A type II toxin-antitoxin system is responsible for the cell death at low temperature in Pseudomonas syringae Lz4W lacking RNase R.

RNase R (encoded by the rnr gene) is a highly processive 3' → 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.

Exoribonuclease RNase R protects Antarctic Pseudomonas syringae Lz4W from DNA damage and oxidative stress.

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.

Xanthomonas oryzae pv. oryzae Exoribonuclease R Is Required for Complete Virulence in Rice, Optimal Motility, and Growth Under Stress.

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.

Replication arrest is a major threat to growth at low temperature in Antarctic Pseudomonas syringae†Lz4W.

Chromosomal damage was detected previously in the recBCD mutants of the Antarctic bacterium Pseudomonas syringae 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.

rnr gene from the antarctic bacterium Pseudomonas syringae Lz4W, encoding a psychrophilic RNase R.

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 ∼25°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 (Mg²âº and Mn²âº) 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.

ATPase activity of RecD is essential for growth of the Antarctic Pseudomonas syringae Lz4W at low temperature.

RecD is essential for growth at low temperature in the Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W. To examine the essential nature of its activity, we analyzed wild-type and mutant RecD proteins with substitutions of important residues in each of the seven conserved helicase motifs. The wild-type RecD displayed DNA-dependent ATPase and helicase activity in vitro, with the ability to unwind short DNA duplexes containing only 5' overhangs or forked ends. Five of the mutant proteins, K229Q (in motif I), D323N and E324Q (in motif II), Q354E (in motif III) and R660A (in motif VI) completely lost both ATPase and helicase activities. Three other mutants, T259A in motif Ia, R419A in motif IV and E633Q in motif V exhibited various degrees of reduction in ATPase activity, but had no helicase activity. While all RecD proteins had DNA-binding activity, the mutants of motifs IV and V displayed reduced binding, and the motif II mutant showed a higher degree of binding to ssDNA. Significantly, only RecD variants with in vitro ATPase activity could complement the cold-sensitive growth of a recD-inactivated strain of P. syringae at 4 degrees C. These results suggest that the requirement for RecD at lower temperatures lies in its ATP-hydrolyzing activity.

Biochemical characterization of RecBCD enzyme from an Antarctic Pseudomonas species and identification of its cognate Chi (χ) sequence.

Pseudomonas syringae Lz4W RecBCD enzyme, RecBCDPs, is a trimeric protein complex comprised of RecC, RecB, and RecD subunits. RecBCD enzyme is essential for P. syringae growth at low temperature, and it protects cells from low temperature induced replication arrest. In this study, we show that the RecBCDPs enzyme displays distinct biochemical behaviors. Unlike E. coli RecBCD enzyme, the RecD subunit is indispensable for RecBCDPs function. The RecD motor activity is essential for the Chi-like fragments production in P. syringae, highlighting a distinct role for P. syringae RecD subunit in DNA repair and recombination process. Here, we demonstrate that the RecBCDPs enzyme recognizes a unique octameric DNA sequence, 5'-GCTGGCGC-3' (ChiPs) that attenuates nuclease activity of the enzyme when it enters dsDNA from the 3'-end. We propose that the reduced translocation activities manifested by motor-defective mutants cause cold sensitivity in P. syrinage; emphasizing the importance of DNA processing and recombination functions in rescuing low temperature induced replication fork arrest.

RecD plays an essential function during growth at low temperature in the antarctic bacterium Pseudomonas syringae Lz4W.

The Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W has been used as a model system to identify genes that are required for growth at low temperature. Transposon mutagenesis was carried out to isolate mutant(s) of the bacterium that are defective for growth at 4 degrees but normal at 22 degrees . In one such cold-sensitive mutant (CS1), the transposon-disrupted gene was identified to be a homolog of the recD gene of several bacteria. Trans-complementation and freshly targeted gene disruption studies reconfirmed that the inactivation of the recD gene leads to a cold-sensitive phenotype. We cloned, sequenced, and analyzed approximately 11.2 kbp of DNA from recD and its flanking region from the bacterium. recD was the last gene of a putative recCBD operon. The RecD ORF was 694 amino acids long and 40% identical (52% similar) to the Escherichia coli protein, and it could complement the E. coli recD mutation. The recD gene of E. coli, however, could not complement the cold-sensitive phenotype of the CS1 mutant. Interestingly, the CS1 strain showed greater sensitivity toward the DNA-damaging agents, mitomycin C and UV. The inactivation of recD in P. syringae also led to cell death and accumulation of DNA fragments of approximately 25-30 kbp in size at low temperature (4 degrees ). We propose that during growth at a very low temperature the Antarctic P. syringae is subjected to DNA damage, which requires direct participation of a unique RecD function. Additional results suggest that a truncated recD encoding the N-terminal segment of (1-576) amino acids is sufficient to support growth of P. syringae at low temperature.

Backbone and stereospecific (13)C methyl Ile (δ1), Leu and Val side-chain chemical shift assignments of Crc.

Carbon catabolite repression (CCR) allows bacteria to selectively assimilate a preferred compound among a mixture of several potential carbon sources, thus boosting growth and economizing the cost of adaptability to variable nutrients in the environment. The RNA-binding catabolite repression control (Crc) protein acts as a global post-transcriptional regulator of CCR in Pseudomonas species. Crc triggers repression by inhibiting the expression of genes involved in transport and catabolism of non-preferred substrates, thus indirectly favoring assimilation of preferred one. We report here a nearly complete backbone and stereospecific (13)C methyl side-chain chemical shift assignments of Ile (δ1), Leu and Val of Crc (~ 31 kDa) from Pseudomonas syringae Lz4W.

Draft Genome Sequence of the Antarctic Psychrophilic Bacterium Pseudomonas syringae Strain Lz4W.

The psychrophilic bacterium Pseudomonas syringae strain Lz4W was isolated from soil samples from Antarctica to decipher the mechanisms of low-temperature adaptation. We report here the 4.982-Mb draft genome sequence of P. syringae Lz4W. This sequence will provide insights into the genomic basis of the psychrophilicity of this bacterium.

All three subunits of RecBCD enzyme are essential for DNA repair and low-temperature growth in the Antarctic Pseudomonas syringae Lz4W.

BACKGROUND: The recD mutants of the Antarctic Pseudomonas syringae Lz4W are sensitive to DNA-damaging agents and fail to grow at 4 degrees C. Generally, RecD associates with two other proteins (RecB and RecC) to produce RecBCD enzyme, which is involved in homologous recombination and DNA repair in many bacteria, including Escherichia coli. However, RecD is not essential for DNA repair, nor does its deletion cause any growth defects in E. coli. Hence, the assessment of the P. syringae RecBCD pathway was imperative. METHODOLOGY/PRINCIPAL FINDINGS: Mutational analysis and genetic complementation studies were used to establish that the individual null-mutations of all three genes, recC, recB, and recD, or the deletion of whole recCBD operon of P. syringae, lead to growth inhibition at low temperature, and sensitivity to UV and mitomycin C. Viability of the mutant cells dropped drastically at 4 degrees C, and the mutants accumulated linear chromosomal DNA and shorter DNA fragments in higher amounts compared to 22 degrees C. Additional genetic data using the mutant RecBCD enzymes that were inactivated either in the ATPase active site of RecB (RecB(K29Q)) or RecD (RecD(K229Q)), or in the nuclease center of RecB (RecB(D1118A) and RecB(Delta nuc)) suggested that, while the nuclease activity of RecB is not so critical in vivo, the ATP-dependent functions of both RecB and RecD are essential. Surprisingly, E. coli recBCD or recBC alone on plasmid could complement the defects of the Delta recCBD strain of P. syringae. CONCLUSIONS/SIGNIFICANCE: All three subunits of the RecBCD(Ps) enzyme are essential for DNA repair and growth of P. syringae at low temperatures (4 degrees C). The RecD requirement is only a function of the RecBCD complex in the bacterium. The RecBCD pathway protects the Antarctic bacterium from cold-induced DNA damages, and is critically dependent on the helicase activities of both RecB and RecD subunits, but not on the nuclease of RecBCD(Ps) enzyme.

Auxotrophy in natural isolate: minimal requirements for growth of the Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W.

A chemically defined minimal medium has been developed for growing the Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W, a model system for studying cold adaptation. This natural isolate from Antarctica has an absolute requirement for two branched chain amino acids, isoleucine and valine, in addition to low osmolality of the growth medium. The bacterium contains threonine deaminase but lacks acetohydroxyacid synthase suggesting that a defect lies in the isoleucine and valine biosynthetic pathway causing auxotrophy. Succinate was found to be preferred carbon source over glucose as it could suppress the glucose metabolizing enzymes in the cells, like in other pseudomonads. The development of the minimal medium (MM Lz) for growing the Antarctic P. syringae Lz4W strain would be useful for investigation of the catabolite repression control mechanism at a very low temperature (below 5 degrees C), which is predominant in vast area of our global ecosystems.