jb accepts, published online ahead of print on 15 july...

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Characterization of components of the Staphylococcus aureus messenger RNA 1

degradosome holoenzyme-like complex 2

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Christelle M. Roux1, Jonathon P. DeMuth2 and Paul M. Dunman1,2,† 5

6 1Department of Microbiology and Immunology, University of Rochester Medical Center, 7

Rochester, NY, 14642.; 2Department of Microbiology and Pathology, University of Nebraska 8

Medical Center, Omaha, NE, 68198. 9 10 11 12 †To whom correspondence should be addressed: 13

Paul M. Dunman 14

University of Rochester School of Medicine and Dentistry 15

Box 672 16

601 Elmwood Avenue 17

Rochester, NY 14642 18

Phone: 585 273-3287; Fax: 585 473-9573 19

E-mail: [email protected] 20

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Running Title: S. aureus RNA degradosome. 23

Key Words: Staphylococcus aureus, RNA degradation, Ribonucleases 24

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Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.05485-11 JB Accepts, published online ahead of print on 15 July 2011

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ABSTRACT (50 words) 28

Bacterial two-hybrid analysis identified the Staphylococcus aureus RNA degradosome-like 29

complex to include RNase J1, RNase J2, RNase Y, PNPase, enolase, phosphofructokinase and a 30

DEAD-box RNA helicase. Results also revealed the recently recognized ribonuclease, RnpA, 31

interacts with the S. aureus degradosome and that this interaction is conserved across other 32

Gram-positive organisms. 33

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INTRODUCTION 59

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Escherichia coli bulk RNA degradation is mediated by a holoenzyme complex, the RNA 61

degradosome, which includes the ribonucleases RNase E and polynucleotide phosphorylase 62

(PNPase), an RNA helicase RhlB and the glycolytic enzyme enolase (3, 21). RNase E is 63

considered the key component of the E. coli degradosome; it catalyzes the initiation of mRNA 64

degradation and serves as a scaffold for the assembly of the other degradosome components (2). 65

By comparison, most Gram-positive bacteria do not contain an RNase E amino acid ortholog and 66

much less is known about their mRNA degradation machinery. Nonetheless, recent studies 67

suggest that the Gram-positive organism, Bacillus subtilis, produces two RNase E functional 68

orthogues, ribonucleases J1 (RNase J1) and J2 (RNase J2) (17). Using bacterial two-hybrid 69

analyses, both enzymes have been shown to interact with an RNA degradosome-like complex 70

consisting of PNPase, enolase, a DEAD-box RNA helicase (CshA), as well as 71

phosphofructokinase and ribonuclease Y (6). 72

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Staphylococcus aureus is a Gram-positive bacterial pathogen of immense healthcare concern, 74

which is evolutionarily similar, but divergent, from the Bacillus genus. Herein we used bacterial 75

two-hybrid and biochemical analyses to measure interactions between putative members S. 76

aureus RNA degradosome and compare those results to that of B. subtilis. Results revealed that 77

although many interactions are conserved between both bacterial species, differences are likely 78

to exist. Moreover, we show that the ribonuclease, RnpA, interacts with components of the S. 79

aureus RNA degradosome and that this interaction is conserved within B. subtilis. 80

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RESULTS AND DISCUSSION 83

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Using bacterial two-hybrid analyses, interactions between putative members of the B. subtilis 85

RNA degradosome were recently measured and used to develop the first model of the Gram-86

positive holoenzyme complex [Figure 1A; (6, 13)]. According to the model, the E. coli RNase E 87

orthologs, RNase J1 and RNase J2 interact with one another; RNase J1 also interacts with two 88

additional ribonucleases, PNPase, and RNase Y, as well as with the glycolytic enzyme 89


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phosphofructokinease, each of which binds to the RNA helicase, CshA (Figure 1A). CshA also 90

binds to a second glycolytic enzyme, enolase. Although the mechanism of B. subtilis cellular 91

RNA degradation has yet to be established, RNases J1 and J2 are bifunctional ribonucleases with 92

endonuclease and 5’ 3’ exoribonuclease activities (8, 16, 25). In complex the enzymes act 93

synergistically and may initiate 5’ mRNA digestion and/or generate endonucleoytic products that 94

could be subsequently be digested by RNA helicase facilitated 3’ 5’ PNPase exoribonuclease 95

activity (14, 18). RNase Y is hypothesized to act upon highly structured RNA fragments that are 96

not effectively degraded by RNase J1 (22) and interact with enolase to modulate the mRNA 97

turnover of S. pyogenes virulence factors (10). It is not clear what direct role, if any, 98

phosphofructokinase plays in the RNA degradation process. 99

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S. aureus contains orthologs of each putative B. subtilis RNA degradosome subunit, suggesting 101

the RNA degradation complexes of these two bacterial species may be highly conserved. To 102

evaluate this possibility, the BACTH (Euromedex, Inc, France) bacterial two-hybrid system was 103

used to measure all possible combinations of interactions between S. aureus enolase (SAR0832), 104

phosphofructokinase (SAR1777), DEAD-box RNA helicase (SAR2168), PNPase (SAR1250), 105

RNase J1 (SAR1063), RNase J2 (SAR1251), RNase Y (SAR1262), as well as the recently 106

recognized S. aureus ribonuclease, RnpA (SAR2799) (20). Accordingly, the coding sequence of 107

each gene was amplified from S. aureus strain UAMS-1 and cloned into the pCR®II vector 108

(Invitrogen, CA) (Supplemental Table 1 and Table 2). Genes were subsequently digested with 109

BamHI or XbaI and KpnI and cloned in-frame into the bacterial two-hybrid expression vectors, 110

pKNT25, pKT25, pUT18 and pUT18C to create N- and C-terminal fusions to the catalytic 111

domains, T25 and T18, of Bordetella pertussis adenylate cyclase CyaA (Supplemental Table 2). 112

All combinations of plasmid pairs were transformed into E. coli DHM1 (Δcya) cells. Cellular 113

interactions between T25- and T18-fused proteins were measured as a function of their ability to 114

reconstitute CyaA activity and, consequently, cellular β-galactosidase levels due to cyclic AMP 115

mediated activation of catabolic operons (11). DHM1 cells transformed with BACTH system kit 116

supplied plasmids, pKT25-zip and pUT18C-zip, or the empty vectors, pKT25 and pUT18C, were 117

used as positive and negative controls, respectively. 118

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To measure protein-protein interactions, the β-galactosidase levels of a total of 256 transformants 120

collectively representing all possible plasmid pairs were independently measured (Supplemental 121

Table 3). Transformants were grown statically overnight at 30°C in Luria-Bertani (LB) medium 122

supplemented with 100 µg/ml Ampicillin, 50 µg/ml Kanamycin, and 0.5 mM isopropyl-β-D-123

thiogalactopyranoside (IPTG). An 500 µl aliquot was added to 2 ml of M63 minimal medium 124

(15 mM (NH4)2SO4, 100 mM KH2PO4, 1.8 µM FeSO47H2O, 3 µM vitamin B1, pH 7.0) and the 125

optical density at 600 nm was measured. Cells were subsequently permeabilized with 30 µl of 126

chloroform and 30 µl 0.1% SDS and an aliquot (500 µl) was mixed with 500 µl of PM2 buffer 127

(70 mM Na2HPO4-H2O, 30 mM NaHPO4-H2O, 1 mM MgSO4, and 0.2 mM MnSO4, 100 mM β-128

mercaptoethanol). For β-galactosidase measurements, 250 µl of o-nitrophenol-β-galactoside 129

(ONPG, 4 mg/ml) was added and reactions were stopped after 15 min incubation at 30°C by 130

adding 500 µl of 1 M Na2CO3. The absorbance was read at 420 nm (cleavage of ONPG) and 131

550 nm (cellular debris). All interactions were tested in triplicate and expressed in Miller units 132

(19). Transformants judged to have interacting proteins exhibited a significant increase 133

(Student’s T-test; P value ≤ 0.01) in β-galactosidase activity in comparison to negative control 134

cells. Results are presented in Figure 2 and Supplemental Table 3. 135

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Taken together, results revealed that orthologs of the putative B. subtilis RNA degradosome 137

subunits are also capable of interacting to form a S. aureus degradosome, suggesting a high 138

degree of conservation of the RNA degradosome among Gram-positive bacteria. However, there 139

were several noted differences between individual protein interactions (comparison of Figures 140

1A and 1B). For instance, as observed for B. subtilis, our results indicate that S. aureus 141

ribonucleases RNase J1 and RNase J2 interacted the most strongly and that RNase J1 also 142

interacts with PNPase (6). However, we did not detect significant interactions between S. aureus 143

RNase J1 and RNase Y or phosphofructokinase. Rather, we found that the only other RNase 144

J1interacting protein is the DEAD-box RNA helicase, CshA. In Gram negative bacteria, RNase 145

E interacts with both PNPase and an RNA helicase (3, 12) and initiates RNA degradation by 146

cleaving substrate molecules, which are subsequently degraded in a 3’ 5’ direction by the 147

concerted activity of RNA helicase and PNPase. Accordingly, the interaction between S. aureus 148

RNase J1 (an RNase E functional ortholog) and both CshA and PNPase suggests a conserved 149

mechanism of RNA degradation in Staphylococcus. Consistent with that possibility, we have 150


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previously shown that PNPase contributes to bulk S. aureus mRNA decay (1). Furthermore, the 151

B. subtilis DEAD-box RNA helicase CshA interacts with phosphofructokinase, enolase, PNPase 152

and RNase Y [Figure 1A; (6, 22)]. Similarly, we observed that S. aureus CshA interacts with 153

phosphofructokinase, enolase and RNase Y. But, S. aureus DEAD-box RNA helicase does not 154

appear to interact with PNPase (Figure 1B). 155

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We recently reported that the protein subunit of S. aureus ribonuclease P, RnpA, is involved in 157

bulk RNA degradation and hypothesized that the enzyme is a component of the organism’s RNA 158

turnover machinery (20). Consistent with that prediction, results revealed that S. aureus CshA 159

interacts with RnpA, suggesting that RNA helicase may augment cellular RnpA ribonuclease 160

activity. To investigate whether the observed interaction between S. aureus RnpA and CshA is 161

conserved among Gram positive bacteria, bacterial two-hybrid studies were expanded to include 162

B. subtilis RnpA (BSU41050) and CshA (BSU04580). Accordingly, both B. subtilis genes were 163

cloned in-frame into the bacterial two-hybrid expression vectors for quantitative measurement of 164

β-galactosidase activity (as described above). Interactions were also qualitatively assessed by 165

color change on agar plates containing 0.5 mM IPTG and 40 µg/ml 5-bromo-4-chloro-3-indolyl-166

β-D-galactopyranoside. B. subtilis RNase J1 (BSU14530) and RNase J2 (BSU16780) were used 167

as positive controls, whereas vector alone was used as a negative control (6). As shown in 168

Figure 3, our results confirmed that B. subtilis RNase J1 dimerizes and interacts with RNase J2. 169

Further, as observed for S. aureus, B. subtilis RnpA dimerized and interacted with CshA, but not 170

RNase J1 (Supplemental Table 4). Collectively, these data suggest that RnpA interacts with the 171

DEAD-box RNA helicase, CshA, and that this interaction is conserved among Gram-positive 172

bacteria. 173

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The interaction of the S. aureus DEAD-box RNA helicase, CshA, with three ribonucleases, 175

RNase J1, RNase Y and RnpA, suggests that the helicase is a central component of the 176

organism’s RNA degradosome. Nonetheless, it is intriguing to consider that CshA could also 177

facilitate the activity of endo/exoribonucleases independently of their participation in the 178

degradosome complex. Indeed, E. coli PNPase can degrade RNA in the presence of the DEAD-179

box RNA helicase independently of their binding to RNase E (15). Similarly, consistent with 180

previous observations, our results indicate that each protein examined can multimerize. Indeed, 181


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each of these molecules has been shown to form homodimers [DEAD-box RNA helicase, 182

enolase, RNase Y and RnpA (4, 6, 9, 15)], trimers [PNPase (23)], tetramers 183

[phosphofructokinase and RNase J1/J2 (7, 24)], or a combinations of multimer populations 184

[RNase J1 and J2 form homodimers, homotetramers (17)]. While the goal of the current study is 185

to genetically evaluate the potential interacting partners of the S. aureus degradosome, it is clear 186

that such work must be biochemically complimented to fully recognize the stoichiometry of the 187

RNA degradosome subunits and whether subset populations of proteins exist within the cell 188

(such as helicase/ribonuclease combinations). 189

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As a preliminary means of validating our bacterial two-hybrid analyses, we biochemically 191

confirmed that purified RNase J1 and RNase J2 interact in vitro; both are hypothesized to be 192

essential S. aureus enzymes (5) and, thus, may play major roles in mRNA decay. For 193

purification, the RNase J1 gene was cloned into pTrcHis2A expression vector (Invitrogen) and 194

RNase J2 gene product was cloned into pET-30 (Novagen, WI). Proteins were expressed within 195

E. coli BL21(DE3) cells following the addition of 0.25 mM IPTG for 2 hours at 30°C or 0.5 mM 196

IPTG for 3 hours at 37°C for RNase J1 and RNase J2, respectively. Cultures were centrifuged 197

and cell pellets were resuspended in lysis buffer (50mM Tris-HCl pH7.5, 10% glycerol, 300mM 198

NaCl, 10mM imidazole) supplemented with protease inhibitor cocktail (1/30, P8849, Sigma, 199

MO), 5 µg/ml lysozyme (Sigma, MO) and Benzonase® nuclease, according to the 200

manufacturer’s instructions (Novagen, WI). Cells were disrupted by three cycles of French press 201

(14,000 psi), cellular debris was removed by centrifugation and proteins were purified using 202

HisPur Cobalt columns, according to the manufacturer’s instruction (Pierce, IL). To assess 203

whether S. aureus RNase J1 and RNase J2 interact, 75 µg of each purified protein was incubated 204

individually or in combination for 30 min at 37°C and was subjected to gel filtration on a 205

Superose 6 10/30 column (GE Life Sciences) in PBS buffer (137 mM NaCl, 2.7 mM KCl, 4.3 206

mM Na2HPO4, 1.47 mM KH2PO4, pH7.4). Resulting fractions (25 µl) were loaded on a 10% 207

SDS PAGE and proteins were detected by silver stain according to the manufacturer’s 208

instructions (Bio-Rad Laboratories, CA). As shown in Figure 4A and 4B, RNase J1 209

predominantly eluted as monomers (collected in later fractions and migrated around 67 kDa). 210

Conversely, RNase J2 predominantly formed dimers (collected in earlier fractions; ~140 kDa). 211

Following co-incubation of both RNases together, we observed a shift in the elution and gel 212


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migration of RNase J1. Further, the detection of RNase J1 in earlier fractions coincided with a 213

loss of RNase J2 monomers. Taken together these results suggest that S. aureus RNase J1 and 214

RNase J2 interact during these experimental conditions. 215

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The bacterial two-hybridization system has been an important tool in indentifying components of 217

the putative B. subtilis RNA degradosome. Herein, we have also used this system to determine 218

and draw comparisons between the S. aureus and B. subtilis RNA degradosome-like complex. 219

Results revealed that most interactions are conserved but significant differences are likely to 220

exist. Further, we show that RnpA interacts with the DEAD-box RNA helicase both in S. aureus 221

and B. subtilis. 222

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Acknowledgments 225

We would like to thank Gregory Tombline for technical assistance and John Morrison for critical 226

reading of the manuscript. This research was supported by NIH/NIAID award 1R01 AI073780-227

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FIGURE LEGENDS 244

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Figure 1. B. subtilis and S. aureus mRNA degradosome-like complexes. Panel A. B. subtilis 246

RNA degradosome-like complex (6). Panel B. Deduced S. aureus RNA degradosome-like 247

complex; blue arrows represent conserved interactions between S. aureus and B. subtilis 248

proteins. Arrow thickness indicates the strength of interactions as determined by quantitative β-249

galactosidase activity results (Supplemental Table 2.). 250

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Figure 2. β-galactosidase deduced protein-protein interactions. β-galactosidase activity of 252

transformants containing the indicated S. aureus proteins fused to the N-terminal (N) or C-253

terminal (C) of the T25 and T18 catalytic fragments of CyaA. Grey bars represent statistically 254

significant positive interacting proteins/domains, whereas black bars represent negative 255

interactions. S. aureus proteins tested include enolase (Eno), phosphohfructokinase (Pfk), 256

PNPase (PNP), DEAD-box RNA helicase CshA, RNase J1 (J1), RNase J2 (J2), RnpA and 257

RNase Y (Y). 258

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Figure 3. Bacterial two-hybrid analysis of selected B. subtilis proteins. Panel A. Qualitative 260

analysis of β-galactosidase activity of indicated protein pairs. Panel B. Corresponding 261

quantitative analysis of β-galactosidase activity; * P ≤ 0.01 (Supplemental Table 4). B. subtilis 262

proteins tested are RNase J1 (J1), RNase J2 (J2), CshA and RnpA. 263

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Figure 4. In vitro interaction of purified S. aureus RNase J1 and RNase J2. Panel A. Gel 265

filtration analysis of RNase J1 (dotted line), RNase J2 (dashed line), and RNase J1 + RNase J2 266

(solid line) as measured by O.D.595nm. Molecular weight size standards are shown above the 267

chromatogram. Panel B. Silver stained 10% SDS-PAGE of eluted RNase J1, RNase J2, and 268

RNase J1 + RNase J2; elution fractions are indicated. 269

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Figure 1. Roux et al.

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Figure 1. B. subtilis and S. aureus mRNA degradosome-like complexes. Panel A. B. subtilis RNAdegradosome-like complex (5). Panel B. Deduced S. aureus RNA degradosome-like complex; blue

t h d i t ti b t S d B btili t i A thi k

Bacillus subtilis Staphylococcus aureus

arrows represent shared interactions between S. aureus and B. subtilis proteins. Arrow thicknessindicates the strength of interactions as determined by quantitative β-galactosidase activity results(Supplemental Table 2.). on July 28, 2018 by guest

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β-ga

lact

osid

ase

acti

vity

25 N C N C N C N C N C N C N C N C N C N C N C N C N C N C N C N C


Y (18N) Y (18C)

25

Figure 2. β-galactosidase deduced protein-protein interactions. β-galactosidase activity oftransformants containing the indicated S. aureus proteins fused to the N-terminal (N) or C-terminal (C) of the T25 and T18 catalytic fragments of CyaA. Grey bars represent statisticallysignificant positive interacting proteins/domains, whereas black bars represent negativeinteractions. S. aureus proteins tested include enolase (Eno), phosphohfructokinase (Pfk),PNPase (PNP), DEAD-box RNA helicase CshA, RNase J1 (J1), RNase J2 (J2), RnpA andRNase Y (Y).


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Pos.Neg.A Pos.Neg.A.

B

J1 J2 RnpA CshA J1 J2J1 RnpA

6000

7000

8000

idas

e ac

tivity

er

uni

ts)

3000

B.

*

*

β-ga

lact

osi

(Mill

e

0

1000

2000

J1 J2 RnpA CshA J1 J2

1 R A

*

* *

J1 RnpA

Figure 3. Bacterial two-hybrid analysis of selected B. subtilis proteins. Panel A.Qualitative analysis of β-galactosidase activity of indicated protein-pairs. Panel B.Corresponding quantitative analysis of β-galactosidase activity; * P ≤ 0.01 (SupplementalTable 4). B. subtilis proteins tested are RNase J1 (J1), RNase J2 (J2), CshA and RnpA.) p ( ), ( ), p


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A


670

kDa

158

kDa

44 k

Da

17 k

Da

A.

670

kDa

158

kDa

44 k

Da

17 k

Da

29 30 31 32 33 34 35 36 37J1

J2

B.

Figure 4. In vitro interaction of purified S. aureus RNase J1 andRNase J2. Panel A. Gel filtration analysis of RNase J1 (dotted line), RNaseJ2 (dashed line), and RNase J1 + RNase J2 (solid line) as measured by

J1 + J2

( ), ( ) yO.D.595nm. Molecular weight size standards are shown above thechromatogram. Panel B. Silver stained 10% SDS-PAGE of eluted RNaseJ1, RNase J2, and RNase J1 + RNase J2; elution fractions are indicated.


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