Isolation and phylogenetic analysis of aerobic copiotrophic ultramicrobacteria from urban soil

Free-living, aerobic, copiotrophic ultramicrobacteria (UMB) that passed through a 0.45 &mgr;m membrane filter and had a cell volume of less than 0.3 &mgr;m(3) were isolated from polluted urban soil by using both the direct plating method and the membrane-filter enrichment technique. The efficiency of recovering UMB from the soil was much higher in the latter method than in the former. All of the UMB isolates grew well with a doubling time of less than 6 h either in a complex nutrient medium or a chemically defined medium. The average cell volumes of the UMB isolates, as measured by scanning electron microscopy and epifluorescent microscopy with an image analysis, ranged from 0.07 to 0.22 &mgr;m(3). The cell size was larger at the exponential phase of growth than at the stationary growth stage in general. Ultrathin-section electron microscopy of representatives of the UMB isolates showed that they had complete cell wall structures like typical Gram-negative or -positive bacteria. Phenotypic studies and phylogenetic analyses on the basis of 16S rDNA sequences showed that the UMB isolates were classified into three major groups, the beta and gamma subdivisions of the Proteobacteria and the Actinobacteria (the high G+C DNA group of Gram-positives). However, none of these isolates were assigned to any previously known species. These results demonstrate that free-living, relatively fast-growing, copiotrophic UMB strains undescribed so far are widely distributed in terrestrial environments, including urban soil.