Site-directed mutagenesis of the succinate dehydrogenase subunits B and D from Corynespora cassiicola reveals different fitness costs and sensitivities to succinate dehydrogenase inhibitors

Carboxamide fungicides target succinate dehydrogenase (SDH). Recently published monitoring studies have shown that Corynespora cassiicola isolates are resistant to one or several SDH inhibitors (SDHIs) with amino acid substitutions in the SDH B and D subunits. We confirmed, by site-directed mutagenesis of the sdhB and sdhD genes, that each of the mutations identified in the field strains of C. cassiicola conferred resistance to boscalid and, in some cases, cross-resistance to other SDHIs (fluopyram, carboxin and penthiopyrad). Analyses of the enzyme activity and sdhB and sdhD gene expression show that modifications (SdhB_H278Y and SdhD_H105R) that result in a decline in SDH enzyme activity may be complemented by gene overexpression. The SdhB_H278Y, SdhB_I280V and SdhD_H105R mutants suffered large fitness penalties based on their biological properties, including conidia production and germination, mycelial growth, pathogenicity or survival abilities under environment stress. However, fitness cost was not found in the SdhB_H278R, SdhD_D95E and SdhD_G109V mutants. In the evaluation of resistance to boscalid in 2018 and 2019, the frequency of the SdhD_D95E and SdhD_G109V genotypes in the Liaoning and Shandong provinces changed dramatically compared with 2005–2017, from low resistance frequency (0.53% for D95E and 2.53% for G109V) to dominant resistance frequency (17.28% for D95E and 15.38% for G109V). Considering both the fitness and increased frequency of these genotypes, we may infer that the SdhD_D95E and SdhD_G109V mutants will be the dominant resistance mutants in field.     

Degradation of carboxin and oxycarboxin by microorganisms

Summary Algae, protozoa and photosynthetic bacteria which occur in considerable number in wet soils were examined for their ability to degrade the fungicides in broth culture. Blue green algae, namely, species of Anabaena, Nostoc and Tolypothrix brought about extensive degradation of the fungicides as revealed by thin layer chromatography. Green alga,Chlorella vulgaris also degraded the fungicides. The photosynthetic bacteriumRhodospirillum sp., failed to degrade carboxin beyond sulphoxide stage but degraded oxycarboxin to a greater extent. A protozoan species, Colpoda on the other hand, brought about extensive degradation of carboxin but not of oxycarboxin. These organisms have not been examined before for the detoxification of either carboxin or oxycarboxin.Part of Ph.D. Thesis, submitted to USA, Bangalore-65 under the guidance of the second author.     

Fungicidal control of Rhizoctonia solani in relation to soil texture, organic matter and clay minerals

In pot tests, MEMC, quintozene, captafol, carboxin, thiabendazole, carbendazim, benomyl and thiophanate-methyl used as seed treatments gave much better control of cowpea seedling rot in light-textured sandy and loamy sand soils than in heavy-textured loam and silt loam soils inoculated with Rhizoctonia solani. Disease control by chloroneb was not altered by soil texture. Amendment of sandy soil with montmorillonite reduced disease control with all fungicides, except chloroneb and carboxin; similar amendments with kaolinite decreased efficacy of MEMC and captafol. Green manuring with cluster bean reduced disease control by MEMC, captafol, benomyl and thiophanate-methyl; sunnhemp reduced efficacy of MEMC. Most fungicides gave poor disease control when farm yard manure or biogas sludge was added to soil, the sludge having the more marked effect. All the fungicides tested, except carboxin, were inactivated to different extents by humic acid extracted from farm yard manure.     

Degradation of carboxin and oxycarboxin in different soils

Summary Five different soils varying in physico-chemical properties were used for studying the persistence and degradation of carboxin and oxycarboxin. In one soil only both fungicides were degraded with accumulation of ammonium and nitrite. Under the conditions of forced circulation of air and continuous perfusion, oxycarboxin was found to be more susceptible to degradation than carboxin. Under simulated conditions of rice fields, conversion of carboxin to its sulphoxide and to a non-toxic derivative of oxycarboxin could only be seen in all the soils.The role of clay, humus and organic matter as protectants of fungicides against degradation indicated that the intermediary compound carboxin sulphoxide was strongly adsorbed probably on organic and inorganic colloids of most of the soils. Organic matter free soils delayed the degradation. Carboxin was rapidly converted to its sulphoxide on three forms of monoionic clays whereas oxycarboxin was transformed to an unidentified derivative.Part of Ph.D. thesis submitted to UAS, Bangalore-65.     

Controlling food-contaminating fungi by targeting their antioxidative stress-response system with natural phenolic compounds

The antioxidative stress-response system is essential to fungi for tolerating exposure to phenolic compounds. We show how this system can be targeted to improve fungal control by using compounds that inhibit the fungal mitochondrial respiratory chain. Targeting mitochondrial superoxide dismutase with selected phenolic acid derivatives (e.g., vanillyl acetone) resulted in a 100- to 1,000-fold greater sensitivity to strobilurin or carboxin fungicides. This synergism is significantly greater with strobilurin than with carboxin, suggesting that complex III of the mitochondrial respiratory chain is a better target than complex II for fungal control, using phenolics. These results show certain natural compounds are effective synergists to commercial fungicides and can be used for improving control of food-contaminating pathogens. These results suggest that the use of such compounds for fungal control can reduce environmental and health risks associated with commercial fungicides, lower cost for control, and the probability for development of resistance.     

Risk assessment studies on succinate dehydrogenase inhibitors, the new weapons in the battle to control Septoria leaf blotch in wheat

Chemical control of Septoria leaf blotch, caused by Mycosphaerella graminicola, is essential to ensure wheat yield and food security in most European countries. Mycosphaerella graminicola has developed resistance to several classes of fungicide and, with the efficacy of azoles gradually declining over time, new modes of action and/or improvements in host varietal resistance are urgently needed to ensure future sustainable disease control. Several new‐generation carboxamide fungicides with broad‐spectrum activity have recently been introduced into the cereal market. Carboxamides inhibit succinate dehydrogenase (Sdh) of the mitochondrial respiratory chain (complex II) but, because of their single‐site specificity, these fungicides may be prone to resistance development. The objective of this study was to assess the risk of resistance development to different Sdh inhibitor (SDHI) fungicides in M. graminicola. UV mutagenesis was conducted to obtain a library of carboxin‐resistant mutants. A range of SDHI resistance‐conferring mutations was found in Sdh subunits B, C and D. Pathogenicity studies with a range of Sdh variants did not detect any fitness costs associated with these mutations. Most of the amino acid residues identified (e.g. B‐S221P/T, B‐H267F/L/N/Y, B‐I269V and D‐D129E/G/T) are directly involved in forming the cavity in which SDHI fungicides bind. Docking studies of SDHI fungicides in structural models of wild‐type and mutated Sdh complexes also indicated which residues were important for the binding of different SDHI fungicides and showed a different binding for fluopyram. The predictive power of the model was also shown. Further diagnostic development, enabling the detection of resistant alleles at low frequencies, and cross‐resistance studies will aid the implementation of anti‐resistance strategies to prolong the cost‐effectiveness and lifetime of SDHI fungicides.     

Sensitivity of Clonostachys rosea and Trichoderma spp. as Potential Biocontrol Agents to Pesticides

Clonostachys rosea 47 (CR47), Trichoderma atroviride 59 (TA59), T. atroviride 312 (TA312), Trichoderma harzianum 24 (TH24), Trichoderma longibrachiatum 9 (TL9), T. longibrachiatum 144 (TL144) and Trichoderma viride 15 (TV15) were tested to evaluate their in vitro sensitivity towards five fungicides (carboxin, guazatine, prochloraz, thiram and triticonazole) and four herbicides (chlorsulfuron, chlorotoluron, flufenacet and pendimethalin). All antagonists showed low sensitivity to carboxin and thiram and high sensitivity to prochloraz. For mycelial radial growth, TV15 was highly sensitive to guazatine, prochloraz and triticonazole and TH24 moderately insensitive to carboxin, guazatine and thiram. For conidial germination TL144 was the most sensitive to the fungicides, for mycelial radial growth and conidial germination CR47 was the least sensitive. None of the antagonists showed any mycelial radial growth inhibition in presence of the herbicides at field dose, except for TL144. Most antagonists did not show any conidial germination inhibition by the herbicides. The in vitro toxicity of prochloraz, guazatine and triticonazole towards the antagonists was confirmed by light and scanning electron microscope showing hyphal disruptions and extrusion of cytoplasmic content. A mixture of CR47 and/or TA312 with carboxin, thiram and triticonazole, applied to wheat seeds, was able to control Fusarium culmorum artificially inoculated to wheat seedlings in growth chambers. In the field, the antagonists applied along with triticonazole or thiram, at 1/10 of the field dose to seeds naturally infected by F. culmorum, gave a disease control comparable to that induced by triticonazole at full field dose. Our results demonstrate how an integration of microorganisms with pesticides makes the control of wheat foot rot possible.     

Fungicidal control of damping-off and seedling root rot in Brassica species caused by Rhizoctonia solani in the growth chamber*

Eight fungicides (benodanil, carboxin, cyproconazole, fenpropimorph, fur-mecyclox, iprodione, pencycuron and tolclofos-methyl) were evaluated, under growth chamber conditions, as seed treatments against pre-emergence damping-off and post-emergence seedling root rot in six Brassica species. Five cultivars of B. rapa, four cultivars of B. juncea, four cultivars of B. napus and one cultivar/ strain from each of B. carinata, B. nigra and B. oleracea were grown in soilless mix infested with an isolate of Rhizoctonia solani AG-2-1. B. nigra and B. juncea were considerably less susceptible to R. solani than the four other species. Cyproconazole at 0.05-0.1 g a.i./kg seed and the other fungicides at 2–4 g a.i./ kg seed provided almost complete control of pre-emergence damping-off in most Brassica species and their cultivars. Their efficacy varied against the post-emergence seedling root rot. Furmecyclox, iprodione, tolclofos-methyl and pencycuron consistently gave good control of seedling root rot in all Brassica species and their cultivars. Benodanil and fenpropimorph provided moderate control, and carboxin and cyproconazole gave poor control against root rot. Efficacy of carboxin, cyproconazole, benodanil and fenopropimorph against seedling root rot varied significantly (P ≤ 0.05) among cultivars within a Brassica species.     

Effects of some fungicides used against cereal pathogens on the growth of Rhizobium trifolii and its capacity to fix nitrogen in white clover

Fungicide residues in soils may affect nitrogen fixation by legumes. Effects of nine systemic fungicides, used against cereal pathogens, on Rhizobium trifolii and white clover were measured. Fenarimol and oxycarboxin inhibited growth of R. trifolii in vitro. The weight of clover plants after 10 weeks' growth in soil containing carboxin, oxycarboxin, benodanil, tridemorph and pyracarbolid was reduced. Symbiotic nitrogen fixation was decreased by carboxin, oxycarboxin and tridemorph present in soil at concentrations somewhat greater than that likely to result from several applications of the fungicide. Only tridemorph reduced nodulation. None of the compounds seems likely to affect nitrogen fixation in the field if applied at the recommended rate.     

Carboxin resistance in Paracoccus denitrificans conferred by a mutation in the membrane-anchor domain of succinate:quinone reductase (complex II)

Succinate:quinone reductase is a membrane-bound enzyme of the citric acid cycle and the respiratory chain. Carboxin is a potent inhibitor of the enzyme of certain organisms. The bacterium Paracoccus denitrificans was found to be sensitive to carboxin in vivo, and mutants that grow in the presence of 3′-methyl carboxin were isolated. Membranes of the mutants showed resistant succinate:quinone reductase activity. The mutation conferring carboxin resistance was identified in four mutants. They contained the same missense mutation in the sdhD gene, which encodes one of two membrane-intrinsic polypeptides of the succinate:quinone reductase complex. The mutation causes an Asp to Gly replacement at position 89 in the SdhD polypeptide. P. denitrificans strains that overproduced wild-type or mutant enzymes were constructed. Enzymic properties of the purified enzymes were analyzed. The apparent K _m for quinone (DPB) and the sensitivity to thenoyltrifluoroacetone was normal for the carboxin-resistant enzyme, but the succinate:quinone reductase activity was lower than for the wild-type enzyme. Mutations conferring carboxin resistance indicate the region on the enzyme where the inhibitor binds. A previously reported His to Leu replacement close to the [3Fe-4S] cluster in the iron-sulfur protein of Ustilago maydis succinate:quinone reductase confers resistance to carboxin and thenoyltrifluoroacetone. The Asp to Gly replacement in the P. denitrificans SdhD polypeptide, identified in this study to confer resistance to carboxin but not to thenoyltrifluoroacetone, is in a predicted cytoplasmic loop connecting two transmembrane segments. It is likely that this loop is located in the neighborhood of the [3Fe-4S] cluster. Received: 18 November 1997 / Accepted: 13 February 1998     

Characterization of succinic dehydrogenase mutants of Bacillus subtilis by crossed immunoelectrophoresis.

Eleven succinic dehydrogenase (SDH) mutants in Bacillus subtilis were analyzed by crossed immunoelectrophoresis with antiserum prepared against wild-type B. subtilis cytoplasmic membrane. A precipitate which stained for SDH was found in Triton X-100-solubilized wild-type membranes and in membranes from two of the SDH mutants. The remaining nine mutants did not show an SDH-staining precipitate. The respective mutations in these nine mutants all map in one locus, citF (Ohné et al., J. Bacteriol. 115:738-745, 1973). An SDH-specific antiserum was prepared by immunizing rabbits with the SDH precipitate obtained in crossed immunoelectrophoresis with solubilized wild-type membrane. Using this antiserum, it was shown that all of the nine citF mutants lack an SDH-specific antigen in the membrane but five of the citF mutants have a soluble SDH-specific antigen. No major differences were found in sodium dodecyl sulfatepolyacrylamide gels of membrane proteins from wild-type B. subtilis and from SDH mutants. A model for the organization of SDH in B. subtilis is proposed.     

Identification of three mutant loci conferring carboxin-resistance and development of a novel transformation system in Aspergillus oryzae

Mutants exhibiting resistance to the fungicide, carboxin, were isolated from Aspergillus oryzae, and the mutations in the three gene loci, which encode succinate dehydrogenase (SDH) B, C, and D subunits, were identified to be independently responsible for the resistance. A structural model of the SDH revealed the different mechanisms that confer carboxin-resistance in different mutations. The mutant AosdhB gene (AosdhB(cxr)) was further examined for possible use as a transformant selection marker. After transformation with AosdhB(cxr), carboxin-resistant colonies appeared within 4 days of culture, and all of the examined colonies carried the transgene. Insertion analyses revealed that the AosdhB(cxr) gene was integrated into AosdhB locus via homologous recombination at high efficiency. Furthermore, AosdhB(cxr) functioned as a successful selection marker in a transformation experiment in Aspergillus parasiticus, suggesting that this transformation system can be used for Aspergillus species.     

Action of respiratory inhibitors on the electron transport system of Escherichia coli]

Bacteria of two strains of Escherichia coli (Q13 and MRE 600) were disintegrated by aluminium oxide. The influence of the respiratory inhibitors RF (a protein from reticulocytes), carboxin, Dexon (fungicides), thenoylftrifluoroacetone (TTFA), rotenone, antimycin A, myristic acid and monolaurin was tested on the succinate oxidase and the NADH oxidase system, respectively, of the membrane preparation obtained in this way as well as on the NADH oxidase activity of the cytosol. Among the inhibitors listed, only TTFA (5mM) inhibited the succinate oxidase system and Dexon (10 miconr), monolaurin (100 micron) and myristic acid (100 micron) inhibited the NADH oxidase system of the membranes. KCN (10 micron) inhibited both NADH oxidase systems. The inhibitory effects by monolaurin and myristic acid were prevent by human serum albumin and were markedly weaker than those on beef heart mitochondrial particles under similar conditions. The results argue for a divergent structure of the iron-sulphur proteins in the dehydrogenase regions of the electron transport system in comparison with animal and plant mitochondria and, moreover, confirm the specificity of RF and carboxin as well as the nature of Dexon as a group reagent on pyridine nucleotide dependent flavin enzymes.     

Fungicidal management of wilt complex of scented geranium

Systemic fungicides, carbendazim + mancozeb and carbendazim alone completely inhibited the mycelia growth even at 100 and 200 ppm, whereas among non-systemic fungicides, thiram was found to be the most effective and gave 52.77, 62.77 and 85.00% inhibition at 500, 1000 and 1500 ppm concentrations, respectively. Thiram was found to be most effective fungicides in inhibiting the both fungi (68.84 and 58.69%) followed by the indofil M 45 (47.59 and 51.38%). In pot house condition, carbendazim + mancozeb (0.2%) and carbendazim (0.1%) were most effective fungicides in reducing cent percent disease incidence and severity, but it was at par with thiram (82.35; 87.50). In field condition, mulching with transparent polyethylene mulch (25 μm) resulted in highest survival rate (95.83%) and least mortality rate (4.16%) of rooted geranium cuttings after 30 days of germination. The application of carbendazim (Bavistin) at 0.1% when applied individually was also found almost complete control as is evident from low disease incidence (1.04%) and severity (0.52%), considered to be second best treatment. Apparent infection rate (0.0) was observed in carbendazim + mancozeb and carbendazim followed by copper oxychloride (0.006) and carboxin (0.009). Area under disease progress curve was less than 100 in cabendazim + mancozeb and carbendazim.     

A sterol 14α-demethylase is required for conidiation, virulence and for mediating sensitivity to sterol demethylation inhibitors by the rice blast fungus Magnaporthe oryzae

The Magnaporthe oryzae genome contains two homologous CYP51 genes, MoCYP51A and MoCYP51B, that putatively encode sterol 14α-demethylase enzymes. Targeted gene deletion mutants of MoCYP51A were morphologically indistinguishable from the isogenic wild type M. oryzae strain Guy11 in vegetative culture, but were impaired in both conidiation and virulence. Deletion of MoCYP51B did not result in any obvious phenotypic changes compared with Guy11. The Δmocyp51A mutants were also highly sensitive to sterol demethylation inhibitor (DMI) fungicides, while Δmocyp51B mutants were unchanged in their sensitivity to these fungicides. Expression of both MoCYP51A and MoCYP51B was significantly induced by exposure to DMI fungicides. Analysis of intracellular localization of MoCyp51A showed that MoCyp51A was mainly localized to the cytoplasm of hyphae and conidia. Taken together, our results indicate that MoCYP51A is required for efficient conidiogenesis, full virulence and for mediating DMI sensitivity by the rice blast fungus.     

Flutolanil and carboxin resistance in<Emphasis Type="Italic"> Coprinus cinereus</Emphasis> conferred by a mutation in the cytochrome<Emphasis Type="Italic"> b</Emphasis><Subscript>560</Subscript> subunit of succinate dehydrogenase complex (Complex II)

A gene that confers resistance to the systemic fungicide flutolanil was isolated from a mutant strain of the basidiomycete Coprinus cinereus. The flutolanil resistance gene was mapped to a chromosome of approximately 3.2 Mb, and a chromosome-specific cosmid library was constructed. Two cosmid clones that were able to transform a wild-type, flutolanil-sensitive, strain of C. cinereus to resistance were isolated from the library. Analysis of a subclone containing the resistance gene revealed the presence of the sdhC gene, which encodes the cytochrome b ₅₆₀ subunit of the succinate dehydrogenase (SDH) complex (Complex II) in the mitochondrial membrane. Comparison between the sdhC gene of a wild-type strain and that of a mutant strain revealed a single point mutation, which results in the replacement of Asn by Lys at position 80. Measurements of succinate-cytochrome c reductase activity in the transformants with mutant sdhC gene(s) suggest that flutolanil resistance of the fungus is caused by a decrease in the affinity of the SDH complex for flutolanil. This sdhC mutation also conferred cross-resistance against another systemic fungicide, carboxin, an anilide that is structurally related to flutolanil. In other organisms carboxin resistance mutations have been found in the genes sdhB and sdhD, but this is the first demonstration that a mutation in sdhC can also confer resistance. The mutant gene cloned in this work can be utilized as a dominant selectable marker in gene manipulation experiments in C. cinereus.Communicated by E. Cerdá-Olmedo     

Direct application of carbendazim and propiconazole at field rates to the external mycelium of three arbuscular mycorrhizal fungi species: effect on 32P transport and succinate dehydrogenase activity

 The influence of the systemic fungicides propiconazole (Tilt 250E) and carbendazim (Bavistin) at field application rates on the functioning of three arbuscular mycorrhizal fungi was studied. Short-term fungal ³²P transport and succinate dehydrogenase (SDH) activity in external hyphae of Glomus intraradices Schenck and Smith, G. claroideum Schenck and Smith and G. invermaium Hall in symbiosis with pea (Pisum sativum L.) were measured. In the experimental system used, the hyphae grew into two root-free hyphal compartments (HCs). The fungicides were applied to each HC 24 days after sowing and ³²P was added to one HC of each pot. Four days later, the fungicide effect on fungal P transport was measured as the difference in ³²P content of treated and untreated plants. SDH activity in fungal hyphae was determined in the HCs given no ³²P. Carbendazim severely inhibited ³²P transport and SDH activity in external hyphae at an application rate of 0.5 μg g^–1 soil. The ergosterol inhibitor propiconazole affected none of these parameters. The fungicides had similar effects on all three fungal species, although P transport efficiency and SDH activity differed markedly between the fungi. Accepted: 12 December 1996     

Mutations in the Saccharomyces cerevisiae succinate dehydrogenase result in distinct metabolic phenotypes revealed through (1)H NMR-based metabolic footprinting

Metabolomics is a powerful method of examining the intricate connections between mutations, metabolism, and disease. Metabolic footprinting examines the extracellular metabolome or exometabolome. We employed NMR-based metabolic footprinting and multivariate statistical analysis to examine a yeast model of mitochondrial dysfunction. Succinate dehydrogenase (SDH) is a component of both the tricarboxylic acid cycle and the mitochondrial respiratory chain. Mutations in the human SDH are linked to a variety of cancers or neurodegenerative disorders, highlighting the genotype/phenotype complexity associated with SDH dysfunction. To gain insight into the underlying global metabolic consequences of SDH dysfunction, we examined the metabolic footprints of SDH3 and SDH4 mutants. We identified and quantified 36 metabolites in the exometabolome. Our results indicate that SDH mutations cause significant alterations to several areas of yeast metabolism. Multivariate statistical analysis allowed us to discriminate between the different metabotypes of individual mutants, including mutants that were phenotypically indistinguishable. Metabotypes were highly correlated to mutant growth yields, suggesting that the characterization of metabotypes offers a rapid means of investigating the phenotype of a new mutation. Our study provides novel insight into the metabolic effects of SDH dysfunction and highlights the effectiveness of metabolic footprinting for examining complex disorders, such as mitochondrial diseases.     

Susceptibility of Various Stages of Trichogrammatoidea armigera Nagaraja to Some Pesticides and Effect of Residues on Survival and Parasitizing Ability

The toxicity, persistence and effect on parasitism of 10 insecticides, eight fungicides and one acaricide on Trichogrammatoidea armigera Nagaraja, an egg parasitoid of a Helicoverpa armigera (Hb), were investigated in the laboratory and under field conditions. At field recommended dosages, the fungicides oxycarboxin, copperoxychloride, streptomycin sulphate + tetracycline hydrochloride and 2‐bromo‐2‐nitropropane‐1,3‐diol and the acaricide dicofol were safe, while the insecticide phosalone and fungicide tridemorph were moderately toxic to adults. All other insecticides tested, namely dimethoate, fenitrothion, monocrotophos, phosphamidon, endosulfan, cypermethrin, decamethrin, fenvalerate and fluvalinate, and the fungicides carbendazim, methyl thiophenate and carboxin were toxic to adults. A high level of parasitism was recorded for all fungicide treatments and for dicofol and fluvalinate. The larval stage of the parasitoid was more tolerant than other stages. The residual toxicity of all fungicides, and dicofol, did not affect the ability of the parasitoid to parasitize its host, while the insecticides phosalone and fluvalinate were slightly persistent, favouring 44.7% and 49.3% parasitism after 15 days. Residues of dimethoate, decamethrin, cypermethrin, fenvalerate, monocrotophos and phosphanidon were moderately persistent, while fenitrothion and endosulfan were persistent.     

Biosynthesis and membrane binding of succinate dehydrogenase in Bacillus subtilis

Antibodies specific for the Mr 65,000 (flavoprotein) and the Mr 28,000 subunits of the succinic dehydrogenase (SDH) of Bacillus subtilis were obtained. By using these antibodies it was shown that both subunits accumulated in the cytoplasm during 5-aminolevulinic acid starvation of a 5-aminolevulinic acid auxotroph. In the cytoplasm the subunits were not associated since they precipitated essentially independently of each other with subunit-specific antibody. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the cytoplasmic subunits migrated identically with the corresponding subunits from the purified membrane-bound SDH complex. Cytoplasmic subunits were pulse-labeled with L-[35S]methionine during 5-aminolevulinic acid starvation. The labeled subunits bound to the membrane when heme synthesis was resumed and also when protein synthesis was blocked by chloramphenicol before readdition of 5-aminolevulinic acid. The experiments thus demonstrated a precursor relationship between cytoplasmic subunits and the subunits of the membrane-bound SDH complex. All SDH-negative mutants isolated so far carry mutations in the citF locus. None of the mutants was found to have either the Mr 65,000 or the Mr 28,000 SDH subunits in the membrane. Four citF mutants, however, contained both subunits in the cytoplasm. Three of these mutants lacked spectrally detectable cytochrome b558. The respective mutations mapped at one end of the citF locus. These results strongly support our previous suggestion that cytochrome b558 is (part of) a membrane binding site for SDH in B. subtilis.