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

Abstract

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...

A Novel Cold-Adapted and Salt-Tolerant RNase R from Antarctic Sea-Ice Bacterium Psychrobacter sp. ANT206

“…The psrnr gene was seen to be 2313 bp in length (GenBank accession numbers MK624989) and to encode a protein with 770 amino acids, while the length of the rnr gene from Pseudomonas syringae Lz4W was seen to be 2658 bp, encoding 885 amino acids [12]. Based on sequence alignments with the related RNase Rs, the residues (Thr 646, Phe 654, Leu 665 and His 667) were identified as the catalytic sites in the sequence of PsRNR (Figure 1).…”

“…A growing number of evidence has indicated that RNase R is a cold shock protein (CspA), and its expression regulation is closely related to microbial adaptation [10,11]. It is well-known that the recruitment of RNase R to the degradosome complex is related to its need for low temperatures during growth [12]. It has been discovered that RNase R in Pseudomonas syringae plays an important role in growth at low temperature [13].…”

“…More importantly, RNase R is necessary for maintaining the precise amount of active ribosomal complexes required for proper mRNA translation, and, accordingly, RNase R might be a promising target for antimicrobial treatment [4]. Due to the function of RNase R in RNA metabolism and biological activities [3,12], it might play an important role in the understanding of virulence associated with enterobacteria and have great value in antimicrobial treatment.…”

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

“…This Gram-negative nonfluorescent pseudomonad was isolated from soil samples from the Schirmacher Oasis and Antarctica (6). Our studies have yielded novel insights into various mechanisms of psychrophilic adaptation that include modifications in lipopolysaccharides (7, 8), the RecBCD complex (9, 10, 11), RNA polymerase (12, 13), and a novel RNA degradosome with exoribonuclease RNase R (14, 15, 16). We and others have generated a transposon-mutagenized library of cold-sensitive mutants which do not grow at 4°C but grow at 22°C, and their suppressors, for characterization of essential genes for growth at low temperatures (9, 17; M. K. Ray, unpublished results).…”

Molecular insights into the ecology of a psychrotolerant Pseudomonas syringae

“…It exhibited defects in the degradation of regulatory transfer-messenger RNA (tmRNA) suggesting that it plays a role in the turnover of tmRNAs (Purusharth et al, 2007;Sulthana et al, 2011). Decreased protein synthesis at 4°C in rnr mutants indicated that the accumulation of unprocessed 16S rRNAs in polysomes affects the protein translation machinery at the low temperature and cause cell death at 4°C (Purusharth et al, 2007;Sulthana et al, 2011) (Fig.3a). Ribosomal RNAs and tmRNAs possess different secondary structured elements, including hairpin duplexes and pseudoknots.…”