Impact of sideways and bottom-up control factors on bacterial community succession over a tidal cycle Ashvini Chauhan1, Jennifer Cherrier, and Henry N. Williams Environmental Sciences Institute, Florida A&M University, Frederick S. Humphries Science Research Building, Suite 305-D, Tallahassee, FL 32307 Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved January 22, 2009 (received for review October 21, 2008) In aquatic systems, bacterial community succession is a function of tandem, top-down factors shape bacterial community structure top-down and bottom-up factors, but little information exists on through a variety of processes, including protistan grazing (10) and ‘‘sideways’’ controls, such as bacterial predation by Bdellovibrio- viral lysis (11). Therefore, in all likelihood at any given time, like organisms (BLOs), which likely impacts nutrient cycling within combinations of these factors drive bacterial community succession the microbial loop and eventual export to higher trophic groups. in aquatic ecosystems (12–14). Here we report transient response of estuarine microbiota and BLO Recently, Mou et al. (2) proposed that in marine systems, spp. to tidal-associated dissolved organic matter supply in a river- transient changes in the dissolved organic carbon (DOC) pool are dominated estuary, Apalachicola Bay, Florida. Both dissolved or- less critical in structuring bacterial communities than those that ganic carbon and dissolved organic nitrogen concentrations oscil- result from viral lysis, protistan grazing, or even physicochemical lated over the course of the tidal cycle with relatively higher conditions. Both grazing and viral lyses are selective, such that concentrations observed at low tide. Concurrent with the shift in factors including nonsusceptibility, morphology, size, and motility dissolved organic matter (DOM) supply at low tide, a synchronous offer protection to certain bacterial groups (12). However, other increase in numbers of bacteria and predatorial BLOs were ob- ‘‘sideways’’ factors, which are only beginning to be understood, also likely contribute to shifts in the bacterial composition through served. PCR-restriction fragment length polymorphism of small processes that exert both positive (syntrophy) and/or negative subunit rDNA, cloning, and sequence analyses revealed distinct (allelopathy) effects (15). In this context, one of the largely ignored shifts such that, at low tide, significantly higher phylotype abun- trophic links within the microbial loop processes is obligate and ␥ ␦ ECOLOGY dances were observed from -Proteobacteria, -Proteobacteria, relatively nonspecific predation by Bdellovibrio-like organisms ؉ Bacteroidetes, and high G C Gram-positive bacteria. Conversely, (BLOs), resulting in potential structural and functional successions diversity of ␣-Proteobacteria, ␤-Proteobacteria, and Chlamydiales- of susceptible prey microbiota. Verrucomicrobia group increased at high tides. To identify meta- BLOs can lyse a variety of Gram-negative bacteria (16, 17) and bolically active BLO guilds, tidal microcosms were spiked with six are characterized by a motile free-living attack form and an 13C-labeled bacteria as potential prey and studied using an adap- intraperiplasmic growth phase. Our recent study indicated that tation of stable isotope probing. At low tide, representative of BLOs are more diverse than previously thought (18); most marine higher DOM and increased prey but lower salinity, BLO community bacteria are susceptible to lysis by these predators (16, 18) and also shifted such that mesohaline clusters I and VI were more hence their sideways trophic interactions would likely result in active; with an increased salinity at high tide, halotolerant clusters successions within the microbial food web processes. III, V, and X were predominant. Eventually, 13C label was identified This study was conducted in Apalachicola Bay, a river-dominated from higher micropredators, indicating that trophic interactions subtropical estuary located in the Florida Panhandle (Fig. S1A). A within the estuarine microbial food web are potentially far more combination of riverine discharge, gulf tides, and winds keep this complex than previously thought. system well mixed, likely resulting in dynamic cycling of DOM and inorganic nutrients from both allochthanous (river, wetlands) and Bdellovibrio-like organisms (BLOs) ͉ dissolved organic matter ͉ autochthanous (in situ production) sources. The overall goal of the predator-prey interactions ͉ stable isotope probing ͉ tidal microbiota work presented here was to evaluate how transient changes in both bottom-up factors (supply of bulk DOM, i.e., both DOC and dissolved organic nitrogen [DON]) and sideways factors transiently arine dissolved organic matter (DOM) is one of the largest influence bacterial community composition and associated func- Mactive reservoirs of reduced carbon at the earth’s surface and, tional changes within the predacious BLO guilds. An improved to a large extent, as the primary consumers of this DOM, bacteria understanding of these tightly coupled predator-prey interactions control its fate via assimilation and/or remineralization processes (1, and the effects of DOM supply will lead to a better understanding 2). The fate of DOM is a also a function of physiologic status and of the trophic links within the microbial loop and recycling of taxonomic composition of the autochthanous microbiota as well as nutrients in coastal systems. the relative DOM lability supplied to the system, all of which vary both spatially and temporally in response to physiochemical con- Results ditions (1, 3, 4). DOM that is assimilated into bacterial biomass is Environmental Parameters and Nutrients. Salinity at our study site potentially available for trophic transfer via the microbial loop (5) indicated vertical stratification of water at both high tides but was and as such must be accounted for in estimates of marine carbon flux. Bacterial groups that mineralize DOM are taxonomically diverse Author contributions: A.C., J.C., and H.N.W. designed research; A.C. and J.C. performed research; A.C. contributed new reagents/analytic tools; A.C. and J.C. analyzed data; and (2, 3, 6), which is often a function of niche variability (1–3). A.C., J.C., and H.N.W. wrote the paper. Specifically, estuarine systems exhibit high spatiotemporal and The authors declare no conflict of interest. physiochemical variability, often resulting in short-lived blooms of some bacterial spp. (7). Among other factors, salinity has been This article is a PNAS Direct Submission. found to typically drive bacterial succession in estuarine systems (3) Data deposition: The 16S rRNA gene sequences reported in this paper have been deposited ␣ in GenBank under accession numbers FJ160298–FJ160358 (bacteria) and FJ160359– such that in Chesapeake Bay, -Proteobacteria were predominant in FJ160412 (BLOs). 13C prey bacteria are included under FJ160294–FJ160297, M59161, and the saltwater regions and ␤-Proteobacteria in the freshwater regions DQ912807. (8). Bacterial succession is also a function of bottom-up substrate 1To whom correspondence should be addressed. E-mail: [email protected]. supply (i.e., dissolved and particulate organic and inorganic nutri- This article contains supporting information online at www.pnas.org/cgi/content/full/ ents) from autochthanous and allochthanous sources (4, 9). In 0809671106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809671106 PNAS ͉ March 17, 2009 ͉ vol. 106 ͉ no. 11 ͉ 4301–4306 Downloaded by guest on September 27, 2021 193.00 12.00 Table 1. Relative bacterial and BLO phylotype abundances from 191.00 samples collected over the tidal cycle at Dry Bar in Apalachicola 10.00 Bay, Fl, in December 2006 189.00 M) Closest phylogenetic HT-I OT LT IT HT-II 187.00 μ M) ( μ n 8.00 io taxa/specie from NCBI (%) (%) (%) (%) (%) at r 185.00 t n Bacterial (prey) Communitya: 183.00 6.00 ␣-Proteobacteria 181.00 DON Conce DOC Concentration ( Uncultured Alpha-proteobacteria 12 3 6 0 0 179.00 4.00 Pelagibacter sp. 45 35 29 42 54 SAR116- like sp. 8 18 0 0 2 177.00 Uncultured Rhodobacteraceae sp. 6 0 4 17 0 175.00 2.00 Uncultured Roseobacter sp. 8 5 11 0 15 23:20 2:15 3:50 5:20 8:18 10:50 12:20 14:20 Rhizobium sp. 0 0 0 6 0 Fig. 1. Changes in DOC (diamonds) and DON (triangles) concentrations vs. ␤-Proteobacteria time over the course of the 12-h tidal cycle at Dry Bar in Apalachicola Bay, FL. Uncultured Comamonadaceae 8 2 0 0 0 Time points taken at 2320, 0350, 0818, 1220, and 1420 represent HT-I, OT, LT, Burkholderia sp. 0 0 0 0 2 IT, and HT-II, respectively. Error bars represent Ϯ1 SD of duplicate samples. Limnobacter sp.00060 Ralstonia sp. 3 0 0 0 8 ␥-Proteobacteria uniformly mixed due to strong wind mixing at incoming tide (IT) Uncultured Gamma-proteobacteria 3 18 22 21 4 and low tide (LT), respectively (Fig. S1 B and C). Small changes in Oleiphilus sp. 1 0 3 0 0 salinity (19.5–21.8 ppt) were observed at 0.5 m over the course of ␦-Proteobacteria the tidal cycle (Fig. S1C) and, for the most part, closely followed the Uncultured Desulfobulbus sp. 00400 changes in tide with the sharpest increase observed between LT and Bacteroidetes HT-II, when the winds subsided and the water became stratified Uncultured Flavobacterium sp. 4 12 7 4 0 again. No significant changes in temperature (15.4–16.5 °C) or Uncultured Bacteroides sp. 0 5 12 4 11 dissolved oxygen (8.7–9.4 mg/L) were observed. Chlamydiales-Verrucomicrobia group DOC and DON concentrations oscillated over the course of the Uncultured Verrucomicrobia sp. 2 2 0 0 4 tidal cycle with relatively higher concentrations at LT (187 Ϯ 0.2 ϩ ␮ Ϯ ␮ High- G C Gram-positive bacteria: M C and 10 0.4 M N) than at outgoing tide (OT) or IT (Fig. Uncultured Actinobacterium sp. 0 0 2 0 0 1). NO3 was between 2 and 5 ␮M with lowest at low tide. NH4 concentrations remained below detection limit (data not shown). Predator (BLO) Communityb: The DOC:TDN remained fairly constant over the 12-h sampling ␦-Proteobacteria period between 15 and 17.