BIOTECHNOLOGY LETTERS Volume I5 No.9 (Sept.1993) pp.901 -903 Received 27th August

SEQUENCE AND ACI’IVITY OF AN ENDOGENOUS PROMOTER OF THE ASSIMILATORY SULFITE REDUCTASE GENE FROM DESULFOVIBRIO VULGARIS (HILDENBOROUGH).

Jian Tan and J. A. Cowan* Contribution from Evans Laboratory of Chemistry, The Ohio State University, 120 West 18th Avenue, Columbus, Ohio 43210. SUMMARY. The 5’-flanking region of the assimilatory-type sulfite reductaseencoding gene from the anaerobic bacterium, DesuZfovibrio uuZguris (Hildenborough), has been isolated and sequenced. The promoter element has been identified and compared with the consensus sequence for Escherichiu coli and the sequences of promoter elements identified in other Desulfouibrio strains. The utility of such promoter sequences in E. co&based expression systems is discussed.

INTRODUCTIONa High level expression of heterologous genes in a host organism can be of great value in both pure and applied research. Escherichiu coZi has been used preferentially as a host for expression of foreign genes since its genetics and physiology are well understood. Additional advantages of E. coli-based expression a Abbreviations: aa, amino acid; amp, ampicillin; ATCC, American Type Culture Collection; ATSiR, assimilatory-type sulfite reductase gene; bp, base pair(s); D., Desulfovibrio; DNA, deoxyribonucleic acid; E., Escherichia; kb, kilobase or 1000 bp; IPTG, isopropyl+D-thiogalactopyranoside; MW, molecular weight; OD, optical density; PAGE, polyacrylamide gel electrophoresis; RT, room temperature; SDS, sodium dodecyl sulfate; Tris, 2-amino-2-(hydroxymethyl)-1,3-propanediol.

systems include high rates of cell growth and the low-cost of culture conditions, while gene expression can often be regulated by use of a variety of exogenous promoter sequences. Our laboratory has recently isolated and sequenced the 5’flanking region of the assimilatory-type sulfite reductase gene (ATSiR) from the anaerobic bacterium Desulfmibrio vulgaris (Hildenborough) (Tan et al., 1991) and found this domain to contain the natural promoter sequence of the gene. Herein we report the sequence of this promoter element, compare it with the consensus sequence for E. coli and promoters identified in other Desulfovibrio strains, and finally detail its expression characteristics. The utility of such promoter sequences in E. co&based expression systems is discussed. RESULTS AND DISCUSSION (a) Nucleotide Sequence Analysis. We have previously defined the Shine-Dalgarno sequence and the termination site for the ATSiR gene (Tan et al., 1991). Figure 1 shows the nucleotide sequence of the 5’4’lanking region of this gene that contains the promoter region. The partial restriction map and the sequencing strategy used to acquire the complete nucleotide sequence of the structural gene and flanking sequences have been described previously (Tan et al., 1991).

CTGCGCGGGT6OTCATAGATAC70GC~G~~~~CCAT~QoT~T~GTGA’~...... start

Figure 1. Nucleotide sequence of the 5’- flanking region of the ATSiR gene. The promoter site (0) and Shine-Dalgarno sequence ( ) are indicated. Unless otherwise specified, general recombinant DNA procedures were taken from Maniatis (1982) or Sambrook (1989). Nucleotide sequencing strategies and protocols have been described in detail previously (Tan et al., 1991).

(b) Promoter Region. Based on the homology with the consensus promoter sequence from E.coli (35 sequence, TTGACA; -10 sequence, TATAAT) (Hawley & McClure, 1983), one

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potential promoter site was noted in the S-upstream sequence (boxed off in Figure 1). The promoter site has the -35 sequence (TTGACG) beginning at nucleotide 20, which is highly homologous to the E. coli -35 consensus sequence, and the -10 sequence (CTCAAG) starting at nucleotide 43. Although the sequence (TTCATA) at nucleotide 60 would appear to be more homologous to the E. cdi -10 consensus sequence, the space between this -10 sequence and the -35 sequence is much greater (34 bp) than the typical consensus spacing (15-21 bp) (O’Neill, 1989). 1

2

3

4

5

6

24.000

18.400

Figure 2. SDS-PAGE analysis of the expression of recombinant ATSiR in E. coli (XL-l blue). Lane 1 - protein MW marker; lane 2 - extract of untransformed cells; lane 3 - cells transformed with pBS(+)KS; lane 4 - cells transformed with pBS(+)KS/ATSiR(S/H) containing the gene and 5’-flanking regions; lane 5 - as lane 4 only the medium was supplemented with Fe*+ during growth; lane 6 - purified ATSiR. For each gel electrophoresis experiment an aliquot (1 mL) of cell culture was removed and spun down. Approximately 100 PL of SDS gel loading buffer was added to the cell pellet. After mixing, the suspension was placed in a boiling water bath for 10 min before aliquots (1 PL) were loaded onto Phast gels using an automated gel loading applicator (Pharmacia/LKB). Expression was examined by SDS-PAGE run on a Phast electrophoresis system (Pharmacia/LKB) at 15OCusing precast 20% homogeneous gels (Pharmacia/LKB). Running and staining (Coomassie) conditions followed recommended preprogramed operating conditions. Molecular weight determinations were referenced to commercially available standards (BRL, high molecular weight range).

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(13 Direct Expression of the ATSiR gene in E. coli. The strong homology between regions of the 5’-flanking sequence of the ATSiR gene and the consensus sequences for promoters in E. coli raised the possibility that expression of the cloned gene might be feasible in E. coli using the endogenous D. oulguris promoter. Examination of E. coli extracts by SDS-PAGE did reveal the presence of an intense protein band in cells (XL-l blue) transformed with a plasmid pBS(+)KS/ATSiR(S/H) that contained the ATSiR gene and 5’-flanking regions (Figure 2, lane 4). This protein band is absent in extracts of untransformed cells (Figure 2, lane 2) or cells transformed with the vector lacking the gene insert (Figure 2, lane 3). This additional protein band comigrates with the purified ATSiR enzyme (Figure 2, lane 6). Expression levels were estimated to be at approximately 5% of total cellular protein. By comparing SDS-PAGE gels of both the cell debris and supernatant solution obtained after cell lysis we found that the ATSiR gene was expressed in E, coli as inclusion bodies under most conditions, reflecting the complexity of maturation and assembly of the holoenzyme. Plasmid pBS(+)KS/ATSiR(S/H) was also introduced into other E. coli strains (DH5aMCR and AGl) and similar expression levels were observed in direct comparison with XL-1 blue. This suggests that the endogenous D. vulgaris promoter is active in a variety of E. coli host strains.

Table 1. List of Promoter Sequences (-35 region and -10 region) of Genes Isolated from D. vulgaris and Other Desulfovibrio Species -35

-10

ATSiR gene from D. mdgaris

TTGACG

flavodoxin gene from D. mlgurisa

TTGAAA TT.GCGC TTGACA TCCGCA TTGACA TTCACA TTGACA

CTCAAG TTCATA TATACA TTTTAT TACCAT GATATT TAGGAT TAAATT TATAAT

or or cytochrome c3 gene from D. vulgaris b hydrogenase gene from D. pulguris C cytochrome cc3 gene from D. vulgaris d flavodoxin gene from D. salexigens e consensus promoter sequence from E. coli f

a (Krey et al., 1988). b (Voordouw et al., 1986). C (Voordouw & Brenner 1985). d (Pollock et al., 1991). e (Helms et al., 1990). f (Hawley & McClure, 1983)

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The requirement for the endogenous promoter was examined by monitoring expression of the ATSiR gene in E. coli after deletion of the promoter region. Plasmid pBS(+)KS/ATSiR(E/E) contains the ATSiR gene with the 5’-flanking region eliminated. ATSiR was not expressed in E. coli cells transformed with this construct indicating a requirement for the endogenous promoter for gene expression. Other proteins (for example, cytochrome c3 gene from D. vulgaris; and flavodoxin gene from D. salexigens) have been expressed in E. coli off their own promoter elements. Table 1 clearly demonstrates that these endogenous promoter sequences are all highly homologous to the consensus promoter sequence from E. coli, which most likely accounts for the ease of gene expression in this host organism. (d) Comparison of Expression Activity using the Tat Promoter. The fat promoter, a fusion of the trp and Zacpromoters (Brosius & Holly, 1984), is widely used to express proteins in E. coli. To directly compare levels of protein expression obtained with the endogenous promoter of ATSiR and tat, a plasmid pKK2233/ATSiR(E/H) was constructed that postioned the fuc promoter upstream of the ATSiR gene. E. coli AGl was transformed with pKK2233/ATSiR(E/H) and grown in LB medium with induction by IPTG. The expression level of the ATSiR gene in pKK223-3 using the tat promoter was similar to that obtained with the pBS(+)KS derived expression vector containing the endogenous promoter. Residual expression was observed even in the absence of IPTG since fat is a “leaky” promoter (Amman et al., 1983; Studier & Moffatt, 1986). These results show that the endogenous promoter from D. vulgaris can be used to achieve protein expression levels that are comparable to the tat system. Genes for cloned proteins are often expressed behind a regulated promoter (Remaut et al., 1981), although induction often requires an expensive chemical (such as IPTG) or an inconvenient and potentially harmful temperature shift. Moreover, most promoters are useful in only a limited number of host strains. The endogenous promoter of ATSiR appears to be straightforward to use, inexpensive (requiring no chemical additives), and is active in a variety of E. coli strains. It should therefore be of value for high level expression of cloned genes in E. coli. ACKNOWLEDGMENTS. This work was supported by the National Science Foundation (CHE-8921468). JAC is a Fellow of the Alfred I’. Sloan Foundation and a National Science Foundation Young Investigator.

REFERENCES Amman, E., Brosius, J., Ptashne, M.: Vectors Bearing a Hybrid trp-lac Promoter useful for Regulated Expression of Cloned Genes in Escherichiu coli. Gene (Amst.) 25 1983 167-178. Brosius, J., Holly, A.: Regulation of Ribosomal RNA Promoters with a Synthetic Lac Operator. Proc. Natl. Acad. Sci. USA 81 1984 6929-6933. Hawley, D.K., McClure, W.R.: Compilation and Analysis of Escherichia coli Promoter DNA Sequences. Nucleic Acids Res. 8 1983 2237-2255. Krey, G. D., Vanin, E. F., Swenson, R. P.: Cloning, Nucleotide Sequence, and Expression of the Flavodoxin Gene from Desulfovibrio vulgaris (Hildenborough). J. Biol. Chem. 263 1988 15436-15443. Maniatis, T., Fritsch, E. F., Sambrook, J.: Molecular Cloning: A Laboratory Manul, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. O’Neill, M.C.: Escherichiu coli Promoters. J. Biol. Chem. 264 1989 5522-5530. Pollock, W.B.R., Loutefi, M., Bruschi, M., Rapp-Giles, B.J., Wall, J.D., Voordouw, G.: Cloning, Sequencing and Expression of the Gene Encoding the HighMolecular-Weight Cytochrome c from Desulfovibrio vulgaris (Hildenborough). J. Bacterial. 173 1991 220-228. Remaut, E., Stanssens, l?., Fiers, W.: Plasmid Vectors for High-Efficiency Expression Controlled by the PL Promoter of Coliphage Lambda. Gene 15 1981 81-93. Sambrook, J., Fritsch, E. F., Maniatis, T.: Molecular Cloning; Cold Spring Harbor Laboratory Press, 1989. Studier, F.W., Moffatt, B.A.: Use of Bacteriophage T7 RNA Polymerase to Direct Selective High-Level Expression of Cloned Genes. J. Mol. Biol. 189 1986 113130. Tan, J., Helms, L., Swenson, R.P., Cowan, J.A.: Primary Structure of the Assimilatory-Type Sulfite Reductase from Desdfovibrio vulgaris (Hildenborough): Cloning and Nucleotide Sequence of the Reductase Gene. Biochemistry 30 (1991) 9900-9907. Voordouw, G., Brenner, S.: Cloning and Sequencing of the Gene Encoding Cytochrome c3 from Desulfovibrio vulgaris (Hildenborough). Eur. J. Biochem. 159 1986 347-351. Voordouw, G., Brenner, S.: Nucleotide Sequence of the Gene Encoding the Hydrogenase from Desulfovibrio vulgaris (Hildenborough). Eur. J. Biochem. 148 1985 515-520.