The regulation of tryptophan biosynthesis in Pseudomonas aeruginosa

Summary Eighteen auxotrophs of Pseudomonas aeruginosa requiring l-tryptophan for growth were isolated following nitrosoguanidine mutagenesis. Mutant blocks for each step of tryptophan biosynthesis were identified by enzymological assay. A regulatory mutant was characterized which was simultaneously constitutive for the gene products of trpA, trpB and trpD. Another class of regulatory mutant appears to synthesize tryptophan synthetase (i.e., trpE and trpF subunits) constitutively. The results implicate three control entities in the pathway of tryptophan biosynthesis: (i) The gene products of trpA, trpB and trpD are repressible by tryptophan, the range of enzyme specific activity varying at least fifty-fold. (ii) No regulation of the trpC gene product could be demonstrated, indicating that its synthesis is constitutive. (iii) The gene products of rpE and trpF are inducible by indoleglycerol 3-phosphate; the magnitude of induction can exceed 100-fold. These results together with some genetic data indicate a general similarity in gene-enzyme relationships between P. aeruginosa and P. putida. A number of specific differences that distinguish the two species are noted.A mutant blocked in the common pathway of aromatic biosynthesis was used to prove that enzymes of tryptophan biosynthesis other than tryptophan synthetase are not inducible by precursors of the common pathway such as chorismate. It is concluded that the concentration of tryptophan that signals total repression of the gene products of trpA, trpB and trpD is lower than the concentrations necessary for maximal feedback inhibition of anthranilate synthetase and for abolition of the induction of tryptophan synthetase.     

Regulation of fatty acid degradation in Escherichia coli: dominance studies with strains merodiploid in gene fadR.

Strains stably merodiploid in the 25-min region of the chromosome of Escherichia coli were constructed and used in dominance tests between various wild-type and mutant alleles of the fadR gene. Whereas the monoploid fadR+ and fadR strains were inducible and constitutive, respectively, for the enzymes involved in fatty acid degradation (fad), merodiploids with at least one fadR+ allele were inducible. This observation was true whether the fadR+ allele resided on the main chromosome or on the episome. These results show that fadR+ is trans dominant to fadR, and they are consistent with the proposal that the fadR gene product is a repressor protein. Complementation tests were also performed by constructing 24 merodiploids harboring fadR alleles on both the main chromosome and the episome. All of these fadR/fadR diploids were able to utilize the noninducing substrate decanoate as sole carbon source, suggesting that only one polypeptide is encoded by the fadR gene.     

Regulation of Tryptophan Biosynthetic Enzymes in Neurospora crassa

The formation of enzymatic activities involved in the biosynthesis of tryptophan in Neurospora crassa was examined under various conditions in several strains. With growth-limiting tryptophan, the formation of four enzymatic activities, anthranilic acid synthetase (AAS), anthranilate-5-phosphoribosylpyrophosphate phosphoribosyl transferase (PRAT), indoleglycerol phosphate synthetase (InGPS), and tryptophan synthetase (TS) did not occur coordinately. AAS and TS activities began to increase immediately, whereas PRAT and InGPS activities began to increase only after 6 to 12 hr of incubation. In the presence of amitrole (3-amino-1,2,4-triazole), the formation of TS activity in a wild-type strain was more greatly enhanced than were AAS and InGPS activities. With a tr-3 mutant, which ordinarily exhibits an elevated TS activity, amitrole did not produce an increase in TS activity greater than that observed on limiting tryptophan. With tr-3 mutants, the increased levels of TS activity could be correlated with the accumulation of indoleglycerol in the medium; prior genetic blocks which prevented or reduced the synthesis of indoleglycerol also reduced the formation of TS activity. The addition of indoleglycerol to cultures of a double mutant (tr-1, tr-3) which could not synthesize indoleglycerol markedly stimulated the production of TS activity but not PRAT activity; the production of TS activity reached the same level with limiting or with excess tryptophan. A model explaining these and other related observations on enzyme formation in N. crassa is proposed.     

Isolation of auxotrophs and analysis of regulation of tryptophan biosynthesis in Zymomonas mobilis

Tryptophan auxotrophs were isolated and used to analyze the regulation of tryptophan biosynthesis in Zymomonas mobilis. Twelve tryptophan auxotrophs were cassified as trp E, B or A based on accumulation of, or growth on, indole and anthranilic acid. Trp B mutants were found to accumulate indole when grown on limiting, but not on excess tryptophan, suggesting that tryptophan plays a role in regulating its biosynthesis. Tryptophan synthase and indoleglycerol phosphate synthase specific activities were measured in the wild-type strain and two trp mutants grown in limiting or excess tryptophan. Neither activity was repressed by exogenous tryptophan.Abbreviations CDRP O-(carboxyphenol amino)-1 deoxyribulose 5-phosphate - IGPS indoleglycerol phosphate synthase - TS tryptophan synthase Dedicated in memory of Dr. O. H. Smith     

Limited proteolysis of N-(5′-phosphoribosyl)anthranilate isomerase: Indoleglycerol phosphate synthase from Escherichia coli yields two different enzymically active,functional domains

The native enzyme must be denatured either by sodium dodecyl sulfate or by urea before limited proteolysis can occur. Under these conditions only one or two peptide bonds are hydrolyzed by each of the following proteases: Staphylococcal V8 protease, trypsin and elastase. The amino-terminal amino acid sequences were determined to identify the cleavage sites. The new sequences comprise approximately 20% of the entire polypeptide chain, and show good agreement with the nucleotide sequence of the trpC gene. Both V8 protease² and elastase yield large carboxy-terminal fragments, about two thirds of the size of the parent enzyme, and corresponding small amino-terminal fragments. Trypsin cleaves a single peptide bond in the last one third of the polypeptide chain. After separation of the fragments, removal of dodecyl sulfate and renaturation, only the large fragments fold to stable structures. The small fragments precipitate. The large amino-terminal fragment catalyzes only the synthesis of indoleglycerol phosphate and precipitates when solutions are frozen and thawed. The large carboxy-terminal fragment catalyzes only the isomerization of N-(5′-phosphoribosyl)anthranilate and is stable towards freezing and thawing. These studies prove that the intact bifunctional enzyme consists of two autonomously folding, functional domains. They also support the notion that the bifunctional enzyme may have arisen by the fusion of separate ancestral genes, and that stabilization of the intrinsically labile indoleglycerol phosphate synthase domain by interdomain interactions is functionally advantageous.     

Some Physical Characteristics of the Enzymes of l-Tryptophan Biosynthesis in Higher Plants

Anthranilate synthetase, phosphoribosyltransferase, phosphoribosyl anthranilate isomerase, and indoleglycerol phosphate synthetase were examined in partially purified extracts of the monocotyledon, Zea mays and the dicotyledon, Pisum sativum. The plant extracts were chromatographed on DEAE-cellulose and Sephadex G150. The molecular weights of the enzymes were determined and found to be similar to those observed for many bacteria. None of the plant tryptophan enzyme activities was aggregated in vitro as is also the case with most bacteria. This is in contrast with the complex aggregation patterns observed in other eucaryotic organisms that have been examined (fungi and Euglena gracilis). The tryptophan enzymes from peas and corn were generally similar but some differences in stability were observed.     

Gene structure in the tryptophan operon of Escherichia coli: Nucleotide sequence of trpC and the flanking intercistronic regions

We have determined the DNA sequence for the portion of the Escherichia coli tryptophan (trp) operon spanning trpC, which codes for the bifunctional enzyme N-(5′-phosphoribosyl)-anthranilic acid isomerase/indole-3-glycerol phosphate synthetase. The coding region consists of 1356 nucleotides, directing the synthesis of a polypeptide 452 amino acids in length. The predicted protein sequence is consistent with the amino acid composition of the pure enzyme, and with all known partial peptide sequences derived from this molecule. The enzyme is of particular functional interest, because it contains the catalytic activities for two sequential reactions in tryptophan biosynthesis in a single polypeptide chain.The nucleotide sequences of the junctions between trpC and its flanking genes, trpD and trpB, have also been determined. The trpD-trpC junction consists of six untranslated nucleotides and translation of trpC initiates at the second of two adjacent AUG codons. The trpC termination codon is separated from trpB by 11 nucleotides. The short non-translated regions flanking trpC distinguish it from trpA and trpD, whose initiation codons overlap the termination codons of the preceding genes (trpB and trpE), respectively. These differences in the intercistronic regions may reflect functional relationships between the products of adjacent genes in the operon.     

The location of the feedback-specific region with the pyrimidine-3 locus of Neurospora crassa.

Summary Thepyrimidine-3 locus ofNeurospora crassa specifies two enzyme activities, pyrimidine-specific carbamyl phosphate synthetase (CPSpyr) and aspartate transcarbamylase (ATC). ATC is translationally distal. CPSpyr, but not ATC, is subject to feedback inhibition by uridine triphosphate (UTP). To investigate the location of the feedback-specific region within the locus, inhibition of a number ofpyr-3 alleles by UTP was investigated. All CPS⁺ ATC^- polar alleles, revertants of CPS^- ATC^- polar alleles, and 5-fluorouracil-resistant mutants had normal UTP response. The location of the feedback-specific region is in or close to the CPS-specific region.Supported by Science Research Council Grant B/RG/2981     

Alteration of Tryptophan-mediated Regulation in Neurospora crassa by Indoleglycerol Phosphate

The accumulation of imidazoleglycerol phosphate during growth of Neurospora crassa in the presence of 3-amino-1,2,4-triazole was found to cause derepression of tryptophan synthetase and to inhibit the induction of kynureninase. Accumulation of indoleglycerol phosphate in response to growth in the presence of indole acrylic acid or anthranilic acid was also accompanied by derepressed synthesis of tryptophan synthetase. Enzyme synthesis in mutants (his-7 and trp-4) unable to form these intermediates was not altered under similar conditions. The rate of formation of tryptophan synthetase and kynureninase was found to differ in the presence of tryptophan and indole.     

Chromosome polymorphisms close to the cm-ADE1 locus of Candida maltosa

The imperfect yeast Candida maltosa has an ill-defined genetic constitution; it is nominally diploid, but probably highly aneuploid, in nature. We report on polymorphisms specifically affecting those chromosomes which bear the cm-ADE1 gene. This gene encodes phosphoribosylaminoimidazole-succinocarboxamide synthetase, an enzyme in the adenine biosynthetic pathway. By electrophoretic karyotype analysis, three differently sized chromosomes were demonstrated to carry cm-ADE; the size (but not the number) of these chromosomes was also found to vary, both between strains and during the mitotic growth of a single strain. Four different alleles of cm-ADE1 have been cloned and sequenced from one prototrophic strain. DNA sequence divergence between these different alleles is as high as 8%, with the greatest divergence being found in the upstream region. Mitotic recombination events that led to changes in the karyotype were followed by using cm-ADE1 DNA as an hybridization probe. A recombination hot-spot in the neighbourhood of the gene appears to be responsible for the instability of the chromosomes on which it resides.     

Positive Selection Scanning Reveals Decoupling of Enzymatic Activities of Carbamoyl Phosphate Synthetase in Helicobacter pylori

In an effort to detect factors which may be under positive selection, a survey for such genes in two pathogenic strains of Helicobacter pylori (J99 and 26695) was performed. Based on an analysis of synonymous and nonsynonymous substitutions, we identified 19 candidate genes under positive selection. A search for homologues with known crystallographic structures revealed Escherichia coli carbomoyl phosphate synthetase as a homologue of H. pylori carbamoyl phosphate synthetase. Carbamoyl phosphate synthetase as isolated from E. coli is a heterodimeric enzyme that possesses two different but coupled functionalities and is involved in the first committed step in the separate biosynthetic pathways for arginine and pyrimidine nucleotides. In this study, we provide evidence indicating that one of these functionalities appears to be under selective pressure. Reports from previously published site-directed mutagenesis studies point to a decoupling of amidotransferase and synthetase activities. Implications of these findings for a metabolic enzyme under positive selection are discussed in terms of the mechanisms of H. pylori pathogenesis. Received: 11 June 2001 / Accepted: 12 September 2001     

Amino-terminal sequences of indoleglycerol phosphate synthetase of Escherichia coli and Salmonella typhimurium.

The partial sequences of the first 40 residues of indoleglycerol phosphate synthetase of Escherichia coli and Salmonella typhimurium were determined, and three amino acid differences were observed among the 38 residues compared.     

The sole Serine/Threonine protein kinase and its cognate phosphatase from Aquifex aeolicus targets Pyrimidine biosynthesis

Serine/Threonine kinases participate in complex, interacting signaling pathways in eukaryotes, prokaryotes, and archae. While most organisms contain many different kinases, the extreme hyperthermophile, Aquifex aeolicus encodes a single hypothetical Ser/Thr kinase. A gene homologous to eukaryotic protein phosphatases overlaps the kinase gene by a single base pair. The putative kinase, AaSTPK and phosphatase, AaPPM, were cloned and expressed in E. coli, purified to homogeneity and found to be functional. AaSTPK is a 34-kDa monomer that can use MgATP, MnATP, or MnGTP as co-substrates, although MgATP appears to be the preferred substrate. AaSTPK was autophosphorylated on a threonine residue and was dephosphorylated by AaPPM. AaPPM phosphatase is homologous to the PPM sub-family of Ser/Thr phosphatases and was stimulated by MnCl₂ and CoCl₂ but not MgCl₂. AaSTPK also phosphorylated one threonine residue on the carbamoyl phosphate synthetase, CPS.A subunit. Carbamoyl phosphate synthetase reconstituted with phosphorylated CPS.A had unaltered catalytic activity but allosteric inhibition by UMP and activation by the arginine intermediate, ornithine, were both appreciably attenuated. These changes in allosteric regulation would be expected to activate pyrimidine biosynthesis by releasing the constraints imposed on carbamoyl phosphate synthetase activity by UMP and uncoupling the regulation of pyrimidine and arginine biosynthesis. CPS.A was also dephosphorylated by AaPPM. Aquifex aeolicus occupies the lowest branch on the prokaryotic phylogenetic tree. The Thr/Ser kinase, its cognate phosphatase and a protein substrate may be elements of a simple signaling pathway, perhaps the most primitive example of this mode of regulation described thus far.     

Enzymes of the Tryptophan Pathway in Three Bacillus Species

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.     

Inactivation and partial degradation of phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthetase in nongrowing cultures of Escherichia coli.

The stability of tryptophan biosynthetic enzyme activities was examined in cultures of repressor-negative (trpR) strains of Escherichia coli K-12 incubated under conditions of nutrient starvation of chloramphenicol inhibition. The results show that four of the five activities examined are stable under most nongrowing conditions, whereas one activity, indoleglycerol phosphate (InGP) synthetase, carried by the trpC protein, is unstable under most conditions tested. Phosphoribosylanthranilate (PRA) isomerase activity, which is also carried by the trpC protein, is unstable during starvation for ammonium, cysteine, or sulfate but is stable under other nongrowing conditions where InGP synthetase is not. InGP synthetase activity but not PRA isomerase activity is also diminished about twofold in cultures using glycerol as a carbon-energy source. These results indicate that one or both activities of the trpC protein is specifically inactivated under several culture conditions. Experiments with antibodies to the trpC protein show that sulfate-starved and ammonium-starved cultures contain 20 to 40% less immunologically reactive trpC protein than unstarved cultures. This indicates that the trpC protein is probably partially degraded under these conditions. During recovery from sulfate starvation or ammonium starvation, cultures slowly regain normal levels of InGP synthetase and PRA isomerase activities, suggesting that inactivation may be reversible.     

Nucleotide sequence of the trpC-trpB intercistronic region from Salmonella typhimurium.

We have sequenced a DNA segment that contains the Salmonella typhimurium trpC-trpB junction. A series of 11 amino acids predicted from the sequence are identical to the amino-terminal amino acid sequence of Escherichia coli tryptophan synthetase β (Crawford et al., 1979). Carboxypeptidase A digestion of phosphoribosyl-anthranilate isomerase-indoleglycerolphosphate synthetase identified its carboxy-terminal amino acids allowing us to specify the end of trpC. Nine nucleotides separate the terminator codon of trpC from the initiator codon of trpB. The messenger RNA around the trpB initiation site, as well as around many other prokaryotic ribosome binding sites, has the potential to form stable stem and loop structures. These secondary structures share the property of having most, if not all, of the sequences complementary to the 3′ end of 16 S ribosomal RNA, as well as the initiator codon, included in single-stranded regions.     

Tryptophan genes in Rhizobium--their organization and their transfer to other bacterial genera.

Summary R. leguminosarum trp alleles mapped by R68.45-mediated recombination were located in three distinct chromosomal regions. We isolated three derivatives of R68.45 that carried different trp genes of R. meliloti. Each of the plasmids suppressed all of the R. leguminosarum trp alleles in a particular region. The R-primes were transferred to strains of P. aeruginosa carrying mutations in different trp genes. The plasmid pAJ24JI suppressed trpA, B and F mutants, pAJ73JI suppressed trpC and D and pAJ88JI suppressed a trpE mutant. When the R-primes were transferred to E. coli trp strains they failed to suppress any trp mutants. A derivative of pAJ24JI was isolated which was able to suppress trpA and F mutants of E. coli.     

Interaction of RNA polymerase mutations in haploid and merodiploid cells of Escherichia coli K-12

Summary Combination in the same chromosome of tsX mutation which affects the attachment of RNA polymerase to DNA template with either of two rifampicin resistant mutations (rif-r-1 or rif-r-5) is lethal. However tsX forms viable combination with other rifampicin resistant mutation—rif-r-76. Moreover a partial restoration of rifampicin binding capacity takes place: RNA polymerase from double tsX rif-r-76 mutant binds rifampicin better than the enzyme from tsX ⁺ rif-r-76 cells. No mutual influence of rifampicin resistant and streptolydigin resistant mutations was found.Heterozygous merodiploids (rif-r/rif-s and stl-r/stl-s) demonstrate phenotypic dominance of sensitivity to each of the drugs no matter whether resistant allele is localized in chromosome or in episome. However certain chromosomal mutations which themselves have no apparent effect on RNA polymerase may cause dominance of rif-r allele.About a half of total cellular RNA polymerase in crude extracts of rif-r/rif-s and stl-r/stl-s heterogenotes was found to be drug-resistant, though rif-s allele is dominant phenotypically.The development of T2 phage is completely inhibited by rifampicin in haploid rif-s cells and is only slightly affected in rif-r mutant. A partial resistance of phage development to rifampicin was observed in rif-r/rif-s heterogenotes which confirms that both rif-s and rif-r enzymes are simultaneously present in such cells.Sensitive and resistant RNA polymerase function independently when mixture of the two enzymes was incubated with the excess of DNA template. However a competition between the two enzymes for the DNA was observed if the limiting amount of the template is available. The result of this competition to major extent depends on which of the enzymes was added first. It is supposed that in certain conditions normal RNA polymerase may act as a repressor of the mutant enzyme: drug-sensitive RNA polymerase may bind to the template in the presence of the drug and thus prevent the function of drug-resistant enzyme. This hypothesis explains phenotypic dominance of sensitive alleles to resistant alleles which leads to inability of heterogenote cells to multiply in the presence of corresponding drugs.     

Activities of enzymes of sugar metabolism in cold-stored tubers of Solanum tuberosum

Storage of tubers of Solanum tuberosum at 10° or 2° for 15 days did not alter significantly the maximum catalytic activities of sucrose phosphate synthetase, sucrose synthetase, glucose-6-phosphate dehydrogenase, aldolase, and glyceraldehydephosphate dehydrogenase. The temperature coefficients of phosphofructokinase, glyceraldehydephosphate dehydrogenase, and pyruvate kinase from the tubers were shown to be higher between 2° and 10° than between 10° and 25°. The rate of sugar accumulation at 2° exceeded the activity of sucrose synthetase but was less than that of sucrose phosphate synthetase. It is suggested that sucrose accumulation at 2° is catalysed by sucrose phosphate synthetase, is not due to changes in the maximum catalytic activities of any of the above enzymes, but may be due, in part, to the susceptibility of key glycolytic enzymes to cold.     

Repression and inhibition of cephalosporin synthetases in Streptomyces clavuligerus by inorganic phosphate

Cephalosporin production by growing cells of Streptomyces clavuligerus was reduced by 100 mM inorganic phosphate. Resting cell production was repressed by prior growth in high phosphate and inhibited by phosphate. The cell-free activity of desacetoxycephalosporin C synthetase (ring expansion activity) was repressed by prior growth in high phosphate and inhibited by phosphate. Isopenicillin N synthetase (cyclase) was inhibited but not repressed. Penicillin epimerase was neither inhibited nor repressed by phosphate.Abbreviations DCW dry cell weight - MOPS 3-(N-morpholino) propane-sulfonic acid