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B a c t e r ~ a l growth rates In sedlments have been calculated fiom measurements of the rate of lncolporatlon of trltlated thymldlne Into d e o x y r ~ b o n u c l e ~ c a ld (DNA) T h e dl lut lon of Isotope In DNA w a s used to est lmate the s u m of the pools of thymldlne In the sed lment and of other cellular precursors of t h y m ~ d ~ n e In DNA Growth rates of bacterla In the surface zone of seagrass bed sedlrnents v a r ~ e d from 3 7 X 10' cell d l v ~ s ~ o n s h ' g ' dry weight of sediment on a hot au tumn day to 3 3 X 10" cell d l v l s ~ o n s h ' g ' In w ~ n t e r By c o m b ~ n ~ n g growth rate measurements uslng the ~ s o t o p e d ~ l u t ~ o n procedure with biomass measurements ~t IS now p o s s ~ b l e to obtaln redsondbly r e l ~ a b l e estimates of b a c t e r ~ a l p roduct~vi ty In sedlments Seagrass beds are highly productive plant communltles, in which much of the primary production 1s not u t~ l ized directly by animals, but enters h ~ g h e r trophlc levels through microorganisms, especially bacteria (Phi l l~ps and McRoy, 1980) To quantify this process we need to know not only the blomass of the bacterial population, but also its growth rate Methods are available for measuring biomass in these sedim e n t ~ , w h ~ c h show that bacteria in seagrass beds of Moreton Bay Queensland constitute dbout 20 O/o of the sedlment organic matter excluding that which seagrass roots contribute (Morla~ty , 1980) The measurement of growth rates of the whole population has not been possible by classical microb~ological techniques By measuring the rate of synthesis of deoxyr~bonuc le~c acid (DNA), we hoped to be able to estimate the growth rate of bactena In the sedlment Bacteria In sedlments take up [ m e t h ~ l ~ H ] thymidine (thymine-2deoxyribose Tdr) and use lt for DNA synthesis (Tobln and Anthony, 1978) Fuhrman and Azam (1980) have used the rate of incorporation of Tdr into DNA in seawater to e s t~ma te growth rate of planktonic bacterm They assumed that by adding a large excess of ~so tope , the contribution of Tdr from other pools or pathways would be negligible In fact this may not be the case as Rosenbaum-Oliver and Zamenhof (1972) found that exogenous Tdr contributed only a portlon of the Tdr in DNA, varylng from 35 % to 63 % In a normal strain of Escherichia coli, depending on the culture conditions. The highest value of 63 % of exogenous Tdr was obtalned with l mg Tdr m l ' of culture medium. If natural populations of bacteria behave similarly, then to estlmate the rate of DNA synthesis, and thus obtain the rate of bacterial division, it is necessary to measure the dilution of added Tdr by pools in the cells and in the sediment. Thymidine-5'triphosphate (dTTP), the final precursor in DNA synthesis, is synthesised only partly from exogenous Tdr and partly via other pathways within the cell (Rosenbaum-Oliver and Zamenhof, 1972). An isotope dilution experiment can be used to d e t e r m ~ n e the effect of added 'cold' precursor on the amount of labelled precursor incorporated into a macromolecule (Forsdyke, 1968). In this paper w e show how, using this technique, the total sum of pools contributing thymine bases to DNA synthesis may be estimated, and thus calculate the growth rate of the bacterial population. MATERIALS AND METHODS We took a series of cores (25 mm diameter) of sediment from a seagrass bed in Moreton Bay, Queensland (Australia), which was dominated by Zostera capricorni, and combined the top 3 mm. For routine isotope d~ lu t ion experiments, a series of 25 mm diameter centrifuge tubes (usually 7 , but more for some experiments) were set up containing 7.4 X 10' Bq (20 Mar Ecol. Prog. Ser 5: 151-156. 1981 t ~ C i ) of tri.tiated Tdr. Unlabelled Tdr was added to give a serles with the ~so tope diluted by progressively more Tdr a s shown in the figures. For some experiments, more tritiated Tdr was used. Portions of the mixed slurry were dispensed with a small plastic spoon into the tubes and incubated at the in s i t u temperature in a water bath on the boat. The incubation was terminated by addition of 2 m1 of 0.6 M NaOH, which gave a final concentration of about 0.4 M NaOH (we now add 0.6 M NaOH conta in~ng l 0 mM Tdr). Sediment was dispensed into control tubes a n d treated with NaOH prior to isotope addition. Normally milder conditions for the extraction of DNA have been used (Thomas et al., 1974; Tobin and Anthony, 1978). We found, however, that the more rigorous conditions described here extracted more DNA from these sediments. Samples were heated at 100 "C for 4 h, centrifuged at 5000 X g for 10 min and the supernatants were dialysed csvelnight against running water, then 100 p1 of Tdr and DNA, each 5 rng ml-l , was added. The pH was adjusted to 0.7 with concentrated HC1. The acidified solutions were cooled for 40 min on ice and the precipitate immediately collected on a Whatman GF/C filter. After washing with 2 m1 cold 5 % (w/v) trichloracetic acid (TCA) the DNA was hydrolysed in 2 m1 5 % TCA at 100 "C for 30 min. After centrifuging, 0.5 m1 of supernatant was counted in 4 m1 scintillant (PCS 11, Amersham Australia Pty Ltd). Forsdyke (1968), in studying RNA synthesis, showed that the observed counts (X) appearing in a macromolecule were given by the relation X = n / ( p + y + l) , where n was the maximum count that would be incorporated with no dilution, and p was the dilution factor due to the endogenous pool of precursors, and ( y + 1) the dilution by the added pool. In the case of DNA synthesis, ( y + l ) is the dilution by added Tdr and p is the dilution by Tdr in the sediment and in the cell. Another factor (p) is added to these pools, the contribution from de novo synthesis via deoxyuridine-5'-monophosphate (dUMP). Thus the equation becomes X = n/ (p + y + U + l ) and is rearranged to give y+ l = (l/x)n (p+u) . Forsdyke (1968) plotted the actual amounts of pyrimidine nucleoside added, rather than the dilution, agalnst ( l l x ) , but as l l x is the dependent variable we have plotted it against the amount of Tdr present. The negative intercept on the abscissa gives the size of the pools that effectively dilute the isotope in d l T P , the final precursor of DNA. The growth rates (G) of the bacterial populations were calculated from the relation G = R X 1.3 X 10IR/ SA, where R = rate of incorporation of Tdr into DNA in disintegrations m u ' (dpm) . m i n ' g ' sediment, and SA = the specific activity of the Tdr, corrected for dilution in dpm mole' . The factor 1.3 X 10'' was derived from the assumption that thyrnine constitutes a n average of 25 % of the bases in bacterial DNA (range 12 O/O to 36 O/") and that the genome size is 2.5 X 10"altons (range 1 X 10" to 3.6 X 10q. Gillis et al., 1970; Wallace and Morowitz, 1973). Bacterial numbers were counted by direct microscopy (Moriarty, 1980).

DNA Synthesis as a Measure of Bacterial Productivity in Seagrass Sediments