Expanding success in the isolation of abundant marine bacteria after reduction in grazing and viral pressure and increase in nutrient availability

Abstract

ABSTRACT Isolation of microorganisms is a useful approach to gathering knowledge about their genomic properties, physiology, and ecology, in addition to allowing the 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 of the northwest Mediterranean to evaluate the impact of grazing, viral mortality, resource competition reduction, and light presence/absence on bacterioplankton growth. Isolates were retrieved using two growth media, and their full 16S rRNA gene was sequenced to assess their identity and calculate their culturability across seasons and experimental conditions. A total of 1,643 isolates were obtained, mainly affiliated to the classes Gammaproteobacteria (44%), Alphaproteobacteria (26%), and Bacteroidia (17%). Isolates pertaining to class Gammaproteobacteria were the most abundant in all experiments, while Bacteroidia were 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 data set, with dramatic increases in the 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 data set. We also show that top-down and bottom-up controls differentially affect taxa in terms of culturability. Our results indicate that culturing marine bacteria using agar plates can be successful in certain ecological situations. IMPORTANCE Bottom-up and top-down controls greatly influence marine microbial community composition and dynamics, which in turn have effects on their culturability. We isolated 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 culturing marine bacteria. Our data hint at factors influencing culturability and underpin bacterial cultures as a powerful way to discover new taxa.

Keywords

• bacterial isolates
• microcosm experiments
• culturability