Microbes Environ. Vol. 26, No. 4, 339–346, 2011 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME11212 Seasonal Variations in the Community Structure of Actively Growing Bacteria in Neritic Waters of Hiroshima Bay, Western Japan AKITO TANIGUCHI1†, YUYA TADA1, and KOJI HAMASAKI1* 1Atmosphere and Ocean Research Institute, The University of Tokyo, 5–1–5 Kashiwanoha, Kashiwa, Chiba 277–8564, Japan (Received May 6, 2011—Accepted June 30, 2011—Published online July 27, 2011) Using bromodeoxyuridine (BrdU) magnetic beads immunocapture and a PCR-denaturing gradient gel electrophoresis (DGGE) technique (BUMP-DGGE), we determined seasonal variations in the community structures of actively growing bacteria in the neritic waters of Hiroshima Bay, western Japan. The community structures of actively growing bacteria were separated into two clusters, corresponding to the timing of phytoplankton blooms in the autumn–winter and spring–summer seasons. The trigger for changes in bacterial community structure was related to organic matter supply from phytoplankton blooms. We identified 23 phylotypes of actively growing bacteria, belonging to Alphaproteobacteria (Roseobacter group, 9 phylotypes), Gammaproteobacteria (2 phylotypes), Bacteroidetes (8 phylotypes), and Actinobacteria (4 phylotypes). The Roseobacter group and Bacteroidetes were dominant in actively growing bacterial communities every month, and together accounted for more than 70% of the total DGGE bands. We revealed that community structures of actively growing bacteria shifted markedly in the wake of phytoplankton blooms in the neritic waters of Hiroshima Bay. Key words: actively growing bacteria, bromodeoxyuridine, PCR-DGGE, seasonal variation Phytoplankton blooms are significant events in coastal eco- nities are still largely unknown. Actively growing bacteria, systems, greatly impacting the structure and composition of defined in this study as rapidly dividing bacteria, should organic matter fields (23); bacterial community structures, in utilize increased levels of organic matter to maintain their particular, must respond and adapt to perturbations (37). Sev- rapid growth rates, and thus, should be lysed by viruses eral reports have demonstrated that phytoplankton blooms and/or grazed by protozoa (34, 49). Because of this, actively lead to marked changes in the abundance, composition, and growing bacteria should be regarded as key components driv- activities of bacterial communities (13, 40). Riemann et al. ing biogeochemical processes in oceanic environments. (38) showed that bacterial abundance, growth rates, and enzy- Bromodeoxyuridine (BrdU), a halogenated nucleoside matic activities markedly increased after manipulated diatom which can serve as a thymidine analog, is useful for analyzing bloom. Additionally, they showed that Alphaproteobacteria DNA synthesis in actively growing bacterial communities and Bacteroidetes were rapidly growing key bacteria during (e.g., 16, 33, 44, 46, 50). An immunocapture technique the decay phase of a bloom. using BrdU-labeled DNA has been applied to determine In natural coastal environments, many studies have phylogenetic affiliations and functions of bacterial groups (6, reported the temporal dynamics of bacterial communities 7, 28). Using BrdU magnetic beads immunocapture and PCR- (e.g., 22, 27, 29, 35, 39, 42). Alonso-Sáez et al. (2) reported DGGE (BUMP-DGGE), previous studies investigated phy- that both bacterial abundance and composition vary season- logenetic affiliations and changes in the spatial distributions ally and that Alphaproteobacteria and Bacteroidetes bacte- of actively growing bacteria in coastal and oceanic environ- ria are predominant in coastal waters throughout the year; ments (17, 48). These studies revealed that Roseobacter and their investigations were based on PCR-denaturing gradient Bacteroidetes bacteria dominate actively growing bacteria gel electrophoresis (DGGE) and catalyzed reporter depo- groups in both coastal and oceanic regions. In addition, the sition fluorescence in situ hybridization (CARD-FISH). On results suggest that different water masses are characterized the basis of microautoradiography combined with FISH by different phylogenetic affiliations of bacteria; however, (MAR- or Micro-FISH) analyses, Alonso-Sáez and Gasol these general conclusions must be carefully examined in each (3) showed that substantial changes occur in the activities region and moreover, temporal variations in the structure and and compositions of some bacterial phylogenetic groups composition of actively growing bacterial communities are (e.g., the Roseobacter group and SAR11 bacteria of the not fully understood. Tada et al. (45) investigated seasonal Alphaproteobacteria) during the course of a year. Although variations in phylotype-specific productivity by means of temporal variations in the abundance, activities and compo- BrdU immunocytochemistry-FISH (BIC-FISH). Their results sitions of total bacteria have been reported in these previous showed that the proportion of actively growing bacteria to studies, the dynamics of actively growing bacterial commu- total bacteria varies seasonally, from 15% to 30%, with an annual mean rate of 22%. Additionally, they found that * Corresponding author. E-mail: [email protected]; Roseobacter/Rhodobacter bacteria constituted a constant Tel: +81–4–7136–6171; Fax: +81–4–7136–6171. population of proliferating microbes and that Bacteroidetes † Present address: Graduate School of Agriculture, Kinki University, populations increased markedly after phytoplankton blooms. 3327–204 Nakamachi, Nara 631–8505, Japan The objectives of this study were to determine the detailed 340 TANIGUCHI et al. phylogenetic affiliations of actively growing bacteria and to and compared bacterial community structures between these two reveal their temporal variations throughout the year in the periods. The threshold temperatures were 12°C (January–March vs. neritic waters of Hiroshima Bay, western Japan. Specifically, April–December), 15°C (January–April vs. May–December), 18°C we inquired into the effects of phytoplankton blooms on the (December–May vs. June–November), 20°C (December–June vs. July–November), and 22°C (November–July vs. August–October). community structures of actively growing bacteria, and on the concordance of results obtained by BUMP-DGGE and Sequencing and phylogenetic analyses BIC-FISH for phylotype proportions of actively growing Excised DGGE bands were sequenced directly from PCR bacteria. products that had been reamplified with the primer set described above. Prior to sequencing, the PCR products were analyzed by Materials and Methods DGGE to confirm band positions relative to the original sample. After purification of the PCR products with a Qiaquick PCR Sampling and BrdU labeling purification kit (Qiagen, Germantown, MD, USA), bidirectional Seawater samples were collected from a depth of 5 m at the pier sequencing using the 341F/907R primer set was performed by of Kure Port (34°14'30''N, 132°33'07''E), Hiroshima Bay, western SolGent (http://www.solgent.com/). Sequences were aligned to Japan, once a month in the morning, from July 2005 to June 2006. known sequences in the GenBank database using BLAST (4). Kure Port has two neighboring rivers. An approximately 5-L sample Phylogenetic trees were constructed with the neighbor-joining was pre-filtered through a 200-µm nylon mesh to remove mesozoo- method using MEGA 4 (47). All sequences were checked by the plankton. A 1-L portion of the sample was stored at 4°C for the program Bellerophon (18). analysis of environmental factors (processing within 5 h). A 2-L Nucleotide sequence accession numbers portion of the sample was immediately filtered through a 0.22-µm Sterivex filter (Millipore, Billerica, MA, USA) with a peristaltic The nucleotide sequences were deposited in the nucleotide pump to collect bacterial cells for extraction of DNA and subsequent sequence database, DNA Data Bank of Japan (DDBJ), under analysis of bacterial community structures. The final 2-L portion of accession numbers AB367452 to AB367491. the sample was incubated with BrdU (final concentration, 1 µM; Sigma-Aldrich, St. Louis, MO, USA) in a dark bottle at ambient Results temperatures for 3 h. After incubation, bacterial cells were collected as described above. Immediately after filtration, the Sterivex filters Environmental factors were stored at −20°C until further analysis. Environmental factors, including water temperature, salinity, particulate organic carbon Environmental factors show seasonal variations, as indi- (POC) and nitrogen (PON), chlorophyll a (chl a) concentration, and cated by the data in Table 1. During the study period, water bacterial abundance, were measured as described previously (45). temperatures ranged from 11.0 to 24.8°C and salinity ranged from 31.5 to 34.5, respectively. Conspicuous phytoplankton BUMP-DGGE analysis blooms occurred in September 2005 and February 2006, BUMP-DGGE analysis was performed according to procedures described previously (17), with slightly modifications. Briefly, the generating massive amounts of organic matter. Chl a µ −1 Sterivex filter was subjected to xanthogenate–SDS DNA extraction, concentrations ranged from 1.73 to 18.0 g L , with and 1 µg of the extracted DNA was used for BrdU immunocapture. maximum concentrations occurring in February 2006. POC The total and immunocaptured BrdU-labeled DNA was used as a and PON concentrations varied from 181.3 to 980.1 µg L−1 template for PCR amplification of 16S