Species composition shift of confined bacterioplankton studied at the level of community DNA

Using a new DNA hybridization technique that does not require culturing, we compared the species composition of natural planktonic bacterial assemblages before and after confinement in 20 1 containers for ca 2 d Although confinement is known to cause species shifts, possibly by stimulating growth of certain types of cells near the container wall, we found that such shifts were minor; 5 to 15 % of the communities had changed during confinement. The greatest shifts occurred in the samples that had the fastest bacterial growth rate measured by [Hlthymidine incorporation. Despite the minor changes in species composition, the fraction of the cultivable cells (colony forming units; CFU) increased 4to 23-fold, but amounted to 70 % DNA homology belong to one species (Wayne et al. 1987), strict interpretation of community DNA similarity to within a few percent is not possible, partly because there is a slight cross-hybridization between species (generally < 10 %). Details of the concept, tests and evaluations, and limitations of the method are presented in Lee & Fuhrman (1990). In this study, confinement effects on bacterial species compositions were examined by the community DNA hybridization method. An important difference from previous studies was the use of larger (20 1) volume containers, needed to obtain sufficient DNA for the hybridizations. We also investigated changes of CFU, cell concentrations, and growth rates under confinement. The community DNA hybridization showed ca 5 to 15 % of species composition shift while the CFU were always < 2 % of the total cell count. Materials and methods. Sampling: Naturally occurring marine planktonic bacteria were sampled (Table 1) from an oligotrophic Pacific station (550 km west of San Diego, USA) at 25 m (OpenPac25) and 100 m (OpenPaclOO) water depths, and from a jetty at Playa Del Rey, Santa Monica Bay, Los Angeles, CA (CoastalPac). Other samples included in the community DNA comparisons were from a beach at Crane Neck, Long Island, New York, USA, (LIS) and the Caribbean Sea (Carib; 100 km west of Dominica). To collect the seawater, we used Niskin bottles (OpenPac and Carib samples), or clean plastic buckets thoroughly rinsed with seawater (CoastalPac and LIS samples). The water collected with the buckets was transferred to clean (see below) 20 1 polyethylene containers for transport and storage (1.e. confinement). Confinement experiment: The seawater samples were transferred to and stored in 20 1 polyethylene containers (Cubitainer; Consolidated Plastics Co., Inc., Twinsburg, OH, USA) which were soaked overnight with 10 % HC1 and rinsed thoroughly with the sample seawater. No treatment was given except aging in 20 1 containers. The OpenPac25 sample was stored on the ship deck in the shade for 48 h, and the OpenPaclOO sample was stored in the ship's lab for 32 h. The CoastalPac sample was stored indoors in the lab at University of Southern California for 48 h. Storage temperatures were 14 to 18 'C (OpenPac) and 21 'C (CoastalPac), whereas in situ temperatures were 17 OC (OpenPac25), 13 OC (OpenPaclOO), and 20 'C (CoastalPac). Cell collection for DNA: Bacterial cells were collected by pressure filtration as described previously (Lee & Fuhrman 1990). In brief, the water was first pre-filtered through type AE glass fiber filters (142 mm diameter; Gelman Sciences Inc., Ann Arbor, MI, USA) to remove larger particles and eukaryotic cells, and bacterial cells were collected on 0.22 pm pore size Durapore filters (142 mm diameter; Millipore Corp., Bedford, MA, USA). The filtrations were done in a relatively short period of time (total of 1.5 to 3 h after sample collection, depending on the volume filtered). Subsamples were taken from unfiltered seawater, intermediate filtrate (between AE filter and Durapore filter) and final filtrate. Filtering efficiencies were monitored from subsamples by the acridine orange direct count (AODC) method (Hobbie et al. 1977). Filtration was done in the same way for stored seawater samples at the end of confinement. DNA extraction: We followed the protocol of Fuhrman et al. (1988) to extract DNA from the cells collected on Durapore filters, with one more step of purification with phenol/chloroform/isoamyl alcohol (24: 6: 1 by vol, pH 8.0). After final purification and redissolution in TE (10 mM Tris, 1 mM EDTA, pH 8.0) at the concentrations of 100 to 500 ng p l l , DNA was quantified by Hoechst 33258 dye (bisbenzimde; Sigma Chemical Co., St. Louis, MO, USA) fluorometry (Paul & Myers 1982). Probe and target DNA preparation: Probe DNA was labeled with [alpha-^S]dATP (DuPont, NEN Research Products, Boston, MA, USA) by nick translation and purified as described previously (Lee & Fuhrman 1990). Probes were dried by vacuum centrifugation Lee & Fuhrman: Species composition shift of confined microorganisms 197 Table 1. Dates and locations of natural bacterioplankton samples