Isolation and characterization of members of the phylum Acidobacteria from soils.

摘要

The soil environment is an abundant source of microbial life; recent studies have estimated that in one gram of soil, there are over one million species. These microbes are essential to the environment as major contributors to biogeochemical cyles (e.g. carbon and nitrogen). Unfortunately only 0.1 to 1% of the total microbial community has been cultivated, leaving a wealth of unexplored diversity; one such phylum is the Acidobacteria. The phylum Acidobacteria is an ubiquitous group of microorganisms found in various soil and sediment environments. Historically, it contains eight monophyletic subdivisions and only three cultivated representatives. This dissertation explored members of this unknown phylum by using molecular and growth-based approaches to increase our understanding of acidobacteria ecology in the soil environment.; A phylogenetic survey of soil taken from the KBS LTER revealed the presence of subdivisions 1, 3, 4, 5, and 6, with members of subdivision 4 being more dominant in the conventional agriculture treatment and subdivisions 1 and 6 being more dominant in the successional community treatment. Additionally, the acidobacteria community composition changed in relation to edaphic properties such as soil moisture (subdivision 3), carbon concentration (subdivision 4), soil pH (subdivision 1), methane fluxes (subdivision 1), and nitrous oxide fluxes (subdivision 4). These trends were used to isolate strains of acidobacteria and helped to provide insight into their physiology.; Successful attempts were made to cultivate members of this phylum with soil collected from the KBS LTER using cultivation strategies designed to mimic the native soil environment. After ca. 30 days of growth, total recoveries ranged from ca. 3 to 6% of the total microbial community; soils containing high moisture content had significantly increased total recovery. In order to screen for the presence of acidobacteria on cultivation plates, a facile high-throughput method called Plate Wash PCR (PWPCR) was developed to rapidly screen enrichment plates using phylum-specific 16S rRNA gene primers. Additionally, PWPCR revealed that acidobacteria were more frequently detected with elevated levels of carbon dioxide (significantly), the presence of the catalase enzyme, low nutrients, and low oxygen concentrations.; The use of these cultivation strategies along with PWPCR was instrumental in isolating more than a dozen members of the phylum Acidobacteria from subdivisions 1 and 3. Colonies of these strains were approximately 1 mm in diameter and either white, pale yellow or pink in color, the latter due to a carotenoid that was synthesized preferentially under 20% as compared to 2% oxygen. Strains were Gram negative, aerobic, chemoorganotrophic, nonmotile rods that produced an extracellular matrix causing cells to clump in liquid culture. All strains contained either 1 or 2 copies of the 16S ribosomal RNA encoding gene, which along with a relatively slow doubling time suggests an oligotrophic lifestyle. Genotypic, physiological, and morphological data revealed the presence of a novel genus in subdivision 1, Terriglobus, which contained pigmented strains of acidobacteria. The physiological and nutritional characteristics of these acidobacteria are consistent with their potential widespread distribution in soil.