Mitochondrial DNA in yeast recombination and subsequent modification following mating between a grande and a suppressive petite

Summary The fluorinated pyrimidines 5-fluorouracil (5FU) and 5-fluorocytosine (5FC) induce the cytoplasmic petite mutation in the yeastSaccharomyces cerevisiae with high efficiency. It was found that in order to induce the mutation, 5FC must first be deaminated to 5FU. However, mutagenesis does not depend on the further conversion of 5FU to its deoxyriboside (5FUDR) and subsequent blockade of intracellular thymidine synthesis, since 5FUDR itself was found not to be mutagenic, and 5FU-induced mutagenesis was not antagonised by supplying thymidine monophosphate (dTMP) to a dTMP permeable strain. In any case, observations of the molecular changes accompanying petite induction in log phase cells ruled out the possibility that mutagenesis resulted simply from the dilution out of replication-blocked mitDNA molecules, since the appearance of mutants coincided with the synthesis of altered mitDNA molecules. In different strains, the resulting defective molecules were either maintained, giving rise to suppressive ^– petites, or completely degraded, to give pure clones of neutral ⁰ mutants. It is suggested that this degradative process was a consequence of the incorporation of 5FU into RNA.     

Studies on energy-linked reactions: isolation, characterisation and genetic analysis of trialkyl-tin-resistant mutants of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae resistant to triethyl tin sulphate have been isolated and are cross-resistant to other trialkyl tin salts. Triethyl-tin-resistant mutants fall into two general phenotypic classes: class 1 and class 2. Class 1 mutants are cross-resistant to a variety of inhibitors and uncoupling agents which affect mitochondrial membranes (oligomycin, ossamycin, valinomycin, antimycin, erythromycin, chloramphenicol, '1799', tetrachlorotrifluoromethyl benzimidazole carbonylcyanide-m-chlorophenylhydrazone and cycloheximide). Class 2 mutants are specifically resistant to trithyl tin and the uncoupling agent "1799' [bis-(hexafluoroacetonyl)-acetone]. Triethyl tin at neutral pH values is a specific inhibitor of mitochondrial energy conservation reactions and prevents growth on oxidisable substrates such as glycerol and ethanol. Triethyl-tin-resistant mutants grow normally on glucose and ethanol in the presence of triethyl tin (10 muM). Biochemical studies indicate that the mutation involves a modification of the triethyl tin binding site on the mitochondrial inner membrane, probably the ATP-synthetase complex. Triethyl tin resistance/sensitivity in yeast is determined by cytoplasmic (mitochondrial) and nuclear genes. The mutants fall into a nuclear and a cytoplasmic (mitochondrial) class corresponding to the phenotypic cross-resistance classes 1 and 2. In the cytoplasmic mutants the triethyl tin resistance segregates mitotically and the resistance determinat is deleted by the action of ethidium bromide during petite induction. Recombination studies indicate that the triethyl tin mutations are not allelic with the other mitochondrial mutations at the loci RI, RIII and OLI. This indicates that the binding or inhibitory sites of oligomycin and triethyl tin are not identical and that the triethyl tin binding site is located on a different mitochondrial gene product to those which are involved in oligomycin binding. Interaction and cooperative effects between different binding sites on the mitochondrial inner membrane have been demonstrated in studies of the effect of the insertion of the TETr phenotype into mitochondrial oligomycin-resistant mutants and provide an experimental basis for complementation studies at the ATP-synthetase level.     

Biogenesis of mitochondria 44

Summary A comparative study of eight independently isolated mitochondrial oligomycin resistant mutants obtained from three laboratories show a variety of phenotypes based on cross resistance to venturicidin and sensitivity to low temperature. Analysis of recombination between pairs of markers indicate the existence of at least three genetic classes; class A, cross resistant to venturicidin and including the mutations O III, [oli1-r], [OLG1-R], [tso-r]; class B, mutations O _I, [oli17-r], [OLG2-R]; and class C, the mutation O _II. The recombination data is consistent with mutations of each class residing in three separate genes, although mutations of class A and B show very close linkage.Recombination in non-polar crosses has demonstrated that markers of all three classes are linked to the mik1 locus in the configuration (AB)-mik1-C. The mapping of this segment with respect to other markers of the mitochondrial genome and the order of classes A and B was established by analyses of co-retention frequencies of markers in primary petite isolates as well as by analysis of marker overlap of genetically and physically defined petite genomes. The unambiguous order ery1-A-B-mik1-C-par was obtained. DNA-DNA hybridization studies using mtDNA isolated from selected petites confirms this map and estimates the physical separation of markers. A reasonable correlation exists in this region of the genome between distances estimated physically by hybridization and genetically by frequency of recombination in non-polar crosses.It is postulated that the oligomycin-mikamycin linkage group represents a cluster of genes involved in determining a number of mitochondrial membrane proteins associated with the mitochondrial ATPase and respiratory complex III.This work was supported by the Australian Research Grants Committee, Project D65/15930     

The selection of wild-type revertants from methotrexate permeability mutants

In a previous report, we described the selection and partial characterization of three distinct classes of methotrexate (Mtx)-resistant Chinese hamster ovary cells (CHO) (1). Class I cells contained a structural alteration in dihydrofolate reductase. Class II cells showed a alteration affecting the permeability of the drug. Class III cells, selected from class I cells, had an increased activity of the altered enzyme. In the work described here, the sensitivity of these lines to the diaminopyrimidines has been investigated. Class I cells are as sensitive, class II cells are 5- to 10-fold more sensitive, and class III cells are 10- to 30-fold more resistant than wild-type cells. The increased sensitivity of the class II cells provided an opportunity to select for revertants of these mutants and such phentotypic wild-type revertant cells have been selected using one diaminopyrimidine, pyrimethamine. Such cells have drug sensitivities and permeability characteristics similar to wild-type cells. A second class has been identified which has wild-type drug sensitivities to the diaminopyrimidines but Mtx class II resistance to Mtx, and drug permeabilities characteristic of Mtx-resistant class II cells.     

Isolation and characterization of adhesin-defective TnphoA mutants of septicaemic porcine Escherichia coli of serotype O115:K-:F165.

Non-enterotoxigenic porcine Escherichia coli strains belonging to the serogroup O115 have been associated with septicaemia and diarrhoea. Putative factors important in the pathogenicity of E. coli of serogroup O115 include fimbrial antigen F165, haemagglutination (MRHA), lipopolysaccharide, serum resistance, capsule and production of aerobactin. Using TnphoA transposon insertion mutagenesis, two classes of mutants were obtained from E. coli of serotype O115:F165 with respect to the phenotypic expression of fimbrial antigen F165 and MRHA of sheep erythrocytes: class I, F165-MRHA-, serum resistant; class II, F165+MRHA-, serum resistant. In a chicken lethality model, class I mutants were either virulent or of intermediate virulence, while class II mutants were of intermediate virulence. Alkaline phosphatase activity of class I and class II TnphoA mutants showed similar environmental regulation to that of fimbrial antigen F165. Moreover, class I and class II mutants were mutated in the prs-like locus, and lacked a 18.5 kDa and/or a 17.5 kDa fimbrial band.     

Impact of mitochondrial activity on the cell wall composition and on the resistance to tannic acid in Saccharomyces cerevisiae

In screening for resistance to tannic acid, mutants of Saccharomyces cerevisiae with an altered cell wall composition were recently isolated. Here we show that these mutants were all respiratory deficient. Cytoplasmic petite mutants isolated after ethidium bromide mutagenesis were resistant to tannic acid and had cell wall characteristics similar to the mutants isolated by screening for tannic acid resistance as shown by the lower sensitivity to zymolyase, a cell wall hydrolyzing enzyme, and by a changed sensitivity to calcofluor white, a molecule interfering with the cell wall assembly. Reintroducing active mitochondria to a tannic-acid-resistant mutant reduced the tannic acid resistance and zymolyase resistance to the wild-type level, showing that a mitochondrial mutation was responsible for the changes in cell wall composition and in tannic acid sensitivity.     

Mitochondrially encoded resistance to paromomycin in Saccharomyces cerevisiae: Reinvestigation of a controversy

We have reinvestigated the nature of mitochondrially inherited resistance to paromomycin in Saccharomyces cerevisiae. Resistance to this antibiotic can arise by a nucleotide alteration in the gene coding for 15 S ribosomal RNA at a recognition site for the restriction endonuclease ThaI (CGCG), as has been observed by Li (M. Li, K. Lyon, N. Martin and A. Tzagoloff (1981). “Abstracts, Cold Spring Harbor Meeting on Mitochondrial Genes,” p. 56. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.). We have confirmed this finding and document here also a new type of paromomycin resistance that is unrelated to this ThaI restriction site. Certain petite mutants derived from different locations of the mtDNA of S. cerevisiae KL14-4A can elicit resistance to paromomycin when crossed with a wild-type sensitive strain. These petite mutants lack detectable sequence homology with the 15 S ribosomal RNA gene and they have no extensive sequence homology with each other. We have constructed paromomycin-resistant diploids by crossing such KL14-4A petite mutants with a sensitive wild-type strain. The diploids that receive the paromomycin-resistant allele from a petite mutant retaining the 15 S ribosomal RNA gene no longer contained the ThaI site. However, diploids that become resistant after a cross with petite mutants retaining fragments from other mtDNA regions than the 15 S ribosomal RNA, still contain the ThaI site. This shows that paromomycin resistance can occur in the presence of the ThaI site. After sporulation, suitable paromomycin-resistant haploids were crossed with each other and sensitive recombinant diploids were found, indicating the existence of more than one form of paromomycin resistance. Possible explanations for this novel type of paromomycin resistance and the unorthodox way in which it arises, are presented.     

Effects of mancozeb and other dithiocarbamate fungicides on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: the role of mitochondrial petite mutants in dithiocarbamate tolerance

Saccharomyces cerevisiae as model system was used to evaluate the occurrence of resistant mutants and adaptation mechanism to mancozeb (MZ), a widespread fungicide of the dithiocarbamate class with a broad spectrum of action and multiple cell targets. We were unable to isolate mutants resistant to inhibitory concentration of MZ but found an unusually large number of mitochondrial defective petite mutants among cells incubated in the presence of subinhibitory MZ concentration. Similar results were obtained with two other dithiocarbamate fungicides. Comparison of wild type and petite mutants showed that the latter were more resistant to toxic effects of MZ, highlighting the role of mitochondria in MZ-tolerance. The data suggest that petite cells, arising by exposure to sub-inhibitory MZ concentration, are not induced by fungicides but are spontaneous mutants already present in the population before the contact with the fungicide.     

Ribosomal and non-ribosomal resistance to oxazolidinones: species-specific idiosyncrasy of ribosomal alterations

A derivative of Mycobacterium smegmatis, which carries only one functional rRNA (rrn) operon, was used to isolate mutants resistant to the ribosome-targeted antibiotic linezolid. Isolation and characterization of linezolid-resistant clones revealed two classes of mutants. Ribosomes from class I mutants are resistant to oxazolidinones in an in vitro peptidyl transferase assay, indicating that resistance maps to the ribosome component. In contrast, ribosomes from class II mutants show wild-type susceptibility to a linezolid derivative in vitro, pointing to a non-ribosomal mechanism of resistance. Introduction of a wild-type ribosomal RNA operon into linezolid-resistant strains restored linezolid sensitivity in class I mutants, indicating that resistance (i) maps to the rRNA and (ii) is recessive. Sequencing of the entire rrn operon identified a single nucleotide alteration in 23S rRNA of class I mutant strains, 2447G --> T (Escherichia coli numbering). Introduction of mutant rrl2447T into M. smegmatis rrn- resulted in a linezolid-resistant phenotype, demonstrating a cause-effect relationship of the 2447G --> T alteration. The 2447G --> T mutation, which renders M. smegmatis linezolid resistant, confers lethality in E. coli. This finding is strong evidence of structural and pos-sibly functional differences between the ribosomes of Gram-positive and Gram-negative bacteria. In agreement with the results of the in vitro assay, class II mutants show a wild-type sequence of the complete rRNA operon. The lack of cross-resistance of the class II mutants to other antibiotics suggests a resistance mechanism other than activation of a broad-spectrum multidrug transporter.     

Studies on the induction of petite mutants in yeast by analogues of berenil

Summary Compound Hoe 15 030 is an analogue of berenil which is as effective as berenil in inducing petite mutants in Saccharomyces cerevisiae. Hoe 15 030 has greater stability than berenil in aqueous solution, and is less toxic to yeast at high drug concentrations. Mutants of S. cerevisia strain J69-1B have been isolated which are resistant to the petite inducing effects of Hoe 15 030. Three mutant strains (HR7, HR8 and HR10) were characterized and each was shown to carry a recessive nuclear mutation determining resistance to Hoe 15 030. The degree of resistance to Hoe 15 030 is different for each mutant, and each was found to be co-ordinately cross-resistant both to berenil and to another analogue of berenil, Hoe 13 548. However, the three mutants show no cross-resistance to other unrelated petite inducing drugs, including ethidium bromide, euflavine and 1-methyl phenyl neutral red.Further studies on the mutants revealed that each strain exhibits characteristic new properties indicative of changes in mitochondrial membrane functions concerned with the replication (and probably also repair) of mitochondrial DNA. Thus, mutant HR7 is hypersensitive to petite induction by the detergent sodium dodecyl sulphate under conditions where the parent J69-1B is unaffected by this agent. Mutant HR8 is even more sensitive to sodium dodecyl sulphate than is HR7, and additionally shows a markedly elevated spontaneous petite frequency. Isolated mitochondria from strains HR8 and HR10 (but not HR7) show resistance to the inhibitory effects of Hoe 15 030 on the replication of mitochondrial DNA in vitro.     

Induction of petite yeast mutants by membrane-active agents.

Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.     

Induction of petite yeast mutants by membrane-active agents

Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.     

Impaired nitrogen fixation and glutamine synthesis in methionine sulfoximine sensitive (MSs) mutants of Rhizobium phaseoli

Summary Random Tn5 mutagenesis of antibiotic-resistant derivatives of Rhizobium phaseoli CFN42 yielded several independent mutants that were sensitive to methionine sulfoximine (MS^s), a specific inhibitor of glutamine synthetase (GS). These MS^s mutants were analyzed for GSI and GSII activities and for their symbiotic properties. Four classes of MS^s mutants have been distinguished. Class I strains are impaired in their synthesis of glutamine and in their symbiotic properties. Class II strains have wild type levels of GSI and GSII activities but have a reduced capacity to fix nitrogen. Class III strains have lost GSII activity, but their symbiotic properties are wild type. In class IV mutants neither glutamine synthesis nor symbiotic properties are affected. Mutants of classes I, III, and IV all have the Tn5 inserted into the chromosome, whereas in class II mutants the Tn5 is located in plasmid p42e, a plasmid different from the previously identified symbiotic plasmid p42d.     

Interactions among genes controlling sensitivity to radiation and alkylation in yeast

Summary In the simple eucaryote Saccharomyces cerevisiae there are at least three phenotypically distinct classes of mutants sensitive to inactivation by radiations and alkylating agents: class I mutants are sensitive to ultraviolet light and nitrogen mustard (HN2); class II mutants are sensitive to X-rays and methylmethane sulphonate (MMS); and class III mutants are sensitive to all four of these agents. We have constructed doubly mutant strains of types (I, I), (I, II), (I, III), and (II, III) and have measured their sensitivity to UV, X-rays, HN2 and MMS in order to characterize the interactions of the various mutant gene pairs. Class (I, III) double mutants proved to be supersensitive to UV and HN2 and class (II, III) double mutants proved to be supersensitive to X-rays and MMS. All other double mutants showed little or no enhancement of sensitivity over their most sensitive single mutant parents. Mutants of class I are known to be defective in excision repair and our results are consistent with the idea that there exist at least two additional pathways for dark repair in yeast, one capable of repairing X-ray and MMS damage to DNA, and another, possibly analogous to post-replication repair in bacteria, that competes with the other two for damaged regions in DNA.     

5-Fluoropyrimidine-resistant mutants of pneumococcus

Three classes of 5-fluorpyrimidine-resistant mutants of Diplococcus pneumoniae have been characterized. The mutant strain upp is resistant to high concentrations of the fluoropyrimidine bases fluorouracil (FU) and fluorocytosine (FC); strain upp has a defective uridine monophosphate pyrophosphorylase. The mutant strain udk is resistant to inhibition by fluorouridine (FUR) and exhibits defective uridine kinase activity. The mutant strain fun is resistant to inhibition by the nucleosides fluorodeoxyuridine, fluorodeoxycytidine, and FUR, but shows normal activity for all pyrimidine pathway enzymes tested. This strain may be defective in the activity of a transport system that governs the cellular uptake of pyrimidine ribo- and deoxyribonucleosides. Biochemical studies on wild-type and fluoropyrimidine-resistant pneumococci are discussed with respect to the transport and early metabolism of preformed pyrimidine precursors by this organism.     

Characterization of RNA-binding properties of three types of RNA-binding proteins in Anabaena sp. PPC 7120.

The Rbp proteins in cyanobacteria are RNA-binding proteins with a single RNA recognition motif or RRM. A comprehensive assembly of genomic data suggests that there are two major classes of Rbp proteins (classes I and II) that diverged before the diversification of cyanobacteria. Class I proteins are further classified into two types with or without a C-terminal glycine-rich domain. The results of selection from a random RNA pool suggest that RbpA1 (class I) has affinity to C-rich and G-rich sequences. In vitro RNA binding assay with homopolymers indicated that class II protein has low affinity to poly(G) in contrast with class I proteins. Site-specific mutagenesis analysis of the RRM in RbpA1 showed that the aromatic residues Tyr4 or Phe46 are important in RNA binding as well as maintenance of secondary structure. We also tested various truncated proteins lacking the C-terminal domain as well as point mutants. Most of these proteins exhibited decreased affinity to RNA. Circular dichroism analysis as well as chromatographic analysis showed that Tyr4 and Phe46 are also important in maintaining the structure of RbpA1 protein. The C-terminal glycine-rich domain itself does not contribute much to the RNA-binding, but Arg83 which is located close to the C-terminal end of RRM is important in the RNA-binding.     

Characterization of the diphtheria toxin-resistance system in Chinese hamster ovary cells.

Variations in two general classes of diphtheria toxin-resistant mutants which may be selected from Chinese hamster ovary (CH0-K1) cells and the conditions for their selection are described. The resistance of class I mutants can be overcome with increasing concentrations of toxin. Their entire complement of EF-2 is susceptible to ADP-ribosylation by toxin. Class I includes those strains in which resistance resides at the level of the plasma membrane. The resistance of class II, translational, mutants cannot be overcome by high concentrations of toxin, as all, or a portion, of their EF-2 is insensitive to the action of diphtheria toxin and Pseudomonas exotoxin A. Adjustment of the concentration of toxin used to select resistant mutants can be used to regulate the class of mutant recovered. Metabolic cooperation between cells does not affect recovery of either class I or class II mutants. Resistance is stable in class I strains, but class IIb strains, which possess 50% resistant and 50% sensitive EF-2, display a transient high level of resistance which is retained for varying lengths of time following exposure to toxin. Class IIa strains, which possess 100% resistant EF-2, grow normally in saturating concentrations of toxin, but class IIb strains grow at a reduced rate. Evidence is presented which suggests that the gene for EF-2 is functionally diploid in CHO-K1 cells.     

Deletion mapping of mitochondrial transfer RNA genes in Saccharomyces cerevisiae by means of cytoplasmic petite mutants

Summary Mitochondrial transfer RNA genes have been ordered relative to the position of five mitochondrial drug resistance markers, namely, chloramphenicol (C), erythromycin (E), oligomycin I and II (O_I, O_II), and paromomycin (P). Forty-six petite yeast clones that were genetically characterized with respect to these markers were used for a study of these relationships. Different regions of the mitochondrial genome are deleted in these individual mutants, resulting in variable loss of genetic markers. Mitochondrial DNA was isolated from each mutant strain and hybridized with eleven individual mitochondrial transfer RNAs. The following results were obtained: i) Of the seven petite clones that retained C, E, and P resistance markers (but not O_I or O_II), four carried all eleven transfer RNA genes examined; the other three clones lost several transfer RNA genes, probably by secondary internal deletion; ii) Prolyl and valyl transfer RNA genes were located close to the P marker, whereas the histidyl transfer RNA gene was close to the C marker; iii) Except for a glutamyl transfer RNA gene that was loosely associated with the O_I region, no other transfer RNA genes were found in petite clones retaining only the O_I and/or the O_II markers; and iv) Two distinct mitochondrial genes were found for glutamyl transfer RNA, they were not homologous in DNA sequence and were located at two separate loci.The data indicate that the petite mitochondrial genome is the result of a primary deletion followed by successive additional deletions. Thus an unequivocal gene arrangement cannot be readily established by deletion mapping with petite mutants alone. Nevertheless, we have derived a tentative circular map of the yeast mitochondrial genome from the data; the map indicates that all but one of the transfer RNA genes are found between the C and P markers without forming a tight cluster. The following arrangement is suggested:-P-pro-val-ile-(phe, ala, tyr, asp)-glu₂-(lys-leu)-his-C-E-O_I-glu₁-O_II-P-.Supported in part by Cancer Center CCRC 111B-3. Present address: Laboratoire de Biologie Generale, Universite Paris-Sud Orsay, 91405, FranceThe Franklin McLean Memorial Research Institute is operated by the University of Chicago for the U.S. Energy Research and Development Administration under Contract E(11-1)69     

Pseudomonas syringae pv. phaseolicola genomic clones harboring heterologous DNA sequences suppress the same phaseolotoxin-deficient mutants

Cosmid cloning and mutagenesis were used to identify genes involved in the production of phaseolotoxin, the chlorosis-inducing phytotoxin of Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight of bean (Phaseolus vulgaris L.). Eight stable clones were isolated from a genomic cosmid library by en masse mating to 10 ethyl methanesulfonate (EMS)-induced Tox- mutants. In cross-matings, each suppressed all 10 mutants as well as an additional 70 EMS-induced Tox- mutants (and one UV-induced Tox- mutant). On the basis of restriction endonuclease analysis and hybridization studies, the clones were grouped into three classes. Clones in a particular class shared common fragments, whereas clones in different classes did not. Clones from class I (but not classes II and III) also suppressed Tn5-induced Tox- mutants. Interposon mutagenesis and marker exchange of a representative clone from class III into the wild-type genome did not alter its Tox+ phenotype, indicating that this clone does not harbor structural or regulatory genes involved in phaseolotoxin production. We suggest that the genome of P. syringae pv. phaseolicola contains a "hot spot" in one of the functions involved in toxin production which is affected by EMS and UV and that heterologous clones are able to suppress the Tox- phenotype because their inserts encode products that are able to substitute for the product of the mutated gene. Alternatively, the inserts may contain sequences which titrate a repressor protein. In either case, the data suggest that suppression of EMS- and UV-induced mutants occurs when heterologous clones are present in multiple copies.