The ISME Journal (2007) 1, 361–371 & 2007 International Society for Microbial Ecology All rights reserved 1751-7362/07 $30.00 www.nature.com/ismej ORIGINAL ARTICLE Improvements of high-throughput culturing yielded novel SAR11 strains and other abundant marine bacteria from the Oregon coast and the Bermuda Atlantic Time Series study site Ulrich Stingl, Harry James Tripp and Stephen J Giovannoni Department of Microbiology, Oregon State University, Corvallis, OR, USA The introduction of high-throughput dilution-to-extinction culturing (HTC) of marine bacterioplank- ton using sterilized natural sea water as media yielded isolates of many abundant but previously uncultured marine bacterial clades. In early experiments, bacteria from the SAR11 cluster (class Alphaproteobacteria), which are presumed to be the most abundant prokaryotes on earth, were cultured. Although many additional attempts were made, no further strains of the SAR11 clade were obtained. Here, we describe improvements to the HTC technique, which led to the isolation of 17 new SAR11 strains from the Oregon coast and the Sargasso Sea, accounting for 28% and 31% of all isolates in these experiments. Phylogenetic analysis of the internal transcribed spacer (ITS) region showed that the isolates from the Oregon coast represent three different subclusters of SAR11, while isolates from the Sargasso Sea were more uniform and represented a single ITS cluster. A PCR assay proved the presence of proteorhodopsin (PR) in nearly all SAR11 isolates. Analysis of PR amino-acid sequences indicated that isolates from the Oregon coast were tuned to either green or blue light, while PRs from strains obtained from the Sargasso Sea were exclusively tuned to maximum absorbance in the blue. Interestingly, phylogenies based on PR and ITS did not correlate, suggesting lateral gene transfer. In addition to the new SAR11 strains, many novel strains belonging to clusters of previously uncultured or undescribed species of different bacterial phyla, including the first strain of the highly abundant alphaproteobacterial SAR116 clade, were isolated using the modified methods. The ISME Journal (2007) 1, 361–371; doi:10.1038/ismej.2007.49; published online 5 July 2007 Subject Category: microbial ecology and functional diversity of natural habitats Keywords: SAR11; cultivation; marine bacterioplankton; microdiversity; proteorhodopsin Introduction the SAR11 clade (Rappe´ et al., 2002), the marine group I Crenarchaeota (Konneke et al., 2005) and the Marine bacterioplankton play an important role in OM43 clade (Connon and Giovannoni, 2002) were marine food chains and global nutrient cycling obtained in pure culture. Much of this success can (Arrigo, 2005). Phylogenetic analysis based on 16S be attributed to using high-throughput culturing rRNA genes indicates that bacterioplankton consist (‘HTC’; Connon and Giovannoni, 2002), a method mainly of about 10–15 abundant clades, which using dilution-to-extinction culturing with pristine usually make up about 90% of all prokaryotic gene sea water as media, mainly based on the ideas and clones in libraries prepared from ocean surface findings reported by Button et al. (1993). waters (Rappe´ and Giovannoni, 2003). Progress has With the recent increase in genomic and meta- been made in recent years at cultivating members genomic data, bacterial speciation and the microbial of the numerically abundant clades; for example, species concept are intensively discussed in the recent literature (see Konstantinidis et al., 2006; Staley, 2006). The SAR11 clade is one of the most Correspondence: Dr U Stingl, Department of Microbiology, abundant microorganisms on earth (Morris et al., Oregon State University, 220 Nash Hall, Corvallis, OR 97331, 2002) and contains well-defined subclusters. The USA. E-mail: [email protected] SAR11 clade consists of at least four different Received 15 April 2007; revised and accepted 28 May 2007; subclusters that can be discriminated by their 16S published online 5 July 2007 rRNA sequence (Morris et al., 2005). Abundances New isolates of the SAR11 clade U Stingl et al 362 of these subclusters change spatially with depth Methods (Field et al., 1997; Morris et al., 2005) and temporally with season (Morris et al., 2005), thus Sampling and media implying different adaptations to the environment Inoculum was sampled with Niskin bottles in May for the different subgroups. These attributes make 2006 at station NH-5, 5 miles off the coast along the the SAR11 clade a good model for studying bacterial Newport, Oregon hydroline, from a depth of 10 m, speciation. and in July 2006 at the BATS from 10 and 200 m The 16S rRNA gene is a slowly evolving evolu- depth. Samples from BATS were collected in a tionary marker that may not resolve recent specia- rinsed 1-liter Teflon bottle and shipped overnight on tion events. Sequencing and phylogenetic analysis ice to Oregon State University. Samples from the of the internal transcribed spacer (ITS) region, Oregon coast were collected in the same bottles the non-coding region between the 16S rRNA and and transported on ice to Oregon State University. the 23S rRNA, has indicated a fine-scale micro- Preparation of the media followed the protocol of diversity within the SAR11 clade (Garcia-Martinez low-nutrient heterotrophic media (‘LNHM’; Connon and Rodriguez-Valera, 2000; Brown and Fuhrman, and Giovannoni, 2002), with sea water collected at 2005). A key question in the debate on bac- NH-5 in May 2006. The media was amended with terial speciation is whether the microdiversity in DMSP (100 nM), a mix of vitamins (Rappe´ et al., mM mM clusters of environmental 16S rRNA sequences 2002), NH4Cl (10 ), K2HPO4 (1 ) and a mixture observed in many environments, including marine of carbon compounds (‘MC’; 0.001% (w/v); Rappe´ habitats (Thompson et al., 2004; Venter et al., 2004; et al., 2002). Sogin et al., 2005, 2006; Pommier et al., 2007), coincides with functional divergence. This question Preparation of Teflon plates has not been answered adequately for most of Custom-made 24-well Teflon plates with a well the major marine bacterial clades (Giovannoni, volume of 6 ml were obtained from Cowie-Tech 2005; Martiny et al., 2006). Answering this ques- (Wilmington, DE, USA). To prevent metal contam- tion will require culturing and comparative inations from the autoclave steam, plates were genomic and metabolic studies of the representa- sterilized using a combination of microwaving and tives of the divergent subclades. Thus far, cultiva- UV irradiation. The plates were rinsed five times tion of SAR11 strains by HTC has only been with nanopure water and soaked overnight in 10% successful in a single experiment, which resul- omnipure HCl. After soaking, plates were rinsed five ted in the isolation of strains with identical 16S times with nanopure, filled up with nanopure, rRNA gene sequences (Rappe´ et al., 2002). No and put into a seal-a-meal bag and microwaved at further cultured strains of this clade have been maximum power for 10 min. The boiling water was reported yet. discarded, the bags were sealed and UV-irradiated Apart from a genome analysis (Giovannoni et al., for at least 60 min on each side. 2005b), physiological data on the SAR11 cluster are scarce, but recent data showed that members of the SAR11 clade play an important role in the Inoculation marine sulfur cycle and can take up and degrade The cell densities of the inocula were determined by dimethylsulfoniopropionate (DMSP; Malmstrom 40,6-diamidino-2-phenylindole (DAPI) counting et al., 2004; Howard et al., 2006). To improve as described by Connon and Giovannoni (2002). culturing efficiencies of SAR11, we modified the Dilutions of the inoculum were performed in media. HTC method by adding DMSP to the media as well Wells were filled with 5 ml media including inocu- as by using ultra-clean Teflon plates as culture lum. Final inoculation densities were calculated vessels. For easy, fast and reliable screening of the based on the dilution factor of the original inoculum cultures, we established a flow cytometry method (Table 1). Incubation of the plates was performed at using a commercial cell counter designed primarily 161C in the dark. for eukaryotic cells. The goals of this study were to improve existing methods for culturing marine bacteria to (1) isolate Screening of the cultures new SAR11 strains from different environments and Plates were screened after 4, 8 and 12 weeks with a (2) isolate abundant yet uncultured marine bacteria Guava EasyCyte cell counter (Guava Technologies, for further physiological studies. Hayward, CA, USA). Samples of 200 ml were Two HTC experiments with inoculum from the transferred to a 96-well plate and stained with ultra-oligotrophic subtropical gyre at the Bermuda SYBR Green1 (final dilution 1:2000, Invitrogen, Atlantic Time Series study site (BATS) and from Eugene, OR, USA) for 60 min. The samples were the nutrient-rich upwelling area at the Oregon run for 5 s each with the green photomultiplier set at coast were performed. The cultures obtained were 700 V; the two other photomutlipliers were set to a screened by sequencing of 16S rRNA genes, and, low value (400 V). The limit of detection of this for SAR11 cultures, ITS and proteorhodopsin (PR) method was tested with a pure culture of Candi- genes were sequenced and analyzed. datus Pelagibacter ubique and determined to be The ISME Journal New isolates of the SAR11 clade U Stingl et al 363 Table 1 Setup and results of dilution-to-extinction experiments Location and depth Inoculation density Number of Positive wells Pure cultures SAR11 isolates (cells/ml) inoculated wells Oregon, NH-5, 10 m 1 96 52 43 12 Oregon, NH-5, 10 m 3 96 96 ND ND BATS, 10 m 1 168 4 4 2 BATS, 10 m 3 168 11 9 3 BATS, 200 m 1 168 1 1 0 BATS, 200 m 5 168 3 2 0 Abbreviations: BATS, Bermuda Atlantic Time Series study site; ND, not determined, microscopic analysis showed many mixed cultures.