RESUMEN
Genome-scale engineering enables rational removal of dispensable genes in chassis genomes. Deviating from this approach, we applied greedy accumulation of deletions of large dispensable regions in the Bacillus subtilis genome, yielding a library of 298 strains with genomes reduced up to 1.48 Mb in size. High-throughput physiological phenotyping of these strains confirmed that genome reduction is associated with substantial loss of cell fitness and accumulation of synthetic-sick interactions. Transcriptome analysis indicated that <15% of the genes conserved in our genome-reduced strains exhibited a twofold or higher differential expression and revealed a thiol-oxidative stress response. Most transcriptional changes can be explained by loss of known functions and by aberrant transcription at deletion boundaries. Genome-reduced strains exhibited striking new phenotypes relative to wild type, including a very high resistance (increased >300-fold) to the DNA-damaging agent mitomycin C and a very low spontaneous mutagenesis (reduced 100-fold). Adaptive laboratory evolution failed to restore cell fitness, except when coupled with a synthetic increase of the mutation rate, confirming low evolvability. Although mechanisms underlying this emergent phenotype are not understood, we propose that low evolvability can be leveraged in an engineering strategy coupling reductive cycles with evolutive cycles under induced mutagenesis.
Asunto(s)
Bacillus subtilis , Genoma Bacteriano , Genoma Bacteriano/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Fenotipo , Mutagénesis , Tasa de MutaciónRESUMEN
The transcriptional regulator Spx plays a key role in maintaining the redox homeostasis of Bacillus subtilis cells exposed to disulfide stress. Defects in Spx were previously shown to lead to differential expression of numerous genes but direct and indirect regulatory effects could not be distinguished. Here we identified 283 discrete chromosomal sites potentially bound by the Spx-RNA polymerase (Spx-RNAP) complex using chromatin immunoprecipitation of Spx. Three quarters of these sites were located near Sigma(A)-dependent promoters, and upon diamide treatment, the fraction of the Spx-RNAP complex increased in parallel with the number and occupancy of DNA sites. Correlation of Spx-RNAP-binding sites with gene differential expression in wild-type and Δspx strains exposed or not to diamide revealed that 144 transcription units comprising 275 genes were potentially under direct Spx regulation. Spx-controlled promoters exhibited an extended -35 box in which nucleotide composition at the -43/-44 positions strongly correlated with observed activation. In vitro transcription confirmed activation by oxidized Spx of seven newly identified promoters, of which one was also activated by reduced Spx. Our study globally characterized the Spx regulatory network, revealing its role in the basal expression of some genes and its complex interplay with other stress responses.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , ARN Polimerasas Dirigidas por ADN/metabolismo , Diamida/toxicidad , Genoma Bacteriano , Regiones Promotoras Genéticas , Regulón , Estrés Fisiológico/genética , Reactivos de Sulfhidrilo/toxicidadRESUMEN
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
The ability to use lactate as a sole source of carbon and energy is one of the key metabolic signatures of Shewanellae, a diverse group of dissimilatory metal-reducing bacteria commonly found in aquatic and sedimentary environments. Nonetheless, homology searches failed to recognize orthologs of previously described bacterial d- or l-lactate oxidizing enzymes (Escherichia coli genes dld and lldD) in any of the 13 analyzed genomes of Shewanella spp. By using comparative genomic techniques, we identified a conserved chromosomal gene cluster in Shewanella oneidensis MR-1 (locus tag: SO_1522-SO_1518) containing lactate permease and candidate genes for both d- and l-lactate dehydrogenase enzymes. The predicted d-LDH gene (dld-II, SO_1521) is a distant homolog of FAD-dependent lactate dehydrogenase from yeast, whereas the predicted l-LDH is encoded by 3 genes with previously unknown functions (lldEGF, SO_1520-SO_1518). Through a combination of genetic and biochemical techniques, we experimentally confirmed the predicted physiological role of these novel genes in S. oneidensis MR-1 and carried out successful functional validation studies in Escherichia coli and Bacillus subtilis. We conclusively showed that dld-II and lldEFG encode fully functional d-and l-LDH enzymes, which catalyze the oxidation of the respective lactate stereoisomers to pyruvate. Notably, the S. oneidensis MR-1 LldEFG enzyme is a previously uncharacterized example of a multisubunit lactate oxidase. Comparative analysis of >400 bacterial species revealed the presence of LldEFG and Dld-II in a broad range of diverse species accentuating the potential importance of these previously unknown proteins in microbial metabolism.
Asunto(s)
Genoma Bacteriano , Lactatos/metabolismo , Shewanella/genética , Bacillus subtilis/genética , Biocatálisis , Escherichia coli/genética , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , Shewanella/metabolismo , EstereoisomerismoRESUMEN
Bacillus subtilis is a widely used bacterial model to decipher biofilm formation, genetic determinants and their regulation. For several years, studies were conducted on colonies or pellicles formed at the interface with air, but more recent works showed that non-domesticated strains were able to form thick and structured biofilms on submerged surfaces. Taking advantage of time-lapse confocal laser scanning microscopy, we monitored bacterial colonization on the surface and observed an unexpected biphasic submerged biofilm development. Cells adhering to the surface firstly form elongated chains before being suddenly fragmented and released as free motile cells in the medium. This switching coincided with an oxygen depletion in the well which preceded the formation of the pellicle at the liquid-air interface. Residual bacteria still associated with the solid surface at the bottom of the well started to express matrix genes under anaerobic metabolism to build the typical biofilm protruding structures.
RESUMEN
A comparative genomic approach was used to reconstruct transcriptional regulation of NAD biosynthesis in bacteria containing orthologs of Bacillus subtilis gene yrxA, a previously identified niacin-responsive repressor of NAD de novo synthesis. Members of YrxA family (re-named here NiaR) are broadly conserved in the Bacillus/Clostridium group and in the deeply branching Fusobacteria and Thermotogales lineages. We analyzed upstream regions of genes associated with NAD biosynthesis to identify candidate NiaR-binding DNA motifs and assess the NiaR regulon content in these species. Representatives of the two distinct types of candidate NiaR-binding sites, characteristic of the Firmicutes and Thermotogales, were verified by an electrophoretic mobility shift assay. In addition to transcriptional control of the nadABC genes, the NiaR regulon in some species extends to niacin salvage (the pncAB genes) and includes uncharacterized membrane proteins possibly involved in niacin transport. The involvement in niacin uptake proposed for one of these proteins (re-named NiaP), encoded by the B. subtilis gene yceI, was experimentally verified. In addition to bacteria, members of the NiaP family are conserved in multicellular eukaryotes, including human, pointing to possible NaiP involvement in niacin utilization in these organisms. Overall, the analysis of the NiaR and NrtR regulons (described in the accompanying paper) revealed mechanisms of transcriptional regulation of NAD metabolism in nearly a hundred diverse bacteria.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , NAD/metabolismo , Regulón , Proteínas Represoras/metabolismo , Thermotoga maritima/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Sitios de Unión , Genoma Bacteriano , Genómica , Proteínas de Transporte de Membrana/clasificación , Proteínas de Transporte de Membrana/genética , Niacina/metabolismo , Elementos Reguladores de la Transcripción , Proteínas Represoras/clasificación , Proteínas Represoras/genética , Transcripción GenéticaRESUMEN
Bacteria are sophisticated systems with high capacity and flexibility to adapt to various environmental conditions. Each prokaryote however possesses a defined metabolic network, which sets its overall metabolic capacity, and therefore the maximal growth rate that can be reached. To achieve optimal growth, bacteria adopt various molecular strategies to optimally adjust gene expression and optimize resource allocation according to the nutrient availability. The resulting physiological changes are often accompanied by changes in the growth rate, and by global regulation of gene expression. The growth-rate-dependent variation of the abundances in the cellular machineries, together with condition-specific regulatory mechanisms, affect RNA metabolism and fate and pose a challenge for rational gene expression reengineering of synthetic circuits. This article is part of a Special Issue entitled: RNA and gene control in bacteria, edited by Dr. M. Guillier and F. Repoila.
Asunto(s)
Bacterias/genética , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/genética , Adaptación Fisiológica , Bacterias/crecimiento & desarrollo , Bacterias/metabolismo , Guanosina Tetrafosfato/metabolismo , ARN Bacteriano/metabolismoRESUMEN
Bacillus subtilis mutants lacking the SMC-ScpAB complex are severely impaired for chromosome condensation and partitioning, DNA repair, and cells are not viable under standard laboratory conditions. We isolated suppressor mutations that restored the capacity of a smc deletion mutant (Δsmc) to grow under standard conditions. These suppressor mutations reduced chromosome segregation defects and abrogated hypersensitivity to gyrase inhibitors of Δsmc. Three suppressor mutations were mapped in genes involved in tRNA aminoacylation and maturation pathways. A transcriptomic survey of isolated suppressor mutations pointed to a potential link between suppression of Δsmc and induction of the stringent response. This link was confirmed by (p)ppGpp quantification which indicated a constitutive induction of the stringent response in multiple suppressor strains. Furthermore, sublethal concentrations of arginine hydroxamate (RHX), a potent inducer of stringent response, restored growth of Δsmc under non permissive conditions. We showed that production of (p)ppGpp alone was sufficient to suppress the thermosensitivity exhibited by the Δsmc mutant. Our findings shed new light on the coordination between chromosome dynamics mediated by SMC-ScpAB and other cellular processes during rapid bacterial growth.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Segregación Cromosómica/genética , Cromosomas Bacterianos/genética , Arginina/análogos & derivados , Arginina/genética , Proteínas de Ciclo Celular/genética , Reparación del ADN/genética , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica/genética , Mutación/genética , ARN de Transferencia/genética , Eliminación de Secuencia/genéticaRESUMEN
RNA-binding protein Hfq is a key component of the adaptive responses of many proteobacterial species including Escherichia coli, Salmonella enterica and Vibrio cholera. In these organisms, the importance of Hfq largely stems from its participation to regulatory mechanisms involving small non-coding RNAs. In contrast, the function of Hfq in Gram-positive bacteria has remained elusive and somewhat controversial. In the present study, we have further addressed this point by comparing growth phenotypes and transcription profiles between wild-type and an hfq deletion mutant of the model Gram-positive bacterium, Bacillus subtilis. The absence of Hfq had no significant consequences on growth rates under nearly two thousand metabolic conditions and chemical treatments. The only phenotypic difference was a survival defect of B. subtilis hfq mutant in rich medium in stationary phase. Transcriptomic analysis correlated this phenotype with a change in the levels of nearly one hundred transcripts. Albeit a significant fraction of these RNAs (36%) encoded sporulation-related functions, analyses in a strain unable to sporulate ruled out sporulation per se as the basis of the hfq mutant's stationary phase fitness defect. When expressed in Salmonella, B. subtilis hfq complemented the sharp loss of viability of a degP hfq double mutant, attenuating the chronic σE-activated phenotype of this strain. However, B. subtilis hfq did not complement other regulatory deficiencies resulting from loss of Hfq-dependent small RNA activity in Salmonella indicating a limited functional overlap between Salmonella and B. subtilis Hfqs. Overall, this study confirmed that, despite structural similarities with other Hfq proteins, B. subtilis Hfq does not play a central role in post-transcriptional regulation but might have a more specialized function connected with stationary phase physiology. This would account for the high degree of conservation of Hfq proteins in all 17 B. subtilis strains whose genomes have been sequenced.
Asunto(s)
Bacillus subtilis/metabolismo , Proteína de Factor 1 del Huésped/metabolismo , Fenotipo , Bacillus subtilis/genética , Proteína de Factor 1 del Huésped/genética , TranscriptomaRESUMEN
Adaptation of cells to environmental changes requires dynamic interactions between metabolic and regulatory networks, but studies typically address only one or a few layers of regulation. For nutritional shifts between two preferred carbon sources of Bacillus subtilis, we combined statistical and model-based data analyses of dynamic transcript, protein, and metabolite abundances and promoter activities. Adaptation to malate was rapid and primarily controlled posttranscriptionally compared with the slow, mainly transcriptionally controlled adaptation to glucose that entailed nearly half of the known transcription regulation network. Interactions across multiple levels of regulation were involved in adaptive changes that could also be achieved by controlling single genes. Our analysis suggests that global trade-offs and evolutionary constraints provide incentives to favor complex control programs.
Asunto(s)
Adaptación Fisiológica , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Redes Reguladoras de Genes , Glucosa/metabolismo , Malatos/metabolismo , Redes y Vías Metabólicas/genética , Algoritmos , Proteínas Bacterianas/metabolismo , Simulación por Computador , Interpretación Estadística de Datos , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Metaboloma , Metabolómica , Modelos Biológicos , Operón , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/fisiología , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Transcripción Genética , Transcriptoma , Adaptación Fisiológica , Algoritmos , Sitios de Unión , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulón , Factor sigma/metabolismo , Regiones Terminadoras GenéticasRESUMEN
The DNA regions upstream from the genes encoding polypeptides of Bacillus thuringiensis subsp. israelensis larvicidal crystals (cry4A, cry4B, cry11A) contain sequences with similarities to the spo0A box of Bacillus subtilis (or '0A' box) and the promoter recognized by the sigma H-associated RNA polymerase of B. subtilis. Expression of cry-lacZ transcriptional fusions was analysed in various B. thuringiensis genetic backgrounds. The early transcription of the toxin genes was not sporulation-dependent, whereas the late-stage expression at t4-6 was sigma E-dependent. Primer extension analysis confirmed that the cry4- and cry11-type toxin genes were weakly transcribed during the transition phase; expression analysis of a cry11A'-lacZ transcriptional fusion in B. subtilis sporulation mutants confirmed the involvement of the sigma H-RNA polymerase. Primer extension analysis showed that in B. thuringiensis subsp. israelensis, the cry4A and cry11A gene transcription observed at the end of the growth stage was turned off at the beginning of the sporulation phase. The DNA region located upstream from the cry11A gene promoter including the putative '0A' box was deleted. This led to a derepression of the expression of the cry11A operon. These results suggest that the cry4A, cry4B and cry11A toxin genes of B. thuringiensis subsp. Israelensis are transcribed during the transition phase by the RNA polymerase associated with the sigma H factor and are subject to Spo0A repression.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Toxinas Bacterianas , Endotoxinas/biosíntesis , Regulación Bacteriana de la Expresión Génica , Factores de Transcripción/genética , Transcripción Genética , Bacillus subtilis/genética , Toxinas de Bacillus thuringiensis , Secuencia de Bases , Endotoxinas/genética , Genes Bacterianos , Genes Reporteros , Proteínas Hemolisinas , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Eliminación de Secuencia , Homología de Secuencia de Ácido Nucleico , Esporas Bacterianas , beta-Galactosidasa/biosíntesisRESUMEN
To study the functions of the uncharacterized open reading frames identified in the Bacillus subtilis genome, several vectors were constructed to perform insertional mutagenesis in the chromosome. All the pMUTIN plasmids carry a lacZ reporter gene and an inducible Pspac promoter, which is tightly regulated and can be induced about 1000-fold. The integration of a pMUTIN vector into the target gene has three consequences: (1) the target gene is inactivated; (2) lacZ becomes transcriptionally fused to the gene, allowing its expression pattern to be monitored; (3) the Pspac promoter controls the transcription of downstream genes in an IPTG-dependent fashion. This last feature is important because B. subtilis genes are often organized in operons. The potential polar effects generated by the integration of the vectors can be alleviated by addition of IPTG. Also, conditional mutants of essential genes can be obtained by integrating pMUTIN vectors upstream of the target gene. The vectors are currently being used for systematic inactivation of genes without known function within the B. subtilis European consortium. pMUTIN characteristics and the inactivation of eight genes in the resA-serA region of the chromosome are presented.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Vectores Genéticos , Mutagénesis Insercional , Sistemas de Lectura Abierta/genética , Bacillus subtilis/enzimología , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Operón Lac , Datos de Secuencia Molecular , Regiones Operadoras Genéticas , Operón/genética , Plásmidos/genética , Regiones Promotoras Genéticas , Regiones Terminadoras Genéticas , Transcripción Genética , beta-Galactosidasa/metabolismoRESUMEN
Structural maintenance of chromosome (SMC) and the SMC-interacting kleisin protein families have key functions in the chromosome organization of most organisms. Here, we report that the Bacillus subtilis kleisin, ScpA, can form a ternary complex with the SMC and ScpB proteins in a yeast tri-hybrid assay, supporting the notion of a bacterial cohesin/condensin-like complex. Furthermore, ScpA interacts in two-hybrid assays with the AddAB complex, essential for recombinational repair, with DegS, a two-component sensor kinase, as well as with other potential transcription regulators. Point mutations in scpA allowing growth under conditions not permissive for the spcA null and not affecting chromosome condensation were isolated. Among these mutations, some affected DNA repair and gene regulation, thus separating ScpA functions in these two pathways from its functions in chromosome condensation and segregation. Some separation-of-function mutations in scpA caused a deficiency in the repair of mitomycin C DNA lesions that was suppressed by increasing the intracellular dosage of the interacting AddAB complex. Another mutation in scpA deregulated the expression of genes encoding degradative enzymes that are known to be controlled by the interacting DegS kinase. We propose that the SMC-ScpA-ScpB complex could: (i) recruit the AddAB helicase/nuclease to act in post-replicative repair; and (ii) form a complex with the DegS sensor kinase that inhibits its kinase activity. Moreover, our results indicate that the role of cohesin and condensin complexes in DNA repair and gene regulation is evolutionary conserved.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparación del ADN , Regulación Bacteriana de la Expresión Génica , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas de Ciclo Celular/genética , Cromosomas Bacterianos/genética , Daño del ADN , ADN Bacteriano/genética , Exodesoxirribonucleasas/metabolismo , Mutación , Mutación Puntual , Regulón , Técnicas del Sistema de Dos HíbridosRESUMEN
The spore-forming bacterium Bacillus subtilis is capable of assembling multicellular communities (biofilms) that display a high degree of spatiotemporal organization. Wild strains that have not undergone domestication in the laboratory produce particularly robust biofilms with complex architectural features, such as fruiting-body-like aerial projections whose tips serve as preferential sites for sporulation. To discover genes involved in this multicellular behavior and to do so on a genome-wide basis, we took advantage of a large collection of mutants which have disruptions of most of the uncharacterized genes in the B. subtilis genome. This collection, which was generated with a laboratory strain, was screened for mutants that were impaired in biofilm formation. This subset of mutated genes was then introduced into the wild strain NCIB 3610 to study their effects on biofilm formation in liquid and solid media. In this way we identified six genes that are involved in the development of multicellular communities. These are yhxB (encoding a putative phosphohexomutase that may mediate exopolysaccharide synthesis), sipW (encoding a signal peptidase), ecsB (encoding an ABC transporter subunit), yqeK (encoding a putative phosphatase), ylbF (encoding a regulatory protein), and ymcA (a gene of unknown function). Further analysis revealed that these six genes play different roles in B. subtilis community development.
Asunto(s)
Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Biología Computacional/métodos , Ecosistema , Eliminación de Gen , MutaciónRESUMEN
A protein-interaction network centered on the replication machinery of Bacillus subtilis was generated by genome-wide two-hybrid screens and systematic specificity assays. The network consists of 91 specific interactions linking 69 proteins. Over one fourth of the interactions take place between homologues of proteins known to interact in other organisms, indicating the high biological significance of the other interactions we report. These interactions provide insights on the relations of DNA replication with recombination and repair, membrane-bound protein complexes, and signaling pathways. They also lead to the biological role of unknown proteins, as illustrated for the highly conserved YabA, which is shown here to act in initiation control. Thus, our interaction map provides a valuable tool for the discovery of aspects of bacterial DNA replication.
Asunto(s)
Bacterias/genética , Replicación del ADN/fisiología , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Eliminación de Gen , Vectores Genéticos , Biblioteca Genómica , Transformación BacterianaRESUMEN
Structural maintenance of chromosomes (SMC) proteins are present in all eukaryotes and in many prokaryotes. Eukaryotic SMC proteins form complexes with various non-SMC subunits, which affect their function, whereas the prokaryotic homologues had no known non-SMC partners and were thought to act as simple homodimers. Here we describe two novel families of proteins, widespread in archaea and (Gram-positive) bacteria, which we denote 'segregation and condensation proteins' (Scps). ScpA genes are localized next to smc genes in nearly all SMC- containing archaea, suggesting that they belong to the same operon and are thus involved in a common process in the cell. The function of ScpA was studied in Bacillus subtilis, which also harbours a well characterized smc gene. Here we show that scpA mutants display characteristic phenotypes nearly identical to those of smc mutants, including temperature- sensitive growth, production of anucleate cells, formation of aberrant nucleoids, and chromosome splitting by the so-called guillotine effect. Thus, both SMC and ScpA are required for chromosome segregation and condensation. Interestingly, mutants of another B. subtilis gene, scpB, which is localized downstream from scpA, display the same phenotypes, which indicate that ScpB is also involved in these functions. ScpB is generally present in species that also encode ScpA. The physical interaction of ScpA and SMC was proven (i) by the use of the yeast two-hybrid system and (ii) by the isolation of a complex containing both proteins from cell extracts of B. subtilis. By extension, we speculate that interaction of orthologues of the two proteins is important for chromosome segregation in many archaea and bacteria, and propose that SMC proteins generally have non-SMC protein partners that affect their function not only in eukaryotes but also in prokaryotes.