Tryptophan Auxotrophs Research Articles

Screening a wide host‐range, waste‐water metagenomic library in tryptophan auxotrophs of Rhizobium leguminosarum and of Escherichia coli reveals different classes of cloned trp genes

A metagenomic cosmid library was constructed, in which the insert DNA was derived from bacteria in a waste-water treatment plant and the vector was the wide host-range cosmid pLAFR3. The library was screened for clones that could correct defined tryptophan auxotrophs of the alpha-proteobacterium Rhizobium leguminosarum and of Escherichia coli. A total of 26 different cosmids that corrected at least one trp mutant in one or both of these species were obtained. Several cosmids corrected the auxotrophy of one or more R. leguminosarum trp mutants, but not the corresponding mutants in E. coli. Conversely, one cosmid corrected trpA, B, C, D and E mutants of E. coli but none of the trp mutants of R. leguminosarum. Two of the Trp+ cosmids were examined in more detail. One contained a trp operon that resembled that of the pathogen Chlamydophila caviae, containing the unusual kynU gene, which specifies kynureninase. The other, whose trp genes functioned in R. leguminosarum but not in E. coli, contained trpDCFBA in an operon that is likely co-transcribed with five other genes, most of which had no known link with tryptophan synthesis. The sequences of these TRP proteins, and the products of nine other genes encoded by this cosmid, failed to affiliate them with any known bacterial lineage. For one metagenomic cosmid, lac reporter fusions confirmed that its cloned trp genes were transcribed in R. leguminosarum, but not in E. coli. Thus, rhizobia, with their many sigma-factors, may be well-suited hosts for metagenomic libraries, cloned in wide host-range vectors.

Analysis of indole-3-butyric acid-induced adventitious root formation on Arabidopsis stem segments

Root induction by auxins is still not well understood at the molecular level. In this study a system has been devised which distinguishes between the two active auxins indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA). IBA, but not IAA, efficiently induced adventitious rooting in Arabidopsis stem segments at a concentration of 10 microM. In wild-type plants, roots formed exclusively out of calli at the basal end of the segments. Root formation was inhibited by 10 microM 3,4,5-triiodobenzoic acid (TIBA), an inhibitor of polar auxin transport. At intermediate IBA concentrations (3-10 microM), root induction was less efficient in trp1, a tryptophan auxotroph of Arabidopsis with a bushy phenotype but no demonstrable reduction in IAA levels. By contrast, two mutants of Arabidopsis with measurably higher levels of IAA (trp2, amt1) show root induction characteristics very similar to the wild type. Using differential display, transcripts specific to the rooting process were identified by devising a protocol that distinguished between callus production only and callus production followed by root initiation. One fragment was identical to the sequence of a putative regulatory subunit B of protein phosphatase 2A. It is suggested that adventitious rooting in Arabidopsis stem segments is due to an interaction between endogenous IAA and exogenous IBA. In stem explants, residual endogenous IAA is transported to the basal end of each segment, thereby inducing root formation. In stem segments in which the polar auxin transport is inhibited by TIBA, root formation does not occur.

Isolation and Characterization of FecA- and FeoB-Mediated Iron Acquisition Systems of the SpirocheteLeptospira biflexaby Random Insertional Mutagenesis

The specific mechanisms by which Leptospira spp. acquire iron from their ecological niches are unknown. A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. In this study, we have developed a system for random transposon mutagenesis of Leptospira biflexa using a mariner transposon, Himar1. To demonstrate the validity of Himar1 in vivo transposon mutagenesis in L. biflexa, a screen of mutants for clones impaired in amino acid biosynthesis was first performed, enabling the identification of tryptophan and glutamate auxotrophs. To investigate iron transporters, 2,000 L. biflexa transposon mutants were screened onto media with and without hemin, thus allowing the identification of five hemin-requiring mutants, and the putative genes responsible for this phenotype were identified. Three mutants had distinct insertions in a gene encoding a protein which shares homology with the TonB-dependent receptor FecA, involved in ferric citrate transport. We also identified two mutants with a Himar1 insertion into a feoB-like gene, the product of which is required for ferrous iron uptake in many bacterial organisms. Interestingly, the growth inhibition exhibited by the fecA and feoB mutants was relieved by deferoxamine, suggesting the presence of a ferric hydroxamate transporter. These results confirm the importance of iron for the growth of Leptospira and its ability to use multiple iron sources.

Starvation survival response of Mycobacterium tuberculosis.

The ability of Mycobacterium tuberculosis auxotrophs to survive long-term starvation was measured. Tryptophan and histidine auxotrophs did not survive single-amino-acid starvation, whereas a proline auxotroph did. All three auxotrophs survived complete starvation. THP-1 cells were also able to restrict the growth of the tryptophan and histidine auxotrophs.

Construction and complementation of the first auxotrophic mutant in the spirochaete Leptospira meyeri.

In bacteria, the first reaction of the tryptophan biosynthetic pathway involves the conversion of chorismate and glutamine to anthranilate by the action of anthranilate synthase, which is composed of the alpha (trpE gene product) and beta (trpG gene product) subunits. In this study, the tryptophan biosynthetic gene trpE of the spirochaete Leptospira meyeri was interrupted by a kanamycin-resistance cassette by homologous recombination. The trpE double cross-over mutant was not able to grow on solid or in liquid EMJH medium. In contrast, the trpE mutant showed a wild-type phenotype when tryptophan or anthranilate was added to the media, therefore showing that disruption of the L. meyeri trpE gene resulted in tryptophan auxotrophy. The authors have also characterized a second selectable marker that allows the construction of a spectinomycin-resistant L. meyeri-E. coli shuttle vector and the functional complementation of the L. meyeri trpE mutant.

ISOLATION OF ALGAL GENES BY FUNCTIONAL COMPLEMENTATION OF YEAST1

Algal cDNAs were isolated and characterized by functional complementation of yeast auxotrophs. Two cDNA libraries, one derived from the diatom Phaeodactylum tricornutum Bohlin and the other from the dinoflagellate Crypthecodinium cohnii Biecheler, were constructed using the Saccharomyces cerevisiae expression vector pFL-61. These libraries were used for functional complementation of auxotrophic markers in two yeast strains. Yeast tryptophan auxotrophs, complemented by the P. tricornutum library, contained a plasmid that encoded a two-domain protein associated with tryptophan synthesis, indole-3-glycerol phosphate synthase-N-(5′-phosphoribosyl) anthranilate isomerase. Another cDNA originating from the C. cohnii library rescued S. cervisiae from a defect in adenine biosynthesis. This cDNA encoded a fusion of phosphoribosylamidoimidazole-succinocarboxamide synthetase and phosphoribosylaminoimidazole carboxylase, which correspond to the yeast ade1 and ade2 genes, respectively. These results demonstrate that heterologous functional complementation can be used to identify algal genes and may provide advantages over other gene discovery methods.

Immunosuppressant-like effects of phenylbutyrate on growth inhibition of Saccharomyces cerevisiae.

Phenylbutyrate (4-phenylbutyric acid; PB) and its metabolite, phenylacetate, are effective anti-neoplastic agents in tissue culture and have shown promise in clinical trials for a variety of neoplasms. PB is a drug of remarkably low toxicity that acts in vitro as a differentiating agent, causing reversion of the transformed phenotype by an unknown mechanism. We attempted to identify the cellular target(s) for PB using Saccharomyces as a model. PB inhibits growth of yeast on rich medium at concentrations of 0.1-1.0 mM, concentrations similar to plasma concentrations observed in human trials. Yeast cells treated with 1 mM PB remain over 90% viable for 24 h. PB inhibits tryptophan uptake, and resistance to PB can be conferred by tryptophan prototrophy, by supplementing tryptophan auxotrophs with the high levels of tryptophan, by overexpression of the aromatic amino acid permeases Tat1p or Tat2p, and by disruption of TAT1. Since tryptophan auxotrophy and transport influences resistance to PB, phytosphingosine, and the immunosuppressant FK506, these drugs might affect the same pathway. We isolated and characterized a mutant resistant to 1 mM PB and identified the mutant as bul1. A chromosomal BUL1 deletion displayed all phenotypes shown by the PB-resistant mutant.

High-level ethanol production from starch by a flocculent Saccharomyces cerevisiae strain displaying cell-surface glucoamylase.

A Strain of host yeast YF207, which is a tryptophan auxotroph and shows strong flocculation ability, was obtained from SaccharomYces diastaticus ATCC60712 and S. cerevisiae W303-1B by tetrad analysis. The plasmid pGA11, which is a multicopy plasmid for cell-surface expression of the Rhyzopus oryzae glucoamylase/alpha-agglutinin fusion protein, was then introduced into this flocculent yeast strain (YF207/pGA11). Yeast YF207/pGA11 grew rapidly under aerobic condition (dissolved oxygen 2.0 ppm), using soluble starch. The harvested cells were used for batch fermentation of soluble starch to ethanol under anaerobic condition and showed high ethanol production rates (0.71 g h(-1) l(-1)) without a time lag, because glucoamylase was immobilized on the yeast cell surface. During repeated utilization of cells for fermentation, YF207/pGA11 maintained high ethanol production rates over 300 h. Moreover, in fed-batch fermentation with YF207/pGA11 for approximately 120 h, the ethanol concentration reached up to 50 g l(-1). In conclusion, flocculent yeast cells displaying cell-surface glucoamylase are considered to be very effective for the direct fermentation of soluble starch to ethanol.

Spectroscopic and kinetic characterization of the bifunctional chorismate synthase from Neurospora crassa: evidence for a common binding site for 5-enolpyruvylshikimate 3-phosphate and NADPH.

Chorismate synthase catalyzes the anti-1,4-elimination of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate, a central building block in aromatic amino acid biosynthesis. The enzyme has an absolute requirement for reduced FMN, which in the case of the fungal chorismate synthases is supplied by an intrinsic FMN:NADPH oxidoreductase activity, i.e. these enzymes have an additional catalytic activity. Therefore, these fungal enzymes have been termed "bifunctional." We have cloned chorismate synthase from the common bread mold Neurospora crassa, expressed it heterologously in Escherichia coli, and purified it in a three-step purification procedure to homogeneity. Recombinant N. crassa chorismate synthase has a diaphorase activity, i.e. it catalyzes the reduction of oxidized FMN at the expense of NADPH. Using NADPH as a reductant, a reduced flavin intermediate was observed under single and multiple turnover conditions with spectral features similar to those reported for monofunctional chorismate synthases, thus demonstrating that the intermediate is common to the chorismate synthase-catalyzed reaction. Furthermore, multiple turnover experiments in the presence of oxygen have provided evidence that NADPH binds in or near the substrate (5-enolpyruvylshikimate 3-phosphate) binding site, suggesting that NADPH binding to bifunctional chorismate synthases is embedded in the general protein structure and a special NADPH binding domain is not required to generate the intrinsic oxidoreductase activity.

Indolic constituents and indole-3-acetic acid biosynthesis in the wild-type and a tryptophan auxotroph mutant of Arabidopsis thaliana.

The tryptophan auxotroph mutant trp3-1 of Arabidopsis thaliana (L.) Heynh., despite having reduced levels of L-tryptophan, accumulates the tryptophan-derived glucosinolate, glucobrassicin and, thus, does not appear to be tryptophan-limited. However, due to the block in tryptophan synthase, the mutant hyperaccumulates the precursor indole-3-glycerophosphate (up to 10 mg per g FW). Instability of indole-3-glycerophosphate leads to release of indole-3-acetic acid (IAA) from this metabolite during standard workup of samples for determination of conjugated IAA. The apparent increase in "conjugated IAA" in trp3-1 mutant plants can be traced back entirely to indole-3-glycerophosphate degradation. Thus, the levels of neither free IAA nor conjugated IAA increase detectably in the trp3-1 mutant compared to wild-type plants. Precursor-feeding experiments to shoots of sterile-grown wild-type plants using [2H]5-L-tryptophan have shown incorporation of label from this precursor into indole-3-acetonitrile and indole-3-acetic acid with very little isotope dilution. It is concluded that Arabidopsis thaliana shoots synthesize IAA from L-tryptophan and that the non-tryptophan pathway is probably an artifact.

Physiological evidence for differently regulated tryptophan-dependent pathways for indole-3-acetic acid synthesis in Azospirillum brasilense.

Disruption of ipdC, a gene involved in indole-3-acetic acid (IAA) production by the indole pyruvate pathway in Azospirillum brasilense Sp7, resulted in a mutant strain that was not impaired in IAA production with lactate or pyruvate as the carbon source. A tryptophan auxotroph that is unable to convert indole to tryptophan produced IAA if tryptophan was present but did not synthesise IAA from indole. Similar results were obtained for a mutant strain with additional mutations in the genes ipdC and trpD. This suggests the existence of an alternative Trp-dependent route for IAA synthesis. On gluconate as a carbon source, IAA production by the ipdC mutant was inhibited, suggesting that the alternative route is regulated by catabolite repression. Using permeabilised cells we observed the enzymatic conversion of tryptamine and indole-3-acetonitrile to IAA, both in the wild-type and in the ipdC mutant. IAA production from tryptamine was strongly decreased when gluconate was the carbon source.

The tryptophan synthase-encoding trpB gene of Aspergillus nidulans is regulated by the cross-pathway control system.

The tryptophan synthase-encoding gene, trpB, of Aspergillus nidulans was cloned and characterized. It was mapped to chromosome I, between the gene medA, which is required for sexual and asexual development, and an ORF encoding a protein with significant similarity to subunit B of vacuolar ATP synthases. The 5' untranslated region was found to be at least 142 nucleotides (nt) long, the poly(A) addition site was localized at position + 216 relative to the stop codon by sequencing of several independent cDNA clones. The trpB gene contains two exons separated by an intron of 105 nt, which is located close to the 5' end of the ORF. Directly upstream of the transcriptional start site, one well conserved potential binding site for the cross-pathway control transcriptional activator CPCA was found. The level of trpB transcript was shown to be regulated by cross-pathway control. A knockout mutant for trpB displays tryptophan auxotrophy, no trpB transcript is detectable, and development is perturbed to an extent that is dependent on the amount of tryptophan added to the medium. The trpB gene encodes a protein of 723 amino acids, with a calculated molecular weight of 77.6 kDa. The deduced amino acid sequence shows 72.6% similarity to the tryptophan synthase of Neurospora crassa. Most amino acid residues essential for catalytic activity in the tryptophan synthase of Salmonella typhimurium are conserved. The linker region joining the two domains of the enzyme is 13 residues longer than the longest connector found so far in tryptophan synthases from fungi.

A counterselection for the tryptophan pathway in yeast: 5-fluoroanthranilic acid resistance.

The ability to counterselect, as well as to select for, a genetic marker has numerous applications in microbial genetics. Described here is the use of 5-fluoroanthranilic acid for the counterselection of TRP1, a commonly used genetic marker in the yeast Saccharomyces cerevisiae. Counterselection using 5-fluoroanthranilic acid involves antimetabolism by the enzymes of the tryptophan biosynthetic pathway, such that trp1, trp3, trp4 or trp5 strains, which lack enzymes required for the conversion of anthranilic acid to tryptophan, are resistant to 5-fluoroanthranilic acid. Commonly used genetic procedures, such as selection for loss of a chromosomally integrated plasmid, and a replica-plating method to rapidly assess genetic linkage in self-replicating shuttle vectors, can now be carried out using the TRP1 marker gene. In addition, novel tryptophan auxotrophs can be selected using 5-fluoroanthranilic acid.

Membrane protein-ligand interactions in Escherichia coli vesicles and living cells monitored via a biosynthetically incorporated tryptophan analogue.

This paper presents a deceptively straightforward experimental approach to monitoring membrane protein-ligand interactions in vesicles and in living Escherichia coli cells. This is achieved via the biosynthetic incorporation of 7-azatryptophan, a tryptophan analogue with a red-shifted absorption spectrum, allowing collection of the emission signal of the target protein in a high tryptophan background via red-edge excitation. The approach is demonstrated for the mannitol permease of E. coli (EII(mtl)), an integral membrane protein of 637 amino acids, including four tryptophans, and single-tryptophan mutants of EII(mtl). By using a tryptophan auxotroph, a high level of 7-azatryptophan incorporation in EII(mtl) was achieved. The change in emission signal of the purified enzyme upon mannitol binding (-28%) was 4-fold larger than with EII(mtl) containing tryptophan, demonstrating the known higher sensitivity of this analogue for changes in the microenvironment [Schlesinger, R. (1968) J. Biol. Chem. 243, 3877-3883]. Changes in emission signal could also be monitored (-5%) when the enzyme was situated in vesicles, although it constituted only 10-15% of the total cytoplasmic membrane fraction. Of the five single-tryptophan mutants, the emission signal of the mutant with 7-azatryptophan at position 198 was the most sensitive for mannitol binding. Changes in emission signal not only were observed in vesicles (-18%) but also could be monitored in viable cells (-5%). The fact that only modest expression levels and no protein purification are needed makes this a useful approach for the characterization of numerous protein systems under in vitro and in vivo conditions.

The expression of the trpD, trpC and trpBA genes of Streptomyces coelicolor A3(2) is regulated by growth rate and growth phase but not by feedback repression.

Transformation of tryptophan auxotrophs of Streptomyces coelicolor A3(2) and subsequent analysis have allowed the identification of four tryptophan biosynthetic genes. Subcloning, complementation of trp strains, nucleotide sequencing of 5.1 kb and 1.95 kb of DNA and subsequent homology comparisons identified the trpC, trpB and trpA genes and trpD gene respectively. The arrangement of genes in the trpCBA cluster is unusual in that trpC is separated by a small open reading frame, trpX, from the potentially translationally coupled trpB and trpA genes. Sequence analysis of the trpD gene revealed the presence of a large mRNA loop structure directly upstream of the trpD-coding region. S1 nuclease mapping studies of trpCXBA have revealed two major potential transcription start points, one just upstream of the trpC gene and the other located upstream of the trpX gene. S1 nuclease mapping of the trpD region revealed four fragment end-points. Quantitative S1 nuclease protection assays and a promoterless catechol dioxygenase reporter gene have revealed that the expression of all these genes is growth phase dependent and growth rate dependent, expression being maximal during early exponential phase and dropping off sharply in late exponential phase. This growth phase-dependent and growth rate-dependent regulation is the first reported in streptomycete primary metabolism.

Biosynthesis and characterization of 4-fluorotryptophan-labeled Escherichia coli arginyl-tRNA synthetase.

Escherichia coli 4-fluorotryptophan-substituted arginyl-tRNA synthetase was biosynthetically prepared and purified from a tryptophan auxotroph which could overproduce this enzyme. A method was developed to separate 4-fluorotryptophan from tryptophan and to determine accurately their contents in the 4-fluorotryptophan-containing proteins. It was confirmed that more than 95% of the tryptophan residues in the purified 4-fluorotryptophan-substituted arginyl-tRNA synthetase were replaced by 4-fluorotryptophan. Studies on the effect of the 4-fluorotryptophan replacement on properties of the enzyme showed that, when compared with the native enzyme, both the specific activity and the first-order rate constant of the fluorinated enzyme decreased by approximately 20% with just slightly higher Km values. CD studies, however, did not reveal any difference between the secondary structure of the native and fluorinated enzymes. In addition, thermal unfolding studies showed that the 4-fluorotryptophan replacement did not significantly affect the thermal stability of the enzyme. We may conclude that the substitution of 4-fluorotryptophan in arginyl-tRNA synthetase had no substantial effect on the structure and function of the enzyme. Finally, a preliminary study of 19F nuclear magnetic resonance spectroscopy of the fluorinated enzyme has shown promising prospect for further investigation of its structure and function with NMR.

The highly conserved, coregulated SNO and SNZ gene families in Saccharomyces cerevisiae respond to nutrient limitation.

SNZ1, a member of a highly conserved gene family, was first identified through studies of proteins synthesized in stationary-phase yeast cells. There are three SNZ genes in Saccharomyces cerevisiae, each of which has another highly conserved gene, named SNO (SNZ proximal open reading frame), upstream. The DNA sequences and relative positions of SNZ and SNO genes have been phylogenetically conserved. This report details studies of the expression of the SNZ-SNO gene pairs under various conditions and phenotypic analysis of snz-sno mutants. An analysis of total RNA was used to determine that adjacent SNZ-SNO gene pairs are coregulated. SNZ2/3 and SNO2/3 mRNAs are induced prior to the diauxic shift and decrease in abundance during the postdiauxic phase, when SNZ1 and SNO1 are induced. In snz2 snz3 mutants, SNZ1 mRNA is induced prior to the diauxic shift, when SNZ2/3 mRNAs are normally induced. Under nitrogen-limiting conditions, SNZ1 mRNAs accumulate in tryptophan, adenine, and uracil auxotrophs but not in prototrophic strains, indicating that induction occurs in response to the limitation of specific nutrients. Strains carrying deletions in all SNZ-SNO gene pairs are viable, but snz1 and sno1 mutants are sensitive to 6-azauracil (6-AU), an inhibitor of purine and pyrimidine biosynthetic enzymes, and methylene blue, a producer of singlet oxygen. The conservation of sequence and chromosomal position, the coregulation and pattern of expression of SNZ1 and SNO1 genes, and the sensitivity of snz1 and sno1 mutants to 6-AU support the hypothesis that the associated proteins are part of an ancient response to nutrient limitation.

Inhibition of Amino Acid Transport by Sphingoid Long Chain Bases in Saccharomyces cerevisiae

Sphingoid long chain bases have many effects on cells including inhibition or stimulation of growth. The physiological significance of these effects is unknown in most cases. To begin to understand how these compounds inhibit growth, we have studied Saccharomyces cerevisiae cells. Growth of tryptophan (Trp-) auxotrophs was more strongly inhibited by phytosphingosine (PHS) than was growth of Trp+ strains, suggesting that PHS diminishes tryptophan uptake and starves cells for this amino acid. This hypothesis is supported by data showing that growth inhibition is relieved by increasing concentrations of tryptophan in the culture medium and by multiple copies of the TAT2 gene, encoding a high affinity tryptophan transporter. Measurement of tryptophan uptake shows that it is inhibited by PHS. Finally, PHS treatment induces the general control response, indicating starvation for amino acids. Multiple copies of TAT2 do not protect cells against two other cationic lipids, stearylamine, and sphingosine, indicating that the effect of PHS on tryptophan utilization is specific. Other data demonstrate that PHS reduces uptake of leucine, histidine, and proline by specific transporters. Our data suggest that PHS targets proteins in the amino acid transporter family but not other distantly related membrane transporters, including those necessary for uptake of adenine and uracil.

The cis-diol dehydrogenase cbaC gene of Tn 5271 is required for growth on 3-chlorobenzoate but not 3,4-dichlorobenzoate

The nucleotide sequence of cbaC, the 1-carboxy-3-chloro-4,5-dihydroxycyclohexa-2,6-diene ( cis-diol) dehydrogenase gene from the 3-chlorobenzoate (3-Cba) catabolic transposon Tn 5271 was determined. The functional significance of the deduced open reading frame was evaluated by deletion of an internal BstEII restriction site in cbaC and by the creation of nested deletions using exonuclease III. Expression studies were carried out with Alcaligenes sp. strain BR6024, a chloramphenicol-resistant, tryptophan auxotroph derived from the wild-type isolate BR60. BR6024 hosts carrying complete cbaAB (3-Cba 3,4-(4,5)-dioxygenase and reductase) genes, with deletions of cbaC, metabolized 3Cba to the cis-4,5-diol metabolite. These mutants failed to grow on 3-Cba, however they grew on 3,4-dichlorobenzoate, accumulating 5-chloroprotocatechuate transiently. These results indicated the cbaC dehydrogenase was not required for re-aromatization of the unstable 3,4-dCba cis-4,5-diol metabolite. Spontaneous elimination of HCl from this metabolite is proposed to generate 5-chloroprotocatechuate, which is a substrate for the protocatechuate meta-ring fission pathway in Alcaligenes sp. BR60. The relationship of the deduced amino acid sequence of cbaC with 15 other oxidoreductases and sequences of unknown function from bacteria, plants and animals revealed a conserved N-terminal GxxGxG dinucleotide-binding domain and a conserved region with a H(x 11)KHVLxEKPxA consensus flanked by α-helical domains. o-Phthalate cis-diol dehydrogenase ( Pseudomonas putida), glucose-fructose oxidoreductase ( Zymomonas mobilis), myo-inositol 2-dehydrogenase ( Bacillus subtilis) and d-galactose 1-dehydrogenase ( Pseudomonas fluorescens) are related proteins. These dehydrogenases are unrelated to the Type I, II and III dehydrogenase superfamilies that include the cis-diol dehydrogenases involved in benzoate, toluene, biphenyl and naphthalene catabolism (Type II) and benzene catabolism (Type III).

An allelic series of blue fluorescent trp1 mutants of Arabidopsis thaliana.

Nine blue fluorescent mutants of the flowering plant Arabidopsis thaliana were isolated by genetic selections and fluorescence screens. Each was shown to contain a recessive allele of trp1, a previously described locus that encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase (PAT, called trpD in bacteria). The trp1 mutants consist of two groups, tryptophan auxotrophs and prototrophs, that differ significantly in growth rate, morphology, and fertility. The trp1 alleles cause plants to accumulate varying amounts of blue fluorescent anthranilate compounds, and only the two least severely affected of the prototrophs have any detectable PAT enzyme activity. All four of the trp1 mutations that were sequenced are G to A or C to T transitions that cause an amino acid change, but in only three of these is the affected residue phylogenetically conserved. There is an unusually high degree of sequence divergence in the single-copy gene encoding PAT from the wild-type Columbia and Landsberg erecta ecotypes of Arabidopsis.