The Bacillus subtilis acyl-lipid desaturase (⌬5-Des) is an iron-dependent integral membrane protein, able to selectively introduce double bonds into long chain fatty acids. Structural information on membrane-bound desaturases is still limited, and the present topological information is restricted to hydropathy plots or sequence comparison with the evolutionary related alkane hydroxylase. The topology of ⌬5-Des was determined experimentally in Escherichia coli using a set of nine different fusions of N-terminal fragments of ⌬5-Des with the reporter alkaline phosphatase (⌬5-Des-PhoA). The alkaline phosphatase activities of cells expressing the ⌬5-Des-PhoA fusions, combined with site-directed mutagenesis of His residues identified in most desaturases, suggest that a tripartite motif of His essential for catalysis is located on the cytoplasmic phase of the membrane. These data, together with surface Lys biotinylation experiments, support a model for ⌬5-Des as a polytopic membrane protein with six transmembrane-and one membrane-associated domain, which likely represents a substrate-binding motif. This study provides the first experimental evidence for the topology of a plasma membrane fatty acid desaturase. On the basis of our results and the presently available hydrophobicity profile of many acyl-lipid desaturases, we propose that these enzymes contain a new transmembrane domain that might play a critical role in the desaturation of fatty acids esterified in glycerolipids.The fatty acyl desaturases encompass a family of enzymes, representatives of which are found in all lower eukaryotes, plants and animals (for a review see Ref. 1), and some prokaryotes such as cyanobacteria (2) and bacilli (3), which have the function of introducing double bonds into fatty acyl chains.Although they all utilize molecular oxygen and reducing equivalents obtained from an electron transport chain, and the basic mechanism of the desaturation reaction may be very similar in all cases (4), fatty acid desaturases can be classified into three main subfamilies (1): (i) the soluble acyl carrier protein desaturases that introduce double bonds into fatty acids esterified to acyl carrier protein and are found in the stroma of plant plastids (4); (ii) the acyl-lipid desaturases, which are membranebound enzymes associated with the endoplasmic reticulum (1), the plant chloroplast membrane (5), the cytoplasmic membrane of some bacilli (6), and the plasmatic and thylacoid membranes of cyanobacteria (7) that desaturate fatty acids esterified in glycerolipids; (iii) the acyl-CoA desaturases, which introduce double bonds into fatty acids esterified to CoA (8) and are associated to the endoplasmic reticulum membrane of animals and fungi (1).The soluble and the membrane-bound desaturases show different consensus motifs (for a review, see 4). Database searching for these motifs reveals that they belong to two distinct multifunctional classes of iron-dependent enzymes, each of which includes desaturases, hydroxylases, and epoxidases that act on fatty acids ...

Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0FϳP), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNAbinding domain of ComA, rather than Spo0FϳP, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the aminoterminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms. T he initiation of sporulation in the Gram-positive organismBacillus subtilis is regulated by the complex phosphorelay signal transduction system. In this system, sporulation-activating signals are sensed by multiple sensor histidine kinases whose activation results in autophosphorylation and phosphoryl transfer to an intermediate component, the Spo0F response regulator. From phosphorylated Spo0F (Spo0FϳP), the phosphoryl group is then transferred to the Spo0B phosphotransferase, which then relays it to the Spo0A response regulator and transcription factor. The phosphorylation level of Spo0A in the cell is the determining factor of whether sporulation will initiate or not in response to the activating signals sensed by the kinases (7,15,22,23).In order to counteract the kinase activities and prevent untimely initiation of sporulation, a number of phosphatases exist to respond to physiological states antithetical to sporulation, such as growth and competence for DNA transformation. There are two families of phosphatases, classified by structure and substrate specificity: the Spo0E-like family and the Rap family (30,32).The members of the Spo0E family of phosphatases (Spo0E, YisI, and YnzD) are small proteins (56 to 85 amino acids) identified by a conserved sequence motif, SQ/RE/DLD, in which the aspartate in the fifth position is the essential catalytic residue.

The Poaceae constitute a taxon of flowering plants (grasses) that cover almost all Earth’s inhabitable range and comprises some of the genera most commonly used for human and animal nutrition. Many of these crops have been sequenced, like rice, Brachypodium, maize and, more recently, wheat. Some important members are still considered orphan crops, lacking a sequenced genome, but having important traits that make them attractive for sequencing. Among these traits is apomixis, clonal reproduction by seeds, present in some members of the Poaceae like Eragrostis curvula . A de novo , high-quality genome assembly and annotation for E . curvula have been obtained by sequencing 602 Mb of a diploid genotype using a strategy that combined long-read length sequencing with chromosome conformation capture. The scaffold N50 for this assembly was 43.41 Mb and the annotation yielded 56,469 genes. The availability of this genome assembly has allowed us to identify regions associated with forage quality and to develop strategies to sequence and assemble the complex tetraploid genotypes which harbor the apomixis control region(s). Understanding and subsequently manipulating the genetic drivers underlying apomixis could revolutionize agriculture.

Sporulation is a complex developmental system characterizing