Widespread distribution and high abundance of Rhizobium radiobacter within Mediterranean subsurface sediments

Eastern Mediterranean sediments are characterized by the occurrence of distinct, organic-rich layers, called sapropels. These harbour elevated microbial numbers in comparison with adjacent carbon-lean intermediate layers. A recently obtained culture collection from these sediments was composed of 20% of strains closely related to Rhizobium radiobacter, formerly classified as Agrobacterium tumefaciens. To prove and quantify the in situ abundance of R. radiobacter, a highly specific quantitative polymerase chain reaction (PCR) protocol was developed. To convert quantification results into cell numbers, the copy number of rrn operons per genome was determined. Southern hybridization showed that our isolates contained four operons. Finally, quantitative PCR was applied to 45 sediment samples obtained across the eastern Mediterranean. Rhizobium radiobacter was present in 38 of 45 samples indicating an almost ubiquitous distribution. In total, 25-40 000 cells per gram of sediment were detected, corresponding to 0.001-5.1% of the bacterial cells. In general, the relative and absolute abundance of R. radiobacter increased with depth and was higher in sapropels than in intermediate layers. This indicates that R. radiobacter forms an active population in up to 200 000 years old sapropels. The present study shows for the first time that a cultivated subsurface bacterium is highly abundant in this environment.