RESUMEN
Ribosomes are evolutionary conserved ribonucleoprotein complexes that function as two separate subunits in all kingdoms. During translation initiation, the two subunits assemble to form the mature ribosome, which is responsible for translating the messenger RNA. When the ribosome reaches a stop codon, release factors promote translation termination and peptide release, and recycling factors then dissociate the two subunits, ready for use in a new round of translation. A tethered ribosome, called Ribo-T, in which the two subunits are covalently linked to form a single entity, was recently described in Escherichia coli. A hybrid ribosomal RNA (rRNA) consisting of both the small and large subunit rRNA sequences was engineered. The ribosome with inseparable subunits generated in this way was shown to be functional and to sustain cell growth. Here, we investigated the translational properties of Ribo-T. We analyzed its behavior during amino acid misincorporation, -1 or +1 frameshifting, stop codon readthrough, and internal translation initiation. Our data indicate that covalent attachment of the two subunits modifies the properties of the ribosome, altering its ability to initiate and terminate translation correctly.
Asunto(s)
Biosíntesis de Proteínas , Ribosomas/metabolismo , Codón de Terminación , Sistema de Lectura Ribosómico , Iniciación de la Cadena Peptídica Traduccional , Terminación de la Cadena Péptídica Traduccional , ARN de Transferencia/metabolismoRESUMEN
In eukaryotes, translation termination is performed by eRF1, which recognizes stop codons via its N-terminal domain. Many previous studies based on point mutagenesis, cross-linking experiments or eRF1 chimeras have investigated the mechanism by which the stop signal is decoded by eRF1. Conserved motifs, such as GTS and YxCxxxF, were found to be important for termination efficiency, but the recognition mechanism remains unclear. We characterized a region of the eRF1 N-terminal domain, the P1 pocket, that we had previously shown to be involved in termination efficiency. We performed alanine scanning mutagenesis of this region, and we quantified in vivo readthrough efficiency for each alanine mutant. We identified two residues, arginine 65 and lysine 109, as critical for recognition of the three stop codons. We also demonstrated a role for the serine 33 and serine 70 residues in UGA decoding in vivo. NMR analysis of the alanine mutants revealed that the correct conformation of this region was controlled by the YxCxxxF motif. By combining our genetic data with a structural analysis of eRF1 mutants, we were able to formulate a new model in which the stop codon interacts with eRF1 through the P1 pocket.
Asunto(s)
Codón de Terminación , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Modelos Moleculares , Mutagénesis , Resonancia Magnética Nuclear Biomolecular , Factores de Terminación de Péptidos/química , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Prion proteins are found in mammals and yeast, and can transmit diseases and encode heritable phenotypic traits. In Saccharomyces cerevisiae, eRF3, Rnq1, Ure2 and Swil are functional proteins with a soluble conformation that can switch to a non-functional, amyloid conformation denoted as [PSI+], [PIN+], [URE3] and [SWI+], respectively. The prion [PSI+] corresponds to an aggregated conformation of the translational release factor eRF3, which suppresses nonsense codons. [PSI+] modifies cellular fitness and induces several phenotypes according to the genetic background. An elegant series of studies has demonstrated that several [PSI+]-induced phenotypes occur as a consequence of decreased translational termination efficiency. However, the genes whose expression levels are controlled by [PSI+] remain largely unknown. Here, we show that [PSI+] enhances expression of antizyme, a negative regulator of cellular polyamines, by modulating the +1 frameshifting required for its expression. Our study also demonstrates that [PSI+] greatly affects cellular polyamines in yeast. We show that modification of the cellular content of polyamines by the prion accounts for half of the [PSI+]-induced phenotypes. Antizyme is the first protein to be described for which expression of its functional form is stimulated by [PSI+].
Asunto(s)
Epigénesis Genética , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/metabolismo , Poliaminas/metabolismo , Priones/química , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Espacio Intracelular/metabolismo , Modelos Biológicos , Fenotipo , Saccharomyces cerevisiae/citologíaRESUMEN
In bacteria, trans-translation rescues stalled ribosomes by the combined action of tmRNA (transfer-mRNA) and its associated protein SmpB. The tmRNA 5' and 3' ends fold into a tRNA-like domain (TLD), which shares structural and functional similarities with tRNAs. As in tRNAs, the UUC sequence of the T-arm of the TLD is post-transcriptionally modified to m (5)UψC. In tRNAs of gram-negative bacteria, formation of m (5)U is catalyzed by the SAM-dependent methyltransferase TrmA, while formation of m (5)U at two different positions in rRNA is catalyzed by distinct site-specific methyltransferases RlmC and RlmD. Here, we show that m (5)U formation in tmRNAs is exclusively due to TrmA and should be considered as a dual-specific enzyme. The evidence comes from the lack of m (5)U in purified tmRNA or TLD variants recovered from an Escherichia coli mutant strain deleted of the trmA gene. Detection of m (5)U in RNA was performed by NMR analysis.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , ARN Bacteriano/metabolismo , ARN de Transferencia/metabolismo , Uridina/química , ARNt Metiltransferasas/metabolismo , Secuencia de Bases , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Datos de Secuencia Molecular , Enzimas Multifuncionales/química , Enzimas Multifuncionales/genética , Enzimas Multifuncionales/metabolismo , Conformación de Ácido Nucleico , Procesamiento Postranscripcional del ARN , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Ribosómico 23S/genética , ARN Ribosómico 23S/metabolismo , ARN de Transferencia/química , ARN de Transferencia/genética , Uridina/genética , Uridina/metabolismo , ARNt Metiltransferasas/química , ARNt Metiltransferasas/genéticaRESUMEN
Methyltransferases that use S-adenosylmethionine (AdoMet) as a cofactor to catalyse 5-methyl uridine (m(5)U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, and are also found in certain Archaea. These enzymes belong to the COG2265 cluster, and the Gram-negative bacterium Escherichia coli possesses three paralogues. These comprise the methyltransferases TrmA that targets U54 in tRNAs, RlmC that modifies U747 in 23S rRNA and RlmD that is specific for U1939 in 23S rRNA. The tRNAs and rRNAs of the Gram-positive bacterium Bacillus subtilis have the same three m(5)U modifications. However, as previously shown, the m(5)U54 modification in B. subtilis tRNAs is catalysed in a fundamentally different manner by the folate-dependent enzyme TrmFO, which is unrelated to the E. coli TrmA. Here, we show that methylation of U747 and U1939 in B. subtilis rRNA is catalysed by a single enzyme, YefA that is a COG2265 member. A recombinant version of YefA functions in an E. coli m(5)U-null mutant adding the same two rRNA methylations. The findings suggest that during evolution, COG2265 enzymes have undergone a series of changes in target specificity and that YefA is closer to an archetypical m(5)U methyltransferase. To reflect its dual specificity, YefA is renamed RlmCD.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Metiltransferasas/metabolismo , ARN Ribosómico 23S/metabolismo , Uridina/análogos & derivados , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Biocatálisis , Metiltransferasas/química , Datos de Secuencia Molecular , ARN Ribosómico 23S/química , Alineación de Secuencia , Uridina/metabolismoRESUMEN
The epigenetic factor [PSI+] in the yeast Saccharomyces cerevisiae is due to the prion form of Sup35p. The N-terminal domain of Sup35p (N), alone or together with the middle-domain (NM), assembles in vitro into fibrils that induce [PSI+] when introduced into yeast cells. The Sup35p C-terminal domain (C), involved in translation termination, is essential for growth. The involvement of Sup35p C-terminal domain into [PSI+] propagation is subject to debate. We previously showed that mutation of threonine 341 within Sup35p C-domain affects translation termination efficiency. Here, we demonstrate that mutating threonine 341 to aspartate or alanine results in synthetic lethality with [PSI+] and weakening of [PSI+] respectively. The corresponding Sup35D and Sup35A proteins assemble into wild-type like fibrils in vitro, but with a slower elongation rate. Moreover, cross-seeding between Sup35p and Sup35A is inefficient both in vivo and in vitro, suggesting that the point mutation alters the structural properties of Sup35p within the fibrils. Thus, Sup35p C-terminal domain modulates [PSI+] prion propagation, possibly through a functional interaction with the N and/or M domains of the protein. Our results clearly demonstrate that Sup35p C-terminal domain plays a critical role in prion propagation and provide new insights into the mechanism of prion conversion.
Asunto(s)
Mutación Missense , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Priones/genética , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sustitución de Aminoácidos , Viabilidad Microbiana , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Desnaturalización Proteica , Multimerización de ProteínaRESUMEN
In eubacteria, the post-transcriptional modification of the wobble cytidine of the CAU anticodon in a precursor tRNA(Ile2) to a lysidine residue (2-lysyl-cytidine, abbreviated as L) allows the amino acid specificity to change from methionine to isoleucine and the codon decoding specificity to shift from AUG to AUA. The tilS gene encoding the enzyme that catalyses this modification is widely distributed. However, some microbial species lack a tilS gene, indicating that an alternative strategy exists to accurately translate the AUA codon into Ile. To determine whether a TilS-dependent bacterium, such as Bacillus subtilis, can overcome the absence of lysidine in its tRNA(Ile2) (CAU), we analysed the suppressor mutants of a tilS-thermosensitive allele. These tilS-suppressor mutants carry a substitution of the wobble guanosine into thymidine in one of the tRNA(Ile1) genes (the original GAT anticodon is changed to a TAT). In absence of TilS activity, the AUA codons are translated into isoleucine by the suppressor tRNA(Ile1), although a low level of AUA codons is also mistranslated into methionine. Results are in agreement with rare cases of eubacteria (and archaea), which naturally lack the tilS gene (or tiaS in archaea) but contain a tRNA(Ile2) gene containing a TAT instead of a CAT anticodon.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Bacillus subtilis/enzimología , ARN de Transferencia/genética , Sustitución de Aminoácidos , Aminoacil-ARNt Sintetasas/genética , Anticodón/genética , Bacillus subtilis/genética , Calor , Lisina/análogos & derivados , Lisina/química , Lisina/genética , Biosíntesis de Proteínas/genética , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/genética , Supresión GenéticaRESUMEN
We have generated a protein-protein interaction network in Bacillus subtilis focused on several essential cellular processes such as cell division, cell responses to various stresses, the bacterial cytoskeleton, DNA replication and chromosome maintenance by careful application of the yeast two-hybrid approach. This network, composed of 793 interactions linking 287 proteins with an average connectivity of five interactions per protein, represents a valuable resource for future functional analyses. A striking feature of the network is a group of highly connected hubs (GoH) linking many different cellular processes. Most of the proteins of the GoH have unknown functions and are associated to the membrane. By the integration of available knowledge, in particular of transcriptome data sets, the GoH was decomposed into subgroups of party hubs corresponding to protein complexes or regulatory pathways expressed under different conditions. At a global level, the GoH might function as a very robust group of date hubs having partially redundant functions to integrate information from the different cellular pathways. Our analyses also provide a rational way to study the highly redundant functions of the GoH by a genetic approach.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Mapeo de Interacción de Proteínas/métodos , Análisis por Conglomerados , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Técnicas del Sistema de Dos HíbridosRESUMEN
Translation termination in eukaryotes is completed by two interacting factors eRF1 and eRF3. In Saccharomyces cerevisiae, these proteins are encoded by the genes SUP45 and SUP35, respectively. The eRF1 protein interacts directly with the stop codon at the ribosomal A-site, whereas eRF3-a GTPase protein-probably acts as a proofreading factor, coupling stop codon recognition to polypeptide chain release. We performed random PCR mutagenesis of SUP45 and screened the library for mutations resulting in increased eRF1 activity. These mutations led to the identification of two new pockets in domain 1 (P1 and P2) involved in the regulation of eRF1 activity. Furthermore, we identified novel mutations located in domains 2 and 3, which confer stop codon specificity to eRF1. Our findings are consistent with the model of a closed-active conformation of eRF1 and shed light on two new functional regions of the protein.
Asunto(s)
Terminación de la Cadena Péptídica Traduccional , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia Conservada , Eliminación de Gen , Prueba de Complementación Genética , Modelos Moleculares , Mutación , Factores de Terminación de Péptidos/metabolismo , Fenotipo , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Important regions of rRNA are rich in nucleotide modifications that can have strong effects on ribosome biogenesis and translation efficiency. Here, we examine the influence of pseudouridylation and 2'-O-methylation on translation accuracy in yeast, by deleting the corresponding guide snoRNAs. The regions analyzed were: the decoding centre (eight modifications), and two intersubunit bridge domains-the A-site finger and Helix 69 (six and five modifications). Results show that a number of modifications influence accuracy with effects ranging from 0.3- to 2.4-fold of wild-type activity. Blocking subsets of modifications, especially from the decoding region, impairs stop codon termination and reading frame maintenance. Unexpectedly, several Helix 69 mutants possess ribosomes with increased fidelity. Consistent with strong positional and synergistic effects is the finding that single deletions can have a more pronounced phenotype than multiple deficiencies in the same region. Altogether, the results demonstrate that rRNA modifications have significant roles in translation accuracy.
Asunto(s)
Biosíntesis de Proteínas , ARN Ribosómico/química , Saccharomyces cerevisiae/genética , Secuencia de Bases , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Nucleótidos/química , ARN de Hongos/químicaRESUMEN
BACKGROUND: When a stop codon is located in the ribosomal A-site, the termination complex promotes release of the polypeptide and dissociation of the 80S ribosome. In eukaryotes two proteins eRF1 and eRF3 play a crucial function in the termination process. The essential GTPase Sup35p, the eRF3 release factor of Saccharomyces cerevisiae is highly conserved. In particular, we observed that all eRF3 homologs share a potential phosphorylation site at threonine 341, suggesting a functional role for this residue. The goal of this study was to determine whether this residue is actually phosphorylated in yeast and if it is involved in the termination activity of the protein. RESULTS: We detected no phosphorylation of the Sup35 protein in vivo. However, we show that it is phosphorylated by the cAMP-dependent protein kinase A on T341 in vitro. T341 was mutated to either alanine or to aspartic acid to assess the role of this residue in the activity of the protein. Both mutant proteins showed a large decrease of GTPase activity and a reduced interaction with eRF1/Sup45p. This was correlated with an increase of translational readthrough in cells carrying the mutant alleles. We also show that this residue is involved in functional interaction between the N- and C-domains of the protein. CONCLUSION: Our results point to a new critical residue involved in the translation termination activity of Sup35 and in functional interaction between the N- and C-domains of the protein. They also raise interesting questions about the relation between GTPase activity of Sup35 and its essential function in yeast.
Asunto(s)
GTP Fosfohidrolasas/genética , Mutación , Terminación de la Cadena Péptídica Traduccional/genética , Priones/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , GTP Fosfohidrolasas/metabolismo , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Fosforilación , Priones/metabolismo , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
In eukaryotes, release factors 1 and 3 (eRF1 and eRF3) are recruited to promote translation termination when a stop codon on the mRNA enters at the ribosomal A-site. However, their overexpression increases termination efficiency only moderately, suggesting that other factors might be involved in the termination process. To determine such unknown components, we performed a genetic screen in Saccharomyces cerevisiae that identified genes increasing termination efficiency when overexpressed. For this purpose, we constructed a dedicated reporter strain in which a leaky stop codon is inserted into the chromosomal copy of the ade2 gene. Twenty-five antisuppressor candidates were identified and characterized for their impact on readthrough. Among them, SSB1 and snR18, two factors close to the exit tunnel of the ribosome, directed the strongest antisuppression effects when overexpressed, showing that they may be involved in fine-tuning of the translation termination level.
Asunto(s)
Terminación de la Cadena Péptídica Traduccional , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Carboxiliasas/genética , Codón de Terminación , ADN de Hongos/genética , Expresión Génica , Genes Fúngicos , Genes Reporteros , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Sustancias Macromoleculares , Modelos Moleculares , Mutagénesis , Factor 1 de Elongación Peptídica/genética , Factor 1 de Elongación Peptídica/metabolismo , ARN de Hongos/química , ARN de Hongos/genética , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/genética , Ribosomas/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The aim of this approach was to identify the major determinants, located at the 5' end of the stop codon, that modulate translational read-through in Saccharomyces cerevisiae. We developed a library of oligonucleotides degenerate at the six positions immediately upstream of the termination codon, cloned in the ADE2 reporter gene. Variations at these positions modulated translational read-through efficiency approximately 16-fold. The major effect was imposed by the two nucleotides immediately upstream of the stop codon. We showed that this effect was neither mediated by the last amino acid residues present in the polypeptide chain nor by the tRNA present in the ribosomal P site. We propose that the mRNA structure, depending on the nucleotides in the P site, is the main 5' determinant of read-through efficiency.
Asunto(s)
Nucleótidos de Adenina/genética , Codón de Terminación/genética , Extensión de la Cadena Peptídica de Translación/genética , Secuencias Reguladoras de Ácido Ribonucleico/genética , Saccharomyces cerevisiae/genética , Composición de Base , Secuencia de Bases , Sitios de Unión , Biblioteca de Genes , Genes Fúngicos/genética , Genes Reporteros/genética , Conformación de Ácido Nucleico , Oligorribonucleótidos/química , Oligorribonucleótidos/genética , Oligorribonucleótidos/metabolismo , ARN de Hongos/química , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In the model fungus Podospora anserina, the PaYIP3 gene encoding the orthologue of the Saccharomyces cerevisiae YIP3 Rab-GDI complex dissociation factor expresses two polypeptides, one of which, the long form, is produced through a programmed translation frameshift. Inactivation of PaYIP3 results in slightly delayed growth associated with modification in repartition of fruiting body on the thallus, along with reduced ascospore production on wood. Long and short forms of PaYIP3 are expressed in the mycelium, while only the short form appears expressed in the maturing fruiting body (perithecium). The frameshift has been conserved over the evolution of the Pezizomycotina, lasting for over 400 million years, suggesting that it has an important role in the wild.
Asunto(s)
Ascomicetos/metabolismo , Sistema de Lectura Ribosómico/fisiología , Proteínas Fúngicas/metabolismo , Ascomicetos/genética , Sistema de Lectura Ribosómico/genética , Proteínas Fúngicas/genética , Podospora/genética , Podospora/metabolismoRESUMEN
The [PSI+] determinant in Saccharomyces cerevisiae is the prion protein corresponding to the eRF3 translation termination factor. Numerous infectious proteins have been described in yeast, in comparison of the unique PrP protein in higher eukaryotes. The presence of the PrP prion is associated with mammalian diseases. Whether fungal prions are beneficial or deleterious are still under discussions. The review focuses on [PSI+]-induced phenotypes and the resulting physiological consequences to shed light on the cellular changes occurring in a [PSI+] cell and its possible role in nature. To date, only two genes directly regulated at the translational level by [PSI+] have been identified. Yet, through all the published works, obtaining a consensus for the described [PSI+] phenotypes appeared a tricky task. They are highly dependent on the prion variant and the genetic background of the strain. The [PSI+] prion might generate diverse modifications not only at the translational, but also at the transcriptional levels, and the phenotypic heterogeneity is the result of these complex combinations of the genotypic expression.
Asunto(s)
Proteínas Fúngicas/metabolismo , Factores de Terminación de Péptidos/metabolismo , Priones/metabolismo , Amiloide/química , Amiloide/metabolismo , Proteínas Fúngicas/química , Factores de Terminación de Péptidos/química , Priones/química , Biosíntesis de ProteínasRESUMEN
In Saccharomyces cerevisiae, the essential IMP3 gene encodes a component of the SSU processome, a large ribonucleoprotein complex required for processing of small ribosomal subunit RNA precursors. Mutation of the IMP3 termination codon to a sense codon resulted in a viable mutant allele producing a C-terminal elongated form of the Imp3 protein. A strain expressing the mutant allele displayed ribosome biogenesis defects equivalent to IMP3 depletion. This hypomorphic allele represented a unique opportunity to investigate and better understand the Imp3p functions. We demonstrated that the +1 frameshifting was increased in the mutant strain. Further characterizations revealed involvement of the Imp3 protein in DNA repair and telomere length control, pointing to a functional relationship between both pathways and ribosome biogenesis.
Asunto(s)
Proteínas Ribosómicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alelos , Daño del ADN , Reparación del ADN , Mutación del Sistema de Lectura , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Modelos Genéticos , Mutación , Polirribosomas/metabolismo , Estructura Terciaria de Proteína , Proteínas Ribosómicas/fisiología , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Telómero/ultraestructura , Técnicas del Sistema de Dos HíbridosRESUMEN
In Bacillus subtilis, although many genetic tools have been developed, gene replacement remains labour-intensive and not compatible with large-scale approaches. We have developed a new one-step gene replacement procedure that allows rapid alteration of any gene sequence or multiple gene sequences in B. subtilis without altering the chromosome in any other way. This novel approach relies on the use of upp, which encodes uracil phosphoribosyl-transferase, as a counter-selectable marker. We fused the upp gene to an antibiotic-resistance gene to create an 'upp-cassette'. A polymerase chain reaction (PCR)-generated fragment, consisting of the target gene with the desired mutation joined to the upp-cassette, was integrated into the chromosome by homologous recombination, using positive selection for antibiotic resistance. Then, the eviction of the upp-cassette from the chromosome by recombination between short repeated chromosomal sequences, included in the design of the transforming DNA molecule, was achieved by counter-selection of upp. This procedure was successfully used to deliver a point mutation, to generate in-frame deletions with reduced polar effects, and to combine deletions in three paralogous genes encoding two-component sensor kinases. Also, two chromosome regions carrying previously unrecognized essential functions were identified, and large deletions in two dispensable regions were combined. This work outlines a strategy for identifying essential functions that could be used at genome scale.