetobutylicum involves various stress proteins and therefore, over-expression of genes encoding stress proteins constitutes an option to improve solvent tolerance. Over-expression of groESL, grpE and htpG, significantly improved butanol tolerance of C. acetobutylicum. Whereas the wild type and vector control strain did not survive 2 % (v/v) butanol for 2 h, the recombinant strains showed 45 % (groESL), 25 % (grpE) and 56 % (htpG), respectively, of the initial c.f.u. after 2 h of butanol exposure. As previously, over-expression of groESL led to higher butanol production rates, but the novel strains over-expressing grpE or htpG produced only 51 and 68 %, respectively, of the wild type butanol concentrations after 72 h clearly differentiating butanol tolerance and production. Not only butanol tolerance but also the adaptation to butanol in successive stress experiments was significantly facilitated by increased levels of GroESL, GrpE and HtpG. Re-transformation and sequence analyses of the plasmids confirmed that not the plasmids, but the host cells evolved to a more robust phenotype.

Structural basis for the cooperation of Hsp70 and Hsp110 chaperones in protein folding.

Protein folding by Hsp70 is tightly controlled by cochaperones, including J-domain proteins that trigger ATP hydrolysis and nucleotide exchange factors (NEFs) that remove ADP from Hsp70. Here we present the crystal structure of the yeast NEF Sse1p (Hsp110) in complex with the nucleotide-binding domain (NBD) of Hsp70. Hsp110 proteins are homologous to Hsp70s and consist of an NBD, a beta sandwich domain, and a three helix bundle domain (3HBD). In the complex, the NBD of Sse1p is ATP bound, and together with the 3HBD it embraces the NBD of Hsp70, inducing opening and the release of bound ADP from Hsp70. Mutations that abolish NEF activity are lethal, thus defining nucleotide exchange on Hsp70 as an essential function of Sse1p. Our data suggest that Sse1p does not employ the nucleotide-dependent allostery and peptide-binding mode of canonical Hsp70s, and that direct interactions of substrate with Sse1p may support Hsp70-assisted protein folding in a cooperative process.

Fes1p acts as a nucleotide exchange factor for the ribosome-associated molecular chaperone Ssb1p.

The HspBP1 homolog Fes1p was recently identified as a nucleotide exchange factor (NEF) of Ssa1p, a canonical Hsp70 molecular chaperone in the cytosol of Saccharomyces cerevisiae. Besides the Ssa-type Hsp70s, the yeast cytosol contains three additional classes of Hsp70, termed Ssb, Sse and Ssz. Here, we show that Fes1p also functions as NEF for the ribosome-bound Ssb Hsp70s. Sequence analysis indicated that residues important for interaction with Fes1p are highly conserved in Ssa1p and Ssb1p, but not in Sse1p and Ssz1p. Indeed, Fes1p interacts with Ssa1p and Ssb1p with similar affinity, but does not form a complex with Sse1p. Functional analysis showed that Fes1p accelerates the release of the nucleotide analog MABA-ADP from Ssb1p by a factor of 35. In contrast to the interaction between mammalian HspBP1 and Hsp70, however, addition of ATP only moderately decreases the affinity of Fes1p for Ssb1p. Point mutations in Fes1p abolishing complex formation with Ssa1p also prevent the interaction with Ssb1p. The ATPase activity of Ssb1p is stimulated by the ribosome-associated complex of Zuotin and Ssz1p (RAC). Interestingly, Fes1p inhibits the stimulation of Ssb1p ATPase by RAC, suggesting a complex regulatory role of Fes1p in modulating the function of Ssb Hsp70s in co-translational protein folding.

Molecular chaperones of the Hsp110 family act as nucleotide exchange factors of Hsp70s.

Hsp70 molecular chaperones function in protein folding in a manner dependent on regulation by co-chaperones. Hsp40s increase the low intrinsic ATPase activity of Hsp70, and nucleotide exchange factors (NEFs) remove ADP after ATP hydrolysis, enabling a new Hsp70 interaction cycle with non-native protein substrate. Here, we show that members of the Hsp70-related Hsp110 family cooperate with Hsp70 in protein folding in the eukaryotic cytosol. Mammalian Hsp110 and the yeast homologues Sse1p/2p catalyze efficient nucleotide exchange on Hsp70 and its orthologue in Saccharomyces cerevisiae, Ssa1p, respectively. Moreover, Sse1p has the same effect on Ssb1p, a ribosome-associated isoform of Hsp70 in yeast. Mutational analysis revealed that the N-terminal ATPase domain and the ultimate C-terminus of Sse1p are required for nucleotide exchange activity. The Hsp110 homologues significantly increase the rate and yield of Hsp70-mediated re-folding of thermally denatured firefly luciferase in vitro. Similarly, deletion of SSE1 causes a firefly luciferase folding defect in yeast cells under heat stress in vivo. Our data indicate that Hsp110 proteins are important components of the eukaryotic Hsp70 machinery of protein folding.

Demasking biological oscillators: properties and principles of entrainment exemplified by the Neurospora circadian clock.

Oscillations are found throughout the physical and biological worlds. Their interactions can result in a systematic process of synchronization called entrainment, which is distinct from a simple stimulus-response pattern. Oscillators respond to stimuli at some times in their cycle and may not respond at others. Oscillators can also be driven if the stimulus is strong (or if the oscillator is weak); i.e., they restart their cycle every time they receive a stimulus. Stimuli can also directly affect rhythms without entraining the underlying oscillator (masking): Drivenness and masking are often difficult to distinguish. Here we use the circadian biological clock to explore properties of entrainment. We confirm previous results showing that the residual circadian system in Neurospora can be entrained in a mutant of the clock gene frequency (frq(9), a strain deficient in producing a functional FRQ protein). This finding has implications for understanding the evolution of circadian programs. By comparing data sets from independent studies, we develop a template for analyzing, modeling, and dissecting the interactions of entrained and masked components. These insights can be applied to oscillators of all periodicities.

Regulation of Hsp70 function by HspBP1: structural analysis reveals an alternate mechanism for Hsp70 nucleotide exchange.

HspBP1 belongs to a family of eukaryotic proteins recently identified as nucleotide exchange factors for Hsp70. We show that the S. cerevisiae ortholog of HspBP1, Fes1p, is required for efficient protein folding in the cytosol at 37 degrees C. The crystal structure of HspBP1, alone and complexed with part of the Hsp70 ATPase domain, reveals a mechanism for its function distinct from that of BAG-1 or GrpE, previously characterized nucleotide exchange factors of Hsp70. HspBP1 has a curved, all alpha-helical fold containing four armadillo-like repeats unlike the other nucleotide exchange factors. The concave face of HspBP1 embraces lobe II of the ATPase domain, and a steric conflict displaces lobe I, reducing the affinity for nucleotide. In contrast, BAG-1 and GrpE trigger a conserved conformational change in lobe II of the ATPase domain. Thus, nucleotide exchange on eukaryotic Hsp70 occurs through two distinct mechanisms.

Entrainment dissociates transcription and translation of a circadian clock gene in neurospora.

Circadian systems coordinate the daily sequence of events in cells, tissues, and organisms. In constant conditions, the biological clock oscillates with its endogenous period, whereas it is synchronized to the 24 hr light:dark cycle in nature. Here, we investigate light entrainment of Neurospora crassa to photoperiods that mimic seasonal changes. Clock gene (frequency, or frq) RNA levels directly reflect the light environment in all photoperiods, whereas the FRQ protein follows neither RNA levels nor light transitions. Induction of frq RNA and protein can be dissociated by as much as 6 hr, depending on photoperiod. The phase of entrainment at the physiological level (e.g., asexual spore development) correlates with FRQ protein. Thus, a dissociation of transcription, translation, and protein stability is fundamental to circadian entrainment of Neurospora. Our findings suggest that simple feedback models are insufficient to explain the molecular circadian mechanisms under entrained conditions and that clock control of light input pathways involves posttranscriptional regulation. The regulators mediating the dissociation between RNA and protein levels are still unknown and will be the key to understanding both circadian timing at the molecular level and how the clock exerts control over many cellular processes.

Combining theoretical and experimental approaches to understand the circadian clock.

This review is intended as a summary of our work carried out as part of the German Research Association (DFG) Center Program on Circadian Rhythms. Over the last six years, our approach to understanding circadian systems combined theoretical and experimental tools, and Gonyaulax and Neurospora have proven ideal for these efforts. Both of these model organisms demonstrate that even simple circadian systems can have multiple light input pathways and more than one rhythm generator. They have both been used to elaborate basic circadian features in conjunction with formal models. The models introduce the "zeitnehmer," i.e., a clock-regulated input pathway, to the conceptual framework of circadian systems, and proposes networks of individual feedbacks as the basis for circadian rhythmicity.

Light reception and circadian behavior in 'blind' and 'clock-less' mutants of Neurospora crassa.

The filamentous fungus Neurospora crassa is a model organism for the genetic dissection of blue light photoreception and circadian rhythms. WHITE COLLAR-1 (WC-1) and WC-2 are considered necessary for all light responses, while FREQUENCY (FRQ) is required for light-regulated asexual development (conidia formation); without any of the three, self-sustained (circadian) rhythmicity in constant conditions fails. Here we show that light-regulated and self-sustained development occur in the individual or mutant white collar strains. These strains resemble wild type in their organization of the daily bout of light-regulated conidiation. Molecular profiles of light- induced genes indicate that the individual white collar-1 and white collar-2 mutants utilize distinct pathways, despite their similar appearance in all aspects. Titration of fluence rate also demonstrates different light sensitivities between the two strains. The data require the existence of an as-yet-unidentified photoreceptor. Furthermore, the extant circadian clock machinery in these mutant strains supports the notion that the circadian system in Neurospora involves components outside the WC-FRQ loop.