hydrolysis of uniformly labelled protein and methylated protein was nearly equal, percent ... gests that methylated amino acids (primarily lysine) were not.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1992, p. 1374-1375
Vol. 58, No. 4
0099-2240/92/041374-02$02.00/0
Copyright © 1992, American Society for Microbiology
Bacterial Hydrolysis of Protein and Methylated Protein and Its Implications for Studies of Protein Degradation in Aquatic Systems RICHARD G. KEILt* AND DAVID L. KIRCHMAN
College of Marine Studies, University of Delaware, Lewes, Delaware 19958 Received 24 September 1991/Accepted 21 January 1992
Ribulose 1,5-bisphosphate carboxylase was radiolabelled by in vitro translation, resulting in uniformly labelled ribulose 1,5-bisphosphate carboxylase, and also by reductive methylation. We investigated the degradation of the two forms of radiolabelled protein by natural bacterial populations. Although total hydrolysis of uniformly labelled protein and methylated protein was nearly equal, percent assimilation, respiration, and release as low-molecular-weight material were different. Radioactivity from uniformly labelled protein was approximately equally assimilated into cells, respired as 3H20, and released as low-molecularweight material, but radioactivity from the methylated protein was nearly all released as low-molecular-weight material, and little was assimilated or respired. and filtered through a 1.0-p.m Nuclepore filter. This filtration separated the bacterial assemblage from most bactivores (8) and removed particulate matter which may have adsorbed the protein. RuBPcase was added to subsamples of the seawater (final concentration, 20 ,ug liter-'), and bacteria were allowed to utilize the protein. Over time, 10-ml samples of the seawater were withdrawn and filtered onto 0.45-pum Millipore filters to determine uptake. The 0.45-p.m filtrate was filtered through a 10,000-Da ultrafilter (Amicon) to estimate the release of LMW material. After ultrafiltration, the seawater was evaporated and the amount of 3H20 generated was determined by difference. After 17 h, the percentage of the added label incorporated into biomass was 32% for the uniformly labelled RuBPcase, but was not significantly different from zero for the protein radiolabelled via reductive methylation (Fig. 1). This suggests that methylated amino acids (primarily lysine) were not assimilated. The amount of labelled protein released as LMW material was 18 to 26% for the uniformly labelled protein and 63% for the 3H-methylated protein (Table 1). Some 20 to 22% of the uniformly labelled protein was respired to 3H20, whereas only 4% of the 3H-methylated protein was respired (Table 1). Thus, after 17 h, total utilization of the uniformly labelled protein was 75%, whereas 67% of the 3H-methylated protein was utilized in this time frame. This corresponds to turnover rates of 4.4 + 0.4 and 3.9 + 0.3% h-1 for the uniformly labelled protein and the methylated protein, respectively. Two experiments with single time points (25 March 1990 and 4 April 1990) showed similar results (Table 1). Not all amino acids resulting from hydrolysis of methylated protein were released, even though nearly all 3H from the 3H-methylated protein was released as LMW material and very little was respired or assimilated. More likely, the unlabelled nonmodified amino acids were assimilated or respired in preference to the methylated amino acids. Some N-methyl-substituted amino acids are used by bacteria as osmolytes and presumably would not be assimilated unless there was a change in the osmotic stress on the bacteria (2). Thus, using methylated amino acids is disadvantageous when studying the fate of protein amino acids after hydrolysis by bacteria. On the other hand, it may be useful under certain circumstances. For example, if only total hydrolysis rates are required, it may be advantageous to use protein
Previous investigations of protein utilization by heterotrophic bacteria in aquatic systems have used proteins radiolabelled (i) uniformly along the amino acid chain (1, 7), (ii) by iodination of tyrosine residues (5), or (iii) by methylation of exposed amine groups (3, 5, 10). Although a method such as iodination or methylation can quickly and easily radiolabel proteins with high specific activity, previous studies have not considered the possible effect of the adduct on utilization of the protein by bacteria. When marine heterotrophic bacteria are incubated in the presence of iodinated (5), methylated (3, 5, 10), or glucosylated (7) protein, lowmolecular-weight (LMW) radioactive material of approximately the same molecular size as amino acids accumulates extracellularly and is not appreciably utilized by microorganisms (3, 5, 7, 10). In this study, we compared the utilization of methylated and uniformly labelled proteins by marine bacteria, and we show that, while total hydrolysis of the two types of radiolabelled proteins is nearly equal, the fate of the radiolabel is highly dependent on the method of labelling. Our experiments are reminiscent of previous studies on the fate of glucose labelled at different carbon positions (4), but our study differs substantially in that labeling does not change the glucose molecule whereas methylation does change the protein. The goal of our study was to evaluate the possible effect of methylation on bacterial protein degradation. We used ribulose 1,5-bisphosphate carboxylase (RuBPcase) as a model protein because it is one of the most abundant proteins found in nature (6) and was determined to be a good tracer of protein degradation by marine bacteria (7). RuBPcase was radiolabelled by reductive methylation (11), which adds 3H-methyl groups to E-amines of lysine. It had a specific activity of 9 [Ci ag-1. RuBPcase was also uniformly labelled with H-amino acids by in vitro translation (7, 9). Chloroplasts were isolated from 10-day-old pea seedlings and used to generate radiolabelled RuBPcase (7, 9). Two different preparations had specific activities of 0.48 and 0.98 ,uCi ugEstuarine water was collected on 9 May 1990 at high tide .
* Corresponding author. t Present address: School of Oceanography, WB-10, University of Washington, Seattle, WA 98195.
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VOL. 58, 1992
NOTES
lated and observed that glucosylated protein was utilized 5 to 40 times more slowly than the unmodified protein (7). Because the sites of methylation and glucosylation are similar, perhaps methylation of e-amines decreases the utilization of protein, as was observed when the a-amines were glucosylated. We could not test this hypothesis directly because the error in our total utilization measurements was
Uniformly t~abelled
Co
5U
1375
20
too great.
10
3H-methylated
0
5
15
10
20
Time (h)
FIG. 1. Bacterial assimilation of 3H-RuBPcase over time. V, protein labelled by reductive methylation; 0, 0, protein labelled by in vitro translation.
radiolabelled via reductive methylation because total hydrolysis is nearly equal to LMW material production, which can be easily estimated by using ultrafiltration or trichloroacetic acid precipitation (3, 10). For most studies, however, uniformly labelled protein is probably better. Although total hydrolysis of methylated proteins was nearly equal to that of the uniformly labelled protein, it did appear slightly lower in all three experiments. The difference between the utilization rates of the two labelled proteins was not significant; however, the consistent observation of an apparently lower protein utilization rate after methylation is thought-provoking. In another study, we compared the utilization of uniformly labelled protein with that of uniformly labelled protein that had been abiotically glucosyTABLE 1. Assimilation, respiration, and production of LMW compounds from the algal protein RuBPcase after 7 (March), 10 (April), and 17 (May) h of incubation in the presence of natural bacterial populationsa RuBPcase
% of added RuBPcase LMW Assimilation material Respiration
Total
0 13 ± 9
26 ± 11 9+8
10 + 10
30 ± 12 32 ± 10
1±6 24 6
42 ± 5 12 7
3 + 11 16 8
46 ± 10 52 8
1 ± 1 30 ± 9 34 ± 11
63 ± 8 26 + 6 18 ± 7
4± 3 20 ± 14 24 ± 10
68 + 12 76 ± 29 74 ± 28
March
3H-methyl-RuBPcase 3H-RuBPcaseb
4 ± 9
April
3H-methyl-RuBPcase 3H-RuBPcasec May 3H-methyl-RuBPcase 3H-RuBPcaseb 3H-RuBPcasec
p.g
a The initial protein concentration was 20 liter-'. Values are means of three replicates ± standard deviation. b Uniformly labelled, with a specific activity of 0.48 ,uCi I Uniformly labelled, with a specific activity of 0.98 ,Ci pg-1.
tug-'.
The radiolabelled material released in other studies (3, 5, 7, 10) was probably methylated or iodinated amino acids. The assimilation of these molecules apparently differs from that of unmodified amino acids because they were not appreciably assimilated after release (3, 5, 7, 10) and free amino acids did not inhibit their eventual uptake (3). This lack of assimilation during degradation of methylated or iodinated proteins suggests that previous conclusions about the relationship between protein hydrolysis and assimilation based on these radioactive proteins should be reevaluated. We thank M. Hoch, M. Montgomery, M. Samuelsson, and T. Nagata for critical and helpful comments on an early draft of the manuscript. This research was supported by ONR contract N0014-87-K-0108. REFERENCES 1. Coffin, R. B. 1989. Bacterial uptake of dissolved free and combined amino acids in estuarine waters. Limnol. Oceanogr. 34:531-542. 2. Csonka, L. N., and A. D. Hanson. 1991. Prokaryotic osmoregulation: genetics and physiology. Annu. Rev. Microbiol. 45: 569-606. 3. Griffith, P. C., and M. Fletcher. 1991. Hydrolysis of protein and model dipeptide substrates by attached and nonattached marine Pseudomonas sp. strain NCIMB 2021. Appl. Environ. Microbiol. 57:2186-2191. 4. Hamilton, R. D., and K. E. Austin. 1967. Assay of relative heterotrophic potential in the sea: the use of specifically labelled glucose. Can. J. Microbiol. 13:1165-1173. 5. Hollibaugh, J. T., and F. Azam. 1983. Microbial degradation of dissolved proteins in seawater. Limnol. Oceanogr. 28:11041116. 6. Jensen, R. G., and J. T. Bahr. 1977. Ribulose 1,5-bisphosphate carboxylase. Annu. Rev. Plant Physiol. 28:379-400. 7. Keil, R. G. 1991. Ph.D. dissertation. University of Delaware, Lewes. 8. Kirchman, D. L., R. G. Keil, and P. A. Wheeler. 1989. The effect of amino acids on ammonium utilization and regeneration by heterotrophic bacteria in the subarctic Pacific. Deep Sea Res. 36:1763-1776. 9. Mullet, J. E., R. R. Klein, and A. R. Grossman. 1986. Optimization of protein synthesis in isolated higher plant chloroplasts. Eur. J. Biochem. 155:331-338. 10. Samuelsson, M.-A., and D. L. Kirchman. 1990. Degradation of adsorbed protein by attached bacteria in relationship to surface hydrophobicity. Appl. Environ. Microbiol. 56:3643-3648. 11. Tack, B. F., J. Dean, D. Eilat, P. E. Lorentz, and A. Schecter. 1980. Tritium labelling of proteins to high specific activity by reductive methylation. J. Biol. Chem. 255:8842-8847.
