Gene, 87 (1990) 127-131 Elsevier

127

GENE 03442

Promoter elements and regulation of expression of the cysD gene of Escherichia coil K-12

(Recombinant DNA;/~-galactosidase assay; dideoxynucleotide sequencing; "1"4DNA polymerase mapping; unidirectional deletion)

Madhu S. Malo and Richard E. Loughlin

Department of Biochemistry, Universityof Sydney, Sydney, N.S.W. 2006 (Australia) Tel. (02)692-2597 Received by H.M. Krisch: 29 July 1989 Revised: 17 October 1989 Accepted: 30 October 1989

SUMMARY

The cysD gene, involved in cysteine biosynthesis in Escherichia coli and Salmonella typhimur~um, is positively regulated by the CysB regulatory protein. The cysD promoter ofE. coli K- 12 in a 492-bp PstI-EcoRI fragment was sequenced. The in vivo transcription start point (tsp) for the cysD gene was determined by the methods of T4 DNA polymerase mapping and mung-bean nuclease mapping. The -10 region of the cysD promoter (TATAGT) is closely homologous to the -10 consensus sequence (TATAAT) for E. coil promoters. The -35 region of this promoter (TTCATF) is less closely related to the -35 consensus sequence (TTGACA). Several mutants were obtained by using a chain-termination method for generating unidirectional deletions. Evidence is presented for a possible CysB protein binding site around -89, thought to be involved in regulation of expression of the cysD gene.

INTRODUCTION

The cysD gene of Escherichia coil K-12 encodes SAT (EC 2.7.7.4), which catalyses the first enzymic reaction in the biosynthesis of cysteine from sulfate (Kredich, 1983). This

Correspondence to: Dr. R.E. Loughlin, Department of Biochemistry,

University of Sydney, Sydney, N.S.W. 2006 (Australia) Tel. (02)692-3466; Fax 2.692-4571. Abbreviations: aa, amino acid(s); pGal, p-galactosidase; bp, base pair(s); dd, dideoxy; dNTP, any deoxynucleoside triphosphate; ds, doublestrand(ed); kb, kilobase(s) or 1000 bp; MB, mang-bean nuelease; Pollk, Klenow (large) fragment of Escherichia coli DNA polymerase I; nt, nucleotide(s); ONP, o-nitrophenol; RBS, ribosome-binding site; S.A., specific activity; SAT, sulfate adenylate transferase; SD, ShineDalgarno sequence; ss, single-strand(ed); tsp, transcription start point(s); u, unit(s); XGal, 5-bromo.4-chloro-3-indolyl-p-D-galactopyranoside; ', indicates that the gene has been truncated at the indicated end. 0378-1! 19/90/503.50 © 1990ElsevierScience PublishersB.V.(BiomedicalDivision)

gene is regulated in a positive manner by the protein encoded by the cysB regulatory gene of the cysteine regulon (Mascarenhas and Yudkin, 1980). A model for regulation has been proposed by Trudinger and Loughlin (1981) in which both O-acetylserine and cystine bind at the same site on the CysB protein such that when O-acetylserine is bound, the complex functions as an activator but when cystine is bound the complex formed is inactive. DNA from the specialized transducing phage ~dcysJIHD (Fimmel and Loughlin, 19"/7)was cloned into protein-fusion vectors and one of the resulting constructs was pRL6, in which the expression ofpGal is regulated by the promoter for the cysD gene (Hunt et al., 1987). In this paper we describe the nucleotide sequence of the 492-bp PstI-EcoRI fragment carrying the cysD promoter and determination of the tsp for the cysD gene by I"4 DNA polymerase mapping. We suggest a putative CysB protein binding site as being involved in the regulation of the cysD gene.

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Fig. 1. Transcript mapping for the cysD gene. The method followed was that ofT4 DNA polymerase mapping described by Hu and Davidson (1986) except that hybridizations were carried out at 60°C instead of 370C The primer used was the 16-mer synthetic primer (5'-OH-TCTTCCTAAACGCCCG-OH-3') for ss pRLI27 as shown in Fig. 3. Lanes: 1, Primer extension products where the RNA used was extracted from a strain carrying pRLI30 grown under conditions of derepression; 2, control with RNA extracted from the same strain grown under conditions of repression; 3, control with no RNA, where the same amount of primer was hybridized to the same amount of template DNA and extended as for lanes I and 2. Lanes A, C, G, and T, dideoxy sequencing reactions using the same primer for ss pRLI27 and the same template DNA as were used for lanes 1, 2 and 3. Fig. 2. Cysteine control of pGal in deletion mutants. (A) Principles of the chain-termination method: (a) an M 13 sequencing primer (small closed box) is annealed to an ss MI3 derivative containing an insert (hatched box) bounded by two restriction sites, A and B, and then a sequencing reaction is performed; (b) the chain-terminated products are then digested by S 1 nuclease, which generates ds fragments ofdiffcrent lengths. The fragments are then digested with restriction enzyme B preferably generating sticky ends for efficient ligation with specific orientation; (e) the ds fragments are then ligated which results in constructs with one nick and with inserts of different sizes. (B) Construction and properties of deletion mutants. Primer hybridization and sequencing reactions were carried out after the dd sequencing method described by Sanger et el. (1977). The primer was then extended by adding 1/JI (1 u) Pollk, 1/41 (20/~Ci) of [0c-a2P]dCTP and 2/~! of the mix for 'ddA' reaction for sequencing (containing ddATP, dATP, dGTP and dTTP from Amersham sequencing kit) and incubated at 37°C for 15 min. The extended primer-ss-DNA complex was digested with 20 u S 1 nuclease at 370C for 30 min. The DNA was extracted and then digested with EcoRI to remove DNA of vector origin. The sample thus obtained was ligated to the vector pRLI24 codigested with Smal+ EcoRI, and JPAI01 was transformed. Mutants were isolated by scoring the appearance of blue coloration of the replicated colonies on the derepressed plates prespread with XGal as described in Male and Loughlin (1988). (a) and (b)Cysteine control of/~Oal in the wild-type pRLI27, and the deletion mutants./1Gel S.A., determined as nmol of ONP produced per min per mg of protein, is plotted against map position (bp, from restriction digests) of the inserts end-points under conditions of (a)derepression (closed circle) and (b) repression (open circle), respectively. (e) Linear maps of the plasmids with only the insert drawn to scale. The hatched boxes represent the inserts and the broken horizontal lines represent vector DNA. tsp ( + 1) represents the tsp from the cysD gene. E, EcoRI; P, PstI. The arrow shows the direction of transcription from the cysD promoter for expression of the promoterless hybrid galK'-'iacZ gene in the vector.

129 by the dd method of Sanger et al. (1977). The cysD promoter mutants pRLI31 (see section e; Fig. 3) and pRLI41 (see section e; Figs. 2B and 3) were also sequenced by this method. The 492-bp sequence is shown in Fig. 3.

EXPERIMENTAL AND DISCUSSION

(a) Subeloning and sequencing of the cysD promoter The 0.49-kb PstI-EcoRI bacterial fragment containing the cysD promoter was subcloned from pRL6 into the promoter-detection vectors pRL124 and pRL126 to give pRLI27 and pRL130, respectively (Malo and Loughlin, 1988). Both strands of the Pstl-EcoRI bacterial fragment containing the cysD promoter were sequenced using ss plasmid and phage (M 13 derivatives) DNA with [~3sS ]dCTP

(b) Transcription start Imint for the cysD geae JPAI01 (Malo and Loughlin, 1988) carrying pRLI30 containing the cysD promoter was grown under conditions of repression (grown on cysteine) and derepression (grown on djenkolic acid) and RNA was prepared by the method

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492

Fig. 3. The nt sequence of the cysD promoter and nearby regions. Also shown are the cysK promoter regions of 5. o,ph~urium and £. coli K-12 (Byrne et al., 1988) from + 1 to -100. The cysK sequence front S. typhimurium (S.t.K) is shown above the cysD sequence from E. coli K-12 (E.c.D) and aligned by aligning the -10 hexamers. The E. coli cysK sequence (E.c.K) is shown below the cysD sequence and was aligned the same way. Relative gaps, each depicted by a dash ( - ), were introduced in the sequences to improve the matching of sequences. Vertical lines between corresponding sequences indicate identical nt (other alignments are not shown). The antisense strand (equivalent to mRNA) is shown. Each tsp is underlined; csp for cysD is marked + ! above the sequence and the direction of transcription from each promoter is indicated by a horizontal arrow. The regions assigned as the -10 and -35 regions of the promoters are underlined. S/D is the proposed SD (Shine and Dalgarno, 1975) in the cysD transcript and its corresponding nt sequence is underlined and labelled. The predicted N-terminal aa sequence of SAT (the cysD product) is shown for 27 aa above the respective DNA codons. Upstream deletion mutants are indicated by a horizontal line above the sequences and the triangle before each mutant name indicates that the deletion encompasses DNA up to the Pstl site. For the upstream deletion mutant pRLi41, the end-point, determined from sequencing, is marked by a vertical line whereas for pRLI45 and pRLI38, the end.points, inferred from restriction fragments, would lie below the dotted regions of the horizontal line. The substitution mutant pRLI31 (see section e) has also been marked and the nt involved are indicated by larger characters. After each mutant name, the residual cysD promoter activity is shown in parentheses. The cysD promoter activities ofeach mutant are also shown in Fig. 2. The restriction sites Pstl and EcoRl are underlined. Other restriction sites are not shown. The primer for ss pRLI27 is underlined and labelled. Stars mark numbered nt.

130 of Aiba et al. (1981). The transcript was mapped using T4 DNA polymerase (Fig. 1) based on the method of Hu and Davidson (1986). The sense DNA template of ss pRL127 and RNA were hybridized at 60°C and the 16-mer primer for ss pRLI27 was then annealed at 60°C to the template upstream of the tsp. The primer was then extended with dNTPs and T4 DNA polymerase to the 5' end of the hybridized RNA. The major band (among a cluster of 3 bands) in lane 1, as indicated by an arrow, is not accompanied by any corresponding band in the control lanes 2 and 3. This band aligns with the T residue at the nt position 372 (Figs. I and 3). As the primer extension and sequencing reactions for the standards were performed using the same primer and template, the tsp should be the following C at nt position 373, which has been marked as + I in Fig. 3. Bands appearing at the same level in all three lanes are probably pause products due to secondary structures. More of these bands were found and the tsp band disappeared when hybridizations were carried out at 37 °C (data not shown). Transcript mapping by Burke's (1984) method using MB in place of S 1 nuclease gave the same tsp as was found with T4 DNA polymerase (data not shown). The result shown in Fig. 1 proves that the cysD gene is regulated at the level of transcription since the quantity of the transcript is increased during derepression compared to that during repression. The ATG codon starting at nt + 40 is preceded by the RBS (AAGGA) starting at nt + 29 (Fig. 3). The reading frame starting from this ATG is open to the end ofthe sequence data and is likely to be the reading frame of SAT.

protein is probably neither a new RNA polymerase nor a new ¢ factor but is an accessory protein which helps RNA polymerase holoenzyme (containing the ¢7o subunit) in transcription initiation. The strategy for construction of overlapping unidirectional deletions using a chain-termination method is described in Fig. 2A and the constructs obtained are shown in Fig. 2B. The S.A. of/1Gal for each plasmid determined under conditions of derepression and repression is also shown in Fig. 2B. Repeated//Gal assays showed that the upstream deletion mutant pRL141 (Fig. 2B) has lost 35% of the cysD promoter activity and sequence analysis revealed that in pRL141 the region upstream from -89 was deleted (Fig. 3). These data suggest that in pRL141 part of the activator (CysB protein)-binding site has been deleted. In pRLI38, the deletion end-point is near the 5' end of the -35 region and the loss of promoter activity is consistent with the loss of the proposed activator binding site further upstream. We compared the sequence of the cysD promoter region between + 1 and -100 with corresponding regions of the cysK promoters of S. typhimurium and E. coli K-12 (Byrne et al., 1988) as shown in Fig. 3. Two small gaps were introduced in the sequences ofcysD and cysK and extensive matching was found in all three sequences between -46 and -93 ofthe cysD promoter region. We suggest a binding site, shown in Fig. 3, for the CysB protein on the basis ofthe loss of promoter function for pRLI41 and pRLI38 and the observed sequence similarity.

ACKNOWLEDGEMENTS

(c) Elements and mutants of the cysD promoter We assigned the sequence TATAGT as the -10 region of the cysD promoter as the tsp is 9 bp downstream, within the range of 5-9 bp and as it closely matches to the -10 consensus sequence (TATAAT) for E. coli promoters (Harley and Reynolds, 1987). Our previous result of 95~ loss ofpromoter activity in pRLI31 (Fig. 3), which was the result of the substitution of the T at nt -9 by a G (Malo and Loughlin, 1988), is also consistent with the assignment of this sequence (TATAGT) as the -10 region. The sequence TrCATI" around 35 bp upstream from the tsp ( + 1) of the cysD promoter has some similarity with the -35 consensus sequence (TrGACA) and contains the most conserved TT residues of the consensus -35 region. The spacing between the assigned -10 and -35 regions is 17 bp, which is optimal for E. coil promoters. As the change at nt -9 caused a marked loss of promoter function in pRLI31 and as the wild-type -10 region (TATAGT) closely matches to the -10 consensus region (TATAAT), which is recognised by the ~7o subunit of RNA polymerase (Helmann and Chamberlin, 1988), we infer that ~7o recognises the -10 region of the cysD promoter. This suggests that the CysB

M.S.M. was supported by the Conmed Supplementary Scholarship from the University of Sydney.

REFERENCES Aiba, H., Adhya, S. and de Crombrugghe, B.: Evidence for two functional £al promoters in intact Escherichia coil cells. J. Biol. Chem. 256 (1981 ) 11905-11910. Burke, J.F.: High-sensitivity S 1 mapping with singie-stranded [32P]DNA probes synthesized from bacteriophage M 13mp templates. Gene 30 (1984) 63-68. Byrne, C.R., Monroe, R.S., Ward, K.A. and Kredich, N.M.: DNA sequences of the cysK regions of Salmonella typhimurium and Escherichia colt and linkage of the cysK regions to ptsH. J. Bacteriol. 170 (1988) 3150-3157. Fimmel, A.L. and Loughlin, R.E.: Isolation of a ~dcys transducing bacteriophage and its use in determining the regulation of cysteine messenger ribonucleic acid synthesis in Escherichia coil K-12. J. Bacteriol. 132 (1977) 757-763. Harley, C.B. and Reynolds, R,P.: Analysis ofE. coli promoter sequences. Nucleic Acids Res. 15 (1987) 2343-2361. Helmann, J.D. and Chamberlin, MJ.: Structure and function of bacterial sigma factors. Annu. Rev. Biochem. 57 (1988) 839-872.

131 Hu, M.C.-T. and Davidson, N.: Mapping transcription start points on cloned genomic DNA with T4 DNA polymerase: a precise and convenient technique. Gene 42 (1986) 21-29. Hunt, C.L., Colless, V., Smith, M.T., Molasky, D.O., Malo, M.S. and Loughlin, R.E.: Lambda transducing phage and clones carrying genes of the cysllHDC gene cluster of Escherichia coli KI2. J. Gen. Microbiol. 133 (1987) 2707-2717. Kredich, N.M.: Regulation ofcysteine biosynthesis in Escherichia coli and Salmonella typhimurium. In Herrmann, K.M. and Somerville, R.L. (Eds.), Amino Acids Biosynthesis and Genetic Regulation. Addison-Wesley Publishing Co., Reading, MA, 1983, pp. 115-132. Malo, M.S. and Loughlin, R.E.: Promoter-detection vectors for Escherichia coilwith multiple useful features. Gene 64 (1988) 207-2 ! 5.

Mascarenhas, D.M. and Yudkin, M.D.: Identification of a positive regulatory protein in Escherichia cob': The product of the cysB gene. Mol. Gen. Genet. 177 (1980) 535-539. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Shine, J. and Dalgarno, L.: Determinant ofcistron specificityin bacterial ribosomes. Nature 254 (1975) 34--38. Trudinger, P.A. and Loughlin, R.E.: Metabolism of simple sulphur compounds. In Florkin, M., Stotz, E.H., Neuberger, A. and Van Deenen, LL.M. (Eds.), Comprehensive Biochemistry. Vol. 19A: Amino Acid Metabolism and Sulphur Metabolism. Elsevier/North Holland Biomedical Press, Amsterdam, 1981, pp. 165-256.