Vol. 173, No. 6

JOURNAL OF BACTERIOLOGY, Mar. 1991, p. 2120-2124

0021-9193/91/062120-05$02.00/0

Purification and Characterization of the DNA-Dependent RNA Polymerase from Clostridium acetobutylicum ANDREAS PICH AND HUBERT BAHL*

Institut fur Mikrobiologie, Georg-August-Universitat Gottingen, Grisebachstrasse 8, W-3400 Gottingen, Federal Republic of Germany Received

5

September 1990/Accepted 27 December 1990

The DNA-dependent RNA polymerase (EC 2.7.7.6) from Clostridium acetobutylicum DSM 1731 has been purified to homogeneity and characterized. The purified enzyme was composed of four subunits and had a molecular mass of 370,000 Da. Western immunoblot analysis with polyclonal antibodies against the if70 subunit

of Escherichia coli RNA polymerase identified the 46,000-Da subunit as an immunologically and probably functionally related protein. The other three subunits of 128,000, 117,000, and 42,000 Da are tentatively analogous to the ,, O', and a subunits, respectively, of other eubacterial RNA polymerases. The RNA polymerase activity was completely dependent on Mg2+, nucleoside triphosphates, and a DNA template. The presence of Mg2' or Mn2+ in buffers used for purification or storage caused irreversible inactivation of the RNA polymerase. of the RNA polymerase from C. acetobutylicum DSM 1731 (ATCC 4259) was performed at 4°C under aerobic conditions. The enzyme was purified from 20 g (wet weight) of C. acetobutylicum cells. These were grown in 20-liter batches on CBM medium (19) at 37°C to an optical density at 600 nm of 2.0. At this growth phase the cells produced acids (acetate and butyrate) and no solvents as shown by gas chromatography (3). Furthermore, no spores or granulose-containing cells could be detected (3). The cells were harvested at 4°C in a continuous-flow centrifuge (Heraeus Sepatech, Osterode, Federal Republic of Germany), washed twice with buffer A (60 mM Tris-HCI [pH 7.8], 20 mM NH4Cl, 1 mM EDTA, 10 mM 2-mercaptoethanol, 10% glycerol), and suspended in 200 ml of buffer A containing 2 mg of lysozyme (Biomol, Hamburg, Federal Republic of Germany) per ml and 150 ,ug of phenylmethylsulfonyl fluoride per ml. After incubation in a water bath shaker (New Brunswick Scientific, Edison, N.J.) for 2 h at 37°C, the cells were disrupted by two passages through a French press cell (SLM Instruments, Hannover, Federal Republic of Germany) at 1,300 lb/in2. The first two purification steps, polyethyleneimine and ammonium sulfate precipitations, were used to remove cellular debris, nucleic acids, and basic proteins from the crude extract. For this purpose 20 ml of 5% polyethyleneimine was added to the crude extract. After the mixture was on ice for 20 min, it was centrifuged in a Sorvall RC5B centrifuge with a GSA rotor (DuPont, Bad Homburg, Federal Republic of Germany) at 12,000 rpm for 20 min. The supernatant was discarded; the pellet was washed three times with buffer A and then extracted three times with 100 ml of buffer A containing 1.2 M NH4C1. The extract was brought to 65% saturation with ammonium sulfate by adding the solid salt. The sample was stirred on ice for 3 h and then centrifuged in a GSA rotor at 12,000 rpm for 60 min. The resulting pellet was dissolved in a small volume of buffer B (buffer A with 40% glycerol instead of 10% glycerol) and then dialyzed against buffer B. Only after this treatment was it possible to assay the RNA polymerase. To determine enzyme activity, the assay mixture contained 100 mM TrisHCI (pH 10), 50 mM NH4Cl (for calf thymus DNA, 100 mM NH4CI), 20 mM MgCl2, 1 mM each ATP, CTP, GTP, and UTP (10 Ci of 3H per mol), 500 ,ug of bovine serum albumin

Clostridium acetobutylicum, a gram-positive, strictly anaerobic sporeformer, belongs to one of the largest genera of bacteria. The impressive metabolic capacity of the many different species has a high biotechnological potential (1, 9). To better understand the molecular mechanisms involved in the regulation of metabolic pathways, several groups have started to analyze the genetics of clostridia (21, 27). Much work has been done on C. acetobutylicum. This might be due to the fact that this organism has been used for the industrial production of useful solvents from renewable biomass and that it is able to change its fermentative metabolism, depending on the environmental conditions (4, 12). During growth, C. acetobutylicum ferments sugars to acetate, butyrate, H2, and CO2. At the end of the exponential growth phase the residual sugars and most of the preformed acids are converted to butanol and acetone. Furthermore, the switch from acid to solvent formation seems to be associated with cell differentiation (storage of granulose, sporulation) (15, 16). Altogether, C. acetobutylicum provides an attractive system to study the expression and regulation of genes during the developmental process of this organism. However, despite research for decades, the mechanisms for the initiation of solvent production and/or sporulation at the molecular level are not known. One important factor for the regulation of gene expression is the RNA polymerase. It is now known that the bacterial transcription machinery comprises a system of multiple RNA polymerase holoenzymes composed of a common core enzyme and several different sigma factors (7, 11). The various holoenzymes recognize specific promoter sequences and control the expression of various sets of genes. The possibility cannot be excluded that the regulation of the sequential gene expression during the developmental process of C. acetobutylicum involves alternative sigma factors. Therefore, we have started to analyze the DNA-dependent RNA polymerase from C. acetobutylicum. In this paper we describe the purification and characterization of the major form of that enzyme. Purffication procedure. The entire purification procedure

*

Corresponding author. 2120

,ug of calf thymus DNA per ml, 50 ,ug of per ml, and 300 ,ug of C. acetobutylicum poly(dA-dT) DNA per ml, or 200 DNA per ml. The reaction was allowed to proceed for 20 min ,ul. The reaction was stopped at40°C in a final volume of 100 by adding ice-cold trichloracetic acid (final concentration, 7.5%) containing 200 mM sodium pyrophosphate. The acidprecipitated material was collected on GF 50 filters (Schleicher und Schull, Dassel, Federal Republic of Germany). The filters were washed with 7.5% trichloracetic acid containing 200 mM sodium pyrophosphate and dried, and the radioactivity was measured in a scintillation counter (Beckman, Palo Alto, Calif.). The protein concentrations were measured by the method of Lowry et al. (17). One unit of RNA polymerase activity incorporated 1 nmol of UMP into RNA in 10 min under standard assay conditions. The dissolved and dialyzed ammonium sulfate precipitate was loaded onto a DEAE-cellulose column. The column was washed with 200 ml of buffer B, and the enzyme was eluted by a linear NH4Cl gradient (0.02 to 1.2 M, 600 ml of buffer B). The enzyme activity eluted in a single peak between 0.36 and 0.5 M salt (data not shown). The peak activity fractions were pooled and dialyzed against buffer B. The following affinity chromatography steps on heparin-agarose and single-stranded DNA-cellulose removed most of the protein contaminations from the RNA polymerase of C. acetobutylicum. The pool from the DEAE-cellulose column was loaded onto a heparinagarose column (20 ml). The column was washed with buffer B, and the enzyme was eluted with a linear NH4Cl gradient (0.02 to 1.2 M, 160 ml of buffer B). The most active fractions were pooled, dialyzed against buffer B, and applied to a single-stranded DNA-cellulose column (10 ml). The enzyme was eluted with a linear ammonium chloride gradient from 0.02 to 1.2 M NH4Cl in buffer B (100 ml). The peak activity fractions were pooled, dialyzed against buffer B, and concentrated to a volume of 0.5 ml with a Centriprep 30 one-way concentrator (Amicon, Witten, Federal Republic of Germany). During both affinity chromatographies the enzyme eluted in a single peak between 0.36 and 0.6 M NH4Cl with the heparin-agarose material and between 0.5 and 0.6 M NH4Cl with the DNA-cellulose column (Fig. 1). To remove remaining contaminating proteins the concentrated active fractions (0.5 ml) from the DNA-cellulose column were loaded onto a 10-ml linear sucrose (10 to 30%)-glycerol (5 to 10%) gradient in buffer A containing 0.5 M NH4Cl but no glycerol. The centrifugation was carried out in a TH641 rotor (Beckman) at 41,000 rpm for 24 h. The gradient was partitioned in 0.4-ml fractions, which were analyzed by gel electrophoresis and enzyme activity measurements. The extent of purification was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 2) (14); the overall purification procedures are summarized in Table 1. The enzyme specific activity was enhanced 17-fold relative to that of the ammonium sulfate precipitate, with a 12.5% yield. The RNA polymerase protein was purified far more than 17-fold (estimated over 100-fold) (Fig. 2). Therefore, great losses in activity in the later purification steps must have occurred, which would explain the relatively low increase in specific activity of the purified enzyme. Composition of the RNA polymerase. The final preparation of the RNA polymerase from C. acetobutylicum contained a single protein as shown by gel electrophoresis under nondenaturing conditions (18). Gel filtration on a Superose 12 column with a fast-protein liquid chromatography system (Pharmacia, Freiburg, Federal Republic of Germany) revealed a molecular mass of 370,000 Da for that enzyme (data not shown). On polyacrylamide gels in the presence of

2121

NOTES

VOL. 173, 1991

= 12000 0

0

0 00

8000

4'

C.)0.

0

20

40

60

80

4000

4

00

0

10

fractions 4)

0,3 -

-6000

cB , x, ~ ~~~-1 ~

0;

4000

0c 00

0.

0

0

0,2100 0,0

C.) -

-

2

2000

C) E

.

0

0

0I 60 40 I8040 60 80

20 20

n

fractions

FIG. 1. Elution profile of RNA polymerase on heparin agarose (A) and single-stranded DNA-cellulose (B). The activity of the

enzyme was determined under the standard assay conditions described in the text; 20-,ul samples of the fractions were tested. The protein content was monitored (measured as A280). Symbols: protein; 0, enzyme activity.

sodium dodecyl sulfate, the subunit patterns of the enzyme exhibited two large components (A and B), with molecular masses of 128,000 and 117,000 Da, respectively, and two light components (C and D) of 46,000 and 42,000 Da, k20a

2

3

4

5

6

200 -A

-11

97-

6X-

43-

29 -

0

...9_

1-) Molm

.....

18 -

FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of samples from the purification steps of RNA polymerase. The gel. Lanes: 1, crude proteins were separated on a 12.5% acrylamide 3, DEAE extract; 2, polyethyleneimine-ammonium sulfate precipitate; pool; 4, heparin pool; 5, DNA-cellulose pool; 6, density gradient.

2122

J. BACTERIOL.

NOTES

TABLE 1. Purification of the DNA-dependent RNA polymerase from C. acetobutylicum"

Enzyme

Vol p(ml)activity ~(U)

Pur Purification step (ml)

Crude extract PP/AS DEAE column Heparin column DNA column Density gradient a

b

130 58 37 56 35 3

ND 7,163 5,848 5,065 2,630 897

Protein

(mg) 402 232.6 78.1 63.5 6.0 1.7

Sp act (U/mg of

Purificationb

protein)

(fold)

30.8 74.9 79.8 435.4 527.0

2.43 2.92 14.14 17.10

Yield (%)

100 81.6

70.7 36.7 12.5

ND, Not determined; PP/AS, polyethyleneimine and ammonium sulfate precipitation. Based on specific activity; see the text.

respectively. On Western immunoblots (26), antibodies against the major Escherichia coli sigma factor, a70, reacted strongly with the C subunit of the purified RNA polymerase from C. acetobutylicum; this polypeptide was tentatively designated a46 (Fig. 3). Therefore, the size of the sigma factor of the RNA polymerase from growing C. acetobutylicum cells is in the same range as the major form of sigma factors from other gram-positive bacteria, for example, Bacillus subtilis (43,000 Da) (2) and Lactobacillus curvatus (44,000 Da) (22). Cross-reaction of antibodies to E. coli a70 has been observed with sigma factors from other RNA polymerases (13). Very probably, the peptides A and B correspond to the /,P' subunits and peptide D corresponds to the a subunit of other eubacterial RNA polymerases. The tentative subunits and I' are smaller than the corresponding subunits of all other eubacterial RNA polymerases that have been described. Conditions for optimal enzyme activity. RNA synthesis by the purified RNA polymerase from C. acetobutylicum was absolutely dependent on the presence of a double- or singlestranded DNA template. It also required bivalent cations for activity. The highest activity was obtained with 20 mM Mg2" on C. acetobutylicum DNA and poly(dA-dT) DNA. With calf thymus DNA as a template, maximal activity was achieved with 10 mM Mg2" (Fig. 4 and 5). The addition of 3 mM Mn2" to the RNA polymerase assay resulted in comparable activity values on poly(dA-dT) DNA but in reduced activity on calf thymus DNA and C. acetobutylicum DNA (Fig. 4 and 5). Mg2" could also be replaced by Co2" (10% of

-ew

-

the activity observed with Mg2" on C. acetobutylicum DNA) but not by other bivalent cations, such as Ni2" and Zn2+. The optimal activity at different ionic strengths depended on the nature of the template. At 20 mM Mg2+, optimal activity was obtained in the presence of 50 mM NH4Cl on poly(dA-dT) DNA and C. acetobutylicum DNA and in the presence of 100 mM NH4C1 on calf thymus DNA. The RNA polymerase from L. curvatus exhibits optimal activity in the presence of Mn2+ under all conditions and is known as an Mn2+-dependent enzyme (22). Since Mn21 activated the enzyme of C. acetobutylicum only on poly(dAdT) DNA with the same efficiency as Mg2' and with considerably lower efficiency on other DNA templates, it is considered as an Mg2'-dependent RNA polymerase like the enzymes from E. coli (5) and B. subtilis (2). To elucidate the pH optimum we used morpholinoethanesulfonic acid buffer (pH 5.0 to 7.0), Tris-HCl (pH 7.0 to 10.0), and 2-amino-2-methyl-1-propanol buffer (pH 10.0 to 12.0) (all from Sigma Chemicals, Deisenhofen, Federal Re-

A

-I)

0

FIG. 3. Western immunoblot analysis of RNA polymerase subunits. Samples (5 ,ug) of purified enzyme were separated in a 12.5% acrylamide gel, transferred to nitrocellulose sheets, and analyzed with polyclonal antibodies against the a70 subunit of the E. coli RNA polymerase. Lanes: 1, proteins stained with Coomassie brilliant blue; 2, antibody reaction.

50

100

150

200

250

salt/bivalent cation [m] FIG. 4. Effect of ionic strength and manganese and magnesium concentration on RNA polymerase activity with poly(dA-dT) DNA as a template. The effects of manganese and magnesium were determined in the presence of 50 mM NH4Cl, and the effect of NH4Cl was determined in the presence of 10 mM Mg2". Symbols: *, manganese; r, magnesium; O, NH4Cl.

NOTES

VOL. 173, 1991

,:, 8000 ,5:

c

0

20

bivalent catons

40 [mM]

0

100

200

salt [mM]

FIG. 5. Effect of template DNA on RNA polymerase activity at different concentrations of Mg2' and Mn2+, with 100 mM NH4Cl for calf thymus DNA and 50 mM NH4Cl for C. acetobutylicum DNA (A) and at different ionic strengths with 20 mM MgCl2 (B). Assay conditions were as described in the text. (A) Symbols: 0, manganese, C. acetobutylicum DNA; 0, magnesium, C. acetobutylicum DNA; A, manganese, calf thymus DNA; A, magnesium, calf thymus DNA. (B) Symbols: 0, C. acetobutylicum DNA; A, calf thymus DNA.

public of Germany). The pH optimum of the RNA polymerase activity in vitro was found to be at 10 with C. acetobutylicum and poly(dA-dT) DNA as a template (data not shown). A broad temperature optimum of the RNA polymerase was found between 40 and 45°C (data not shown). The enzymatic reaction proceeded linearly at a temperature of 40°C for 25 to 30 min. Therefore the activity tests were usually carried out for 20 min at pH 10 with 20 mM Mg2' and 50 mM NH4CI (100 mM NH4Cl on calf thymus DNA) and at 40°C. The significance of the basic pH optimum and the high temperature optimum found in vitro is not clear for the in vivo environment, since C. acetobutylicum grows optimally at 37°C and has an internal pH of 7 and lower (10). Incubation of the RNA polymerase of C. acetobutylicm with rifampin (100 ,ug/ml) for 5 min resulted in a complete inhibition of the enzyme. Stability of RNA polymerase. The RNA polymerase from C. acetobutylicum was quite as labile as the enzyme from L. curvatus, and the recommendations concerning the isolation of such an unstable RNA polymerase given by Stetter and Zillig (22) were followed. Particularly notable is the fact that all buffers used for purification and storage had to be free of Mg2+ or Mn2+. Both cations caused significant losses of activity within several hours at 4°C and within several days even at -70°C. The enzyme activity could not be recovered by the addition of EDTA or by dilution in Mg2+- or Mn2+_ free buffer. In contrast, the RNA polymerase of B. subtilis is stabilized in the presence of Mg2+ (6). No physical changes could be observed during prolonged incubation with Mg2+ or Mn2 On the other hand, one of these cations was absolutely required for enzyme activity, in addition to nucleoside triphosphate and DNA. Furthermore, for the stability of the enzyme the addition of glycerol (40%) was absolutely necessary. Lower concentrations of glycerol, for example, 10%, caused losses of enzyme activity of 50% within a few days at 4°C and within 2 weeks at -20°C. The purified RNA poly.

2123

merase (2 to 8 mg of protein per ml) of C. acetobutylicum could be stored for several months at -20°C in buffer B containing 200 mM NH4Cl. The DNA-dependent RNA polymerase from C. acetobutylicum is one of the first transcriptases described from a member of the diverse group of Clostridium species. Recently the purification of the RNA polymerase from the pathogen Clostridium perfringens was reported (8), but a detailed characterization of that enzyme was not given. Nevertheless, the subunit composition and ratio of both clostridial enzymes corresponded to those of enzymes from many other eubacteria, including E. coli (5), B. subtilis (2), and L. curvatus (22). The purification procedure described herein and the characterization of the RNA polymerase from actively growing acid-producing C. acetobutylicum cells is the necessary basis for further studies. Recently, a connection between the heat shock response and the initiation of solvent formation in C. acetobutylicum was proposed (20, 25). In the regulation of the heat shock response of E. coli (23) and the sporulation process of B. subtilis (24), alternate sigma factors play an important role. It will be of interest to know whether the RNA polymerase from C. acetobutylicum cells, which switch from acid to solvent production, initiate sporulation, and/or are exposed to other forms of stress, contains different sigma factors controlling the expression of respective genes. We are grateful to W. Zillig for advice and helpful discussions and to G. Gottschalk for support and critically reading the manuscript. We thank C. A. Gross for the antibodies against u70 of E. coli. This work was supported by a grant of the Deutsche Forschungsgemeinschaft. REFERENCES 1. Andreesen, J. R., H. Bahl, and G. Gottschalk. 1989. Introduction

2.

3.

4.

5.

6. 7.

to the physiology and biochemistry of the genus Clostridium, p. 27-63. In N. P. Minton and D. J. Clarke (ed.), Biotechnology handbooks, vol. 3. Clostridia. Plenum Publishing Corp., New York. Avila, J., J. M. Hermoso, E. Vinuela, and M. Salas. 1971. Purification and properties of DNA-dependent RNA polymerase from Bacillus subtilis vegetative cells. Eur. J. Biochem. 21:526-535. Bahl, H., W. Andersch, K. Braun, and G. Gottschalk. 1982. Effect of pH and butyrate concentrations on the production of acetone and butanol by Clostridium acetobutylicum grown in continuous culture. Eur. J. Appl. Microbiol. Biotechnol. 14:1720. Bahl, H., and G. Gottschalk. 1988. Microbial production of butanol/acetone, p. 1-30. In H. J. Rehm and G. Reed (ed.), Biotechnology, vol. 6b. VCH Verlagsgesellschaft, Weinheim, Germany. Burgess, R. R. 1969. A new method for the large scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase. J. Biol. Chem. 244:6188-6201. Doi, R. H. 1982. RNA polymerase of Bacillus subtilis, p. 71-110. In D. A. Dubnau (ed.), The molecular biology of the bacilli. Academic Press, Inc., New York. Doi, R. H., and L.-F. Wang. 1986. Multiple procaryotic ribonucleic acid polymerase sigma factors. Microbiol. Rev. 50:227-

243. 8. Garnier, T., and S. T. Cole. 1988. Studies of UV-inducible promoters from Clostridium perfringens in vivo and in vitro. Mol. Microbiol. 2:607-614. 9. Gottschalk, G., J. R. Andreesen, and H. Hippe. 1981. The genus Clostridium, p. 1767-1803. In M. P. Starr, H. Stolp, H. G. Truper, A. Balows, and H. G. Schlegel (ed.), The prokaryotes. Springer-Verlag, Berlin. 10. Gottwald, M., and G. Gottschalk. 1985. The internal pH of Clostridium acetobutylicum and its effect on the shift from acid

2124

NOTES

to solvent formation. Arch. Microbiol. 143:4246. 11. Helmann, J. D., and M. J. Chamberlin. 1988. Structure .and function of bacterial sigma factors. Annu. Rev. Biochem. 57: 839-872. 12. Jones, D. T., and D. R. Woods. 1986. Acetone-butanol fermentation revisited. Microbiol. Rev. 50:484-524. 13. Klimpel, K. W., S. H. Lesley, and V. L. Clark. 1989. Identification of gonococcal RNA polymerase by immunoblot analysis: evidence for multiple sigma factors. J. Bacteriol. 171:3713-3718. 14. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 15. Long, S., D. T. Jones, and D. R. Woods. 1984. The relationship between sporulation and solvent production in C. acetobutylicum P262. Biotechnol. Lett. 6:529-534. 16. Long, S.,,D. T. Jones, and D. R. Woods. 1984. Initiation of solvent production, clostridial stage, and endospore formation in Clostridium acetobutylicum P262. Appl. Environ. Microbiol. 20:493498. 17. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275. 18. Margolis, J., and K. G. Kennrick. 1967. Polyacrylamide gel electrophoresis across a molecular sieve gradient. Nature (London) 214:1334-1336. 19. O'Brien, R. W., and J. G. Morris. 1971. Oxygen and the growth and the metabolism of Clostridium acetobutylicum. J. Gen.

J. BACTERIOL.

Microbiol. 68:307-318. 20. Pich, A., F. Narberhaus, and H. Bahl. 1990. Induction of heat shock proteins during solvent formation in Clostridium acetobutylicum. Appl. Microbiol. Biotechnol. 33:697-704. 21. Rogers, P. 1986. Genetics and biochemistry of Clostridium relevant to development of fermentation processes. Adv. Appl. Microbiol. 31:1460. 22. Stetter, K. O., and W. Zillig. 1974. Transcription in Lactobacillaceae. DNA-dependent RNA polymerase from Lactobacillus curvatus. Eur. J. Biochem. 48:527-540. 23. Straus, D. B., W. A. Walter, and C. A. Gross. 1987. The heat shock response of E. coli is regulated by changes in the concentration of a32. Nature (London) 329:3487-3492. 24. Tatti, K. M., H. L. Carter, A. Moir, and C. P. Moran, Jr. 1989. Sigma H-directed transcription of citG in Bacillus subtilis. J. Bacteriol. 171:5928-5932. 25. Terracciano, J. S., E. Rapaport, and E. R. Kashket. 1988. Stress and growth phase-associated proteins of Clostridium acetobutylicum. Appl. Environ. Microbiol. 54:1899-1995. 26. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 27. Young, M., N. P. Minton, and W. L. Staudenbauer. 1989. Recent advances in the genetics of the clostridia. FEMS Microbiol. Rev. 63:301-326.

Purification and characterization of the DNA-dependent RNA polymerase from Clostridium acetobutylicum.

The DNA-dependent RNA polymerase (EC 2.7.7.6) from Clostridium acetobutylicum DSM 1731 has been purified to homogeneity and characterized. The purifie...
929KB Sizes 0 Downloads 0 Views