Expression of glutamine synthetase and glutamate dehydrogenase by marine bacterioplankton: Assay optimizations and efficacy for assessing nitrogen to carbon metabolic balance in situ

Expression of glutamate metabolism enzymes, glutamate dehydrogenase (GDH), and glutamine synthetase (GS) are proposed to yield information on the nitrogen (N) to carbon (C) metabolic balance within bacterioplankton communities. Whole-cell assay conditions were optimized, and reactions were linear with time and biomass. Enzyme activities were assayed in seawater cultures from four ecosystems of contrasting trophic state amended with different regimes of glucose, amino acids, and ammonium (NH4 +) to validate expression patterns of GDH and GS in various combinations of N and C limitation or excess. In three of four experiments, glucose amendment enhanced GS expression by 2-fold but repressed GDH by 10% to 40% relative to the control. In contrast, addition of amino acids or NH4 + resulted in 20% to 90% repression of GS and enhanced GDH expression by 20% to 900%. The GDH:GS activity ratio (×10–3) ranged from 6 to 22 in glucose added treatments and 63 to 264 in NH4 +-amended treatments and appears to be a more sensitive index of bacterial N bioavailability relative to C supply than either enzyme alone. Cluster analysis was used to identify the condition of ambient bacterioplankton by matching enzyme expression with the validation results. This enzymatic approach overcomes some of the biases and limitations of other methods for assessing N metabolism in marine bacteria while providing unique information to further understand constraints of bacterioplankton respiration, production, and N flux. *Current address: Pennsylvania State University, York, PA 17403, USA. Acknowledgments Thanks to Roland Ferry for assisting with logistics during the southeast Florida cruise and to the captains and crews of the OSV Anderson and RV Bellows. Special thanks to Luis Cifuentes and Lynn Roelke for assisting with TDN analyses. This research was largely supported by a cooperative agreement between Texas A&M University and the U.S. EPA, Gulf Ecology Research Laboratory and U.S. EPA, Region IV, Water Management Division. Additional support came from an NSF Fellowship in Marine Biotechnology (OCE-9321698) to M.P. Hoch. Limnol. Oceanogr.: Methods 4, 2006, 308–328 © 2006, by the American Society of Limnology and Oceanography, Inc. LIMNOLOGY and OCEANOGRAPHY: METHODS and protist growth (Hoch et al. 1994). These limitations may bias interpretation of in situ biogeochemical processes if not recognized. Alternatively, ambient rate measurements of N uptake and NH4 + regeneration via short-term incubations with tracer compounds provide opportunities to observe and test conditions that influence N metabolism with little disturbance to other plankton processes. For example, studies of NH4 + uptake and its inhibition by amino acids, particularly glutamine, suggests N starved bacterioplankton possess a high affinity NH4 + transport system and NH4 + assimilation enzymes similar to enteric bacteria (Fuhrman et al. 1988; Kirchman et al. 1989). However, these rate measurements are technically more challenging and costly than seawater cultures, particularly if multiple compounds are studied simultaneously. Physiological approaches to understanding bacterioplankton N metabolism in situ have largely focused on N-substrate use and defining N replete versus N deplete conditions. Aminopeptidase activity has greatly contributed to the understanding of protein degradation and use by bacterioplankton (Someville and Billen 1982; Smith et al. 1992). Recently, enhanced leucineaminopeptidase activity (LAPA) in total plankton communities was proposed as an indicator of N-depleted plankton conditions (Sala et al. 2001). Whether LAPA increases specifically in N-depleted bacterioplankton remains uncertain given that other studies have identified the inverse relationship (Christian and Karl 1998, Jørgensen et al. 1999). The C:N ratio of bacterial biomass has also been proposed to increase with more N deplete metabolic conditions (Fagerbakke et al. 1996). This parameter reflects a coupling of N and C metabolism, but the range in biomass C:N ratio is small for natural bacterioplankton (3.8 to 9.9) and may not vary significantly (Fukuda et al. 1998). Another direct approach to exploring bacterioplankton N and C metabolism is to analyze concentrations and isotope dilution of intracellular amino acids (Simon 1991; Simon and Rosenstock 1992), especially glutamate, which couples N and C metabolism via NH4 + assimilation. However, an assumption that high intracellular glutamate concentration indicates NH4 + as primary N source under N deplete conditions has not been verified. Glutamate can also be produced by NH4 + assimilation under N replete conditions and catabolism of numerous other nitrogenous metabolites (Hudson and Daniel 1993). However, the relative activities of enzymes involved in glutamate biosynthesis and catabolism could be a useful indicator of N bioavailability in heterotrophic marine bacterioplankton. Prokaryotes have two major pathways for intracellular NH4+ assimilation that involve glutamate (Merrick and Edwards 1995). All prokaryotes studied have a high affinity pathway (Km ≤ 0.1 mM intracellular NH4 +) whereby ammonia is captured by glutamate to form glutamine via the glutamine synthetase (GS) reaction: glutamate + NH3 + ATP → glutamine + ADP + Pi. The GS reaction can couple with the glutamate synthase (GOGAT) reaction, which catalyzes transamination between glutamine and α-ketoglutarate to form two glutamates: glutamine + α-ketogluterate + NADPH → 2 glutamate + NADP+. The glutamate dehydrogenase (GDH) reaction is an alternate lower affinity (Km ≥ 1.0 mM intracellular NH3) pathway for many prokaryotes, whose reaction is similar to the net GS/ GOGAT reaction except ATP is not required for ammonia assimilation: NH3 + α-ketogluterate + NAD(P)H ↔ glutamate + NAD(P) +. The reverse reaction permits oxidative deamination of glutamate in addition to the reductive amination of α-ketogluterate to glutamate. Generally, the anabolic reaction requires the NADPH as coenzyme and the catabolic reaction uses NADH (Hudson and Daniel 1993). There are multiple isozymes for the NH4 + assimilation enzymes. Four GS isozymes have been identified (Pesole et al. 1995; Eisenburg et al. 2000). Some bacteria have multiple copies of their GS gene and even more than one GS isozyme. The transcriptional regulatory mechanisms are also diverse among GS genes and even within the glnA gene family of enterics (Reitzer 2003), cyanobacteria (Herrero et al. 2001), and bacilli (Fisher 1999; Hu et al. 1999). Post-translational regulation of GS activity is by allosteric inhibition, and the GSI-β isozyme is also subject to covalent modification (adenylylation or ADPribosylation). Conditions controlling gene expression and enzyme activity are consistant for GS isozymes. Many prokaryotes have a constitutive level of GS expression, which can be greatly enhanced when N bioavailability is low relative to the organic C substrate supply and vice versa (Merrick and Edwards 1995). There are four GDH isozymes that vary in subunit number and size as well as coenzyme specificity (Hudson and Daniel 1993; Anderson and Roger 2003). Like GS types, there are different regulatory motifs of gene expression for GDH isozymes, but in contrast to GS, expression of GDH isozymes is increased when N substrates are in excess of organic C substrates. These conditions include high (mM) NH4 + or N-rich amino acids like arginine, ornthine, or histidine (Smith et al. 1975; Hudson and Daniel 1993; Baggio and Morrison 1996). Excess organic C and energy substrates like glucose will repress GDH, particularly catabolic GDH types (e.g., Bonete et al. 1996; Belitsky et al. 2004). Overall, the conditions that lead to GDH expression are associated with GS repression and vice versa, as has been demonstrated for putative marine pseudomonads and marine vibrios (Brown et al. 1972; Hoch et al. 1992). Thus, measurements of the GS and GDH specific activities in natural bacterioplankton may provide community level information on the average balance of N to C metabolism. Activities of GS and GDH have been measured in a few studies of N metabolism in natural phytoplankton communities (Ahmed et al. 1977; Dorch et al. 1985; Clayton and Ahmed 1987; Mulholland et al. 2001) and one experimental study of heterotrophic bacterioplankton (Jørgensen et al. 1999). In the Hoch et al. Bacterioplankton GS and GDH expression