Vol. 59: 283–293, 2010 AQUATIC MICROBIAL ECOLOGY Published online April 21 doi: 10.3354/ame01398 Aquat Microb Ecol High diversity of Rhodobacterales in the subarctic North Atlantic Ocean and gene transfer agent protein expression in isolated strains Yunyun Fu1,*, Dawne M. MacLeod1,*, Richard B. Rivkin2, Feng Chen3, Alison Buchan4, Andrew S. Lang1,** 1Department of Biology, Memorial University of Newfoundland, 232 Elizabeth Ave., St. John’s, Newfoundland A1B 3X9, Canada 2Ocean Sciences Centre, Memorial University of Newfoundland, Marine Lab Road, St. John’s, Newfoundland A1C 5S7, Canada 3Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 236-701 East Pratt St., Baltimore, Maryland 21202, USA 4Department of Microbiology, University of Tennessee, M409 Walters Life Sciences, Knoxville, Tennessee 37914, USA ABSTRACT: Genes encoding gene transfer agent (GTA) particles are well conserved in bacteria of the order Rhodobacterales. Members of this order are abundant in diverse marine environments, fre- quently accounting for as much as 25% of the total bacterial community. Conservation of the genes encoding GTAs allows their use as diagnostic markers of Rhodobacterales in biogeographical stud- ies. The first survey of the diversity of Rhodobacterales based on the GTA major capsid gene was con- ducted in a warm temperate estuarine ecosystem, the Chesapeake Bay, but the biogeography of Rhodobacterales has not been explored extensively. This study investigates Rhodobacterales diver- sity in the cold subarctic water near Newfoundland, Canada. Our results suggest that the subarctic region of the North Atlantic contains diverse Rhodobacterales communities in both winter and sum- mer, and that the diversity of the Rhodobacterales community in the summer Newfoundland coastal water is higher than that found in the Chesapeake Bay, in either the summer or winter. Approxi- mately one-third of GTA sequences retrieved from the subarctic waters were most closely related to those from bacteria isolated from sea ice or cold regions. Distinguishable diversity patterns were found between the temperate and subarctic waters, providing further support for niche adaptation of specific Rhodobacterales members to unique environments. We also demonstrate that a number of Rhodobacterales strains, from both the subarctic and temperate locations, express the GTA major capsid protein. This provides robust evidence that the widespread conservation of GTA genes in the Rhodobacterales may result in the production of functionally similar and active GTA systems in these bacteria in different environments. KEY WORDS: Rhodobacterales · Gene transfer agent · GTA · Major capsid protein Resale or republication not permitted without written consent of the publisher INTRODUCTION ton (Buchan et al. 2005). All complete Rhodobac- terales genome sequences contain gene transfer Estimates in marine microbial populations suggest agent (GTA) gene clusters (Lang & Beatty 2007, Biers members of the α-proteobacterial order Rhodobac- et al. 2008). GTAs are bacteriophage-like particles terales, specifically the Roseobacter clade, can com- that package and transfer genomic DNA between prise upwards of 25% of total marine bacterioplank- bacterial cells in a process analogous to transduction. *These authors contributed equally **Corresponding author. Email: [email protected] © Inter-Research 2010 · www.int-res.com 284 Aquat Microb Ecol 59: 283–293, 2010 GTAs are distinguished from transducing phages in Bay and that g5 sequence diversity was higher in several ways, including the apparently random and these natural communities than appreciated from obligate packaging of fragments of the producing previous isolates. cell’s genome, and the fact that the size of DNA We have exploited the GTA gene conservation to packaged in the particles is insufficient to encode the explore the diversity of Rhodobacterales in microbial full GTA structure (Lang & Beatty 2007, Stanton communities in the subarctic western North Atlantic 2007). This mechanism of genetic exchange has been Ocean. The results presented here demonstrate that identified in diverse prokaryotic species: the purple the cold productive subarctic waters of coastal New- non-sulfur α-proteobacterium Rhodobacter capsula- foundland (Canada) harbor a remarkably diverse col- tus (Marrs 1974), the sulfate-reducing δ-proteobac- lection of Rhodobacterales, and approximately one- terium Desulfovibrio desulfuricans (Rapp & Wall third of the GTA gene sequences retrieved from these 1987), the spirochete Brachyspira hyodysenteriae waters were most similar to those from bacteria iso- (Humphrey et al. 1997), the methanogenic archaeon lated from sea ice or cold regions. The propensity of Methanococcus voltae (Bertani 1999), and the marine the characterized representatives of ecologically Rhodobacterales bacterium Ruegeria (formerly Sili- important Rhodobacterales bacteria to display the cibacter) pomeroyi (Biers et al. 2008). With the genetic potential for production of GTAs warrants fur- exception of the R. pomeroyi GTA (Biers et al. 2008), ther investigation of this property. Therefore, we have all of these particles structurally resemble tailed also cultured several strains from this coastal environ- phages (reviewed in Stanton 2007). ment and demonstrated GTA capsid protein expres- The role of GTAs in natural environments is not yet sion amongst these, as well as in strains recovered understood, but they exist in the above-mentioned from the Chesapeake Bay. These findings show that, (and presumably other) phylogenetically diverse not only the genetic potential for GTA production, but prokaryotes, suggesting this mode of DNA transfer also the expression of the GTA capsid protein is wide- may be important in shaping some microbial genomes spread, both taxonomically and biogeographically, and communities. An outcome of GTA production for among members of the Rhodobacterales. This research the producing species is homologous recombination. provides further evidence that GTA-mediated gene Therefore GTA activity could provide benefits to the transfer could be an important phenomenon in natural producing species as a whole (Vos 2009), even if it is microbial communities. detrimental to the individual producing cell that pre- sumably must lyse to allow GTA release. The release of GTAs is known to occur by lysis only in Brachyspira MATERIALS AND METHODS hyodysenteriae (Humphrey et al. 1997, Matson et al. 2005), but it is anticipated that cell lysis is the likely Sample collection. Microbial communities were col- mechanism of release for all GTAs because there is no lected onto 0.2 µm polycarbonate filters (Millipore) known release of tailed phage particles from cells from 500 ml surface water samples collected at Tap- other than through lysis. per’s Cove (47° 39’ 54’’ N, 52° 43’ 38’’ W) on July 24, The Rhodobacter capsulatus GTA (RcGTA) gene 2007, and at Logy Bay (47° 38’ 14’’ N, 52° 39’ 36’’ W) cluster comprises 16 open reading frames (ORFs) in on December 11, 2008. These are adjacent locations ~14 kb on the bacterial chromosome (Lang & Beatty that experience very similar climate and hydrographic 2000, Lang & Beatty 2007). Several of these ORFs forcing, and the inshore branch of the Labrador have recognizable homology to known bacteriophage Current influences both bays. DNA was extracted from genes, such that the cluster appears to be an isolated the filters as previously described (Kan et al. 2006). head- and tail-encoding unit. Of these 16 ORFs, 3 Filters were thawed and each one was combined with are particularly well conserved between different 2 ml pre-lysis buffer (0.1 M Tris-HCL; 0.1 M EDTA; species (Lang et al. 2002): g2, predicted to encode 0.8 M sucrose; pH 8) and 10 µl lysozyme (200 µg ml–1) the large terminase protein, g5, encoding the major and incubated at 37°C for 30 min. This incubation was capsid protein, and g9, predicted to encode the major followed by addition of 200 µl of 10% (w/v) cetyl- tail protein (Lang & Beatty 2000, Lang & Beatty trimethylammonium bromide (CTAB) in 1.4 M NaCl 2007). Due to the conservation of GTA genes in the and incubation at 65°C for 30 min. Samples were then Rhodobacterales order, the GTA major capsid pro- sequentially extracted with phenol-chloroform-iso- tein-encoding gene, g5, has been used as a marker amyl alcohol (25:24:1) and chloroform-isoamyl alco- to estimate Rhodobacterales diversity in estuarine hol (24:1), followed by isopropanol precipitation. microbial communities (Zhao et al. 2009). This work Following a wash with 70% ethanol, the pellets demonstrated that there was a distinct temporal and were air-dried and dissolved in 200 µl Tris-EDTA spatial pattern of Rhodobacterales in the Chesapeake (TE) buffer. Fu et al.: Rhodobacterales diversity and gene transfer agents 285 Water samples for enumerating bacterial abundance Sigma-Aldrich). Each culture was then grown in liquid were fixed with 0.2 µm filtered formaldehyde (final broth at 25°C for 1 d and DNA was extracted using the concentration 3.7%) and filtered onto 0.2 µm poly- Puregene DNA Purification kit (Qiagen). The DNA carbonate membrane filters. Cells were stained with from these isolates was used as templates for PCR with 1 µg ml–1 DAPI (4’,6-diamidino-2-phenylindole) and the g5 primers (Zhao et al. 2009) as described in the counted by epifluorescence microscopy at 1250× mag- previous section, purified and directly sequenced nification using UV light excitation. For each sample, using the g5 primers. The primers 27F (5’-AGA GTT at least 1000 cells were enumerated. TGA TCM TGG CTC AG-3’) (Giovannoni 1991) and PCR amplification, cloning and sequencing of GTA Roseo536R (5’-CAA CGC TAA CCC CCT CCG-3’) g5 genes. Degenerate primers for amplification of (Brinkmeyer et al. 2000) were used to amplify a GTA g5 genes (encoding the major capsid protein) ~500 bp region of the 16S rRNA gene. The 16S rDNA described previously (Zhao et al. 2009) were used in PCR reaction components were the same as for the g5 the present study: MCP-109F, 5’-GGC TAY CTG GTS reactions with the following thermocycling conditions: GAT CCS CAR AC-3’ and MCP-368R, 5’-TAG AAC 30 s at 98°C, followed by 35 cycles of 98°C for 10 s, AGS ACR TGS GGY TTK GC-3’.