AQUATIC MICROBIAL ECOLOGY Vol. 41: 15–23, 2005 Published November 11 Aquat Microb Ecol Marine bacterial microdiversity as revealed by internal transcribed spacer analysis Mark V. Brown1, 2,*, Jed A. Fuhrman1 1Department of Biological Sciences and Wrigley Institute for Environmental Studies, University of Southern California, Los Angeles, California 90089-0371, USA 2Present address: NASA Astrobiology Institute, University of Hawaii, Honolulu, Hawaii 96822, USA ABSTRACT: A growing body of evidence suggests analysis of 16S rRNA gene sequences provides only a conservative estimate of the actual genetic diversity existing within microbial communities. We examined the less conserved internal transcribed spacer (ITS) region of the ribosomal operon to determine the impact microdiversity may have on our view of marine microbial consortia. Analysis of over 500 ITS sequences and 250 associated 16S rRNA gene sequences from an oceanic time series station in the San Pedro Channel, California, USA, revealed that the community in this region is com- posed of large numbers of distinct lineages, with more than 1000 lineages estimated from 3 clusters alone (the SAR11 clade, the Prochlorococcus low-B/A clade 1, and the Roseobacter NAC11-7 clade). Although we found no instances where divergent ITS sequences were associated with identical 16S rRNA gene sequences, the ITS region showed much greater pairwise divergence between clones. By comparison to our 16S rRNA gene–ITS region linked database, we were able to place all ITS sequences into a phylogenetic framework, allowing them to act as an alternative molecular marker with enhanced resolution. Comparison of SAR11 clade ITS sequences with those available in GenBank indicated phylogenetic groupings based not only on depth but also on geography, potentially indicating localized differentiation or adaptation. KEY WORDS: Microbial ecology · Microdiversity · Intergenic transcribed spacer Resale or republication not permitted without written consent of the publisher INTRODUCTION play a role in rRNA processing and may encode for up to 2 tRNA genes, it appears for the most part to lack the The ribosomal operon, which in prokaryotes is gen- requirement for structural integrity that constrains erally composed of 16S and 23S ribosomal RNA (rRNA) mutation in the ribosomal molecules. Consequently, genes separated by an internal transcribed spacer this region displays a great deal of length and (ITS) region, is part of the slowly evolving bacterial sequence variation which may provide high taxonomic ‘core genome’, the set of genes present in all bacteria. resolution and serve as a fast molecular chronometer Several reports have indicated that organismal gene by which initial genome diversification and evolution- content closely reflects phylogeny, and that incremen- ary speciation may be detected (Schloter et al. 2000). In tal diversification of the core genome reflects the the present study, we examined the ITS sequence of all diversification of the ‘accessory’ genome over time clones retrieved during a temporal and spatial analysis (Feil 2004). However, it remains unclear as to what of a marine time series station in the San Pedro Chan- level phylogenetic markers, if any, may accurately nel, California, USA. Our aim was to verify the extent correspond to genomic diversification. In several cases of microdiversity commonly observed in environmen- (e.g. Rocap et al. 2002, Seurinck et al. 2003, Jaspers & tal surveys that employ 16S rRNA gene sequence Overmann 2004) genetic clusters defined by ribosomal analysis (e.g. Acinas et al. 2004) using an alternative, ITS sequences have been identified as ‘ecotypes’ less conserved marker, and examine what effects this (closely related organisms inhabiting different niches) finer resolution may have on our current understand- within an environment. Although the ITS region does ing of marine microbial diversity. *Email: [email protected] © Inter-Research 2005 · www.int-res.com 16 Aquat Microb Ecol 41: 15–23, 2005 MATERIALS AND METHODS final step at 72°C for 7 min. Amplification products were purified using a Qiagen MinElute PCR purifica- Study site and sample collection. Coastal marine tion column, quantified using Pico Green fluorescence water was collected as part of the San Pedro Ocean (Molecular Probes) in a Bio-Rad VersaFluor™ fluoro- Time Series (SPOTS) monthly sampling at a station meter and ligated into pGEM®-T Easy (Promega) plas- situated approximately midway between San Pedro mid vectors. Ligation reactions were transformed into Harbor and Catalina Island in the San Pedro Channel, JM109 High Efficiency Competent Cells (Promega) California, USA (33° 33’ N, 118° 24’ W). Months and and colorimetric screening was used to determine depths analyzed for this study included: October 2000, clones containing recombinant vectors. Plasmid DNA 5 m; April 2001, 5 and 14 m (corresponding to the was isolated from 3 ml overnight cultures using the chlorophyll a maximum); August 2001, 5 m; December QIAprep Spin Miniprep Kit (Qiagen). 2001, 5 m; February 2002, 43 m (corresponding to the Plasmid DNA was sequenced with the DYEnamic ET chlorophyll a maximum); May 2003, 150, 500, and Terminator Cycle Sequencing Kit (Amersham Bio- 890 m. The time series station displays a complex sciences) and 5 pmol of sequencing primer. Sequenc- hydrographic regime, with the deeper waters (parti- ing reactions were prepared and cleaned according to cularly below 700 m) being almost anoxic, whilst the the manufacturer’s instructions. Electrophoresis was euphotic zone is alternately influenced by oligotrophic carried out on an ABI 377XL automated sequencer. Ini- eddies moving inshore and up the coast or by nutrient- tial sequencing was done with the universal 16S rRNA rich water up-welled near Point Conception to the gene primer 1392F (5’-GYACACACCGCCCGT-3’), to north and swept southward in the California Current. encompass most of the ITS region. Further sequencing Samples were obtained using 20 l Niskin bottles, was done on multiple representative clones with sequentially filtered through a 142 mm Whatman GF/F primers M13F (5’-GTAAAACGACGGCCAGT-3’), filter followed by a 142 mm Durapore 0.22 µm pore size M13R (5’-CAGGAAACAGCTATGAC-3’), universal filter using a positive pressure filtration system, and 536F (5’-CAGCMGCCGCGGTAATWC-3’) and one of filters were stored at –80°C until processing. Auxiliary 1520r a (5’-AAGGAGGTGATCCAGCC-3’), b (5’-TA data obtained from the water samples used to create GGAGGTGATCCAGCC-3’), c (5’-AAGGAGGTAAT- clone libraries, including temperatures, salinities, CCAGCC-3’), d (5’-AAGGAGGTGATCCAACC-3’), chlorophyll and nutrient data, can be accessed at e (5’-AAGGAGGTGTTCCAGCC-3’), f (5’-AAGGA www.usc.edu/microbialobservatory. GATGTTCCAGCC-3’), g (5’-AAAGAGATATTCCAG Clone library construction and analysis. Commu- CC-3’), h (5’-AAGGAGGTATTCCAGCC-3’), i (5’- nity DNA was extracted from Durapore filters using ATGG AGGTGATCCAGCC-3’), j (5’-AAGGAGGT- hot SDS lysis followed by phenol:chloroform:isoamyl- GATCCATCC-3’), k (5’-AAGGAGATAATCCAGCC- alcohol extractions and ethanol precipitation as de- 3’), l (5’-TAGGAGGTGATCCATCC-3’) to provide full scribed by Fuhrman et al. (1988). Monthly depth and length 16S rRNA gene sequences. To conserve temporal variability of samples were initially screened resources, not all 16S rRNA gene sequences were com- using Automated rDNA Intergenic Spacer Analysis pleted. Where 2 or more identical ITS sequences were (ARISA) (Fisher & Triplett 1999). Clone libraries were found, at least 2 of the associated 16S rRNA gene created from samples that would enable a wide range regions were sequenced. As the objective of the survey of ARISA peak identification, i.e. where peaks repre- was to examine ITS sequence diversity, 16S rRNA senting major bacterial groups varied. PCR amplicons gene sequences were used to ground that diversity in encompassing almost full length 16S rRNA genes a phylogenetic framework. All raw sequence along with the associated ITS spacer region were data were verified manually from electrophero- amplified using the primers 27f, 5’-AGAGTTTGATC grams using the program Chromas version 1.45 MTGGCTCAG-3’ (bacterial-specific 16S rRNA gene) (www.mb.mahidol.ac.th/pub/chromas/chromas.htm). (Lane 1991) and 23Sr, 5’-GGGTTBCCCCATTCRG-3’ Completed clone sequences were checked using (bacterial-specific 23S rRNA gene) (Fisher & Triplett the chimera detection programs Chimera Check 1999). The 100 µl reaction mixture contained a final (http://rdp8.cme.msu.edu/cgis/chimera.cgi?su=SSU) concentration of 1× PCR AmpliTaq Gold‚ buffer and Bellerophon (Huber et al. 2004). Sequences have (Applied Biosystems), 3.5 mM MgCl2 (Applied Bio- been deposited in GenBank under the accession num- systems), 350 µM of each dNTP (Promega), 800 nM of bers DQ009080 to DQ009478. 16S rRNA gene each primer, 40 ng µl–1 BSA, 5 U of the high fidelity sequences were imported into the Hugenholtz align- DNA polymerase AmpliTaq Gold‚, and 0.1 ng µl–1 of ment (Hugenholtz 2002) in ARB (Ludwig et al. 2004) template DNA. The reaction mixture was held at 94°C aligned to their closest relatives using the ARB align- for 10 min followed by 24 cycles of amplification at ment tool and manually corrected. 16S rRNA gene 94°C for 40 s, 55°C for 40 s and 72°C for 3 min with a phylogenetic trees were generated in ARB using the Brown & Fuhrman: ITS analysis of marine microdiversity 17 neighbour-joining function with appropriate taxon In all cases, pairwise comparisons between ITS based (e.g. alpha-Proteobacteria) filters, which utilize sequences were significantly (p < 0.05) lower than only conserved bases within each group. Only full those of the associated 16S rRNA gene sequences. length sequences (those covering the range of the However we did not observe a consistent quantitative filter) were used in the analysis. ITS sequences were relationship, as different taxa displayed ITS sequence aligned in BioEdit (www.mbio.ncsu.edu/BioEdit/ divergence at different rates (Fig. 1). At a 16S rRNA bioedit.html) initially using Clustal W followed by gene pairwise similarity above 0.99, the marine manual editing. Trees inferring ITS sequence relation- cyanobacteria (Fig.