Biological Activity of Volatiles from Marine and Terrestrial Bacteria

The antiproliferative activity of 52 volatile compounds released from bacteria was investigated in agar diffusion assays against medically important microorganisms and mouse fibroblasts. Furthermore, the activity of these compounds to interfere with the quorum-sensing-systems was tested with two different reporter strains. While some of the compounds specific to certain bacteria showed some activity in the antiproliferative assay, the compounds common to many bacteria were mostly inactive. In contrast, some of these compounds were active in the quorum-sensing-tests. γ-Lactones showed a broad reactivity, while pyrazines seem to have only low intrinsic activity. A general discussion on the ecological importance of these findings is given.


Introduction
Marine bacteria are a rich source of natural products with interesting biological and pharmacological activities [1,2]. While most research on marine bacterial metabolites focusses on compounds with moderate polarity isolable from fermentation experiments, relatively few volatile compounds from marine as well as other bacteria are known [3]. Many bacteria, marine or terrestrial, produce volatiles, but the specific function of these compounds is known only in few cases. It seems likely that the volatiles are used either in intra-or inter-specific communication and/or in the chemical defence against other microorganisms. Several studies have shown that bacterial volatiles can indeed influence the growth of other organisms [4]. Bacterial volatiles can impact, most often inhibit, the growth of fungi [4][5][6][7][8][9], but only few studies describe the activities of certain compounds. Compounds identified from fungistatic soil bacteria inhibiting spore germination of certain fungi were acetamide, trimethylamine, 3-methyl-2-pentanone, benzaldehyde, N,N-dimethyloctylamine, benzothiazole, 1-butanamine, methanamine, phenylacetaldehyde, and 1-decene [10,11]. Benzothiazole, cyclohexanol, nonanal, decanal, dimethyl trisulfide, and 2-ethyl-1-hexanol, the latter most likely an anthropogenic artefact, identified from several Pseudomonas strains, were active in inhibiting growth of the plant pathogen, Sclerotinia sclerotiorum [12]. 1-Undecene and dimethyl disulfide (2), released by several bacteria, were also active [4], despite the fact that the latter proved to be inactive in other studies [10][11][12]. Effects on plants have been described for 2,3-butanediol (47) and acetoin (48) which improve plant growth in Arabidopsis thaliana, and also effects on animals have been described [4]. On the other hand, reports on interbacterial effects of volatile compounds are rare. Indole acts as a signalling compound in Escherichia coli [13] and shows effects on other bacteria [14] while volatile carboxylic acids inhibit spore formation in several pathogenic bacteria [15].
More or less all studies performed so far concentrated on the evaluation of the effects of readily available compounds. While these studies showed activity in some cases, questions remain about the activity potential of other components. The volatiles identified so far can be roughly divided into different groups depending on their occurrence. While there are common compounds produced by many different, often unrelated bacteria, e.g., dimethyl disulfide (52), or at least by certain groups as geosmin (49) and 2-methylisoborneol (50) found in actinobacteria, myxobacteria, and cyanobacteria, some are unique for certain strains as the lactones 4-11 [3].
In the present study 52 compounds were tested for their biological effects, covering strain or species specific compounds (1-44) and commonly occurring ones (45-52). In a first set of experiments we established a general profile of the biological activities of these compounds. Therefore, the antiproliferative activity with bacteria, fungi, and murine fibroblasts was tested. The antimicrobial assays were focussed on medically important strains. In a second set of experiments the possibility of interference with well known bacterial chemical communication channels was probed. In this context, the compounds were investigated in an assay on bacterial crosstalk [16] to demonstrate whether they can influence the known bacterial quorum-sensing signalling pathway via N-acylhomoserine lactones (AHLs).
The compounds tested ( Figure 1) were identified from different marine bacteria (see Table 1 for references). Several compounds originally known from terrestrial bacteria are also included to compare activities between compounds originating from terrestrial or marine sources. Nevertheless, there is no clear cut difference in chemistry between terrestrial and bacterial microorganisms.
The results discussed so far were obtained with compounds produced only by specific bacterial strains. In contrast, the compounds common to many bacteria showed no inhibitory activity. This is even true for components as dimethyl disulfide (52) which showed activity in other assays [4].    (1) Occurs in unclassified myxobacteria [31]. (2) Occurs in Myxococcus xanthus DK 1622 [31].
Additionally, the cytotoxic activity against L-929 mouse fibroblasts was investigated in concentration dependant assays. The obtained minimal inhibition concentration (MIC) values indicated moderate or no activity. The most active compounds were furfuryl isovalerate (42, MIC 62 µmol/L), the ketone 19 (MIC 73 µmol/L), the lactones 10 and 11 (MIC 260 and 280 µmol/L), as well as the pyrazine 28 (MIC 320 µmol/L). All these compounds were also active in at least some antimicrobial assays except 28.
All compounds were also investigated for their activity in N-acylhomoserine lactone (AHL) mediated bacterial communication systems (quorum-sensing). These test were performed using green fluorescent protein (gfp) equipped reporter strains capable of sensing AHLs with certain side chain specificity. The used E. coli MT102 (pJBA132) reporter shows the highest sensitivity for N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-AHL) [32], while the Pseudomonas putida F117 (pKR-C12) reporter is very sensitive to N-(dodecanoyl)homoserine lactone (C12-AHL) [33]. The corresponding AHLs were added to the sensor strains together with the test compound and the resulting reduction or increase of fluorescence was recorded ( Table 2). Values of more than 50% were considered significant.
Several compounds were able to modify the sensor response. The lactones 5-9 inhibited the response of the C12-AHL sensor. Especially the δ-lactone 9 was highly active. In contrast, the 3-oxo-C6-AHL sensor showed a different behavior. In particular 4 and 7 stimulated this sensor. This influence may be due to the structural similarity between the lactones and the AHLs. Ketones also proved to be active, with 15,16,18,19, and the related alcohol 3 reducing activity of the C12-AHL sensor, while 17 did so with the 3-oxo-C6-AHL sensor, as did the pyridine derivative 41. The sulfur containing ester 34 and furfuryl isovalerate (42), also one of the most active compounds in the antimicrobial assays, were the only compounds able to reduce the activity of both sensors. In this assay also compounds common to many different bacteria showed some activity, reducing the effectivity of the 3-oxo-C6-AHL sensor. These compounds were the alcohols 45 and 46, and both enantiomers of methylisoborneol (50 and 51). In summary, 25% of the compounds were able to reduce activity of the C12-AHL sensor, while 19% show inhibitory activity against 3-oxo-C6-AHL and 13% enhanced its activity. Table 2. Acylhomoserine lactone assay using N-3-oxohexanoylhomoserine lactone for E. coli MT102 (C 6 -AHL) and N-dodecanoylhomoserine lactone for P. putida F117 (C 12 -AHL). The maximum reduction relative to the native ligand is reported (%). Negative values indicate no inhibition, but increase of the fluorescence induction. Here the maximum induction rate relative to the native ligand (100%) is given.

No Compound
Conc.  The concentration of the compounds used in the sensor tests was quite high and dilution experiments usually led to reduced activity (see Table 2). The concentration of compounds released by bacteria can be quite high close to the bacterial membrane, their point of release. Therefore, nearby other bacteria might well experience relatively high concentrations of such metabolites, pointing to the possibility that these compounds might have ecological relevance for known bacterial communication channels.
In summary, the results show that bacterial volatiles can, despite their relatively small size, influence different biological traits. Often it is not clear why bacteria produce volatiles. One interesting observation is that some special compounds produced by only few organisms can have antimicrobial activity and therefore might ensure survival of the bacterium when under competition with other bacteria. Contrary to polar and larger compounds they are transported by air and are useful for bacteria under certain conditions, but can also travel in water with high velocity because of their small size. The volatiles can also interfere with bacterial communication channels and are ideally suited for being signalling compounds by themselves. Their potential receptors are not known because only few bacterial communication mechanisms have been established so far (see Introduction). Finally, in some cases they may also serve as a means of detoxification, as recently proposed for some pyrazines [34]. Interestingly, the pyrazines show almost no antimicrobial activity. The activity of the volatiles against proliferative cells can be regarded as quite low with the exception of furfuryl isovalerate, a surprisingly active compound in several aspects.
Finally, there is no clear difference in activity between volatiles produced by marine or terrestrial bacteria. The current known origin of the these compounds from bacteria either of terrestrial or marine habitats is probably pure chance. Most likely terrestrial strains will be found in the future that produce compounds only known from marine sources so far, e.g., sulfur compounds 37-40, and vice versa.
In summary, we have shown that bacterial volatiles can have a multitude of activities. Their production has to be taken into account in future studies, especially when researching aspects of the bacterial chemical communication systems and their chemical defence.

Antimicrobial assay
Antimicrobial activities were determined by agar diffusion assays using paper discs of 6 mm diameter soaked with 20 µL of methanolic solution of the test compound. Microorganisms from the HZI collection were grown on standard media and seeded into liquid agar medium to a final O.D. of 0.01 (bacteria) or 0.1 (yeasts). Spores of fungi were seeded according to experience. Plates were incubated at 30 • C and the diameter of resulting inhibition zones were measured after 1 or 2 days.

Cell proliferation assay
L-929 mouse fibroblasts were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) and cultivated at 37 • C and 10% CO 2 in DME medium (high glucose) supplemented with 10% fetal calf serum. Inhibition of proliferation was measured in microtiter plates. 60 µL of serial dilutions of the test compounds were given to 120 µL of the suspended cells (50,000/mL) in wells of 96-well plates. After 5 days growth each well was judged visually under the microscope. The lowest concentration of a compound that led to a clearly reduced growth is given as minimal inhibitory concentration (MIC).

Acylhomoserine lactone sensors
The AHL biosensor strains E. coli MT102 (pJBA132) and Pseudomonas putida F117 (pRK-C12) were grown in Luria-Bertani (LB) medium with 25 µg/mL tetracycline at 37 • C and with 20 µg/mL gentamycin at 30 • C, respectively. Overnight cultures (50 mL) of the strains were diluted with the same volume of fresh medium, incubated for about one more hour and adjusted to an OD620 of 1.0. Aliquots (100 µL) of these cultures were transferred by pipet into 96-well microtiter plates which contained 90 µL of LB medium, and 5 µL of the test compounds in the concentrations reported in Table 2. To test the compounds for inhibitory activity on quorum-sensing, the sample wells contained 5 µL of the corresponding AHL N-3-oxohexanoylhomoserine lactone for E. coli MT102, N-dodecanoylhomoserine lactone for P. putida F117) at a concentration of 0.25 µg/mL. Samples containing only the corresponding AHL without test compound were used as positive control and samples containing solvent (10 µL) alone were used as negative control. The microtiter plates were incubated at 30 • C with shaking. After 0, 4, 8, and 24 h growth at an OD 620 nm , fluorescence (excitation wavelength 485 nm, detection wavelength of 535 nm) was determined in a Victor 1420 Multilabel Counter (Perkin Elmer). Fold induction of fluorescence in the test samples was calculated by dividing their specific fluorescence (gfp535/OD620) by the specific fluorescence of the negative control. Relative activity was calculated by comparing the fold induction of positive control samples (only homoserine lactones) to samples containing samples with test compounds and the corresponding homoserine lactone.