JOURNAL OF BACrERIOLOGY, June 1978, p. 1002-1012 0021-9193/78/0134-1002$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 134, No. 3 Printed in U.S.A.

In Vitro Synthesis and Regulation of the Biotin Enzymes of Escherichia coli K-12 OM PRAKASH AND MAX A. EISENBERG* Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Received for publication 20 December 1977

The synthesis and regulation of two of the enzymes of the biotin operon of Escherichia coli, 7,8-diaminopelargonic acid aminotransferase and dethiobiotin synthetase, were studied in vitro in a coupled transcription-translation system. These enzymes are encoded by genes located on opposite strands of the divergently transcribed operon (A. Guha, Y. Saturen, and W. Szybalski, J. Mol. Biol. 56:53-62, 1971). The kinetics of synthesis of both the enzymes were determined, and the efficiency of the system was 0.3 to 0.4% that of the in vivo rate of synthesis in derepressed cells. Guanosine 3'-diphosphate 5'-diphosphate at 0.2 mM concentration stimulated the synthesis of 7,8-diaminopelargonic acid aminotransferase two- to threefold but had no effect on dethiobiotin synthetase synthesis. Biotin, which was most effective as the corepressor in vivo, also functioned in vitro at physiological concentrations in conjunction with a crude repressor protein isolated from a lysogen carrying the bioR gene. However, the two strands showed differential repression. At a repressor concentration where 7,8-diaminopelargonic acid aminotransferase synthesis was completely repressed, the repression of dethiobiotin synthetase was only 20% and did not exceed 50% with increasing repressor concentrations. Although the exact reason for the partial repression remains to be resolved, our data clearly suggest that the biotin operon is regulated from two separate operators.

Divergent transcription of the genes of the biotin locus at min 17 in the chromosomal map of Escherichia coli (2) was first demonstrated by Guha et al. (13). According to their transcriptional map, gene A is transcribed leftward on the I strand and genes B to D rightward on the r strand (Fig. 1). Biotin biosynthesis from these genes is regulated by biotin through repression (3), and early biochemical evidence suggested that the repression of biotin enzymes is coordinate (9). The repressive action of biotin is mediated through the bioR gene situated close to the bfe locus in E. coli (10, 24). Coordinate repression of the leftward and rightward transcriptional units by biotin was also demonstrated by Guha et al. (13) by the hybridization of bio-specific mRNA, isolated from E. coli grown under derepressed and repressed conditions, with the complementary DNA strands of a Abio phage. However, when biotin was replaced by the biotin analogs, adehydrobiotin and homobiotin, only transcription from the I strand was markedly reduced, indicating differential repression of the two strands (35). In contrast, Eisenberg (7), using the enzyme levels as a direct measure of repression, failed to observe differential repression

with the same analogs. Ketner and Campbell (17), on the basis of biochemical studies with regulatory mutants in the biotin locus, have indicated that the leftward and rightward transcriptions may not be under completely independent control. Their conclusion was based on the finding that promoter-up mutations for leftward transcription increased bioA transcription four- to sixfold and diminished bioB to bioD transcription by two-thirds. However, polar insertions into the bio operator-promoter region simultaneously inactivated transcriptions from both strands, whereas operator mutations resulted in simultaneous derepression. They proposed a model with two overlapping operons in which the promoter sites were face to face with no region in common. More recently, Das Gupta et al. (6) presented additional evidence based on electron microscopic studies on the binding of RNA polymerase to the promoter sites of the biotin operon, which lends support to their earher model in which the two promoters are located back to back. Although the genetic and biochemical studies of the biotin gene cluster of E. coli have been extensive, the nature of the repressor, the true corepressor, and their mechanism of interactions

1002

BIOTIN ENZYMES OF E. COLI K-12

VOL. 134, 1978 i strand op A

B 'po

r strand

F

C

D

o-

FIG. 1. Proposed model for the divergent transcription of the biotin locus of E. coli (13). The capital letters represent genes coding for the following enzymes: 7,8-diaminopelargonic acid aminotransferase (A); biotin synthetase (B); 7-keto-8-aminopelargonic acid synthetase (F); step prior to 7-keto-8-aminopelargonic acid synthetase (C); and dethiobiotin synthetase (D).

with an operator site(s) still remain to be resolved. Thus, we felt that an approach through an in vitro coupled transcription-translation system might provide some answers to these questions. The development of such in vitro systems has been very useful in the elucidation of the regulatory control mechanisms of several E. coli genomes (40). In this communication we report some of the general properties of the in vitro coupled transcription-translation system which supports the DNA-dependent synthesis of two of the enzymes of the biotin locus: 7,8-diaminopelargonic acid (DAPA) aminotransferase and dethiobiotin (DTB) synthetase encoded by A and D genes, respectively. We have also been able to demonstrate a differential repression of the synthesis of both the enzymes with a partially purified biotin repressor protein, suggesting independent regulation. A part of this work was presented at the 75th Annual Meeting of the American Society for Microbiology, New York, N.Y., April 27 to May 2, 1975. MATERLALS AND METHODS Reagents. tRNA was purchased from Miles Laboratories, Inc. Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) was purchased from P-L Biochemicals, Inc. Crystalline d-biotin, S-adenosyl-L-methionine, phospho(enol)pyruvate, and the sodium salts of all the nucleoside triphosphates were purchased from Sigma Chemical Co. Avidin was purchased from Worthington Biochemicals Corp. Folinic acid (leucovorin) was kindly provided by G. Zubay. Biotin sulfone and biocytin were prepared according to the procedures of Hofmann et al. (14) and Weijlard et al. (36), respectively. Biocytin was further purified by the method of McCormick and Fory (23). The a-dehydrobiotin and a-methylbiotin were gifts from L. J. Hanka. a-Dehydrobiotin was used after chromatography on a Dowex1-formate column. Homobiotin was a gift from W. E. Scott. All other chemicals were of the highest grades commercially available. Bacterial strains. The biotin deletion mutant of E. coli K-12, strain PR-7 (ns pnp-7) (28), was the source of S-30 extracts for most of the studies. In order

1003

to study the effect of added ppGpp on enzyme synthesis, S-30 extracts from E. coli K-12 strain 514 (F- Alac relA trp rpsL), kindly provided by N. Kelker, were used. The biotin deletion mutants of both strains were generated by nitrous acid mutagenesis as described by Alper and Ames (1). The colonies surviving on 2-

deoxy-D-galactose and chlorate under anaerobic conditions were selected for biotin auxotrophy by replica plating on media with and without biotin. These biostrains were phenotypically UV' and Gal-. Since rifampin or chloramphenicol was added to the reaction mixtures to terminate transcription or translation, respectively, it was essential to use bioassay organisms (bioA109 and bioD302, point mutants of E. coli, strain Y10-1) resistant to these antibiotics. Rifampin-resistant strains were obtained by the procedure previously described (10). Chloramphenicol resistance was introduced by transduction with PlCMcIrlOO according to the method of Rosner (32) for phage curing. Thermalresistant isolates were selected for chloramphenicol resistance. The strain bioB105, a point mutant of E. coli K-12 that grows only on biotin, was the assay organism for biotin estimation. The stock cultures of all the mutants were maintained at -20°C in nutrient medium containing 50% glycerol. The strain H105 (XcI857s7, AcI857s7drifdl8), kindly provided by J. B. Kirschbaum, was the source of repressor protein. The defective phage in the double lysogen carries a fragment of E. coli chromosome starting in bfe and extending through rif (18). The mapping data for bioR gene from two laboratories did not agree as to the position of bioR in relation to bfe (10, 24). Transduction studies with Adrifdl8 showed that phage did carry bioR (unpublished data), and hence the position of bioR is clockwise to bfe in accordance with the results of Pai and Yau (24). Isolation of phage DNA. DNA carrying the biotin gene cluster was isolated from a biotin-transducing, plaque-forming phage, Abiotl24, kindly provided by K. Krell. The entire biotin locus of E. coli in this phage replaces a segment of the A DNA from the attA through part of the cl gene (13). Stocks of Abiotl24 were prepared on strain T5-2, a biotin deletion mutant of E. coli K-12, Y10-1. The cells were grown at 37°C in a medium containing 1% tryptone, 0.5% yeast extract, 0.5% NaCl, and 10 mM MgSO4 until a cell density of 5 x 108 per ml was reached when the appropriate amount of phage was added to give a multiplicity of infection of 0.1. Incubation was continued with vigorous aeration, and cell lysis was monitored by the decrease in optical density. When a constant reading was reached, deoxyribonuclease I was added to the incubation mixture to reduce the

viscosity of the solution. The cell debris was then removed by centrifugation at 8,000 rpm for 10 min, and polyethylene glycol (10% wt/vol) and 5 M NaCl (10% vol/vol) were added to precipitate the phage. Phage were separated by centrifugation at 8,500 rpm for 15 min in a Sorvall SS-34 rotor, and resuspended in phage buffer [10 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.3, 2 mM MgSO4, and 68 mM NaCl], and 7.76 g of CsCl was added per 10 ml. The solution was first clarified by centrifugation at 5,000 rpm and then centrifuged in nitrocellulose tubes at 34,000 rpm in a Beckman type 40 rotor for 20

1004

PRAKASH AND EISENBERG

h at 5°C. Phage bands from a number of tubes were pooled, and sufficient CsCl solution (p = 1.5) was added to provide a suitable volume for recentrifugation at 34,000 rpm. The purified phage was then dialyzed for 15 h against 0.015 M sodium citrate, pH 7.0, containing 0.15 M NaCl. Phage DNA was extracted according to the method of Zubay (40). Stocks of AcI857s7 DNA were prepared in the manner described above after heat induction of the lysogen, H105 (AcI857s7), kindly furnished by J. B. Kirschbaum. Preparation of S-30 extracts. Cells of the appropriate strain were grown in 50-liter batches in a model F-130 fermentor (New Brunswick Scientific Co.) at 34°C in a medium containing, per liter: KH2PO4, 5.6 g; K2HPO4, 28.9 g; bactotryptone, 10 g; yeast extract, 5 g; glucose, 8 g; MgSO4 7H20, 0.2 g; thiamine hydrochloride, 5 mg; and biotin, 0.5 mg. The inoculum was prepared by growing the bacteria at 34°C in 2 liters of a medium containing 1% tryptone, 0.5% yeast extract, 0.5% NaCI, and 10 mM MgSO4 until a Klett reading at 66 nm of 180 was attained. The cell growth was monitored until a Klett value at 66 nm of 45 to 50 was reached. The medium was rapidly chilled by adding ice, and the cells were collected by continuous-flow centrifugation (CEPA, Carl Padberg, Germany). The final yield of cells was approximately 35 g of wet weight. The S-30 extracts were prepared essentially according to the procedure of Zubay (40) with some modifications. The cells were processed immediately rather than first freezing at -90°C and were broken in a Sorvall Omnimix using glass beads (Minnesota Mining Superbrite 100) at maximum speed for 3 min (12). The dialysis was carried out against 500 ml of buffer for 5 h with three changes. The dialyzed preparation was distributed in plastic vials in 0.5-ml portions and rapidly frozen in an acetone-dry ice mixture. The vials were stored at -90°C. The protein content of the extracta was estimated by the biuret procedure (21). Partial purification of biotin repressor. Strain H105 (Xc1857a7, AcI857s7rifd18) was grown to midlog at 34°C in a model F-130 fermentor in 50 liters of media containing, per liter: bactotryptone, 10 g; yeast extract, 5 g; NaCl, 5 g; MgSO4 7H20, 2.5 g; and biotin, 25 ,ug. The lysogen was induced at 42°C for 30 min, and the incubation was continued for an additional 3 h at 34°C before the culture was harvested by continuous-flow centrifugation. The cells were spread in a thin layer and frozen at -90°C. Fifty grans of cells was suspended in 1.3 volumes of breaking buffer [0.2 M tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.6, at 4°C, 0.2 M potassium chloride, 0.01 M magnesium acetate, 0.3 mM dithiothreitol, and 5% glycerol] and disrupted by sonic treatment (Bronson model W140D) for 10 min. The broken-cell suspension was stirred at 0°C for 1 to 2 h to decrease the viscosity and centrifuged for 3 h in a Beckman type 30 rotor at 30,000 rpm. The supernatant was subjected to ammonium sulfate fractionation, and the protein fraction precipitating between 30 to 60% saturation was dissolved in buffer I (0.01 M phosphate buffer, pH 7.6, 5% glycerol, and 0.1 mM dithiothreitol) and dialyzed against the same. The dialyzed preparation was applied to a 150-mi diethylaminoethyl (DEAE)-cellulose column (DE-32, Whatman Inc.) previously equilibrated with buffer I, and the column

J. BACTERIOL.

was washed with the same buffer. Most of the biotin binding activity as determined by the procedure of Bourgeois and Jobe (4) eluted in the first major peak. The active fractions were pooled and brought to 60% saturation with solid ammonium sulfate. The precipitated proteins were collected, dissolved in buffer II (0.01 M phosphate, pH 6.9, 5% glycerol, and 0.1 mM dithiothreitol), and dialyzed against the same buffer. The dialyzed sample was applied to a 50-ml phosphoceliulose column (P-11, Whatman Inc.) previously equilibrated with buffer II. The column was first washed with the equilibrating buffer until the absorbancy at 280 nm was below 0.1 and then eluted with buffer II containing 0.25 M NaCl. Only one protein peak was observed which showed both biotin binding and repressor activities. The active fractions were stored at -20°C. The high degree of inhibition observed with the crude extracts and frequently with the DEAE fractions precluded the use of the in vitro system for assaying the repressor activity during the early stages of purification. In vitro protein synthesis. Unless otherwise stated, all experiments were carried out with S-30 extracts from the PR-7 strain. The synthesizing mixture in a final volume of 0.25 ml was essentially that described by Zubay (40) with the following modifications; cyclic AMP was omitted, and 8 to 10 ,mol of magnesium acetate and 4 to 5 ,imol of calcium acetate per ml of synthesizing mixture were used. Routinely, 60 pg of Xbiotl24 DNA per ml was added unless otherwise indicated. Synthesis was initiated by the addition of 0.4 ml of S-30 extract per ml, corresponding to 7 to 8 mg of protein, and the incubation was carried out at 34°C for 20 min with shaking (240 rpm, gyratory waterbath shaker). The reaction was terminated by the addition of chloramphenicol (100 ug/ml). The reaction mixture without DNA served as the control. Enzyme ssays. The DAPA aminotransferase and DTB synthetase enzymes synthesized in the in vitro system were estimated by a modification of the bioassay procedures of Eisenberg and Stoner (11) and Eisenberg and Krell (8), which permitted the quantitative detection of as little as 1 pmol of reaction products. After termination of protein synthesis by the addition of the antibiotic, the activity of each enzyme was determined by the addition of a mixture of the appropriate reagents for each bioassay, and the incubation was continued for an additional hour at 37°C. The reaction mixture for DAPA aminotransferase assay contained, in a volume of 0.2 ml, 12.5 mM dithiothreitol, 0.5 mM pyridoxal 5'-phosphate, 6.25 mM S-adenosylmethionine, and 0.025 mM 7-keto-8-aminopelargonic acid. The DTB synthetase assay contained, in a volume of 0.1 ml, 50 mM adenosine 5'-triphosphate, 0.2 M NaHCO3, and 0.1 mM DAPA. The enzymatic reactions were terminated by the addition of 0.1 ml of 0.06 N ZnSO4 and heating for 1 min in a boiling water bath. An equivalent amount of Ba(OH)2 was then added to each tube, and the protein precipitates were removed by centrifugation. The clear supernatants were taken to dryness in vacuo over P206, the residues were resuspended in 0.04 ml of water, and 0.02-ml samples were applied to 6-mm filter disks for microbiological assy, using the appropriate Rif' Cml' assay organism. All results are averages of duplicate determinations.

VOL. 134, 1978 One unit is defined as that amount of enzyme which produces 1 nmol of DAPA or DTB in 60 min under the standard assay conditions used. In vitro repression studies. The conditions for protein synthesis were the same as described previously except that to maximize interaction of repressor, corepressor, and operator, all the components except the S-30 and nucleoside triphosphates were preincubated in a final volume of 0.15 ml for 3 min at 0°C. After addition of 0.08 ml of S-30, the reaction was started by the addition of 0.02 ml of nucleoside triphosphates. The reaction mixtures minus repressor, corepressor, and both served as controls. When only the DAPA aminotransferase activity was assayed, the residue was resuspended in phosphate-buffered saline (2 mM phosphate buffer, pH 7, with 0.9% NaCl) containing excess avidin in order to bind all the biotin or the biotin analog added to the reaction mixture. For the determination of DAPA aminotransferase and DTB synthetase activities in the same sample, the enzyme synthesis was carried out in a volume of 0.5 ml, and the samples were exhaustively dialyzed for 20 h against 2 mM tris(hydroxymethyl)aminomethanehydrochloride, pH 7.4, containing 0.006 M 2-mercaptoethanol and i0-' M pyridoxal 5'-phosphate to remove biotin prior to the enzyme assays. Only trace amounts of biotin were sometimes detected in the dialyzed samples as determined by bioassay with strain bioB10. The dialyzed samples were divided equally into two portions, and the enzyme activities were determined by bioassay procedures as previously described. The results of the DTB synthetase synthesis were corrected for biotin contamination when required, whereas the samples for DAPA aminotransferase were treated with avidin as indicated above.

RESULTS Since the S-30 extract was prepared from a strain of E. coli that contains a deletion of the

biotin locus, detectable enzyme activity after a period of incubation must represent true de novo synthesis of the enzyme. The results presented in Table 1 indicate the synthesis of both DAPA aminotransferase and dethiobiotin synthetase in the presence of ADNA template carrying the biotin locus. No enzyme synthesis can be detected in the absence of the Abio DNA or in the presence of wild-type ADNA. The absence of detectable activity in these controls also indicates no biotin contamination in the components of the system as well as no biotin release from any of the biotin-containing enzymes in the S-30 extract during the course of the reaction. Both transcription and translation are essential, since antibiotics that interfere with transcription (rifampin) or translation (chloramphenicol) completely abolish the synthesis of both enzymes when added prior to the addition of S-30 extract. The products of the enzyme reactions were identified by paper chromatography (Table 2) as well as by the growth response of the appropriate biotin mutants. As with other in vitro systems, the synthesis

BIOTIN ENZYMES OF E. COLI K-12

1005

TABLE 1. Cell-free synthesis of DAPA aminotransferaBe and DTB synthetase" Incubation system

DAPA ami-DTsyte notranserase systhe DtB (U/mi) tse(/) 0.53 0.21 0.00 0.00 0.00 0.00

Complete -Abiotl24 DNA -Abiotl24 DNA + AcI857s7 DNA + Rifampin 0.00 0.00 + Chloramphenicol 0.00 0.00 a In vitro synthesis and enzyme assays were performed as described in the text. The final Mg2+ concentration in this and all other experiments was 13 mM. The concentration of A phage DNA was 60 ug/ml. Rifampin (10 jig/ml) and chloramphenicol (100 pg/ml), where indicated, were added prior to the addition of S-30 extract. The extent of enzyme synthesis is presented as units per milliliter of incubation mixture. TABLE 2. Chromatographic characterization of DAPA aminotransferase and DTB synthetase productsa Source

RI

In vitro products DAPA aminotransferase 0.31 DTB synthetase 0.57 Reference compounds DAPA + reaction mixture 0.29 DTB + reaction mixture 0.57 a The products of in vitro synthesized enzymes were analyzed by ascending paper chromatography on Whatman 3MM filter paper with n-butanol-glacial acetic acid-water (60:15:25) as the solvent system. The chromatograms were developed by placing the paper onto the surface of a minimal agar medium inoculated with the appropriate assay organism.

of both enzymes is markedly dependent on the divalent ion concentration in the reaction mixture. The optimum Mg and Ca ion concentrations were found to be 13 and 4 mM, respectively. The optimum Mg ion concentration varied slightly with some S-30 preparations. In the absence of Ca ions, the activity of the system was markedly affected, decreasing by almost 85%. A calcium requirement for the functional and chemical stability of mRNA was reported by K. Jacobs and D. Schlessinger (Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, H65, p. 106). Cyclic AMP, which has been shown to be required for the maximal expression of a number of inducible operons (30), was without effect when added to the reaction mixture at a final concentration of 1o-3 M. Cyclic GMP, likewise, did not influence the synthesis of the two enzymes. Guanosine 3'-diphosphate 5'-diphosphate at a 0.2 mM concentration stimulated the synthesis of DAPA aminotransferase two- to

1006

PRAKASH AND EISENBERG

threefold with reLA S-30 extracts, whereas no dependence of DTB synthetase synthesis was observed. Addition of ppGpp with S-30 extracts from the rel strain had no effect on the synthesis of either of the enzymes. Kinetic studies. In order to obtain some measure of the efficiency of the in vitro system, it was necessary to optimize conditions for the synthesis and assay of both enzymes. Synthesis of.dethiobiotin synthetase was linear with increasing DNA concentration, reaching a maximum at about 60 ,ug per ml (Fig. 2). Synthesis of DAPA aminotransferase, however, was sigmoidal; it was linear only over the range of 15 to 40 yg of DNA per ml, but it also reached a maximum at about 60 lag of DNA per ml. Nonlinearity at the lower DNA concentrations was observed consistently, regardless of the source of the S-30 extracts. As shown in Fig. 3, the time course for each enzyme assay after protein synthesis is linear up to 80 min, so that initial rate studies would be a true measure of the amount of active enzyme synthesized. Using the manimum concentration of DNA and a 60-min time period for assay, it was possible to study the kinetics of enzyme synthesis. The in vitro-coupled transcription-translation reaction was permitted to go on for the time intervals indicated in Fig. 4 before the reaction was terminated with the addition of chloramphemcol and the bioassay reagents were added. Both enzymes were readily detected as early as 5 min. The rate of

J. BACTrERIOL.

E

a,

/

0.4

N/ W 0.2

0.

0NCUsaToN TINE (m10) FIG. 3. Kinetics of DAPA aminotransferase (0) and DTB synthetase (A) activities. Standard conditions of protein synthesis and enzyme assay were used except with a DNA concentration of 55 pg/ml. The enzyme assays were carried out for the times

indicated.

0

r

0

0.4

0.6

0~~~~~~~~ wz 0.2

0.4

CA/ z

0.2

I/

0

20

40

60

so

SYNTHESIS TIME (min)

FIG. 4. Kinetics of in vitro synthesis ofDAPA aminotransferase (0) and DTB synthetase (A). Standard conditions of protein synthesis and enzyme assay were used with the protein synthesis stopped at the indicated times by the addition of chloramphenicol. The DNA concentration in the reaction mixture was 60 pg/ml.

A^ /

60 synthesis of each enzyme was constant up to 20 e0 min and then fell to 0. The reason for this abrupt (pg/mi) FIG. 2. Synthesis of DAPA aminotransferase (0) termination is not known, but the fact that the and DTB synthetase (A) as a function of DNA con- enzymatic activity does not decrease over the centration. Standard conditions ofprotein synthesis 90-mm period would suggest that the two enand enzyme assay were used with the various DNA zymes are stable under these conditions. concentrations indicated. Comparison of in vitro and in vivo rates 0.0

20

D

DNA

VOL. 134, 1978

of enzyme synthesis. From the initial slopes of the curves shown in Fig. 4, it was possible to estimate the rate of synthesis per gene and compare it with the in vivo rate. The in vivo rate of synthesis of each enzyme was estimated from the rate of derepression of exponentially growing biotin auxotroph, bioB105, in which synthesis of biotin from dethiobiotin is blocked (7). This corresponded to 7.5 x 10"° molecules of DAPA aminotransferase and 7 x 1010 molecules of DTB synthetase per mg of cell protein per min. These estimates were based on the known molecular weights of the two enzymes and the calculated specific activities of 6.8 and 5.5 U/,ug of the respective pure proteins (33, 19). The assumption was made that the turnover numbers of the enzymes in the crude extracts and pure proteins are identical. To determine the number of molecules synthesized per cell per min, we used the value of 2.4 mg of protein per 1010 cells, which is based on our determination of the total cell protein by the procedure of Lowry et al. (22) and viable cell count. Thus each cell produced 18 molecules of DAPA aminotransferase and 16.8 molecules of DTB synthetase, respectively. On the assumption that cells with a generation time of 60 min contain two chromosomes (5), each with a copy of the bioA and bioD genes, then approximately nine molecules of each enzyme were synthesized per gene per mi. Our in vitro protein-synthesizing system produced approximately 2.9 x 1010 molecules of DAPA aminotransferase and 4.3 x 1010 molecules of DTB synthetase per ml per min, respectively, under optimum conditions. Assuming a

BIOTIN ENZYMES OF E. COLI K-12

low the course of the purification procedure. A fraction with the highest biotin-binding activity was obtained which exhibited poor repression and marked inhibition of the in vitro system. A more appropriate source of the repressor protein was found to be the lysogen H105 (AcI857s7, AcI857s7drifdl8) isolated by Kirschbaum and Konrad (18). Since this phage was found to carry the bioR gene, heat induction resulted in a large increase in repressor protein. A partially purified preparation as described in the text was used for the repression studies, and a DNA concentration of 33 ,ug/ml was maintained in order to gain a more favorable repressor-to-DNA ratio. The results, shown in Table 3, indicate that, in the absence of biotin, the addition of repressor produces a 48% decrease in enzyme synthesis; but when biotin was also added, there was a further decrease in synthesis corresponding to 86% of that observed with repressor alone. Biotin by itself did not affect the enzyme synthesis. Addition of avidin to the complete system before initiating protein synthesis resulted in about 70% derepression. However, the inhibitory effect of repressor on enzyme synthesis was not alleviated by pretreating the repressor preparation with avidin in the reaction mixture prior to initiating protein synthesis, indicating that residual-free biotin is not the responsible factor. The unavailability of bound biotin to avidin cannot be excluded. The effect of repressor concentration on enzyme synthesis is presented in Fig. 5. The DAPA TABLE 3. In vitro repression of DAPA aminotransferase synthesisa

molecular weight for Abiotl24 DNA of 30 x 106

and

that it contains one copy of each the rate of synthesis per gene per min was calculated to be 0.024 and 0.036 molecules for DAPA aminotransferase and DTB synthetase, respectively. This gives an efficiency (expressed as the percentage of the in vivo rate per genome) of 0.27% for DAPA aminotransferase and 0.43% for DTB synthetase-synthesis. These values may be underestimated, since in our calculations we have assumed that all DNA molecules are active as templates. In vitro repression of DAPA aminotransferase. The fact that DAPA is not combinable with avidin made it feasible for us to use our bioassay method to study the repression of DAPA aminotransferase synthesis with biotin as the corepressor. Preliminary experiments with S-30 extract from a bioR+ strain were unsuccessful in demonstrating repression in the presence of biotin. Assuming that the repressor concentration was too low in the extracts, we proceeded to carry out fractionation on a large scale. The biotin-binding assay was used to folassung

gene,

1007

DAPA amiIncubation system

Complete - Biotin - Repressor - Biotin - repressor + Avidin - Biotin + avidin

notransferase

(U/mi) 0.02 0.14 0.27 0.27

I

.b.

tionb (%) sin() sionRe(%) ton(% 86 48 0

0.10 29 0.13 52 a The repression studies were carried out as described in the text. DNA and biotin concentrations in the synthesizing mixtures were 33,Ug/ml and 200 nM, respectively. 80 il (0.16 mg of protein) of repressor preparation was used per ml of synthesizing mixture. Avidin (1 U/ml) was added after 3 min of incubation at 0°C and incubated for an additional 2 min before

initiating protein synthesis. b Percent decrease in enzyme synthesis in the presence of repressor alone as compared with synthesis observed in the absence of both repressor and biotin. Percent decrease in enzyme synthesis in the presence of both repressor and biotin as compared with synthesis observed in the presence of repressor alone. '

1008

PRAKASH AND EISENBERG

J. BACTERIOL.

1k 20

20

40

6o

so

REPRESSOR (hal/mil)

FIG. 5. DAPA aminotransferase synthesis as a function of repressor concentration. Each synthesizing mixture with (0) or without (A) biotin (200 nM) contained the repressor concentrations indicated. Other experimental conditions were as described in the text. Maximal enzyme synthesis refers to the units of DAPA aminotransferase synthesized in the absence of repressor and biotin.

aminotransferase synthesis fell rapidly with increasing repressor concentration up to 40 ll/ml with little change thereafter. The inhibitory effect of the repressor protein almost paralleled the repressor activity. The effect of biotin concentration on repression was studied with varying concentrations of biotin and a constant amount of repressor protein in the synthesizing mixtures (Fig. 6). The addition of biotin alone had no effect on enzyme synthesis, as already indicated, whereas in the presence of repressor, the enzyme synthesis dropped as the biotin concentration was increased and reached an almost constant level above 120 nM. The specificity of biotin as the corepressor is attested to by the results presented in Table 4 showing the effect of several structurally related biotin analogs on the repression of DAPA aminotransferase synthesis. None of the compounds tested produced any appreciable repression, even when used at a concentration 200 times higher than biotin. To determine whether the synthesis of DAPA aminotransferase was prevented at the transcriptional level, biotin was added to the synthesizing mixtures containing repressor protein at various time intervals after initiating protein synthesis. The reaction was then continued for a total of 20 min to obtain maximal enzyme synthesis and stopped by the addition of chloramphenicol. The results are presented in Fig. 7. The zero-time points represent the results of the almost simultaneous addition of the nucleoside triphosphates with biotin, rifampin, or avidin. It is evident that the addition of biotin even at this point represses the system to almost 90%. As the time interval increased the degree of repression decreased and was essentially abolished within

5 to 6 min. To gain some insight into the kinetics of transcription, rifampin was added under the same conditions instead of biotin. There was approximately a 1-min lag period before the enzyme synthesis began to increase rapidly and reached a maximum in 5 min, indicating that the majority of RNA polymerase molecules are actively engaged in transcription at this stage. The data indicate that the biotin-repressor complex is acting at the transcriptional level, since the repression was maximal during the early stages oftranscription when the number of RNA polymerase molecules actively engaged in transcription was low and decreased as the initiation sites became progressively saturated with RNA polymerase. The addition of avidin immediately after initiating protein synthesis partially derepresed the system. However, after 1 min, practically no effect was observed, suggesting that the biotin in the complex is not accessible to avidin. In vitro repression of DTB synthetase. The in vitro studies on the repression of DTB synthetase synthesis could not be carried out with the procedure used for DAPA aminotransferase, since DTB is avidin combinable. However, exhaustive dialysis of the reaction mixtures prior to enzyme assays removed the added biotin and permitted repression studies on DTB synthetase synthesis. Results of these studies are presented in Table 5, and for comparison, the DAPA aminotrnsferase activities in the same samples are also included. No measurable amounts of biotin were detected in any of the samples after dialysis. The data clearly indicate that under the conditions where DAPA aminotransferase synthesis is almost completely repressed, the repression of DTB synthetase is only about 50% in both repressor preparations. Lowering the repressor concentration to one-

BIOTIN CONCENTRATION InM)

FIG. 6. Effect of biotin concentration on the repression of DAPA aminotransferase synthesis. Each synthesizing mixture with (0) or without (A) repressor (80 id/ml) contained biotin as indicated. Other experimental conditions were as described in the text.

BIOTIN ENZYMES OF E. COLI K-12

VOL. 134, 1978

TABLE 4. Effect of biotin analogs on DAPA aminotransferase synthesise Concn

(g/ml)

Biotin or analog

Repressionb (%)

0.02 83 Biotin 4.0 16 Biotin sulfone 4.0 24 Biocytin 4.0 4 a-Methylbiotin Homobiotin 4.0 16 4.0 9 a-Dehydrobiotin aThe repression studies were carried out as described in the text except that various analogs were used in place of biotin. The samples were treated with sufficient amounts of avidin before applying to the filter disks to avoid interference in the bioassay procedure. b Percent decrease in enzyme synthesis as compared with synthesis observed in the presence of repressor alone.

1009

half did not significantly change the repression of DAPA aminotransferase synthesis, indicating that the repressor is present in saturating concentrations. However, the repression of DTB synthetase under the same conditions was reduced to one-half the value. On the other hand, a threefold increase in repressor concentration did not materially increase the degree of repression over that of the original preparation. The biotin concentration in these experiments was not limiting, since almost 10 times the concentration required for more than 80% repression of DAPA aminotransferase synthesis (Fig. 6) was used. Since both the repressor fractions at different stages of purification exhibited similar repression patterns, it would suggest that the same protein is involved in the repression of the synthesis of both the enzymes.

DISCUSSION In this communication, we have demonstrated the in vitro synthesis and repression of two of the biotin enzymes of E. coli, DAPA aminotransferase and DTB synthetase, which are encoded on the I and r strands, respectively. The synthesizing efficiency for both enzymes is 0.3 to 0.4% of the in vivo rate, which is higher than the 0.11% observed for galactokinase (37) and 0.09% for N-a-acetylornithinase (34) systems but lower than the 5% reported for arabinose isomerase (38), 2

5 TIME OF ADOITION imin) 3

4

FIG. 7. Kinetics of repression and Itranscription. Biotin (2X0 nM), 0; rifampin (10 pg/ml) A;roravidin (0.5 U/ml), El was added to the synthesi'z;n ormixtue. containing repressor (80 id/ml) or repre tin at the indicated time intervals. Oth4 tal conditions were as in the text. Ma;rimal enzyme synthesis refers to the units of DAPA aminotransferase synthesized in the presence of rej pressor alone.

2 to 10% for

and 10% for

7

tryptophan synthetase (26), 6-phosphogluconate dehydrogenase

Our studies have revealed a number of differin the in vitro mode of synthesis and

ences

ezin erexperimn X

repression

of the two enzymes. The DNA satu-

curve (Fig. 2) is sigmoidal for DAPA aninotransferase but not for DTB synthetase synthesis. We have ruled out the following contributing factors: interference in the enzyme as-

ration

TABLE 5. In vitro repression of DAPA aminotransferase and DTB synthetase synthesis with partially purified repressor proteina DAPA aminotransferase Purification step

Volume of repressor fraction (pl/ml)

synthetase

DTB synthetase

Inhibition Repression Repression (%) (%) (%) (O 40 0 20 DEAE-cellulose (combined wash) 24 83 80 20 0 44 98 40 82 22 3 0 Phosphocellulose (0.25 N NaCl eluate) 80 25 95 28 48 8 51 240 24 96 The in vitro repression studies were carried out as described in the text. The DNA and biotin concentrations in the synthesizing mixtures were 27 #g/ml and 800 nM, respectively. The above repressor fractions, isolated from another batch of cells, were less inhibitory than those used in the preceding studies. Protein concentrations of DEAE-cellulose (combined wash) and phosphocellulose (0.25 N NaCl eluate) fractions were 5 and 1 mg per ml, respectively. Inhibition

1010

PRAKASH AND EISENBERG

say by the components of the in vitro synthesizing mixture, and slow association of the enzyme subunits into the active dimeric form (33) at low enzyme concentrations. This phenomenon must, therefore, be ascribed to some step in the synthesis of this enzyme. The DAPA aminotransferase synthesis was stimulated two- to threefold by the addition of ppGpp when an S-30 extract from a relA strain was used. No effect, however, was observed on DTB synthetase synthesis, suggesting that the ppGpp effect in our system is gene specific. A similar observation was made in the divergently transcribed argECBH gene cluster (25) where argH gene expression is stimulated and argE gene expression is inhibited (29). The repression of the A and D gene expression was also different. Normally, 20 nM biotin almost completely represses the synthesis of both enzymes in vivo (7). Our earlier studies on biotin transport in E. coli K-12 strain Y10-1 indicated a 16-fold increase in the internal biotin concentration when the external concentration was 20 nM (27). In the in vitro system, maximum repression of DAPA aminotransferase synthesis was observed at 120 nM biotin with S-30 preparation from strain PR-7 (Fig. 6). Although the internal biotin concentration of this strain was not measured, it would appear that the concentration required for complete repression of DAPA aminotransferase synthesis is within the physiological range. On the other hand, DTB synthetase synthesis was only partially repressed (48%) even at 800 nM biotin (Table 5). When the repressor concentration was increased threefold, the repression still did not exceed 51%. Thus, neither the biotin nor the repressor concentration can account for the partial repression of this enzyme. Two possible explanations can account for the failure to observe complete repression of DTB synthetase synthesis in our in vitro system. (i) Transcription may originate at an internal promoter site(s) within bioB to bioD genes. Imamoto and Ito (15) reported that in trp operon, transcription could be initiated at internal promoters and was not repressible by tryptophan. Examination of our system by their procedure did not reveal any internal promoter(s) in the biotin operon. The electron microscopic studies of Das Gupta et al. (6) also suggest the absence of internal promoter sites, since only two strong RNA polymerase binding sites were observed within the regulatory region ofthe biotin operon. (ii) Initiation of transcription may occur at a phage promoter site instead of the proper promoter site and read through the bacterial genes as reported in the case of tip operon (39). Previous studies by Krell et al. (20) on biotin escape synthesis in induced A lysogens showed that a

J. BACTERIOL.

deletion in the b2 region was required for transcriptional escape synthesis of DTB synthetase. The escape synthesis of DAPA aminotransferase which is under replicative control was less marked. Approximately 170 molecules of DTB synthetase were synthesized per min, compared with only 1 molecule of DAPA aminotransferase. The Abiotl24 DNA template used in our experiments has an intact b2 region, and the ratio of DTB synthetase to DAPA aminotransferase molecules synthesized per gene in vitro is 1.5 compared with 0.9 in a fully derepressed cell in vivo. Thus, while extensive readthrough from A promoter site(s) did not appear to be occurring in our system, a small amount of readthrough even with an intact b2 region could account for the partial repression of DTB synthetase synthesis. The effect of biotin at the transcriptional level was clearly demonstrated by Vrancic and Guha (35) in an in vivo system using the hybridization technique. This is further corroborated by our in vitro studies which demonstrate that the repressor-biotin complex can successfully compete with RNA polymerase for the regulatory region on the template when biotin was added initially (Fig. 7). As the time interval for biotin addition after initiation of the reaction increased, the repression decreased, and by 5 min, biotin had little effect when the majority of RNA polymerase molecules were engaged in transcription. Similar observations were made by Rose and Yanofsky (31) for the tip operon. The fact that the rifampin curve leveled off after 5 min suggested that transcription had stopped. However, since the rate of enzyme synthesis is linear for 20 min when chloramphenicol is used to terminate protein synthesis, the data would indicate that translation ofthe message may be occurring more than once. Vrancic and Guha (35) also observed differential repression by the biotin analogs, a-dehydrobiotin and homobiotin, which completely repressed only the transcription on the I strand. Eisenberg (7), however, reported coordinate repression of the two enzymes with biotin and a-dehydrobiotin. The various biotin analogs tested in the in vitro system for corepressor activity using the DAPA aminotransferase level as an indicator proved to be ineffective when used at 200 times the biotin concentration necessary for complete repression (Table 4). Thus, the in vitro results do not correlate with either of the above in vivo studies. A differential effect on the leftward and rightward transcriptions is obtained with varying repressor concentrations (Table 5), suggesting that there should be two binding sites for the repressor-corepressor complex; i.e., the regulation of transcription from two strands is independent.

BIOTIN ENZYMES OF E. COLI K-12

VOL. 134, 1978

It is very unlikely that there are two different repressor proteins, each encoded by a separate gene, since one single mutation has been mapped near the bfe locus which affects the expression of both the A and D genes (10, 24). Further experiments using better-defined systems and highly purified repressor preparations should help elucidate the regulatory mechanisms mvolved in this bidirectionally oriented operon and resolve the differences observed in our in vitro studies with the in vivo findings of various investigations. ACKNOWLEDGMENTS We thank L. J. Hanka of Upjohn Co., Kalamazoo, Mich., for his generous supply of a-dehydrobiotin and a-methylbiotin and W. E. Scott of Hoffman-LaRoche Laboratories, Nutley, N.J., for homobiotin. We also thank Sue-Chi Hsiung for her technical assistance. This investigation was supported by Public Health Service grant AM-14450 from the National Institute of Arthritis, Metabolism and Digestive Diseases.

UITERATURE CITED 1. Alper, M. D., and B. N. Ames. 1975. Positive selection of mutants with deletions of the gal-chl region of the Salmonella chromosome as a screening procedure for mutagens that cause deletions. J. Bacteriol. 121:259-266. 2. Bachmann, B. J., K. B. Low, and A. L Taylor. 1976. Recalibrated linkage map of Escherichia coli K-12. Bacteriol. Rev. 40:116-167. 3. Birnbaum, J., C. H. Pai, and H. C. Lichstein. 1967. Biosynthesis of biotin in microorganisms. V. Control of vitamer production. J. Bacteriol. 94:1846-1853. 4. Bourgeois, S., and A. Jobe. 1970. Superrepressors of the lac operon, p. 325-341. In J. R. Beckwith and D. Zipser (ed.), The Lac operon. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 5. Cooper, S., and C. E. Helmstetter. 1968. Chromosome replication and the division cycle of Escherichia coli B/r. J. Mol. Biol. 31:618-540. 6. Das Gupta, C. K., A. Vrancic, and A. Guha. 1977. Isolation and characterization of the biotin genes of Escherichia coli K-12. Gene 1:331-345. 7. Eisenberg, M. A. 1975. Mode of action of a-dehydrobiotin, a biotin analogue. J. Bacteriol. 123:248-254. 8. Eisenberg, M. A., and K.-Kreli. 1969. Dethiobiotin synthesis from 7,8-diaminopelargonic acid in cell-free extracts of a biotin auxotroph of Escherichia coli K-12. J. Biol. Chem. 244:5503-5509. 9. Eisenberg, M. A., and R. Maseda. 1966. An early intermediate in the biosynthesis of biotin. Incorporation studies with [1,7-'4C2] pimelic acid. Biochem. J. 101:601-606. 10. Eisenberg, M. A., B. Mee, 0. Prakash, and M. R. Eisenberg. 1975. Properties of a-dehydrobiotin-resistant mutants of Escherichia coli K-12. J. Bacteriol. 122:66-72. 11. Eisenberg, M. A., and G. L. Stoner. 1971. Biosynthesis of 7,8-diaminopelargonic acid, a biotin intermediate, from 7-keto-8-aminopelargonic acid and S-adenosyl-Lmethionine. J. Bacteriol. 108:1135-1140. 12. Gold, L. M., and M. Schweiger. 1971. Synthesis of bacteriophage-specific enzymes directed by DNA in vitro. Methods Enzymol. 20:537-542. 13. Guha, A., Y. Saturen, and W. Szybalski. 1971. Divergent orientation of transcription from the biotin locus of Escherichia coli. J. Mol. Biol. 56:53-62.

1011

14. Hofmann, K., D. B. Melville, and V. duVigneaud. 1941. Characterization of the functional groups ofbiotin. J. Biol. Chem. 141:207-214. 15. Imamoto, F., and J. Ito. 1968. Simultaneous initiation of transcription and translation at internal sites in the tryptophan operon of Escherichia coli. Nature (London) 220:27-31. 16. Isturi, T., and R. E. Wolf, Jr. 1975. In vitro synthesis of a constitutive enzyme of Escherichia coli, 6-phosphogluconate dehydrogenase. Proc. Natl. Acad. Sci. U.S.A. 72:4381-4384. 17. Ketner, G., and A. Campbell. 1975. Operator and promoter mutations affecting divergent transcription in the bio gene cluster of Escherichia coli. J. Mol. Biol.

96:13-27. 18. Kirschbaum, J. B., and E. B. Konrad. 1973. Isolation of a specialized lambda transducing bacteriophage carrying the beta subunit gene for Escherichia coli ribonucleic acid polymerase. J. Bacteriol. 116:517-526. 19. Krell, K., and M. A. Eisenberg. 1970. The purification and properties of dethiobiotin synthetase. J. Biol.

Chem. 245:6558-6566.

20. Krell, K., M. E. Gottesman, J. S. Parks, and M. A. Eisenberg. 1972. Escape synthesis of the biotin operon in induced Ab2 lysogens. J. Mol. Biol. 68:69-82. 21. Layne, E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol. 2:447-453. 22. 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. 23. McCormick, D. B., and W. Fory. 1970. Purification of biocytin by ion-exchange chromatography. Methods

Enzymol. 18A:413-414. 24. Pai, C. H., and H. C. Yau. 1975. Chromosomal location of mutations affecting the regulation of biotin synthesis in Escherichia coli. Can. J. Microbiol. 21:1116-1120. 25. Panchal, C. J., S. N. Bagchee, and A. Guha. 1974. Divergent orientation of transcription from the arginine gene ECBH cluster of Escherichia coli. J. Bacteriol. 117:675-680. 26. Pouwels, P. H., and J. van Rotterdam. 1972. In vitro synthesis of enzymes of the tryptophan operon of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 69:1786-1790. 27. Prakash, O., and M. A. Eisenberg. 1974. Active transport of biotin in Escherichia coli K-12. J. Bacteriol. 120:785-791. 28. Reiner, A. M. 1969. Characterization of polynucleotide phosphorylase mutants of Escherichia coli. J. Bacteriol. 97:1437-1443. 29. Reiness, G., H-L Yang, G. Zubay, and M. Cashel. 1975. Effects of guanosine tetraphosphate on cell-free synthesis of Escherichia coli ribo8omal RNA and other gene products. Proc. Natl. Acad. Sci. U.S.A. 72:2881-2885. 30. Rickenberg, H. V. 1974. Cyclic AMP in procaryotes. Annu. Rev. Microbiol. 28:353-369. 31. Rose, J. K., and C. Yanofsky. 1974. Interaction of the operator of the tryptophan operon with repressor. Proc. Natl. Acad. Sci. U.S.A. 71:3134-3138. 32. Rosner, J. L. 1972. Formation, induction and curing of bacteriophage P1 lysogens. Virology 48:679-689. 33. Stoner, G. L., and M. A. Eisenberg. 1975. Purification and properties of 7,8-diaminopelargonic acid aminotransferase. An enzyme in the biotin biosynthetic pathway. J. Biol. Chem. 250:4029-4036. 34. Urm, E., N. Kelker, H. Yang, G. Zubay, and W. Maas. 1973. In vitro repression of N-a-acetyl-L-ornithinase synthesis in Escherichia coli. Mol. Gen. Genet. 121:1-7. 35. Vrancic, A., and A. Guha. 1973. Evidence of two operators in the biotin locus of Escherichia coli. Nature (London) New Biol. 245:106-108. 36. Weijlard, J., G. Purdue, and M. Tishler. 1954. Im-

1012

PRAKASH AND EISENBERG

proved synthesis of biocytin. J. Am. Chem. Soc. 76:2606. 37. Wetekam, W., K. Staack, and R. Ehring. 1971. DNAdependent in vitro synthesis ofenzymes of the galactose operon ofEcherichia coli. Mol. Gen. Genet. 112:14-27. 38. Wilcox, G., P. Meuri, R. Base, and K. Engleberg. 1974. Regulation of the L-arabinose operon BAD in

J. BACTERIOL.

vitro. J. Biol. Chem. 249:2946-2962. 39. ZaIkin, IL, C. Yanobky, and C. L Squires. 1974. Regulated in vitro synthesis of Escherichia coli tryptophan operon messenger ribonucleic acids and enzymes. J. Biol. Chem. 249:4656475. 40. Zubay, G. 1973. In vitro synthesis of protein in microbial systems. Annu. Rev. Genet. 7:267-287.