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

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.

Genomic and phenotypic characterization of 26 novel marine bacterial strains with relevant biogeochemical roles and widespread presence across the global ocean

“…Subsamples of 1 mL were stored in cryovials at −80°C with 75 μL DMSO until the time of isolation. The isolation was carried out between November 2020 and January 2022 using samples from the initial and final times of the experiments, as reported in Rey-Velasco et al (2023) . Briefly, aliquots of 100 μL of undiluted, 1:10 and 1:100 diluted seawater were spread in triplicate on Marine Agar 2216 (MA; Difco) and Marine Reasoner’s 2A Agar (mR2A) plates and incubated at room temperature (20–25°C) until no more colonies appeared (maximum 30 days).…”

“…The amplification and sequencing of the 16S rRNA gene were carried out as described in Rey-Velasco et al (2023) . Briefly, the DNA of each isolate was extracted from 100 μL of liquid culture by thermal shock and was used for PCR amplification of the nearly complete 16S rRNA gene with primers 27Fmod (5′-AGR GTT TGA TCM TGG CTC AG-3′) and 1492Rmod (5′-TAC GGY TAC CTT GTT AYG ACT T-3′) from Page et al (2005) .…”

“…An initial classification of the near-complete 16S rRNA gene sequences was made with the SINA aligner ( Pruesse et al, 2012 ) against the SILVA database (release 138.1). Isolate sequences were also submitted to BLASTn v. 2.12.0+ ( Altschul et al, 1990 ) against a subset of the RDP database (release 11; Cole et al, 2014 ) containing only cultured taxa, and another one containing only uncultured taxa, as shown in Figure 1 of Rey-Velasco et al (2023) . The sequences that presented less than 97% identity to cultured taxa according to these data were assessed against type strains using the Identifier tool of EzBioCloud ( Yoon et al, 2017 ).…”

“…We have recently obtained a large culture collection from Blanes Bay (NW Mediterranean) seawater microcosm experiments run in the four astronomical seasons in which we tested the impact of the reduction in grazing and viral pressure, the increase in nutrient availability, and the presence/absence of light on the culturability of marine bacteria ( Rey-Velasco et al, 2023 ). In this culturing effort, we could isolate taxa that represented the top-abundant fractions of the population after some of the manipulations, and we isolated a set of bacteria with less than 97% identity in their 16S rRNA gene sequences to cultured taxa in public databases.…”