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In Pseudomonas aeruginosa, the operon encoding tryptophan synthase (trpBA) is positively regulated by the TrpI protein and an intermediate in tryptophan biosynthesis, indoleglycerol phosphate (InGP). A gene fusion in which the trpBA promoter directs expression of the Pseudomonas putida xylE gene was constructed. By using a P. putida F1 todE mutant carrying this fusion on a plasmid, three cis-acting mutations that increased xylE expression enough to allow the todE strain to grow on toluene were isolated. The level of xylE transcript from the trpBA promoter was increased in all three mutants. All three mutations are base substitutions located in the -10 region of the trpBA promoter; two of these mutations make the promoter sequence more like the Escherichia coli RNA polymerase sigma 70 promoter consensus sequence. The activities of the wild-type and mutant trpBA promoters, as monitored by xylE expression, were assayed in P. putida PpG1 and in E. coli. The up-regulatory phenotypes of the mutants were maintained in the heterologous backgrounds, as was trpI and InGP dependence. These results indicate that the P. aeruginosa trpBA promoter has the key characteristics of a typical E. coli positively regulated promoter. The results also show that the P. aeruginosa and P. putida trpI activator gene products are functionally interchangeable.

Expression of the trpBA gene pair of Pseudomonas aeruginosa is regulated by the endogenous level of indoleglycerol phosphate (InGP) and the trpI gene product. The TrpI protein binds to the -77 to -32 region of the trpBA promoter. This region is divisible into two sites: site I, which is protected by TrpI in the presence and absence of InGP; and site II, which is protected by TrpI only in the presence of InGP. Recently, the trpI gene was subcloned into an expression vector and the protein was overproduced in Escherichia coli. The TrpI protein was purified to 80 to 95% purity. The molecular weight of native TrpI protein is estimated to be 129,000 by gel exclusion chromatography, and therefore it is likely a tetramer composed of 31,000-dalton monomers. Gel retardation assays with the purified TrpI protein demonstrated that InGP increases the affinity of TrpI for sites I and II approximately 17- and 14-fold, respectively. Binding of TrpI to site I is site II independent. However, the protein has low intrinsic affinity for site II and its binding to site II is site I dependent. Therefore, binding of TrpI to site II probably requires its interaction with a second TrpI molecule at site I.

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.

McDonald and Burke (J. Bacteriol. 149:391-394, 1982) previously cloned a sulfanilamide-resistance gene, sul, residing on a 4.9-kb segment of Bacillus subtilis chromosomal DNA, into plasmid pUB110. In this study we determined the nucleotide sequence of the entire 4.9-kb fragment. Genes identified on the fragment include pab, trpG, pabC, sul, one complete unidentified open reading frame, and one incomplete unidentified open reading frame. The first three of these genes, pab, trpG, and pabC, are required for synthesis of p-aminobenzoic acid. The trpG gene encodes an amphibolic glutamine amidotransferase required for synthesis of both p-aminobenzoate and anthranilate, the latter an intermediate in the tryptophan biosynthetic pathway. The pabC gene may encode a B. subtilis analog of enzyme X, an enzyme needed for p-aminobenzoate synthesis in Escherichia coli. The sul gene probably encodes dihydropteroate synthase, the enzyme responsible for formation of 7,8-dihydropteroate, the immediate precursor of folic acid. All six of the cloned genes are arranged in a single operon. Since all four of the identified genes are needed for folate biosynthesis, we refer to this operon as a folic acid operon. Expression of the trpG gene is known to be negatively controlled by tryptophan. We propose that this regulation is at the level of translation. This hypothesis is supported by the finding of an apparent Mtr-binding site which overlaps with the trpG ribosome-binding site.

In Rhizobium meliloti, the genes involved in biosynthesis of the amino acid tryptophan are found at three separate chromosomal locations. Of the three gene clusters, trpE(G), trpDC, and trpFBA, only the trpE(G) gene is regulated by the end product of the pathway, tryptophan. We found that trpE(G) mRNA contains a leader transcript that terminates at a stem-loop structure in a putative transcription attenuator. The level of this leader transcript was constant regardless of the amount of tryptophan in the growth medium. However, the level of full-length trpE(G) mRNA decreased as the amount of tryptophan increased. The beta-galactosidase activity of an R. meliloti strain carrying a trpL'-'lacZ fusion remained constant at different tryptophan concentrations, but the beta-galactosidase activity of the same strain carrying a trpE(G)'-'lacZ fusion decreased as the tryptophan concentration increased. These data indicate that transcription of the R. meliloti trpE(G) gene is regulated only by attenuation. We also found that the product of the trpE(G) gene, anthranilate synthase, is feedback inhibited by tryptophan.

Pseudomonas putida possesses seven structural genes for enzymes of the tryptophan pathway. All but one, trpG, which encodes the small (beta) subunit of anthranilate synthase, have been mapped on the circular chromosome. This report describes the cloning and sequencing of P. putida trpE, trpG, trpD, and trpC. In P. putida and Pseudomonas aeruginosa, DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also contains trpD and trpC. In P. putida, trpE is 2.2 kilobases upstream from the trpGDC cluster, whereas in P. aeruginosa, they are separated by at least 25 kilobases (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983). The DNA sequence in P. putida shows an open reading frame on the opposite strand between trpE and trpGDC; this putative gene was not characterized. Evidence is also presented for sequence similarities in the 5' untranslated regions of trpE and trpGDC in both pseudomonads; the function of these regions is unknown, but it is possible that they play some role in regulation of these genes, since all the genes respond to repression by tryptophan. The sequences of the anthranilate synthase genes in the fluorescent pseudomonads resemble those of p-aminobenzoate synthase genes of the enteric bacteria more closely than the anthranilate synthase genes of those organisms; however, no requirement for p-aminobenzoate was found in the Pseudomonas mutants created in this study.

Two pairs of related but easily distinguishable genes for the two subunits of anthranilate synthase have been identified in Pseudomonas aeruginosa. These were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance cassette, and returned to the P. aeruginosa chromosome, replacing the wild-type gene. Gene replacement implicated only one of the pairs in tryptophan biosynthesis. This report describes the cloning and sequencing of the tryptophan-related gene pair, designated trpE and trpG, and presents experiments implicating their gene products in tryptophan production. DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also includes trpD and trpC. Complementation of Trp auxotrophs by R-prime plasmids (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983) has shown that a large cluster of pyocin R2 genes is flanked at one end by trpE and the other end by trpDC; the physical map that was obtained shows the distance between trpE and trpDC to be about 25 kilobases. Our restriction map of the trpE and trpGDC regions agrees with data presented by Shinomiya et al.

Two anthranilate synthase gene pairs have been identified in Pseudomonas aeruginosa. They were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance gene, and returned to P. aeruginosa, replacing the wild-type gene. One anthranilate synthase enzyme participates in tryptophan synthesis; its genes are designated trpE and trpG. The other anthranilate synthase enzyme, encoded by phnA and phnB, participates in the synthesis of pyocyanin, the characteristic phenazine pigment of the organism. trpE and trpG are independently transcribed; homologous genes have been cloned from Pseudomonas putida. The phenazine pathway genes phnA and phnB are cotranscribed. The cloned phnA phnB gene pair complements trpE and trpE(G) mutants of Escherichia coli. Homologous genes were not found in P. putida PPG1, a non-phenazine producer. Surprisingly, PhnA and PhnB are more closely related to E. coli TrpE and TrpG than to Pseudomonas TrpE and TrpG, whereas Pseudomonas TrpE and TrpG are more closely related to E. coli PabB and PabA than to E. coli TrpE and TrpG. We replaced the wild-type trpE on the P. aeruginosa chromosome with a mutant form having a considerable portion of its coding sequence deleted and replaced by a tetracycline resistance gene cassette. This resulted in tryptophan auxotrophy; however, spontaneous tryptophan-independent revertants appeared at a frequency of 10(-5) to 10(6). The anthranilate synthase of these revertants is not feedback inhibited by tryptophan, suggesting that it arises from PhnAB. phnA mutants retain a low level of pyocyanin production. Introduction of an inactivated trpE gene into a phnA mutant abolished residual pyocyanin production, suggesting that the trpE trpG gene products are capable of providing some anthranilate for pyocyanin synthesis.

Twelve tryptophan auxotrophs of Rhizobium leguminosarum were characterized biochemically. They were grown in complex and minimal media with several carbon sources, in both limiting and excess tryptophan. Missing enzyme activities allowed assignment of all mutant to the trpE, trpD, trpB, or trpA gene, confirming earlier results with the same mutants (Johnston et al., Mol. Gen. Genet. 165:323-330, 1978). In regulatory experiments, only the first enzyme of the pathway, anthranilate synthase, responded (about 15-fold) to tryptophan excess or limitation.

Eight trp mutations (four trpE, two trpB, one trpC, and one trpD) have been mapped in Bacillus megaterium QM B1551 and were found to be linked to two hisH mutations and unlinked to several other his mutations.

In Acinetobacter calcoaceticus the seven genes coding for the enzymes responsible for tryptophan synthesis map at three chromosomal locations. Two three-gene clusters, one (trpGDC) specifying the small subunit of anthranilate synthase, phosphoribosyl transferase, and indoleglycerol phosphate synthase and the other (trpFBA) specifying phosphoribosyl anthranilate isomerase and both tryptophan synthase subunits, are not linked to each other or to the trpE gene specifying the large anthranilate synthase subunit. When regulation of trp gene expression is studied in the wild type, only the level of the trpF gene product decreases upon addition of tryptophan to the medium. Tryptophan starvation of tryptophan auxotrophs, however, results in increased levels of all the tryptophan enzymes; this and additional evidence suggests that the expression of all the trp genes is subject to repression. The trpGDC genes are coordinately controlled, and the trpE gene is regulated in parallel with them. The trpFBA genes are controlled neither coordinately nor in parallel with the other trp genes, but respond proportionally when compared with each other. So far, two types of constitutive mutants have been found. The first class of mutants apparently occurs in the structural gene for a repressor protein; this repressor locus is unlinked to any of the biosynthetic trp genes and affects only the expression of trpE and the trpGDC cluster. The second class contains mutants closely linked to the trpGDC region; they overproduce only the gene products of this cluster.

The Pseudomonas aeruginosa tryptophan synthase genes, trpA and trpB, which are induced by their substrate indoleglycerol phosphate, were cloned along with their controlling region into the BamHI site of pBR322 to produce the 10.7-megadalton plasmid pZAZ5. SalI partial digestion and ligation yielded a smaller plasmid, pZAZ167, with the chromosomal insert reduced in size from 8.1 to 3.4 megadaltons. Both pZAZ5 and pZAZ167 display Pseudomonas-like regulation of the trpA and trpB genes. Deletion of an EcoRI fragment or a BglII fragment from pZAZ167 yielded plasmids pZAZ168 and pZAZ169; the former expresses trpB but not trpA, and the latter has lost both activities. A deleted form of pZAZ5 designated pZAZ101 was obtained by excising a BglII-BamHI segment and religating the trip gene segment in the opposite orientation. This plasmid expresses trpA and trpB constitutively. The physical maps of these plasmids establish the gene order: promoter-trpB-trpA.

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.

Studies of a trpA mutant constitutive for tryptophan synthase production support the hypothesis of autogenous regulation (R. F. Goldberger, 1974; A. R. Proctor and I. P. Crawford, 1975) of the Pseudomonas putida trpAB loci.

Tryptophan biosynthetic enzyme levels in wild-type Vibrio harveyi and a number of tryptophan auxotrophs of this species were coordinately regulated over a 100-fold range of specific activities. The tryptophan analog indoleacrylic acid evoked substantial derepression of the enzymes in wild-type cells. Even higher enzyme levels were attained in auxotrophs starved for tryptophan, regardless of the location of the block in the pathway. A derepressed mutant selected by resistance to 5-fluorotryptophan was found to have elevated basal levels of trp gene expression; these basal levels were increased only two- to threefold by tryptophan limitation. The taxonomic implications of these and other biochemical results support previous suggestions that the marine luminous bacteria are more closely related to enteric bacteria than to other gram-negative taxa.

Cross-reacting beta(2) subunits (CRMs) were purified from eight trpB missense mutants to test for complementation in vitro after urea dissociation and reaggregation. One CRM (B290, demonstrating "repairability," i.e., the appearance of enzymatic activity on combination with alpha subunits) was clearly positive with four others, all "non-repairable" CRMs resulting from mutations at three different but neighboring sites. One complementing pair, B290-B248, was studied in more detail and found, upon mixing purified proteins, to give complementation in the absence of denaturants. Complementation activity was low in each case. To study the mechanism of the modest increases in activity, we used a reduced beta(2) subunit as an artificial CRM to form hybrids where both the amount of activity due to complementation and the amount of hybrid could be measured. (In a reduced beta(2) subunit, the two pyridoxal phosphate cofactors have been chemically reduced by sodium borohydride and are covalently attached to lysine residues. This abolishes activity in the tryptophan synthetic reaction and causes the protein to migrate much faster than normal in acrylamide gel electrophoresis.) Reduced beta(2) subunit formed hybrid dimers with the non-repairable CRMs B244 and B248 at pH 6.0, but no enzymatic activity appeared. On the other hand, when reduced beta(2) subunit was mixed with B290 CRM at pH 6.0 to 6.6, an activity increase was seen that was proportional to the amount of hybrid. We conclude that hybrid formation is essential for complementation and that the mechanism of complementation in this system is the correction of a repairable active site on the B290 beta chain by a conformational change occuring when hybrid dimer is formed. This type of complementation must be restricted to a small class of CRMs having a conformationally deformed active site. From the amount of hybrid present and the increase in activity, a specific activity of 50 U/mg was calculated for the hybrid containing reduced and B290 beta chains. This value is slightly less than but close to the activity of the hybrid formed between reduced and normal beta chains, shown earlier to have half the specific activity of the normal dimer.

We determined the DNA sequence of the Rhizobium meliloti gene encoding anthranilate synthase, the first enzyme of the tryptophan pathway. Sequences similar to those seen for the two subunits of the enzyme as found in all other procaryotic species studied are present in a single open reading frame of 729 codons. This apparent gene fusion joins the C terminus of the large subunit (TrpE) to the N terminus of the small subunit (TrpG) through a short connecting segment. We designate the fused gene trpE(G). The gene is flanked by a typical rho-independent terminator at the 3' end and a complex regulatory region at the 5' end resembling those of operons under transcriptional attenuation control. The location of the promoter was determined by S1 nuclease protection, using Rhizobium mRNA. Although this promoter was inactive in Escherichia coli, mutations eliciting activity were easily obtained. One of these was a C----T change at position -9 in the -10 region. The +1 position of the mRNA is the first base of the initiation codon of the leader peptide, implying that unlike trpE(G), which has a normal Shine-Dalgarno sequence, the leader peptide gene lacks a ribosome-binding site.

Two labeled peptides were recovered from tryptic digests of the NaB3H4-reduced, performic acid-oxidized beta2 protein of Serratia marcescens tryptophan synthase. These two pyridoxyl peptides were identical except for the presence or absence of an NH2-terminal arginyl residue. Tryptic digestion of nonreduced, performic acid-oxidized protein allowed isolation of the peptides that comprise the two halves of the pyridoxyl peptide. The partial primary structure for this region of the protein was shown to be Arg-Glx-Asx-Ler-Leu-His(Gly,Gly,Ala,His)Lys(Pxy)-Thr-Asx-Glx-Val(Leu,Gly,Glx,Ala,Leu,Leu,Ala)Lys. All the data available indicate that the sequence is identical with the homologous region from the Escherichia coli enzyme.

5-Methyltryptophan-resistant mutants derived from Bacillus subtilis strain 168 synthesize all of the tryptophan biosynthetic enzymes constitutively and excrete tryptophan. These mutants can be divided into three classes: class 1, low enzyme level and low rate of tryptophan excretion; class 2, high enzyme level and intermediate rate of tryptophan excretion; and class 3, high enzyme level and high rate of tryptophan excretion. A bradytrophic requirement for phenylalanine is correlated with the rate of tryptophan excretion. The phenylalanine requirement is relieved when the rate of tryptophan excretion is reduced by either (i) lowering the level of the tryptophan enzymes, (ii) reducing the supply of a tryptophan precursor (chorismate), or (iii) stopping tryptophan synthesis by a mutational block in the pathway. All of the mutants map in a region of the chromosome previously reported as the mtr locus. Our data show that synthesis of the tryptophan enzymes is controlled through the mtr locus but not influenced by precursors of tryptophan.

Auxotrophs of Acinetobacter calcoaceticus blocked in each reaction of the synthetic pathway from chorismic acid to tryptophan were obtained after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. One novel class was found to be blocked in both anthranilate and p-aminobenzoate synthesis; these mutants (trpG) require p-aminobenzoate or folate as well as tryptophan (or anthranilate) for growth. The loci of six other auxotrophic classes requiring only tryptophan were defined by growth, accumulation, and enzymatic analysis where appropriate. The trp mutations map in three chromosomal locations. One group contains trpC and trpD (indoleglycerol phosphate synthetase and phosphoribosyl transferase) in addition to trpG mutations; this group is closely linked to a locus conferring a glutamate requirement. Another cluster contains trpA and trpB, coding for the two tryptophan synthetase (EC 4.2.1.20) subunits, along with trpF (phosphoribosylanthranilate isomerase); this group is weakly linked to a his marker. The trpE gene, coding for the large subunit of anthranilate synthetase, is unlinked to any of the above. This chromosomal distribution of the trp genes has not been observed in other organisms.

The tryptophan synthetic pathway was characterized in three species of Bacillus, B. subtilis, B. pumilus, and B. alvei. They share the common features of a pathway which is subject to tryptophan repression, contains no unexpected complexes among the five enzymes, exhibits dissociable anthranilate synthase enzymes which do not require phosphoribosyl transferase for amidetransfer activity, contains separate indoleglycerol phosphate synthase and phosphoribosylanthranilate isomerase enzymes, and contains similar tryptophan synthetase multimers. In looking at these characteristics in detail however, differences among the three species became apparent, as, for example, in the complementation observed between the alpha and beta(2) components of tryptophan synthetase, and the dissociation patterns of the large and small components of anthranilate synthase. The results demonstrate some pitfalls in attempting to compare multimeric enzymes in crude extracts from different organisms.

The two protein components of Pseudomonas putida tryptophan synthetase have been purified to homogeneity. Although there is general similarity between the Pseudomonas enzyme and that of the enteric bacteria, many differences were found. Components from Escherichia coli and P. putida do not stimulate each other enzymatically, and the enzymes differ in their response to monovalent cations. Serine deamination occurs best with the intact enzyme of P. putida, not with the beta(2) subunit alone as in E. coli. The amino acid compositions of the alpha subunits differ appreciably. These findings extend earlier studies showing differences between enteric organisms and pseudomonads in the regulation and genetic organization of the enzymes of the tryptophan pathway.

Rohlfing, S. R. (Western Reserve University, Cleveland, Ohio), and I. P. Crawford. Purification and characterization of the beta-galactosidase of Aeromonas formicans. J. Bacteriol. 91:1085-1097. 1966.-The beta-galactosidase of Aeromonas formicans was purified by diethylaminoethyl cellulose chromatography and gel filtration on Sephadex G-200. The properties of the enzyme molecule were compared with purified beta-galactosidase from Escherichia coli. The sedimentation coefficients and electrophoretic mobilities of the two enzymes were not significantly different; the electrophoretic mobility of urea-produced subunits of the two enzymes was also similar. The stabilities of the two enzymes to denaturing agents provided measurable differences; E. coli beta-galactosidase is relatively more heat-stable and more resistant to the action of urea. The amino acid compositions of the two proteins revealed significant differences in several amino acids, particularly alanine, arginine, glycine, and leucine. The comparisons cited suggest that A. formicans and E. coli are not completely unrelated, for their beta-galactosidases show considerable structural similarity.

The first four enzymatic activities of the tryptophan synthetic pathway in Pseudomonas putida were found on separate molecules. Gel filtration and density gradient centrifugation experiments did not disclose any associations or aggregations among them. These findings contrast with the situation found in the enteric bacteria, where the first two activities are found in an aggregate and the third and fourth are catalyzed by a single enzyme. Tryptophan synthetase, the last enzyme of the pathway, consists of two dissociable components. The affinity of these components is less in P. putida than is the case in Escherichia coli.

Extracts of Chromobacterium violaceum catalyzed all of the reactions involved in synthesizing tryptophan from chorismic acid. Tryptophan auxotrophs which had lost any of these activities did not produce the characteristic purple pigment, violacein, when grown on a medium in which tryptophan was limiting. Gel filtration of extracts allowed us to estimate molecular weights for the tryptophan enzymes. All of the enzymes appeared to have molecular weights below 100,000. No enzymes were observed to occur in aggregates. The specific activities of the enzymes of the tryptophan pathway did not change when mutants were grown under conditions of limiting or excess tryptophan. The first enzyme in the pathway, anthranilate synthetase, was subject to feedback control by the end product, tryptophan. Tryptophan acted as a noncompetitive inhibitor with respect to glutamine, one of the substrates for anthranilate synthetase, and as a competitive inhibitor of the reaction when chorismate, the other substrate, was varied. The nonlinearity observed in the Lineweaver-Burk plot in the latter case suggests that there may be more than one chorismate-binding site on anthranilate synthetase.

The beta(2) subunits of tryptophan synthetase, formula alpha(2)beta(2), from Escherichia coli, Shigella dysenteriae, Enterobacter aerogenes, Salmonella typhimurium, and Serratia marcescens were compared by three criteria. (i) alphabeta association constants for the various beta(2) subunits and E. coli alpha subunit varied between 3.6 x 10(8)m(-1) for E. coli and 0.33 x 10(8)m(-1) for S. marcescens; values for the other organisms were intermediate. (ii) Antiserum neutralization of the beta(2) subunit enzyme activity using anti-E. coli beta(2) serum showed significant cross-reaction among the organisms (E. coli, 1.0; S. dysenteriae, 0.98; S. typhimurium, 0.67; E. aerogenes, 0.61; S. marcescens, 0.42). (iii) Quantitative microcomplement fixation showed E. coli beta(2) and S. marcescens beta(2) subunits to have an index of dissimilarity of 1.8 while the other organisms had intermediate indexes. Similar complement fixation data were obtained with antisera from separate rabbits and from first course and boost sera. These findings suggest that the general surface structure and the respective alpha subunit binding site of the beta(2) subunits from these Enterobacteriaceae have been strongly conserved.

There is 50% identity in the sequences of the first 50 residues of the alpha chains of Escherichia coli and Pseudomonas putida. No deletions or additions of residues are found in this region, except for the N-terminal methionine residue which is missing in the polypeptide isolated from P. putida. Most of the residues which differ are chemically dissimilar, and half of them are specified by codons which differ by more than a single base. The two residues known by mutational analysis to be essential for catalysis in E. coli are conserved in P. putida. The potential taxonomic usefulness of information of this sort is analyzed.

Three indole analogues, 5-methylindole, 5-fluoroindole, and 7-methylindole, and the tryptophan analogue 5-fluorotryptophan were found to inhibit the growth of wild-type Pseudomonas putida. Mutants resistant to these analogues were obtained. Some of the 5-fluoroindole- and 5-fluorotryptophan-resistant strains exhibit an abnormality in the regulation of certain trp genes. These strains excrete anthranilate when grown in minimal medium in the presence or absence of the inhibitor. In these strains, the trpA, B, and D gene products, the first, second, and fourth enzymes of the tryptophan pathway, are produced in 20-fold excess over the normal wild-type level. The other enzymes of the pathway are unaffected. Exogenous tryptophan is still able to repress the expression of the trpABD cluster somewhat. Similarity between the 5-fluoroindole- and 5-fluorotryptophan-resistant strains suggests that the former compound becomes effective through conversion to the latter. Repression and derepression experiments with two anthranilate-excreting, 5-fluoroindole-resistant strains showed coordinate variation of the affected enzymes. The locus conferring resistance and excretion is not linked by transduction to any of the trp genes.