Analysis of bacterial biotin-proteins

Biochimica et Biophysica Acta, 379 (1975) 496--503 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 36953 ANALYSIS OF B A C T E R I A L BIOTIN-PROTEINS

R. RAY FALL*, A. W. ALBERTS and P. R. VAGELOS Department of Biological Chemistry, Division of Biology and Biomedical Sciences, Washington University, St. Louis, Mo. 63110 (U.S.A.) (Received July 9th, 1974)

SUMMARY The biotin-protein populations in several bacterial strains were analyzed by solubilization of [3H]biotin-labeled cells with sodium dodecylsulfate followed by electrophoresis on polyacrylamide gels containing the detergent. A variety of patterns of biotin-labeled polypeptide chains was seen, ranging from a single biotin-protein in Escherichia coli, corresponding to the biotin carboxyl carrier protein component of acetyl-CoA carboxylase, to multiple species in Enterobacter aerogenes, Pseudomonas citronellolis, Bacillus cereus, Propionibacterium shermanii, Lactobacillus plantarum, and Mycobacterium phlei, which probably represent subunits of multiple biotin-dependent enzymes present in these organisms. In the case ofPseudomonas citronellolis two major biotin-containing polypeptides with approximate molecular weights of 65 000 and 25 000 were shown to correspond to the biotin carboxyl carrier components of pyruvate carboxylase and acetyl-CoA carboxylase, respectively. Thus in the case of Pseudomonas citronellolis two different biotin-dependent enzymes in the same cell do not share common biotin carboxyl carrier subunits.

INTRODUCTION It is now well established that the biotin-dependent enzymes, such as acetylCoA, propionyl-CoA, fl-methylcrotonyl-CoA, and pyruvate carboxylases, and urea amidolyase, catalyze analagous two-step reactions which involve a covalently bound biotin residue which serves as a "CO2 carrier" (see refs 1 and 2 for a review): Enzyme-biotin -k ATP + HCO3- ~- Enzyme-biotin-CO2- -k ADP ÷ P~

(1)

Enzyme-biotin-CO2- -k acceptor ~- Enzyme-biotin -k acceptor-CO2-

(2)

In addition, a methylmalonyl-CoA: puryvate transcarboxylase [3] has been described which involves an enzyme-bound biotin "CO2 carrier"; and certain decarboxylases apparently also catalyze biotin-dependent reactions [4, 5]. * Current address: Department of Chemistry, University of Colorado, Boulder, Colo. 80302, U.S.A.

497 In the case of Escherichia coli acetyl-CoA carboxylase, the role of a biotincontaining "CO2 carrier" in Reactions 1 and 2 has been clearly elucidated following resolution of the enzyme into three functional subunits [6, 7]: biotin carboxylase, which catalyzes Reaction 1; a transcarboxylase, which catalyzes Reaction 2 with acetyl-CoA as acceptor; and a biotin carboxyl carrier protein, which contains a covalently bound biotin residue and acts as the "CO2 carrier" between the biotin carboxylase and transcarboxylase subunits. A biotin carboxyl carrier protein subunit has also been isolated from Propionibacterium shermanii methylmalonyl-CoA:pyruvate transcarboxylase by Gerwin et al. [8]. The similarity in mechanism of biotin-dependent enzymes has led to the suggestion that organisms which contain multiple biotin-dependent enzymes might share common subunits [7, 8]. For example, it seems plausible that acetyl-CoA, propionylCoA, and pyruvate carboxylases in the same cell might share common biotin carboxylase and biotin carboxyl carrier protein subunits, while differing in the transcarboxylase subunit which specifies the acceptor (i.e. acetyl-CoA, propionyl-CoA or pyruvate). In order to test such a possibility we initiated a study to examine the biotin proteins in various bacteria by analysis of [3H]biotin-labeled whole cell extracts using sodium dodecylsulfate-polyacrylamide gel electrophoresis, with the view in mind of looking for evidence for sharing of a common biotin carboxyl carrier protein subunit in organisms containing multiple biotin-dependent enzymes. Previous examinations of [all]biotin-labeled E. coli cell extracts by such a technique revealed that this organism contains only one major biotin-containing polypeptide corresponding to the biotin carboxyl carrier protein component of acetyl-CoA carboxylase [9] (see Fig. 1A). This observation is consistent with the known absence of pyruvate and propionyl-CoA carboxylases in this organism [2]. Interestingly, most of the other microorganisms examined revealed more complex patterns of biotin-containing polypeptides, and representative experiments are described below. One of the microorganisms studied, Pseudomonas citronellolis is known to contain both acetyl-CoA carboxylase [2] and pyruvate carboxylase [10]. The latter enzyme has been highly purified and partially characterized [11, 12]. In this report we describe a preliminary comparision of the biotin carboxyl carrier protein components of P. citronellolis pyruvate and acetyl-CoA carboxylases. MATERIALS AND METHODS Various bacterial strains were grown in biotin-free media which were supplemented with D-[3H]biotin (2.6 Ci/mmole) at a concentration of 20/~g/1. D-[aH]Biotin was prepared and purified as previously described [13]. E. coli strain 8, a gift of Dr E. C. Lin, was grown as previously described [13]. P. citronellolis (ATCC 13674) was grown at 30 °C in Medium 56 of Monod et al. [14] containing 0.5~ ammonium acetate. For purifications of P. citronellolis acetyl-CoA and pyruvate carboxylases, 200 1 of this medium was used for growth of cells, and at approximately 3/4 log phase cells were harvested and the cell paste was stored at --20 °C. Enterobacter aerogenes (ATCC 13048) was grown at 37 °C in Medium 56 containing 0.2 ~ glucose. Bacillus cereus (ATCC 14579) was grown at 30 °C in Medium 56 containing 0.2~ glucose and 0.2 ~o Casamino acids (vitamin free, Difco). Mycobacterium phlei (ATCC 356), a gift of Dr J. Law, was grown at 30 °C as described by Brennan and Ballou [15]. Lacto-

498 bacillus plantarum (ATCC 8014) was grown semi-anaerobically at 37 °C in biotinassay medium (Difco). Propionibacterium shermanii (ATCC 31673) was grown at 30 °C in the medium described by Delwiche [16], modified so that casein hydrolysate was replaced by Casamino acids (vitamin free, Difco). For analysis of cellular [3H]biotin-proteins bacteria were harvested at approximately 3/4 log phase, and washed at 4 °C with 0.062 M Tris-HCl, pH 6.8. Cells were then suspended in 0.062 M TrisHC1, pH 6.8, containing 2 ~ sodium dodecylsulfate and 2 ~ 2-mercaptoethanol, and sonicated for 2 min. The [3H]biotin-proteins in these extracts were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis as described below. Sodium dodecylsulfate extracts of L. plantarum, P. shermanii, and M. phlei had to be centrifuged before electrophoresis in order to remove insoluble material; this resulted in sodium dodecylsulfate extracts containing 75-90~ of the [3H]biotin present in the original resuspended cells. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of whole cell extracts and purified P. citronellolis fractions was conducted essentially as described by Laemmli [17] except that gels and samples were adjusted to contain 6 M urea in order to help prevent protein precipitation during electrophoresis. Samples were heated at 100 °C for 2 min before application to the gels. Radioactivity in gels containing [3H]biotin-proteins, and the migration of proteins of known molecular weight were determined as previously described [18]. Acetyl-CoA carboxylase and pyruvate carboxylase from P. citronellolis were resolved from one another by modifications of existing procedures [11, 13]. Cells were homogenized, crude extract prepared, and the protein fraction precipitating between 0 and 55 ~ saturation with (NH4)2SO4 was obtained, as previously described for the purification of the components of E. coli acetyl-CoA carboxylase [18]. This fraction, which contained both acetyl-CoA carboxylase and pyruvate carboxylase, was fractionated with alumina CV gel [6]. This resulted in a non-adsorbed fraction containing approximately 80 ~o of the pyruvate carboxylase activity, and a fraction eluted with 0.4 M potassium phosphate, pH 7.7, which contained the bulk of the acetyl-CoA carboxylase activity. The latter fraction was partially stimulated by the transcarboxylase component of E. coli acetyl-CoA carboxylase, and by the non-adsorbed fraction. suggesting that the P. citronellolis acetyl-CoA carboxylase is similar in behavior to the E. coli enzyme during alumina Cy gel fractionation [6, 19]. Pyruvate carboxylase and transcarboxylase in the non-adsorbed fraction were further purified by chromatography on hydroxylapatite as previously described for the purification of the E. coli transcarboxylase component [19]; pyruvate carboxylase was eluted with 0.2 M potassium phosphate, pH 7.0, while the transcarboxylase component was eluted with 0.4 M potassium phosphate, pH 7.0. Following (NH4)2SO4 fractionation and dialysis, chromatography of the pyruvate carboxylase fraction on DEAE-cellulose as described by Seubert and Weicker [11 ] resulted in an enzyme preparation with specific activity of 1.5 units per mg protein and which was free of acetyl-CoA carboxylase activity. The acetyl-CoA carboxylase fraction was further purified on hydroxylapatite and the fraction eluting with 0.4 M potassium phosphate, pH 7.0, had a specific activity of 0.8 unit per mg protein. This fraction was free of pyruvate carboxylase activity, and stimulated the fraction eluted from alumina C~ gel by 3-fold. Acetyl-CoA carboxylase was assayed as previously described [9]. Pyruvate carboxylase was assayed spectrophotometrically essentially as described by Seubert

499 and Weicker [11] except that for crude extracts a modified H14CO; fixation assay (minus acetyl-CoA) was used [20]. RESULTS A N D DISCUSSION

In order to analyze the total biotin-protein population of bacterial cells, we subjected whole cells labeled with [3H]biotin (by growth in [all]biotin-containing media) to solubilization with 2 ~ sodium dodecylsulfate, followed by electrophoresis

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Fig. 1. Resolution of bacterial [3H]biotin-polypeptides by sodium dodecylsulfate-polyacrylamide gel electrophoresis. 15% gels containing 0.1% sodium dodecylsulfate and 6 M urea were used as described in Materials and Methods. Samples contained 0.1-0.3 mg protein (20 000-100000 dpm [3H]biotin). The bacterial strains are indicated on the figures and described more fully in Materials and Methods. The molecular weight markers are bovine serum albumin (68 000), ovalbumin (43 000), E. coli biotin carboxyl carrier protein (22 500), and a subtilisin peptide of E. coli biotin carboxyl carrier protein (9100; see ref. 13).

500 on sodium dodecylsulfate-polyacrylamide gels. The sodium dodecylsulfate gel electropherograms of the [all]biotin polypeptides solubilized by this procedure are shown in Figs 1 and 2A. The migration positions of proteins of known molecular weight are indicated on the figures. As previously noted [9] and illustrated in Fig. IA, E. coli contains only one major biotin-protein, corresponding to the biotin carboxyl carrier protein component (mol. wt 22 500) of acetyl-CoA carboxylase. In contrast, all of the other bacteria examined contained two or more major [3H]biotin-containing polypeptide chains. The other two Gram-negative bacteria examined, E. aerogenes (Fig. 1B) and P. citronellolis (Fig. 2A), both contained two major [3H]biotin-polypeptides corresponding to approximate molecular weights of 90 000 and 25 000, and 60 000 and 25 000, respectively. Analysis ofP. citronellolis biotin-proteins (described below) and previous work on E. coli biotin carboxyl carrier protein [9, 18] suggest that the [aH]biotin-polypeptide chain with molecular weight ~ 25 000 common to E. coli, E. aerogenes and P. citronellolis corresponds to the biotin carboxyl carrier protein component of acetyl-CoA carboxylase in these organisms. The role of the higher molecular weight biotin-polypeptide (mol. wt ~ 60 000) ofP. citronellolis is described below: that of E. aerogenes (mol. wt ~ 90 000) is unidentified. It should be emphasized that the molecular weight ranges for the [3H]biotin-polypeptides described here should be considered as approximations, due to experimental variability in the electrophoresis of whole cell extracts. However, the general patterns illustrated were consistently seen from preparation to preparation. The biotin-polypeptides of several Gram-positive bacteria solubilized with sodium dodecylsulfate were examined in the same way. B. cereus (Fig. 1C) and L. plantarum (Fig. 1E) exhibited similar patterns with major [aH]biotin-polypeptides in the molecular weight range 20 000-25 000, and minor [3H]biotin-polypeptides with

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Fig. 2. Resolution of P. citronellolis [3H]biotin-polypeptides by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Details are described in the text. Fig. 2A, electropherogram of [3H]biotinlabeled whole cell extract. Fig. 2B, electropherogram of purified [3H]biotin-labeled pyruvate carboxylase (e~-O), and [3H]biotin-labeled acetyl-CoA carboxylase ( © . -- (3); open circles which coincided with closed circles are not indicated. Samples contained 2-10 mg protein (approx. 200 000 dpm).

501 molecular weight ~ 90 000. A partially purified acetyl-CoA carboxylase preparation from L. plantarum was enriched in the molecular weight 20 000-25 000 peak (Fall, R. R. and Vagelos, P. R., unpublished) suggesting that this biotin-polypeptide corresponds to the biotin carboxyl carrier protein component of acetyl-CoA carboxylase as in the Gram-negative bacteria examined. The higher molecular weight [3H]biotinpolypeptides in B. cereus and L. plantarum are unidentified. However, both of these organisms contain pyruvate carboxylase activity (Alberts, A. W., unpublished) and this radioactive peptide may represent the biotin-polypeptide of pyruvate carboxylase in the two bacteria. The patterns of biotin-polypeptides from two other Gram-positive bacteria, P. shermanii (Fig. ID) and M. phlei (Fig. IF), were different than those of the other bacteria examined. P. shermanii contains a major biotin-polypeptide with a molecular weight in the range 10 000-13 000, as well as minor species with molecular weights < 10 000 and 75 000-80 000. The major biotin-polypeptide was shown to migrate identically on sodium dodecylsulfate-polyacrylamide gels with the 1.3-S biotin carboxyl carrier protein component (mol. wt 12 000) of P. shermanii transcarboxylase (ref. 8; a gift of Dr H. G. Wood). The minor species of biotin-polypeptides are unidentified. It is interesting to note the lack of a major biotin-polypeptide in the molecular weight range 20 000-25 000, characteristic of all the biotin carboxyl carrier protein components of the bacterial acetyl-CoA carboxylases discussed above. This is consistent with the finding that acetyl-CoA carboxylase has not yet been found in propionibacteria [21]. The pattern of [3H]biotin-polypeptides solubilized from M. phlei is shown in Fig. IF. Three peaks were seen corresponding to approximate molecular weights of 80 000, 55 000 and 25 000. Erfle [22] has isolated a biotin-dependent carboxylase from M. phlei which carboxylates acetyl-CoA and propionyl-CoA at similar rates, with propionyl-CoA exhibiting a somewhat lower Kin. This enzyme, unlike the E. coli type of acetyl-CoA carboxylase, purifies as an intact complex, analogous to the mammalian and yeast acetyl-CoA carboxylases [2]. When the partially purified [3H]biotin-labeled enzyme was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis only a [3H]biotin-polypeptide with molecular weight ~ 55 000 was seen (Fall, R. R. and Vagelos, P. R., unpublished). Thus it appears that the M. phlei acetyl-CoA/ propionyl-CoA carboxylase is of the mammalian or yeast type (aggregated) and has a fundamentally different biotin carboxyl carrier protein subunit than the E. coli tyFe acetyl-CoA carboxylase (disaggregated). Erfle [22] was unable to detect any disaggregated type acetyl-CoA carboxylase in M. phlei; however, it is worth noting that M. pklei (Fig. IF) contains a [3H]biotin-polypeptide with molecular weight ~ 25 000, characteristic of the biotin carboxyl carrier protein component of E. coli, L. plantarum and P. citronellolis acetyl-CoA carboxylases. Thus it is possible that M. phlei contains a disaggregated acetyl-CoA carboxylase (Fall, R. R., unpublished). The biotin-polypeptide in M. phlei with molecular weight ~ 80 000 is unidentified. It was clear from preliminary studies such as those described above that several patterns of biotin-polypeptides were present in this small sampling of bacterial strains. We turned to a study of bacterium for which two different biotin-dependent carboxylases could be readily obtained and compared. P. citronellolis contains pyruvate carboxylase [10] and acetyl-CoA carboxylase [2] activities. The [3H]biotin-polypeptide pattern of sodium dodecylsulfate-solubilized whole P. citronellolis cells shown in Fig.

502 2A reveals two major peaks with molecular weights of approx. 60 000 and 25 000. The purification behavior of acetyl-CoA carboxylase components was similar to that described for E. coli [6, 7]. Treatment of a crude preparation of the enzyme with alumina C~ gel resulted in a partial separation of the transcarboxylase component from the fraction containing biotin carboxylase plus biotin carboxyl carrier protein, so that the latter fraction alone exhibited acetyl-CoA carboxylase activity, but it was stimulated 3-fold by addition of the transcarboxylase component or by addition of the purified E. coli transcarboxylase component. In addition, a purified P. citronellolis biotin carboxyl carrier protein component was an effective CO2 acceptor and donor when mixed with purified E. coli biotin carboxylase and transcarboxylase components. It seems likely that P. citronellolis acetyl-CoA carboxylase is very similar to the E. coli enzyme. The purified P. citronellolis [3H]biotin-labeled fraction containing biotin carboxyl carrier protein and biotin carboxylase was free from detectable pyruvate carboxylase activity, and when subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, a single [3H]biotin-polypeptide peak with molecular weight ~ 25 000 was seen (Fig. 2B). The [3H]biotin-labeled pyruvate carboxylase from P. citronellolis was purified essentially as described by Seubert and Weicker [11] with some modification (see Materials and Methods). The final preparation was free from detectable acetyl-CoA carboxylase components. Electrophoresis of the purified enzyme on sodium dodecylsulfate-polyacrylamide gels revealed a single [3H]biotin-pclypeptide peak with molecular weight ~ 60 000 (Fig. 2B). This is consistent with the results of Barden and Taylor [23] who found a molecular weight of 65 000 for the biotin-containing subunit of P. citronellolis pyruvate carboxylase. These results show that at least for P. citronellolis two different biotin-dependent enzymes in the same cell do not share common biotin carboxyl carrier protein subunits. Whether this is true for other organisms is not yet known. It is also possible that cells which contain distinct acetyl-CoA and propionyl-CoA carboxylases may share common subunits, especially since these two enzymes are probably more closely related than acetyl-CoA carboxylase and pyruvate carboxylase. ACKNOWLEDGMENTS Supported in part by G r a n t GB-38676XI from the National Science Foundation and Grant R01-HLI0406 from the National Institutes of Health. REFERENCES 1 Moss, J. and Lane, M. D. (1971) Adv. Enzymol. 35, 321~142 2 Alberts, A. W. and Vagelos, P. R. (1972) in The Enzymes (Boyer, P. D., ed.), Vol. 6, p. 37, 3rd edn., Academic Press, New York 3 Wood, H. G., Lochmuller, H., Reipertinger, C. and Lynen, F. (1963) Biochem. Z. 337, 247-266 4 Stern, J. R. (1967) Biochemistry 6, 3545-3551 5 Galivan, J. H. and Allen, S. H. G. (1968) Arch. Biochem. Biophys. 126, 838-847 6 Alberts, A. W. and Vagelos, P. R. (1968) Proc. Natl. Acad. Sci. U.S. 59, 561-568 7 Alberts, A. W., Nervi, A. M. and Vagelos, P. R. (1969) Proc. Natl. Acad. Sci. U.S. 63, 1319-1326 8 Gerwin, B. I., Jacobson, B. E. and Wood, H. G. (1969) Proc. Natl. Acad. Sci. U.S. 64, 1315-1322 9 Fall, R. R., Nervi, A. M., Alberts, A. W. and Vagelos, P. R. (1971) Proc. Natl. Acad. Sci. U.S. 68, 1512-1515

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Seubert, W. and Remberger, U. (1961) Biochem. Z. 334, 401-414 Seubert, W. and Weicker, H. (1969) Methods Enzymol. 13, 258-262 Taylor, B. L., Barden, R. E. and Utter, M. F. (1972) J. Biol. Chem. 247, 7383-7390 Fall, R. R. and Vagelos, P. R. (1973) J. Biol. Chem. 248, 2078-2088 Monod, J., Cohen-Bazire, G. and Cohen, M. (1951) Biochim. Biophys. Acta 7, 585-599 Brennan, P. and Ballou, C. E. (1967) J. Biol. Chem. 242, 3046-3056 Delwiche, E. A. (1949) J. Bacteriol. 58, 395-398 Laemmli, U. K. (1970) Nature 227, 680-685 Fall, R. R. and Vagelos, P. R. (1973) J. Biol. Chem. 247, 8005-8015 Alberts, A. W., Gordon, S. G. and Vagelos, P. R. (1971) Proc. Natl. Acad. Sci. U.S. 68, 1259-1263 Sundaram, T. K., Cazzulo, J. J. and Kornberg, H. L. (1969) Biochim. Biophys. Acta 192, 355-357 Wood, H. G. (1972) Enzymes 6, 83-115 Erfle, J. D. (1973) Biochim. Biophys. Acta 316, 143-155 Barden, B. E. and Taylor, B. L. (1973) Fed. Proc. 32, 510