Elias Balbinder

Pressure effects on the association of the α and β2 subunits of tryptophan synthetase from Escherichia coli and Salmonella typhimurium

Abstract Sucrose density gradient centrifugation was employed to study the association of the α and β 2 subunits of the enzyme tryptophan synthetase from Escherichia coli and Salmonella typhimurium . In both cases, the fully associated enzyme ( α 2 β 2 ) showed a sedimentation coefficient of 6.4 S, in agreement with the values reported by other workers for the E. coli enzyme. The substrate, l -serine, and the cofactor, pyridoxal phosphate, were required for complex formation in both cases. Generation of moderately high pressures by increasing the centrifuge speed from 39,000 rpm to 50,000 rpm was found to interfere with complex formation in both species at 5 °C. This effect was reversed by a temperature increase from 5 °C to 20 °C or by low concentrations of a nonpolar solvent, ethanol, at 5 °C. These results indicate that hydrophobic bonding plays an important role in the formation of the active tryptophan synthetase α 2 β 2 complex. Monovalent and divalent cations also interfered with the formation of the α 2 β 2 complex, indicating the possibility that ionic bonds are also involved.

Purification of anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase from Salmonellatyphimurium using affinity chromatography: Resolution of monomeric and dimeric forms

Abstract Anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PRT) as the unaggregated enzyme, has been purified to 90–95 percent homogeneity using affinity and ion-exchange chromatography. The affinity matrix consisted of anthranilate coupled to succinylamidohexamethylimino-Sepharose via its free amino group. Electrophoresis of the purified PRT on gels of varying polyacrylamide concentrations resolved a faster-moving minor band at concentrations above 8 percent. Both the major and minor bands were found to possess PRT activity. The minor band was found to have a molecular weight one-half that of the major band. Electrophoresis of the enzyme in the presence of sodium dodecyl sulfate resulted in almost complete conversion to the lower molecular weight form.

Use of affinity matrices in determining steric requirements for substrate binding: Binding of anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase from Salmonella typhimurium to sepharose-anthranilate derivatives

Abstract The binding of anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PRT) in crude extracts of Salmonella typhimurium to Sepharose-anthranilic acid derivatives has been studied. When anthranilic acid was covalently bound to hydrocarbon “arms” by its free amino group or phenyl moiety, the derivatives thus prepared bound PRT preferentially. PRT activity could be eluted from these adsorbents using buffers of increasing pH. The specific activity of PRT eluted from these Sepharose-anthranilate adsorbents was 10- to 12-fold that of the original extract. Anthranilic acid bound to aminohexamethylimino-Sepharose by its free carboxyl group did not bind PRT effectively. Results indicated that the carboxyl group of anthranilic acid must be free in order for PRT to bind the Sepharose-anthranilate derivatives. Throughout this study, control columns consisting of Sepharose derivatives containing the appropriate hydrocarbon “arms” alone were used to investigate the possibilities of nonspecific enzyme binding. The findings obtained stress the necessity of using such controls in any affinity studies of this type. The prospect of utilizing the techniques of affinity chromatography to examine the requirements for the binding of proteins to specific ligands is discussed.

Evidence for the order promoter-operator-first structural gene in the tryptophan operon of Salmonella☆

Abstract Genetic evidence suggests that the order, with respect to the first structural gene, of the genetic elements controlling the expression of the tryptophan ( trp ) operon of Salmonella tryphimurium is P1-trpO-trpA (promoter-operator-first structural gene). Two independent lines of evidence are consistent with this order: (1) the deletion supX38 , which removes P1, fails to recombine with some trpO c mutations but recombines with one of them suggesting that it covers only part of trpO ; (2) we have been unable to find supX to trpO deletions after several attempts, but have easily obtained multisite mutations with the characteristics of deletions extending from trpO to trpA .

The association of tryptophan synthetase subunits from Escherichia coli and Salmonella typhimurium in homologous and heterologous combinations.

The association of α and β 2 subunits of tryptophan synthetase from Escherichia coli and Salmonella typhimurium in homologous and heterologous combinations was studied by sucrose density gradient centri-fugation. Under conditions allowing for optimal association of subunits derived from the same source, subunit association in the mixture E. coli α– S. typhimurium β 2 was weaker than normal while in the reciprocal combination of S. typhimurium α– E. coli β 2 it was tighter than normal. These observations suggest that a certain degree of binding between the α and β 2 subunits of tryptophan synthetase could have had a selective advantage during the evolutionary divergence of the species of Entero-bacteriaceae, so that a mutation leading to the substitution of an amino acid involved in α–β 2 association in one of the subunits could have been compensated by a mutation in the complementary one.

A mutation to 5-methyltryptophan dependence in the trp operon of Salmonella typhimurium. IV. Isolation and characterization of trp promoter mutations.

SummarySeveral revertants to 5-methyltryptophan (MT) independence of the MT-dependent mutation trpA515 were altered in the trp promoter trpP1 (Callahan et al., 1978). This suggested the possibility that by screening MT-independent revertants for a class which would produce no prototrophic recombinants in spot transduction tests against the deletion supX38, which covers the trpP0 region but does not extend into the first structural gene of the trp operon (trpA), we could recover mutations of the trp promoter. Of two thousand revertants screened in this manner, ten carried deletions extending into trpP0. Nine of these extended into trpA and one, trpP0 (A?) 1351 apparently did not. These results show that genetic screening of MT-independent revertants of SO495 (trp A515) provides a means of isolating trp promoter mutations. The mutation trpP1443 which had been previously isolated from the MT-independent revertant trpP1443trpA515 (Callahan et al., 1978) was characterized genetically and biochemically and shown to have the attributes of a promoter mutation. It was found to map at the extreme operator end of the tryptophan operon. Strain SO443 (genotype trpP1443) showed “leaky” growth on medium not containing tryptophan, was subject to normal repression by tryptophan, but under derepression produced undetectable amounts of trpmRNA and the trp enzymes did not increase appreciably in level. The trpP1443 mutation is cis-dominant. Strain SO443 could revert to full prototrophy. A detailed study of the revertants thus obtained disclosed, in addition to true revertants at the trpP1443 site (class 1), three additional classes. Class 2 revertants were resistant to low concentrations of MT (10 μg/ml) and showed that they had retained the trpP1443 mutation in genetic crosses. The mutation conferring simultaneously full prototrophy and MT resistance was closely linked to trpP1443. Class 3 revertants were resistant to high concentrations (100 μg/ml) of MT and thus far have not shown the presence of trpP1443 in crosses. The mutation responsible for the reversion to prototrophy and resistance to the analogue is clearly within the early portion of the trp operon and could be at the trpP1443 site itself. Revertants of class 4 seem to be caused by mutations at an unlinked suppressor locus (or loci).The recovery of four classes of prototrophic revertants of SO443 shows that a mutation of the promoter can be corrected not only by a true reversion to the wild type but in at least two additional ways. Reversions of class 2 can represent the creation of a new transcription start downstream from the promoter mutation itself and there is precedent for this class of event. Revertants of class 3 could represent a different event of this type, or a mutation at the trpP1443 site which simultaneously affects the operator function. Since there have been reports based on work in Escherichia coli that the trp promoter and operator overlap, this possibility should be seriously considered. Revertants of class 4 constitute an entirely novel and interesting class.

Molecular characterization of a stable Flac plasmid

Abstract F lac S is a thermostable extrachromosomal element isolated in Salmonella typhimurium which is altered in its replication as compared to its precursor Fts114 lac . Sedimentation of both these plasmids in alkaline sucrose gradients has indicated a difference in their sizes. Contour length measurements of open circular plasmid DNA molecules photographed in the electron microscope have revealed the estimated molecular weight of Fts114 lac to be 81 × 106 daltons while that of F lac S is 109 × 106 daltons. F lac S may carry a segment of S. typhimurium chromosomal or cryptic plasmid DNA.

Differences Between the Anthranilate-5- phosphoribosylpyrophosphate Phosphoribosyltransferases of Salmonella typhimurium Strains LT2 and LT7

The anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferases (PRT), coded by the second structural gene (trpB) of the tryptophan (trp) operon in strains LT2 and LT7 of Salmonella typhimurium, differ from each other in a number of parameters. These include the apparent Km values for their substrates anthranilic acid and 5-phosphoribosylpyrophosphate, thermostability, sensitivity to substrate inhibition by anthranilic acid, as well as end-product inhibition by tryptophan and specific activity. The PRT of strain LT7 further differs from that of strain LT2 in that its apparent Km for 5-phosphoribosylpyrophosphate is three to seven times higher when associated with anthranilate synthase in the enzyme complex which catalyses the first two steps of tryptophan biosynthesis than in its free uncomplexed form, which the PRT of strain LT2 shows the same apparent Km for this substrate in both its free and complexed forms. These results confirm and extend the finding of Stuttard (1975) that strains LT2 and LT7 differ genetically form each other at a single site within region II of the trpB gene.

A mutation to 5-methyltryptophan dependence in the trp operon of Salmonella typhimurium

SummaryThe AS-PRT enzyme complex which catalyzes the first two steps in the biosynthesis of tryptophan in S. typhimurium consists of two polypetide subunits: anthranilate synthetase (component I or AS-CoI) and anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PRT). These polypeptides are the products of the first two structural genes of the trp operon, trpA and trpB respectively. The PRT component has two functions: the aminoterminal 40% of the polypeptide is necessary for glutamine amidotransferase activity (GAT) while the carboxy-terminal 60% carries out the PRT activity proper. The mutant strain SO495 has a mutation, trpA515, which confers a unique phenotype: while the strain is capable of utilizing anthranilic acid (AA) a substrate of PRT, as a growth factor, it can only do so in the presence of the analogue 5-methyltryptophan (MT) normally a potent growth inhibitor. Previous evidence indicates that SO495 may possess a somewhat altered PRT, and that its activity could be inhibited by an altered, enzymatically inactive AS made in this strain under derepression. Some experiments designed to test these possibilities are described in this paper. Various properties of the PRTs of the MT-dependent mutant and several of its MT-independent revertants were examined and compared. These included the determination of their apparent Kms for the substrates anthranilic acid (AA) and phosphoribosyl pyrophosphate (PRPP) and the presence or absence of GAT activity. In addition, the possibility that a complex consisting of PRT and an enzymatically inactive AS-CoI was present in some of the revertant strains only when grown under derepressing conditions was investigated by gel chromatography. The results showed that the MT-dependent strain SO495 and the MT-independent revertants have PRTs which differ from each other as well as from wild type LT7 PRT. In MT-independent revertants which retain the trpPO region and most of trpA, PRT can form a loose aggregate which elutes from Bio-Gel columns as three fast moving peaks. This loose aggregate is absent when the strains are grown under repressing conditions and is always absent in strains which lack most of the trpA gene. These results support the idea that the dependence of strain S0495 on MT for utilization of AA as a growth factor has to do with the inhibition of the altered PRT made in this mutant by an altered AS-CoI polypeptide which is synthesized only under derepression. They also suggest that translation of trpB starts from different points in the wild type, S0495 and the MT-independent revertants.

A mutation to 5-methyltryptophan dependence in the tryptophan (trp) operon of Salmonella typhimurium

SummaryMutants of S. typhimurium with a defect in the first structural gene of the trp operon can utilize anthranilic acid (AA) as a growth factor. Among a group of 5-methyltryptophan (MT) resistant derivatives of trpA mutants we encountered several with a novel phenotype: they actually grew better in the presence of MT than in its absence. Normally MT inhibits growth of S. typhimurium at the concentration we employed due to its ability to act as co-repressor of the trp operon, and as a feedback inhibitor of anthranilate synthetase (AS) the first enzyme for tryptophan biosynthesis. Mutations to MT-dependence were only found in strains carrying extremely polar trpA mutations. In all cases analyzed, mutations causing MT-dependence mapped at the extreme operator distal end of trpA. The mutation trpA515 responsible for MT-dependence in strain SO61 (genotype trpA49trpA515) was, recombined away from the polar mutation. The strain thus obtained, SO495 was totally dependent on MT for growth on AA supplement. Strain SO495 lacks AS and under repressing grwoth conditions synthesizes the trp enzymes constitutively at 2–3 times the basal level. Under derepression, while the levels of the distal enzymes, as represented by tryptophan synthetase-β subunit (TSβ), did not increase there was a marked drop in the activity of anthranilate-PRPP phosphoribosyltransferase, (PRT) the enzyme catalyzing the second step of tryptophan biosynthesis. trpA515 was found to revert to prototrophy at a low frequency (about 10−8) which was not increased by chemical mutagens or ultraviolet radiation. In contrast, it was found to revert to MT-independence (growth on AA in the absence of MT) at a fairly high spontaneous frequency (about 10−6) and this frequency could be increased approximately tenfold by mutagens causing base substitutions or deletions but not by frameshift mutagens. About one hundred MT-independent revertants of trp A515 were mapped and found to fall into three general classes: (A) mutations at or near the trpA515 site (B) secondary mutations located upstream from trpA515, (C) deletions of various sizes. Based on a detailed genetic and physiologocal study of twelve representative MT-independent revertants, it appears that trpA515 may be caused by the insertion of a piece of DNA with some of the properties described for the IS elements found in Escherichia coli. The trpA515 insertion should contain (in this order), a transcription terminator, a low efficiency promoter and, probably, a translation start signal.