Isolation and characterization of catabolite-resistant mutants in the D-serine deaminase system of Escherichia coli K-12.

Two classes of D-serine deaminase (Dsdase)-specific secondary mutants of Escherichia coli K-12 were isolated from a Dsdase low constitutive nonhyperinducible mutant as types which could grow in the presence of both D-serine and glucose. These strains contain cis dominant, nonsuppressible mutations in the dsdO (operator-initiator) region. In the first class of mutants (e.g., FB4010), Dsdase synthesis is completely insensitive to catabolite repression, and synthesis occurs at a high constitutive rate in the absence of cyclic adenosine 5'-monophosphate. In the second class (e.g., FB4005), Dsdase synthesis is partially insensitive to catabolite repression, and catabolite repression is reversed by the addition of cyclic adenosine 5'-monophosphate. Dsdase synthesis in strain FB4005 is partially independent of the cyclic adenosine 5'-monophosphate binding protein, as constitutive synthesis is reduced only 65% (relative to the cap+ strain) in strains unable to synthesize the cyclic adenosine 5'-monophosphate binding protein. Surprisingly, the constitutive rate of Dsdase synthesis is fourfold higher in all mutants of both classes than in the parent, indicating a close interrelationship between the sites of response to induction and catabolite repression.     

Isolation and characterization of D-serine deaminase constitutive mutants by utilization of D-serine as sole carbon or nitrogen source.

Mutants constitutive for D-serine deaminase (Dsdase) synthesis were isolated by utilizing D-serine as sole nitrogen or carbon source in the chemostat. This method generated only regulatory constitutive (dsdC) mutants. The altered dsdC gene product in these strains is apparently able to bind D-serine more efficiently than the wild-type dsdC+ gene product--a selective advantage. Constitutive synthesis of Dsdase in all of these dsdC mutants is extremely sensitive to catabolite repression, and catabolite repression is reversed by the addition of D-serine. Of the 15 mutants generated by this method, none are suppressible by supD, supE, or supF. Mutations to a low level of constitutivity (maximal specific activity of 9) occur much more frequently than mutations to a high level (maximal specific activity of 79). High level constitutive synthesis of Dsdase results from the synthesis of an altered dsdC gene product--not from loss of ability to form the dsdC product. Dsdase synthesis is not regulated by the nitrogen supply in the medium, as nitrogen starvation does not result in the derepression of Dsdase synthesis.     

Role of small molecules in regulation of D-serine deaminase synthesis.

Cyclic AMP is required for optimal synthesis of D-serine deaminase synthesis from dsdO+ templates and for optimal hyperinducible synthesis from low constitutive dsdO templates both in vitro and in vivo. Neither D-serine, cyclic AMP, nor dsdC activator has an effect on expression of a high constitutive dsdO template. The synthesis of the dsdC activator itself in vitro is independent of cyclic AMP. Guanosine tetraphosphate does not have a significant effect on in vitro D-serine deaminase synthesis from dsdO+ or dsdO templates. A previously described class of dsdO mutants showing partial catabolite sensitivity of constitutive D-serine deaminase synthesis proved to be low dsdO types. They all contain a low constitutive dsdC mutation; the two effects are additive with regard to level of constitutivity, but only that portion of synthesis attributable to the dsdC mutation is cyclic AMP dependent.     

Catabolite repression in the D-serine deaminase system of Escherichia coli K-12

The induced synthesis of d-serine deaminase in Escherichia coli is subject to three catabolic effects: inhibition on inducer uptake, transient repression, and catabolite repression. Inhibition on d-serine uptake is not significant at the d-serine concentration normally used for induction. Transient repression and catabolite repression of d-serine deaminase synthesis are abolished by mutations in dsdCy, which appears to be an operator locus. The decline in the rate of constitutive synthesis observed in dsdCx mutants growing with glycerol as carbon source at temperatures above 37 C is due to catabolite repression. The low level of constitutivity at 37 C and the partial cis dominance of dsdCx mutants are not artifacts of catabolite repression. It is suggested that a product of one of the genes of the dsd operon may regulate the expression of the operon.     

Dominance studies with stable merodiploids in the D-serine deaminase system of Escherichia coli K-12

An episome, F32, which carries the genetic markers dsdA(+), the presumed structural gene for d-serine deaminase, dsdC(+), a regulatory locus governing the synthesis of d-serine deaminase, aroC(+), and purC(+) was obtained from strain AB311 of Escherichia coli K-12, and was used to construct appropriate merodiploids with dsdC markers. In all dsdC / dsdC(+) diploids examined, dsdC was found to be cis dominant, trans recessive, to dsdC(+). In two cases, however, the cis dominance was only partial. Moreover, complementation was observed between one of the dsdC markers which is fully cis dominant and one which is partially cis dominant. Because of the size of the dsdC region, the phenotypes of the mutants, and the partial trans dominance of dsdC(+) over some of the dsdC mutations, it is suggested that the dsdC region specifies a product, but that this product does not move with facility through the cytoplasm     

Cyclic Adenosine 3′,5′-Monophosphate in Escherichia coli

The concentration of cyclic adenosine 3',5'-monophosphate (c-AMP) in Escherichia coli growing on different sources of carbon was studied. Cultures utilizing a source of carbon that supported growth relatively poorly had consistently higher concentrations of c-AMP than did cultures utilizing sugars that supported rapid growth. This relationship was also observed in strains defective in c-AMP phosphodiesterase and simultaneously resistant to catabolite repression; in such strains the c-AMP concentration was slightly higher for several sources of carbon tested. Cultures continued to synthesize c-AMP and secreted it into the medium, under conditions that brought about an inhibition of the intracellular accumulation of the cyclic nucleotide. Transient repression of the synthesis of beta-galactosidase was not associated with an abrupt decrease in the cellular concentration of c-AMP.     

Physical mapping of the Escherichia coli D-serine deaminase region: contiguity of the dsd structural and regulatory genes.

The genes dsdA, dsdO, and dsdC have been located on a 3.0-kilobase pair (kb) fragment of the Escherichia coli chromosome by a combination of techniques. The loci were first cloned onto lambda and various plasmid vectors. dsd hybrid plasmids were then digested with restriction enzymes, and the fragments were recloned to test for the presence of dsdC or dsdA. In one case, a 4.2-kb restriction fragment containing the dsdA operon was used to form a heteroduplex with a well-defined lambda dsd deoxyribonucleic acid. The results show that dsdA, dsdO, and at least 0.6 kb of dsdC are present on this piece of deoxyribonucleic acid. On the basis of the mapping analysis and the molecular weight of D-serine deaminase, 1.9 kb of the 4.2-kb fragment is accounted for by the three dsd loci. We conclude that dsdO and dsdC are contiguous. A detailed dsd restriction map is presented.     

Effect of point mutations in the lac promoter on transient and severe catabolite repression of the lac operon of Escherichia coli

1. Experiments were devised to show whether the point mutations L8 and L29 in the lac promoter alleviate transient repression. 2. Several recombinants were picked from matings between a single F(-)p(+) strain and Hfr strains carrying mutations L8 and L29. All of the 19 p(-) recombinants tested proved to suffer no transient repression, whereas all of the eight p(+) recombinants tested suffered prolonged transient repression. 3. A diploid strain was constructed in which more than 90% of the thiogalactoside transacetylase is synthesized from the episome with a wild-type lac promoter, whereas 100% of the beta-galactosidase is synthesized from the chromosome with a promoter carrying mutation L8. In this diploid the synthesis of thiogalactoside transacetylase suffered transient repression but the synthesis of beta-galactosidase did not. 4. Exactly similar results were obtained with a diploid strain in which the chromosomal promoter carried mutation L29. 5. The same diploid strains were used in experiments to show whether mutations L8 and L29 alleviate the severe catabolite repression caused by growth in glucose plus gluconate. In both strains glucose+gluconate repressed the synthesis of beta-galactosidase much less than the synthesis of thiogalactoside transacetylase. 6. These and previously reported results can be explained by assuming (a) that both mutations L8 and L29 render the lac promoter partially, but not completely, insensitive to catabolite repression, and (b) that transient repression is an exceptionally severe form of catabolite repression.     

Stimulation of galactokinase synthesis in Escherichia coli by adenosine 3',5'-cyclic monophosphate

Adenosine 3',5'-cyclic monophosphate (cyAMP) stimulates the rate of synthesis of galactokinase in glycerol-grown Escherichia coli both when production of the enzyme is induced by d-fucose and when it is repressed by glucose in the presence of inducer. cyAMP also stimulates the synthesis of galactokinase in constitutive strains B78A (R(-)) and R10 (O(c)), and overcomes the transient repression of galactokinase synthesis caused by glucose.     

Catabolite repression of the lac operon. Separate repression of two enzymes

1. Catabolite repression of β-galactosidase and of thiogalactoside transacetylase was studied in several strains of Escherichia coli K 12, in an attempt to show whether a single site within the structural genes of the lac operon co-ordinately controls translational repression for the two enzymes. In all experiments the rate of synthesis of the enzymes was compared in glycerol–minimal medium and in glucose–minimal medium. 2. In a wild-type strain, glucose repressed the synthesis of the two enzymes equally. 3. The possibility that repression was co-ordinate was investigated by studies of mutant strains that carry deletions in the genes for β-galactosidase or galactoside permease or both. In all of the strains with deletions, the repression of thiogalactoside transacetylase persisted, and it is concluded that there is no part of the structural gene for β-galactosidase that is essential for catabolite repression of thiogalactoside transacetylase. 4. Subculture of one strain through several transfers in rich medium greatly increased its susceptibility to catabolite repression by glucose. It is concluded that unknown features of the genotype can markedly affect sensitivity to catabolite repression. 5. These results make it clear that one cannot draw valid conclusions about the effect of known genotypic differences on catabolite repression from a comparison of two separate strains; to study the effect of a particular genetic change in a lac operon it is necessary to construct a partially diploid strain so that catabolite repression suffered by one lac operon can be compared with that suffered by another. 6. Four such partial diploids were constructed. In all of them catabolite repression of β-galactosidase synthesized by one operon was equal in extent to catabolite repression of thiogalactoside transacetylase synthesized by the other. 7. Taken together, these results suggest that catabolite repression of β-galactosidase and thiogalactoside transacetylase is separate but equal.     

Uncoupling of Protein and Ribonucleic Acid Synthesis by 5′,5′,5′-Trifluoroleucine in Salmonella typhimurium

The addition of 5',5',5'-trifluoroleucine (fluoroleucine) to leucine auxotrophs of Salmonella typhimurium permitted protein but not ribonucleic acid (RNA) synthesis to continue after leucine depletion. The uncoupling of the formation of these macromolecules by fluoroleucine was apparent if RNA and protein synthesis was measured either by the uptake of radioactive precursors or by direct chemical determinations. The analogue did not appear to be an inhibitor of RNA formation, since it was as effective as leucine in permitting RNA synthesis in a leucine auxotroph upon the addition of small amounts of chloramphenicol. In contrast to these data, fluoroleucine allowed continued protein and RNA formation in a leucine auxotroph of Escherichia coli strain W. In addition, contrary to the results obtained with S. typhimurium, the analogue replaced leucine for repression of the leucine bio-synthetic enzymes as well as the isoleucine-valine enzymes. We propose that these ambivalent effects of fluoroleucine on repression and RNA and protein synthesis in the two strains are due to differences in the ability of the analogue to attach to the various species of leucine transfer RNA.     

Escape synthesis of D-serine deaminase in lambda dsdC integration lysogens.

Heat shock of lysogens that contain lambda thermosensitive repressor mutants integrated into the dsdC gene results in escape synthesis of d-serine deaminase at a high differential rate.