ASSESSING THE INFLUENCES OF PREDATION AND RESOURCE LIMITATION ON MICROBIAL POPULATIONS

Paul W. Lepp and Thomas M. Schmidt

Department of Microbiology and Center for Microbial Ecology, Michigan State University, E. Lansing, MI 48824

SUMMARY

Applying models of predator-prey interactions we are seeking to determine which factors control the population size of Synechococcus along a nutrient gradient in the Gulf of Mexico is limited by available resources or predation. We have found that the synechococci population increases along this gradient. We demonstrate that radiolabeled oligonucleotide probes can be used to determine changes in macromolecular composition, which reflect changes in cellular growth rate. Plots of the synechococci equilibrium population size versus cellular growth rate are being used to determine the relative influence of resource limitation and predation on the size of the synechococci populations.

INTRODUCTION

Current models of predator-prey interactions suggest that a plot of the equilibrium prey population size against the specific growth rate, , has the predictive power to determine whether that population is limited by nutrients, predation or a combination of both. The goal of this research is to determine whether the size of marine Synechococcus populations, along a nutrient gradient in the Gulf of Mexico, are nutrient limited or predator controlled. Because the macromolecular composition of microorganisms varies proportionately with specific growth rate it has been possible to use radiolabeled oligonucleotide probes targeting 16S rRNA to determine relative changes in specific growth rates.

Cyanobacteria of the genus Synechococcus are reported to contribute from 2 to 46% of total primary productivity moving from coastal to oceanic waters (Prezelin et al., 1986). Since their discovery in marine environments, synechococci (Waterbury et al., 1986) have been proven to be both ubiquitous and abundant by epifluorescence microscopy (Waterbury et al., 1986), flow cytometry (Li, 1994) and nucleic acid sequence analysis (Giovannoni et al., 1990; Schmidt et al., 1991). Despite their significant contribution to total primary production and marine community structure, little is known about the mechanisms controlling their population size. An understanding of the mechanisms which control Synechococcus populations is the first step in constructing models of marine trophic interactions.

Synechococcus populations in nature may often be limited in size by available resources. Reports indicate that Synechococcus may at times be limited for light (Mitchell et al., 1991), nitrogen (Glover et al., 1988), phosphorus (Waterbury et al., 1979) and trace metals, in particular Fe2+ (Martin et al., 1994).

Synechococcus populations may also be limited in size by predation. A number of flagellates and protozoa are known predators of Synechococcus in marine environments (Caron et al., 1991). The reported in situ grazing coefficient for flagellates and protozoan specific to Synechococcus range from 0 to 1.7 d-1 (Caron et al., 1991). Grazing pressure can reportedly reduce the population of Synechococcus by 30 to 50% (Caron et al., 1991) and was a possible mechanism in controlling phytoplankton populations in recent experiments (Banse, 1995). Synechococcus populations may also be controlled species specific bacteriophage. Viruses have been reported to reduce phytoplankton primary productivity by as much as 78% (Jiang and Paul, 1994).

Prey-dependent (Power, 1992) and ratio-dependent models (Arditi and Ginzburg, 1989) of predator-prey interactions suggest that the factors controlling synechococci population size may be resolved by plotting equilibrium population size versus specific growth rate.

Under a prey-dependent model bacterial populations free of predator control tend to reach an equilibrium population size dependent on the concentration of the limiting resource. An increases in the equilibrium synechococci population size results from an increase in the concentration of the limiting nutrient, but will not increase the specific growth rate. This situation is graphically represented as the resource limited population in Figure 1. Increased predation results in an increase in the specific growth rate, µ, of the Synechococcus population without affecting equilibrium population size, as depicted in the predator limited population in Figure 1. These alternating mechanisms of control in prey-dependent models predict a change in the factors controlling the equilibrium prey population size that alternates with the number of trophic interactions, as depicted in Figure 1. Models of resource and predator control of Synechococcus populations are currently being tested in our lab using phosphate-limited chemostats.

Ratio-dependent models predict that an increase in available resources leads to a proportional increase in the equilibrium population size at all trophic levels. Because there is a concurrent increase in the predator population, as well as the prey population, there should be an increase in the specific growth rate, µ, in order for the prey population to maintain itself in the face of increased consumption. Thus, the ratio-dependent model would predict an increase in available resources would lead to an increase in both the equilibrium prey population and the specific growth rate, µ, of the prey population. This situation is illustrate in Figure 2.

A measurement of the in situ growth rate is a crucial parameter in determining whether the Synechococcus population is substrate limited or predator controlled. There is a long established correlation between macromolecular composition and the specific growth rate of a microorganism (Rosset et al., 1966). It should therefore be possible to probe the 16S rRNA with species specific radiolabeled oligonucleotide probes to determine relative changes in growth rate among a population.

MATERIALS AND METHODS

Determining the Population Size of Synechococcus. Sampling to determine the equilibrium population size and isolation of nucleic acids was done at 6 stations along a salinity gradient in the Gulf of Mexico between October 23, 1993 and November 3, 1993. The decrease in salinity is indicative of the influence of the nutrient rich waters of the Mississippi River (Whitledge, 1994).

Seawater was prefiltered through a 10 µm cartridge filter (Micron Separations, Inc./Cole-Parmer) at 15 psi before being filtered through a 1 µm cartridge filter (Millipore). Amounts varying from 10 ml to 50 ml of < 1 µm filtered water were filtered onto black polycarbonate membrane filters (Poretics). Utilizing the autofluorescent properties of the photosynthetic pigments the Synechococcus population size was determined by direct epifluorescent counts of 300 - 400 cells distributed among 20 to 30 random fields using a Zeiss 48.77.09 filter set.

Models of Synechococcus PCC 6301Population Control. Stock cultures of Synechococcus PCC 6301 (ATCC 27144) were maintained at room temperature on BG-11 agar at 86.5 µEm-2 s-1.

Batch cultures of Synechococcus PCC 6301 were grown on BG-11 at 37C and 150 rpm. Variation in growth rate was accomplished by changing the available light intensity from 15 µEm-2 s-1 to 64.4 µEm-2 s-1.

Chemostat cultures of Synechococcus PCC 6301 were grown on modified Cg medium as previously described (Ihlenfeldt and Gibson, 1975). Cultures were illuminated at 162 µEm-2 s-1 at 37C and aerated with 40 ml of 3% CO2 per min. Phosphate limited growth was achieved by varying the reservoir concentration from 5 µM K2HPO4 to 15 µM K2HPO4. Growth rate was varied by changing the dilution rate from 20 µl min-1 to 365 µl min-1 in a 250 ml culture vessel. The culture was considered to have reached a stable equilibrium after maintaining the same optical density at 750 nm and 600 nm for a period of three volume changes of the culture vessel.

Chemical Determination of Cellular RNA Content. Cells of Synechococcus PCC 6301 were harvested by centrifuging 10 ml culture at 16,000 x g for 25 min at 4C. Cell pellets were washed twice with ice cold absolute EtOH and resuspended in 0.1 M NaCl. RNA content per cell was determined by a modified orcinol procedure, previously described (Parrott and Slater 1980).

Determination of Cellular 16S rRNA Content. In order to reduce RNase contamination all glassware was DEPC treated and baked at 240C or virgin polypropylene was used whenever possible. A 1.5 ml sample was taken from a log phase batch culture or stable chemostat culture and centrifuged at 16,000 g for 25 min. Nucleic acids were isolated by mechanical disruption (Stahl et al., 1988).

A cyanobacterial-specific oligonucleotide probe (5'-CATTGCGGAAA

ATTCCC-3' 16S rRNA) was radiolabeled with 0.25 mCi [32P] -labeled ATP in 1x kinase buffer (50 mM Tris [pH 7.6], 10 mM MgCl2, 5 mM DTT, 0.1 mM spermidine, 0.1 mM EDTA [pH 7.6], 0.001% NP-40 (Sigma Chemical Co.) and 10 U T4 phosphonucleotide kinase (PNK). The solution was incubated for 30 min at 37C, followed by the addition of 10 U T4 PNK for another 30 min incubation at 37C. The reaction was terminated by the addition of stop buffer (11 nM EDTA, 54 nM NH4OAc). The radiolabeled probe was purified by a triple elution from a TSK-DEAE column with 1 ml 50 mM NH4OAc, once with 1 ml 100 mM NH4OAc and stripping with 1 ml 1 M NH4OAc. The probe was then dried in a speed-vac, suspended in 150 µl H2O, and the specific activity determined by scintillation counting.

Quantitation of 16S rRNA was done by comparison of slot blots on MagnaCharge nylon membranes (Micron Separation, Inc.) with known quantities of purified 16S rRNA. RNA content per cell was calculated assuming rRNA was 34% 16S rRNA by weight and that rRNA constituted 80% of cellular RNA by weight.

RESULTS AND DISCUSSION

Our evidence indicates that Synechococcus population size increases along a salinity gradient that is increasingly influence by the nutrient rich Mississippi River, e.g. the nitrate concentration decreases from 40 mM at the mouth of the Mississippi to <1 mM at off shore stations (Whitledge, 1994). Data on Synechococcus population size were gathered on 10/27/93 - 11/3/93. Six sites ranging in salinity from 20 ppt to 30 ppt were examined. Synechococcus population sizes were found to range from 1.36 x 103 cells ml-1 at the high salinity, low nutrient sample site to 2 x 105 cells ml-1 at the low salinity, high nutrient sample site (Table 1). The increase in available resources may be responsible for the increase in the Synechococcus population, an explanation that is consistent with a resource-control in a prey-dependent model (Figure 1). However, increases in population size alone do not exclude more complex interactions and models, e.g., ratio-dependent models of predator-prey interactions (Arditi and Ginzburg, 1989) (Figure 2). In order to distinguish between alternative models it is necessary to determine cellular growth rate within the population.

A prey-dependent model, exhibiting resource-control, would predict that increases in the equilibrium Synechococcus population size would not be accompanied by an increase in the cellular growth rate from sampling site to sampling site. Rather each of the sampling sites would be expected to have similarly low cellular growth rates (Figure 1).

There is a long established correlation between macromolecular composition and the specific growth rate of a microorganism (Rosset et al., 1966). Chemical measurements of the RNA content per cell, from light-limited batch cultures, increased from 32 fg/cell at a specific growth rate of 0.019 h-1 to 49 fg/cell at a specific growth rate of 0.067 h-1 (Table 2) for the fresh water species Synechococcus sp. PCC 6301, supporting earlier studies (Mann and Carr, 1974). Chemical measurements of the RNA content per cell, from phosphate-limited chemostat cultures of Synechococcus sp. PCC 6301, increased from 21 fg/cell at a specific growth rate of 0.005 h-1 to 58 fg/cell at a specific growth rate of 0.086 h-1 (Table 2), confirming earlier studies (Parrott and Slater, 1980).

However, chemical means of measuring RNA are not specific for a particular species. Rather the method measures all RNA within a sample, regardless of whether the sample consists of a multi-species community or is an axenic culture. For this reason it is not possible to use chemical measurements of total RNA from samples gathered in the Gulf of Mexico to determine the specific growth rate of Synechococcus alone. Species specific radiolabeled oligonucleotide probes targeting 16S rRNA do not suffer from the same shortcomings encountered with chemical measurements of RNA. In both light-limited batch cultures and phosphate-limited chemostat cultures of Synechococcus sp. PCC 6301, values ranging from 5.7 fg 16S rRNA/cell at a specific growth rate of 0.005 h-1 to 18.5 fg 16S rRNA/cell at a specific growth rate of 0.087 h-1 were observed using radiolabeled oligonucleotide probes specific for cyanobacterial 16S rRNA (Table 3). Estimates of total RNA content per cell ranged from 20.6 fg/cell to 69 fg/cell, respectively. This technique offers the advantage of being able to specifically determine the 16S rRNA content of a single species in a non-axenic culture. We are now applying this technique to detect relative changes in the specific growth of synechococci along the salinity gradient the Gulf of Mexico.

ACKNOWLEDGMENTS

This research was supported by U.S. Environmental Protection Agency grant CR 822014-01-0 and the National Science Foundation Center for Microbial Ecology BIR9120006.

REFERENCES

Arditi R and Ginzburg LR (1989). Coupling in predator-prey dynamics: ratio-dependence. J. theor. Biol. 139:311-326.

Banse K (1995). Community response to IRONEX. Sci. 375:112.

Caron DA, Lim EL, Miceli G and Waterbury JB (1991). Grazing and utlization of chroocoid cyanobacteria and heterotrophic bacteria by protozoa in laboratory cutlures and a coastal plankton community. Mar. Ecol. Prog. Ser. 76:205-217.

Giovannoni SJ, Britschgi TB, Moyer CL and Field KG (1990). Genetic diversity in Sargasso Sea bacterioplankton. Nat. 354:60-63.

Glover HE, Prezelin BB, Campbell L, Wyman M and Garside C (1988). A nitrate-dependent Synechococcus bloom in surface Sargasso Sea water. Nature 331:161-163.

Ihlenfeldt MJA and Gibson J (1975). CO2 fixation and its regulation in Anacytis nidulans (Synechococcus). Arch. Microbiol. 102:13-21.

Jiang SC and Paul JH (1994). Seasonal and diel abundance of viruses and occurrence of lysogeny/bacteriocinogeny in the marine environment. Mar. Ecol. Prog. Ser. 104:163-172.

Li WKW (1994). Primary production of prochlorophytes, cyanobacteria, and eucaryotic ultraphytoplankton: measurements from flow cytometric sorting. Limnol. Oceanogr. 39:169-175.

Mann N and Carr NG (1974). Control of macromolecular composition and cell division in the blue-green alga Anacystis nidulans. Journal of General Microbiology 83:399-405.

Martin JH, et al. (1994). Testing the iron hypothesis in ecosystems of the equatorial Pacific Ocean. Nature 371:123-129.

Mitchell BG, Brody EA, Holm-Hansen O, McClain C and Bishop J (1991). Light limitation of phytoplankton biomass and macro-nutrient utilization in the Southern Ocean. Lim. Oce. 36:1662-1677.

Parrott LM and Slater JH (1980). The DNA, RNA and protein composition of the cyanobacterium Anacytis nidulans grown in light and carbon dioxide-limited chemostat. Arch. Micro. 127:53-58.

Power ME (1992). Top-down and bottom-up forces in food webs: do plants have primacy? Ecology 73:733-746.

Prezelin BB, Putt M and Glover HE (1986). Diurnal patterns in photosynthetic capacity and depth-dependent photosynthesis-irradiance relationships in Synechococcus spp. and larger phytoplankton in three water masses in the Northwest Atlantic Ocean. Marine Biology 91:205-217.

Rosset R, Juoien J and Monier R (1966). Ribonucleic acid composition of bacteria as a function of growth rate. J. Mol. Biol. 18:308-320.

Schmidt TM, DeLong EF and Pace NR (1991). Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing. J. Bac. 173:4371-4378.

Stahl D, Flesher B, Mansfield HR and Montgomery L (1988). Use of phylogentically based hybridization probes for studies of ruminal microbial ecology. Appl. Environ. Microbiol. 54:1079-1084.

Waterbury JB, Watson SW, Guillard RRL and Brand LE (1979). Widespread occurence of a unicellular, marine, planktonic, cyanobacterium. Nature 277:293-294.

Waterbury JB, Watson SW, Valois FW and Franks DG (1986). Biological and ecological characterization of the marine unicellular cyanobacterium Synechococcus. Canadian Bulletin of Fisheries and Aquatic Science 214:71-120.

Whitledge TE (1994). Nuctrient concentrations at the Mississippi/Atchafalaya River outflows. Coastal Oceanographic Effects of Sumer 193 Mississippi River Flooding. National Oceanic and Atmospheric Administration. 36-37.

Figure 1. Prey dependent model of equilibrium prey populations (N), along a nutrient gradient, versus specific growth rate of the prey (µ). Each line segment represents an additional trophic level.

Figure 2. Alternative relationships, predicted by ratio-dependent models, between Synechococcus equilibrium population size (N) and specific growth rate (µ).