Methylobacterium Organophilum Research Articles

Phospholipid and lipopolysaccharide normal and hydroxy fatty acids as potential signatures for methane-oxidizing bacteria

The extractable ester-linked and the lipopolysaccharide (LPS) normal and hydroxy fatty acids of the methylotrophic bacteria Methylosinus trichosporium 0B3B, Methylobacterium organophilum XX, grown on methane and methanol, Mb. organophilum RG and Methylomonas sp. were analysed by capillary gas chromotography-mass spectrometry (GC-MS). Precise monounsaturated double bond position and geometry was determined by GC-MS analysis of the derivatized fatty acids. The three species were readily distinguished based on the extractable fatty acid and LPS hydroxy acid profiles. Type I and Type II methylotrophs can be separated based on the presence of 16-carbon and 18-carbon monoenoic fatty acids in the two groups of organisms, respectively. Relatively novel components, 18 : 1ω8c, 18 : 1ω8t, 18 : 1ω7t and 18 : 1ω6c were present in Ms. trichosporium, and 16 : 1ω8c, 16 : 1ω8t, 16 : 1ω7t, 16 : 1ω5c and 16 : 1ω5t were detected in Methylomonas sp. These specific lipids may be used, together with other components, as signatures for these methylotrophic bacteria in manipulated laboratory and environmental samples.

Prokaryotic triterpenoids. New bacteriohopanetetrol cyclitol ethers from the methylotrophic bacterium Methylobacterium organophilum.

Together with bacteriohopanetetrol, which is identical to bacteriohopanetetrol isolated from Bacillus acidocaldarius, three other bacteriohopane derivatives were isolated from the methylotrophic bacterium Methylobacterium organophilum. Three different kinds of polar moieties were found linked to the C-35 hydroxyl group of bacteriohopanetetrol: glucosamine linked through a glycosidic bond and two new polyhydroxylated methylcyclopentanoids differing one from each other by the presence of an amino or a guanidino group and linked through an ether bond. The significance of the presence of these compounds in terms of membrane reinforcement and DNA/triterpenoid interactions is discussed.

2. 2β‐Methylhopanoids from Methylobacterium organophilum and Nostoc muscorum, a new series of prokaryotic triterpenoids

European Journal of BiochemistryVolume 150, Issue 1 p. 33-34 Free Access 2. 2β-Methylhopanoids from Methylobacterium organophilum and Nostoc muscorum, a new series of prokaryotic triterpenoids Philippe Bisseret, Philippe BisseretSearch for more papers by this authorMagali Zundel, Magali ZundelSearch for more papers by this authorMichel Rohmer, Michel RohmerSearch for more papers by this author Philippe Bisseret, Philippe BisseretSearch for more papers by this authorMagali Zundel, Magali ZundelSearch for more papers by this authorMichel Rohmer, Michel RohmerSearch for more papers by this author First published: July 1985 https://doi.org/10.1111/j.1432-1033.1985.tb08983.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume150, Issue1July 1985Pages 33-34 RelatedInformation

Prokaryotic triterpenoids. 2. 2 beta-Methylhopanoids from Methylobacterium organophilum and Nostoc muscorum, a new series of prokaryotic triterpenoids.

2 beta-Methylhopanoids, a new series of triterpenoids was identified from two prokaryotes. 2 beta-Methyldiplopterol was isolated from the methylotrophic bacterium Methylobacterium organophilum, and three different 2 beta-methylbacteriohopanepolyols from the cyanobacterium Nostoc muscorum. The structures of these compounds was deduced by direct comparison with 2 beta-methyldiplopterol synthesized from 22-hydroxyhopan-3-one.

Prokaryotic triterpenoids

The incorporation of L-[methyl-3H,14C]methionine or L-(methyl-2H3)methionine into 2 beta-methyldiplopterol of Methylobacterium organophilum and various 3 beta-methylhopanoids of Acetobacter pasteurianus ssp. pasteurianus showed that all three hydrogen atoms of the transferred methyl group are retained in the triterpenoids. These methylations are compatible with a methylation substrate such as a delta 2-hopanoid in the case of the 2 beta-methylhopanoid biosynthesis and of a delta 2-hopanoid or squalene in the case of the formation of 3 beta-methylhopanoids. The intervention of intermediates possessing an exomethylene group or a cyclopropane ring is excluded.

Construction of broad-host-range cosmid cloning vectors: identification of genes necessary for growth of Methylobacterium organophilum on methanol.

Four new cloning vectors have been constructed from the broad-host-range cloning vector pRK290. These vectors, pLA2901, pLA2905, pLA2910, and pLA2917, confer resistance to kanamycin and tetracycline. The latter two are cosmid derivatives of pLA2901. The new vectors can be mobilized into, and are stably maintained in, a variety of gram-negative bacteria. A Sau3A genomic bank of Methylobacterium organophilum strain xx DNA has been constructed in pLA2917, and complementation analysis, with a variety of mutants unable to grow on methanol, revealed at least five separate regions necessary for growth on methanol. Complementation analysis and Tn5 mutagenesis data suggest that at least three genes are responsible for expression of active methanol dehydrogenase.

Emendation of Methylobacterium Patt, Cole, and Hanson 1976; Methylobacterium rhodinum (Heumann 1962) comb. nov. corrig.; Methylobacterium radiotolerans (Ito and Iizuka 1971) comb. nov. corrig.; and Methylobacterium mesophilicum (Austin and Goodfellow 1979) comb. nov.

The present definition of the genus Methylobacterium excludes organisms which do not utilize methane facultatively. However, reports have indicated that cultures of the type strain of the type species, Methylobacterium organophilum, can lose this feature easily. Furthermore, studies have shown that M. organophilum is phenotypically highly similar to the methane-nonutilizing, pink-pigmented, facultatively methylotrophic bacteria and that the latter should be excluded from the various genera to which they have been assigned previously. Therefore, in accordance with an earlier suggestion, we propose that the description of the genus Methylobacterium be emended to permit the inclusion of methane-nonutilizing organisms which are otherwise highly similar to the type species. We further propose that all of the pink-pigmented, facultatively methylotrophic bacteria, which include the species Pseudomonas rhodos, Pseudomonas radiora, and Pseudomonas mesophilica, be transferred to the genus Methylobacterium.

Genetic analysis of the streptomycin resistance encoded by Tn5

Two different aminoglycoside phosphotransferases were separated in crude extracts of Methylobacterium organophilum (Tn5). One is the aminoglycoside phosphotransferase (3′) type II (APH(3′)II), the only aminoglyconside modifying enzyme expressed by Tn5 in E. coli; the other is a streptomycin phosphotransferase (SPH). Various deletion mutants of Tn5 were cloned into pBR322 (plasmids pX) and co-transferred by the plasmid pJB3J1 into M. organophilum where stable cointegrants were formed. By the analysis of the resulting phenotypes, the streptomycin resistance gene was located in a 1,000 bp segment of the central region of Tn5. This gene is not expressed in E. coli. The possibility that this resistance gene is the structural gene of the SPH, is discussed. The formation of the cointegrants (pJB3J1; pX) is mediated through the IS21 sequence of pJB3J1. From two cointegrants KmR SmR in M. organophilum (pPM111 and pPM113), mutations KmS SmR and Kmr SmS were isolated. Restriction analysis of these mutants showed that the direction of transcription of the kanamycin and of the streptomycin resistance genes is identical. The existence of a particular promoter for the streptomycin resistance gene could not be firmly established because IS21 might have a promoter activity. However the postulated promoter was located toward the 3′ end of the APH(3′)II (Km) gene.

Extension of the model concerning linkage of genes coding for C-1 related functions in Methylobacterium organophilum.

Evidence is presented which suggests that Methylobacterium organophilum contains isoenzymes of phosphoenolpyruvate carboxylase activity. Methanol-grown cells contained an acetyl coenzyme A (CoA)-insensitive activity which precipitated in a 65 to 75% of saturation ammonium sulfate fraction. Succinate-grown cells contained an acetyl-CoA-stimulated activity which precipitated in a 55 to 65% of saturation ammonium sulfate fraction. Mutants unable to grow on methanol appeared to lack acetyl-CoA-insensitive activity. This acetyl-CoA-insensitive phosphoenolpyruvate carboxylase, along with malyl-CoA lyase, is proposed to be encoded by the C-1 operon. The gene for formate dehydrogenase appeared to reside outside the operon and was not inducible by methanol. M. organophilum was unable to grow on formate, and evidence is presented suggesting that formate is unable to induce the enzymes which comprise the serine pathway for formaldehyde fixation. An expanded model for the C-1 operon is presented.

Microbial oxidation of gaseous hydrocarbons: epoxidation of C2 to C4 n-alkenes by methylotrophic bacteria.

Over 20 new cultures of methane-utilizing microbes, including obligate (types I and III) and facultative methylotrophic bacteria were isolated. In addition to their ability to oxidize methane to methanol, resting cell-suspensions of three distinct types of methane-grown bacteria (Methylosinus trichosporium OB3b [type II, obligate]; Methylococcus capsulatus CRL M1 NRRL B-11219 [type I, obligate]; and Methylobacterium organophilum CRL-26 NRRL B-11222 [facultative]) oxidize C2 to C4 n-alkenes to their corresponding 1,2-epoxides. The product 1,2-epoxides are not further metabolized and accumulate extracellularly. Methanol-grown cells do not have either the epoxidation or the hydroxylation activities. Among the substrate gaseous alkenes, propylene is oxidized at the highest rate. Methane inhibits the epoxidation of propylene. The stoichiometry of the consumption of propylene and oxygen and the production of propylene oxide is 1:1:1. The optimal conditions for in vivo epoxidation are described. Results from inhibition studies indicate that the same monooxygenase system catalyzes both the hydroxylation and the epoxidation reactions. Both the hydroxylation and epoxidation activities are located in the cell-free particulate fraction precipitated between 10,000 and 40,000 x g centrifugation.

Alcohol dehydrogenase from Methylobacterium organophilum.

The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 x 10(-5) M for methanol and 8.2 x 10(-5) M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum.

Intracytoplasmic membrane, phospholipid, and sterol content of Methylobacterium organophilum cells grown under different conditions.

Intracytoplasmic membranes were present in Methylobacterium organophilum when cells were grown with methane, but not methanol or glucose, as the sole carbon and energy source. Cells grown with methane as the carbon and energy source and low levels of dissolved oxygen had the greatest amount of intracytoplasmic membrane. Cells grown with increased levels of dissolved oxygen had less intracytoplasmic membrane. The amount of total lipid correlated with the amount of membrane material observed in thin sections. The individual phospholipids varied in amount, but the same four were present in M. organophilum grown with different substrates and oxygen levels. Phosphatidyl choline was present as a major component of the phospholipids. Sterols were present, and they differed from those in the type I methylotroph Methylococcus capsulatus. The relative amounts of different sterols and squalene changed with the substrate provided for growth. The greatest amounts of sterols were found in methane-grown cells grown at low levels of dissolved oxygen. None of the unusual or usual membrane components assayed was uniquely present in the intracytoplasmic membranes.

Linkage Relationships Between Mutants of Methylobacterium organophilum Impaired in their Ability to Grow on One-carbon Compounds

Eight mutant strains of Methylobacterium organophilum which are capable of growing on succinate but not on methanol are described. Three of these contain single enzyme deficiencies while five have pleiotropic deficiencies. Some of the mutant strains are revertible to the wild-type phenotype by N-methyl-N′-nitro-N-nitrosoguanidine and ethyl methanesulphonate and others are not. Crosses between strains were performed by transformation using DNA prepared from cells which were streptomycin-resistant and either methanol+ or methanol−. The frequencies of streptomycin-resistant transformants and methanol+ transformants were determined independently. Four of the methanol− mutant strains appeared to contain mutations which are linked to each other (group 1) while the other four (group 2) appeared to contain mutations not linked to the first four. In reciprocal crosses with the group 2 markers, two appeared to be linked to each other, while the other two were not linked to any mutant tested. The data presented were used to construct a model for the organization of these genes in which the CO-binding cytochrome c is not linked to the five genes for C1-specific enzymes. In this model, the expression of six C1-specific functions is controlled by a common regulatory agent.

Enzyme Regulation in Methylobacterium organophilum

Several enzymes have been assayed in Methylobacterium organophilum grown on different substrates. The enzymes which are involved in growth on C1 compounds were induced by methanol and not repressed by succinate. When succinate-grown bacteria were resuspended in medium containing methanol, four enzymes unique to growth on C1 compounds (hydroxypyruvate reductase, serine-glyoxylate aminotransferase, methanol dehydrogenase and glycerate kinase) were fully induced by the time growth began. When methanol-grown bacteria were resuspended in medium containing succinate, all four enzyme activities decreased. Several mutants unable to grow on C1 compounds were examined for deficiencies in the enzymes specific for growth on these compounds. Seven of the mutants were pleiotropic, and six were not revertible by chemical mutagens, suggesting the possibility of genetic linkage or the presence of a regulon for the genes involved in C1 metabolism.

Genetic Transformation in Methylobacterium organophilum

Several mutants have been isolated from the facultative methylotroph, Methylobacterium organophilum, using either N-methyl-N'-nitro-N-nitrosoguanidine or ultraviolet light as mutagens. One of these isolates, a glutamate auxotroph lacking isocitrate dehydrogenase, has been transformed to prototrophy, using wild-type DNA, at a frequency of 0-5%. Competence and DNA uptake occur only in cultures which are near the end of exponential growth, and maximal transformation requires a DNA concentration of 100 mug ml-1.

Methylobacterium, a New Genus of Facultatively Methylotrophic Bacteria

A new genus and a new species of methane-oxidizing bacteria are described. The colonies produced by these bacteria are pink, circular, and convex with entire margins. Cells are gram negative and are normally found singularly with some rosettes. Negative stains indicate polar flagellation. In thin sections, intracytoplasmic membranes, similar to those described as type II in other methylotrophs, were present when the cells were grown with methane. No such membranes were apparent when the cells were grown with the other carbon and energy sources tested. The serine pathway for formaldehyde incorporation is the pathway of C1 metabolism. The deoxyribonucleic acid base composition is 66 mol% guanine plus cytosine. Methylobacterium is proposed as the name for this new genus of rod-shaped, methane-oxidizing bacteria. The specific epithet in the name of the type species, Methylobacterium organophilum sp. nov., denotes the preference of this organism for organic carbon and energy sources more complex than methane. The type strain of M. organophilum is XX (= ATCC 27886). This bacterium differs from all previously described genera and species of methane-oxidizing bacteria in its ability to utilize a variety of organic substrates with carbon-carbon bonds as sources of carbon and energy. The pathways for methane oxidation and the assimilation of one-carbon units are repressed during growth on complex organic substrates.