Isolation and Characterization of Paracoccus Denitrificans Mutants with Defects in the Metabolism of One-Carbon Compounds NELLIE HARMS,'* GERT E
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JOURNAL OF BACTERIOLOGY, Dec. 1985, p. 1064-1070 Vol. 164, No. 3 0021-9193/85/121064-07$02.00/0 Copyright © 1985, American Society for Microbiology Isolation and Characterization of Paracoccus denitrificans Mutants with Defects in the Metabolism of One-Carbon Compounds NELLIE HARMS,'* GERT E. DE VRIES,2 KICK MAURER,' EDUARD VELTKAMP,2 AND ADRIAAN H. STOUTHAMER' Department of Microbiology' and Department of Genetics,2 Biological Laboratory, Free University, 1007 MC Amsterdam, The Netherlands Received 11 June 1985/Accepted 12 September 1985 Mutants deficient in the metabolism of one-carbon compounds have been obtained by treating Paracoccus denitrificans with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Mutants were selected without enrich- ment procedures by newly developed plate screening tests. The obtained mutants were characterized by their growth responses, cytochrome composition, ehizyme activities, and immunogenic reaction with antisera against methanol dehydrogenase. By these criteria five mttant classes could be distinguished. Class I mutants are involved in the expression of methaniol dehydrogenase. Three mutants of this class have a defect in the structural gene. A double mutant was found with defects in the expression of both methanol dehydrogenase and hydrogenase. Class II mutants have a defect in a regulatory gene involved in the regulation of both methanol dehydrogenase and methylamine dehydrogenase. Class III mutants are deficient in formaldehyde metabolism. A defect may exist in the expression of a second non-NAD-linked formaldehyde dehydrogenase which was postulated to be involved in C1 metabolism. Class IV mutants are deficient in cytochrome c. Mutants of class V have a defect in synthesis of the molybdenum cofactor essential for the function of formate dehydrogenase. Paracoccus denitrificans is a gram-negative bacterium and UV radiation of M. organophilum that were unable to capable of autotrophic growth with methanol, methylamine, grow with methanol (11, 16, 26). Because these organisms do or formate as an energy source. In addition, a variety of not grow autotrophically, specific selection of mutations in substrates can be used heterotrophically (24). Methanol arid methanol dissimilation was not possible, and the majority of methylamine are oxidized to formaldehyde by methanol the mutants isolated were found to have defects in the serine dehydrogenase (MDH) and methylamine dehydrogenase, pathway. However, mutants with defects in MDH or respectively (4, 10, 20). Electrons from methanol flow from cytochrome c from Pseudomonas sp. AMl; M. organo- the MDH cofactor pyrroloquinoline quinone (PQQ) via a philum, and also Hyphomicrobium sp. X have been reported postulated CO-binding c-type cytochrome, cytochromes c (11, 16, 21, 28, 31). Whether these mutants have defects in and aa3 to oxygen (36). In P. denitrificans all formaldehyde the structural MDH gene, in regulatory genes, or in ancillary generated is thought to be oxidized via formate to carbon functions is unclear. Linkage studies in Pseudomonas sp. dioxide by NAD-dependent dehydrogenases. For synthesis AM1 were performed with the broad-host-range plasmid of biomass, carbon dioxide is fixed by ribulose bisphosphate R68.45, which was shown to mobilize the chromosome in carboxylase, the key enzyme of the Calvin cycle (4, 10). this organism (31, 32). Linkage was determined between In methylotrophs using the ribulose bisphosphate cycle, genes involved in the proper expression of MDH, methyl- no data are available on the genetics of methanol oxidation at amine dehydrogenase, and cytochrome c and in the genes this time. The isolation of mutants in other methylotrophs determining resistance to streptomycin, phosphonomycin, has proveti to be difficult. Williams and co-workers were and cycloserine. Linkage relationships in M. organophilum unable to induce mutations by UV irradiation in have been investigated by a gene transformation system Methylomonas albus, Methylococcus capsulatus, and developed by O'Connor et al. (25, 27, 28). The genes coding Methylosinus trichosporium (OB4) and succeeded only with for the enzymes of formaldehyde assimilation and genes the mutagen N-methyl-N'-nitro-N-nitrosoguanidine (NTG) involved in MDH expression were reported to be grouped on (37, 38). They suggested that the lack of error-prone SOS a single operon. However, recent results from Allen and DNA repair mechanisms was responsible for this phenome- metabolism are non. Genetic studies have been performed with the obligate Hanson (1) show that the genes for methanol methylotroph Methylophilus methylotrophus, where carbon not closely linked. Genes involved in methanol oxidation fixation follows the ribulose monophosphate pathway. Tem- have been cloned from Pseudomonas sp. AM1 (13, 14) and perature-sensitive, auxotrophic, and glutamate synthase- M. organophilum (1) after identification by complementation deficient mutants have been isolated with NTG (8, 41). of a variety of mutants. However, no mutants with defects in methanol utilization The efficiency and energetics of methanol metabolism have yet been reported. Studies have focused primarily on have been subject of previous studies on P. denitrificans in the facultative methylotrophs Pseudomonas sp. AM1 and our laboratory. Here we report the isolation and character- Methylobacterium organophilum, where carbon assimilation ization of mutants with defects in the metabolism of one- follows the Icl- variant of the serine pathway. Mutants have carbon compounds. In conjunction with the studies on the been isolated by NTG treatment of Pseudomonas sp. AM1 physiological regulation of MDH induction (G. E. de Vries, N. Harms, and K. Maurer, manuscript in preparation), this study forms the basis for future cloning of P. denitrificans * Corresponding author. genes involved in methanol oxidation. 1064 VOL. 164, 1985 P. DENITRIFICANS MUTANTS IN METHANOL OXIDATION 1065 TABLE 1. Bacterial strainsa triphenyltetrazolium chloride per ml. Among red-staining colonies reducing triphenyltetrazolium chloride to the insol- Strain Relevant phenotype' reference uble formazan, white colonies were visible that were picked from the original glycerol plates and tested further for PD1001 Wild type NCIB 8944 growth on methanol. Controls, with the and (DSM 413) wild type the PD11O1 PD1001, Rif This study previously isolated cytochrome c-deficient mutant PD1201 PD1103 P O1101, Spcr This study (35), indicated that incubation on plates either with or PD1201 PD1001, cyt c- (35) without methanol for periods shorter than 24 h always PD1205 PD1101, MeOH-, allyl OH' This study resulted in red colonies. Beyond this period only the wild PD1210 PD1001, Stri, cyt aa3- NS-3 (39) type retained the capacity to reduce triphenyltetrazolium PD1214 through PD1101, MeOH- This study chloride, demonstrating utilization of methanol. With this PD1221 screening procedure 25,000 colonies were tested, and 15 a The rmutants PD1214 through PD1221 were derived from PD1101, except mutants were found that were unable to grow on methanol. the strains PD1215 and PD1220, which are derivatives of strain PD11Q3. In method B, mutagenlied cells were seeded on 9-cm b Rif, rifampin resistance; Spc, sjpectinomycin resistance; Str, streptomycin methanol-agar plates that, were covered with 4 ml of 0.1% resistance; cyt c -, cytochrome c negative; cyt aa'-, cytochrome aa3 nega- ethanol-MM agar. After incubation for 72 h, pinpoint colo- tive; MeOH-, no growth on methanol; allyl OHr, resistance to allyl alcohol; NS-3, definition. nies were detected among otherwise normal-appearing col- onies. In addition a triphenyltetrazolium chloride overlay as described above was performed, and large nonstaining col- onies were found (method C). Both colony types were MATERIALS AND METHODS purified by restreaking and tested for growth on methanol; Among 21,000 colonies tested, 3 mutants that were unable to Bacterial strains and culture conditions. The P. denitrificans grow on methanol were selected via method B, and 5 bacterial strains used in this study are listed in Table 1. Strains mutants were selected via method C. In method D, mutagen- were maintained on Luria,broth (LB) agar plates or stored in ized cells were plated on glycerol-ethanol-MM agar contain- 50% glycerol at -20°C. The bacteria were grown in batch ing. 0.01% allyl alcohol. A single mutant, PD1205, unable to cultures at 32°C either on LB or on mineral salts medium use methanol as the sole source of energy was found among (MM) containing the following (per liter): KH2PO4, 1.7 g; allyl alcohol-resistant colonies. K2HPO4, 8 g; NH4Cl, 1.0 g; Na2MoO4- 2H20, 145 mg; Revertants. Depending on the existing growth deficiencies, MnSO4, 2 mg; EDTA, 37 mg; Fe citrate, 5 ml of a solution a few drops, of a fresh culture of each mutant were spread containing 4 mM FeSO4, 5 mM citric acid, and 18 mM H2SO4; onto one of the following substrates: methanol, methyl- NaHCO3, 0.5 g; CaCl2, 73 mg; MgCl2, 0.1 g (pH 7.5). amine, choline, or glycerol plus nitrate. In the center of the Filter-sterilized carbon sources were added at 0.5% (wt/vol), plate, 10 ,ul of ethyl methanesulfonate or NTG (1 mg/ml) was except formate, which was added at 0.1% (wt/vol). For placed. After 2 to 5 days colonies appeared which were anaerobic growth, MM was used with KNO3 (25 mM) as the purified by restreaking and tested for the original mutant terminal electron acceptor. For autotrophic growth on phenotype. molecular hydrogen as the sole source of energy, the Enzyme activities. All spectral studies were performed composition of the gas phase was 90% N2, 5% C02, 4% H2, with an Aminco DW-2a spectrophotometer (American In- 1% 02. Chemostat cultures were performed in a Bioflo strument Co.). Enzyme activities were determined by fol- fermentor with a working volume of 250 ml under carbon lowing chaniges in dual-wavelength absorption measure- limitation on MM supplemented with 0.01% Difco yeast ments at 25°C (dichlorophenolindophenol, 612, reference 580 extract, 5 mM glucose, and 7.5 mM methanol at a dilution rate nm; NADH, 340, reference 375 nm). For dye-linked enzyme of 0.01 h-1 at 32°C (pH 7.5).