Manipulation in grazing, viral pressure and resource availability leads to success in the isolation of abundant marine bacteria

Abstract

Isolation of microorganisms is a useful approach to gather knowledge about their genomic properties, physiology, and ecology, in addition to allowing characterization of novel taxa. We performed an extensive isolation effort on samples from seawater manipulation experiments that were carried out during the four astronomical seasons in a coastal site in the NW Mediterranean to evaluate the impact of grazing, viral mortality, resource competition and light on bacterioplankton growth. Isolates were retrieved using two growth media and their full 16S rRNA gene was sequenced to assess their identity and compute their culturability across seasons and experimental conditions. A total of 1643 isolates were obtained, which mainly affiliated to classes Gammaproteobacteria (44%), Alphaproteobacteria (26%) and Bacteroidia (17%). The most commonly isolated genera were Alteromonas and Limimaricola. While isolates varied across culture media, seasons and treatments, those pertaining to class Gammaproteobacteria were the most abundant in all experiments, while Bacteroidia was preferentially enriched in the treatments with reduced grazing. Sixty-one isolates had a similarity below 97% to cultured taxa and are thus putatively novel. Comparison of isolate sequences with 16S rRNA gene amplicon sequences from the same samples showed that the percentage of reads corresponding to isolates was 21.4% within the whole dataset, with dramatical increases in summer virus-reduced (71%) and diluted (47%) treatments. In fact, we were able to isolate the top-10 abundant taxa in several experiments and from the whole dataset. IMPORTANCEThe traditional observation that we can only culture 1% of bacteria for a given environment has recently been questioned on several grounds, among other reasons because it is importantly influenced by environmental conditions. We cultured a high amount of heterotrophic bacterial strains from experiments where seawater environmental conditions had been manipulated and found that decreasing grazing and viral pressure as well as rising nutrient availability are key factors increasing the success in isolating marine bacteria. Our data clearly suggests that the "1% culturability paradigm" needs to be revised and reinforces bacterial cultures as a powerful way to discover new taxa.