337 Gene, 28 (1984) 337-342

Elsevier

GENE 1005

Nucleotide sequence of gene pJkB encoding the minor phosphofructokinase of EsehericAia coii K-12?

(Recombin~t DNA; codon usage; gene cloning; multicopy plasmid vector; allosteric enzyme)

Fevzi Daldal*

Department ofMicrobiology and Molecular Genetics, HarvardMedical School, Boston, MA 02115 (U.S.A.) Tel. (617) 732-1910

(Received December 2nd, 1983)

(Accepted February 17th, 1984)

SUMMARY

Tlx nucieotide sequence of a 1.3-kb DNA fragment cont~ning the e~t~ep~~ gene which codes for Pfk-2 of EsckePichia co&, a minor phosphofructokinase fPfk) enzyme, is reported. The Pfk-2 protein subunit is encoded by 924 bp, has 308 amino acids and an M, of 33 000. Like other weakly expressed E. co& genes the codon usage in the p&B gene is random; there is no strong bias for the usage of major tRNA isoac~eptin~ species, and the codon preference rules of Grosjean and Fiers [Gene, 18 (1982) 199-2091 are followed. This is the first report of the complete gene sequence of a phosphofructokinase.

INTRODUCTION

E. coli K-12 has two Pflc activities. The first is converting fructose-6-phosphate to fructose-1,6-bis- phosphate, an important step in glycolysis (Fraenkel, 1981). About 90% of the activity present in a witd- type strain is Pfk-1, a well-known ahosteric enzyme (Blangy et al., 1968)coded by&U (Thomson, 1977) located at 88 min on the E. cali chromosome. The remaining activity is P&-2 (Fraenkel et al., 1973).

t Dedicated to the memory ofmy friend, Dr. Ahmad I. Bukhari, who passed away on November 19, 1983 after a massive heart attack, at the age of forty. * Present address: Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, NY 11724 (U.S.A.) Tel. (516) 36% 8367.

Abbreviations: bp, base pairs; kb, 1OOObp; N, any nucleotide (A,C,G,T); Pfk, fructose-6-P,]-kinase; SDS, sodium dodecyl sulfate.

Mutations causing either the loss of Pfk-2 or its modi~~ation, have been isolated from several strains (Vinopal and Fraenkel, 1975; Daldal and Fraenkel, 1981) and mapped at the pfkB locus, at 38 min, hence establishing it as the structural gene for Ptk-2, and clones with p&B on a multicopy plasmid have a high level of PIk-2 (Daldal, 1983).

Pfk-2, unlike Pfk- 1, does not show cooperative interaction with fructose-6-phosphate, inhibition by phosphoenolpy~vate or activation by ADP; it is somewhat sensitive to inhibition by ATP (Kotlarz and But, 1981) and by fructose-1,6-bisphosphate (Babul, 1978). No immunoIo~c~ cross-reactivity can be detected between Pfk-1 and Pfk-2 (Kotlarz and But, 1977). In the presence of Pfk-1, Pfk-2 is dispensible. However, in the absence of Pfk-1 (pfkja - pfkB + ), slow growth on sugars depends on Pfk-2, for its loss (pfkA - p$kB- ) causes complete inability to grow (Vinopal and Fraenkel, 1975; Daldal and Fraenkel, 198 1). The presence of a high level of Ptk-2 in such a strain (p&I - p&B 1)

0378-l 119/84/~03.~ 0 1984 Elsevier Science Publishers

338

improves growth markedly (Morrissey and Fraen- kel, 1972; Babul, 1978). Thus, Plk-2 can support substantial glycolytic flux when present in sufftcient amounts. Interestingly, growth is further improved in such strains when they carry a second mutation, p&BlO, affecting the protein itself (Daldal et al., 1982; Daldal and Fraenkel, 1983).

To better understand these metabolic and enzy- mological effects it was necessary to define the var- ious pjkB mutations at the molecular level. I have already reported the cloning of p&B + and its single and double mutant derivatives pfktz 1 and p&B1

p&B IO, the localization ofp@B on a 2.1-kb H&dIII- Pst I fra~ent, the sequencing of the portion of the gene which codes for the 38 amino-terminal residues of Pfk-2 and of the 5’ controlling region, and have demonstrated that pfkB 1 contains a base change at position -12 leading to increased tr~scription (Daldal, 1983). Here I present the entire nucleotide sequence of the p&B gene, the first nucleotide se- quence for a phosphof~ctokinase.

signed to the insert by comparison with pBK322 treated similarly, and were also mapped with respect to the known HindHI, SmaI, WI, PstI and other sites, by second digestions, using a small fraction of labeled material (generally l/100). The remaining fractions were cut with appropriate restriction en- zymes, or were strand-sepa~dted to obtain end- labeled fragments suitable for sequence analysis. They were then purified from 6”/, polyacrylamide gels as described by Maxam and Gilbert (1980) and further purifjed by BND-cellulose mini-column chromatography and used in chemical degradation reactions. For approximately 90% of the presented sequence, a minimum of two independent determi- nations were made (one from &Bl, and the other from pfiB’), both strands were sequenced for at least 90% of the residues and the data includes over- laps at most restriction sites used. The only un- certainty resides in the region from amino acid 273 (bp 1133) to the cat-boxy-terminus of Pllc-2, (bp 1238) which was determined only from one strand for the wild-type and the mutant gene.

(c) Materials MATERIALS AND METHODS

(a) Strains and plasmids

E. coli strains DF961 (F- , pro, thi, end-4 hsd&,

recA56) and DFlOlO (F- dl5(pflc-rha), d(pfkB),

recA56)carryingeither pFD121 (pfkB +)or pFDl10 (pfkB I) plasmids, their growth conditions, plasmid DNA purification, restriction enzyme digestion, aga- rose and polyacrylamide gel eiectrophoresis have been described previously (Daldal, 1983).

(b) Restriction analysis and sequencing

Nucleotide sequence analysis of DNA was per- formed as described by Maxam and Gilbert (1980). The sequencing strategy is shown in Fig. 1. Purified plasmid pFD 121 (P-‘B + > and pFDll0 BABY) which carry the 2.1-kb I;lindIII-PstI insert were di- gested separately with IS&I, AvaII, DdeI, E&rdIII, XmaI, S&I, BarnHI, treated with bacterial alka- line phosphatase, and phosphorylated either with [ Y-~‘P]ATP using T4 polynucleotide kinase, or ]a-32P]NTP and the large fragment of DNA poly- merase. The resulting labeled fragments were as-

BND-cellulose is from Servacel and mini econo- columns from Biorad. All other chemicals were as described (Daldal, 1983).

RESULTS AND DISCUSSION

{a) Cloning and sequencing of the pfkB gene

Thep&B gene has been cloned from chromosomal DNA by complementation of the appropriate mu- tants, and it has been shown by subcloning and by BAL3 1 mediated deletion analysis that the informa- tion necessary for coding Plk-2 is located on an approx. 1300-bp ~~~~s-~~~dIII fragment (Fig. 1) (Daldal, 1983). During the subcloning, it became apparent that the unique Sat1 site was located in p$kB, since subclones carrying either the Pst I-Sal1 or SalI-Hind111 fragments were unable to grow on su- gars and had no detectable phosphofructokinase ac- tivity. Following the strategy shown in Fig. 1, and as described in MATERIALS AND METHODS, SeCtiOn b, the entire nucleotide sequence of the Hint-I,-HftzdIII fragment was determined and is presented in Fig. 2.

339

HinfIg HinfI5

\ \

Hinf 14 HinfI3

AvaIL \. Sal1 \

HinfI2

\

HinfIl

AvolI XmaI AvalI /

Pst I 20 1.5 1.0 Barn HI 0.5 HindlIt kb

AAT DfkB GTA

Sal1 < + XmaI < >

Barn HI Ava II Me < = >

Hind III Hinf I M--m _,-“,

Fig. 1. Restriction enzyme cleavage map and DNA sequencing strategy for the E. colipfkB gene. The position of the leftward oriented

gene is indicated by the ATG-pj7&-TAA line. The direction of transcription for pw is from Hind111 toward Pst I. The arrows indicate

the direction of sequencing and the length of the sequence determined. The sequencing was done separately for the wild-type pjkB +

and the mutant pfkB 1. The Hinfl sites are numbered 1 to 6.

In this region there is an open reading frame, of 308 amino acids, starting at position 3 15 bp, and ending at position 1238 bp. The amino acid sequence at the amino-terminus of this open reading frame matches perfectly with the amino-terminal amino acid se- quence determined by direct analysis of the purified Pfk-2 protein (Daldal, 1983). Therefore, according to the nucleotide sequence presented here, the Ptk-2 protein is 308 amino acids long and has a calculated molecular weight of 32.4 kDal. This is in good agree- ment with the estimated subunit M, of approx. 36 000 using SDS-polyacrylamide gel electrophoresis (Bab- ul, 1978). Of these 308 amino acids, 56 are charged, 27 are basic and 29 acidic, leaving an excess of two negative charges at neutral pH. Several features of the 3 14-bp long 5’ sequence including the promoter and the ribosome binding site have been discussed earlier (Daldal, 1983).

The entire coding sequence was identified for pjlzB + andpfk.B 1 and found to be identical. This tits with the apparent enzymological identity of Ptk-2 from the two genes (Babul, 1978), and suggests that the difference in enzyme amounts might be due to the only known difference between these genes, namely the previously identified C to T change at - 12 ofpfkB promoter (Daldal, 1983).

(b) Codon usage

The codon usage of the pfkB gene (Table I) is random, as observed for other weakly expressed genes in E. coli. Thus, out of 61 possible codons, 58 are used at least once. Five of the six most frequently used codons (more than 10 times) correspond to

major isoaccepting tRNA species and code altogeth- er for approx. 25% of the amino acid residues, a relatively low value when contrasted with the codon usage for highly expressed genes (Clement and Hof- nung, 1981). GCC, which is used 10 times in pjkB,

is decoded by a minor tRNA,,, species. Codon usage inpjlcB fits the rules proposed by Grosjean and Fiers (1982) regarding selection of synonymous cod- ons NNU or NNC which are usually recognized by the same tRNA species. First, in weakly expressed genes, contrary to the highly expressed ones, NNU is preferred over NNC when N is an A or a U; thus in pfkB the following codon usage for Phe = UU(C : l/U : 3), Ile = AU(C : 6/U : 8); Tyr = UA(C : 2/U : 3) and Asp = AA(C : 7/U : 7) is ob- served (Table I). Second, again in weakly expressed genes, NNC is preferred over NNU when N is a C or a G; thus in pjlcB the following codon frequency for Arg = CG(C : 6/U : 4); Pro = CC(C : 3/U : 1); Ala = GC(C : 10/U : 6) and Gly = GG(C : 12/U : 7) is found (Table I). Therefore it seems that thep_fkB gene has a typical codon usage associated with weakly expressed E. coli genes. In addition to this codon usage the weakpfkB promoter causes its low level of transcription. However, the 5’ end of this gene presents a strong ribosome binding site, which is perfectly complementary to the 3’ end of the 16s ribosomal RNA, perhaps promoting efficient ini- tiation of translation (Daldal, 1983).

(c) Comparison with other related genes

At present, the only other available phosphofruc- tokinase protein sequence is that of Bacillus

340

Hind111 1

AAGCTTCATTTATCAAGAG 18 TCCGTACAACAAAAAAAGAGACCATCGCGGTCCCGGAAACTTTCTTAAGGATCAAAGATT 18 AGCGTCCCTGGAAAGGTAACGAATTATAAAAAGGCGCGAATAACTTAGCAATGTATTCTT 158 ATTTCATTTTTTGAATAAGCATGTGGCGAAAACAGATTTTTATTTATATATATTTATCTG 218 CAAAATTTTAAATAAAGCTCCAATAAATCATATTGTTAATTTCTTCACTTTCCGCTGATT 218 -------

-35 MetValArgIleTyrThrLeuThr

CGGTGCCAGACTGAAATCAGCCTATAGGAGGAAATGATGGTACGTATCTATACGTTGACA 338 -10 +1 S.D.

LeuAlaProSerLeuAspSerAlaThrIleThrProGlnIleTyrProGluGluAsnCys CTTGCGCCCTCTCTCGATAGCGCAACAATTACCCCGCAAATTTATCCCGAGGAAAACTGC 398

AlaValProHisArgCysSerAsnProGlyGlyGlyIleAsnValAlaArgAlaIleAla GCTGTACCGCACCGGTGTTCGAACCCGGGCGGCGGCATCAACGTCGCCCGCGCCATTGCC 458

HisLeuGlyGlySerAlaThrAlaIlePheProAlaGlyGlyAlaThrGlyGluHisLeu CATCTTGGAGGCAGTGCCACAGCGATCTTCCCGGCGGGTGGCGCGACCGGCGAACACCTG 518

ValSerLeuLeuAlaAspGluAsnValProValAlaThrValGluAlaLysAspTrpThr GTTTCACTGTTGGCGGATGAAAATGTCCCCGTCGCTACTGTAGAAGCCAAAGACTGGACC 518

ArgGlnAsnLeuHisValHisValGluAlaSerGlyGluGlnTyrArgPheValMetPro CGGCAGAATTTACACGTACATGTGGAAGCAAGCGGTGAGCAGTATCGTTTTGTTATGCCA 638

GlyAlaAlaLeuAsnGluAspGluPheArgGlnLeuGluGluGlnValLeuGluIleGlu GGCGCGGCATTAAATGAAGATGAGTTTCGCCAGCTTGAAGAGCAAGTTCTGGAAATTGAA 698

SerGlyAlaIleLeuValIleSerGlySerLeuProProGlyValLysLeuGluLysLeu TCCGGGGCCATCCTGGTCATAAGCGGAAGCCTGCCGCCAGGTGTGAAGCTGGAAAAATTA 158

ThrGlnLeuIleSerLeuArgLysAsnLysGlySerAlaAlaSerSerThrValLeuGly ACCCAACTGATTTCGCTGCGCAAAAACAAGGGATCCGCTGCATCGTCGACAGTTCTTGGA 818

GlnGlyLeuSerAlaAlaLeuAlaIleGlyAsnIleGluLeuValLysProAsnGlnLys CAGGGCTTAAGTGCAGCACTGGCAATTGGTAACATCGAGTTGGTTAAGCCTAACCAAAAA 818

GluLeuSerAlaLeuValAsnArgGluLeuThrGlnProAspAspValArgLysAlaAla GAACTCAGTGCGCTGGTGAATCGCGAACTCACCCAGCCGGACGATGTCCGCAAAGCCGCG 93 8

GlnGluIleValAsnSerGlyLysAlaLysArgValValValSerLeuGlyProGlnGly CAGGAAATCGTTAATAGCGGCAAGGCCAAACGGGTTGTCGTTTCCCTGGGTCCACAAGGA 998

AlaLeuGlyValAspSerGluAsnCysIleGlnValValProProAlaLeuLysSerGln GCGCTGGGTGTTGATAGTGAAAACTGTATTCAGGTGGTGCCACCAGCGTTGAAAAGCCAG 1058

SerThrValGlyAlaGlyAspArgLeuValGlyAlaMetThrLeuLysLeuAlaGluAsn AGTACCGTTGGCGCTGGTGACAGACTGGTCGGCGCGATGACACTGAAACTGGCAGAAAAT 1118

AlaSerLeuGluGluMetValArgPheGlyValAlaAlaGlySerAlaAlaThrLeuAsn GCCTCTCTTGAAGAGATGGTTCGTTTTGGCGTAGCTGCGGGGAGTGCAGCCACACTCAAT 1178

GlnGlyThrArgLeuCysSerHisAspAspThrGlnLysIleTyrAlaTyrLeuSerArg CAGGGAACACGTCTGTGCTCCCATGACGATACGCAAAAAATTTACGCTTACCTTTCCCGC 1238 ***

TAACAAAAACCCCCAGCATTGGGGGAATCA 1268 Ijinf15

Fig. 2. The nucleotide and amino acid sequence of gene pfiB. The DNA strand running 5’ to 3’ from the Hind111 site at coordinate

0 to the Hinfl, site at coordinate 1268 is shown. The orientation in Fig. 2 is opposite to that in Fig. 1. Promoter (-35, -lo), mRNA

start point ( + 1) and ribosome-binding sites (S.D.) ofpJke are underlined, and have already been described (Daldal, 1983). The 30%amino

acid sequence of the Pk-2 protein starts at position 315 and stops at position 1238.

341

TABLE I

Codon utilization in the E. coli pj7cB gene”

AA Number Codon % AA Number Codon %

Arg 0

6

3

4

1

0

Leu 0

4

16

6

4

4

Ser 1

5

4

2

6

6

Thr 7

6

2

1

Pro 5

3

6

1

Ala 9

10

12

6

Trp 1

Met 4

CGA 0

CGC 1.9

CGG 1.0

CGU 1.3

AGA 0.3

AGG 0

CUA 0

cut 1.3

CUG 5.2

cuu 1.9

UUA 1.3

UUG 1.3

UCA 0.3

ucc 1.6

UCG 1.3

UCU 0.6

AGC 1.9

AGU 1.9

ACA 2.3

ACC 1.9

ACG 0.6

ACU 0.3

CCA 1.6

ccc 1.0

CCG 1.9

ecu 0.3

GCA 2.9

GCC 3.2

GCG 3.9

GCU 1.9

UGG 0.3

AUG 1.3

Gly 6

12

2

7

Val 5

7

5

11

Lys 9

4

Asn I

7

Gln 6

9

His 3

3

Glu 16

6

Asp 4

6

Tyr 2

3

cys 2

2

Phe 1

3

Ile 1

6

8

GGA 1.9

GGC 3.9

GGG 0.6

GGU 2.3

GUA 1.6

GUC 2.3

GUG 1.6

GUU 3.5

AAA 2.9

AAG 1.3

AAC 2.3

AAU 2.3

CAA 1.9

CAG 2.9

CAC 1.0

CAU 1.0

GAA 5.2

GAG 1.9

GAC 1.3

GAU 1.9

UAC 0.6

UAU 1.0

UGC 0.6

UGU 0.6

uuc 0.3

uuu 1.0

AUA 0.3

AUC 1.9

AUU 2.6

a The frequency at which each of the 64 codons is present per

100 codons in the gene for Plk-2 and also the total number of

each codon in the gene is listed. The sum of the entries for each

amino acid gives the amino acid composition of Ptk-2. AA,

amino acid.

stearothennophilus enzyme (Kolb et al., 1980), which was obtained by amino acid sequence analysis. The B. stearothermophilus enzyme, however, kinetically resembles the major E. coli isoenzyme, Pfk-1, in being cooperative for fructose-6-phosphate (Evans and Hudson, 1979). An amino acid sequence com- parison between Pfk-2, as derived from the DNA sequence, and the B. stearothermophilus enzyme

shows no striking homology. Thus, even though both

enzymes recognize the same substrates and pro-

ducts, they have apparently evolved independently.

Indeed, Plk-2 of E. coli has certain enzymatic char- acteristics more closely related to the eukaryotic en- zymes, such as ATP inhibition, phosphoenolpyru- vate insensitivity and fructose-1,6-bisphosphate sen- sitivity. Thus, comparison of E. coli Plk-2 amino acid sequence with the sequences of eukaryotic phosphofructokinases and E. coli Pfk-1 will be of

interest.

ACKNOWLEDGEMENTS

This work was supported by NSF grant PCM-82- 06542 to D.G. Fraenkel. I am grateful to Dan G. Fraenkel for his deep interest, constant advice and help in preparing this manuscript.

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