RESUMEN
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 approximately 4,100 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.
Asunto(s)
Bacillus subtilis/genética , Genes Bacterianos , Bacillus subtilis/citología , Bacillus subtilis/metabolismo , División Celular/genética , Membrana Celular/genética , Coenzimas/genética , Coenzimas/metabolismo , Metabolismo Energético/genética , Genoma Bacteriano , Mutación , Nucleótidos/genética , Nucleótidos/metabolismo , FilogeniaRESUMEN
The precursors of beta-lactamase fusion proteins having the signal peptide of Bacillus subtilis alkaline protease (pAprE-BlaH6) or penicillin binding protein 5(*) (pPBP5(*)-BlaH6) accumulated in B. subtilis cells in the absence of SecA or Ffh. Using the five purified precursors of secretory proteins including the two fusion proteins, B. subtilis Ffh and SecA, we analyzed the protein targeting mechanism of B. subtilis in vitro. B. subtilis SecA recognized the completely translated precursors of secretory proteins to which Ffh also bound. Moreover, B. subtilis SecA-precursor complex formation was enhanced 15-to 30-fold when the precursor and Ffh were incubated first and then SecA was added, but not vice versa. We also found that B. subtilis SecA directly interacted with Ffh in vitro. These results indicate that B. subtilis SecA and Ffh interact to function cooperatively in a protein translocation pathway including other protein factors, and that Ffh, as well as SecB in Escherichia coli, enhances the binding of SecA to presecretory proteins in B. subtilis cells.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Proteínas de Transporte de Membrana , Precursores de Proteínas/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Canales de Translocación SEC , Proteína SecA , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
Bacillus subtilis Ffh protein is a homologue of the 54-kDa subunit of mammalian signal recognition particle (SRP54). It contains three highly hydrophobic regions (h1, h2, and h3) in the C-terminal methionine-rich domain (M-domain). Two of the hydrophobic regions, h2 and h3, are essential for small cytoplasmic RNA (scRNA) binding [Kurita, K., Honda, K., Suzuma, S., Takamatsu, H., Nakamura, K., & Yamane, K. (1996) J. Biol. Chem. 271, 13,140-13,146]. Using purified presecretory proteins and mutant Ffh proteins, we identified a region required for presecretory protein binding in B. subtilis Ffh. Deletion of this region, which consisted of residues Ser311-Gly362 of B. subtilis Ffh, including a hydrophobic sequence (h1), reduced precursor binding activity. In contrast, deletions of residues Leu121-Lys279, Lys364-Met446, or Leu338-Ser397 of B. subtilis Ffh did not. We also analyzed the mutant B. subtilis Ffh proteins, FfhQQQR and FfhQQQQ having wild-type residues 398-401 (Arg-Arg-Lys-Arg) replaced with Gln3Arg and Gln4, respectively. FfhQQQR bound to both scRNA and presecretory protein. Although the FfhQQQQ mutation prevented binding to scRNA, binding to the precursor was not affected. FfhQQQR restored the growth of B. subtilis DF46 strain in which ffh gene expression is regulated by an inducible promoter in the absence of an inducer, whereas FfhQQQQ did not. These results indicate that the region including h1 is required for B. subtilis Ffh to bind to presecretory protein. The results also suggest that scRNA is required for the complete function of the B. subtilis SRP-like particle in vivo, although this protein is intrinsically capable of binding a signal peptide free from scRNA.
Asunto(s)
Bacillus subtilis/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Partícula de Reconocimiento de Señal/genética , Partícula de Reconocimiento de Señal/metabolismo , Secuencia de Aminoácidos , Animales , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/inmunología , Sitios de Unión , División Celular/genética , Citoplasma/genética , Mamíferos , Datos de Secuencia Molecular , Mutación , Precursores de Proteínas/metabolismo , ARN Bacteriano/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Partícula de Reconocimiento de Señal/inmunología , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
We analyzed the binding activity of B. subtilis Ffh to the precursors of secretory proteins by purifying mature and precursor proteins of beta-lactamase derived from pUC18 and its derivatives, of which the signal peptide region was replaced with that of E. coli OmpA, B. subtilis AprE, PBP5* or an alkalophilic Bacillus sp. #1011 CGTase. Each of them was mixed with purified B. subtilis Ffh in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC). The tested precursor proteins, including those of E. coli, of which the signal sequences differ from those of B. subtilis in the number of charged amino acids and hydrophobicity, cross-linked with Ffh, whereas mature proteins did not. The addition of scRNA, the B. subtilis counterpart of mammalian SRP 7S RNA, into the mixture did not affect the complex formation. These findings suggest that B. subtilis Ffh intrinsically binds to several precursor proteins.