Analysis in vivo of factors affecting the control of transcription initiation at promoters containing target sites for trp repressor

An investigation of repression in the trp system of Escherichia coli was undertaken using operon fusions and plasmids constructed via recombinant DNA technology. The promoters of the trp operon and the trpR gene were fused to lacZ, enabling the activity of these promoters to be evaluated under various conditions through measurements of beta-galactosidase production. In confirmation of earlier studies, the trpR gene was shown to be regulated autogenously. This control feature of the trp system was found to maintain intracellular Trp repressor protein at essentially invariant levels under most conditions studied. Increasing the trpR+ gene dosage did not significantly elevate Trp repressor protein levels, nor did the introduction of additional operator "sinks" result in significantly decreased levels of Trp repressor protein. Definite alterations in intracellular Trp repressor protein levels were achieved only by subverting the normal trpR regulatory elements. The placement of the lacUV5 or the lambda PL promoters upstream of the trpR gene resulted in significant increases in repression of the trp system. Substituting the primary trp promoter/operator for the native trpR promoter/operator resulted in an altered regulatory response of the trp system to tryptophan limitation or excess. The regulation of the trpR gene effectively imparts a broad range of expression to the trp operon in a manner finely attuned to fluctuations in intracellular tryptophan levels.     

The effect of nalidixic acid on expression from related E. coli promoters

The effect of the DNA gyrase inhibitor, nalidixic acid, on expression from E. coli promoters was studied using the pKO-1, galactokinase expression vector system. Expression from a series of related hybrid promoters, tet promoter variants and the trp promoter flanked by oligonucleotide blocks was measured after incubation with nalidixic acid. Expression from the pBR322 tet promoter and tet promoter mutants within the -10 region was reduced after the drug treatment. The lacUV5, trp, and tettrp promoters were essentially unaffected while the trplac and the trptet promoters were stimulated. Studies of the trp promoter flanked by upstream or downstream oligonucleotide blocks revealed similar responses to the trp promoter parent control plasmids.     

DNA sequence of the E. coli trpR gene and prediction of the amino acid sequence of Trp repressor.

A DNA sequence of 1041 base pairs from a BamHI fragment containing the E. coli trpR gene has been determined. With this sequence and other experimental evidence, the primary structure (88 amino acids) of the Trp repressor can be predicted. Additional features of the DNA sequences include a 22 base pair region upstream from the proposed structural gene which exhibits striking homology with the trp operator, thus implying that expression of the trpR gene may be under autogenous regulation.     

Role of DNA regions flanking the tryptophan promoter of Escherichia coli. I. Insertion of synthetic oligonucleotides

To examine the effect of altering the nucleotide sequence near the promoter on its activity, pKO-1 vector derivatives have been constructed which allow insertion of DNA fragments at specified sites upstream or downstream from the trp promoter. Oligonucleotides that might be expected to alter the melting properties, or have a tendency to form a distinctive nonstandard structure were introduced. These oligonucleotides had the repeating dinucleotide sequences GC, AT or AG. Sequence analysis of the inserts and studies of the relative galactokinase expression from the altered plasmids indicated that changes upstream from the trp promoter at -39 or beyond had little effect on trp promoter activity, whereas changes at +2 or farther downstream produced up to two-fold increases in gene expression, as compared to the control plasmid.     

Expression of delta-endotoxin gene from Bacillus thuringiensis var. berliner 1715 in Escherichia coli and Bacillus megaterium cells

Effects of the structure of plasmids carrying the cloned delta-endotoxin gene (tox) ot Bacillus thuringiensis and of the culture media on the expression of the gene have been studied. The DNA region located upstream from the crystal protein gene promoter inhibited the expression of the tox gene in Escherichia coli cells, but enhanced the expression in Bacillus megaterium cells grown in LB medium. The upstream DNA region did not affect the tox gene expression when Bacillus megaterium cells were grown in SSM medium.     

New approach to tryptophan production by Escherichia coli: genetic manipulation of composite plasmids in vitro.

For the purpose of studying the production of L-tryptophan by Escherichia coli, the deletion mutants of the trp operon (trpAE1) were transformed with mutant plasmids carrying the trp operon whose anthranilate synthase and phosphoribosyl anthranilate transferase (anthranilate aggregate), respectively, had been desensitized to tryptophan inhibition. In addition to release of the anthranilate aggregate from the feedback inhibition required for plasmids such as pSC101 trp.I15, the properties of trp repression (trpR) and tryptophanase deficiency (tnaA) were both indispensable for host strains such as strain Tna (trpAE1 trpR tnaA). The gene dosage effects on tryptophan synthase activities and on production of tryptophan were assessed. A moderate plasmid copy number, approximately five per chromosome, was optimal for tryptophan production. Similarly, an appropriate release of the anthranilate aggregate from feedback inhibition was also a necessary step to ward off the metabolic anomaly. If the mutant plasmid pSC101 trp-I15 was further mutagenized (pSC101 trp.I15.14) and then transferred to Tna cells, an effective enhancement of tryptophan production was achieved. Although further improvement of the host-plasmid system is needed before commercial production of tryptophan can be realized by this means, a promising step toward this goal has been established.     

Gene cloning, nucleotide sequence, and expression of a cephalosporin-C deacetylase from Bacillus subtilis.

The gene encoding a cephalosporin-C deacetylase (CAH) from Bacillus subtilis SHS 0133 was cloned and sequenced. The nucleotide sequence contained an open reading frame encoding a polypeptide consisting of 318 amino acids, the molecular weight of which was in good agreement with the value obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence contained the common sequence Gly-X-Ser-X-Gly found in many esterases, lipases, and serine proteases. This indicates that CAH is a serine enzyme. A possible promoter sequence which is very similar to the consensus sequences of -35 and -10 regions recognized by B. subtilis RNA polymerase utilizing sigma factor H was found in the 5'-flanking region of the CAH structural gene. Two repeated A+T-rich blocks consisting of 24 bp were also found in the upstream region of the initiation codon. We constructed a series of expression plasmids by inserting the CAH gene into Escherichia coli ATG vectors. The degree of CAH gene expression depended on promoters and vector plasmids, which have different replication origins. The expressed CAH protein was an active form in the soluble fraction obtained after cell disruption. The highest expression level was accomplished with an expression plasmid, pCAH400, which has the trp promoter and the replication origin derived from pAT153. In the fermentation using a 30-liter jar fermentor, the transformant E. coli JM103(pCAH400) produced 440 U of CAH per ml of culture during a 24-h incubation. This value corresponded to 2.1 g of CAH protein in 1 liter of culture broth.     

TnA-directed deletion of the trp operon from RSF2124-trp in Escherichia coli

Plasmid pMT-trp was constructed by digestion of RSF2124-trp with restriction endonuclease PstI and ligation with T4 ligase. In pMT-trp about 78% of the DNA of transposon TnA from RSF2124-trp was deleted, and hence the gene for ampicillin resistance was lost. All Trp- segregants from pMT-trp carriers in Escherichia coli W3110 and its derivatives were found to have lost the entire plasmid. On the other hand, deletion plasmids which had lost the trp operon were found among Trp- segregants from RSF2124-trp carriers, particularly from the mutant strain trpAE1 trpR tnaA. The experimental fact that deletion occurred exclusively in RSF2124-trp suggests that the presence of TnA in the plasmid (RSF2124-trp) was responsible for the deletion.     

trp repressor interactions with the trp aroH and trpR operators. Comparison of repressor binding in vitro and repression in vivo

Interaction of the Escherichia coli trp repressor with the promoter-operator regions of the trp, aroH and trpR operons was studied in vivo and in vitro. The three operators have similar, but non-identical, sequences; each operator is located in a different segment of its respective promoter. In vivo repression of the three operons was measured using single-copy gene fusions to lacZ. The extent of repression varied from 300-fold for the trp operon, to sixfold for the aroH operon and threefold for the trpR operon. To determine whether differential binding of repressor to the three operators was responsible for the differences in repression observed in vivo, three in vitro binding assays were employed. Restriction-site protection, gel retardation and DNase footprinting analyses revealed that repressor binds to the three operators with almost equal affinity. It was also shown in an in vivo competition assay that repressor binds approximately equally well to each of the three operators. It is proposed that the differential regulation observed in vivo may be due to the different relative locations of the three operators within their respective promoters.     

Streptomyces griseus streptomycin phosphotransferase: expression of its gene in Escherichia coli and sequence homology with other antibiotic phosphotransferases and with eukaryotic protein kinases

The aphD gene of Streptomyces griseus, encoding a streptomycin 6-phosphotransferase (SPH), was sub-cloned in the pBR322-based expression vector pRK9 (which contains the Serratia marcescens trp promoter) with selection for expression of streptomycin resistance in Escherichia coli. Two hybrid plasmids, pCKL631 and pCKL711, were isolated which conferred resistance. Both contained a approximately 2 kbp fragment already suspected to include aphD. The properties of in vitro deletion derivatives of these plasmids were consistent with the presumed location of aphD. In vitro deletion of a sequence including most of the trp promoter largely, but not quite completely, abolished the ability of the plasmid to confer streptomycin resistance, confirming that expression was indeed principally from the trp promoter. A polypeptide of approximately 34.5 kDa was present in minicells containing plasmids that conferred streptomycin resistance, but was absent when the plasmids contained in vitro deletions removing streptomycin resistance. Part of the fragment was sequenced and an open reading frame corresponding to aphD identified. A computer-assisted comparison of the deduced SPH sequence with those of other antibiotic phosphotransferases suggested a common structure A-B-C-D-E, where B and D were conserved between all sequences compared while A, C and E divided between the streptomycin and hygromycin B phosphotransferases on one hand and kanamycin/neomycin ones on the other. A composite sequence data base was searched for homologues to consensus matrices constructed from five approximately 12-residue subsequences within blocks B and D. For one subsequence, corresponding to the N-terminal portion of block D, those sequences from the database that yielded the highest homology scores comprised almost entirely either antibiotic phosphotransferases or eukaryotic protein kinases. Possible evolutionary implications of this homology, previously described by other groups, are discussed.     

Repression of 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase (trp) and enzymes of the tryptophan pathway in Escherichia coli K-12

Mutant strains of Escherichia coli K-12 have been isolated in which the synthesis of 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase (trp) is partially constitutive. The mutation causing derepression is closely linked to aroH [the structural gene for DAHP synthetase (trp)] and occurs in a locus designated aroJ. The aroJ mutation is not recessive in an aroJ(+)/aroJ(-) diploid strain, as the synthesis of DAHP synthetase (trp) is still derepressed in this strain. On the basis of its close linkage to aroH and its continued expression in an aroJ(+)/aroJ(-) diploid, it is postulated that aroJ is an operator locus controlling the expression of the structural gene aroH. In support of this conclusion, the synthesis of anthranilate synthetase is still normally repressible in aroJ(-) strains, whereas, in trpR(-) strains, both DAHP synthetase (trp) and anthranilate synthetase are synthesized constitutively. The synthesis of DAHP synthetase (trp) remains repressible in an operator-constitutive mutant of the tryptophan operon. In two trpS mutants which possess defective tryptophanyl transfer ribonucleic acid synthetase enzymes, neither DAHP synthetase (trp) nor anthranilate synthetase derepress under conditions in which the defective synthetase causes a decrease in growth rate. On the other hand, an effect of the trpS mutant alleles on the level of anthranilate synthetase has been observed in strains which are derepressed for the synthesis of this enzyme, because of a mutation in the gene trpR. Possible explanations for this effect are presented.     

A procedure for amino acid sequencing in internal regions of proteins

We describe a novel procedure for determining the amino acid (aa) sequence of the internal regions of proteins. This procedure has been implemented by directly determining the sequence of aa 65-75 of the product of the trpR gene of Escherichia coli, the trp repressor. This method is based on the insertion of the cleavage site of a specific protease (factor Xa) into the protein immediately before the region to be sequenced by Edman degradation. The simplicity of the procedure makes it appealing for studies of protein structure-function relationships, and of the expression of genetic information. The method is particularly useful when there is ambiguity concerning the co-linearity of the aa and nucleotide sequences.