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Sequence of the Escherichia coli K-12 edd and eda genes of the Entner-Doudoroff pathway
Gene, 130 (1993) 155-156 % 1993 Elsevier Science Publishers
GENE
B.V. All rights
reserved.
155
0378-l 119/93/$06.00
07214
Sequence of the Escherichia coli K- 12 edd and eda genes of the Entner-Doudoroff pathway (6-phosphogluconate
dehydratase; 2-keto-3-deoxy-6-phosphogluconate
aldolase)
A.T. Carte?, B.M. Pearsor?, J.R. Dickinsonb and W.E. Lancashire” a AFRC Instituteof Food Research, Norwich Research Park, Norwich, NR4 7UA, UK; b School of Pure and Applied Biology, University of Wales College of Cardiff, P.O. BOX 915, Card@ CFl3TL. UK. Tel. (44-222) 874000; and ’ Whithread PLC Technical Centre, Luton, LCJI 3ET, UK. Tel. (44-582) 424200 Received by K.F. Chater:
27 November
1992; Revised/Accepted:
3 March/3
March
1993; Received at publishers:
15 April 1993
SUMMARY
A 3.5-kb DNA fragment from the Clarke and Carbon Escherichia coli genomic clone, pLC37-44, was sequenced on both strands. Part of the zwf gene, encoding glucose-6-phosphate dehydrogenase, and all of the edd and eda genes, encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, respectively, of the EntnerDoudoroff pathway, were identified. These data are compared with those of Egan et al. [J, Bacterial. 174 (1992) 463846461 and important differences were noted.
The sequence of a 3.5-kb SalI-BamHI fragment from pLC37-44 (Clarke and Carbon, 1976) was analysed using the GCG software package (Devereux et al., 1984). The genes were identified as encoding zwf-edd-eda (Fig. 1). A recently published sequence of the same genes (Egan et al. 1992) differs in several positions from the present data in Fig. 1: (I) Egan et al. (1992) identified promoters of both edd and eda genes and highlighted a 9-bp sequence near to both - 35 regions, as a possible candidate for a gluconate regulatory element. However, our data (nt 600-607): (5’CGTGCGGA) differ at two positions from the sequence of Egan et al. (5’-CGGTGCCGA). Our version appears (as the complement) in the sequence ladder used to size Correspondence to: Dr. A.T. Carter, AFRC Institute of Food Research, Norwich Research Park, Norwich, NR4 7UA, UK. Tel. (44-603) 56122; Fax (44-603) 507723; e-mail: CARTERA(qUK.AC.AFRC.ARCB Abbreviations: aa, amino acid(s); bp, base pair(s); eda, gene encoding 2-keto-3-deoxy-6-phosphogluconate aldolase; edd, gene encoding 6-phosphogluconate dehydratase; GCG, Genetics Computer Group, (Madison, WI, USA); kb, kilobase or 1000 bp; nt, nucleotide(s); RBS, ribosome-binding site(s); ZWJ gene encoding glucose-6-phosphate dehydrogenase.
a primer extension product in Fig. 5 of Egan et al. (1992). These data still do not exclude the possibility that the true element is a consensus of the two versions. (2) The RBS 6-bp upstream from the edd gene is stated by Egan et al. (1992) to be AGAG, i.e., relatively inefficient at initiating translation. Our data indicate a perfect RBS, AGGAG, at this position. (3) We found the edd gene to comprise 603 codons, but Egan et al. (1992) reported 602 codons. A GAP comparison revealed a major difference at codons 252-254. The data of Egan et al. (1992) lack the equivalent of an entire codon: our sequence (nt 1501-l 5 15) is 5’-TCT CCG CTG CGC GAT-3’; whereas that of Egan et al. (1992) is 5’TCT GCC TGC GAT-3’. This translates in our case to SPLRD, whereas the Egan et al. version is SACD. A GAP comparison of E. coli and Zymomonas mobilis edd polypeptides (Egan et al., 1992) shows that the PLR residues are conserved. (4) The TERMINATOR programme revealed a perfect 13-nt inverted repeat (nt 3246-3258 and 3262-3274) separated by a 3-nt loop region, downstream from the eda gene. Egan et al. (1992) also located this region, but did not include the four A and T residues at the base of the
156 643
nt.1
GTCGACV
- - - - GAGTA?E
D
450
(ZWJ
codon 300
-. .-
- - - -TcAGGcc~GT~GG~~cAc-
- - - - -G&C-
-
FN”
2247
250
1 w4
251
G
A
E
Q 602
L
END
213
(da)
253
254
255
256
4%
499
so0
1
2
2569
2556
S
252
603
(eda)
(edd)
-7
Fig. 1. Schematic diagram of the major nt sequence features of the zwf-edd-eda locus, as determined by Egan et al.(1992), showing regions of difference with our data. Numbering above the nt sequence begins at the first nt of a SalI restriction site in zwf and continues to a BamHI site downstream from eda. Coding triplets of ZWJ edd and eda are indicated below by single letter aa nomenclature. END specifies the stop codon( GEI, GE2 (boxed): putative gluconate regulatory elements. Pl > , P2 > , P3 >, P4 > : transcription start points (boxed): of four putative promoters. SD (boxed); RBS for edd and eda. The boxed region encoding aa 252-254 of edd indicates a major area of sequence difference with our data; minor differences appear in boxes above the sequence. (A refers to a nt missing from the data of Egan et al.). The inverted repeats of a potential transcriptional terminator downstream from eda are indicated by convergent arrows. The full sequence data will appear in the EMBL/GenBank Data Libraries under the accession No. X63694.
stem. Interestingly two differences with our data remain complementary to each other. (5) Other minor sequence differences occur as indicated in Fig. 1.
ACKNOWLEDGEMENTS
This research LINK scheme.
was funded by an AFRC/Industrial
REFERENCES Clarke, L. and Carbon, J.: A colony bank containing synthetic ColEl hybrid plasmids representative of the entire E. coli genome. Cell 9 (1976) 91-99. Devereux, J., Haeberli, P. and Smithies, 0.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387-395. Egan, SE., Fliege, R., Tong, S., Shibata, A., Wolf Jr., R.E. and Conway, T.: Molecular characterization of the Entner-Doudoroff pathway in Escherichia coli: sequence analysis and localization of’promoters for the eda-edd operon. J. Bacterial. 174 (1992) 4638-4646.
GENE
B.V. All rights
reserved.
155
0378-l 119/93/$06.00
07214
Sequence of the Escherichia coli K- 12 edd and eda genes of the Entner-Doudoroff pathway (6-phosphogluconate
dehydratase; 2-keto-3-deoxy-6-phosphogluconate
aldolase)
A.T. Carte?, B.M. Pearsor?, J.R. Dickinsonb and W.E. Lancashire” a AFRC Instituteof Food Research, Norwich Research Park, Norwich, NR4 7UA, UK; b School of Pure and Applied Biology, University of Wales College of Cardiff, P.O. BOX 915, Card@ CFl3TL. UK. Tel. (44-222) 874000; and ’ Whithread PLC Technical Centre, Luton, LCJI 3ET, UK. Tel. (44-582) 424200 Received by K.F. Chater:
27 November
1992; Revised/Accepted:
3 March/3
March
1993; Received at publishers:
15 April 1993
SUMMARY
A 3.5-kb DNA fragment from the Clarke and Carbon Escherichia coli genomic clone, pLC37-44, was sequenced on both strands. Part of the zwf gene, encoding glucose-6-phosphate dehydrogenase, and all of the edd and eda genes, encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, respectively, of the EntnerDoudoroff pathway, were identified. These data are compared with those of Egan et al. [J, Bacterial. 174 (1992) 463846461 and important differences were noted.
The sequence of a 3.5-kb SalI-BamHI fragment from pLC37-44 (Clarke and Carbon, 1976) was analysed using the GCG software package (Devereux et al., 1984). The genes were identified as encoding zwf-edd-eda (Fig. 1). A recently published sequence of the same genes (Egan et al. 1992) differs in several positions from the present data in Fig. 1: (I) Egan et al. (1992) identified promoters of both edd and eda genes and highlighted a 9-bp sequence near to both - 35 regions, as a possible candidate for a gluconate regulatory element. However, our data (nt 600-607): (5’CGTGCGGA) differ at two positions from the sequence of Egan et al. (5’-CGGTGCCGA). Our version appears (as the complement) in the sequence ladder used to size Correspondence to: Dr. A.T. Carter, AFRC Institute of Food Research, Norwich Research Park, Norwich, NR4 7UA, UK. Tel. (44-603) 56122; Fax (44-603) 507723; e-mail: CARTERA(qUK.AC.AFRC.ARCB Abbreviations: aa, amino acid(s); bp, base pair(s); eda, gene encoding 2-keto-3-deoxy-6-phosphogluconate aldolase; edd, gene encoding 6-phosphogluconate dehydratase; GCG, Genetics Computer Group, (Madison, WI, USA); kb, kilobase or 1000 bp; nt, nucleotide(s); RBS, ribosome-binding site(s); ZWJ gene encoding glucose-6-phosphate dehydrogenase.
a primer extension product in Fig. 5 of Egan et al. (1992). These data still do not exclude the possibility that the true element is a consensus of the two versions. (2) The RBS 6-bp upstream from the edd gene is stated by Egan et al. (1992) to be AGAG, i.e., relatively inefficient at initiating translation. Our data indicate a perfect RBS, AGGAG, at this position. (3) We found the edd gene to comprise 603 codons, but Egan et al. (1992) reported 602 codons. A GAP comparison revealed a major difference at codons 252-254. The data of Egan et al. (1992) lack the equivalent of an entire codon: our sequence (nt 1501-l 5 15) is 5’-TCT CCG CTG CGC GAT-3’; whereas that of Egan et al. (1992) is 5’TCT GCC TGC GAT-3’. This translates in our case to SPLRD, whereas the Egan et al. version is SACD. A GAP comparison of E. coli and Zymomonas mobilis edd polypeptides (Egan et al., 1992) shows that the PLR residues are conserved. (4) The TERMINATOR programme revealed a perfect 13-nt inverted repeat (nt 3246-3258 and 3262-3274) separated by a 3-nt loop region, downstream from the eda gene. Egan et al. (1992) also located this region, but did not include the four A and T residues at the base of the
156 643
nt.1
GTCGACV
- - - - GAGTA?E
D
450
(ZWJ
codon 300
-. .-
- - - -TcAGGcc~GT~GG~~cAc-
- - - - -G&C-
-
FN”
2247
250
1 w4
251
G
A
E
Q 602
L
END
213
(da)
253
254
255
256
4%
499
so0
1
2
2569
2556
S
252
603
(eda)
(edd)
-7
Fig. 1. Schematic diagram of the major nt sequence features of the zwf-edd-eda locus, as determined by Egan et al.(1992), showing regions of difference with our data. Numbering above the nt sequence begins at the first nt of a SalI restriction site in zwf and continues to a BamHI site downstream from eda. Coding triplets of ZWJ edd and eda are indicated below by single letter aa nomenclature. END specifies the stop codon( GEI, GE2 (boxed): putative gluconate regulatory elements. Pl > , P2 > , P3 >, P4 > : transcription start points (boxed): of four putative promoters. SD (boxed); RBS for edd and eda. The boxed region encoding aa 252-254 of edd indicates a major area of sequence difference with our data; minor differences appear in boxes above the sequence. (A refers to a nt missing from the data of Egan et al.). The inverted repeats of a potential transcriptional terminator downstream from eda are indicated by convergent arrows. The full sequence data will appear in the EMBL/GenBank Data Libraries under the accession No. X63694.
stem. Interestingly two differences with our data remain complementary to each other. (5) Other minor sequence differences occur as indicated in Fig. 1.
ACKNOWLEDGEMENTS
This research LINK scheme.
was funded by an AFRC/Industrial
REFERENCES Clarke, L. and Carbon, J.: A colony bank containing synthetic ColEl hybrid plasmids representative of the entire E. coli genome. Cell 9 (1976) 91-99. Devereux, J., Haeberli, P. and Smithies, 0.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387-395. Egan, SE., Fliege, R., Tong, S., Shibata, A., Wolf Jr., R.E. and Conway, T.: Molecular characterization of the Entner-Doudoroff pathway in Escherichia coli: sequence analysis and localization of’promoters for the eda-edd operon. J. Bacterial. 174 (1992) 4638-4646.