Phylogenetic analysis and development of probes for differentiating methylotrophic bacteria

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.