RecD Plays an Essential Function During Growth at Low Temperature in the Antarctic Bacterium <i>Pseudomonas syringae</i> Lz4W

Regha, Kakkad; Satapathy, Ajit K.; Ray, Malay K.

doi:10.1534/genetics.104.038943

Cited by 29 publications

(64 citation statements)

References 48 publications

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“…The psychrophilic P. syringae strain Lz4W (32) was grown in Antarctic bacterial medium (ABM) composed of 5 g of peptone and 2.5 g of yeast extract liter Ϫ1 or on ABM-agar (1.5%), as described earlier (30). E. coli cells were grown in Luria-Bertani medium (31).…”

Section: Methodsmentioning

confidence: 99%

rnr Gene from the Antarctic Bacterium Pseudomonas syringae Lz4W, Encoding a Psychrophilic RNase R

Sulthana

Rajyaguru

Mittal

et al. 2011

Appl Environ Microbiol

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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 His 6 -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 ϳ25°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 (Mg 2؉ and Mn 2؉ ) 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-␥S 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.RNA processing and degradation play a crucial role in the regulation of gene expression. Although a number of proteins, such as endoribonucleases, exoribonucleases, helicases, and RNA-binding proteins, work in a concerted manner to bring about processing and/or degradation of target RNAs, exoribonucleases play a major role in these processes (2, 11). Bacterial exoribonucleases generally show the 3Ј-5Ј polarity of degradation, with one notable exception (20). RNase R, which is a member of RNB/RNR superfamily of exoribonucleases, exhibits (3Ј35Ј) polar activity with a hydrolytic mode of action and plays an important role in bacterial physiology (11). It was first discovered as a product of vacB, an essential gene for virulence in Shigella flexneri, and later rediscovered in Escherichia coli as RNase R for its action on rRNA (10). This highly processive enzyme has been implicated in quality control of rRNA, mRNA degradation and tmRNA processing. Subsequently, RNase R was shown to be upregulated in response to multiple stress conditions such as cold shock, stationary phase, and starvation in E. coli (1, 5, 7). Although most of these studies were performed in E. coli, the gene (rnr) encoding RNase R has been observed in all sequenced bacterial genomes including in Mycoplasma that has the smallest genome of all freeliving organisms. Recently, studie...

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Section: Methodsmentioning

confidence: 99%

rnr Gene from the Antarctic Bacterium Pseudomonas syringae Lz4W, Encoding a Psychrophilic RNase R

Sulthana

Rajyaguru

Mittal

et al. 2011

Appl Environ Microbiol

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“…4 For growth analysis, bacterial cells from overnight culture were inoculated into fresh medium at a dilution of 1:100, and the turbidity of the cultures at 600 nm (A 600 ) was measured at various time intervals. For complementation studies, the plasmids were introduced into P. syringae strain by conjugation with E. coli S17-1 (19).…”

Section: Methodsmentioning

confidence: 99%

“…The resultant plasmid pBS-rnr was linearized with HincII, and a tetracycline resistance gene was cloned within the rnr to generate pBSrnr-tet plasmid. The source of tetracycline resistance gene (tet) cassette (2.4 kilobase pair) was pTc28 plasmid (19), from which it was cleaved out as XbaIHindIII fragment and made blunt-ended using Klenow enzyme before ligating to the HincII site of pBS-rnr. Plasmid construct was confirmed by sequencing and PCR analysis.…”

Section: Methodsmentioning

confidence: 99%

“…For each time point, CFUs on plates (in triplicates) were counted. The cells in cultures at each time point were also examined microscopically (19) by staining them with Syto9 and propidium iodide (PI) using LIVE/DEAD Baclight bacterial viability kit (Molecular Probes, Eugene, OR). Isolation and Separation of Ribosome-Ribosomes were isolated and separated on sucrose gradient in presence of 10 mM or 0.3 mM MgCl 2 depending upon the experimental requirement as described earlier (23).…”

Section: Methodsmentioning

confidence: 99%

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Exoribonuclease R in Pseudomonas syringae Is Essential for Growth at Low Temperature and Plays a Novel Role in the 3′ End Processing of 16 and 5 S Ribosomal RNA

Rajyaguru

Madhuri

Ray

2007

Journal of Biological Chemistry

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The (3 3 5) exoribonuclease RNase R interacts with the endoribonuclease RNase E in the degradosome of the coldadapted bacterium Pseudomonas syringae Lz4W. We now present evidence that the RNase R is essential for growth of the organism at low temperature (4°C). Mutants of P. syringae with inactivated rnr gene (encoding RNase R) are cold-sensitive and die upon incubation at 4°C, a phenotype that can be complemented by expressing RNase R in trans. Overexpressing polyribonucleotide phosphorylase in the rnr mutant does not rescue the cold sensitivity. This is different from the situation in Escherichia coli, where rnr mutants show normal growth, but pnp (encoding polyribonucleotide phosphorylase) and rnr double mutants are nonviable. Interestingly, RNase R is not cold-inducible in P. syringae. Remarkably, however, rnr mutants of P. syringae at low temperature (4°C) accumulate 16 and 5 S ribosomal RNA (rRNA) that contain untrimmed extra ribonucleotide residues at the 3 ends. This suggests a novel role for RNase R in the rRNA 3 end processing. Unprocessed 16 S rRNA accumulates in the polysome population, which correlates with the inefficient protein synthesis ability of mutant. An additional role of RNase R in the turnover of transfer-messenger RNA was identified from our observation that the rnr mutant accumulates transfer-messenger RNA fragments in the bacterium at 4°C. Taken together our results establish that the processive RNase R is crucial for RNA metabolism at low temperature in the coldadapted Antarctic P. syringae.Regulated degradation of RNA within cells is mostly an outcome of coordinated and combined activities of endoribonucleases, exoribonucleases, and RNA helicases. In bacteria, few of these enzymes interact with each other to form the RNA degradosome complex (1-3). The degradosome has been shown to be involved in both mRNA and rRNA degradation (4, 5). In the Escherichia coli degradosome, RNase E (a 5Ј end-dependent endoribonuclease) associates with polynucleotide phosphorylase (PNPase, 2 a 3Ј 3 5Ј exoribonuclease), RhlB (a DEAD box RNA helicase), and enolase (an enzyme of glycolytic pathway) to constitute the "core" complex. Additionally, DnaK, poly(A) polymerase and polyphosphate kinase have also been reported to be a part of this complex (6). A similar kind of RNA degrading complex has also been reported from Rhodobacter capsulatus (7). On the other hand, we have recently shown that exoribonuclease RNase R interacts with RNase E in the degradosome of the cold-adapted Antarctic bacterium Pseudomonas syringae Lz4W (8). This bacterium does have a pnp gene that is expressed but does not form a part of this complex.3 PNPase and RNase R differ in their mode of action, the former exhibiting phosphorolytic activity and the latter exhibiting hydrolytic activity.RNase R is one of the eight exoribonucleases reported in E. coli and distributed widely in different prokaryotes (9). Although all of these exoribonucleases display 3Ј 3 5Ј activity on RNA substrate, RNase R is the most highly processive enzyme among ...

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“…However, recombination in recD cells becomes heavily dependent on the activities of other exonucleases, in particular, RecJ and exonuclease VII, both capable of ssDNA degradation in the 5Ј33Ј direction (80,83,183,184,187). RecD function also appears to be important when restriction alleviation mechanisms are impaired (194), for the degradation of restricted phage (255), and for survival in high-pressure or low-temperature environments (36,229). In species that lack RecBCD homologues, the function of an apparently solo RecD-like protein is needed for resistance to oxidants (250,327), gamma rays, and UV light (250) or for pilin variation (56).…”

Section: Discoverymentioning

confidence: 99%

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

Dillingham

Kowalczykowski

2008

Microbiol Mol Biol Rev

488

580

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SUMMARY The RecBCD enzyme of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. It also degrades linear double-stranded DNA, protecting the bacteria from phages and extraneous chromosomal DNA. The RecBCD enzyme is, however, regulated by a cis-acting DNA sequence known as Chi (crossover hotspot instigator) that activates its recombination-promoting functions. Interaction with Chi causes an attenuation of the RecBCD enzyme's vigorous nuclease activity, switches the polarity of the attenuated nuclease activity to the 5′ strand, changes the operation of its motor subunits, and instructs the enzyme to begin loading the RecA protein onto the resultant Chi-containing single-stranded DNA. This enzyme is a prototypical example of a molecular machine: the protein architecture incorporates several autonomous functional domains that interact with each other to produce a complex, sequence-regulated, DNA-processing machine. In this review, we discuss the biochemical mechanism of the RecBCD enzyme with particular emphasis on new developments relating to the enzyme's structure and DNA translocation mechanism.

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RecD Plays an Essential Function During Growth at Low Temperature in the Antarctic Bacterium Pseudomonas syringae Lz4W

Cited by 29 publications

References 48 publications

rnr Gene from the Antarctic Bacterium Pseudomonas syringae Lz4W, Encoding a Psychrophilic RNase R

rnr Gene from the Antarctic Bacterium Pseudomonas syringae Lz4W, Encoding a Psychrophilic RNase R

Exoribonuclease R in Pseudomonas syringae Is Essential for Growth at Low Temperature and Plays a Novel Role in the 3′ End Processing of 16 and 5 S Ribosomal RNA

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

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