THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 31, Issue of November 5, p p . 20922-20927,1991 Printed in U.S.A.

Coexistence of the Genes for Putrescine Transport Protein and Ornithine Decarboxylase at 16 min on Escherichia coli Chromosome*

(Received for publication, January 17, 1991)

Keiko Kashiwagi, Tomoko Suzuki, Fumihiro Suzuki, Takemitsu Furuchi, Hiroshi Kobayashi, and Kazuei IgarashiS From the Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Chiba 260, Japan

The nucleotide sequence of one of the putrescine transport operons (pPT71), located at 16 min of the Escherichia coli chromosome, was determined. It con- tained the genes for an induced ornithine decarboxyl- ase and a putrescine transport protein. The gene for the ornithine decarboxylase contained a 2,196-nucleo- tide open reading frame encoding a 732-amino acid protein whose calculated M, was 82,414, and the pre- dicted amino acid sequence from the open reading frame had 65% homology with that of a constitutive ornithine decarboxylase encoded by the gene at 64 min. The ornithine decarboxylase activity was observed in the cells carrying pPT71 cultured at pH 5.2, but not in the cells cultured at pH 7.0. The gene for the putrescine transport protein contained a 1,3 17-nucleotide open reading frame encoding a 439-amino acid protein whose calculated M, was 46,494. The hydropathy pro- file of the putrescine transport protein revealed that it consisted of 12 putative transmembrane spanning seg- ments linked by hydrophilic segments of variable length. The transport protein was in fact found in the membrane fraction. When the gene for the putrescine transport protein was linked to the tet promoter of the vector instead of its own promoter, the putrescine transport activity increased greatly. The results sug- gest that the gene expression of the operon is repressed strongly under standard conditions.

Polyamine contents in cells are regulated by both polyamine biosynthesis and polyamine transport. In Escherichia coli, polyamine uptake is energy-dependent, and the putrescine transport system is different from the spermidine and sperm- ine transport system (1, 2). Furthermore, two transport sys- tems for putrescine have been suggested in E. coli K12 grown in a low osmolarity medium (3). To further clarify the prop- erties of polyamine transport systems in E. coli, we isolated mutants in which polyamine transport was deficient, and clones of polyamine transport genes. We succeeded in obtain- ing two polyamine transport-deficient mutants (KK313 and NH1596) and three clones of polyamine transport genes (pPT104, pPT79, and pPT71) (4). E . coli KK313 was deficient

* This work was supported by a Grant-in-Aid for Scientific Re- search from the Ministry of Education, Science and Culture, Japan, and by a Research Aid of the Inoue Foundation for Science, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted t o the GenBankl”/EMBL Data Bank with accession number(& M64495.

$ To whom correspondence should be addressed.

in spermidine transport, and E. coli NH1596 was deficient in spermidine transport and had a 90% decreased putrescine transport activity. The system encoded by pPT104 clone mapped at 15 min on the E. coli chromosome can catalyze both spermidine and putrescine transport, but the systems encoded by pPT79 clone mapped at 19 min and by pPT71 clone mapped at 16 min can catalyze only putrescine trans- port. The putrescine transport system encoded by pPT71 showed a relatively low activity compared to that encoded by pPT79 (4). In order to determine the characteristics of the putrescine transport protein and the reason why the system encoded by pPT71 showed a low activity, we tried to identify the nucleotide sequence of the gene encoded by pPT71. The results indicated that the operon contained the genes for an induced ornithine decarboxylase and a putrescine transport protein. Since the gene for a constitutive ornithine decarbox- ylase has already been mapped at 64 min on E. coli chromo- some (5 , 6), the gene at 16 min was the second one to be identified for ornithine decarboxylase.

EXPERIMENTAL PROCEDURES

Bacterial Strains and Culture Conditions-A polyamine-requiring mutant, E. coli MA261 (speB spec serA thr leu thi, Ref. 7), provided by Dr. W. K. Maas, New York University School of Medicine, and its polyamine transport-deficient mutant NH1596 (4) were grown in medium A in the absence of putrescine as described previously (8). 15. coli DR112 (speA speB thi, Ref. 9), provided by Dr. D. R. Morris, University of Washington, was also grown in medium A to measure the normal ornithine decarboxylase activity. speA, speB, and spec are the genes for arginine decarboxylase, agmatine ureohydrolase, and ornithine decarboxylase, respectively. For the induction of ornithine decarboxylase at acidic pH, the cells were grown in 10 ml of LB-broth with aeration at 37 “C for 24 h, and then were transferred to 100 ml of induction medium (1% nutrient broth, 0.05% (NH4)PSOl, 0.1% NaCI, 0.1% K,HPO,, 0.05% sodium citrate, and 0.8% L-ornithine, adjusted to pH 5.2) (10). The cultivation was continued at 37 “C without aeration for 24 h. Chloramphenicol (30 pg/ml) or ampicillin (100 pg/ml) was added for the growth of the strains containing plasmids.

Plasmids-Plasmids pPT71.10 and pPT71.20 were constructed by inserting a 4.3-kb’ BamHI fragment of pPT71 (4) in alternative directions into a BamHI site ofpACYC184 (11) (Fig. 1). Short plasmid pPT71.11 was made by deletion of the Hind111 fragment (one Hind111 site was from the vector) of pPT71.10. pPT71.30 was constructed by inserting a 2.0-kb BamHI-AsuII fragment into the EcoRV site of pACYC184 after blunting the ends with a Klenow fragment. pODC1.l had a PstI fragment of pODCl (12) in pACYC184. Plasmid pT7-5, a modified plasmid of pT7-1 (13) in which the orientation of the 0- lactamase gene is reversed with respect to the T7 promoter (obtained from Dr. S. Tabor, Harvard Medical School), was used to construct pT7-46K by inserting a PstI-ClaI fragment of pPT71.10 (&I site was from pACYC184) into the same restriction sites of pT7-5.

Assay of Putrescine Uptake Activity-This was performed as de-

’ The abbreviations used are: kb, kilobase(s); Pipes, 1,4-pipera- zinediethanesulfonic acid Hepes, 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid.

20922

Transport and Biosynthesis of Polyamines 20923

PUT Uptake Activity (mSllrrNW p o t a h )

1. pPT7 1 - 3. pPT71.11

4. pPT71.20 I

5. pPT7 1.30

E B A I R H B a B S E

P Y. 7

n

3.96

3.49

0.1 6

0.96

11.12

6. NH1596 0.16

7. MA261 2.24

FIG. 1. Structure of subclones and their putrescine trans- port activity. The thick horizontal bar was from the vector pACYC184, and the horizontal line was from E. coli DNA. The cloning of pPT71, consisting of 7.1 kb, was performed as described previously (4). The position of the genes for ornithine decarboxylase (0) and putrescine transport protein (0) was shown on the basis of the results of Fig. 2. The arrows indicate the direction of transcription. As, AsuII; B, BamHI; Bs, BstEII; C, ClaI; E, EcoRI; Eu, EcoRV; H, HindIII; Ps, PstI; S, SaZI; Xb, XbaI. The physical map constructed by Kohara et al. (34) of the X phage clone 8A8 at 16 min on E. coli chromosome was also shown in the upper portion.

scribed previously (2). The putrescine uptake activity (nmol/min/mg protein) was expressed as the average value of three experiments with different incubation times (1, 3, or 5 min). The uptake was normally linear up to 5 min as shown in the previous communication (2). When the putrescine transport activity was high, the cell suspension was diluted by buffer A (2) so as to have a linear uptake for 5 min. Protein content was determined by the method of Lowry et al. (14).

DNA Sequencing-The 4.3-kb BamHI fragment of pPT71 was inserted into a BamHI site of M13mp19 (15) in both directions, and unidirectional deletion mutants were prepared according to the method of Henikoff (16). The DNA was sequenced primarily by the dideoxy method of Sanger et al. (17), using the M13 phage system (15), with commercial and synthesized primers.

Analysis of Hydropathy and Sequence Homology of Proteins-Av- erage hydropathy profiles of the proteins were obtained according to the method of Kyte and Doolittle (18) with a DNASIS program (Hitachi Software Engineering Co. Ltd.) using a window length of 7. A model of the secondary structure of the putrescine transport protein was then constructed according to these profiles. Sequence homolo- gies of the proteins were analyzed according to the method of Needle- man and Wunsch (19) using a DNASIS program.

Assay /or Ornithine Decarboxylase-The assay for ornithine decar- boxylase was performed as described previously (20) except that the reaction mixture (0.35 ml) contained 100 mM Hepes-KOH (pH 7.1) and 0.1 mM GTP. For measurement of the pH curve, a series of 100 mM Pipes-KOH with values of 6.0, 6.5, 7.0, or 7.5 and 100 mM Tris- HC1 with pH values of 7.5, 8.0,8.5,9.0, or 9.5 was used instead of 100 mM Hepes-KOH (pH 7.1). The activities of ornithine decarboxylase with 100 mM Pipes-KOH and Tris-HC1 at pH 7.5 were almost the same.

Northern Blot Analysis of RNA-The RNA sample was isolated from E. coli cells according to the method of Emory and Belasco (21). Electrophoresis of the RNA, the hybridization of RNA to DNA probes (1 X lo6 cpm/ml), and its autoradiography were performed as de- scribed previously (22). The DNA probes used were the 1.5-kb ClaI- AsuII fragment of the ornithine decarboxylase gene and the 1.0-kb HindIII-BstEII fragment of the putrescine transport protein gene (see Fig. 1).

Expression of Plasmid-encoded Proteins in Maxicell Procedure- Expression was performed according to the method of Sancar et al. (23, 24). E. coli CSR603 (reCAI uruA6 phr-I) was transformed by pACYC184, pPT71, or pPT71.10, and the cells were grown in 10 ml of K medium (24) containing 30 pg/ml chloramphenicol until AeOo reached a level of 0.15. The culture was transferred into a Petri dish (9 cm diameter) and irradiated by a 15-watt UV lamp from a 50-cm distance for 5 s. The protein of cells was labeled with ["'Slmethionine (1.85 MBq, 39.8 TBq/mmol) a t 37 "C for 1 h. The protein samples

were prepared as described previously (25) and sodium dodecyl sul- fate-gel electrophoresis was performed according to the method of Laemmli (26) using 12% acrylamide slab gels. After staining the proteins with Coomassie Brilliant Blue R-250, fluorography was performed using ENLIGHTNINGTM (Du Pont-New England Nu- clear) (27, 28).

Expression of a Putrescine-Transport Protein in a T7-Promoter Expression System-E. coli HT551 (A(speE-speD) zad22O:TnlO panB6 A(&-pro)[F', lacFz AM15, pro']) (29) was transformed by pT7-5 or pT7-46K. Cells were grown in 5 ml of medium B (M9 plus 50 pg/ml each of 18 amino acids except methionine and cysteine, 2 pg/ml thiamin, and 5 pg/ml calcium pantothenate) containing 100 pg/ml ampicillin a t 37 "C until A,,o reached a level of 0.3 (6 X 10' cells/ml). The cells were infected with phage M13mGP1-2 (1.6 X 10" plaque-forming units/ml) (30) and incubated at 37 "C for 15 min without agitation. They were then incubated with isopropyl 0-D- thiogalactopyranoside (1.5 mM) for 30 min to induce T 7 RNA polym- erase encoded by M13mGP1-2, and further incubated with rifampicin (200 pg/ml) for 30 min to inhibit the activity of the host RNA polymerase. The proteins were labeled by the incubation of cells with ["Slmethionine (2.96 MBq, 39.8 TBq/mmol) a t 37 "C for 30 min. After the cells were mixed with unlabeled cells (4 X lo9 cells) and collected by centrifugation at 20,000 X g for 10 min, the cell extract (30,000 X g supernatant) wasprepared by sonication (31). The extract was then centrifuged at 105,000 X g for 1 h to obtain cytosol and ribosome-containing membrane fractions. Samples from each frac- tion were prepared for gel electrophoresis (25), and fluorography was performed as described above.

RESULTS

Subcloning of pPT71"We subcloned pPT71, which con- tains the 7.1-kb fragment, in order to locate the putrescine transport gene (Fig. 1). Similar putrescine transport activity was observed with both pPT71 and pPT71.10 containing 4.3 kb of the B a m H I fragment. The putrescine transport activity increased greatly with pPT71.30 containing 2.0 kb of the AsuII-BamHI fragment, although the direction of the gene was reversed. These results indicated that the gene for pu- trescine transport protein is located in the AsuII-BamHI fragment.

Nucleotide Sequence of the Gene Encoding the Putrescine Transport Protein-The nucleotide sequence of the 4.3 kb of the BarnHI fragment was determined (Fig. 2). The open reading frame for the putrescine transport protein consisted of 439 amino acids, and its molecular weight was estimated as 46,494. In addition to the gene for putrescine transport protein, the operon contained another open reading frame which can encode 732 amino acids (Mr 82,414) upstream of the gene for putrescine transport protein. The operon con- tained another open reading frame which can encode 732 amino acids (M, 82,414) upstream of the gene for putrescine transport protein. The operon contained the candidates for the -10 and -35 regions of the promoter (32) and the p - independent transcriptional termination signal (33). From a comparison of our restriction enzyme map and the physical map constructed by Kohara et al. (34), it was shown that the orientation of the operon was counterclockwise on the phys- ical map of E. coli (Fig. 1).

Characteristics of the Putrescine Transport Protein-When the hydropathy of the protein along the amino acid sequence was evaluated, it became apparent that the protein contained 12 putative transmembrane spanning segments linked by hydrophilic segments of variable length (Fig. 3). Based on this analysis, a model of the secondary structure of the protein was constructed (Fig. 4).

The putrescine transport system encoded by pPT71 showed a lower activity compared to that encoded by pPT79 (4). We tried to clarify the reason for this low activity. When the gene for the putrescine transport protein was linked to the tet promoter of the vector instead of to the promoter of

20924

FIG. 2. Nucleotide sequence of the genes for ornithine decarboxylase and putrescine transport protein. The deduced amino acid sequence is shown under the nucleotide sequence. The putative -10 and -35 regions of the promoter and Shine-Dargarno sequence (40) are underlined. The inverted repeats in the promoter region and the putative termination signal of RNA synthesis are indicated by horizontal arrows below the sequences.

I ndor

- ~ ~ , ~ ~ ~ , ~ ~ ~ ~ ~ , ~ ~ ~ ~ ~ ~ ~ ~ sd

its gene, the activity increased from 0.96 to 11.12 nmol/min/ mg protein (the data of pPT71.20 and pPT71.30 in Fig. 1). Therefore, this would indicate that the promoter activity of the operon is probably very low.

Decarboxylase-We next tried to identify the 82K protein encoded by the open reading frame existing upstream of the putrescine transport gene. Hydropathy of the protein showed

FIG. 3. Hydropathy profile of putrescine transport protein it to be hydrophilic (Fig. 5 ) . When the amino acid sequences encoded by pPT71. were analyzed with a DNASIS program, homology of consti-

~2

- 4 - 5

~3 Identification of the 82K Protein as an Induced Ornithine

0 100 200

Amino acid residue number

Transport and Biosynthesis of Polyamines 20925

N P "

FIG. 4. Model of the secondary structure of the putrescine trans- port protein. Putative transmembrane segments are shown in large boxes. Basic amino acids are shown in circles and acidic amino acids are shown in squares.

P L

I W I

S G G

w w N

M V T

I L T

Q L V

V + G

4 9 Q S

" G

T S I I

S I w

V L T

V A G

w s A

A L W

A __t

F

SA G n F

T ~ L L P C G S f- Y G

A V S

A I I

V A N

I A L

S L V

Y G T

Y _t

N A n

7 L G

A q V

" w

F

W F G

I 1 C

G L V

P V I

V I G

v w T

I

i s N S F I

F G Q N GI@TG G

S Q

index

:I ' '

- 4 - 5 1 I

0 l@Q 200 300 400 500

I NJCX

3

- 4

500 -5 1

680 7 1 800 900 1000

Amino acid residue number

FIG. 5. Hydropathy profile of induced ornithine decarbox- ylase encoded by pPT7 1.

tutive ornithine decarboxylase' encoded by the gene mapped at 64 min on the E. coli chromosome and the 82K protein was found to be 65% (Fig. 6). Homology was mainly observed in the central portion and carboxyl-terminal of the protein. The amino acid sequence of the coenzyme (pyridoxal 5"phos- phate) binding site (VHKQQAG) was exactly the same as that of constitutive ornithine decarboxylase (Fig. 6).

Ornithine decarboxylase activity was observed when E. coli NH1596 carrying pPT71 was grown at pH 5.2 (Table I). However, when the cells were grown under standard condi- tions a t pH 7.0, the activity was not observed (Table I). Ornithine decarboxylase activity was also observed with E. coli NH1596 carrying pPT71.11, but not with E. coli carrying pPT71.30 (plasmid structures, see Fig. 1). The optimal pH of ornithine decarboxylase was 7.0, not 8.5 (Fig. 7), the same as that of induced ornithine decarboxylase at the acidic pH reported previously (35). Both ornithine decarboxylases could catalyze the decarboxylation of lysine at the rate of about 0.3% compared to that of ornithine as reported previously (20). These results indicate that the 82K protein encoded by the operon is an induced ornithine decarboxylase.

The question then arose as to whether the genes for orni-

' S. M. Boyle, personal communication.

A P

F F S

V A G

s s I

M A T

W L A

L F c

L

F v

N m E N

A I

V N

S T V

Y I I

A V A

L C T

G G L

A V I

P

T

vG d S Q F N

Q 0 I V V I

t- G

S Y S L

Y A F

Y S n

G A V

A F V

N F A

V

Q @

VANVPP BL

P G-COOH

q V L

thine decarboxylase and putrescine transport protein were transcribed together. As DNA probes for both ornithine de- carboxylase and putrescine transport protein genes hybridized the same RNA sized about 4.0 kb, this indicated that both genes constitute an operon (data not shown).

Expression of Induced Ornithine Decarboxylase and Putres- cine Transport Protein-The expression of induced ornithine decarboxylase and putrescine transport protein was examined in maxicells (Fig. 8A) . Chloramphenicol acetyltransferase ( M , 25K) and a M, 38K protein were expressed in E. coli CSR603 containing vector pACYC184 and clones pPT71 and pPT71.10. However, two proteins with apparent molecular weights of 82K and 36K were expressed only in maxicells containing the clones pPT71 and pPT71.10, although the degree of expression was low (Fig. 8A, lunes 2 and 3 ) . The 82K protein corresponded with the induced ornithine decar- boxylase. Since the apparent molecular weights of the mem- brane proteins were generally smaller than their true molec- ular weights (36-38), the 36K protein was judged to be the putrescine transport protein whose calculated molecular weight was 46K.

To confirm that the 36K protein was the real putrescine transport protein, the T7 promoter expression system was used with pT7-46K, in which only the gene of the putrescine transport protein was included. As shown in Fig. SB, lanes 2 and 4, the 36K protein was expressed in the membrane fraction, but not in the cytosol fraction. The 36K protein was not expressed in the membrane and cytosol fractions of E. coli HT551 transformed by control plasmid pT7-5 (Fig. 8B, lanes 1 and 3 ) .

DISCUSSION

In this study, we found a new operon consisting of the genes for ornithine decarboxylase and putrescine transport protein at 16 min on the E. coli chromosome. Both of these genes are probably important for the maintenance of cellular polya- mines under certain circumstances. However, under standard conditions, their expression is repressed. Since two inverted repeat structures exist at the promoter region (Fig. 2), a repressor may recognize the inverted repeat(s).

The gene for constitutive ornithine decarboxylase is located at 64 min on the E. coli chromosome (5,6). The characteristics of ornithine decarboxylase encoded by the gene at 16 min are different from those of constitutive ornithine decarboxylase,

20926 Transport and Biosynthesis of Polyamines A:

E:

A:

E:

A:

E:

A:

E:

A :

E:

A:

E:

A:

E:

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M-KSMNIA-A-S-SELVSRLSSHRRWALGDTDFTDVAAWITAAD-SRSGI--L-ALLKRTGFHLPVFLYSE---H-AVE-LPAGVTAVI-N--CN-EQQWLE-LESAACQYEENLLPP

. .

FFRAL---VDYVNQGNSAFDCPGHQ-CGEFFRRHPAGNQFVEYFGEALFRADLCNADVAMGDLLIHEGAPCIAQQH-AAKVFNADKTYFVLNGTSSSNKWLNALLTPGDLVLFDRNNHK

FYDTLTQYVE-M--GNSTFACPGHQHGA-FFKKHPAGRHFIDFFGENVFRADMCNADVKLGDLLIHEGSAKDAQ-KFAAKVFHAKTYFVLNGTSAANKWTNALLTRGDLVLFDRNNHK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

SNHHGALLQAGATPVYLETANPYGFIGGIDAHCF-EESYLRELIAEVAPQRAKE-ARPFRLAVIQLGTYDGTIYNARQWDKIGHLCDYILFDSAWVGYEQFIPMMADCSPLLLDLNEN

SNHHGALIQAGATPVYLEASRNPFGFIGGIDAHCFNEE-YLRQQIRDVAPEKA-DLPRPYRLAIIQLGTYDGTVYNARQVIDTVGHLCDYILFDSAWVGYEQFIPMMADSSPLLLELNEN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Q I A V D L R F F Q F V - P C E H m S F E G Y A - E N Q Y F V D P C K L L L T T P G V P E K C D L N S I L F L L T P A E D M A K - - L Q Q L V A L L V R F E - K L L E S D A P L A E V

VLASDRRFFSFERGA-K~GFEGYAADQ-YFVDPCKLLLTT~IDAETGEY-SDFGVPATILAHYLRENGIVPEKCDLNSILFLLTPAE--SHEKLAQLVAMLAQFEQHIED-DSPLVEV . . . . . . ........................................................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

LPSIYKQHEE-RYAGYTLRQLCQEMHDLY-ARH~KQLQKE~KEHFPRVSMNPQEA~AYLRGEVELVRLPDAEGRIAAEGALPYPPGVL~VPGEIWGGAVLRYFSALEEGINLLPG

LPSVY-NKYPVRYRDYTLRQLCQEMHDLWSm)-VKDLQKA~QQSFPSWMNPQDAHSAYIRGDVEL~IRDAEGRIAAEGALPYP~VLCW~EVWGGAVQRYFLALEEGVNLLPG . . . . ................ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

FAPELQGVYIE-EHDG-RKQVWCYVIKPRDAQSTLLKGEKL 732

FSPELQGVYSETDADGVKR-LYGYVLK 711 . . . . . . . . . . . . . . .....................

FIG. 6. Comparison of amino acid sequences of induced (A) and constitutive (B) ornithine decarbox- ylases. The same amino acids are indicated by colons, and the equivalent amino acids by periods. The cofactor binding site is boxed.

TABLE I Ornithine decarboxylase activity of E. coli DRI 12, NH1596, and

NH1596 carving D P T ~ I or DODCI. I ~

Ornithine decarboxylase activity

Strain Synthetic Rich me- medium, dium, pH 7.0 eH 5.2

nmol/rnin/rng protein DR112 31.4 f. 0.14h 38.9 f 0.18 NH1.596 0.18 -C 0.08 0.59 f 0.11 NH1596/pPT71 0.28 f 0.09 39.7 f 4.8 NH1596/pODCl.l‘ 575 f 44 692 f 53

I’ Strain possessing the wild type ornithine decarboxylase activity.

‘ Plasmid pODC1.l has a gene for constitutive ornithine decarbox- ylase in pACYC184 vector. This plasmid was prepared from pODCl (12), provided by S. M. Boyle, Virginia/Maryland Regional College of Veterinary Medicine.

Mean f S.D.

-loo- .”/” iODC cODC

- 8

‘5 50 - c r

.- .- 2

0 ’ 6 7 8 9 1 0

PH

FIG. 7. pH profile of the activity of induced and constitutive ornithine decarboxylase. 0, constitutive ornithine decarboxylase (100% activity; 594 nmol/min/mg protein); 0, induced ornithine decarboxylase (100% activity; 43.2 nmol/min/mg protein).

A B

110

102

225

217

343

335

461

453

575

567

69 3

685

1 2 3 1 2 3 4

FIG. 8. Expression of induced ornithine decarboxylase and putrescine transport protein in maxicells (A) and T7 promoter expression system (B) . Experiments were performed as described under “Experimental Procedures.” In A: I , E. coli CSR603/ pACYC184; 2, E. coli CSR603/pPT71; c, E. coli CSR603/pPT71.10; In B: I , E. coli HT551/pT7-5, membrane protein; 2, E. coli HT551/ pT7-46K. membrane protein; 3, E. coli HT551/pT7-5, cytoplasmic protein; 4, E. coli HT551/pT7-46K, cytoplasmic protein. Numbers on the left and right represent M,, a, induced ornithine decarboxylase; b, putrescine transport protein.

as the optimal pH of the activity was 7.0 rather than 8.5, and the enzyme was inducible a t low environmental pH. The enzyme resembles the so-called “biodegradative” ornithine decarboxylase reported previously (35), which was produced when E. coli UW44 was cultured at pH 5.2, and had a specific activity of 9.3 pmol/min/mg protein (35). That value was 230- fold greater than that of E. coli NH1596/pPT71. It has been reported that biodegradative ornithine decarboxylase cannot be found in most strains of E. coli, including E.coli K12 (39). Therefore, it remains to be clarified whether the weak activity of our induced enzyme has any relationship to the much higher activity of biodegradative ornithine decarboxylase in E. coli UW44.

We would like to propose that, hereafter, the genes for the induced ornithine decarboxylase and the putrescine transport protein be named speF and pot& respectively. We are cur- rently in the process of investigating the details of how putrescine is recognized by the putrescine transport protein.

Transport and Biosynthesis of Polyamines 20927

Acknowledgments-We thank Drs. S. M. Boyle, W. K. Maas, D. R. Morris, C. W. Tabor, H. Tabor, and S. Tabor for their kind gifts of E. coli strains and plasmids pT7-5 and pODC1.

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