Cloning of the trp gene cluster from a tryptophan-hyperproducing strain of Corynebacterium glutamicum: identification of a mutation in the trp leader sequence.

Corynebacterium glutamicum ATCC 21850 produces up to 5 g of extracellular L-tryptophan per liter in broth culture and displays resistance to several synthetic analogs of aromatic amino acids. Here we report the cloning of the tryptophan biosynthesis (trp) gene cluster of this strain on a 14.5-kb BamHI fragment. Subcloning and complementation of Escherichia coli trp auxotrophs revealed that as in Brevibacterium lactofermentum, the C. glutamicum trp genes are clustered in an operon in the order trpE, trpD, trpC, trpB, trpA. The cloned fragment also confers increased resistance to the analogs 5-methyltryptophan and 6-fluorotryptophan on E. coli. The sequence of the ATCC 21850 trpE gene revealed no significant changes when compared to the trpE sequence of a wild-type strain reported previously. However, analysis of the promoter-regulatory region revealed a nonsense (TGG-to-TGA) mutation in the third of three tandem Trp codons present within a trp leader gene. Polymerase chain reaction amplification and sequencing of the corresponding region confirmed the absence of this mutation in the wild-type strain. RNA secondary-structure predictions and sequence similarities to the E. coli trp attenuator suggest that this mutation results in a constitutive antitermination response.     

Tryptophan-transducing bacteriophages: in vitro studies with restriction endonucleases HindII + III and Escherichia coli ribonucleic acid polymerase.

The HindII + III restriction enzyme fragmentation pattern of various lambda-phi80trp deoxyribonucleic acid molecules is presented. An analysis of deoxyribonucleic acid molecules carrying deletions ending within the trp regulatory elements and a deoxyribonucleic acid molecule carrying a deletion within trpE indicates that a fragment of 8.3 X 10(5) daltons contains at least part of the trp promoter, the entire trp leader region, and part of the trpE gene. The observation that ribonucleic acid polymerase, when present in the HindII + III digestion mixture, results in the fusion of this 8.3 X 10(5)-dalton fragment to the preceding bacterial fragment suggests that HindII + III cuts within trpP.     

Sequence changes preceding a Shine-Dalgarno region influence trpE mRNA translation and decay

In studies with a trpE promoter-strength measuring system we observed that constructs containing the Escherichia coli trp promoter and its adjacent transcribed region yielded lower levels of trpE protein than were expected. To analyze this observation we introduced mutational changes in the nucleotide sequence preceding the trpE Shine-Dalgarno region and examined their effects on trpE mRNA synthesis, translation and decay. We found that certain deletion, insertion and substitution mutations in the pre-Shine-Dalgarno region caused a two- to fivefold increase in trpE enzyme activity. These increases were accompanied by increases in steady-state levels of trpE mRNA. Pulse-chase analyses of trpE mRNA degradation revealed that the observed steady-state trpE mRNA levels correlated with changes in trpE mRNA stability. These findings are interpreted in terms of alternative models in which the primary effect of mutational changes that elevate trpE expression is to increase trpE mRNA translation, versus increasing trpE mRNA stability.     

Evolutionary divergence of the Citrobacter freundii tryptophan operon regulatory region: comparison with other enteric bacteria

The regulatory region of the trp operon of Citrobacter freundii was sequenced and compared with the corresponding regions of other enteric bacteria. Significant differences were noted in the promoter region. These differences are presumably responsible for the weak expression of the cloned trp operon in Escherichia coli. The presumed operator region, although nonfunctional in E. coli, has dyad symmetry, but the sequence of the symmetrical region differs appreciably from those of operators that can be regulated by the E. coli trp repressor. The sequence of the trp leader region of C. freundii resembles that of other enteric bacteria, suggesting that the C. freundii operon is also regulated by attenuation. Comparison of the sequence of the initial portion of trpE with the homologous regions of E. coli and Salmonella typhimurium indicates that the three organisms probably are evolutionary equidistant.     

Tryptophan gene cluster of Methanobacterium thermoautotrophicum Marburg: molecular cloning and nucleotide sequence of a putative trpEGCFBAD operon.

A recombinant cosmid carrying the Methanobacterium thermoautotrophicum Marburg trp genes was selected by complementation of Escherichia coli trp mutations. A 7.3-kb fragment of the cloned archaeal DNA was sequenced. It contained the seven trp genes, arranged adjacent to each other in the order trpEGCFBAD. No gene fusions were observed. The trp genes were organized in an operonlike structure, with four short (5- to 56-bp) intergenic regions and two overlapping genes. There was no indication for an open reading frame encoding a leader peptide in the upstream region of trpE. The gene order observed in the M. thermoautotrophicum trp operon was different from all known arrangements of the trp genes in archaea, bacteria, and eucarya. The encoded sequences of the Methanobacterium Trp proteins were similar in size to their bacterial and eucaryal counterparts, and all of them contained the segments of highly similar or invariant amino acid residues recognized in the Trp enzymes from bacteria and eucarya. The TrpE, TrpG, TrpC, TrpA, and TrpD proteins were 30 to 50% identical to those from representatives of other species. Significantly less sequence conservation (18 to 30%) was observed for TrpF, and TrpB exhibited a high degree of identity (50 to 62%) to the sequences of representatives of the three domains. With the exception of TrpB, the beta subunit of tryptophan synthase, tryptophan was absent from all Trp polypeptides.     

Complementation of a trpE deletion in Escherichia coli by Spirochaeta aurantia DNA encoding anthranilate synthetase component I activity.

A 2.7-kilobase Sau3A fragment of Spirochaeta aurantia DNA cloned in pBR322 complemented a trpE deletion in Escherichia coli. Deletion analysis and Tn5 mutagenesis of the resulting plasmid pBG100 defined a 2-kilobase-pair region that was required for both the complementation and the synthesis of 59,000- and 47,000-molecular-weight polypeptides (59K and 47K polypeptides) in maxicells. Both the 59K and the 47K polypeptides appear to be encoded by a single gene. A maxicell analysis of pBG100::Tn5 mutants suggests that the 47K polypeptide is not sufficient for the trpE complementation. In vitro and in vivo anthranilate synthetase (AS) assays indicate that the complementing activity encoded by pBG100 was functionally analogous to the AS component I of E. coli in that it utilized NH3 but not glutamine as the amino donor. pBG100 did not encode a glutamine amidotransferase activity, although the AS component I it encoded was capable of interacting with E. coli AS component II to catalyze the glutamine-requiring reaction. Expression appeared to depend on a promoter in the cloned S. aurantia DNA.     

Nucleotide sequence of the Bacillus subtilis trpE and trpD genes

Several overlapping portions of the tryptophan (trp) operon of Bacillus subtilis have been cloned into plasmid pBR322. The nucleotide sequence of the region comprising the trpE and trpD genes and a portion of the trpC gene has been determined. When the deduced amino acid sequences of these genes are compared with their counterparts in Escherichia coli, several regions of striking homology are seen. The probable initiation codons for the trpE, D and C genes are each preceded by a recognizable Shine-Dalgarno sequence. The coding sequences for the trpE and trpD genes and for the trpD and trpC genes overlap slightly, leaving no intercistronic regions between the genes.     

(Beta alpha)8-barrel proteins of tryptophan biosynthesis in the hyperthermophile Thermotoga maritima.

To better understand the evolution of a key metabolic pathway, we have sequenced the trpCFBA gene cluster of the hyperthermophilic bacterium Thermotoga maritima. The genes were cloned by complementation in vivo of trp deletion strains of Escherichia coli. The new sequences, together with earlier findings, establish that the trp operon of T.maritima has the order trpE(G.D)CFBA, which might represent the ancestral organization of the tryptophan operon. Heterologous expression of the trp(G.D) and trpC genes in E.coli and N-terminal sequencing of their polypeptide products showed that their translation is initiated at the rate start codons TTG and ATC, respectively. Consequently, the N-terminus of the trp(G.D) fusion protein is 43 residues shorter than previously postulated. Amino acid composition and sequence analyses of the protein products of T.maritima trpC (indoleglycerol phosphate synthase), trpF (phosphoribosyl anthranilate isomerase) and trpA (alpha-subunit of tryptophan synthase) suggest that these thermostable (beta alpha)8-barrel proteins may be stabilized by additional salt bridges, compared with the mesostable forms. Another notable feature is the predicted lack of the N-terminal helix alpha 0 in the alpha-subunit of tryptophan synthase.     

Nucleotide sequence and analysis of a gene encoding anthranilate synthase component I in Spirochaeta aurantia.

A Spirochaeta aurantia DNA fragment containing the trpE gene and flanking chromosomal DNA was cloned, and the sequence of the trpE structural gene plus 870 bp upstream and 1,257 bp downstream of trpE was determined. The S. aurantia trpE gene codes for a polypeptide of 482 amino acid residues with a predicted molecular weight of 53,629 that showed sequence similarity to TrpE proteins from other organisms. The S. aurantia TrpE polypeptide is not more closely related to the other published spirochete TrpE sequence (that of Leptospira biflexa) than to TrpE polypeptides of other bacteria. Two additional complete open reading frames and one partial open reading frame were identified in the sequenced DNA. One of the complete open reading frames and the partial open reading frame are upstream of trpE and are encoded on the DNA strand opposite that containing trpE. The other open reading frame is downstream of trpE and on the same DNA strand as trpE. On the basis of the results of a protein sequence data base search, it appears that trpE is the only tryptophan biosynthesis gene in the sequenced DNA. This is in contrast to L. biflexa, in which trpE is separated from trpG by only 64 bp.     

A new series of trpE vectors that enable high expression of nonfusion proteins in bacteria.

Expression of recombinant proteins in bacteria has facilitated the characterization of many gene products. However, the biochemical characterization of recombinant proteins is limited since the bacterially expressed proteins are often synthesized as fusion polypeptides. The presence of bacterial sequences in fusion proteins further limits the use of these proteins for generating antibodies since the bacterial sequences are also antigenic. We describe two new bacterial expression vectors based on the pATH series of plasmids. These vectors were made by precisely deleting all of the trpE coding sequences found in pATH. The new vectors have enabled us to express eukaryotic genes as nonfusion polypeptides. These altered plasmids can be used to insert any DNA sequence of interest through a multiple cloning site located just 3' of an ATG start codon. Protein expression is still under the control of the trp operon and is carried out at great efficiency when the bacteria are tryptophan deprived. Studies presented here test the expression system with neurofilament subunits, NF-L and NF-H. Large amounts of recombinant nonfusion proteins were produced. Also, a time course of induction shows that the production of the nonfusion proteins was under the control of the trp operon which is readily inducible after tryptophan starvation and addition of indoleacrylic acid. These vectors may be useful for the overexpression of many proteins in a form closely approximating their native state.     

Construction of new cloning vehicles with genes of the tryptophan operon of Escherichia coli as genetic markers

In vitro recombination techniques were used to construct a series of new cloning vehicles with genes of the tryptophan (trp) operon of Escherichia coli as selective marker. To construct these plasmids we have made a restriction cleavage map of the trp operon for the enzymes AosI, AvaI, BglI, BglII, HindIII, HpaI, PvuII, SalI, SstI and XhoI. The constructed plasmids pHP39, pEP392, pEP3921 and pEP3923 are derived from the amplifiable plasmid pBR345 and carry two or more genes of the trp operon, which are controlled by the trp regulatory elements. Plasmid pEP3921 (7.0 kb) carries intact trpE and trpA genes and contains single BglII and SstI sites in trpE, a single HindIII site located between trpE and trpA, and single EcoRI, SalI and XhoI sites located outside the trp genes. Plasmid pEP121 (12 kb) is similar to pEP3921, but has an extra selective marker conferring bacterial resistance to ampicillin. Plasmid pEP3923 (7.4 kb) comprises intact trpB and trpA genes and single BglII, HindIII, EcoRI, SalI and XhoI sites. Plasmids pHP39 (9.8 kb) and pEP392 (9.8 kb) carry an intact trp operon and have two and one EcoRI site, respectively. Plasmid pHP3 (18 kb) carries an intact trp operon and markers for tetracycline and ampicillin resistance.