AQUATIC MICROBIAL ECOLOGY Vol. 19: 57-66.1999 Published September 6 Aquat Microb Ecol 1 ~ Bromodeoxyuridine as an alternative to 3~-thymidinefor measuring bacterial productivity in aquatic samples Grieg F. Steward*,Farooq Azam Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093-0202, USA ABSTRACT: Measuring bacterial productivity with radiolabeled substrates such as tritiated thymidine (3H-TdR)poses logistical difficulties and has high associated costs due to strict regulations on the trans- port, use, and disposal of radioactivity. The TdR analog 5-bromo-2'-deoxyuridine (BrdU) can be detected in~n~unochenlicallyand has been used for many years as a non-radioactive alternative for measuring DNA synthesis in cultures. The goal of this study was to determine whether a non-radioactive im- munoassay for BrdU could be used to quantitatively measure bacterial productivity in natural aquatic samples. The first step was to determine the relative reliability of BrdU incorporation as an indicator of DNA synthesis in natural communities. Incorporation rates of 3H-~rd~and 3H-TdR in samples of coastal seawater and a freshwater lake were found to be highly correlated (I = 0.98, n = 50, p < 0.0001) with an average BrdU:TdR incorporation ratio of 0.71 i 0.24 (mean i SD).The results indicated that, despite an apparent kinetic discrimination, BrdU could accurately predict TdR incorporation over a wide range of bacterial productivity (0.45to 349 pm01 TdR 1" h-'). A filter-based chemiluminescent immunoassay was then developed and used to estimate BrdU incorporation in natural seawater and freshwater samples non-radioactively. Estimated rates of BrdU incorporation were within 0.5 to 30 % of 3H-~dRincorpora- tion rates. The assay showed a linear chemiluminescent response spanning at least 1.5 orders of magni- tude and a detection Limit of S? fmol of incorporated BrdU. These results suggest that a BrdU-based immunoassay has the potential to serve as a simple, sensitive, and quantitative non-radioactive alterna- tive to 3H-TdR for routine measurements of bacterial productivity in the field or laboratory. KEY WORDS: Bacterial production . Tritiated thymidine . Bromodeoxyuridine . Non-radioactive . Immunoassay - Method INTRODUCTION topes, there are significant, and occasionally insur- mountable, logistical difficulties and constraints on The importance of bacteria in carbon and nutrient their application in field studies. For example, ship- fluxes in the ocean is now well established (Fuhrman board use of 3H often requires a separate, specialized 1992, Ducklow & Carlson 1993, Azam 1998) and 2 of laboratory, elaborate precautions for isotope use and the most fundamental and commonly measured vari- disposal, and constant monitoring to ensure that the ables in aquatic microbial ecology are bacterial abun- ship remains free of contamination. In addition, any dance and production. Incorporation of 3H-thymidine personnel handling the isotopes are required to have (Fuhrman & Azam 1982) and 3H-leucine (Kirchman et specialized training in radiation safety. The relatively al. 1985, Sirnon & Azam 1989) are by far the most large quantities of 'H typically used for bacterial pro- widely used method for measuring bacterial productiv- ductivity measurements also pose a significant conta- ity. However, because these methods employ radioiso- mination risk for other scientific studies which rely on sensitive and accurate measurements of the extremely 'Present address: Monterey Bay Aquarium Research Insti- low levels of 3H naturally occurring in seawater. For tute, PO Box 628, Moss Landing, California 93059-0628, cruises deploying from foreign ports, transport of USA. E-mail: [email protected] radioisotopes through foreign countries is often neces- 0 Inter-Research 1999 58 Aquat Microb Ecol 19: 57-66, 1999 sary, but logistically difficult due to variable and some- mine whether BrdU incorporation could serve as a times very stringent regulations. In some cases, the use proxy for TdR incorporation in natural aquatic bacter- of radioisotopes may simply be prohibited, for example ial communities and, if so, (2) develop a non-radio- at field stations or on vessels (especially ships of oppor- active assay to quantify BrdU incorporation in those tunity) not licensed, designed, or equipped for radioac- communities. To accomplish these objectives, the in- tive work. Due to strict regulation, the costs of working corporation hnetics of BrdU and TdR were first com- with radioisotopes can also be substantial. pared using radiolabeled substrates. A non-radioactive Despite the difficulties of working with radioactivity, assay was then developed by combining and adapting this method has remained popular because it is direct, common techniques in molecular biology and im- quantitative, sensitive and relatively simple in applica- munochemistry for use with aquatic microbial commu- tion. Non-radioactive alternatives matching these cri- nities. teria have been lacking. Two approaches which have been explored are the frequency of dividing cells (FDC; Hagstrom et al. 1979) and, more recently, bacte- MATERIALS AND METHODS rial rRNA content, determined either in bulk as an RNA:DNA ratio (Kerkhof & Ward 1993) or at the single Reagents and solutions. Specialized reagents for the reli level (DeLong et al. 1989, Kemp et al. 1993, ~Herniluminescent irnmunoassajr, which included Poulsen et al. 1993). The FDC approach requires BrdU, monoclonal anti-BrdU Fab fragments conju- microscopic examination of large numbers of individ- gated to alka!ine phosphatase (anti-BrdU-AP),block- ual cells in each sample to determine the percentage ing reagent (powder), Nucleases, Incubation Buffer, which are in the process of dividing. FDC has been and the chemiluminescent substrate CDP-Starm, were found to be roughly correlated with 3H-TdR incorpora- obtained from Boehringer-Mannheim. Blocking re- tion (Riemann et al. 1984). but this method has not agent was stored at room temperature (19 to 24"C), the found widespread use because it is somewhat more nuclease stock as aliquots at -20°C, and all other difficult and time consuming and not always readily reagents at 4°C. Nuclease stock and Incubation Buffer interpretable in terms of growth rates for mixed assem- were obtained as components of the BrdU Labeling blages (Moriarty 1986). Approaches based on RNA: and Detection Kit I11 and their exact formulations are DNA ratios or rRNA content have shown promise. The considered proprietary. However, the nuclease stock is latter even has the potential to provide species specific hown to contain exonuclease 111 and 1 or more restric- growth rates. However, questions still remain about tion endonucleases, similar to original reports on the quantitatively relating RNA content to growth rates in use of nucleases in BrdU detection techniques (Dol- natural mixed assemblages. beare & Gray 1988, Bayer et al. 1990, Dinjens et al. There are compelling reasons, therefore, for explor- 1992). ing and developing other non-radioactive methods Other solutions were prepared as follows: SET (20% for measuring bacterial productivity. One alternative Sucrose, 50 mM EDTA, 50 mM Tris; pH 8), Lysis Solu- which has not yet been exploited is the use of the TdR tion ( 1.5 M NaCl and 0.5 M NaOH), Neutralization analog 5-bromo-2'-deoxyuridine (BrdU) which can be Buffer (1.5 M NaCl, 0.5 M Tris Cl; pH 7.8), Detection detected non-radioactively by a wide range of im- Buffer (0.1M NaC1, 0.1 M Tris Cl; pH 9.5),Maleic Acid munochemical techniques (Mazzotti et al. 1990, Dover Buffer (100 mM maleic acid, 150 mM NaCl; pH 73,TE & Pate1 1994, Dakhama & Hegele 1996).BrdU incorpo- (10 mM Tris, 1 mM EDTA; pH 8.0), and 10x Blocking ration into bacterial DNA has been used in numerous Buffer (10% w/v Blocking Reagent in Maleic Acid physical, biochemical, and molecular biological studies Buffer). Blocking Buffer was prepared by adding in a limited number of species (e.g. Hanawalt 1967, Blocking Reagent to Maleic Acid Buffer, heating to Binnie & Coote 1986, Yamamoto & Fujiwara 1990, 65°C in a microwave, then stirring on a hot plate to dis- Lewis & Errington 1997) and more recently as a quali- solve. All solutions except the Lysis Solution were tative indicator of DNA synthesis in natural communi- autoclaved. Blocking Buffer was stored at 4°C and all ties (Borneman 1999, Urbach et al. 1999), but has not others at room temperature. been reported previously as a quantitative measure of Field sampling. Coastal seawater samples were col- bacterial productivity. lected by submersion of a weighted polycarbonate The continuing advances in sensitivity and simplicity flask on a nylon line from the end of the pier at Scripps of non-radioactive irnmunoassays together with the Institution of Oceanography between November 1996 persistent difficulties of radioisotope use in the field and August 1998. In situ temperatures ranged from 16 have made the development of a BrdU-based assay for to 22°C. Scripps Pier samples were transported directly aquatic bacterial productivity a realistic and appealing to the laboratory and maintained at in situ temperature prospect. The objectives of this study were to (1) deter- (+2"C). Samples from Mission Bay (San Diego) and Steward & Azam: Measuring bacterial productivity with bromodeoxyuridine 59 Lake Hodges (Escondido, CA) were collected by hand procedures (Sambrook et al. 1989). The DNA pellets submersion of polycarbonate bottles at approximately were resuspended in TE and stored at 4OC. Total DNA 3 to 5 m from shore in August 1998. In situ temperature was measured by fluorometry using picogreen (Molec- of both the bay and lake was 29°C. Bay and lake sam- ular Probes) and BrdU incorporation was determined ples were transported to the laboratory in an insulated by counting aliquots in a scintillation counter. The con- chest at ambient temperature (ca 24°C) and used in centration of incorporated BrdU was calculated using labeling experiment within 3 h of collection. All sam- the measured incorporated activity and the known ples were obtained from 0 to 0.5 m depth.