Neurospora crassa is a central organism in the history of twentieth-century genetics, biochemistry and molecular biology. Here, we report a high-quality draft sequence of the N. crassa genome. The approximately 40-megabase genome encodes about 10,000 protein-coding genes-more than twice as many as in the fission yeast Schizosaccharomyces pombe and only about 25% fewer than in the fruitfly Drosophila melanogaster. Analysis of the gene set yields insights into unexpected aspects of Neurospora biology including the identification of genes potentially associated with red light photobiology, genes implicated in secondary metabolism, and important differences in Ca(2+) signalling as compared with plants and animals. Neurospora possesses the widest array of genome defence mechanisms known for any eukaryotic organism, including a process unique to fungi called repeat-induced point mutation (RIP). Genome analysis suggests that RIP has had a profound impact on genome evolution, greatly slowing the creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related genes

We present an analysis of over 1,100 of the ∼10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease

Histidine kinases allow bacteria, plants, and fungi to sense and respond to their environment. The 2.6 A resolution crystal structure of Thermotoga maritima CheA (290-671) histidine kinase reveals a dimer where the functions of dimerization, ATP binding, and regulation are segregated into domains. The kinase domain is unlike Ser/Thr/Tyr kinases but resembles two ATPases, Gyrase B and Hsp90. Structural analogies within this superfamily suggest that the P1 domain of CheA provides the nucleophilic histidine and activating glutamate for phosphotransfer. The regulatory domain, which binds the homologous receptor-coupling protein CheW, topologically resembles two SH3 domains and provides different protein recognition surfaces at each end. The dimerization domain forms a central four-helix bundle about which the kinase and regulatory domains pivot on conserved hinges to modulate transphosphorylation. Different subunit conformations suggest that relative domain motions link receptor response to kinase activity.

The Tar receptor is a transmembrane protein that regulates bacterial chemotaxis in response to changes in the level of aspartic acid in the medium. The extracellular portion of the protein can bind aspartate, and the cytoplasmic portion modulates CheA kinase activity. The receptor can either activate or inhibit the kinase. The cytoplasmic portion of the receptor can be modified by c.rboxymethylation of specific glutamic acid residues. To test the effects of differential methylation on receptor function, we prepared membranes from cells that have specifically modified forms of the receptor and tested the relative ability of each of these forms to activate or In Escherichia coli and Salmonella typhimurium, a variety of chemical stimuli are sensed by any one of four different transmembrane receptors. These proteins are part of a signal transducing system that the cell uses to compare the current level of a specific ligand with the concentration experienced in the recent past and to adjust swimming behavior appropriately. Signal transduction in bacterial chemotaxis involves two highly integrated processes, which have been referred to as excitation and adaptation. The excitation process reflects the instantaneous changes in the state of specific transmembrane receptors. Ligand release from the periplasmic portion of one of these receptors (e.g., the aspartate receptor Tar) results in transmembrane signal transmission, which presumably causes the signaling properties of the cytoplasmic domain of the receptor to change. The cytoplasmic portion of the transmembrane receptor interacts with two soluble proteins, CheA and CheW. The CheA protein kinase is activated in this process and phosphorylates the CheY protein, which acts as a "second messenger" to change the bias of flagellar rotation and thus presumably induces a brief period of tumbling (5, 6). Binding of ligand to receptor can also "inhibit" CheA kinase activity and thus stabilize the direction of flagellar rotation, eliminating episodes of "tumbling" and resulting in longer periods of "smooth" swimming (7).The second process that can be distinguished experimentally is adaptation. It is brought about by action of the CheR and CheB proteins (8-10). CheR catalyzes the S-adenosylmethionine-dependent carboxymethylation of specific glutamic acid residues on the cytoplasmic portion of the receptor. This reaction is reversed by the CheB protein, which catalyzes hydrolysis ofthese methylesters. The Tar chemoreceptor contains four glutamate residues, which are subject to reversible modification. In the newly synthesized transmembrane protein, they are present as two glutamines and two glutamates (Q295, E302, Q309, and E491 of Tar) (11, 12). Deamidation of the glutamines to glutamates is catalyzed by a second enzymatic activity of the CheB protein (11, 13). Activation of the methylesterase activity of CheB results from its phosphorylation by the CheA kinase (14-17). Binding of attractant ligands leads to increased steady-state levels of methylation of the receptors,...

We have used surface plasmon resonance biosensor technology to monitor the assembly and dynamics of a signal transduction complex which controls chemotaxis in Escherichia coli. A quaternary complex formed which consisted of the response regulator CheY, the histidine protein kinase CheA, a coupling protein CheW and a membrane-bound chemoreceptor Tar. Using various experimental conditions and mutant proteins, we have shown that the complex dissociates under conditions that favour phosphorylation of CheY. Direct physical analysis of interactions among proteins in this signal transduction pathway provides evidence for a previously unrecognized binding interaction between the kinase and its substrate. This interaction may be important for enhancing substrate specificity and preventing 'crosstalk' with other systems. The approach is generally applicable to furthering our understanding of how signalling complexes transduce intracellular messages.