Actively Growing Bacteria in the Inland Sea of Japan, Identified by Combined Bromodeoxyuridine Immunocapture and Denaturing Gradient Gel Electrophoresis

Abstract

A fundamental question in microbial oceanography concerns the relationship between prokaryote diversity and biogeochemical function in an ecosystem context. We combined bromodeoxyuridine (BrdU) magnetic bead immunocapture and PCR-denaturing gradient gel electrophoresis (BUMP-DGGE) to examine phylotype-specific growth in natural marine assemblages. We also examined a broad range of marine bacterial isolates to determine their abilities to incorporate BrdU in order to test the validity of the method for application to diverse marine assemblages. We found that 27 of 29 isolates belonging to different taxa could incorporate BrdU. BUMP-DGGE analysis revealed phylogenetic affiliations of DNA-synthesizing, presumably actively growing bacteria across a eutrophic to mesotrophic transect in the Inland Sea of Japan. We found that the BrdU-incorporating (growing) communities were substantially different from the total communities. The majority (34/56) of phylotypes incorporated BrdU and were presumably growing, and these phylotypes comprised 10 alphaproteobacteria, 1 betaproteobacterium, 11 gammaproteobacteria, 11 Cytophaga-Flavobacterium-Bacteroides group bacteria, and 1 unclassified bacterium. All BrdU-responsive alphaproteobacteria were members of the Rhodobacterales, suggesting that such bacteria were dominant in the growing alphaproteobacterial populations in our samples. The BrdU-responsive gammaproteobacteria belonged to the Oceanospirillales, the SAR86 cluster, the Pseudomonadales, the Alteromonadales, and the Vibrionales. Thus, contemporaneous cooccurrence of diverse actively growing bacterial taxa was a consistent pattern in our biogeochemically varied study area.Bacteria in seawater play important roles in the ocean's food webs. Their activities and responses to organic substrates significantly influence the flux of organic matter and oceanic biogeochemical cycles (3, 4). Because only a small fraction of bacteria in any seawater sample can be isolated and cultivated, culture-independent methods employing molecular approaches have been used to great advantage during the last two decades to study the natural assemblages of marine bacteria (11,19,39). These and other studies have revealed unexpected diversity and dynamics of bacterial community structure in seawater (8,16,47). However, the great challenge remains to relate diversity to ecological function and biogeochemical activities of bacteria. In recent studies workers have combined fluorescence in situ hybridization (FISH) with microautoradiography and found differences in the uptake of specific organic substrates by different phylogenetic groups of bacteria (7,8,40). Stable-isotope probing has enabled PCR-based DNA analysis of bacteria that incorporate specific substrates (45).Bromodeoxyuridine (BrdU), a halogenated nucleoside that can serve as a thymidine (TdR) analog, has been widely used as an alternative to tritiated TdR ([ 3 H]TdR) incorporation to label proliferating cells in cell biology (2, 59). BrdU incorporated into de novo DNA can be detected using ...

Detection of UVBR-sensitive and -tolerant bacteria in surface waters of the western North Pacific

“…Considering that PCR-DGGE bands indicate the predominant community members [18] and that the BrdU-immunocapture technique can detect actively growing bacteria specifically [25], the difference between the total and the BrdU-immunocaptured bacterial community indicated that more than half of the growing bacterial phylotypes were minor populations. The results obtained in this study are similar to those of Hamasaki et al [33] who used the same technique. The low abundance of actively growing bacteria could be caused by the differences in phylotype-specific mortality by viral infection, selective ingestion of actively growing bacteria by heterotrophic nanoflagellates [44,45], and/or UVBR [13,15,46].…”

“…This technique has recently been used for quantification of DNA-synthesizing bacteria in seawater [26][27][28][29][30][31]. PCR-DGGE combined with the immunocapturing method succeeded in distinguishing actively growing bacteria from the bacterial community in the ocean [32][33][34].…”

Altitudinal patterns of diversity and functional traits of metabolically active microorganisms in stream biofilms

“…We recognise the potential caveats of BrdU incorporation to detect metabolically active microorganisms. There is evidence in fact that a certain fraction of active microbial taxa may be missed because of their intrinsic inability to incorporate BrdU (e.g., Hamasaki et al, 2007). Furthermore, despite the fact that we amended in excess, we cannot exclude that some taxa used metabolites from others.…”

Improved group-specific PCR primers for denaturing gradient gel electrophoresis analysis of the genetic diversity of complex microbial communities

“…However, with the possible exception of the high G þ C-containing Actinobacteria, this method has limited application and does not separate bacteria by phylogeny. Combining bromodeoxyuridine immunocapture and DGGE has been proposed to separate the DNA of the actively growing bacteria from the rest of the environmental DNA (Hamasaki et al, 2007). However, this results in the analysis of subgroups of bacteria that are not defined on phylogenetic criteria, and does not allow screening of the whole range of genetic bacterial diversity.…”