Response of Desulfovibrio vulgaris to alkaline stress.

The response of exponentially growing Desulfovibrio vulgaris Hildenborough to pH 10 stress was studied using oligonucleotide microarrays and a study set of mutants with genes suggested by microarray data to be involved in the alkaline stress response deleted. The data showed that the response of D. vulgaris to increased pH is generally similar to that of Escherichia coli but is apparently controlled by unique regulatory circuits since the alternative sigma factors (sigma S and sigma E) contributing to this stress response in E. coli appear to be absent in D. vulgaris. Genes previously reported to be up-regulated in E. coli were up-regulated in D. vulgaris; these genes included three ATPase genes and a tryptophan synthase gene. Transcription of chaperone and protease genes (encoding ATP-dependent Clp and La proteases and DnaK) was also elevated in D. vulgaris. As in E. coli, genes involved in flagellum synthesis were down-regulated. The transcriptional data also identified regulators, distinct from sigma S and sigma E, that are likely part of a D. vulgaris Hildenborough-specific stress response system. Characterization of a study set of mutants with genes implicated in alkaline stress response deleted confirmed that there was protective involvement of the sodium/proton antiporter NhaC-2, tryptophanase A, and two putative regulators/histidine kinases (DVU0331 and DVU2580).

Regulation of tryptophan synthase gene expression in Chlamydia trachomatis.

We previously reported that Chlamydia trachomatis expresses the genes encoding tryptophan synthase (trpA and trpB). The results presented here indicate that C. trachomatis also expresses the tryptophan repressor gene (trpR). The complement of genes regulated by tryptophan levels in C. trachomatis is limited to trpBA and trpR. trp gene expression was repressed if chlamydiae-infected HeLa cells were cultured the presence of tryptophan and induced if grown in tryptophan-depleted medium or in the presence of IFN-gamma. Furthermore, expression of the trp genes in strains which encode a functional tryptophan synthase is repressed when infected cells are cultured in the presence of the tryptophan precursor indole. Results from experiments with cycloheximide, an inhibitor of eukaryotic protein synthesis, indicate that in addition to the absolute size of the intracellular tryptophan pool, host competition for available tryptophan plays a key role in regulating expression of the trp genes. The tryptophan analogue, 5-fluorotryptophan, repressed trp gene expression and induced the formation of aberrant organisms of C. trachomatis. The growth-inhibitory properties of 5-fluorotryptophan could be reversed with exogenous tryptophan but not indole. In total, our results indicate that the ability to regulate trp gene expression in response to tryptophan availability is advantageous for the intracellular survival of this organism. Furthermore, the fact that C. trachomatis has retained the capacity to respond to tryptophan limitation supports the view that the in vivo antichlamydial effect of IFN-gamma is via the induction of the tryptophan-degrading enzyme, indoleamine 2,3-dioxygenase.

Effective structure of a leader open reading frame for enhancing the expression of GC-rich genes.

To overexpress broad kinds of GC-rich genes in Escherichia coli, we examined how the structures of leader open reading frames (leader ORFs) affect the expression of GC-rich genes, such as polA, trpA, and trpB, from Thermus thermophilus. When a leader ORF overlapped with the polA-initiation codon by 1 bp in the TGATG motif, gene expression increased by more than 3-fold compared to when a leader ORF was several-bp distant from the initiation codon. A 4-bp overlap with the ATGA motif was more effective than a 1-bp overlap with the TGATG motif. When a 4-bp overlapping leader ORF was placed in front of the successive trpB and trpA genes, the trpA gene was poorly expressed whereas the trpB gene was overexpressed. Mutation analysis revealed that the expression of the trpA gene was strongly enhanced by replacing G and C in the translation termination region of the leader ORF with A and T. In contrast, other mutations, such as alterations between synonymous codons in the trpA-coding region, produced diminished gene expression. Using the most effective leader ORF obtained from these results, new expression vectors were constructed.

The Rhizobium etli trpB gene is essential for an effective symbiotic interaction with Phaseolus vulgaris.

A mutant strain (CTNUX4) of Rhizobium etli carrying Tn5 unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a trpB (tryptophan synthase)-homologous gene. When tested on the roots of Phaseolus vulgaris, strain CTNUX4 was able to induce only small, slightly pink, ineffective (Fix-) nodules. However, under free-living conditions, strain CTNUX4 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) unless tryptophan was added to the growth medium. These data and histological observations indicate that the lack of tryptophan biosynthesis affects the symbiotic behavior of R. etli.

Identification of the gene encoding the tryptophan synthase beta-subunit from Chlamydomonas reinhardtii.

We report the isolation of a Chlamydomonas reinhardtii cDNA that encodes the beta-subunit of tryptophan synthase (TSB). This cDNA was cloned by functional complementation of a trp-operon-deleted strain of Escherichia coli. Hybridization analysis indicated that the gene exists in a single copy. The predicted amino acid sequence showed the greatest identity to TSB polypeptides from other photosynthetic organisms. With the goal of identifying mutations in the gene encoding this enzyme, we isolated 11 recessive and 1 dominant single-gene mutation that conferred resistance to 5-fluoroindole. These mutations fell into three complementation groups, MAA2, MAA7, and TAR1. In vitro assays showed that mutations at each of these loci affected TSB activity. Restriction fragment-length polymorphism analysis suggested that MAA7 encodes TSB. MAA2 and TAR1 may act to regulate the activity of MAA7 or its protein product.

5-Fluoroindole resistance identifies tryptophan synthase beta subunit mutants in Arabidopsis thaliana.

A study of the biochemical genetics of the Arabidopsis thaliana tryptophan synthase beta subunit was initiated by characterization of mutants resistant to the inhibitor 5-fluoroindole. Thirteen recessive mutations were recovered that are allelic to trp2-1, a mutation in the more highly expressed of duplicate tryptophan synthase beta subunit genes (TSB1). Ten of these mutations (trp2-2 through trp2-11) cause a tryptophan requirement (auxotrophs), whereas three (trp2-100 through trp2-102) remain tryptophan prototrophs. The mutations cause a variety of changes in tryptophan synthase beta expression. For example, two mutations (trp2-5 and trp2-8) cause dramatically reduced accumulation of TSB mRNA and immunologically detectable protein, whereas trp2-10 is associated with increased mRNA and protein. A correlation exists between the quantity of mutant beta and wild-type alpha subunit levels in the trp2 mutant plants, suggesting that the synthesis of these proteins is coordinated or that the quantity or structure of the beta subunit influences the stability of the alpha protein. The level of immunologically detectable anthranilate synthase alpha subunit protein is increased in the trp2 mutants, suggesting the possibility of regulation of anthranilate synthase levels in response to tryptophan limitation.

Immunological characterization and chloroplast localization of the tryptophan biosynthetic enzymes of the flowering plant Arabidopsis thaliana.

In order to study the tryptophan biosynthetic enzymes of the plant Arabidopsis thaliana, polyclonal antibodies were raised against five of the tryptophan biosynthetic pathway proteins: anthranilate synthase alpha subunit, phosphoribosylanthranilate transferase, phosphoribosylanthranilate isomerase, and the tryptophan synthase alpha and beta subunits. Immunoblot analysis of Arabidopsis leaf protein extracts revealed that the antibodies identify the corresponding proteins that are enriched in Arabidopsis chloroplast fractions. Precursors of phosphoribosylanthranilate isomerase and tryptophan synthase alpha subunit were synthesized by in vitro translation. The precursors were efficiently imported and processed by isolated spinach chloroplasts, and the cleavage sites within the precursors were determined. These results provide the first direct evidence that the tryptophan biosynthetic enzymes from Arabidopsis are synthesized as higher molecular weight precursors and then imported into chloroplasts and processed into their mature forms.

Thermal inactivation of tryptophan synthase. Stabilization by protein-protein interaction and protein-ligand interaction.

This study investigates effects of ligands on thermal inactivation of the tryptophan synthase alpha and beta 2 subunits alone and in the alpha 2 beta 2 complex. Addition of pyridoxal phosphate to the apo-beta 2 subunit increases the temperature of one-half inactivation (Ti) from 52 to 77 degrees C. Ligands that promote association of the alpha and holo-beta 2 subunits markedly stabilize the more temperature-labile alpha subunit in the alpha 2 beta 2 complex from irreversible thermal denaturation. The combination of a beta 2 subunit ligand (L-serine) with an alpha subunit ligand (alpha-glycerol 3-phosphate) raises the inactivation temperature (Ti) of the alpha subunit in the holo-alpha 2 beta 2 complex from 54 to 66 degrees C. In contrast, values of Ti for inactivation of the alpha and beta subunits in the holo-alpha 2 beta 2 complex are more similar to respective values for the isolated alpha subunit (50 degrees C) and holo-beta 2 subunit (77 degrees C). Surprisingly, the addition of L-serine results in a larger decrease in the Ti of the beta 2 subunit in the holo-alpha 2 beta 2 complex (78 degrees C-->64 degrees C) than in Ti of the holo-beta 2 subunit alone (77 degrees C-->71 degrees C). The observation that ligands have different effects on the isolated and associated subunits provides evidence that the alpha and beta 2 subunits do not fully dissociate during thermal inactivation of the alpha 2 beta 2 complex at pH 7.8 and at approximately 0.1 ionic strength. Our results demonstrate that linkage between protein-ligand interactions and protein-protein interactions affects the conformational stability of the tryptophan synthase alpha 2 beta 2 complex.

Expression patterns of duplicate tryptophan synthase beta genes in Arabidopsis thaliana.

Expression of the two Arabidopsis thaliana genes encoding tryptophan synthase beta (TSB1 and TSB2) was investigated by gene-specific RNA blot hybridization and reporter gene analysis. TSB1 mRNA abundance varies in an organ-specific manner, whereas TSB2 mRNA does not. Quantitative analysis of transgenic plants expressing TSB1 and TSB2 translational fusions to the beta-glucuronidase (GUS) gene (gusA) indicates that TSB1-GUS activity is 15-fold higher than TSB2-GUS. Histochemical analysis of these transgenic A. thaliana plants indicates that GUS expression occurs in a developmentally regulated manner. GUS activity driven from the TSB1 promoter is predominantly associated with the stem, root tips, foliar vasculature, mesophyll cells, base of developing seed pods, and tips of anther filaments in plants 15 d and older. Sections through the vegetative stem reveal GUS staining in all cell types including the shoot apical meristem. Although TSB2-GUS expression is consistently detected in root tips and at the base of developing seed pods, it is observed later in plant development than is TSB1-GUS expression.

In vitro gene expression for the localization of antigenic determinants: application to the E. coli tryptophan synthase beta 2 subunit.

Gene expression of the beta subunit of E. coli tryptophan synthase in an E. coli cell-free transcription-translation system proceeds by pauses and produces a discrete but quite continuous pattern of nascent chains starting from the N terminus and ranging in size up to the 44 kDa end product corresponding to the completed beta chains. Using specific immunoadsorption of [35S]Met radiolabelled nascent chains by different monoclonal antibodies directed against the beta 2 subunit of E. coli tryptophan synthase, the size of the smallest N-terminal fragment reacting with each antibody has been determined by SDS electrophoretic analysis of the immunoadsorbed polypeptides. The immunoadsorption assay is performed in solution under conditions avoiding the usual drawbacks of solid phase immunoassay. This approach, in combination with the results obtained with a DNA fragment library permitted us to localize the antigenic determinants recognized by the monoclonal antibodies. The proposed method could help to localize rapidly the C-terminal boundary of an epitope, before starting systematic and precise mapping by other approaches. Moreover, the method described may be of general interest for the rapid production of a large set of C-terminal truncated polypeptides for studies of antigen-antibody recognition.

Development of a prokaryotic expression vector that exploits dicistronic gene organization.

For unknown reasons, levels of expression of foreign genes inserted into expression vectors in Escherichia coli have frequently been undetectable. The most critical step in the successful production of foreign proteins seems to be the initiation of translation. Since most prokaryotic genes are transcribed in a polycistronic form, we have devised a new prokaryotic expression system utilizing dicistronic gene organization. Downstream from a strong promoter and the gene encoding glutathione S-transferase from Schistosoma japonicum, various foreign genes were connected via a ribosome-binding site, a stop codon and a start codon. The VH domain of an immunoglobulin fused to the alpha subunit of tryptophan synthase, FK506-binding protein, cyclophilin, and a domain of a major histocompatibility complex antigen were successfully produced in E. coli as discrete polypeptides by this method.

Overexpression and purification of the separate tryptophan synthase alpha and beta subunits from Salmonella typhimurium.

To obtain high levels of expression of the free alpha and beta subunits of tryptophan synthase from Salmonella typhimurium, we have used two plasmids (pStrpA and pStrpB) that carry the genes encoding the alpha and beta subunits, respectively. The expression of each plasmid in Escherichia coli CB149 results in overproduction of each subunit. We also report new and efficient methods for purifying the individual alpha and beta subunits. Microcrystals of the beta subunit are obtained by addition of polyethylene glycol 8000 and spermine to crude bacterial extracts. This crystallization procedure is similar to methods used previously to grow crystals of the S. typhimurium tryptophan synthase alpha 2 beta 2 complex for X-ray crystallography and to purify this complex by crystallization from bacterial extracts. The results suggest that purification by crystallization may be useful for other overexpressed enzymes and multienzymes complexes. Purification of the alpha subunit utilizes ammonium sulfate fractionation, chromatography on diethylaminoethyl-Sephacel, and high-performance liquid chromatography on a Mono Q column. The purified alpha and beta subunits are more than 95% pure by the criterion of sodium dodecyl sulfate gel electrophoresis. The procedures developed can be applied to the expression and purification of mutant forms of the separate alpha and beta subunits. The purified alpha and beta subunits provide useful materials for studies of subunit association and for investigations of other properties of the separate subunits.

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene in Escherichia coli.

The application of an inducible regulation system using the tryptophanase operon promoter (TPase promoter; Ptna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of Ptna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, L-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-Ptna TS was increased 500-fold compared to that of the cultured host strain. About 1 mol L-tryptophan/l reaction mixture was formed from indole and L-serine at 37 degrees C for 3.5 h.

Effects of nutritional conditions on plasmid stability and production of tryptophan synthase by a recombinant Escherichia coli.

Effects of nutritional conditions and insertion direction of the tryptophan synthase (TSase) gene into a plasmid vector on the plasmid stability and the production of TSase in high cell concentration cultures were examined using recombinant Escherichia coli (E. coli K12 IFO 3301) harboring pBR322trpAB(1) (the TSase gene was inserted at the EcoRI site of pBR322 in the clockwise direction) and pBR322trpAB(2) (counterclockwise direction). As to the effects of the insertion direction, the cells harboring pBR322trpAB(2) were slightly lower in the growth rate and the plasmid stability than those harboring pBR322trpAB(1). However, the former was higher in the productivity of TSase and the final cell concentration attained than the latter. On the other hand, the addition of organic nutrients, especially yeast extract, to TK-25 medium was very effective to improve the plasmid stability. Among the components of yeast extract, L-glutamic acid was found to be effective to improve both the plasmid stability and the production of TSase. When 1 g/l of L-glutamic acid was added to TK-25 medium, a mineral synthetic medium developed for a high concentration culture, 115g (dry basis)/l of recombinant cells were obtained in 14 hr and the expression of TSase was maintained at 240-300 U/mg-protein during the cultivation.

Evolution of the tryptophan synthetase of fungi. Analysis of experimentally fused Escherichia coli tryptophan synthetase alpha and beta chains.

During evolution of fungi, the separate tryptophan synthetase alpha and beta polypeptides of bacteria appear to have been fused in the order alpha-beta rather than the beta-alpha order that would be predicted from the order of the corresponding structural genes in all bacteria. We have fused the tryptophan synthetase polypeptides of Escherichia coli in both orders, alpha-beta and beta-alpha, with and without a short connecting (con) sequence, to explore possible explanations for the domain arrangement in fungi. We find that proteins composed of any of the four fused polypeptides, beta-alpha, beta-con-alpha, alpha-beta, and alpha-con-beta, are highly active enzymatically. However, only the alpha-beta and alpha-con-beta proteins are as active as the wild type enzyme. All four fusion proteins appear to be less soluble in vivo than the wild type enzyme; this abnormal characteristic is minimal for the alpha-con-beta enzyme. The alpha and beta domains of the four fusion polypeptides were not appreciably more heat labile than the wild type polypeptides. Competition experiments with mutant tryptophan synthetase alpha protein, and the fusion proteins suggest that in each fusion protein the joined alpha and beta domains have a functional tunnel connecting their alpha and beta active sites. Three tryptophan synthetase beta'-alpha fusion proteins were examined in which the carboxyl-terminal segment of the wild type beta polypeptide was deleted and replaced by a shorter, unnatural sequence. The resulting deletion fusion proteins were enzymatically inactive and were found predominantly in the cell debris. Evaluation of our findings in relation to the three-dimensional structure of the tryptophan synthetase enzyme complex of Salmonella typhimurium (5) and the results of mutational analyses with E. coli suggest that tryptophan synthetase may have evolved via an alpha-beta rather than a beta-alpha fusion because in beta-alpha fusions the amino-terminal helix of the alpha chain cannot assume the conformation required for optimal enzymatic activity.

[Enhancing effect of chuangxinmycin on synthesis of enzymes in tryptophan synthesis pathway of chuangxinmycin-resistant mutant strain of E. coli].

Chuangxinmycin (CXM) and L-trp cause repression of trp-operon in wild strain of E. coli and indolepropanic acid (IPA) causes derepression of this strain. In CXM-resistant mutant strain, CXM and IPA both cause derepression, and interaction of CXM and IPA is competitive. However, L-trp results in neither repression nor derepression in the mutant, but it can counteract the activity of CXM or IPA. These results suggest that there are repression and derepression sites on repressor of E. coli. CXM can bind to both sites, but mainly to repression site. The structure of repression site of the CXM-resistant mutant is altered, so that CXM can not bind to the repression site and binds mainly to the derepression site, thus leading to the derepression of trp-operon.

Molecular characterization of TRP1, a gene coding for tryptophan synthetase in the basidiomycete Coprinus cinereus.

We utilized a cloned gene (TRP5) encoding tryptophan synthetase (TSase) from Saccharomyces cerevisiae to identify and clone the corresponding gene (TRP1) from the basidiomycete Coprinus cinereus. The primary nucleotide (nt) sequence of this gene was determined and compared to sequences from other filamentous fungi, as well as to other genes coding for TSase. A transformation assay was used to demonstrate that 321 nt, which do not include CAAT or TATAAA elements and precede the translation initiation codon, are sufficient for expression in a variety of chromosomal locations. The coding region (2584 nt) is interrupted at nine positions, and putative splicing signals (5'-GTRNGT...YAG-3') are present in each case. The predicted translation product contains 702 amino acids (aa) and is very similar to other TSases, except in the region of aa 257-296 that connects the alpha and beta functional domains. Both the number and the identity of the aa differ in this region between C. cinereus. S. cerevisiae, and Neurospora crassa. Comparison of exon boundaries in the C. cinereus sequence to the three-dimensional structure of Salmonella typhimurium TSase indicates that there is no simple correlation between exons and major functional domains in this protein.

Solubilization and renaturation of overexpressed aggregates of mutant tryptophan synthase alpha-subunits.

Certain Escherichia coli tryptophan synthase mutant alpha-subunits encoded from mutagenized trpA-containing plasmids were overexpressed as insoluble aggregates which were seen as large, intracellular inclusion bodies. The insoluble aggregates were solubilized to various degrees by several neutral, chaotropic salts. The order of effectiveness of these salts (KSCN, NaI greater than NaNO3, LiBr greater than CaCl2) followed that for the Hofmeister series. Optimum conditions for the use of KSCN resulted in a maximum 70 to 75% solubilization of the aggregate forms for all mutant alpha-subunits examined. Removal of KSCN by dialysis resulted in the recovery of biological activity and of certain characteristic structural properties. Such salts may be a useful alternative for other recombinant protein aggregates which resist complete renaturation by commonly used treatments with guanidine or urea.

Sequence of the Pseudomonas aeruginosa trpI activator gene and relatedness of trpI to other procaryotic regulatory genes.

In Pseudomonas aeruginosa, the trpI gene product regulates the expression of the trpBA gene pair encoding tryptophan synthase. trpI and trpBA are transcribed divergently. The trpI DNA sequence and deduced amino acid sequence were determined. The trpI start codon was found to be 103 base pairs from that of trpB. trpI encodes a 293-residue protein and the size of the trpI gene product, measured on sodium dodecyl sulfatepolyacrylamide gels, was close to that calculated from the amino acid sequence. The amino acid sequence of trpI resembles that of Enterobacter cloacae ampR, the regulatory gene for the ampC cephalosporinase. The N-terminal portions of trpI and ampR resemble corresponding portions of ilvY, metR, and lysR in Escherichia coli and nodD in Rhizobium meliloti. This resemblance may help to define a trpI-related family of activator proteins sharing a common structural plan.