Phylogenetic analysis and development of probes for differentiating methylotrophic bacteria

Abstract

Fifteen small-subunit rRNAs from methylotrophic bacteria have been sequenced. Comparisons of these sequences with 22 previously published sequences further defined the phylogenetic relationships among these bacteria and illustrated the agreement between phylogeny and physiological characteristics of the bacteria. Phylogenetic trees were constructed with 16S rRNA sequences from methylotrophic bacteria and representative organisms from subdivisions within the class Proteobacteria on the basis of sequence similarities by using a weighted least-mean-square difference method. The methylotrophs have been separated into coherent clusters in which bacteria shared physiological characteristics. The clusters distinguished bacteria which used either the ribulose monophosphate or serine pathway for carbon assimilation. In addition, methanotrophs and methylotrophs which do not utilize methane were found to form distinct clusters within these groups. Five new deoxyoligonucleotide probes were designed, synthesized, labelled with digoxigenin-11-ddUTP, and tested for the ability to hybridize to RNA extracted from the bacteria represented in the unique clusters and for the ability to detect RNAs purified from soils enriched for methanotrophs by exposure to a methane-air atmosphere for one month. The 16S rRNA purified from soil hybridized to the probe which was complementary to sequences present in 16S rRNA from serine pathway methanotrophs and hybridized to a lesser extent with a probe complementary to sequences in 16S rRNAs of ribulose monophosphate pathway methanotrophs. The nonradioactive detection system used performed reliably at amounts of RNA from pure cultures as small as 10 ng.

Methane oxidation potential and preliminary analysis of methanotrophs in blanket bog peat using molecular ecology techniques

“…Regions diagnostic for each of the available representatives of the five genera and for the identification of RUMP containing methanotrophs were selected for probe design. A probe for methylotrophs containing the serine pathway was also made, this being the complement of the 9a probe previously described by Brusseau et al [16] and Tsien et al [30]. 16s rRNA gene probe sequences and control organisms are shown in Table 1.…”

Bacterial populations occurring in a trichloroethylene‐contaminated aquifer during methane injection

“…3). The D2 and D3 bands were closely related to genera Methylomonas and Methylobacter, respectively, which are known to belong to type I methanotrophs (Brusseau et al, 1994). When the samples from three different depths were analysed in triplicate, the bands marked as D2 and D3 in Fig.…”

Genetic diversity of carbofuran-degrading soil bacteria

“…Homology research and sequence comparisons were achieved on the Infobiogen server (Villejuif, France) with Fasta version 3.3t04 [21] and Align version 2.0u [22] programs. Reference strains of carbofuran-degrading bacteria, Sphingomonas CF06 [23], WM111 [5] and ER2 [7] were included in the phylogenetic tree as well as some reference methylotrophic strains [24].…”

“…Carbofuran-degrading micro-organisms generally utilize methylamine for growth and thus belong to the heterogeneous group of bacteria known as facultative methylotrophs. Several reports have stressed the high degree of diversity among these organisms which can belong to the K-, L-and Q-subdivisions of the Proteobacteria [7,24]. Fig.…”

“…The ribotypes and isolates sequenced are indicated, respectively, by letters and isolate codes. Bold names correspond to reference methylotrophic bacteria as described by Brusseau et al [24]. Underlined bacteria codes or names correspond to reference carbofuran-degrading bacteria, Sphingomonas CF06 [23], WM111 [5] and ER2 [7].…”