1 Study of rnr gene from the Antarctic bacterium Pseudomonas syringae Lz4W 1 that encodes a ...

31 Pages·2011·0.76 MB·English

AEM Accepts, published online ahead of print on 16 September 2011
Appl. Environ. Microbiol. doi:10.1128/AEM.05683-11
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Study of rnr gene from the Antarctic bacterium Pseudomonas syringae Lz4W
2 that encodes a psychrophilic RNase R
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4 Shaheen Sulthana1, Purusharth I Rajyaguru1,2, Pragya Mittal, and Malay K Ray*
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6 Centre for Cellular and Molecular Biology (CCMB),
7 Council of Scientific & Industrial Research (CSIR), D
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8 Uppal Road, Hyderabad 500007, India w
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10 1These two authors have contributed equally to the work. d
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11 2Present Address: Department of Molecular and Cellular Biology, University of Arizona, d
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12 Tucson, Arizona, 85721-0106, USA.
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14 *Corresponding Author: p
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15 Dr. Malay K Ray a
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16 Centre for Cellular and Molecular Biology
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17 Uppal Road, s
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18 Hyderabad 500007 .o
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19 India
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21 Tel. [phone] p
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22 Fax. [phone] il
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23 E-mail: [email protected] ,

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26 Running Title: Psychrophilic RNase R
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28 Key words: Psychrotroph/Psychrophile, Pseudomonas syringae, Exoribonuclease RNase R, rnr t
29 gene, RNA degradation.
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34 Abstract
35 RNase R is a highly processive, hydrolytic 3′-5′ exoribonuclease belonging to the
36 RNB/RNR superfamily which plays significant roles in RNA metabolism in bacteria. The
37 enzyme was observed to be essential for growth of the psychrophilic Antarctic bacterium
38 Pseudomonas syringae Lz4W at low temperature. Here we present results pertaining to the
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39 biochemical properties of RNase R and the RNase R-encoding gene (rnr) locus from this o
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40 bacterium. By cloning and expressing a 6×His tagged form of the P. syringae RNase R lo
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41 (RNaseRPs) we show that the enzyme is active at 0-4°C but exhibits optimum activity at e
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42 ~25°C. The enzyme is heat-labile in nature, losing activity upon incubation at 37°C and ro
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43 above, a hallmark of many psychrophilic enzymes. The enzyme requires divalent cations h
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44 (Mg2+ and Mn2+) for activity, and the activity is higher in 50-150 mM KCl when it largely :/
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45 remains as monomer. On synthetic substrates RNaseRPs exhibited maximum activity on m
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46 poly(A) and poly(U) in preference over poly(G) and poly(C). The enzyme also degraded s
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47 structured malE-malF RNA substrates. The analysis of the cleavage products shows that the r
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48 enzyme, apart from releasing 5′-nucleotide monophosphates by the processive o
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49 exoribonuclease activity, produces four-nucleotide end-products, as opposed to two- p
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50 nucleotide products, of RNA chain by Escherichia coli RNase R. Interestingly, three 1
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51 ribonucleotides (ATP, GTP, and CTP) inhibited the activity of RNaseRPs in vitro. The ability 20
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52 of the non-hydrolysable ATP-γS to inhibit RNaseRPs activity suggests that nucleotide
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53 hydrolysis is not required for inhibition. This is the first report on biochemical property of a g
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54 psychrophilic RNase R from any bacterium. t
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56 Introduction
57 RNA processing and degradation play a crucial role in the regulation of gene expression.
58 Although a number of proteins such as endoribonucleases, exoribonucleases, helicases, and
59 RNA-binding proteins work in a concerted manner to bring about processing/degradation of
60 target RNAs, the exoribonucleases play a major role in these processes (1, 11). Bacterial
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61 exoribonucleases generally show the 3′-5′ polarity of degradation, with one notable exception o
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62 (19). RNase R which is a member of RNB/RNR superfamily of exoribonucleases exhibits lo
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63 (3′→5′) polar activity with hydrolytic mode of action, and plays an important role in bacterial e
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64 physiology (11). It was first discovered as a product of vacB, an essential gene for virulence fr
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65 in Shigella flexneri, and later rediscovered in E. coli as RNase R for its action on ribosomal
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66 RNA (rRNA) (10). This highly processive enzyme has been implicated in quality control of :
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67 rRNA, mRNA degradation and tmRNA processing. Subsequently RNase R was shown to be
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68 upregulated in response to multiple stress conditions such as cold shock, stationary phase and s
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69 starvation in E. coli (2, 5, 7). Although most of these studies were performed in E. coli, the o
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70 gene (rnr) encoding RNase R has been observed in all sequenced bacterial genomes o
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71 including in Mycoplasma that has the smallest genome of all free-living organisms. Recently,
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72 studies have been initiated on the RNase R from many other bacteria (6, 12-14, 19, 24, 33). In il 1
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73 our studies with the psychrotrophic Pseudomonas syringae we observed that RNase R is a 2
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74 component of the novel degradosome, a multi-subunit RNA degrading complex that also 9

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75 comprises the endoribonuclease RNase E and RhlE helicase (25). More importantly, we g
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76 discovered that rnr knockout mutants of the psychrotroph were severely cold-sensitive. These s
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77 mutants were also defective in the processing of 3′-ends of 16S and 5S rRNAs. The study
78 implicated a new role for RNase R in rRNA processing, not yet shown in any other bacteria
79 (26). Recruitment of RNase R to the degradosome complex, its need during growth at low
80 temperature, and its novel role in rRNA processing prompted us to undertake biochemical
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81 characterization of this enzyme from P. syringae. Towards this goal we have characterized
82 the rnr gene locus and the RNase R enzyme from P. syringae Lz4W. This is the first report
83 on the characterization of this exoribonuclease from any psychrotrophic organism. We
84 present here the general properties of the RNaseRPs enzyme with respect to substrate
85 specificity, temperature optima, metal ion requirement, and activity on structured RNA
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86 substrates. o
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87 Materials and Methods lo
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88 Bacterial strains and growth conditions. The psychrophilic P. syringae Lz4W (32) was e
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89 grown in Antarctic Bacterial Medium (ABM) composed of 5 g l-1 peptone and 2.5 g l-1 yeast fr
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90 extract, or on ABM-agar (1.5%), as described earlier (30). E. coli cells were grown in Luria-
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91 Bertani medium (31). When required, growth media were supplemented with antibiotics: :
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92 ampicillin- 100, kanamycin- 50, and tetracycline- 20 μg ml-1. For growth analysis, bacterial em
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93 cells from overnight cultures were inoculated into fresh medium at a dilution of 1:100, and s
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94 the turbidity of the cultures at 600 nm (OD ) was measured at various time intervals. o
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95 Enzymes, reagents and general molecular biology techniques. All chemicals were o
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96 reagent grade. Nucleotides and the ATP analogue, ATP-γS, were purchased from Roche. p
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97 Restriction enzymes, T4 polynucleotide kinase, and other DNA modifying enzymes were 1
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98 bought from New England Biolabs (NEB, USA), unless otherwise mentioned. 2
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99 Oligonucleotides were bought from a commercial source (BioServe Biotechnology, India).
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100 Protein markers were from Amersham Biosciences. RNA markers were from Ambion (USA). g
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101 General molecular biology techniques including the isolation of genomic DNA, restriction s
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102 analysis, polymerase chain reaction (PCR), ligation, transformation, RNA isolation, reverse
103 transcription PCR (RT-PCR) analysis were performed as described (31). Plasmids were
104 isolated using plasmid isolation kit (Qiagen). DNA sequencing reactions were carried out
105 using double-stranded plasmid DNA as templates and ABI PRISM Dye terminator cycle
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106 sequencing method (Perkin-Elmer) and analyzed on an automated DNA sequencer (ABI
107 model 3700, Applied Biosystem).
108 Cloning, nucleotide sequencing, and RT-PCR analysis of rnr locus from P. syringae.
109 The amplification and cloning of the RNase R encoding gene (rnr) of P. syringae has been
110 reported earlier (25). We amplified the rnr upstream region by PCR, using a set of forward
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111 (Prnr_Fw: 5′CTACCAATTTC(C/A)CCA(C/T)CTGGGC-3′) and reverse (Prnr_RP: o
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112 5′GGGATCGAGGGTTTGCCAATCGGCCAT-3′) primers, and cloned it in pMOSBlue lo
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113 plasmid (Amersham) to generate pMOSrnr. The rnr downstream region was amplified by e
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114 using the forward primer (RR_FP8: 5′ GCCGAGCTGCGCAAAAGTCGTGAATTG-3′) fr
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115 located within the rnr gene and the reverse primer
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116 (S6_RP3:CGTTGTARCGGAAGTTGTCTTCCAGCTC-3′) corresponding to a conserved :
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117 region of rpsF gene that encodes S6 ribosomal protein. All PCR amplified products were
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118 cloned in the EcoRV site of pMOSBlue and sequenced. The overlapping nucleotide s
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119 sequences were aligned to get the complete sequence of the rnr locus of P. syringae o
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120 (GenBank accession no. HQ122447). o
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121 The primers Rnr_RTFP (5′-CTAAAGACGCAGGCAAGCCTTCCAAGC-3′) and
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122 Trm_RTRP (5′-TTACATCAGCCACAACACCCTGGTGC-3′) were employed for RT-PCR il 1
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123 analysis of co-transcripts from the rnr-trmH operon. 2
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124 Overexpression and purification of P. syringae RNase R (RNaseRPs) protein. For 9

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125 biochemical characterization of RNaseRPs protein, the rnr gene of P. syringae was subcloned g
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126 from pMOSrnr into the pET28a expression vector (Novagen). Briefly, the rnr gene was s
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127 excised out of pMOSrnr and ligated between the Nde1 and Sac1 sites of pET28a. The
128 resultant pETrnr-His was then analyzed by nucleotide sequencing to confirm the rnr ORF is
129 in-frame with the vector-encoded six histidine (6×His) tag at the N-terminal end of the
130 recombinant RNase R.
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131 RNaseRPs was expressed by IPTG induction of E. coli BL21(DE3) harbouring the pETrnr-
132 His plasmid. To increase the yield of RNaseRPs in soluble cytoplasmic fractions, 37o grown
133 cells (OD ~0.6) were shifted to 15oC in which they were kept shaking in the presence of
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134 IPTG (0.2 mM) for overnight (~16 hrs). His-tagged RNaseRPs was then isolated from the cell
135 lysates under native conditions using Ni-NTA-agarose (Qiagen) chromatography following
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136 the supplier’s instructions. All buffers contained protease inhibitors (complete, EDTA free o
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137 cocktail, Roche). Final purification of the protein for biochemical analysis was made by gel lo
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138 filtration on Superose 12 column. Purified proteins were kept at -20 oC in the buffer de
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139 containing 25 mM Tris-Cl pH 7.5, 150 mM KCl, and 10% glycerol. fr
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140 RNA substrates and exoribonuclease activity assays. Poly(A) and other synthetic RNA
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141 substrates were labeled at their 5′-ends using [γ-32P]ATP and T4 polynucleotide kinase. The :
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142 unincorporated nucleotides were removed by gel filtration on Sephadex G50 columns. For m
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143 preparing the 32P-labeled malE-malF RNA substrate, the plasmid containing the malE-malF s
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144 intergenic region under the control of T7 promoter (17) was linearized by digestion with o
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145 HindIII and used as template for transcription by T7 RNA polymerase in the presence of [α- o
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146 32P]UTP using in vitro transcription kit (MAXIscript, Ambion). The unincorporated
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147 radioactive nucleotides were removed by using NucAway spin columns (Ambion) and the il 1
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148 transcripts (318 nt) were checked by 8M urea-PAGE as described (25). 2
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149 RNase R assays were typically carried out in 20 µl reaction mixtures containing 25 mM 9

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150 Tris-HCl (pH7.0), 100 mM KCl, 0.25 mM MgCl2 and 5 mM DTT, 10 pmol of 32P-labeled g
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151 substrate and the indicated amount of enzyme. Unless otherwise mentioned, the reactions s
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152 were performed for 5 minutes at 22oC and stopped by adding 10 mM EDTA, 0.2% SDS and
153 1 mg ml-1 proteinase K. Two volumes of formamide dye mix that contained 5 mM EDTA,
154 10% SDS, 0.025% bromophenol blue and 95% formamide were then added to above and
155 boiled for 5 minutes and snap-frozen. RNA degradation products were resolved on
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156 denaturing gels (8M urea - 8% polyacrylamide). For resolving smaller products, sequencing
157 gels (8M urea - 20% polyacrylamide) was used. The cleavage products were visualized by
158 Phosphorimager (Fuji) and quantified by using Image Gauge software (Fuji). For comparing
159 relative activities, it was ensured that substrates are not completely digested. The data were
160 obtained from the ratio of signal intensities of products arising due to degradation and the
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161 total input of substrates (combined signals from degraded and undegraded smears of o
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162 heterogeneous substrates), and then normalized for protein amounts and reaction time. Data lo
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163 points are presented as mean of values derived from at least two independent experiments. As e
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164 the synthetic substrates used in assays were heterogeneous, their concentrations were fr
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165 calculated by taking their average size: 150 nt for poly(A), poly(G), and poly(C), and 100 nt
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166 for poly(U). :
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167 Other methods. Proteins were quantified by dye binding method of Bradford (4), using
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168 bovine serum albumin (BSA) as standard. SDS-PAGE, transfer of proteins onto Hybond C s
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169 membrane, and Western analyses of proteins using RNase R specific polyclonal antibodies or o
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170 anti-His-Tag antibodies (Santa Cruz Biotechnology) were carried out as described earlier o
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171 (25). The analysis of molecular mass of RNase R by gel filtration under different salt
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172 conditions was performed on Superdex 200 column (Amersham Biosciences). The protein il 1
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173 molecular markers for gel-filtration analysis were aldolase, catalase, ferritin, and 2
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174 thyroglobulin which had the molecular mass of 158, 232, 440, and 669 kDa respectively. 9

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175 Results g
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176 The rnr gene locus of the Antarctic P. syringae. Bioinformatic analysis of genome s
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177 sequences of different bacteria available in the NCBI data base (www. ncbi.nlm.nih.gov)
178 suggested that the rnr (encoding exoribonuclease R) locus is highly conserved among
179 different Pseudomonas species, in which the trmH gene (encoding a putative tRNA/rRNA
180 methyl transferase) constitutes the second gene of an rnr-trmH operon. Downstream of the
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181 dicistronic operon had another highly conserved gene (rpsF) encoding the S6 ribosomal
182 protein in these bacteria. For the characterization of rnr locus from the Antarctic P. syringae
183 Lz4W we employed the cloned rnr gene in pMOSBlue plasmid (named as pMOSrnr) as
184 described under Materials and the Methods. The nucleotide sequence analysis of the cloned
185 DNA confirmed that it encodes RNase R homologue (RNaseRPs) of the organism. We then
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186 characterized the entire rnr locus of P. syringae Lz4W by cloning and sequencing of the o
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187 downstream and upstream regions of the rnr gene (Fig. 1). The downstream region was lo
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188 amplified by using a set of forward primer (RR_FP8) located within the rnr and reverse e
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189 primer (S6_RP3) corresponding to a conserved region within the rpsF gene that encodes S6 fr
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190 ribosomal protein. The amplified DNA was cloned and sequenced. As expected, this
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191 amplified DNA segment contained the trmH gene, apart from the partial sequences of rnr and :
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192 rpsF genes. Analysis of the sequence also showed that the reading frames of RNase R and
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193 TrmH (encoding the proteins of 885 and 255 amino acids respectively) of P. syringae s
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194 overlaps with each other by 4 nucleotides, as in the case of other Pseudomonas species. The o
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195 trmH gene was however separated from the downstream rpsF gene by 306 nucleotides, which o
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196 contained two stem-loop structures (Fig. 1B) that may function as a transcriptional
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197 terminator. RT-PCR analyses of cellular RNAs indicated that rnr and trmH genes are co- il 1
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198 transcribed (Supplementary Fig. S1). 2
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199 We also cloned and sequenced the upstream region of rnr using the primers set (Prnr_Fw 9

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200 and Prnr_RP) as described under Materials and Methods. The nucleotide sequence analysis g
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201 indicated that the upstream region contains not only a putative rnr promoter but also s
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202 divergently transcribing genes for two tRNALeu (Fig. 1). The putative rnr promoter consisting
203 of ‘-10’ (TATTA) and ‘-35’ (TGAAT) boxes separated by 16 nucleotides was located 80
204 nucleotides upstream of the ‘ATG’ start codon of RNase R reading frame. A six-nucleotide
205 sequence (AAGGTG) showing imperfect complementarity to the 16S rRNA 3′-end
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206 (3′UUCCUG-5′) of P. syringae (24) was observed 11 nucleotides upstream of the ‘ATG’
207 codon, which is likely to act as the Shine-Dalgarno (SD) sequence for translation initiation.
208 On the other hand, a five-nucleotide putative SD sequence (AAGGT) which is perfectly
209 complementary to the 3′-end of 16S rRNA sequence was located six nucleotides upstream of
210 ‘ATG’ start codon of trmH reading frame of the bi-cistronic operon.
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211 The deduced amino acid sequence of RNaseRPs was 71 to 89 % identical (the lowest with o
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212 P. aeruginosa and highest with P. fluorescence) to the homologues of different Pseudomonas lo
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213 species and only about 54% identical (70% similar) to E. coli RNase R (RNaseREc). Clustal de
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214 W analysis of the homologues from different γ-proteobacteria indicated that RNase R of fr
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215 different Pseudomonas species form a distinct cluster which is separated from the clusters h
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216 representing E. coli/Salmonella group and Vibrio and Photobacterium group (Fig. S2). :
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217 Similar clustering of the homologues were also observed with TrmH; the deduced amino acid m
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218 sequences of TrmH of P. syringae Lz4W displayed 80 to 90% identity (representing P. s
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219 aeruginosa and P. fluorescence respectively) with the homologue from E. coli showing 56 % o
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220 identity. o
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221 Cloning, expression and purification of RNaseRPs. For biochemical characterization of
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222 RNaseRPs, E. coli BL21 harboring pETrnr-His was induced by IPTG at low temperature as il 1
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223 described in Materials and Methods. The cells produced His-tagged RNase R protein, which 2
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224 migrated near the 97 kDa marker band in SDS-PAGE (Fig. 2A). This was consistent with the 9

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225 calculated molecular mass of the RNaseRPs protein (~98.7 kDa). The over-expressed protein g
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226 was purified using Ni-NTA chromatography under native conditions as described under s
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227 Materials and Methods. A few minor proteins were co-eluted with RNaseRPs from the Ni-
228 NTA column (Fig. 2B). When these eluted fractions were subjected to Western analysis using
229 polyclonal anti-His antibody, the proteins cross-reacted with the antibody (data not shown),
230 suggesting that the co-eluted proteins probably represent the degraded RNase R fragments
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231 which have retained the His-tag. The intact RNaseRPs protein was separated and purified
232 from the degraded protein fragments by glycerol gradient centrifugation, or by gel-filtration
233 on Superose 12 as shown in Fig. 2C. The purified protein fractions that produced a single
234 band on SDS-PAGE were used for subsequent biochemical analysis. The yield of purified
235 RNaseRPs was ~10 mg per 1 liter of culture.
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236 Substrate preference and end product analysis of RNaseRPs activity. The o
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237 exoribonuclease activity of purified His-RNaseRPs was examined on 32P end-labeled synthetic nlo
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238 RNA substrates such as poly(A), poly(G), poly(U) and poly(C) to check any nucleotide e
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239 preference. Maximum activity was observed on poly(A) as seen with other enzymes of RNB fr
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240 family (9, 34). A quantitative estimation of degradation activity, as described under Materials
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241 and Methods, showed following specific activities (pmole min-1 per nanogram protein): 1.9 :
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242 poly(A), 1.7 poly(U), 0.29 poly(C), and 0.05 poly(G). Thus, poly(A) and poly(U) were better
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243 than poly(G) and poly(C) as substrates for degradation. We then analyzed the products of s
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244 RNaseRPs activity on 5′[32P]-end labeled poly(A) (Fig. 3A) and internally 32P-labeled malE- .o
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245 malF RNA (Fig. 3B). The cleavage product of malE-malF was nucleotide monophosphates o
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246 (Fig. 3B) as expected for exonucleolytic degradation. However, as depicted in Fig. 3A, the
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247 predominant limit product was a tetramer produced from 5′[32P]-end labeled poly(A) il 1
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248 degradation. A certain amount of pentamer was also observed. This is interesting because the 2
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249 limit product produced by E. coli RNase R (RNaseREc) activity is a dinucleotide (9, 21). We 9

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250 also noticed that the activity of P. syringae degradosome complex (glycerol gradient purified) g
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251 on poly(A) also produced tetramer as major product, with only a trace amount of trimer s
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252 (lanes 4 & 5, Fig. 3A). This suggests that degradation of RNA chains mediated by
253 degradosome is also incomplete, implying a requirement for oligoribonuclease (16).
254 Temperature optima and thermal stability of RNaseRPs. We examined the effect of
255 temperature on the activity of purified RNaseRPs on poly(A) substrate. The activity assays
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