INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

There is current interest in the role of acetone in atmospheric chemistry and in determining natural and anthropogenic sources of acetone. Acetone has been found in the upper troposphere and lower stratosphere in surprisingly large amounts and may be an important contributor to the formation of odd hydrogen radicals (OH plus HO₂) and the sequestration of nitrogen oxides as peroxyacetylnitrate (2, 38, 44). While some of the acetone found in the atmosphere results from photochemical reactions of natural and anthropogenic hydrocarbons, direct emissions from biological sources may be important (10, 45). The atmospheric oxidation of various biogenic hydrocarbons, such as 2-methyl-3-buten-2-ol and various monoterpenes, also gives rise to the secondary production of acetone (14, 27).

There are several known biological sources of acetone. Those best characterized include enzymatic decarboxylation of acetoacetate in certain bacteria, such as clostridia (16) and Bacillus polymyxa (24), and nonenzymatic decarboxylation of acetoacetate in animals (47). Other biogenic acetone sources of uncertain magnitude and mechanism include seedlings of oil seed plants (39), branches of various plants (22), conifer buds (31), and wounded pasture grasses and clover (10, 25). Recently, we described the production of acetone in marine Vibrio species (40). Acetone formation was dependent on the presence of L-leucine in the growth medium and was repressed by glucose. These findings suggested that an inducible leucine catabolic pathway, similar to that described for pseudomonads (37), might give rise to this ketone. In the pathway worked out in Pseudomonas putida (36), leucine oxidation occurs in the sequence shown in Fig. 1 (right), giving rise to acetoacetate and acetyl-coenzyme A (CoA). In P. putida, the acetoacetate is further metabolized to acetyl-CoA via succinyl-CoA transferase and acetoacetyl-CoA thiolase (49). Possibly, a similar leucine catabolic pathway may function in vibrios, but with enzymatic or nonenzymatic decarboxylation of the acetoacetate formed.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Metabolic pathway for leucine catabolism in Pseudomonas species (right, solid arrows; redrawn from reference 36) and possible pathways for leucine-dependent acetone formation in Vibrio species (left and right, dashed arrows). Pathways A and B are discussed in the text. The isopropyl moiety of leucine is shown fully deuterated to help trace the origin of acetone in each pathway as discussed in the text. Abbreviations: HIV, 3-hydroxyisovaleric acid; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; IV-CoA, isovaleryl-CoA; -KIC, -ketoisocaproic acid; MC-CoA, 3-methylcrotonyl-CoA; MG-CoA, 3-methylglutaconyl-CoA; FAD, flavin adenine dinucleotide.

Other pathways for leucine catabolism in bacteria result in the production of either 3-methyl-1-aminobutane or 3-methylbutanal. Proteus vulgaris is known to have a neutral amino acid decarboxylase that is very active with L-leucine yielding 3-methyl-1-aminobutane (12). However, the metabolic role of this decarboxylation pathway is uncertain (5). Studies with P. vulgaris and Streptococcus lactis have also revealed significant metabolism of L-leucine via transamination to -ketoisocaproic acid, followed by decarboxylation to 3-methylbutanal (33, 43). Whether 3-methyl-1-aminobutane and 3-methylbutanal are further metabolized in these bacteria is uncertain. Here we have examined the underlying mechanism of acetone formation in Vibrio, Listonella, and Photobacterium isolates metabolizing L-leucine. In contrast to what was found for the leucine-degradative pathway in pseudomonads and the acetoacetate decarboxylation pathway for acetone formation in bacilli and clostridia, it appears that these marine bacteria form acetone by a new type of pathway.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Chemicals. L-Leucine-5,5,5-d₃ (98 atom% deuterium [D]) was obtained from Cambridge Isotope Laboratories (Andover, Mass.). (Isopropyl-d₇) L-leucine (98.4 atom% D) was obtained from CDN Isotopes (Quebec, Canada). 3-Hydroxyisovaleric acid was obtained from TCI America (Portland, Oreg.). All of the other chemicals were of analytical grade and were obtained from Sigma Chemical Co. (St. Louis, Mo.) or Aldrich Chemicals (Milwaukee, Wis.).

Bacterial strains and media. The sources of strains used here are summarized in Table 1. Additional marine Vibrio isolates were obtained from estuary sediments at two coastal sites in southern California (Del Mar and Los Penasquitos marsh) by plating on TCBS agar (35) as described previously (40). The strains were characterized as Vibrio splendidus by the methods described earlier (40) and the methods of Ortigosa et al. (41). All strains were maintained frozen at 70°C in 7% (vol/vol) dimethyl sulfoxide.

1

Analytical methods. Acetone in bacterial cultures was routinely assayed by a headspace method (gas chromatography-photoionization detection [GC-PID] method). Samples (0.5 to 1 ml) of cell-free medium, obtained by centrifugation, were placed in 4.8-ml vials sealed with Teflon-lined septa and allowed to equilibrate for 1 h at 30°C. A sample of the headspace (250 µl) was removed with a gas-tight syringe and injected onto a gas chromatograph (Photovac model 10S) equipped with a capillary column (CPSil-5; 6 ft) and a photoionization detector. The carrier gas was purified nitrogen (8 cm³ min¹), and the column was typically operated at 23°C; under these conditions acetone typically eluted at 115 ± 5 s. For each experiment the headspace of fresh solutions of acetone ranging from 10 µM to 10 mM were sampled identically to establish a calibration curve. For verification of acetone formation, samples of culture supernatants were treated with 2,4-dinitrophenyl hydrazine (DNPH) and the resulting hydrazone derivative of acetone was analyzed by high-pressure liquid chromatography (HPLC) as described previously (40). For some cultures acetone formation was also verified by GC-mass spectrometry (MS) analysis (see below).

Bacterial conversion of L-leucine, deutero-L-leucine, and leucine metabolites to acetone. For these experiments V. splendidus D2 or P5 cells were grown at room temperature on MT leucine medium to logarithmic phase (OD₆₀₀ of 0.5 to 0.7) and the cells were harvested by centrifugation and washed twice with marine salts (MT medium with tryptone deleted). Washed cells were suspended in approximately 1/10 the original volume of marine salts, adjusted to contain 2 mM L-leucine or potential leucine metabolites including 3-methyl-1-aminobutane, 3-methylbutanal, sodium 3-hydroxy-3-methylbutanoate, or sodium -ketoisocaproate (each at 2 mM), and then incubated with shaking at room temperature for 1 to 3 h. Aliquots were taken for measurement of cell density (OD₆₀₀) and acetone content; for the latter, cells were removed by centrifugation, 0.5-ml samples of the supernatants were equilibrated in sealed vials for 1 h at 30°C, and then headspace acetone was determined as described above. In some experiments cells were incubated with potential inhibitors added in water, or in dimethylformamide (20 µl per 2 ml of incubation mixture) in the case of rotenone. For inhibition by a N₂ atmosphere, cell suspensions were bubbled with N₂ gas for 1 min before and after L-leucine or -ketoisocaproate was added and the vial was quickly sealed with a Teflon-lined septum and then incubated and processed as described above.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Leucine-dependent acetone formation is widespread in the Vibrionaceae. Previously we had isolated Vibrio spp. from estuarine samples and determined that acetone formation in these isolates was dependent on the presence of L-leucine and was repressed by glucose. These cultures did not survive storage, so new isolates were obtained from similar coastal estuary sites in southern California. Again, enrichment for Vibrio isolates by plating on TCBS agar (35) led to a high frequency of isolation of colonies that exhibit L-leucine-dependent acetone formation. Isolates D2 and P5 were further examined here. They were each identified as a Vibrio sp. by the methods we described previously (40) and were most similar to nonluminescent V. splendidus (phenon 3), as determined by the taxonomic methods of Ortigosa et al. (41).

Effects of growth conditions on acetone formation in V. splendidus and P. angustum. Previously we found that Vibrio isolates produced acetone primarily in the stationary phase of growth (40); however these experiments were complicated by the oxygen depletion that occurred during the incubation of culture samples in sealed vials. Here we grew V. splendidus P5 and P. angustum 25915 in a stirred fermentor under controlled aerobic conditions and at various times obtained samples, removed bacteria by centrifugation, and analyzed acetone in the resulting supernatants. Figure 2A illustrates that for V. splendidus P5 grown in marine broth, acetone was produced in stationary cells; some carryover of acetone from the inoculum was seen at early times. The decline in acetone after 8 h of culture can be attributed to volatilization of the ketone by the large volume of air passed through the culture. This experiment, repeated twice, also illustrated that acetone was not produced in response to a low pH, since the pH of the culture remained in the range of 7.7 to 7.9. To examine whether acetone formation might be due to excretion of acetoacetate into the medium with accompanying nonenzymatic decarboxylation, samples throughout the fermentation were analyzed for acetone in the presence and absence of acetoacetate decarboxylase (23). There was no significant difference in acetone levels in these samples (data not shown), indicating that acetone and not acetoacetate was released from the cells.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Acetone formation in V. splendidus P5 and P. angustum cultured in a stirred fermentor. (A) V. splendidus P5 grown in marine broth (conditions: 400 rpm, 2 liters of air min1, 30°C). (B) V. splendidus P5 grown in MT leucine broth (conditions: 300 rpm, 2 liters of air min1, 26°C). (C) P. angustum 25915 grown in MT leucine broth (conditions same as those for panel B). In each case the liquid volume was 1 liter and acetone in the culture fluid was determined from aliquots removed and analyzed as described in Materials and Methods.

Specificity and stoichiometry of L-leucine conversion to acetone. Since leucine-degrading pseudomonads can metabolize either D- or L-leucine (36), we tested whether this was also true for Vibrio isolates. V. splendidus P5 was cultured in MT leucine medium, and log-phase cells were washed twice with marine salts and resuspended in marine salts with 2 mM leucine (D, L, or DL isomers). Acetone formation at 1 and 2.5 h was measured, and only the L isomer and the DL racemate were metabolized in this Vibrio strain. No acetone was detected in cells incubated with the D isomer.

Bacterial conversion of putative leucine metabolites to acetone. It was expected that vibrios might degrade leucine by the well-known pseudomonad pathway, leading to the production of acetoacetic acid and acetyl-CoA (Fig. 1). However, to explain acetone formation in this case, a plausible pathway would involve acetoacetate decarboxylation to acetone rather than acetoacetic acid metabolism by the glyoxylate pathway (36). As shown above, we had ruled out the secretion of acetoacetic acid into the medium. Several attempts to demonstrate acetoacetic acid or acetoacetate decarboxylase in extracts of V. splendidus D2 or P5, by the methods of Kimura et al. (24), were unsuccessful.

Effects of inhibitors of leucine-dependent acetone formation. To gain some insight into the metabolic pathway involved in L-leucine conversion to acetone the effects of the following metabolic inhibitors were tested: azide, rotenone and a nitrogen atmosphere (to block electron transport), and arsenite (to block branched-chain keto acid dehydrogenase) (9, 17). These experiments were conducted with V. splendidus P5, P. angustum, and L. pelagia (Table 3). The most potent inhibition was seen with removal of oxygen (i.e., a nitrogen atmosphere); for all three bacterial species inhibition ranged from 92 to 97% in different experiments. Since sodium azide and rotenone, electron transport inhibitors, had little or no inhibitory effect, these findings suggest that the oxygen dependence of acetone formation may be indicative of an oxidase or oxygenase reaction. Inhibition by arsenite ranged from 41 to 65%, which is consistent with inhibition of a lipoamide-dependent keto acid dehydrogenase of the type used by P. putida PpG2 (46) in the conversion of -ketoisocaproic acid to isovaleryl-CoA (Fig. 1). Very similar results were seen with V. splendidus P5 incubated with -ketoisocaproic acid in place of L-leucine.

Bacterial conversion of deutero-L-leucine to acetone. In order to directly test whether acetone formation occurs by the pseudomonad pathway or some other pathway, (isopropyl-d₇)-L-leucine with a label in the isopropyl moiety was incubated with washed cells that had been pregrown on MT leucine broth. Deuterated acetone that accumulated in the headspace was analyzed by GC-MS; control incubation mixtures contained nondeuterated L-leucine. These experiments were conducted with L. pelagia, P. angustum, and V. splendidus strains, and the results are summarized in Table 4. The positions of the deuterium labels and expected labeled products of the pseudomonad and vibrio pathways are shown in Fig. 1. For all three bacterial strains the acetone produced by incubation with (isopropyl-d₇)-L-leucine led to >90% enrichment of the molecular ion (m/z 64) over the nondeuterated acetone (m/z 58). This result is indicative of the retention of all six methyl deuterium atoms in the acetone produced. This labeling pattern is inconsistent with metabolism of (isopropyl-d₇)-L-leucine by the pseudomonad pathway (ignoring deuterium isotope effects), which would be expected to remove a methyl deuterium during the 3-methylcrotonyl-CoA carboxylase reaction, resulting in the eventual formation of acetoacetate with five deuterium atoms (Fig. 1). Decarboxylation of acetoacetate (enzymatic or nonenzymatic) would produce acetone with five deuterium atoms (molecular ion m/z 63). In the experiments shown in Table 4 no significant amount of molecular ion at m/z 63 was seen. Analysis of the major acetone fragment ion due to loss of methyl would be expected to give m/z 43 for the nondeuterated fragment and m/z 46 for the trideuterated fragment; only the latter pattern was seen for all three strains of Vibrionaceae (Table 4).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We have been interested in identifying new biological sources of the volatile organic compound acetone, which, as described in the introduction, has a significant impact on the chemistry of the atmosphere. Here we have extended our earlier finding that marine vibrios produce acetone in the presence of L-leucine (40). Now we show that this metabolic trait is widespread in the Vibrio genus and other Vibrionaceae, such as Listonella and Photobacterium. However, Shewanella isolates, which some include in the Vibrionaceae (11), produced little or no acetone in the presence of L-leucine. Perhaps these results are indicative of differences in metabolism in aerobic versus anaerobic environments. The metabolism of L-leucine was shown here to be an aerobic process. Shewanella grows naturally in anaerobic environments (6, 28).

Given these results it is possible that leucine catabolism in marine environments gives rise to some of the acetone found in seawater. Small amounts of acetone at concentrations of 3 to 50 nM have been measured in open seawater (50); however these levels of the compound might be derived from partitioning with atmospheric acetone (4). Measurement of acetone in estuaries which contain abundant Vibrio populations (41) is complicated by the presence of anthropogenic sources of the ketone (50). At this point it is not possible to predict whether bacterial conversion of leucine in marine systems would be a significant contributor to atmospheric acetone.

When acetone formation in vibrios was discovered to be dependent on the presence of L-leucine, we assumed that the pathway was similar to those seen in animals, plants, and pseudomonads, involving 3-methylcrotonyl-CoA as an intermediate (Fig. 1) (1, 37). In animals L-leucine is a ketogenic amino acid that is metabolized to acetoacetate, which can break down nonenzymatically to produce acetone (47). However, we could not detect substantial pools of acetoacetate or acetoacetate decarboxylase in V. splendidus cultures. To further determine if the 3-methylcrotonyl-CoA-type pathway is operative in vibrios, we incubated cells with a deuterated L-leucine substrate, (isopropyl-d₇)-L-leucine, containing fully deuterated methyl groups. If acetone is derived from the isopropyl end of the leucine molecule, one would expect to see acetone with five deuterium atoms since a proton is removed at the 3-methylcrotonyl-CoA carboxylase step (Fig. 1). This was not the case for L. pelagia 33784, P. angustum 25915, or V. splendidus P5, and instead the acetone produced from (isopropyl-d₇)-L-leucine retained all six methyl group deuterium atoms (Table 4).

For this metabolic transformation to occur, an oxygen atom must be introduced at C-4 of the leucine carbon skeleton to provide the keto oxygen of the resulting acetone. There are two precedents for the transformation of leucine to metabolites with this type of oxygen substitution; both involve -ketoisocaproic acid as an intermediate. First, transformation might occur as a result of an -ketoisocaproate dioxygenase reaction, such as that described for rat liver (42). A putative bacterial dioxygenase would convert -ketoisocaproic acid to 3-hydroxyisovaleric acid as shown in Fig. 1 (pathway A), and subsequently its oxidative cleavage, probably as the acyl-CoA derivative, could produce acetone and acetyl-CoA. Acetone would be released as a by-product, and the acetyl-CoA would be metabolized by the acetate assimilation pathway known to occur in most free-living vibrios (19). Consistent with pathway A is the finding that production of acetone from L-leucine or -ketoisocaproic acid was dependent on oxygen but not blocked by electron transport inhibitors. Although an -ketoisocaproate dioxygenase has apparently not been demonstrated in bacteria, it is noteworthy that a related enzyme, 4-hydroxyphenylpyruvate dioxygenase, has been described for the Vibrionaceae, V. cholerae, and Shewanella colwelliana (26). Both enzymes catalyze a similar reaction: the atoms of molecular oxygen are introduced into both the -keto carbon and the side chain accompanied by decarboxylation of the keto acid (30, 42). The failure of 3-hydroxyisovaleric acid to support acetone formation in whole cells is inconsistent with pathway A, although this result might be explained by lack of uptake of the acid.

A second scheme for introduction of the requisite oxygen atom would involve production of isovaleryl-CoA, and 3-methylcrotonyl-CoA as in the pseudomonad leucine catabolic pathway (37), followed by a hydration reaction, such as that catalyzed by enoyl-CoA hydratase, resulting in the formation of 3-hydroxyisovaleryl-CoA (Fig. 1, pathway B). The latter metabolic sequence has recently been demonstrated in the yeast G. reesii, which converts isovaleric acid via isovaleryl-CoA and 3-methylcrotonyl-CoA to 3-hydroxyisovaleryl-CoA (29). As in pathway A, 3-hydroxyisovaleryl-CoA could be cleaved to produce acetone and acetyl-CoA. The operation of pathway B is supported by the finding that acetone production was inhibited by arsenite, a known inhibitor of lipoic acid-dependent enzymes such as branched-chain keto acid dehydrogenase; however, arsenite is not very specific, inhibiting a variety of enzymes (48). The role of oxygen in acetone formation in pathway B could be explained by a branched-chain acyl-CoA oxidase reaction. This type of enzyme has been described in relation to leucine catabolism in plant peroxisomes (15); in the Pseudomonas leucine degradation pathway isovaleryl-CoA is oxidized by a flavin-dependent acyl-CoA dehydrogenase (37) (Fig. 1).

Clarification of the leucine degradation pathway in vibrios will require demonstration of key enzymes such as an -ketoisocaproate dioxygenase, isovaleryl-CoA oxidase, and the putative 3-hydroxyisovaleryl-CoA lyase. The last enzyme would use a -lyase mechanism analogous to the well-known 3-hydroxy-3-methylglutaryl-CoA lyase, which catalyzes a Claisen-type cleavage reaction (Fig. 1) (20). However, this type of lyase has apparently not been previously described. We have noted that growth of V. splendidus P5 on leucine but not glucose led to the induction of isocitrate lyase (data not shown). This finding is consistent with the idea that leucine catabolism gives rise to an acetate equivalent that is processed by the glyoxylate pathway known to exist in Vibrio species (21).

Clarification of the vibrio leucine degradation pathway is in progress. It will be interesting to see if this new pathway is present in other biological systems, such as the buds, stems, and leaves of plants, that can produce significant amounts of acetone by an unknown mechanism (10, 22, 25, 31, 39).