Development of a group-specific 16S rRNA-targeted probe set for the identification of Marinobacter by fluorescence in situ hybridization

Members of the Marinobacter genus play an important role in hydrocarbon degradation in the ocean - a topic of special significance in light of the recent Deepwater Horizon oil spill of 2010. The Marinobacter group has thus far lacked a genus level phylogenetic probe that would allow in situ identification of representative members. Here, we developed two new 16S rRNA-targeted oligonucleotide probes (Mrb-0625-a and Mrb-0625-b) to enumerate Marinobacter species by fluorescence in situ hybridization (FISH). In silico analysis of this probe set demonstrated 80% coverage of the Marinobacter genus. A competitor probe was developed to block hybridization by Mrb-0625-a to six Halomonas species with which it shared a one base pair mismatch. The probe set was optimized using pure cultures, and then used in an enrichment experiment with a deep sea oil plume water sample collected from the Deepwater Horizon oil spill. Marinobacter cells rapidly increased as a significant fraction of total microbial abundance in all incubations of original contaminated seawater as well as those amended with n-hexadecane, suggesting this group may be among the first microbial responders to oil pollution in the marine environment. The new probe set will provide a reliable tool for quantifying Marinobacter in the marine environment, particularly at contaminated sites where these organisms can play an important role in the biodegradation of oil pollutants.