RESUMO
RNase R (encoded by the rnr gene) is a highly processive 3' â 5' exoribonuclease essential for the growth of the psychrotrophic bacterium Pseudomonas syringae Lz4W at low temperature. The cell death of a rnr deletion mutant at low temperature has been previously attributed to processing defects in 16S rRNA, defective ribosomal assembly, and inefficient protein synthesis. We recently showed that RNase R is required to protect P. syringae Lz4W from DNA damage and oxidative stress, independent of its exoribonuclease activity. Here, we show that the processing defect in 16S rRNA does not cause cell death of the rnr mutant of P. syringae at low temperature. Our results demonstrate that the rnr mutant of P. syringae Lz4W, complemented with a RNase R deficient in exoribonuclease function (RNase RD284A), is defective in 16S rRNA processing but can grow at 4 °C. This suggested that the processing defect in ribosomal RNAs is not a cause of the cold sensitivity of the rnr mutant. We further show that the rnr mutant accumulates copies of the indigenous plasmid pLz4W that bears a type II toxin-antitoxin (TA) system (P. syringae antitoxin-P. syringae toxin). This phenotype was rescued by overexpressing antitoxin psA in the rnr mutant, suggesting that activation of the type II TA system leads to cold sensitivity of the rnr mutant of P. syringae Lz4W. Here, we report a previously unknown functional relationship between the cold sensitivity of the rnr mutant and a type II TA system in P. syringae Lz4W.
RESUMO
IMPORTANCE: Bacterial exoribonucleases play a crucial role in RNA maturation, degradation, quality control, and turnover. In this study, we have uncovered a previously unknown role of 3'-5' exoribonuclease RNase R of Pseudomonas syringae Lz4W in DNA damage and oxidative stress response. Here, we show that neither the exoribonuclease function of RNase R nor its association with the RNA degradosome complex is essential for this function. Interestingly, in P. syringae Lz4W, hydrolytic RNase R exhibits physiological roles similar to phosphorolytic 3'-5' exoribonuclease PNPase of E. coli. Our data suggest that during the course of evolution, mesophilic E. coli and psychrotrophic P. syringae have apparently swapped these exoribonucleases to adapt to their respective environmental growth conditions.
Assuntos
Escherichia coli , Exorribonucleases , Exorribonucleases/genética , Exorribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , Regiões Antárticas , Dano ao DNA , Estresse Oxidativo , RNA Bacteriano/genéticaRESUMO
Exoribonuclease R (RNase R) is a 3' hydrolytic exoribonuclease that can degrade structured RNA. Mutation in RNase R affects virulence of certain human pathogenic bacteria. The aim of this study was to determine whether RNase R is necessary for virulence of the phytopathogen that causes bacterial blight in rice, Xanthomonas oryzae pv. oryzae (Xoo). In silico analysis has indicated that RNase R is highly conserved among various xanthomonads. Amino acid sequence alignment of Xoo RNase R with RNase R from various taxa indicated that Xoo RNase R clustered with RNase R of order Xanthomonadales. To study its role in virulence, we generated a gene disruption mutant of Xoo RNase R. The Xoo rnr- mutant is moderately virulence deficient, and the complementing strain (rnr-/pHM1::rnr) rescued the virulence deficiency of the mutant. We investigated swimming and swarming motilities in both nutrient-deficient minimal media and nutrient-optimal media. We observed that RNase R mutation has adversely affected the swimming and swarming motilities of Xoo in optimal media. However, in nutrient-deficient media only swimming motility was noticeably affected. Growth curves in optimal media at suboptimal temperature (15°C cold stress) indicate that the Xoo rnr- mutant grows more slowly than the Xoo wild type and complementing strain (rnr-/pHM1::rnr). Given these findings, we report for the first time that RNase R function is necessary for complete virulence of Xoo in rice. It is also important for motility of Xoo in media and for growth of Xoo at suboptimal temperature.
Assuntos
Oryza , Xanthomonas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Exorribonucleases/metabolismo , Oryza/microbiologia , Doenças das Plantas/microbiologia , VirulênciaRESUMO
Low temperatures constrain cellular life due to reductions in nutrient uptake, enzyme kinetics, membrane permeability, and function of other biomacromolecules. This has implications for the biophysical limits of life on Earth, and the plausibility of life in extraterrestrial locations. Although most pseudomonads are mesophilic in nature, isolates such as the Antarctic Pseudomonas syringae Lz4W exhibit considerable psychrotolerance, with an ability to grow even between 4 and 0°C. In this review, we explore the molecular traits and characteristic phenotypes of P. syringae Lz4W that enable life at low temperatures. We describe adaptations that enhance membrane fluidity; examine genes involved in cellular function and survival in the cold; assess capability for energy generation at low temperature; and detail the mechanics of DNA repair and RNA processing at low temperature, and speculate that P. syringae Lz4W can also synthesize glycerol to maintain flexibility of macromolecular systems. In the range 4 to 0ºC, there are considerable changes in the properties and behaviour of water. Specifically, density can have adverse impacts on plasma-membrane functions, cytoplasmic viscosity, protein behaviour, and other essential properties of cellular system. We identified a combination of adaptations that may be peculiar to cold-tolerant P. syringae, including increase of unsaturated fatty acids in the plasma membrane; a RNA polymerase able to function at 0°C; RecBCD- and RuvAB-dependent reestablishment of replication fork; and efficiencies of degradosome machinery and RNA processing by RNaseR at low temperature. Several unresolved questions are discussed in the context of astrobiology, and further work needed on the psychrotolerance of P. syringae.
Assuntos
Temperatura Baixa , Pseudomonas syringae , Aclimatação , Reparo do DNA/genética , Fenótipo , Pseudomonas syringae/genéticaRESUMO
Many organisms are subject to selective pressure that gives rise to unequal usage of synonymous codons, known as codon bias. To experimentally dissect the mechanisms of selection on synonymous sites, we expressed several hundred synonymous variants of the GFP gene in Escherichia coli, and used quantitative growth and viability assays to estimate bacterial fitness. Unexpectedly, we found many synonymous variants whose expression was toxic to E. coli Unlike previously studied effects of synonymous mutations, the effect that we discovered is independent of translation, but it depends on the production of toxic mRNA molecules. We identified RNA sequence determinants of toxicity and evolved suppressor strains that can tolerate the expression of toxic GFP variants. Genome sequencing of these suppressor strains revealed a cluster of promoter mutations that prevented toxicity by reducing mRNA levels. We conclude that translation-independent RNA toxicity is a previously unrecognized obstacle in bacterial gene expression.
Assuntos
Códon/metabolismo , Escherichia coli/metabolismo , Mutação , RNA Bacteriano/metabolismo , Códon/genética , Escherichia coli/genética , RNA Bacteriano/genéticaRESUMO
Chromosomal damage was detected previously in the recBCD mutants of the Antarctic bacterium Pseudomonas syringaeâ Lz4W, which accumulated linear chromosomal DNA leading to cell death and growth inhibition at 4°C. RecBCD protein generally repairs DNA double-strand breaks by RecA-dependent homologous recombination pathway. Here we show that ΔrecA mutant of P. syringae is not cold-sensitive. Significantly, inactivation of additional DNA repair genes ruvAB rescued the cold-sensitive phenotype of ΔrecBCD mutant. The ΔrecA and ΔruvAB mutants were UV-sensitive as expected. We propose that, at low temperature DNA replication encounters barriers leading to frequent replication fork (RF) arrest and fork reversal. RuvAB binds to the reversed RFs (RRFs) having Holliday junction-like structures and resolves them upon association with RuvC nuclease to cause linearization of the chromosome, a threat to cell survival. RecBCD prevents this by degrading the RRFs, and facilitates replication re-initiation. This model is consistent with our observation that low temperature-induced DNA lesions do not evoke SOS response in P. syringae. Additional studies show that two other repair genes, radA (encoding a RecA paralogue) and recF are not involved in providing cold resistance to the Antarctic bacterium.
Assuntos
Ciclo Celular/efeitos da radiação , Replicação do DNA/efeitos da radiação , Pseudomonas syringae/fisiologia , Pseudomonas syringae/efeitos da radiação , Regiões Antárticas , Temperatura Baixa , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Deleção de Genes , Pseudomonas syringae/enzimologia , Pseudomonas syringae/isolamento & purificaçãoRESUMO
RNase R is a highly processive, hydrolytic 3'-5' exoribonuclease belonging to the RNB/RNR superfamily which plays significant roles in RNA metabolism in bacteria. The enzyme was observed to be essential for growth of the psychrophilic Antarctic bacterium Pseudomonas syringae Lz4W at a low temperature. We present results here pertaining to the biochemical properties of RNase R and the RNase R-encoding gene (rnr) locus from this bacterium. By cloning and expressing a His6-tagged form of the P. syringae RNase R (RNase R(Ps)), we show that the enzyme is active at 0 to 4°C but exhibits optimum activity at â¼25°C. The enzyme is heat labile in nature, losing activity upon incubation at 37°C and above, a hallmark of many psychrophilic enzymes. The enzyme requires divalent cations (Mg²âº and Mn²âº) for activity, and the activity is higher in 50 to 150 mM KCl when it largely remains as a monomer. On synthetic substrates, RNase R(Ps) exhibited maximum activity on poly(A) and poly(U) in preference over poly(G) and poly(C). The enzyme also degraded structured malE-malF RNA substrates. Analysis of the cleavage products shows that the enzyme, apart from releasing 5'-nucleotide monophosphates by the processive exoribonuclease activity, produces four-nucleotide end products, as opposed to two-nucleotide products, of RNA chain by Escherichia coli RNase R. Interestingly, three ribonucleotides (ATP, GTP, and CTP) inhibited the activity of RNase R(Ps) in vitro. The ability of the nonhydrolyzable ATP-γS to inhibit RNase R(Ps) activity suggests that nucleotide hydrolysis is not required for inhibition. This is the first report on the biochemical property of a psychrophilic RNase R from any bacterium.