Cultivation-Independent Characterization of Methylobacterium Populations in the Plant Phyllosphere by Automated Ribosomal Intergenic Spacer Analysis

Abstract

Bacteria of the genus Methylobacterium are widespread in the environment, but their ecological role in ecosystems, such as the plant phyllosphere, is not very well understood. To gain better insight into the distribution of different Methylobacterium species in diverse ecosystems, a rapid and specific cultivationindependent method for detection of these organisms and analysis of their community structure is needed. Therefore, 16S rRNA gene-targeted primers specific for this genus were designed and evaluated. These primers were used in PCR in combination with a reverse primer that binds to the tRNA Ala gene, which is located upstream of the 23S rRNA gene in the 16S-23S intergenic spacer (IGS). PCR products that were of different lengths were obtained due to the length heterogeneity of the IGS of different Methylobacterium species. This length variation allowed generation of fingerprints of Methylobacterium communities in environmental samples by automated ribosomal intergenic spacer analysis. The Methylobacterium communities on leaves of different plant species in a natural field were compared using this method. The new method allows rapid comparisons of Methylobacterium communities and is thus a useful tool to study Methylobacterium communities in different ecosystems.Bacteria of the genus Methylobacterium are facultative methylotrophs capable of growth on one-carbon compounds, such as methanol, methylamine, formaldehyde, and formate (16). The members of this genus of Alphaproteobacteria are ubiquitous in nature. They have been detected in soil, dust, freshwater, lake sediments, and the air and on plants (16). They have also been found in association with humans (2), and some members of this genus have increasingly been reported to be a cause of opportunistic infections in immunocompromised patients (22). The genus Methylobacterium currently comprises 28 described species (28, 60). Several new species have been described during the last few years, including species from environments that have been analyzed for several decades, such as the plant phyllosphere (23,27,28,38,58). This suggests that the diversity of Methylobacterium is still not fully known. The high diversity of species in the genus Methylobacterium and their broad distribution in nature raises questions of whether different species are adapted to certain environments and whether they play similar ecological roles in different environments. To address these questions, a better understanding of the distribution of Methylobacterium strains between and within different ecosystems and analysis of Methylobacterium community compositions and dynamics in natural habitats are necessary.One of the major habitats of Methylobacterium is represented by plants. Members of this genus have been detected by cultivation-dependent methods (8,12,39,45,59) and, to a lesser extent, by cultivation-independent methods (3,24,25,47,48) as epiphytic and endophytic colonizers of the plant phyllosphere, as intracellular colonizers of the buds of Scots pine (47), and as root-nod...

Site and plant species are important determinants of the Methylobacterium community composition in the plant phyllosphere

“…It has been shown that different plant species grown at the same site harbored communities of different complexity, a finding that is supported by the results of cultivation-dependent studies Raja et al, 2008). Individuals of the same plant species harbored very similar Methylobacterium communities, which differed from those of some other plant species grown at the same site, suggesting a certain degree of plant species specificity (Knief et al, 2008).…”

“…The presence of Methylobacterium strains in the phyllosphere has been shown in numerous studies, but information on the composition of this community on plants and the factors that affect composition is limited and derives mostly from cultivationdependent methods, based on the analysis of a restricted number of isolates. Recently, Methylobacterium communities were for the first time analyzed by a cultivation-independent method (Knief et al, 2008). It has been shown that different plant species grown at the same site harbored communities of different complexity, a finding that is supported by the results of cultivation-dependent studies Raja et al, 2008).…”

Isolation of Optically Targeted Single Bacteria by Application of Fluidic Force Microscopy to Aerobic Anoxygenic Phototrophs from the Phyllosphere

“…The isolation of Methylobacterium spp. was not unexpected, since these previously described AAnPs (61,62) are known to occur in high numbers in the phyllosphere (1,2,4,(65)(66)(67)(68) and harbor genes for a photosynthetic reaction center as well as genes for bacteriochlorophyll biosynthesis based on completely genome-sequenced strains of this genus (with the exception of the nonpigmented nodulating root symbiont Methylobacterium nodulans) (69,70). However, the exclusive isolation of methylobacteria was not anticipated, because the diversity observed at the genomic level by metagenome studies suggested a higher diversity of pufM genes than was expected to be derived solely from Methylobacterium spp.…”

Abundance and Composition of Epiphytic Bacterial and Archaeal Ammonia Oxidizers of Marine Red and Brown Macroalgae

“…For biofilm detachment from the algal surfaces, the cut algae were immersed in 2 ml of 0.1 M sodium pyrophosphate (Na 4 P 2 O 7 · 10H 2 O). Biofilm was dislodged by two rounds of sonication for 20 s at maximum intensity (Selecta, Barcelona, Spain), followed by 30 s on ice (23). Algal suspensions were centrifuged at 10,000 ϫ g for 2 min, and supernatants were discarded.…”