Volatile ketone formation in bacteria: release of 3-oxopentanoate by soil pseudomonads during growth on heptanoate.

During enrichments to search for soil bacteria that form the volatile ketones, acetone and butanone, we isolated pure cultures of Pseudomonas aureofaciens, P. fluorescens, and P. putida that excrete the beta-keto-acid, 3-oxopentanoate (3-OPA), when grown on heptanoic acid as carbon source. Analysis of 3-OPA used enzymatic decarboxylation by acetoacetate decarboxylase to yield butanone, which was detected by headspace gas chromatography. The formation of 3-OPA was strongly dependent on heptanoic acid concentration, the level of oxygen, and the state of growth, and was not seen with even-chain or other odd-chain fatty acids. Uptake of 3-OPA during stationary phase of growth is probably related to polyhydroxyalkanoate (PHA) formation in these isolates. A model for formation and release of 3-OPA is proposed.

Acetone formation in the Vibrio family: a new pathway for bacterial leucine catabolism.

There is current interest in biological sources of acetone, a volatile organic compound that impacts atmospheric chemistry. Here, we determined that leucine-dependent acetone formation is widespread in the Vibrionaceae. Sixteen Vibrio isolates, two Listonella species, and two Photobacterium angustum isolates produced acetone in the presence of L-leucine. Shewanella isolates produced much less acetone. Growth of Vibrio splendidus and P. angustum in a fermentor with controlled aeration revealed that acetone was produced after a lag in late logarithmic or stationary phase of growth, depending on the medium, and was not derived from acetoacetate by nonenzymatic decarboxylation in the medium. L-Leucine, but not D-leucine, was converted to acetone with a stoichiometry of approximately 0.61 mol of acetone per mol of L-leucine. Testing various potential leucine catabolites as precursors of acetone showed that only alpha-ketoisocaproate was efficiently converted by whole cells to acetone. Acetone production was blocked by a nitrogen atmosphere but not by electron transport inhibitors, suggesting that an oxygen-dependent reaction is required for leucine catabolism. Metabolic labeling with deuterated (isopropyl-d(7))-L-leucine revealed that the isopropyl carbons give rise to acetone with full retention of deuterium in each methyl group. These results suggest the operation of a new catabolic pathway for leucine in vibrios that is distinct from the 3-hydroxy-3-methylglutaryl-coenzyme A pathway seen in pseudomonads.

Three distinct phases of isoprene formation during growth and sporulation of Bacillus subtilis.

During growth on a glucose-tryptone medium, Bacillus subtilis 6051 (Marburg strain) exhibited three phases of isoprene (2-methyl-1, 3-butadiene) formation, corresponding to (i) glucose catabolism and secretion of acetoin, (ii) catabolism of acetoin, and (iii) the early stages of sporulation. These results establish an experimental system for studying the biological role of isoprene formation.

Characterization of a Pseudomonas putida allylic alcohol dehydrogenase induced by growth on 2-methyl-3-buten-2-ol.

We have been working to develop an enzymatic assay for the alcohol 2-methyl-3-buten-2-ol (232-MB), which is produced and emitted by certain pines. To this end we have isolated the soil bacterium Pseudomonas putida MB-1, which uses 232-MB as a sole carbon source. Strain MB-1 contains inducible 3-methyl-2-buten-1-ol (321-MB) and 3-methyl-2-buten-1-al dehydrogenases, suggesting that 232-MB is metabolized by isomerization to 321-MB followed by oxidation. 321-MB dehydrogenase was purified to near-homogeneity and found to be a tetramer (151 kDa) with a subunit mass of 37,700 Da. It catalyzes NAD+-dependent, reversible oxidation of 321-MB to 3-methyl-2-buten-1-al. The optimum pH for the oxidation reaction was 10.0, while that for the reduction reaction was 5.4. 321-MB dehydrogenase oxidized a wide variety of aliphatic and aromatic alcohols but exhibited the highest catalytic specificity with allylic or benzylic substrates, including 321-MB, 3-chloro-2-buten-1-ol, and 3-aminobenzyl alcohol. The N-terminal sequence of the enzyme contained a region of 64% identity with the TOL plasmid-encoded benzyl alcohol dehydrogenase of P. putida. The latter enzyme and the chromosomally encoded benzyl alcohol dehydrogenase of Acinetobacter calcoaceticus were also found to catalyze 321-MB oxidation. These findings suggest that 321-MB dehydrogenase and other bacterial benzyl alcohol dehydrogenases are broad-specificity allylic and benzylic alcohol dehydrogenases that, in conjunction with a 232-MB isomerase, might be useful in an enzyme-linked assay for 232-MB.

Methanol Emission from Leaves (Enzymatic Detection of Gas-Phase Methanol and Relation of Methanol Fluxes to Stomatal Conductance and Leaf Development).

We recently reported the detection of methanol emissions from leaves (R. MacDonald, R. Fall [1993] Atmos Environ 27A: 1709-1713). This could represent a substantial flux of methanol to the atmosphere. Leaf methanol production and emission have not been investigated in detail, in part because of difficulties in sampling and analyzing methanol. In this study we used an enzymatic method to convert methanol to a fluorescent product and verified that leaves from several species emit methanol. Methanol was emitted almost exclusively from the abaxial surfaces of hypostomatous leaves but from both surfaces of amphistomatous leaves, suggesting that methanol exits leaves via stomates. The role of stomatal conductance was verified in experiments in which stomates were induced to close, resulting in reduced methanol. Free methanol was detected in bean leaf extracts, ranging from 26.8 [mu]g g-1 fresh weight in young leaves to 10.0 [mu]g g-1 fresh weight in older leaves. Methanol emission was related to leaf development, generally declining with increasing leaf age after leaf expansion; this is consistent with volatilization from a cellular pool that declines in older leaves. It is possible that leaf emission could be a major source of methanol found in the atmosphere of forests.

Bacteria produce the volatile hydrocarbon isoprene.

Various bacterial species, both Gram-negative and Gram-positive, were found to produce the volatile hydrocarbon isoprene (2-methyl-1,3-butadiene). Out of the tested cultures, Bacillus produced the most isoprene. The production of isoprene from bacteria was confirmed by gas chromatography-mass spectrometry. Media and growth effects on isoprene production were investigated: growth in rich media led to higher levels of isoprene than growth in minimal media, and highest isoprene emission rates were seen in log-phase cultures. Temperature profiles for bacterial isoprene production showed an optimum of 45 degrees C and were suggestive of an enzymatic mechanism for isoprene formation.

Marine Vibrio species produce the volatile organic compound acetone.

While screening aerobic, heterotrophic marine bacteria for production of volatile organic compounds, we found that a group of isolates produced substantial amounts of acetone. Acetone production was confirmed by gas chromatography, gas chromatography-mass spectrometry, and high-performance liquid chromatography. The major acetone producers were identified as nonclinical Vibrio species. Acetone production was maximal in the stationary phase of growth and was stimulated by addition of l-leucine but not the other common amino acids, suggesting that leucine degradation leads to acetone formation. Acetone production by marine vibrios may contribute to the dissolved organic carbon associated with phytoplankton, and some of the acetone produced may be volatilized to the atmosphere.

Kinetics of appearance and disappearance of classes of bacterial ice nuclei support an aggregation model for ice nucleus assembly.

The kinetics of appearance and disappearance of three classes of ice nuclei in Pseudomonas syringae was investigated under conditions where high-level expression of the ice nucleation phenotype was obtained. The appearance of types 1, 2, and 3 ice nuclei, catalyzing nucleation at -2 to -5, -5 to -7, and -7 to -10 degrees C, respectively, was investigated during low-temperature induction in wild-type strains and in a unique, detergent-sensitive mutant that contained no type 3 ice nuclei when grown at 32 degrees C. Nuclei appeared in the following order: type 3, then type 2 and type 1. The disappearance of classes of ice nuclei was monitored during high-temperature treatment of fully induced cells; nuclei disappeared in the order type 1, type 2, and type 3. Although analysis of nucleation events is complicated by masking and unmasking of ice sites in the same cells, these temporal sequences of ice nucleus appearance or disappearance are consistent with an aggregation model for ice nucleus assembly (A. G. Govindarajan and S. E. Lindow, Proc. Natl. Acad. Sci. USA 85:1334-1338, 1988; G. Warren and P. Wolber, Mol. Microbiol. 5:239-243, 1991).

High-level expression of ice nuclei in a Pseudomonas syringae strain is induced by nutrient limitation and low temperature.

Attempts were made to maximize the expression of ice nuclei in Pseudomonas syringae T1 isolated from a tomato leaf. Nutritional starvation for nitrogen, phosphorous, sulfur, or iron but not carbon at 32 degrees C, coupled to a shift to 14 to 18 degrees C, led to the rapid induction of type 1 ice nuclei (i.e., ice nuclei active at temperatures warmer than -5 degrees C). Induction was most pronounced in stationary-phase cells that were grown with sorbitol as the carbon source and cooled rapidly, and under optimal conditions, the expression of type 1 ice nuclei increased from < 1 per 10(7) cells (i.e., not detectable) to 1 in every cell in 2 to 3 h. The induction was blocked by protein and RNA synthesis inhibitors, indicative of new gene expression. Pulse-labeling of nongrowing cultures with [35S]methionine after a shift to a low temperature demonstrated that the synthesis of a new set of "low-temperature" proteins was induced. Induced ice nuclei were stable at a low temperature, with no loss in activity at 4 degrees C after 8 days, but after a shift back to 32 degrees C, type 1 ice nuclei completely disappeared, with a half-life of approximately 1 h. Repeated cycles of low-temperature induction and high-temperature turnover of these ice nuclei could be demonstrated with the same nongrowing cells. Not all P. syringae strains from tomato or other plants were fully induced under the same culture conditions as strain T1, but all showed increased expression of type 1 ice nuclei after the shift to the low temperature. In support of this view, analysis of the published DNA sequence preceding the translational start site of the inaZ gene (R. L. Green and G. Warren, Nature [London] 317:645-648, 1985) suggests the presence of a gearbox-type promoter (M. Vincente, S. R. Kushner, T. Garrido, and M. Aldea, Mol. Microbiol. 5:2085-2091, 1991).