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Nucleotide Sequence of the Trp Promoter/Operator/Leader Region of Shigella Dysenteri

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Nucleotide Sequence of the Trp Promoter/Operator/Leader Region of Shigella Dysenteri Proc. Natl. Acad. Sct. USA Vol. 75, No. 11, pp. 5580-5584, November 1978 Genetics Naturally occurring promoter down mutation: Nucleotide sequence of the trp promoter/operator/leader region of Shigella dysenteriae 16 (evolutionary sequence alterations/regulatory mutations/nutritional deficiencies) GIUSEPPE MIOZZARI AND CHARLES YANOFSKY Department of Biological Sciences, Stanford University, Stanford, California 94305 Contributed by Charles Yanofsky, June 8, 1978 ABSTRACT The promoter/operator/leader region of the MATERIALS AND METHODS trp operon of Shigell dysenteriae 16 has single base pair dif- ferences from the corresponding region of Escherichia coli at All E. coli strains used are derivatives of strain W3110. Strain positions -24 and -13. The difference at -13 was shown to be SH16 of S. dysenteriae was originally obtained from S. E. Luria. responsible for the 90% reduction in promoter function char- acteristic of the trp operon of S. dysenteriae. The base pair The E. coli/S. dysenteriae hybrid strains SDEC11 and difference at position -13 also renders the operator partially SDEC1ITP have been described (9). Hybrid strains generated constitutive. This allows the organism to maintain relativel in the course of this work are described in the legend to Table high repressed levels of the trp enzymes and increases the rel 1. All hybrid strains were generated by P1-transduction. The ative importance of attenuation as a transcription control mechanism. These findings and the earlier observation that the construction of the plaque-forming trp transducing phages trpE protein of S. dysenteriae is only slightly active explain the 080htrpSD11 and 080trpSDllTP is described in the text. low in vivo expression of the tip operon of this organism. Plasmid pGM1 1 carries the initial portion of the trp operon of Nutritional studies sugest that operons involved in other amino S. dysenteriae. It was constructed by inserting the fragments acid biosynthetic pathways in S. dysenteriae 16 may be simi- generated by EcoRI digestion of 080htrpSD 1-DNA into the larly partially inactivated. EcoRI site of pVH51 (miniColEl; ref. 13). pGM11 was isolated Both DNA-DNA (1) and DNA-RNA (2) hybridization data and from the ligation reaction mixture by selecting tip+ transfor- protein primary structure comparisons (3, 4) indicate a close mants of an E. coli recipient strain, W3110/trpLD102, in relationship between the enteric bacteria Shigella dysenteriae which the initial portion of the trp operon has been deleted (14). and Escherichia coli. They share susceptibility to some of the Procedures for the isolation of phage and plasmid DNA and the same bacteriophages (5), and hybrid recombinants can be conditions for ligations and transformations have been described readily obtained from crosses of the two organisms (5-10). (15, 16). Despite these similarities, some features of the nutrition and Cells for the determination of enzyme activities were grown biochemistry of E. coli and S. dysenterae are quite different. in glucose minimal medium (17) supplemented with 100 jig of In particular, many strains of S. dysenteriae have been reported L-tryptophan per ml. The conditions for the assay of the glu- to require various amino acids or vitamins, or both, for growth tamine- and ammonia-stimulated reactions of anthranilate (11, 12). As described in this report, our laboratory strain, SH16, synthetase and of phosphoribosylanthranilate transferase have will only grow well on a glucose minimal medium when it is been described (9). Protein was determined by the method of supplemented with several amino acids. Lowry et al. (18). Enzyme specific activities are given as units The structural, functional, and regulatory properties of the of S. dysenteriae trp operon were studied by Manson and Ya- per milligram protein. nofsky (9, 10). They analyzed transduction hybrids in which DNA sequence analysis was performed by the method of the entire cysB-trp region of the E. col chromosome was re- Maxam and Gilbert (19). Details of this and related procedures placed by the corresponding region from S. dysenteriae. Al- such as phosphatase treatment, polynucleotide kinase and re- though the order of the tip structural genes of S. dysenteriae striction endonuclease reactions, agarose and polyacrylamide is identical with that in E. coli, tryptophan biosynthesis is largely gel electrophoresis, restriction site mapping, and extraction of blocked in the hybrids Deletion mapping identified two sites DNA from agarose and acrylamide gels are given in Bennett within the first structural gene of the operon that are responsible et al. (20) and Brown et al. (21). The conditions for RNA for a severe decrease in the activity of the enzyme complex that polymerase and Trp repressor protection of restriction sites in catalyzes the initial reaction in tryptophan biosynthesis. In the trp promoter/operator region against cleavage of restriction addition, a linked site that maps outside the structural gene endonucleases are given in Bennett et al. (22). region of S. dysenteriae acts to decrease the maximal rates of Restriction endonuclease EcoRI was purchased from Miles trp mRNA and enzyme synthesis to 10% of those of wild-type Research Laboratories (Elkhart, IN). Restriction endonucleases E. colh. Thus, the tryptophan auxotrophy of S. dysenteriae arises Alu I, Hha I, HincII, HindIII, Sal I, and T4 DNA ligase were from the combination of a catalytically inefficient "first" en- obtained from New England Biolabs (Beverly, MA). Hpa I and zyme and decreased expression of the operon. Hpa II were prepared by the method of Sharp et al. (23); Hinfl In this report we present the DNA nucleotide sequence of was prepared by an unpublished procedure. T4 polynucleotide the trp promoter/operator/leader region of S. dysenternae and kinase was a gift of A. Maxam. Calf intestinal alkaline phos- of a regulatory "revertant" that has promoter activity compa- phatase was purchased from Boehringer Mannheim (India- rable to that of E. coli. napolis, IN). E. coli RNA polymerase and partially purified Trp The publication costs of this article were defrayed in part by page repressor were gifts of F. Lee. charge payment. This article must therefore be hereby marked "ad- These studies were performed in accordance with the Na- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate tional Institutes of Health "Guidelines for Recombinant DNA this fact. Research." 5580 Downloaded by guest on September 28, 2021 Genetics: Miozzari and Yanofsky Proc. Natl. Acad. Sci. USA 75 (1978) 5581 RESULTS promoter/operator region in 080htrpEA190 and the presumed Strain SDEC11 and its spontaneous tryptophan-independent corresponding fused Hpa I fragment from 080htrpSDII. derivative, SDEC11 TP, are transductiQn hybrids in which the Comparative digestions of either whole phage DNA or isolated entire cysB-trp-tonB region of E. coli is replaced by the cor- restriction fragments with various endonucleases allowed us responding sequences from S. dysenterzae (9). The initial to derive the restriction maps shown in the upper portion of Fig. portions of the trp operons of the two hybrid strains were 2. The homology of the two E. coli Hpa I fragments with the transferred onto phage 080htrpEA190AOC1418 by genetic large fragment from S. dysenteriae is confirmed by the pres- exchange (24). In the resulting transducing phages, ence of common restriction sites. However, there are several 080htrpSD11 and 080htrpSD11TP, deletion AtrpOC1418 is differences clustered around the region corresponding to the replaced by trpPOLED and at least the initial part of trpC from initial portion of the trp operon of E. coli; in addition to the Hpa the hybrids. I site difference, S. dysenteriae DNA has a number of restric- In an attempt to identify the S. dysenteriae trp promoter/ tion sites in this region that are absent from E. coli DNA. In operator region, the DNA of 080htrpSD11 and of particular, there is a recognition sequence for EcoRI about 500 080htrpSD 1 TP was digested with Hpa I and the restriction base pairs preceding the region containing the Hpa I site in E. pattern was compared with that obtained with an Hpa I digest coli. The position of this EcoRI site preceding the trp pro- of the DNA of the parent phage 080htrpEA190. This phage moter/operator region was confirmed by digesting phage k080trpSD1 DNA with EcoRI and cloning the fragment con- carries the entire trp operon of E. coli. The trp promoter/ taining the S. dysenteriae trp genes into the EcoRI site of operator region of E. coli has been shown to contain a charac- plasmid pVH51 (miniColEl; ref. 13). The resulting plasmid, teristic recognition sequence for Hpa I (GTTWAAC) at positions pGM11, allows expression of trpE and trpD, the first two -14 to -9 preceding the transcription initiation site (22). This structural genes of the trp operon, and, therefore, probably site can be protected from Hpa I cleavage by either RNA carries the S. dysenteriae trp promoter. Restriction analysis polymerase or Trp repressor, generating a large protected band revealed that the plasmid carries a single 6.5-megadalton insert composed of the two Hpa I fragments on either side of the that is identical in size to the EcoRI fragment that starts 500 base cleavage site (22) (Fig. 1). Comparison of lanes 1 and 2 in Fig. pairs before the presumptive trp promoter/operator region in 1 indicates that '80htrpSDll lacks one of the Hpa I recognition 480htrpSD11 and spans the entire trp operon. Because of the sites present in the parent phage 480htrpEA190. The Hpa I ease with which large amounts of plasmid DNA can be obtained restriction pattern of 080htrpSD11 DNA is, however, indis- and because of the smaller size of the plasmid, all subsequent tinguishable from the restriction pattern of 080htrpEA190 experiments involving the S. dysenteriae wild-type trp operon DNA digested in the presence of RNA polymerase or Trp re- were performed with pGM11 DNA. pressor (Fig. 1, lanes 4-7).
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