Enzymological Basis of Reluctant Auxotrophy for Phenylalanine and Tyrosine in Pseudomonas aeruginosa

Dual biosynthetic pathways to L-phenylalanine and L-tyrosine exist in Pseudomonas aeruginosa blocked

Presumably the presence of a mutation that interrupts one pathway is masked by the presence of the alternative pathway. However, a leaky phenylalanine auxotroph (doubling time of 140 min in minimal glucose medium compared to 57 min with wild type) was isolated which completely lacked aminotransferase DE I. This is one of four aromatic aminotransferases of overlapping specificity, each capable of transamination with prephenate, phenylpyruvate, or 4-hydroxyph qylpyruvate. A suppressor mutation in the genetic background of the phenylalanine bradytroph was equated with the constitutive synthesis of aminotransferase HA I, normally a catabolic enzyme induced in wild type in the presence of either L-tyrosine or L-phenylalanine.
The synthesis of aminotransferase HA II is repressed in the presence of phenylalanine and tyrosine, a result which suggests its probable role in aromatic amino acid biosynthesis.
Aminotransferase HA III is unregulated by aromatic metabolites and is thought to function primarily in branched-chain amino acid metabolism.
Although the suppressor mutation restores the wild type growth rate in minimal glucose medium, aromatic biosynthesis is highly stressed in this strain as revealed by its hypersensitivity to antimetabolite analogues of phenylalanine and tyrosine.
In fact, the DE I aminotransferase deficiency is no longer suppressed when fructose, a carbon source previously shown in wild type to render aromatic biosynthesis limiting to growth, is used.
It is likely that the phenylalanine bradytroph can be utilized as the genetic background for the isolation of otherwise silent mutations that inactivate the various biosynthetic enzymes of tyrosine and phenylalanine biosynthesis.
Although phenylalanine and tyrosine are synthesized exclu-* These investigations were supported by Grant PCM 761993 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18  various (and probably all) species of bluegreen algae form L-tyrosine from the amino acid intermediate, pretyrosine (1,2). In some organisms pretyrosine may also function as a precursor of L-phenylalanine, as described recently in Pseudomonas aeruginosa (3). This bacterium was shown to possess dual enzymatic routes to convert prephenate to L-phenylalanine via either phenylpyruvate or pretyrosine, and likewise to possess enzymes converting prephenate to tyrosine via either 4-hydroxyphenylpyruvate or pretyrosine. This enzymatic multiplicity for tyrosine and phenylalanine synthesis in P. aeruginosa is illustrated in Fig. 1.
Mutagenic treatment of wild type cultures with nitrosoguanidine has routinely produced nutritional mutants at high frequency in our laboratory. Thus, we readily obtained a full set of tryptophan auxotrophs, determined their enzymological deficiencies (4), and these have now been mapped on the P. aeruginosa chromosome.' Other aromatic mutants blocked before shikimate or between shikimate and chorismate have been obtained (5)* with no difficulty. Nevertheless, we have not managed to isolate mutants having absolute growth requirements for either L-phenylalanine or L-tyrosine. This is consistent with the presence in vivo of a second enzymatic option to either L-tyrosine or to L-phenylalanine in any given mutant.
The fractional contributions of the two enzymatic routes to L-phenylalanine or to L-tyrosine under various physiological conditions is uncertain. If any of these four two-step sequences are inadequate in the absence of the alternative pathway to supply the total end product required for protein synthesis, then the imposition of a mutant block in the alternative sequence might produce a phenotype of leaky auxotrophy. Several phenylalanine bradytrophs were indeed obtained, and one was selected for detailed enzymological examination.  (12).

RESULTS
Recovery of Aromatic Auxotrophs in P. aeruginosa-Tightly blocked phenylalanine or tyrosine auxotrophs were not obtained. We did, however, encounter leaky mutants whose growth was restored to the rate of wild type in the presence of L-phenylalanine.
Such mutants are recognized if survivors of mutagenesis are screened for faint growth as early as possible following the replica plating technique. Growth data obtained with one such phenylalanine bradytroph, our isolate number NP 72, are illustrated in Table I. In minimal glucose medium, the bradytroph grows about 40% as fast as wild type. The wild type rate of growth was achieved in the presence of L-phenylalanine or phenylpyruvate. L-Tyrosine and its keto acid precursor, 4-hydroxyphenylpyruvate, also increased the growth rate significantly, but did not restore the growth rate seen in the wild type parent. Spontaneous revertants were readily obtained.
Enzymological Deficiency of Phenylalanine Bradytroph NP 72-When crude extracts of the mutant were compared with wild type extracts for differences in enzyme activities, none were apparent. However, in view of the recent appreciation that enzyme studies with crude extracts are exceedingly complicated owing to the presence of two enzymatic pathways to L-tyrosine and L-phenylalanine (3), fractionation procedures were essential. Using procedures described before (31, mutant NP 72 was found to possess unaltered enzymes for both species of chorismate mutase, prephenate dehydratase, pretyrosine dehydratase, prephenate dehydrogenase, and pretyrosine dehydrogenase. However, when appropriate chromatographic procedures were used to separate the four aromatic aminotransferase activities previously characterized in wild type, aminotransferase DE I was clearly absent in phenylalanine bradytroph NP 72 (Fig. 2). A revertant derivative of the phenylalanine bradytroph, isolate number NP 76, is seen to have arisen as a The data given in Fig. 2 are consistent with an inducible regulation of aminotransferase HA I synthesis. The basal, uninduced level is probably that found in the bradytroph cultured in minimal glucose medium, a condition of end product limitation for the mutant. Presumably, the endogenous level of phenylalanine or tyrosine of wild type (or both) ( Fig. 2) are sufficient to partially induce the synthesis of aminotransferase HA I. The data given in Table II illustrate the variation of aminotransferase HA I activity over a 7-fold range. The suppressed mutant (right column) displays constitutive synthesis of aminotransferase HA I. In wild type and in the bradytroph, the presence of L-phenylalanine results in full induction of the HA I enzyme while L-tyrosine promotes partial induction. In the two mutant strains lacking aminotransferase DE I, a certain proportionality between growth rate ( Table I) and level of aminotransferase HA I (Table II) exists.
The data given in Fig. 2 are consistent with repression control of aminotransferase HA II synthesis. Thus, in the bradytroph where both phenylalanine and tyrosine are limiting to growth (Table I), the level of aminotransferase HA II was elevated significantly.
When the bradytroph was grown in r.-phenylalanine-supplemented medium, the level of aminotransferase HA II was repressed to the level of measured in wild type (data not shown). Repression of the synthesis of aminotransferase HA II over about a 6-fold range in the bradytroph is shown in Table III. Apparently in wild type the endogenous levels of end products during growth in minimal glucose are sufficient for maximal repression of aminotransferase HA II.
The level of aminotransferase DE I in wild type is unaffected by culture in the presence of L-phenylalanine or L-tyrosine (or both) (in comparison with unsupplemented cultures). The specific activity of aminotransferase HA III was unaffected by the presence or absence of aromatic amino acids in all these strains.

Total
Aminotransferase Activities with Aromatic Substrates in Wild Type and Mutant Strains-In Fig. 3, the combined activities of the four aminotransferase species with prephenate, phenylpyruvate, or 4-hydroxyphenylpyruvate in wild type are shown schematically, total activity being proportional to the height of the leftward bars. In the bradytroph and its suppressed revertant, the total activity shown is the sum of the three aromatic aminotransferases that remain in these strains, taking into account inducible changes in aminotransferase HA I and repressive changes in aminotransferase HA II.   to Biosynthetic Function -Aminotransferase DE I is reasonably deduced to be a biosynthetic enzyme since its loss by mutation leads to slowed growth in the absence of end products. Aminotransferase HA I is catabolic in function since it is induced by phenylalanine and tyrosine. The kinetic data illustrated in Fig. 4  and tyrosine synthesis, as suggested by the analysis depicted in Fig. 3. An apparently greater deficiency in endogenous phenylalanine synthesis is suggested by the better growth response to Lphenylalanine or phenylpyruvate (Table I) than to L-tyrosine or 4-hydroxyphenylpyruvate.
However, this probably reflects the excellent induction of aminotransferase HA I synthesis by L-phenylalanine, but not by L-tyrosine (Table II) In minimal glucose medium the growth rate of wild type P. aeruginosa is retarded weakly by P-X-thienylalanine, which is nevertheless one of the most effective phenylalanine antimetabolites for this organism (8). The pseudo wild type derivative (NP 76) of the phenylalanine bradytroph grows in glucose minimal medium at the same rate as wild type (Table I). However, mutant NP 76 is hypersensitive to growth inhibition by p-2-thienylalanine (Table V), indicating that phenylalanine biosynthesis is more stressed in the revertant strain than in wild type. The growth rat&s of the two strains are only slightly different in the presence of 4-aminophenylalanine, an antimetabolite of Ltyrosine in P. aeruginosa (8). Strikingly, the revertant does not achieve the wild type rate of growth in minimal fructose. In fact, the growth rate of the revertant is as slow as that of the bradytroph.
Clearly, the suppressor mutation present in the revertant would never have been detected if selection had been carried out on minimal fructose medium. Presumably aminotransferase HA I cannot operate effectively in the biosynthetic direction under the metabolic conditions that exist during growth on fructose. tion -The simultaneous presence of separate enzymatic pathways leading to a given major metabolite may be more common in nature than previously suspected, and even E. coli is not altogether free of such pathway multiplicity (13). The physiological and evolutionary implications of metabolic ambiguity for synthesis of small molecules has been discussed elsewhere (14). It seems likely that difficulties encountered in the isolation of amino acid auxotrophs from various groups of bacteria of wide distribution in nature may be explained by the compensatory presence in "silent" mutants of a second pathway to a given end product. Aromatic biosynthesis in pseudomonad microorganisms is an excellent example. Since the phenylpyruvate and 4-hydroxyphenylpyruvate pathways of E. coli were readily demonstrated in P. aeruginosa, the additional presence of the pretyrosine pathways to phenylalanine and tyrosine has remained unsuspected until recently. In such systems, single mutations may at best be expressed as leaky auxotrophs (if the presence of only one intact sequence to end product results in a growth-limiting supply of that end product), or perhaps as mutants that are hypersensitive to inhibition of growth by end product analogues (if the presence of only one intact sequence to end product results in a significantly decreased endogenous pool size of end product). If the presence of a mutation is recognized, either by leaky auxotrophy or by analogue hypersensitivity, a second mutagenesis using this strain should produce tightly blocked derivatives carrying an additional block in the second pathway. When genetic backgrounds containing such combinations of sequentially introduced mutations are subsequently separated by recombination, it is likely that their individual phenotypes may be similar to wild type.

In Vito
Function of Aminotransferase Reactions-The extent to which a given aminotransferase may be shared to carry out transamination reactions in different biochemical pathways is not very well understood. The broadly overlapping specificities of most aminotransferase proteins in vitro may or may not reflect the spectrum of reactions actually catalyzed in uiuo. Even when a particular transaminase reaction can only be catalyzed by one aminotransferase, that aminotransferase may nevertheless function in another pathway, as with histidinol phosphate aminotransferase in B.
subtilis (15). Under particular, specialized conditions an aminotransferase may function in transamination reactions that do not ordinarily occur in wild type, as with prephenate transaminase in N. crassa (12).
ciencies by the expression of phenotypes that may not be apparent when isolated in wild type backgrounds.
Specialization ofAromatic Aminotransferases in P. aeruginosa -Aminotransferase DE I is clearly essential for normal aromatic biosynthesis in wild type since its loss by mutation results in growth-limiting rates of phenylalanine or tyrosine synthesis. The DE I-negative bradytroph is deficient in overall aminotransferase activity with phenylpyruvate and 4-hydroxyphenylpyruvate, but not in overall aminotransferase activity with prephenate.
Since intracellular levels of prephenate should be elevated in the bradytroph owing to relaxation of the regulation of 3-deoxy-Dar&no-heptulosonate-7-phosphate synthetase (18) in response to end product limitation, intracellular concentrations of pretyrosine may even exceed that of wild type. If so, then pretyrosine dehydratase and pretyrosine dehydrogenase must be rate-limiting reactions for growth in the bradytroph when cultured in minimal glucose medium.
Aminotransferase HA I appears to overlap the catalytic characteristics of aminotransferase DE I most effectively. Since the synthesis of species HA I is induced in the presence of either L-phenylalanine or L-tyrosine, its normal function in uiuo is undoubtedly catabolic. The inducible regulation of aminotransferase HA I prevents the bradytroph lacking species DE I from exploiting the potential of species HA I to catalyze the reactions normally carried out by species DE I. This interpretation is further supported by the ability of a mutation to constitutive synthesis of aminotransferase HA I to suppress the DE I deficiency. Thus, it appears that elevated levels of a degradative aminotransferase in the absence of its normal substrate (e.g. L-phenylalanine) and the probable increase of its normal product (e.g. phenylpyruvate) functions in the backward direction in recruitment to biosynthetic function. The ability of a constitutively synthesized aminotransferase HA I to functionally replace the biosynthetic aminotransferase DE I may suggest the evolutionary origin of these aminotransferase proteins from a common protein. The identical molecular weights (70,000) of aminotransferases HA I and DE I (3) are consistent with this possibility. Although a constitutively synthesized aminotransferase HA I can replace aminotransferase DE I when glucose is the carbon source for growth, it cannot do so when aromatic biosynthesis is further stressed by growth on fructose. The combined activities of aminotransferase enzymes, having different, but overlapping specificities has long been assumed in many biochemical systems to account for the lack of mutant phenotypes that might arise from aminotransferase deficiencies. The fractional contribution of various aminotransferases to particular transamination reactions in uiuo can best be approached through the systematic and sequential elimination of individual aminotransferases by mutation. Thus, in E. coli each of three aminotransferases (specified by genes tyrB, ihE, and as&) is sufficient alone to function as phenylpyruvate aminotransferase (16). Absence of aminotransferases specified by tyrB or aspC does not lead to nutritional requirements unless the genetic background is also deficient in iluE (16). A fourth aminotransferase of E. coli appears to participate primarily in L-valine and L-alanine biosynthesis (17).

Aminotransferase
HA II is repressible over a 6-fold range, and this regulation implicates its role in aromatic amino acid biosynthesis. Enzyme HA II works much better as prephenate aminotransferase than as either phenylpyruvate aminotransferase or 4-hydroxyphenylpyruvate aminotransferase (3). The enzymological analysis of the wild type and mutant strains is suggestive of a primary role of aminotransferase HA II as prephenate aminotransferase.
The synthesis of aminotransferase HA III does not appear to vary in response to excess or limitation of aromatic end products. The dramatically better function of L-leucine as an amino-donor reactant in comparison with L-glutamate