Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among Ϸ4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

The unusual heme distal site structure observed shows that previously undescribed molecular mechanisms of ligand stabilization are operative in VtHb. The polypeptide chain disorder observed in the CE region indicates a potential site of interaction with the FAD/NADH reductase partner, in analogy with observations in the chimeric flavohemoglobin from Alcaligenes eutrophus.

form an intersubunit salt bridge. The mutants R292D and D297R are totally inactive. The crystal structure of R292D reveals that the mutant enzyme retains the T-state quaternary structure. However, the mutation induces a reorganization of the interface with the creation of a network of interactions similar to that observed in the crystal structures of R-state yeast and M1 PK proteins. Furthermore, in the R292D structure, two loops that are part of the active site are disordered. The K382Q and R431E mutations were designed to probe the binding site for fructose 1,6-bisphosphate, the allosteric activator. R431E exhibits only slight changes in the regulatory properties. Conversely, K382Q displays a highly altered responsiveness to the activator, suggesting that Lys 382 is involved in both activator binding and allosteric transition mechanism. Taken together, these results support the notion that domain interfaces are critical for the allosteric transition. They couple changes in the tertiary and quaternary structures to alterations in the geometry of the fructose 1,6-bisphosphate and substrate binding sites. These site-directed mutagenesis data are discussed in the light of the molecular basis for the hereditary nonspherocytic hemolytic anemia, which is caused by mutations in human erythrocyte PK gene. ) for its activity. The reaction is essentially irreversible under physiological conditions and is critical for the control of the metabolic flux in the second part of glycolysis. Moreover, the substrate PEP and the product pyruvate are involved in a variety of metabolic pathways. Such a central position in the cellular metabolism is reflected in the regulatory properties of PK, which is a typical allosteric protein (1). The activity is controlled by several physiological effectors, including H ϩ , Mg 2ϩ

No abstract

The gene coding for amylase (EC.3.2.1.1) has been isolated and sequenced from Bacillus subtilis by cloning in lambda Charon4A and pBR322. The entire coding sequence and large preceding and following regions, comprising the presumed transcriptional and translational regulatory regions, were sequenced. The coding sequence shows a large open reading frame with a translated molecular weight of 72,800 and a presumed signal sequence of approximately thirty-two amino acids. When the intact gene is present in Escherichia coli, it confers the ability to degrade starch, indicating that the gene is expressed in a functional state.

Bacillus subtilis implements several adaptive strategies to cope with nutrient limitation experienced at the end of exponential growth. The DegS-DegU two-component system is part of the network involved in the regulation of postexponential responses, such as competence development, the production of exoenzymes, and motility. The degU32(Hy) mutation extends the half-life of the phosphorylated form of DegU (DegU-P); this in turn increases the production of alkaline protease, levan-sucrase, and other exoenzymes and inhibits motility and the production of flagella. The expression of the flagellum-specific sigma factor SigD, of the flagellin gene hag, and of the fla-che operon is strongly reduced in a degU32(Hy) genetic background. To investigate the mechanism of action of DegU-P on motility, we isolated mutants of degU32(Hy) that completely suppressed the motility deficiency. The mutations were genetically mapped and characterized by PCR and sequencing. Most of the mutations were found to delete a transcriptional termination signal upstream of the main flagellar operon, fla-che, thus allowing transcriptional readthrough from the cod operon. Two additional mutations improved the A -dependent promoter sequence of the fla-che operon. Using an electrophoretic mobility shift assay, we have demonstrated that purified DegU binds specifically to the P A promoter region of the fla-che operon. The data suggest that DegU represses transcription of the fla-che operon, and they indicate a central role of the operon in regulating the synthesis and assembly of flagella.Swimming motility in bacteria depends upon the presence on the cell surface of the flagellar organelle, composed of the basal body, the hook, and the filament. The production of flagella is of such adaptive value that most bacterial species are endowed with flagella, despite the high energy requirement for the synthesis of the numerous flagellin monomers that are necessary to build and maintain the flagellar filament.In enterobacteria, the genes involved in flagellar formation are organized into regulons which are arranged into three hierarchical classes. The first class is constituted by the flhDC master operon, whose expression is necessary to turn on class II genes, coding for components of the export machinery and for the hook and basal body. The class II gene fliA encodes 28 , the transcription factor for the class III genes, which include flagellar filament structural genes and the chemotaxis signal transduction system (7,19). In addition, many global regulators, such as CAP, H-NS, H-HU, Lrp, etc., have been reported to affect flagellar synthesis and assembly (5, 13, 24, 34).In Bacillus subtilis, a bona fide master operon is missing and all genes corresponding to the enteric class II are clustered in a single fla-che operon. The expression of the operon depends upon a A -recognized promoter (fla-che P A ), with an additional D -dependent promoter (P D-3 ) playing a minor role (1, 9). Deletion of the fla-che P A promoter renders the cells completely nonmotile, ...

Calcium carbonate precipitation, a widespread phenomenon among bacteria, has been investigated due to its wide range of scientific and technological implications. Nevertheless, little is known of the molecular mechanisms by which bacteria foster calcium carbonate mineralization. In our laboratory, we are studying calcite formation by Bacillus subtilis, in order to identify genes involved in the biomineralization process. A previous screening of UV mutants and of more than one thousand mutants obtained from the European B. subtilis Functional Analysis project allowed us to isolate strains altered in the precipitation phenotype. Starting from these results, we focused our attention on a cluster of five genes (lcfA, ysiA, ysiB, etfB, and etfA) called the lcfA operon. By insertional mutagenesis, mutant strains carrying each of the five genes were produced. All of them, with the exception of the strain carrying the mutated lcfA operon, were unable to form calcite crystals. By placing transcription under IPTG (isopropyl-␤-D-thiogalactopyranoside) control, the last gene, etfA, was identified as essential for the precipitation process. To verify cotranscription in the lcfA operon, reverse transcription-PCR experiments were performed and overlapping retrocotranscripts were found comprising three adjacent genes. The genes have putative functions linked to fatty acid metabolism. A link between calcium precipitation and fatty acid metabolism is suggested.