The (ADP-ribosyl)ation reaction in thermophilic bacteria
Maria Rosaria Faraone-Mennella a,∗, Anna De Maio a, Anna Petrella a, Maria Romano a,Patrizia Favaloro a, Agata Gambacorta b, Licia Lama b, Barbara Nicolaus b, Benedetta Farina a
a Dipartimento di Biologia Strutturale e Funzionale, Facolta’ di Scienze M.F.N., Universita “Federico II”, Via Cynthia, 80126 Napoli, Italyb Istituto di Chimica Biomolecolare, CNR, Pozzuoli, Napoli, Italy
Mono(ADP-ribosyl)ation is a reversible post-translationalmodification of proteins, catalyzed by mono(ADP-ribose)transferases (ADPRTs; ARTs; EC 2.4.2.31), that transfer a sin-gle ADP-ribose unit from NAD+ to specific acceptor proteins[6,15]. This reaction is referred to as ubiquitous in all livingmesophilic organisms. Extracellular mono(ADP-ribosyl)ationis described as being typical of bacterial (ADP-ribosyl)atingtoxins (cholera, diphtheria, pertussis, etc.) which modify eu-karyotic host cell proteins [5,15]. Intracellular mono(ADP-ribosyl)ation involves acceptor proteins inside the cell and reg-ulates physiological processes like signal transduction, musclecell differentiation, protein trafficking and secretion [6,14]. Ina recent study, the discovery of over 20 new putative bacterialmono(ADP-ribose) transferases, including new potential tox-ins, was reported [19]. ADPRT genes were also demonstrated in
humans, where four functional toxin-related ARTs were foundto correspond to extracellular enzymes (ectoenzymes) [3,6].
All these (ADP-ribosyl)transferases were reported to bepart of a novel protein family [6] like that described for eu-karyotic poly(ADP-ribose)polymerases (PARPs; EC 2.4.2.30),which use NAD+ to synthesize complex ADP-ribose poly-mers and regulate the main cell functions [1]. At present,it is generally accepted that phylogenetically, mono(ADP-ribosyl)ation is an ancient mechanism, and although the re-lated enzymes often fail to show amino acid sequence sim-ilarity, prokaryotic and eukaryotic ADPRTs are thought tobe evolutionarily related [19]. As an example, in Rhodospi-rillum rubrum mono(ADPribosyl)ation regulates nitrogen fix-ation by reversibly inhibiting nitrogenase activity [14]. Inthis bacterium, as in a few free-living diazotrophs, the nitro-genase complex is rapidly, reversibly inactivated by (ADP-ribosyl)ation of iron protein to avoid unproductive nitrogenfixation during energy-limiting or nitrogen-sufficient condi-tions [12]. The (ADP-ribosyl)ation system has been iden-tified in R. rubrum and R. capsulatus (purple, non-sulfurphotosynthetic bacteria), Azospirillum brasilense and Azospi-rillum lipoferum (microaerophilic, associative bacteria), and
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Chromatium vinosum (a purple sulfur bacterium) [12]. Al-though there is little apparent variation in the sequencesand structures of nitrogenases, only these species have apost-transcriptional fast-acting nitrogenase-regulating scheme,since most nitrogen-fixing bacteria control nitrogenase atthe transcriptional level [12]. The (ADP-ribosyl)ation systemof R. rubrum involves an NAD+-dependent enzyme, dini-trogenase reductase (ADP-ribosyl)transferase (DRAT), andits partner, dinitrogenase reductase-activating glycohydrolase(DRAG) [12,14]. This reaction is very similar to that of choleratoxin, although no amino acid sequence similarity has beenfound between the (ADP-ribosyl)transferases from the twosources. Similarly, we have recently described an active (ADP-ribosyl)ating system in the lowest eukaryote Saccharomycescerevisiae, that lacks any nucleotide sequence correspond-ing to ADPRT/PARP genes [11], and we previously discov-ered that in the thermophilic archaeon Sulfolobus solfataricus,a eukaryotic-like (ADP-ribosyl)ating enzyme is present, despitethe fact that no gene sequence comparable to known (ADP-ribosyl)ating enzymes was found [9].
Thermophily and thermostability are unique features of pro-teins and other macromolecules from thermophilic microor-ganisms. The occurrence of an (ADP-ribosyl)ating system inS. solfataricus prompted us to investigate whether this reactionis common to other thermophilic organisms and whether it hasthe same features as those studied in the archaeon [4,9,10].
We have undertaken screening of (ADP-ribosyl)ating activ-ities in some thermophilic bacteria by selecting examples withdifferent physiological properties. The present study was per-formed by analyzing species of Alicyclobacillus, Bacillus andThermus genera. All these bacteria are thermophilic and wereisolated from different geothermal environments in differentparts of the world. The Alicyclobacillus species were also aci-dophilic and one of them was isolated in Italy while the secondone came from the Antarctic [7,8,13,16–18].
Enzymatic levels, reaction products and protein acceptors ofADP-ribose were studied in cells grown at logarithmic and sta-tionary growth phases under batch and fermenter conditions.
2. Materials and methods
2.1. Materials
All reagents were of the highest purity available. β-NAD+,ADP-ribose (ADPR), AMP, bovine serum albumin (BSA),DNase I, RNase A, proteinase K, snake venom phosphodi-esterase, phenyl methyl sulfonyl fluoride (PMSF), and ethyl-enediaminetetracetate (EDTA, disodium salt) were purchasedfrom Sigma-Aldrich (Italy); relabeled NAD+ and the polyvinyli-dene difluoride (PVDF) membrane were from Amersham Bio-sciences (Uppsala, Sweden); kits for chemiluminescence werefrom Pierce and antibodies from Santa Cruz Biotechnology,U.S. (primary) and Pierce (secondary). Rabbit anti-ADPRT an-tibodies were a kind gift of Dr. Joel Moss, National Institutesof Health, Bethesda, MD, USA. PEI-cellulose was from Merck(Germany).
2.2. Cell culture and sample preparation
The microorganisms, Alicyclobacillus acidocaldarius strainPisciarelli, Alicyclobacillus acidocaldarius subsp. rittman-nii strain MR1 (DSM 11297), Thermus thermophilus strainHB-8 (ATCC 27634), Thermus thermophilus strain Samu(DSM15284) and Bacillus thermantarcticus strain M1 (DSM9572), were grown at their optimal growth conditions in com-plex media as previously described [7,13,16–18]. Cells weregrown either in batch or in a fermenter with mechanical agi-tation and an air flux of 5 ml/l, and collected during both latethe logarithmic and stationary phases of growth. Growth wasfollowed by measuring the absorbance at 540 nm. After twowashes in isotonic solution, cells were collected by centrifu-gation at 9000 g for 30 min. The homogenate preparation wascarried out as previously described [9,17]. Briefly, harvestedcells were lysed with several freezing/thawing cycles in 20 mMTris–HCl, pH 7.5 (1:1, w/v), followed by ultrasonic treatment(Heat System Instruments) for 4 min, mixed with sand andmechanically crushed with a pestle. After homogenization inOmni-Mixer Sorvall for 10 min, sand was washed twice withthe same buffer, collecting supernatants. Pooled supernatantswere indicated to be “homogenates”.
2.3. Enzyme assay
The enzymatic activity was routinely assayed in sealed vialsat optimal growth temperature, as described in [9]. Briefly, thereaction mixture (final volume 62.5 µl) contained 100 mM Tris–HCl buffer, pH 7.7/5 mM NaF, 10 µl of 0.64 mM [32P] NAD(The Radiochemical Center, Amersham; 10 000 cpm/nmol)and defined amounts (20 µg protein) of bacterial prepara-tions. After incubation for 10 min at 65 ◦C, the reactionwas stopped by transfer onto ice and addition of 20% (w/v)trichloroacetic acid (final concentration). The mixture was fil-tered through Millipore filters (HAWP0001, 0.45 µ) and washedwith 10% trichloroacetic acid. The activity was measured asacid-insoluble radioactivity by liquid scintillation in a Beckmancounter (model LS 1701). One enzymatic milliunit catalyzesthe synthesis of 1 nmol ADP-ribose/min under standard condi-tions.
2.4. Electrophoretic analyses and immunoblotting
Polyacrylamide gel (12%) electrophoreses were carried outin 0.1% sodium dodecyl sulfate (SDS) [10]. Staining was in0.1% Coomassie G in 10% acetic acid/30% methanol. Westernblot was performed by electrotransferring proteins from gel toa PVDF membrane in a Bio-Rad apparatus at 200 mA for 2 h at4 ◦C, followed by immunoblotting according to the proceduredescribed in [10]. Rabbit anti-ADPRT and rabbit anti-humanPARP-1 catalytic site primary antibodies were used at 1:1000(v/v) dilution; goat anti-rabbit secondary antibodies (1:2000,v/v) were horseradish peroxidase-conjugated and were revealedby measuring chemiluminescence with a Quantity One Pro-gram in a P-imager (Bio-Rad).
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2.5. ADP-ribose acceptor proteins
Homogenates (500 µl) were adjusted to 0.4 mM Tris–HClbuffer (pH 7.5), 100 mM MgCl2, 1 mM PMSF, 100 mM NaCl,and, after addition of DNase I (2 µg/ml), were incubated for30 min at 37 ◦C. Thereafter, RNase A (20 µg/ml) was addedand incubation went on for a further 30 min. The reaction wasstopped with 200 mM EDTA, and mixtures were centrifugedat 2800 rpm and 4 ◦C for 20 min. Supernatants (crude extracts)were collected.
Reaction mixtures were prepared as for the enzyme as-say, incubating crude extracts (40 µg protein) with [32P] NAD(40 000 cpm/nmol) in a final volume of 125 µl. Proteins wereprecipitated with 20% trichloroacetic acid (final concentration),washed twice in 80% ethanol, suspended in sample buffer forSDS–PAGE and electrophoresed as described in the previoussection. Radioactivity of the dried gel, stained in Coomassie,was revealed by a P-imager (mod. 583, Bio-Rad).
2.6. Reaction products
Sensitivity of the ADPR-protein bond to alkali was testedas a function of time by incubating crude extracts (50 µg pro-tein) with [32P] NAD+ (40 000 cpm/nmol) under standard con-ditions. Thereafter reaction mixtures were adjusted to 0.1 MNaOH and alkali-treated at specific times up to 3 h. Residualradioactivity was measured after 20% trichloroacetic acid pre-cipitation as described above.
Reaction products were identified after detachment fromproteins by incubating [32P] ADP-ribose-protein adducts withproteinase K (0.2 µg/ml) at 37 ◦C overnight. One aliquot(3000 cpm) of protease digest was incubated with snake venomphosphodiesterase (0.1 mg/ml) at 25 ◦C overnight and chro-matographed on a PEI-cellulose thin layer (cm 20 × 20). Thesolvent system was 0.05 M NH+
4 -bicarbonate.
2.7. Other procedures
The protein concentration was determined by BCA assay ac-cording to Pierce instructions and using bovine serum albuminas a standard.
3. Results
3.1. ADP-ribosylating activity and growth conditions
Homogenates obtained from cell cultures under differentgrowth conditions were assayed for (ADP-ribosyl)ating activ-ity at 65 ◦C and pH 7.7 (Fig. 1). Enzyme activity (mU/g cells,wet weight) was virtually comparable under both batch andfermenter growth conditions for all microorganisms, exceptfor Bacillus thermantarcticus strain M1, where in batch-growncells, the enzymatic activity doubled with respect to that foundin cells grown in a fermenter. No relevant differences wereobserved between logarithmic and stationary growth phase.T. thermophilus strain Samu and B. thermantarcticus strain M1showed the highest activity.
3.2. Electrophoretic and immunochemical analyses
All homogenates from batch-grown cells at stationary phaseshowed heterogeneous and varying protein patterns (Fig. 2A).Immunoblotting of these samples was performed with bothanti-ADPRT and anti-PARP antibodies with similar results. Im-munoreactivity with anti-human PARP catalytic site antibodiesis shown in Fig. 2B. In almost all samples a single immune bandwas evident and localized in a 43–53 kD range. T. thermophilusstrain HB-8, T. thermophilus strain Samu and Alicyclobacillusacidocaldarius strain Pisciarelli showed a single band close tothat of purified PARP from S. solfataricus (46.5 kD), used asa reference sample [9]. A slight molecular mass increase was
Fig. 1. (ADP-ribosyl)ating activity in cell homogenates from thermophilic bacteria. Activity was assayed at 65 ◦C. Mean values of four different determinationsin duplicate. sl: logarithmic growth phase under static conditions; ss: stationary growth phase under static conditions; dl: logarithmic growth phase under dynamicconditions; ds: stationary growth phase under dynamic conditions. M1: B. thermantarcticus strain M1; MR1: A. acidocaldarius subsp. rittmannii strain MR1;Bac: A. acidocaldarius strain Pisciarelli; Tth: T. thermophilus strain HB-8; Samu: T. thermophilus strain Samu.
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Fig. 2. (A) SDS–PAGE (12%) and (B) immunoblot of protein homogenates (20 µg) from different microorganisms. Immunoblotting was performed with polyclonalrabbit anti-human PARP-1 antibodies. Samples were prepared from batch-grown cells at stationary phase. Abbreviations of bacteria are as in Fig. 1. Sso: S. solfa-taricus is shown for comparison.
observed for B. thermantarcticus strain M1 and for A. acido-caldarius strain MR1, which showed a second immunosignalat 92 kD.
Protein patterns and enzyme expression were analyzed incrude extracts from homogenates of cells grown under differ-ent conditions (Fig. 3). T. thermophilus strain HB-8, T. ther-mophilus strain Samu and A. acidocaldarius strain Pisciarelliconfirmed the presence of a single immunoband at 46–47 kDat all growth phases (Fig. 3B). Only A. acidocaldarius strainPisciarelli showed a strong increase in band intensity fromlogarithmic to stationary growth phase under batch conditions(Fig. 3B).
B. thermantarcticus strain M1 gave an immunoband ataround 50 kD. Enzyme levels were comparable at differentgrowth phases, but the signal was more intense using fermentergrowth. Moreover, batch-grown cells gave a second band at90 kD, not detectable in samples under fermenter conditions.The latter pattern (two immunosignals) was markedly evidentin both fermenter and batch-grown cells from A. acidocaldariusstrain MR1, with no significant difference between logarithmicand stationary phase (Fig. 3B).
3.3. ADPR acceptor proteins
Few relevant qualitative or quantitative differences were ob-served in the electrophoretic patterns of proteins extracted fromalmost all homogenates of cells obtained under any growth con-ditions (Fig. 4). Only for B. acidocaldarius strain Pisciarelli didbatch growth alter to a greater extent the quality and number ofproteins present in crude homogenates from both the logarith-mic and stationary growth phase (Fig. 4A, lanes 1, 2).
In all bacteria, the 32P-(ADP-ribosyl)ation pattern of pro-teins from homogenates did not vary during growth phases un-der either batch or fermenter conditions (Fig. 4B). A large num-ber of proteins of different molecular weights were evidencedby autoradiography, mainly in B. thermantarcticus strain M1
and A. acidocaldarius strain MR1. In T. thermophilus strainHB-8, most radioactivity was at the top of the gel and the au-toradiographic pattern showed slight similarity to that obtainedfor 32P-proteins of T. thermophilus strain Samu, although withmore marked labeling in the latter.
Among different strains belonging to the same genus (A. aci-docaldarius strain MR1 and A. acidocaldarius strain Piscia-relli), qualitative and quantitative differences were observed(Fig. 4).
3.4. Reaction products
In all analyzed microorganisms, the ADPR-protein bondwas only in part labile to alkali, with the highest resistancefor T. thermophilus strain HB-8 (data not shown). Therefore,to detach the whole (ADPR)n bound to proteins, the adductswere incubated with proteinase K and the products were ana-lyzed by thin-layer chromatography before and after incubationwith snake venom phosphodiesterase (PDE) (Fig. 5, lane 5).In all samples, the protein-free product co-migrated with stan-dard ADP-ribose, and no contamination by NAD+ was evident(Fig. 5, odd lanes). After PDE digestion, radioactivity shiftedin correspondence with authentic AMP, the expected productof phosphodiesterase activity (Fig. 5, even lanes).
4. Discussion
The present results show that, as previously reported inthe thermophilic archaeon S. solfataricus [9,10], a thermophilicADP-ribosylating system also occurs in thermophiles belong-ing to the Bacteria domain, and they confirm the widespreadoccurrence of this reaction in living organisms. Enzyme mole-cular masses are in the same range (45–50 kD) for all ana-lyzed microorganisms, and we present evidence that proteinactivity/expression is generally independent of growth condi-tions. Indeed, assay of the enzyme in the whole homogenates
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Fig. 3. (A) SDS–PAGE (12%) and (B) immunoblot of proteins (20 µg) ex-tracted from homogenates of thermophilic microorganisms at different phasesand grown either in batch or fermenter. Immunoblotting was performed withpolyclonal rabbit anti-human PARP-1 antibodies. Logarithmic (1) and station-ary (2) phases of batch growth; logarithmic (3) and stationary (4) phases offermenter growth. Abbreviations of bacteria are as in Fig. 1. DM: molecularweight markers.
from all microorganisms using the same amount of proteins ledto results shown in Fig. 1, which are also supported by elec-trophoretic patterns not showing high qualitative and quantita-tive protein variability (Fig. 3A). The high working temperature(65 ◦C) is the main feature distinguishing the involved enzymesfrom heat-labile eukaryotic ADPRTs [1]. The fact that, in the
Fig. 4. (A) SDS–PAGE (12%) and (B) autoradiography of 32P-ADP-ribose/protein adducts in extracts (40 µg) from bacterial cells at different growthphases and under static or dynamic conditions. Logarithmic (1) and station-ary (2) phases of cells grown in batch; logarithmic (3) and stationary (4) growthphases of cells from fermenter. Abbreviations of bacteria are as in Fig. 1.
studied bacteria, protein targets of ADP-ribose vary in a growthphase-independent manner (Fig. 4B) suggests their possible in-volvement in different cell processes, and this will be the objectof future investigations in our laboratory.
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Fig. 5. Autoradiography of protein-free 32P-products after thin-layer chro-matography. The experiment was performed with batch-grown cells at sta-tionary phase, incubated with 32P-NAD and treated as described in Sec-tion 2.6. Odd lanes: Enzymatic products (1000 cpm) after digestion of32P-(ADPR)n/protein adducts with proteinase K. (1) M1: B. thermantarticusstrain M1; (3) MR1: MR1: A. acidocaldarius subsp. rittmannii strain MR1;(5) Bac: A. acidocaldarius strain Pisciarelli; (7) Tth: T. thermophilus strainHB-8; (9) Samu: T. thermophilus strain Samu. Even lanes: products (1000 cpm)from proteinase K digestion, followed by incubation with phosphodiesterase.(2) M1; (4) MR1; (6) Bac; (8) Tth; (10) Samu. M: mixture of authentic ADPR,AMP, 32P-NAD (400 cpm). Unlabeled standards were revealed by UV light.
On the basis of the available data, it is possible to define thesystem of the analyzed thermophilic bacteria as mono(ADP-ribosyl)ation. In fact, although most samples reacted with bothanti-ADPRT and anti-PARP antibodies, reaction product analy-sis unequivocally identified the la belled, enzymatically synthe-sized compound as mono(ADP-ribose) (Fig. 5). Mono(ADP-ribosyl)ation is an ancient mechanism of protein regulation, andit is possibly an evolutionary link between prokaryotic and eu-karyotic ADPRTs [6,19]. Mesophilic mono(ADP-ribosyl)ationwas first identified as a reaction catalyzed by bacterial toxinsable to deregulate physiological functions by modifying hostcell proteins [5,15]; thereafter, however, eukaryotic endogenousARTs were shown to reversibly control physiological processessuch as the immune response, cell adhesion, and signal and en-ergy metabolism [6]. As regards the thermophilic bacteria westudied, the biochemical evidence here described enables us todefine the thermophilic bacterial enzymes as endogenous AD-PRTs. As stated above, the possible role of the reaction remainsto be explored, since the pattern of protein acceptors of ADP-ribose is heterogeneous and, as already shown for nitrobacteria,one function can be regulated by various mechanisms even indifferent species of the same genus [12].
At present, we attribute these enzymes to endocellular AD-PRTs rather than to ecto (membrane-anchored)-enzymes onthe basis of the biochemical parameters analyzed thus far. Upuntil several years ago, genomic analysis was often consid-ered decisive for confirming the absence of the occurrenceof an enzyme in a given organism. On this basis, eukaryoticorganisms like Saccharomyces and Arabidopsis were consid-ered ADPRT- or PARP-lacking because no nucleotide sequencewas found which matched the ADPRT/PARP genes [6,15]. Re-cently, Pallen et al. demonstrated the occurrence of >20 puta-tive ADPRTs, sharing with all ARTs common active-site fea-tures, but lacking sequence homology [19]. This finding is inline both with the opinion that in organisms which are evolu-tionarily distant, proteins with the same function do not nec-essarily share high similarity in primary structure, and withour previous discovery that in both the thermophilic archaeon
S. solfataricus and the yeast S. cerevisiae, a eukaryotic-like(ADP-ribosyl)ating enzyme is present, although no sequencehomology with ADPRT/PARP enzymes was found [6,9,12].Similarly, nitrogenase-modifying DRAT catalyzes the same re-action as cholera toxin, though there is no obvious amino acidsequence similarity between these enzymes [2,12]. The samequestion arises for the bacterial thermo-ADPRTs describedhere: the biochemical evidence we present needs to be sup-ported by genome sequence and/or purified enzyme structureanalysis. Thereafter, clarifying their biological function(s) willhelp to evaluate any possible biotechnological interest of bacte-rial thermophilic mono(ADP-ribosyl)transferases.
Acknowledgements
We wish to thank Valeria Calandrelli and Ida Romano forcell cultures. This work was supported in part by a grant fromRegione Campania (Legge 41, annualita’ 1999) and PNRA.
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