Mutants of Salmonella typhimurium responding to cysteine or methionine: their nature and possible role in the regulation of cysteine biosynthesis.

Nineteen mutants of Salmonella typhimurium responding to either cysteine or methionine (cym) have been identified amongst cysteine (cys) and methionine (met) auxotrophs. Their growth responses to known intermediates in the related pathways of cysteine and methionine biosynthesis and complementation patterns in abortive transduction tests divided the mutants into six groups. Results of conjugation, cotransduction and deletion mapping experiments substantiated these groups, each of which carried a lesion within known cys genes. Enzyme assays on cym mutants from five of the six groups confirmed their cys gene deficiencies. Growth response and enzyme assay data were not consistent with mutants being leaky cys mutants (spared by methionine). None of eight cym mutants tested were able to convert [35S]methionine into [35S]cysteine. Selenate specifically inhibits the early enzymes of cysteine synthesis. In cym mutants this inhibition was relieved by cysteine but not by methionine, indicating that cym mutants require active cys enzymes for growth on methionine. There was evidence that methionine stimulated in vivo activity of cys enzymes in a cym mutant. Resistance to inhibition by 1,2,4-triazole results in reduced levels of the O-acetyl serine sulphydrylase. In cym mutants triazole resistance gave unstable suppression of the cym phenotype. Cym mutants may result from mutation in regulatory regions common to each of the cys genes, with the precise role of methionine as yet unknown.     

Genetics of sulfate transport by Salmonella typhimurium

Sixty-four mutants were isolated from the LT-2 wild-type strain of Salmonella typhimurium by selecting for chromate resistance. The majority of lesions were shown to lie in the cysA gene. (i) The mutants cannot take up sulfate, a finding which verifies the role of cysA in sulfate transport. In addition, 52 sulfate-transport mutants isolated without chromate selection were defective in the cysA gene. (ii) Most had less than 25% of the binding activity of the wild-type strain. (iii) Most had normal sulfite reductase (H(2)S-nicotinamide adenine dinucleotide phosphate oxidoreductase, EC 1.8.1.2) activity. (iv) Their sulfate-binding protein (binder) appears electrophoretically and immunologically normal. (v) Amber cysA mutants also make apparently normal binder in small amounts. (vi) All classical cysA mutants tested, including two with long deletions, had normal binding activity. From these observations, it is suggested that the cysA gene does not code for the binder. But many mutations in this gene reduce the binding activity in some unknown way. Other mutants, identified as cysB mutants, had neither binding nor uptake activities and their sulfite reductase activities were similarly reduced, thus confirming the regulatory role of the cysB gene. When binder was detectable, it had wild-type properties. No mutations in the binder gene were found among more than 100 mutants examined. Thus, sulfate binding has not been established as a part of sulfate transport. However, the production of binder is intimately connected with cysA, the established sulfate transport gene, and is regulated by the same mechanism that regulates both transport and the rest of the cysteine biosynthetic pathway.     

Regulation of O-acetylserine sulfhydrylase B by L-cysteine in Salmonella typhimurium.

A technique based on resistance to azaserine was used to isolate mutants lacking O-acetylserine sulfhydrylase B, one of two enzymes in Salmonella typhimurium capable of synthesizing L-cysteine from O-acetyl-L-serine and sulfide. The mutant locus responsible for this defect has been designated cysM, and genetic mapping suggests that cysM is very close to and perhaps contiguous with cysA. Strains lacking either O-acetylserine sulfhydrylase B or the second sulfhydrylase, O-acetylserine sulfhydrylase A (coded for by cysK), are cysteine prototrophs, but cysK cysM double mutants were found to require cysteine for growth. O-Acetylserine sulfhydrylase B was depressed by growth on a poor sulfur source, and depression was dependent upon both a functional cysB regulatory gene product and the internal inducer of the cysteine biosynthetic pathway, O-acetyl-L-serine. Furthermore, a cysBc strain, in which other cysteine biosynthetic enzymes cannot be fully repressed by growth on L-cystine, was found to be constitutive for O-acetylserine sulfhydrylase B as well. Thus O-acetylserine sulfhydrylase B is regulated by the same factors that control the expression of O-acetylserine sulfhydrylase A and other activities of the cysteine regulon. It is not clear why S. typhimurium has two enzymes whose physiological function appears to be to catalyze the same step of L-cysteine biosynthesis.     

Cysteine auxotrophs of Salmonella typhimurium which grow without cysteine in a hydrogen/carbon dioxide atmosphere.

Cysteine auxotrophs of Salmonella typhimurium mutated in cysB, cysI or cysJ grew with sulphate as a sulphur source when incubated under a hydrogen/carbon dioxide atmosphere. Yields obtained under these conditions were equivalent to those characteristic of wild-type S. typhimurium. The same mutants failed to grow with sulphate as a sulphur source when incubated aerobically. Auxotrophs mutated in cysA, cysC, cysD, cysE, cysG and cysH required cysteine for growth under both incubation conditions. The results suggest that mutations in cysB (regulation of the several cys operons) and also cysI and cysJ (sulphite reductase activity) can be circumvented during anaerobic growth under hydrogen.     

Bacteriophage PMB12 conversion of the sporulation defect in RNA polymerase mutants of Bacillus subtilis.

The pseudotemperate phage PMB12 was isolated from soil on the basis of its ability to enhance the rate of sporulation of Bacillus subtilis 168. PMB12 was subsequently shown to convert the sporulation defect in two genetically distinct classes of sporulation mutants. One class includes those rifampin-resistant mutants that are also spore-negative (mutated at the rif locus). The other class includes a strain carrying the sporulation mutation spoCM-1. The spoCM-1 mutation is linked to cysA15 by PBS1 transduction but is distinct from the rif locus. Several other sporulation mutants were not converted by PMB12. PMB12 is related to phage PBS1. However, PBS1 did not convert the above sporulation mutants. The replication of PBS2, a clear-plaquing derivative of PBS1, is rifampin insensitive, apparently due to a phage-induced rifampin-insensitive RNA polymerase. PMB12 replication is also rifampin insensitive.     

The structure of the cysCDHIJ region in unstable cysteine or methionine requiring mutants of Salmonella typhimurium.

Summary A genetic method was devised to test the hypothesis that in some cysteine or methionine requiring (cym) mutants of Salmonella typhimurium suppression of auxotrophy is due to an insertion at the site of the cym mutation. It was found that suppressed strains have an insertion of about 9 kb in the cysCDHIJ region and that in unstable suppressed strains it is the instability of this insertion which results in the segregation of cym auxotrophs.     

Mutations affecting the initiation of plasmodial development in Physarum polycephalum

In the heterothallic myxomycete Physarum polycephalum, uninucleate amoebae normally differentiate into syncytial plasmodia following heterotypic mating. In order to study the genetic control of this developmental process, mutations affecting the amoebal-plasmodial transition have been sought. Numerous mutants characterized by self-fertility have been isolated. The use of alkylating mutagens increases the mutant frequency over the spontaneous level but does not alter the mutant spectrum. Three spontaneous and 14 induced mutants have been analyzed genetically. In each, the mutation appears to be linked to the mating type locus. In three randomly selected mutants, the nuclear DNA content is the same in amoebae and plasmodia, indicating that amoebal syngamy does not precede plasmodium development in these strains. These results indicate that a highly specific type of mutational event, occurring close to or within the mating type locus, can abolish the requirement for syngamy normally associated with plasmodial differentiation. These mutations help define a genomic region regulating the switch from amoebal to plasmodial growth.     

Kinetic properties of polymorphic variants and pathogenic mutants in human cystathionine gamma-lyase

Human cystathionine-gamma-lyase (CGL) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme, which functions in the transsulfuration pathway that converts homocysteine to cysteine. In addition, CGL is one of two major enzymes that can catalyze the formation of hydrogen sulfide, an important gaseous signaling molecule. Recently, several mutations in CGL have been described in patients with cystathioninuria, a rare but poorly understood genetic disease. Moreover, a common single nucleotide polymorphism in CGL, c.1364G>T that converts serine at position 403 to isoleucine, has been linked to elevated plasma homocysteine levels. In this study, we have characterized the pathogenic T67I and Q240E missense mutations and the polymorphic variants at amino acid residues 403 using kinetic and spectrophotometric methods. We report that the polymorphism does not influence the cofactor content of the enzyme or its steady-state kinetic properties. In contrast, the T67I mutant exhibits a 3.5-fold decrease in V max compared to that of wild-type CGL, while the Q240E mutant exhibits a 70-fold decrease in V max. The K Ms for cystathionine for both pathogenic mutants are comparable to that of wild type CGL. The PLP content of the T67I and Q240E mutants were about 4-fold and 80-fold lower than that of wild-type enzyme, respectively. Preincubation of the T67I mutant with PLP restored activity to wild-type levels while the same treatment resulted in only partial restoration of activity of the Q240E mutant. These results reveal that both mutations weaken the affinity for PLP and suggest that cystathionuric patients with these mutations should be responsive to pyridoxine therapy.     

Involvement of cysB and cysE genes in the sensitivity of Salmonella typhimurium to mecillinam.

cysB and cysE strains were obtained as spontaneous mecillinam-resistant mutants of Salmonella typhimurium. The resistance to mecillinam was caused by the cys mutations which also conferred tolerance to lethal cell shape mutations. Most, but not all, cysB and cysE mutations from other origins displayed the same behavior. Resistance was abolished by O- and N-acetylserine in cysE mutants; by thiosulfate, sulfite, and sulfide in cysB mutants; and by cysteine in both types of mutants. It is concluded that an event involved in mecillinam action requires the inducer and the activator protein of the cysteine regulon.     

Fine-structure genetic map of the cysB locus in Salmonella typhimurium.

A genetic map of the cysB region of the Salmonella typhimurium chromosome was constructed using bacteriophage P22-mediated transduction. Strains bearing delta (supX cysB) mutations were employed to divide this regulatory locus into 12 segments containing a total of 39 single-site mutations. Twenty-five of these single-site mutations were further ordered by reciprocal three-point crosses. The results do not support the concept of multiple cistrons at cysB and suggest that the abortive transductants previously observed in crosses between certain cysB mutants were due to intracistronic complementation. The prototrophic cys-1352 mutation, which causes the constitutive expression of the cysteine biosynthetic enzymes, was found to lie within the cysB region itself. It is bracketed by mutations, which lead to an inability to derepress for these enzymes and result in auxotrophy for cysteine.     

The genetics of polyamine synthesis in Neurospora crassa

New mutations of the polyamine pathway of Neurospora crassa fell into three categories. The majority affected ornithine decarboxylase and lay at the previously defined spe-1 locus. One mutation, JP100, defining the new spe-2 locus, eliminated S-adenosyl-methionine decarboxylase and led to putrescine accumulation. Revertants of this mutation suggested that the locus encodes the enzyme. Two other mutations, LV105 and JP120, defined a third locus, spe-3. Strains with these mutations also accumulated putrescine and were presumed to lack spermidine synthase activity, which catalyzes the formation of spermidine from putrescine and decarboxylated S-adenosylmethionine. The three spe loci lay within about 20 map units of one another on the right arm of Linkage Group V in the order: centromere-spe-2-spe-1-spe-3. The requirement for spermidine for growth was much less in spe-2 and spe-3 mutants than in spe-1 mutants, which do not accumulate putrescine. This suggested that putrescine fulfills many, but not all, of the functions of spermidine, or that high levels of putrescine render spermidine more effective in its essential roles.     

Mutational analysis of the Vibrio fischeri LuxI polypeptide: critical regions of an autoinducer synthase.

Synthesis of the Vibrio fischeri autoinducer, a signal involved in the cell density-dependent activation of bioluminescence, is directed by the luxI gene product. The LuxI protein catalyzes the synthesis of N-acyl-homoserine lactones from S-adenosylmethionine and acylated-acyl carrier protein. We have gained an appreciation of the LuxI regions and amino acid residues involved in autoinducer synthesis by isolating and analyzing mutations generated by random and site-specific mutagenesis of luxI. By random mutagenesis we isolated 13 different single amino acid substitutions in the LuxI polypeptide. Eleven of these substitutions resulted in no detectable autoinducer synthase activity, while the remaining two amino acid substitutions resulted in reduced but detectable activity. The substitutions that resulted in no detectable autoinducer synthase activity mapped to two small regions of LuxI. In Escherichia coli, wild-type luxI showed dominance over all of the mutations. Because autoinducer synthesis has been proposed to involve formation of a covalent bond between an acyl group and an active-site cysteine, we constructed site-directed mutations that altered each of the three cysteine residues in LuxI. All of the cysteine mutants retained substantial activity as an autoinducer synthase in E. coli. Based on the analysis of random mutations we propose a model in which there are two critical regions of LuxI, at least one of which is an intimate part of an active site, and based on the analysis of site-directed mutations we conclude that an active-site cysteine is not essential for autoinducer synthase activity.     

Optimal conditions for selecting specific auxotrophs of Saccharomyces cerevisiae using temperature-sensitive suicide mutants.

Of 48 temperature-sensitive mutants of Saccharomyces cerevisiae examined, five belonging to the same complementation group were found to undergo extensive loss of viability at the restrictive temperature. These mutants were protected from the lethal effects of exposure to a non-permissive temperature by starving for an auxotrophic requirement. By analogy with the method described by Littlewood [6] for selecting antibiotic-sensitive mutants, these temperature-sensitive mutants were found suitable in enriching for specific auxotrophs. Optimal conditions have been determined for selecting specific auxotrophs after mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine. These enable 20-fold enrichment and at least in the case of mutation to adenine dependence the method does not appear to favour mutations at any particular locus.     

Pleiotropic effect of a rifampin-resistant mutation in Bacillus subtilis.

Rifampin-resistant (Rifr) mutants were isolated spontaneously from Bacillus subtilis strain 168. A fraction of the mutants did not grow on a minimal medium. A high concentration of one of the L-amino acids (glutamic acid, glutamine, arginine, proline, aspartic acid, or asparagine) was required to restore their growth on the medium. Further analysis of one of the mutants (strain RF 161) suggested that the mutant is unable to use ammonia as a nitrogen source and requires amino acids instead. Activity of glutamate synthase was not detected in the crude extract of the mutant. The Rifr mutation was closely located to cysA and the drug resistance was cotransformed with the property of amino acid requirement at 100% frequency. All revertants to prototrophy tested showed the rifampin-sensitive (Rifs) property. The activity of the DNA-dependent RNA polymerase of the mutant was resistant to rifampin. It is concluded that some alteration of RNA polymerase may cause absence of the activity of an enzyme involved in the nitrogen metabolism.     

Genetic analysis of ribonucleic acid polymerase mutants of Bacillus subtilis

Deoxyribonucleic acid-dependent ribonucleic acid polymerase mutants of Bacillus subtilis strain Marburg were isolated after mutagenesis of spores with ethyl methane sulfonate. Genetic analysis by PBS1-mediated transduction and by transformation indicated that mutations responsible for all of the four phenotypic classes studied (rifampin resistance, streptovaricin resistance, streptolydigin resistance, and temperature sensitivity) were clustered close to the cysA14 locus. Three-factor transformation analysis has indicated the most probable marker order as follows: Rif(R)(Stv)(R)-Std(R)-Ts(418)-Ts(427). In addition, further characterization of the classical group I reference marker, cysA14, is reported.     

Pleiotropic hydrogenase mutants of Escherichia coli K12: growth in the presence of nickel can restore hydrogenase activity

Anaerobic growth in the presence of 0.6 mM NiCl2 was able to restore hydrogenase and benzyl-viologen-linked formate dehydrogenase activities to a mutant (FD12), which is normally defective in these activities. This mutant carries a mutation located near minute 58 in the genome. Hydrogenase isoenzyme I and II activities were restored along with the hydrogenase activity that forms part of the formate hydrogen lyase system. A plasmid (pRW1) was constructed, containing a 4.8 kb chromosomal DNA insert, which was able to complement the lesion in mutant FD12. Further mutants with mutations near 58 minutes on the chromosome, and which lacked hydrogenase and formate dehydrogenase activities were isolated. These mutants were divided into three groups. Class I mutants were restored to the wild-type phenotype either by growth with 0.6 mM NiCl2 or following transformation with pRW1. Class II mutants were also complemented by pRW1 but were unaffected by growth with NiCl2. Class III mutants were unaffected by both pRW1 and growth with NiCl2. The cloned 4.8 kb fragment of chromosomal DNA therefore encodes two genes essential for hydrogenase activity. Restriction analysis indicates that the cloned DNA is the same as a fragment that has previously been cloned and which complements the hydB locus (Sankar et al. (1985) J. Bacteriol., 162, 353-360). None of the three classes of mutants possess mutations in hydrogenase structural genes.     

Reversions of two proline-requiring auxotrophs of Haemophilus influenzae by n-methyl-n'-nitro-n-nitrosoguanidine and hydrazine.

New mutation detection systems are described for Haemophilus influenzae. They involve two independently isolated proline auxotrophs which appear to be mutants at different sites in a proline locus (proB) that is very closely linked to a locus (thd) for thymidine requirement. One of the mutants, proB1, appears to revert to prototrophy only by mutations at the locus. The other, proB2, reverts both by mutation at the locus and by unlinked suppressors. The latter account for about 90% of the reversions induced by MNNG and by HZ. The close linkage of proB to thd was used to distinguish between true revertants and suppressors by a transformation test. A comparison was made between the mutation induction kinetics of the different classes of revertants and mutations to novobiocin resistance with MNNG and HZ. The very different induction kinetics for these two mutagens previously reported for the novobiocin resistance system were also found for the proline systems. There were some differences between the detection systems, however, in the frequency of induced mutation relative to the spontaneous frequency and, in one case, in the form of the induction curve. It is concluded that the major features of the induction curves reflect the amount of damage done to DNA and so are general for all systems, but that there are some features which are locus-or site-specific.     

Genetic Analysis of a New Mutation Conferring Cysteine Auxotrophy in Saccharomyces Cerevisiae: Updating of the Sulfur Metabolism Pathway

We have identified a mutation in a gene of Saccharomyces cerevisiae, STR1, that leads to a strict nutritional requirement for cysteine. The str1-1 mutation decreases to an undetectable level the cystathionine gamma-lyase activity. This enzyme catalyzes one of the two reactions involved in the transsulfuration pathway that yields cysteine from homocysteine with the intermediary formation of cystathionine. The phenotype induced by this mutation implies that, in S. cerevisiae, the sulfur atom of sulfide resulting from the reductive assimilation of sulfate is incorporated into a four carbon backbone yielding homocysteine, which, in turn, is the precursor of the biosynthesis of both cysteine and methionine. This also reveals that the direct synthesis of cysteine by incorporation of the sulfur atom into a three carbon backbone as found in Escherichia coli does not occur in S. cerevisiae. The study of the meiotic progeny of diploid strains heterozygous at the STR1 locus has shown that the str1-1 mutation undergoes a particularly high frequency of meiotic gene conversion.     

The role of the G2 box,a conserved motif in the histidine kinase superfamily,in modulating the function of EnvZ

Sulphur is essential for some of the most vital biological activities such as translation initiation and redox maintenance, and genes involved in sulphur metabolism have been implicated in virulence. Mycobacterium tuberculosis has three predicted genes for the prototrophic acquisition of sulphur as sulphate: cysA, part of an ABC transporter, and cysA2 and A3, SseC sulphotransferases. Screening for amino acid auxotrophs of Mycobacterium bovis BCG, obtained by transposon mutagenesis, was used to select methionine auxotrophs requiring a sulphur-containing amino acid for growth. We have characterized one of these auxotrophs as being disrupted in cysA. Both the cysA mutant and a previously identified mutant in an upstream gene, subI, were functionally characterized as being completely unable to take up sulphate. Complementation of the cysA mutant with the wild-type gene from M. tuberculosis restored prototrophy and the ability to take up sulphate with the functional characteristics of an ABC transporter. Hence, it appears that this is the sole locus encoding inorganic sulphur transport in the M. tuberculosis complex.