Peptidase mutants of Salmonella typhimurium

Six peptidase activities have been distinguished electrophoretically in cell extracts of Salmonella typhimurium with the aid of a histochemical stain. The activities can also be partially separated by chromatography on diethylaminoethyl-cellulose. These peptidases show overlapping substrate specificities. Mutants (pepN) of the parent strain leu-485 lacking one of these enzymes (peptidase N) were obtained by screening for colonies that do not hydrolyze the chromogenic substrate l-alanyl-beta-naphthylamide. The absence of this broad-specificity peptidase in leu-485 pepN(-) mutants allowed the selection of mutants unable to use l-leucyl-l-alaninamide as a leucine source. These mutants (leu-485 pepN(-)pepA(-)) lack a broad-specificity peptidase (peptidase A) similar to aminopeptidase I previously described in Escherichia coli. Mutants (pepD) lacking a dipeptidase (peptidase D) have been isolated from a leu-485 pepN(-)pepA(-) parent by penicillin selection for mutants unable to use l-leucyl-l-glycine as a leucine source. Mutants (pepB) lacking a fourth peptidase (peptidase B) have been isolated from a leu-485 pepN(-)pepA(-)pepD(-) strain by penicillin selection for failure to utilize l-leucyl-l-leucine as a source of leucine. Single recombinants were obtained by transduction for each of the peptidases missing in a leu-485 pepN(-)pepA(-)pepD(-)pepB(-) strain. The growth response of these recombinants to leucine peptides shows that all of these peptidases can function in the catabolism of peptides and that they display overlapping substrate specificities in vivo.     

Maltose adaptive mutant of heterotrophic marine bacterium, Alteromonas espejiana Bal-31: changes in the growth and the induction of extracellular alpha-amylase

Growth of the heterotrophic marine bacterium, Alteromonas espejiana Bal-31 was inhibited in the presence of sucrose, maltose and even glucose, but not with starch. Extracellular alpha-amylase was induced with a lag phase of 2 h in the presence of starch. In contrast, cell growth of the S2a mutant was not affected by the addition of maltose, and starch was ineffective in the induction of extracellular alpha-amylase in this mutant. Activity of extracellular alpha-amylase was induced from the S2a mutant with a 4-h lag phase in the presence of maltose, and the high level of enzyme activity was maintained for at least 24 h. Activity of alpha-amylase induced by both wild type starch and S2a mutant maltose cultures were mainly observed in extracellular locations. This activity could be stopped by tetracycline treatment, indicating that enzyme induction was dependant on gene expression and not on enzyme protein secretory mechanisms. Our results showed that the mutation in S2a changed the growth and the modulation of the specific alpha-amylase in response to carbon nutrients.     

The leucine operon carrying theleu-500 promoter mutation is expressed under anaerobic conditions

TheSalmonella typhimurium leu-500 auxotrophic mutant grew when cultivated in minimal medium anaerobically, but not aerobically. This mutant carries an AT CG mutation in the Pribnow box of the promoter region of the leucine operon and was found to be suppressible by anaerobic conditions. Analysis of the anaerobic gases revealed that hydrogen in the anaerobic gas mixture (85% N₂, 10% CO₂, 5% H₂) is essential for the suppression of theleu-500 mutation. Whenleu-500 mutant cells were incubated in the presence of the hydrogen gas, the synthetic rates for the first and last gene products of theleu-500 operon were similar to those of the wild-type cells. It was concluded that the entire leucine operon was efficiently expressed inleu-500 when the cells were grown under the hydrogen gas-containing anaerobic environment. Thus, theleu-500 promoter mutant is a model system for regulation of gene expression by a specific atmospheric environment, i.e., hydrogen gas found in the anaerobic environment.     

The Product of the LEU-3 Cistron as a Regulatory Element for the Production of the Leucine Biosynthetic Enzymes of Neurospora

A class of intracistronic (or closely linked) partial reversions of leu-3 mutations has been found to be conditionally constitutive with respect to the synthesis of isopropylmalate isomerase (specified by the leu-2 cistron) and beta-isopropylmalate dehydrogenase (specified by the leu-1 cistron), two of the enzymes of leucine biosynthesis in Neurospora. The intermediate level of enzyme production by these leu-3(cc) mutants is independent of the obligatory inducer effector, alpha-isopropylmalate, but dependent upon the presence of the branched-chain amino acids, isoleucine, valine and leucine. The properties of leu-3+, leu-3 and leu-3(cc) in heterokaryons indicate that the transnuclear regulatory activity of the leu-3 product varies specifically as a function of available effector molecules. The information presented suggests that the leu-3 cistron is responsible not only for the production of a "positive" regulatory substance necessary for the expression of the leu-1 and leu-2 cistrons, but that it probably serves also a coordinating role in the expression of many of the genes involved in branched-chain amino acid metabolism.     

Evidence for an Altered Operator Specificity: Catabolite Repression Control of the Leucine Operon in Salmonella typhimurium

A mutation, GD-1, in the leucine operon imposed unusual growth characteristics upon a leucine auxotrophic strain bearing the leucine operator mutation, leu-500. The strain with the GD-1 mutation was able to grow on a minimal salts medium when citrate was the sole carbon source, but required leucine when glucose was present. Tests with a large number of carbohydrates suggest that in the strain bearing the GD-1 mutation the leucine biosynthetic enzymes are under catabolite repressor control. Recombination studies indicate that the GD-1 mutation is a secondary alteration of the leucine operator at or very close to the site of the leu-500 mutation. Mutations at the supX locus (previously termed su leu 500 and located on the chromosome between the cysteine B and tryptophan gene clusters) result in elimination of the catabolite repression effect. The data are interpreted as an indication that the GD-1 and leu-500 mutations alter the leucine operator with respect to its specificity of response to repressors.     

Mutation and “Reversion” at the leu-5 locus of Neurospora and its effect on the cytoplasmic and mitochondrial leucyl-tRNA synthetases

The Neurospora mitochondrial and cytoplasmic leucyl-tRNA synthetases differ from each other not only in location but also with respect to tRNA specificity, chromatographic mobility, leucine affinity, and sensitivity to phosphate inhibition. Strain 45208t, which bears a mutation in the leu-5 cistron, produces a cytoplasmic enzyme with reduced affinity for leucine and little if any mitochondrial enzyme activity. Reversion of the 45208t mutation was found to result not only in the reappearance of mitochondrial leucyl-tRNA synthetase activity but also in the production of a cytoplasmic synthetase with an affinity for leucine intermediate between mutant and wild type. The reversion studied, then, did not involve a return to the wild-type nucleotide sequence in the leu-5 cistron. The results obtained lend further support to the conclusion that the leu-5 cistron is involved in specifying, at least in part, the structure of both the cytoplasmic and mitochondrial leucyl-tRNA synthetases, despite the physical and functional differences between them.Research was supported in part by National Science Foundation Grant 27575.     

Biochemical comparison of the Neurospora crassa wild-type and the temperature-sensitive leucine-auxotroph mutant leu-5. Detailed kinetic comparison of the leucyl-tRNA synthetases

The cytoplasmic leucyl-tRNA synthetases were purified from a wild-type Neurospora crassa and from a temperature-sensitive leucine-auxotroph (leu-5) mutant. A detailed steady-state kinetic study of the aminoacylation of the tRNALeu from N. crassa by the purified synthetases was carried out. These enzymes need preincubation with dithioerythritol and spermine before the assay in order to become fully active. The Kappm value for leucine was lowered by high ATP concentrations and correspondingly the Kappm,ATP was lowered by high leucine concentrations. The Kappm,Leu was lowered by high pH, a pK value of 6.7 (at 30 degrees C) was calculated for the ionizable group affecting the Km. At the concentrations of 2 mM ATP, 20 microM leucine, 0.3 microM tRNALeu, and pH 7 the apparent Km values were Kappm,ATP = 1.3 mM, Kappm,Leu = 49 microM and Kappm,tRNA = 0.15 microM. No essentially altered cytoplasmic leucyl-tRNA synthetase was produced by the temperature-sensitive mutant strain when kept at 37 degrees C. In none of these experiments could we find any difference between the wild-type enzyme and the enzyme from the mutant strain (whether grown at permissive temperature, 28 degrees C, or grown at permissive temperature for 24 h followed by growth at 37 degrees C). We therefore think that the small difference in the Km value for leucine of the wild-type and mutant enzyme, established in some earlier investigations, is not due to a difference in the kinetic properties of the enzyme molecules but to an external influence. The almost total lack of the mitochondrial leucyl-tRNA synthetase in the mutant strain besides the leucine autotrophy remains the only difference between the wild-type and mutant strains.     

On the regulation of D-ribose metabolism in E. coli B-r. I. Isolation and characterization of D-ribokinaseless and D-ribose permeaseless mutants

Summary A presumed single site mutation designated leu-500, affects both the basal expression and response to the leucine repression signal by the leucine operon of Salmonella typhimurium. The distantly located supX mutation suppresses the leucine auxotrophy imposed by the leu-500 mutation by raising the level of basal expression while maintaining the abnormal regulation. An additional type of suppressor mutation which is closely linked to the leu-500 locus restores essentially normal regulation but maintains the low repressed expression characteristic of the leu-500 strain. The leucine sufficiency of the leu-500 strain with the linked suppressor and the supX leu-500 strains is temperature conditional in that both types require leucine for growth at 42° but not at 37°. These results, which indicate that a single site mutation can simultaneously affect promoter-like and operator-like function, are discussed in terms of DNA superstructure.This investigation was supported by U.S. Public Health Service Research Grant GM-12551 from the National Institute of General Medical Sciences, and is a portion of the work submitted by Lloyd H. Graf in partial fulfillment of the requirements for the hP. D. degree from Duke University. Lloyd H. Graf was the recipient of a predoctoral fellowship award from the National Institute of General Medical Sciences. R. O. Burns is the recipient of a Research Career Development Award from the National Institutes of General Medical Sciences.     

The effect of amino acids on the temperature sensitive phenotype of the mammalian leucyl-tRNA synthetase mutant tsHl and its revertants.

The temperature sensitive leucyl-tRNA synthetase mutant tsHl and two revertants have been compared to the parental Chinese hamster ovary cells with respect to the effects of amino acid concentrations in the medium on growth. Elevating the leucine concentration 30- or 100-fold allowed tsHl to grow exponentially at 38.5 degrees C, normally the nonpermissive temperature. Partial revertants that had recovered some enzyme activity required smaller supplements for growth. Measurements of the leucine pools indicated that they respond directly to the extracellular leucine concentration and may mediate the effect. Use of combinations of amino acids confirmed that isoleucine has a similar though weaker effect on tsHl and identified an even weaker protection by valine. The triple combination of leucine, isoleucine and valine was a much more efficient medium supplement and three times normal concentrations of these amino acids supported growth of tsHl at 38.5 degrees C. It is postulated that they are acting at their respective aminoacyl-tRNA synthetases to help stabilize a complex which also contains the mutant leucyl-tRNA synthetase. The pool size measurements also showed that the leucine pools of tsHl and a revertant increased 2-fold more in a response to increased temperature than those of WT. It is suggested that this is a regulatory response to low leucyl-tRNA synthetase activity and is important in determining growth phenotypes.     

Isolation and characterization of a laccase-derepressed mutant of Neurospora crassa.

Laccase from the ascomycete Neurospora crassa is an inducible secretory enzyme. Production of this enzyme is repressed in vegetative cultures but can be induced by treatment with low concentrations of cycloheximide. Isolation and characterization of a derepressed mutant, the lah-1 mutant, that is capable of producing laccase in vegetative cultures without induction by cycloheximide are described. The lah-1 mutation is mapped between nit-2 and leu-3 on linkage group I, and it behaved as a recessive mutation in a forced heterokaryon. No differences were detected biochemically or immunologically between the laccase protein produced by the lah-1 mutant in the absence of cycloheximide and that induced with cycloheximide in the wild-type strain. This suggests that both laccases (66 kilodaltons) are products of the same structural gene. Relative amounts of laccase in the culture filtrate of the lah-1 mutant were much higher than those induced with cycloheximide in the wild-type strain, demonstrating high efficiency of the lah-1 mutant in production and secretion of laccase. The time course of laccase production by the lah-1 mutant revealed that expression of 66-kilodalton laccase was repressed in conidia and derepressed during vegetative mycelial growth. This suggests that a multiple regulatory mechanism is involved in the production and/or maturation of Neurospora laccase. The lah-1 mutant may be useful for identifying genes that regulate expression of the laccase gene in N. crassa.     

Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria.

Since both transport activity and the leucine biosynthetic enzymes are repressed by growth on leucine, the regulation of leucine, isoleucine, and valine biosynthetic enzymes was examined in Escherichia coli K-12 strain EO312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the iluB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), NOR THE LEUB gene product, 3-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxido-reductase, EC 1.1.1.85), were significantly affected in their level of derepression or repression compared to the parental strain. A number of strains with alterations in the regulation of the branched-chain amino acid biosynthetic enzymes were examined for the regulation of the shock-sensitive transport system for these amino acids (LIV-I). When transport activity was examined in strains with mutations leading to derepression of the iluB, iluADE, and leuABCD gene clusters, the regulation of the LIV-I transport system was found to be normal. The regulation of transport in an E. coli strain B/r with a deletion of the entire leucine biosynthetic operon was normal, indicating none of the gene products of this operon are required for regulation of transport. Salmonella typhimurium LT2 strain leu-500, a single-site mutation affecting both promotor-like and operator-like function of the leuABCD gene cluster, also had normal regulation of the LIV-I transport system. All of the strains contained leucine-specific transport activity, which was also repressed by growth in media containing leucine, isoleucine and valine. The concentrated shock fluids from these strains grown in minimal medium or with excess leucine, isoleucine, and valine were examined for proteins with leucine-binding activity, and the levels of these proteins were found to be regulated normally. It appears that the branched-chain amino acid transport systems and biosynthetic enzymes in E. coli strains K-12 and B/r and in S. typhimurium strain LT2 are not regulated together by a cis-dominate type of mechanism, although both systems may have components in common.     

Stimulating action of endorphins on the development of sympathetic ganglia in culture

The effect of gamma-and beta-endorphins, leu- and met-enkephalins on the growth and differentiation of nervous tissue was studied in organotypic cultures of the sympathetic ganglia of newborn rats. The growth of explants in living cultures and preparations stained by the Holmes method was analyzed. It was established that endorphins are capable of stimulating the growth of neurites from the explant, of increasing the number of glial and fibroblastoid cells in the zone of the growth. The mean value of the maximal magnitude of the zone of the growth in normalcy was 464 +/- 136 micron, that on addition of leu- and met-enkephalins, gamma- and beta-endorphins 879 +/- 161, 900 +/- 160, 959 +/- 170, and 1056 +/- 137 microns. The growth effect induced by endorphins was demonstrated within a wide dose range--from 10(-7) to 10(-14) M. Naloxone did not inhibit the stimulant action of the peptides. It is suggested that opioid neuropeptides can be used as a source of nonspecific growth factors for nervous tissue.     

Transforming growth factor alpha: mutation of aspartic acid 47 and leucine 48 results in different biological activities.

To study the relationship between the primary structure of transforming growth factor alpha (TGF-alpha) and some of its functional properties (competition with epidermal growth factor (EGF) for binding to the EGF receptor and induction of anchorage-independent growth), we introduced single amino acid mutations into the sequence for the fully processed, 50-amino-acid human TGF-alpha. The wild-type and mutant proteins were expressed in a vector by using a yeast alpha mating pheromone promoter. Mutations of two amino acids that are conserved in the family of the EGF-like peptides and are located in the carboxy-terminal part of TGF-alpha resulted in different biological effects. When aspartic acid 47 was mutated to alanine or asparagine, biological activity was retained; in contrast, substitutions of this residue with serine or glutamic acid generated mutants with reduced binding and colony-forming capacities. When leucine 48 was mutated to alanine, a complete loss of binding and colony-forming abilities resulted; mutation of leucine 48 to isoleucine or methionine resulted in very low activities. Our data suggest that these two adjacent conserved amino acids in positions 47 and 48 play different roles in defining the structure and/or biological activity of TGF-alpha and that the carboxy terminus of TGF-alpha is involved in interactions with cellular TGF-alpha receptors. The side chain of leucine 48 appears to be crucial either indirectly in determining the biologically active conformation of TGF-alpha or directly in the molecular recognition of TGF-alpha by its receptor.     

Modeling the Leigh syndrome nt8993 T-->C mutation in Escherichia coli F1F0 ATP synthase.

The mutations in human mitochondrial DNA at nt8993 are associated with a range of neuromuscular disorders. One mutation encodes a proline in place of a leucine conserved in all animal mitochondrial ATPase-6 subunits and bacterial a subunits of F1F0 ATP synthases. This conserved site is leu-156 and leu-207 in humans and Escherichia coli, respectively. An aleu-207-->pro substitution mutation has been constructed in the E. coli F1F0 ATP synthase in order to model the biochemical basis of the human disease mutation. The phenotype of the aleu-207-->pro substitution has been compared to that of the previously studied aleu-207-->arg substitution (Hartzog and Cain, 1993, Journal of Biological Chemistry 268, 12250-12252). The leu-207-->pro mutation resulted in approximately a 35% decrease in the number of intact enzyme complexes as determined by N, N'-dicyclohexylcarbodiimide-sensitive membrane associated ATP hydrolysis activity and western analysis using an anti-a subunit antibody. A 75% reduction in the efficiency of proton translocation through F1F0 ATP synthase was observed in ATP-driven proton pumping assays. Interestingly, the loss in F1F0 ATP synthase activity resulting from the leu-207-->pro substitution was markedly less dramatic than had been observed for the leu-207-->arg mutation studied earlier. By analogy, the human enzyme may also be affected by the leu-156-->pro substitution to a lesser extent than the leu-156-->arg substitution, and this would account for the milder clinical manifestations of the human leu-156-->pro disease mutations.     

DNA supercoiling and suppression of the leu-500 promoter mutation.

top mutations (formerly supX) eliminate DNA topoisomerase I activity and suppress the leu-500 promoter mutation in Salmonella typhimurium (K. M. Overbye, S. K. Basu, and P. Margolin, Cold Spring Harbor Symp. Quant. Biol. 47:785-791, 1983). Sublethal doses of coumermycin which reduce intracellular levels of supercoiling activity in a top mutant eliminated suppression of the leu-500 mutation. This result provides evidence that increased DNA supercoiling suppresses the leu-500 promoter mutation in top mutants.