Acetate utilization as a survival strategy of peat-inhabiting Methylocystis spp.

Representatives of the genus Methylocystis are traditionally considered to be obligately methanotrophic bacteria, which are incapable of growth on multicarbon substrates. Here, we describe a novel member of this genus, strain H2s, which represents a numerically abundant and ecologically important methanotroph population in northern Sphagnum-dominated wetlands. This isolate demonstrates a clear preference for growth on methane but is able to grow slowly on acetate in the absence of methane. Strain H2s possesses both forms of methane monooxygenase (particulate and soluble MMO) and a well-developed system of intracytoplasmic membranes (ICM). In cells grown for several transfers on acetate, these ICM are maintained, although in a reduced form, and mRNA transcripts of particulate MMO are detectable. These cells resume their growth on methane faster than those kept for the same period of time without any substrate. Growth on acetate leads to a major shift in the phospholipid fatty acid composition. The re-examination of all type strains of the validly described Methylocystis species showed that Methylocystis heyeri H2(T) and Methylocystis echinoides IMET10491(T) are also capable of slow growth on acetate. This capability might represent an important part of the survival strategy of Methylocystis spp. in environments where methane availability is variable or limited.

Methyloferula stellata gen. nov., sp. nov., an acidophilic, obligately methanotrophic bacterium that possesses only a soluble methane monooxygenase.

Two strains of aerobic methanotrophic bacteria, AR4(T) and SOP9, were isolated from acidic (pH 3.8-4.0) Sphagnum peat bogs in Russia. Another phenotypically similar isolate, strain LAY, was obtained from an acidic (pH 4.0) forest soil in Germany. Cells of these strains were Gram-negative, non-pigmented, non-motile, thin rods that multiplied by irregular cell division and formed rosettes or amorphous cell conglomerates. Similar to Methylocella species, strains AR4(T), SOP9 and LAY possessed only a soluble form of methane monooxygenase (sMMO) and lacked intracytoplasmic membranes. Growth occurred only on methane and methanol; the latter was the preferred growth substrate. mRNA transcripts of sMMO were detectable in cells when either methane or both methane and methanol were available. Carbon was assimilated via the serine and ribulose-bisphosphate (RuBP) pathways; nitrogen was fixed via an oxygen-sensitive nitrogenase. Strains AR4(T), SOP9 and LAY were moderately acidophilic, mesophilic organisms capable of growth between pH 3.5 and 7.2 (optimum pH 4.8-5.2) and at 4-33 °C (optimum 20-23 °C). The major cellular fatty acid was 18 : 1ω7c and the quinone was Q-10. The DNA G+C content was 55.6-57.5 mol%. The isolates belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and were most closely related to the sMMO-possessing methanotrophs of the genus Methylocella (96.4-97.0 % 16S rRNA gene sequence similarity), particulate MMO (pMMO)-possessing methanotrophs of the genus Methylocapsa (96.1-97.0 %), facultative methylotrophs of the genus Methylovirgula (96.1-96.3 %) and non-methanotrophic organotrophs of the genus Beijerinckia (96.5-97.0 %). Phenotypically, strains AR4(T), SOP9 and LAY were most similar to Methylocella species, but differed from members of this genus by cell morphology, greater tolerance of low pH, detectable activities of RuBP pathway enzymes and inability to grow on multicarbon compounds. Therefore, we propose a novel genus and species, Methyloferula stellata gen. nov., sp. nov., to accommodate strains AR4(T), SOP9 and LAY. Strain AR4(T) ( = DSM 22108(T)  = LMG 25277(T)  = VKM B-2543(T)) is the type strain of Methyloferula stellata.

Bacterial cultures preferentially removing singly flanked chlorine substituents from chlorobenzenes.

The wide though not ubiquitous distribution of chlorobenzene-dechlorinating bacteria in anaerobic sludge from German sewage plants is demonstrated. The model substrates 1,2,3- and 1,2,4-trichlorobenzene (TCB) were dechlorinated to dichlorobenzenes (DCBs) and monochlorobenzene (MCB) via distinct pathways. For easy visualization and differentiation of the pathways, a novel plotting method was developed. While many of the cultures showed a dechlorination pattern similar to that previously found for Dehalococcoides species, removing doubly flanked rather than singly flanked chlorine substituents from TCBs, some cultures formed 1,2-DCB from 1,2,3-TCB and/or 1,3-DCB from 1,2,4-TCB. Stable cultures preferentially catalyzing the removal of singly flanked chlorines were obtained by repeated subcultivation in sediment-free synthetic medium. This dechlorination pattern is potentially of great benefit for remediation as the accumulation of persistent intermediates such as 1,3,5-TCB from highly chlorinated compounds can be avoided. In addition, the cultures dechlorinated 1,3,5-TCB, pentachlorobenzene (PeCB), and hexachlorobenzene (HCB). Nested PCR demonstrated the presence of low numbers of Dehalococcoides species. However, the observed insensitivity of the dechlorinating bacteria in our cultures to oxygen and sensitivity to vancomycin is not in accordance with the reported properties of Dehalococcoides species, suggesting that other bacteria than Dehalococcoides catalyzed the removal of singly flanked chlorines from TCB.

Two isozymes of particulate methane monooxygenase with different methane oxidation kinetics are found in Methylocystis sp. strain SC2.

Methane-oxidizing bacteria (methanotrophs) attenuate methane emission from major sources, such as wetlands, rice paddies, and landfills, and constitute the only biological sink for atmospheric methane in upland soils. Their key enzyme is particulate methane monooxygenase (pMMO), which converts methane to methanol. It has long been believed that methane at the trace atmospheric mixing ratio of 1.75 parts per million by volume (ppmv) is not oxidized by the methanotrophs cultured to date, but rather only by some uncultured methanotrophs, and that type I and type II methanotrophs contain a single type of pMMO. Here, we show that the type II methanotroph Methylocystis sp. strain SC2 possesses two pMMO isozymes with different methane oxidation kinetics. The pmoCAB1 genes encoding the known type of pMMO (pMMO1) are expressed and pMMO1 oxidizes methane only at mixing ratios >600 ppmv. The pmoCAB2 genes encoding pMMO2, in contrast, are constitutively expressed, and pMMO2 oxidizes methane at lower mixing ratios, even at the trace level of atmospheric methane. Wild-type strain SC2 and mutants expressing pmoCAB2 but defective in pmoCAB1 consumed atmospheric methane for >3 months. Growth occurred at 10-100 ppmv methane. Most type II but no type I methanotrophs possess the pmoCAB2 genes. The apparent K(m) of pMMO2 (0.11 muM) in strain SC2 corresponds well with the K(m(app)) values for methane oxidation measured in soils that consume atmospheric methane, thereby explaining why these soils are dominated by type II methanotrophs, and some by Methylocystis spp., in particular. These findings change our concept of methanotroph ecology.