Occurrence of β-Aminoglutaric Acid in Marine Bacteria
SUSAN M. HENRICHS1* AND RUSSELL CUHEL2
Institute of Marine Science, University of Alaska, Fairbanks, Alaska 99701,1 and Division of Biology and Living Resources, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, Miami, Florida 331492
Received 21 March 1985/Accepted 29 May 1985
β-Aminoglutaric acid, a nonprotein amino acid isomer of glutamic acid, was found in the free amino acid pool of a marine bacterium, Alteromonas luteoviolacea. It was also found in a mixed culture of fermenting bacteria enriched from an anoxic marine sediment.
A nonprotein amino acid, β-aminoglutaric acid [HOOCCH2CH(NH2)CH2COOH], is a major constituent of dissolved free amino acids (DFAA) in marine sediments (2, 6-8). Others (5, 9) report a major unidentified pore water DFAA which, based on chromatographic behavior, is probably β-aminoglutaric acid. These pore waters came from a wide range of depositional environments, including both oxidizing and reducing sediments. β-Aminoglutaric acid has been previously reported to be a constituent of Chondria armata, a red alga (4, 15). C. armata, however, is not a plausible source for β-aminoglutaric acid in pore water samples, and we were unable to find other reports of the occurrence of this amino acid in organisms. A related compound, 3-(N-methylamino)glutaric acid, is found in Prochloron didemnii (14).
Because concentrations of ,-aminoglutaric acid are often relatively high in anoxic sediments, bacteria are a likely source. Free amino acid pools of marine and nonmarine bacteria have been analyzed previously (1, 13, 17) without detection of β-aminoglutaric acid. However, these studies used techniques which might not have resolved β-aminoglutaric acid from glutamic acid. We analyzed the DFAA and insoluble protein amino acids of several marine bacteria to discover whether ,B-aminoglutaric acid is a constituent which was overlooked previously and to confirm bacteria as a potential source in pore waters. The bacteria examined represent several nutritional types: obligately aerobic heterotrophs (Alteromonas luteoviolacea and Pseudomonas halodurans); a heterotrophic sulfate reducer (Desulfovibrio salexigens); an autotrophic ammonia oxidizer (Nitrosococcus oceanus); and facultatively anaerobic bacterial communities from enriched sediments. A. luteoviolacea and P. halodurans were grown in an artificial seawater medium (3) with the addition of 20 mM D-glucose, 1 mM NH4+, 80 p.M P043-, 1 mM S042-, and trace metals. The cells were harvested after 51 h (P. halodurans) or 95 h (A. luteoviolacea) at 22°C. D. salexigens was grown anaerobically in artificial seawater (10) supplemented with Tris buffer (10 mM), NH4Cl (400 ,uM), Lcalcium lactate (10 mM), and trace elements. N. oceanus was grown in the medium described by Watson (16) to a density of 108 cells per ml. In all cases cells were harvested by centrifugation.
A selective enrichment of fermenting cells from the Pettaquamscutt River estuary was done as follows. A sediment sample (which had been stored frozen for 4 months) was thawed, and 0.25 ml was suspended in 10 ml of sterile Sargasso seawater with added NH4′ (0.5 mM), P043- (40 FM), trace metals, and 5 mM of one of the following: glucose, fructose, glycerol, sucrose, galactose, mannitol, 8-gluconolactone, or mannose. The suspensions were sparged with argon and capped. After 5 days, the cultures were centrifuged at 5,000 x g for 10 min and then suspended in 5 ml of artificial seawater. These suspensions were inoculated at 0.25 ml per 25 ml in the above media, except that substrate concentrations were increased to 20 mM. The cells were harvested after 24 days of aerobic growth. Glucose, galactose, fructose, and mannitol substrates yielded enough cellular material for analysis. Pellets resulting from centrifugation were extracted by boiling with doubly distilled water, and the resulting solutions were filtered through Gelman A/E glass fiber filters to remove particulate material. For A. Iuteoviolacea and D. salexigens, material retained by these filters was hydrolyzed in 6 N HCI for 24 h at 110°C, Amino acid analyses of boiling-water extracts and particle hydrolysates were performed as described previously (8; S. M. Henrichs, Ph.D. thesis, Massachusetts Institute of Technology, Cambridge- Woods Hole Oceanographic Institution, 1980). This method involves desalting, derivatization to form the Nheptafluorobutyryl- n-butyl esters of the amino acids, and glass capillary gas chromatography. Data for arginine, histidine, tryptophan, and cysteine are not shown, because these amino acids could not be precisely measured by this technique. Samples containing a peak coeluting with the derivative of authentic β-aminoglutaric acid were subjected to gas chromatography-mass spectrometry to confirm peak identity. The DFAA compositions of the four species of bacteria analyzed are shown in Table 1. β-Aminoglutaric acid was present in A. luteoviolacea at 5 to 6 mol% of the DFAA. It was not detected in the other species. The occurrence of β-aminoglutaric acid was confirmed by mass spectrometry (Fig. 1). The mass spectrum of the compound from A. luteoviolacea was essentially identical to that of β-aminoglutaric acid from marine sediment pore water (6) and to that of authentic β-aminoglutaric acid. The presence of β-aminoglutaric acid in the DFAA of A. luteoviolacea was further confirmed by repeating the analysis after the culture had been maintained in the laboratory for 1 year (c.f. columns A and B, Table 1).
Free amino acids extracted from P. halodurans contained significant amounts of two other nonprotein amino acids, β-alanine and -γ-aminobutyric acid, which have been reported to occur in marine sediment hydrolysates or pore waters (8, 12). Glutamic acid was the major constituent of the DFAA of all bacteria analyzed, as previously reported (1, 13, 17). However, DFAA compositions and concentrations vary with growth conditions (13), and so the data are valid only for the particular conditions given.
Because some bacterial cell walls include nonprotein amino acids (11), hydrolysates of the insoluble residues of A. luteoviolacea and D. salexigens were examined for β-aminoglutaric acid (Table 2). None was detected.
Results of analyses of mixed cultures of fermenters enriched from Pettaquamscutt River estuary sediments are given in Table 3. Pore water DFAA from this highly anoxic sediment (0.6 mM H2S) contained about 1 FiM β-aminoglutaric acid (Henrichs, Ph.D. thesis). β-Aminoglutaric acid made up 4 mol% of the DFAA of bacteria grown on galactose but was not found in bacteria grown on the other substrates. The source of β-aminoglutaric acid in the enrichment culture could not have been A. luteoviolacea, because it is an obligate aerobe.
We conducted an experiment to find whether β-aminoglutaric acid alone could support growth of A. luteoviolacea or P. halodurans. The bacteria were inoculated into the artificial seawater medium described earlier, except that 5 mM of β-aminoglutaric acid was added instead of D-glucose. Neither A. luteoviolacea nor P. halodurans grew with β-aminoglutaric acid as the sole carbon and energy source. Amino acids which did support growth as sole carbon and energy sources at 5 to 10 mM concentrations were alanine, β-alanine, aspartic acid, asparagine, glutamic acid, glutamine, and lysine (P. halodurans) and alanine, asparagine, glutamic acid, and glutamine (A. luteoviolacea). Thus, the structure of β-aminoglutaric acid was definitely a significant factor in the failure of either organism to grow. In conclusion, we have shown biosynthesis of β-aminoglutaric acid by A. luteoviolacea and by an unidentified facultative anaerobe(s). This suggests that bacteria produce β-aminoglutaric acid in sediments, but production by the obligately aerobic A. luteoviolacea indicates that sedimenting particles are also a potential source. High proportions of β-aminoglutaric acid relative to other amino acids in pore waters (2, 6-9) may result from resistance to biodegradation, because neither the nutritionally versatile organism P. halodurans nor a source organism, A. luteoviolacea, could utilize it as a sole substrate for growth.
This work was supported by National Science Foundation grants OCE 79-18665 (to S.M.H.) and OCE 77-12172 (to R.L.C.).