RESUMEN
Streptomyces are renowned for their prolific production of specialized metabolites with applications in medicine and agriculture. These multicellular bacteria present a sophisticated developmental cycle and play a key role in soil ecology. Little is known about the impact of Streptomyces phage on bacterial physiology. In this study, we investigated the conditions governing the expression and production of "Samy", a prophage found in Streptomyces ambofaciens ATCC 23877. This siphoprophage is produced simultaneously with the activation of other mobile genetic elements. Remarkably, the presence and production of Samy increases bacterial dispersal under in vitro stress conditions. Altogether, this study unveiled a new property of a bacteriophage infection in the context of multicellular aggregate dynamics.
Asunto(s)
Profagos , Streptomyces , Streptomyces/virología , Streptomyces/fisiología , Streptomyces/genética , Profagos/genética , Profagos/fisiología , Activación Viral/genéticaRESUMEN
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
Adherent-invasive Escherichia coli (AIEC) strains are frequently recovered from stools of patients with dysbiotic microbiota. They have remarkable properties of adherence to the intestinal epithelium, and survive better than other E. coli in macrophages. The best studied of these AIEC is probably strain LF82, which was isolated from a Crohn's disease patient. This strain contains five complete prophages, which have not been studied until now. We undertook their analysis, both in vitro and inside macrophages, and show that all of them form virions. The Gally prophage is by far the most active, generating spontaneously over 108 viral particles per mL of culture supernatants in vitro, more than 100-fold higher than the other phages. Gally is also over-induced after a genotoxic stress generated by ciprofloxacin and trimethoprim. However, upon macrophage infection, a genotoxic environment, this over-induction is not observed. Analysis of the transcriptome and key steps of its lytic cycle in macrophages suggests that the excision of the Gally prophage continues to be repressed in macrophages. We conclude that strain LF82 has evolved an efficient way to block the lytic cycle of its most active prophage upon macrophage infection, which may participate to its good survival in macrophages.
Asunto(s)
Bacteriófagos , Infecciones por Escherichia coli , Humanos , Escherichia coli , Macrófagos , Mucosa Intestinal , Adhesión BacterianaRESUMEN
Double-strand breaks (DSBs) are the most detrimental DNA damage encountered by bacterial cells. DBSs can be repaired by homologous recombination thanks to the availability of an intact DNA template or by Non-Homologous End Joining (NHEJ) when no intact template is available. Bacterial NHEJ is performed by sets of proteins of growing complexity from Bacillus subtilis and Mycobacterium tuberculosis to Streptomyces and Sinorhizobium meliloti. Here, we discuss the contribution of these models to the understanding of the bacterial NHEJ repair mechanism as well as the involvement of NHEJ partners in other DNA repair pathways. The importance of NHEJ and of its complexity is discussed in the perspective of regulation through the biological cycle of the bacteria and in response to environmental stimuli. Finally, we consider the role of NHEJ in genome evolution, notably in horizontal gene transfer.
Asunto(s)
Bacterias/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Regulación Bacteriana de la Expresión Génica , Transferencia de Gen Horizontal , Genoma Bacteriano , Recombinación HomólogaRESUMEN
Enterococcus faecalis is an opportunistic pathogen that has emerged as a major cause of nosocomial infections worldwide. Many clinical strains are indeed resistant to last resort antibiotics and there is consequently a reawakening of interest in exploiting virulent phages to combat them. However, little is still known about phage receptors and phage resistance mechanisms in enterococci. We made use of a prophageless derivative of the well-known clinical strain E. faecalis V583 to isolate a virulent phage belonging to the Picovirinae subfamily and to the P68 genus that we named Idefix. Interestingly, most isolates of E. faecalis tested-including V583-were resistant to this phage and we investigated more deeply into phage resistance mechanisms. We found that E. faecalis V583 prophage 6 was particularly efficient in resisting Idefix infection thanks to a new abortive infection (Abi) mechanism, which we designated Abiα. It corresponded to the Pfam domain family with unknown function DUF4393 and conferred a typical Abi phenotype by causing a premature lysis of infected E. faecalis. The abiα gene is widespread among prophages of enterococci and other Gram-positive bacteria. Furthermore, we identified two genes involved in the synthesis of the side chains of the surface rhamnopolysaccharide that are important for Idefix adsorption. Interestingly, mutants in these genes arose at a frequency of ~10-4 resistant mutants per generation, conferring a supplemental bacterial line of defense against Idefix.
Asunto(s)
Bacteriófagos/patogenicidad , Enterococcus faecalis/genética , Enterococcus faecalis/virología , Podoviridae/patogenicidad , Bacteriófagos/aislamiento & purificación , Genoma Viral , Fenotipo , Profagos/genética , Aguas del Alcantarillado/virología , Virulencia , Secuenciación Completa del GenomaRESUMEN
Bacteriophages are remarkable for the wide diversity of proteins they encode to perform DNA replication and homologous recombination. Looking back at these ancestral forms of life may help understanding how similar proteins work in more sophisticated organisms. For instance, the Sak4 family is composed of proteins similar to the archaeal RadB protein, a Rad51 paralog. We have previously shown that Sak4 allowed single-strand annealing in vivo, but only weakly compared to the phage λ Redß protein, highlighting putatively that Sak4 requires partners to be efficient. Here, we report that the purified Sak4 of phage HK620 infecting Escherichia coli is a poorly efficient annealase on its own. A distant homolog of SSB, which gene is usually next to the sak4 gene in various species of phages, highly stimulates its recombineering activity in vivo. In vitro, Sak4 binds single-stranded DNA and performs single-strand annealing in an ATP-dependent way. Remarkably, the single-strand annealing activity of Sak4 is stimulated by its cognate SSB. The last six C-terminal amino acids of this SSB are essential for the binding of Sak4 to SSB-covered single-stranded DNA, as well as for the stimulation of its annealase activity. Finally, expression of sak4 and ssb from HK620 can promote low-level of recombination in vivo, though Sak4 and its SSB are unable to promote strand exchange in vitro. Regarding its homology with RecA, Sak4 could represent a link between two previously distinct types of recombinases, i.e., annealases that help strand exchange proteins and strand exchange proteins themselves.
RESUMEN
Non-homologous end-joining (NHEJ) is a double strand break (DSB) repair pathway which does not require any homologous template and can ligate two DNA ends together. The basic bacterial NHEJ machinery involves two partners: the Ku protein, a DNA end binding protein for DSB recognition and the multifunctional LigD protein composed a ligase, a nuclease and a polymerase domain, for end processing and ligation of the broken ends. In silico analyses performed in the 38 sequenced genomes of Streptomyces species revealed the existence of a large panel of NHEJ-like genes. Indeed, ku genes or ligD domain homologues are scattered throughout the genome in multiple copies and can be distinguished in two categories: the "core" NHEJ gene set constituted of conserved loci and the "variable" NHEJ gene set constituted of NHEJ-like genes present in only a part of the species. In Streptomyces ambofaciens ATCC23877, not only the deletion of "core" genes but also that of "variable" genes led to an increased sensitivity to DNA damage induced by electron beam irradiation. Multiple mutants of ku, ligase or polymerase encoding genes showed an aggravated phenotype compared to single mutants. Biochemical assays revealed the ability of Ku-like proteins to protect and to stimulate ligation of DNA ends. RT-qPCR and GFP fusion experiments suggested that ku-like genes show a growth phase dependent expression profile consistent with their involvement in DNA repair during spores formation and/or germination.
RESUMEN
Non-homologous end joining is a ligation process repairing DNA double strand breaks in eukaryotes and many prokaryotes. The ring structured eukaryotic Ku binds DNA ends and recruits other factors which can access DNA ends through the threading of Ku inward the DNA, making this protein a key ingredient for the scaffolding of the NHEJ machinery. However, this threading ability seems unevenly conserved among bacterial Ku. As bacterial Ku differ mainly by their C-terminus, we evaluate the role of this region in the loading and the threading abilities of Bacillus subtilis Ku and the stimulation of the DNA ligase LigD. We identify two distinct sub-regions: a ubiquitous minimal C-terminal region and a frequent basic C-terminal extension. We show that truncation of one or both of these sub-regions in Bacillus subtilis Ku impairs the stimulation of the LigD end joining activity in vitro. We further demonstrate that the minimal C-terminus is required for the Ku-LigD interaction, whereas the basic extension controls the threading and DNA bridging abilities of Ku. We propose that the Ku basic C-terminal extension increases the concentration of Ku near DNA ends, favoring the recruitment of LigD at the break, thanks to the minimal C-terminal sub-region.
RESUMEN
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
In prokaryotes, transcription results from the activity of a 400 kDa RNA polymerase (RNAP) protein complex composed of at least five subunits (2α, ß, ß', ω). To ensure adequate responses to changing environmental cues, RNAP activity is tightly controlled by means of interacting regulatory proteins. Here, we report the affinity-purification of the Bacillus subtilis RNAP complexes from cells in different growth states and stress conditions, and the quantitative assessment by mass spectrometry of the dynamic changes in the composition of the RNAP complex. The stoichiometry of RNA polymerase was determined by a comparison of two mass spectrometry-based quantification methods: a label-based and a label-free method. The validated label-free method was then used to quantify the proteins associated with RNAP. The levels of sigma factors bound to RNAP varied during growth and exposure to stress. Elongation factors, helicases such as HelD and PcrA, and novel unknown proteins were also associated with RNAP complexes. The content in 6S RNAs of purified RNAP complexes increased at the onset of the stationary phase. These quantitative variations in the protein and RNA composition of the RNAP complexes well correlate with the known physiology of B. subtilis cells under different conditions.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Marcadores de Afinidad , Bacillus subtilis/genética , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Cromatografía de Afinidad , ARN Polimerasas Dirigidas por ADN/análisis , Electroforesis en Gel de Poliacrilamida , Complejos Multiproteicos/análisis , Complejos Multiproteicos/metabolismo , Subunidades de Proteína/análisis , Subunidades de Proteína/metabolismo , Proteómica , ARN Bacteriano/análisis , ARN Bacteriano/metabolismo , ARN no Traducido , Factor sigma/análisis , Factor sigma/metabolismo , Espectrometría de Masas en TándemRESUMEN
We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSB(Cter)) as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSB(Cter) interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSB(Cter) deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSB(Cter) acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/genética , Replicación del ADN , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Cromosomas Bacterianos/metabolismo , ADN Bacteriano/genética , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Deinococcus radiodurans is an extraordinarily radioresistant bacterium that is able to repair hundreds of radiation-induced double-stranded DNA breaks. One of the players in this pathway is an X family DNA polymerase (PolX(Dr)). Deletion of PolX(Dr) has been shown to decrease the rate of repair of double-stranded DNA breaks and increase cell sensitivity to gamma-rays. A 3'-->5' exonuclease activity that stops cutting close to DNA loops has also been demonstrated. The present crystal structure of PolX(Dr) solved at 2.46-A resolution reveals that PolX(Dr) has a novel extended conformation in stark contrast to the closed "right hand" conformation commonly observed for DNA polymerases. This extended conformation is stabilized by the C-terminal PHP domain, whose putative nuclease active site is obstructed by its interaction with the polymerase domain. The overall conformation and the presence of non standard residues in the active site of the polymerase X domain makes PolX(Dr) the founding member of a novel class of polymerases involved in DNA repair but whose detailed mode of action still remains enigmatic.
Asunto(s)
Proteínas Bacterianas/química , ADN Polimerasa Dirigida por ADN/química , Deinococcus/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN/efectos de la radiación , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Deinococcus/genética , Rayos gamma , Eliminación de Gen , Estructura Terciaria de Proteína/fisiología , Relación Estructura-ActividadRESUMEN
DNA topoisomerases resolve DNA topological problems created during transcription, replication, and recombination. These ubiquitous enzymes are essential for cell viability and are highly potent targets for the development of antibacterial and antitumoral drugs. Type II enzymes catalyze the transfer of a DNA duplex through another one in an ATP-dependent mechanism. Because of its small size and sensitivity to antitumoral drugs, the archaeal DNA topoisomerase VI, a type II enzyme, is an excellent model for gaining further understanding of the organization and mechanism of these enzymes. We present the crystal structure of intact DNA topoisomerase VI bound to radicicol, an inhibitor of human topo II, and compare it to the conformation of the apo-protein as determined by small-angle X-ray scattering in solution. This structure, combined with a wealth of experimental data gathered on these enzymes, allows us to propose a structural model for the two-gate DNA transfer mechanism.
Asunto(s)
ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/metabolismo , ADN/metabolismo , Catálisis , Cristalografía por Rayos X , Topoisomerasa de ADN IV/química , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Sulfolobus/enzimologíaRESUMEN
In bacteria, several salvage responses to DNA replication arrest culminate in reassembly of the replisome on inactivated forks to resume replication. The PriA DNA helicase is a prominent trigger of this replication restart process, preceded in many cases by a repair and/or remodeling of the arrested fork, which can be performed by many specific proteins. The mechanisms that target these rescue effectors to damaged forks in the cell are unknown. We report that the single-stranded DNA binding (SSB) protein is the key factor that links PriA to active chromosomal replication forks in vivo. This targeting mechanism determines the efficiency by which PriA reaches its specific DNA-binding site in vitro and directs replication restart in vivo. The RecG and RecQ DNA helicases, which are involved in intricate replication reactivation pathways, also associate with the chromosomal replication forks by similarly interacting with SSB. These results identify SSB as a platform for linking a 'repair toolbox' with active replication forks, providing a first line of rescue responses to accidental arrest.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/metabolismo , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , RecQ Helicasas/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Cromosomas Bacterianos/genética , Reparación del ADN/fisiología , Proteínas de Unión al ADN/genética , RecQ Helicasas/genéticaRESUMEN
To evaluate the importance of RecA in DNA double-strand break (DSB) repair, we examined the effect of low and high RecA concentrations such as 2500 and 100 000 molecules per cell expressed from the inducible Pspac promoter in Deinococcus radiodurans in absence or in presence of IPTG respectively. We showed that at low concentration, RecA has a negligible effect on cell survival after gamma-irradiation when bacteria were immediately plated on TGY agar whereas it significantly decreased the survival to gamma-irradiation of DeltaddrA cells while overexpression of RecA can partially compensate the loss of DdrA protein. In contrast, when cells expressing limited concentration of RecA were allowed to recover in TGY2X liquid medium, they showed a delay in mending DSB, failed to reinitiate DNA replication and were committed to die during incubation. A deletion of irrE resulted in sensitivity to gamma-irradiation and mitomycin C treatment. Interestingly, constitutive high expression of RecA compensates partially the DeltairrE sensitization to mitomycin C. The cells with low RecA content also failed to cleave LexA after DNA damage. However, neither a deletion of the lexA gene nor the expression of a non-cleavable LexA(Ind-) mutant protein had an effect on survival or kinetics of DNA DSB repair compared with their lexA+ counterparts in recA+ as well as in bacteria expressing limiting concentration of RecA, suggesting an absence of relationship between the absence of LexA cleavage and the loss of viability or the delay in the kinetics of DSB repair. Thus, LexA protein seems to play no major role in the recovery processes after gamma-irradiation in D. radiodurans.
Asunto(s)
Proteínas Bacterianas/metabolismo , Reparación del ADN , Deinococcus/genética , Rec A Recombinasas/metabolismo , Proteínas Bacterianas/genética , Supervivencia Celular , Daño del ADN , Deinococcus/metabolismo , Deinococcus/efectos de la radiación , Rayos gamma , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Rec A Recombinasas/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
Interactions between dendritic cells and Mycobacterium tuberculosis, the aetiological agent of tuberculosis in humans, are thought to be central to anti-mycobacterial immunity. We have previously shown that M. tuberculosis binds to human monocyte-derived dendritic cells mostly through the C-type lectin DC-SIGN (dendritic-cell-specific intercellular molecule-3-grabbing non-integrin)/CD209, and we have suggested that DC-SIGN may discriminate between mycobacterial species through recognition of the mannose-capping residues on the lipoglycan lipoarabinomannan of the bacterial envelope. Here, using a variety of fast- and slow-growing Mycobacterium species, we provide further evidence that mycobacteria recognition by DC-SIGN may be restricted to species of the M. tuberculosis complex. Fine analyses of the lipoarabinomannan molecules purified from these species show that the structure and amount of these molecules alone cannot account for such a preferential recognition. We propose that M. tuberculosis recognition by DC-SIGN relies on both a potential difference of accessibility of lipoarabinomannan in its envelope and, more probably, on the binding of additional ligands, possibly including lipomannan, mannose-capped arabinomannan, as well as the mannosylated 19 kDa and 45 kDa [Apa (alanine/proline-rich antigen)] glycoproteins. Altogether, our results reveal that the molecular basis of M. tuberculosis binding to DC-SIGN is more complicated than previously thought and provides further insight into the mechanisms of M. tuberculosis recognition by the immune system.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Lectinas Tipo C/metabolismo , Mycobacterium tuberculosis/metabolismo , Receptores de Superficie Celular/metabolismo , Adhesión Bacteriana , Células HeLa , Humanos , Unión Proteica , Especificidad de la Especie , Especificidad por SustratoRESUMEN
DNA polymerases of the X family have been implicated in a variety of DNA repair processes in eukaryotes. Here we show that Deinococcus radiodurans, a highly radioresistant bacterium able to mend hundreds of radiation-induced double-stranded DNA breaks, expresses a DNA polymerase belonging to the X family. This novel bacterial polymerase, named PolX(Dr), was identified as the product of the Deinococcal DR0467 gene. The purified PolX(Dr) protein possesses a DNA polymerase activity that is stimulated by MnCl2, a property of the X family DNA polymerases. Antibodies raised against PolX(Dr) recognized human pol lambda, rat pol beta and yeast Pol4 and, conversely, antibodies raised against these proteins recognized PolX(Dr). This immunological cross-reactivity suggests a high degree of structural conservation among the polymerases of the X family. Lack of PolX(Dr) reduced the rate of repair of double-stranded DNA breaks and increased cell sensitivity to gamma-rays. PolX(Dr) thus appears to play an important role in double-stranded DNA break repair in D. radiodurans.
Asunto(s)
Proteínas Bacterianas/metabolismo , Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Deinococcus/enzimología , Deinococcus/genética , Animales , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , División Celular , ADN Polimerasa Dirigida por ADN/clasificación , ADN Polimerasa Dirigida por ADN/genética , Deinococcus/efectos de la radiación , Rayos gamma , Genoma Bacteriano , Humanos , Ratas , Homología de Secuencia de Ácido Nucleico , Rayos UltravioletaRESUMEN
Deinococcus radiodurans possesses an exceptional capacity to withstand the lethal and mutagenic effects of most form of DNA damage and has received considerable interest for use in both fundamental and applied research. Here we describe vectors that allow regulated expression of Deinococcal genes for functional analysis. The vectors contain the IPTG-regulated Spac system (Pspac promoter and lacI repressor gene), originally designed for Bacillus subtilis, that we have adapted to be functional in D. radiodurans. We show that the Spac system can control the expression of a lacZ reporter gene over two orders of magnitude depending on the inducer concentration and the copy number of the lacI regulatory gene. Furthermore, we demonstrate that the Spac system can be used to regulate the synthesis of a critical repair protein, such as RecA, resulting in a conditional mitomycin-resistant cell phenotype. We have also developed tools for the construction of conditional mutants where the expression of the target gene is regulated by an inducible promoter. The utility of these conditional gene inactivation systems is exemplified by the conditional lethal phenotype of a mutant expressing gyrA from the Pspac promoter.
Asunto(s)
Deinococcus/genética , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Girasa de ADN/genética , Deinococcus/efectos de los fármacos , Deinococcus/crecimiento & desarrollo , Genes Esenciales/genética , Isopropil Tiogalactósido/farmacología , Operón Lac/genética , Mutación , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Rec A Recombinasas/genética , beta-Galactosidasa/metabolismoRESUMEN
Many different modified nucleotides are found in naturally occurring tRNA, especially in the anticodon region. Their importance for the efficiency of the translational process begins to be well documented. Here we have analyzed the in vivo effect of deleting genes coding for yeast tRNA-modifying enzymes, namely Pus1p, Pus3p, Pus4p, or Trm4p, on termination readthrough and +1 frameshift events. To this end, we have transformed each of the yeast deletion strains with a lacZ-luc dual-reporter vector harboring selected programmed recoding sites. We have found that only deletion of the PUS3 gene, encoding the enzyme that introduces pseudouridines at position 38 or 39 in tRNA, has an effect on the efficiency of the translation process. In this mutant, we have observed a reduced readthrough efficiency of each stop codon by natural nonsense suppressor tRNAs. This effect is solely due to the absence of pseudouridine 38 or 39 in tRNA because the inactive mutant protein Pus3[D151A]p did not restore the level of natural readthrough. Our results also show that absence of pseudouridine 39 in the slippery tRNA(UAG)(Leu) reduces +1 frameshift efficiency. Therefore, the presence of pseudouridine 38 or 39 in the tRNA anticodon arm enhances misreading of certain codons by natural nonsense tRNAs as well as promotes frameshifting on slippery sequences in yeast.