Degradation of Gallic Acid and Hydrolysable Polyphenols Is Constitutively Activated in the Freshwater Plant-Associated Bacterium Matsuebacter Sp
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Degradation of gallic acid and hydrolysable polyphenols is constitutively activated in the freshwater plant-associated bacterium Matsuebacter sp. FB25 Nicolai Müller1, 2, Melanie Hempel1, Bodo Philipp2, Elisabeth M. Gross1,* 1Limnology, Department of Biology, University of Konstanz, PO Box 5560 M659, 78457 Konstanz, Germany 2Microbiology, Department of Biology, University of Konstanz, PO Box 5560 M936, 78457 Konstanz, Germany ABSTRACT: Hydrolysable polyphenols are present in Myriophyllum spicatum L. at high concentra- tions of up to 25% of dry matter and are also excreted. Bacteria associated with the submerged macrophyte M. spicatum isolated from the surrounding water column and epiphytic biofilm were tested for their ability to degrade polyphenols. Several bacterial isolates were capable of growing with tannic acid as the sole carbon and energy source, among them Matsuebacter sp. FB25, Agrobac- terium vitis EB26 and Pseudomonas sp. FB22. Cell suspensions of Matsuebacter sp. precultured on succinate were capable of degrading gallic acid, while those of A. vitis were not, indicating the con- stitutive presence of gallate-degrading enzymes in the former. When cells were precultured on gal- lic or tannic acid, cell suspensions of both strains exhibited an enhanced degradation rate of gallic acid. M. spicatum-derived hydrolysable polyphenols, which are comparable in structure to tannic acid, resulted in the same enhanced degradation rate of gallic acid or tellimagrandin II, the major M. spicatum polyphenol, by cell suspensions of Matsuebacter sp. FB25. The presence of polyphenol- degrading bacteria in the vicinity of M. spicatum explains the observed fast disappearance of tellima- grandin II and other hydrolysable polyphenols after excretion and has implications for allelochemical interference with competitors, herbivores and potential pathogenic microorganisms. The presence of Matsuebacter sp. and other polyphenol-degrading strains in such environments suggests a suffi- ciently strong effect of M. spicatum exudates to bring about selection in favour of highly specialised bacteria. KEY WORDS: Hydrolysable polyphenol · Submerged macrophyte · Allelochemistry · Biofilm · Betaproteobacteria · Burkholderiales · Myriophyllum spicatum · Tannin INTRODUCTION hydrolysable polyphenols inhibit algae and cyanobac- teria by interference with extracellular alkaline phos- The freshwater dicotyledonous plant Myriophyllum phatase (Gross et al. 1996) or photosynthesis (Leu et al. spicatum L. (Haloragaceae: Rosidae) produces and 2002). Hydrolysable polyphenols cause a reduced excretes bioactive hydrolysable polyphenols inter- growth of larvae of the aquatic moth Acentria fering with competitors and herbivores. On average, ephemerella (Choi et al. 2002), and inhibit the growth 7 to 10% of the plant dry mass (dm) are polyphenols, of bacteria isolated from the gut of larvae (Walenciak among them tellimagrandin II (β-1,2,3-tri-O-galloyl- et al. 2002). Exudates of M. spicatum also interfere 4,6-(S)-hexahydroxydiphenoyl-D-glucose). Apical shoots with zooplankton (Linden & Lehtiniemi 2005). can even comprise more than 20% of polyphenols Hydrolysable polyphenols are actively excreted by M. based on dry matter. Tellimagrandin II and other spicatum (Gross 2003), but the fate of individual *Corresponding author. Email: [email protected] 84 polyphenols, e.g. tellimagrandin II, is difficult to assess propylene tubes filled with sterile water. Samples were and depends on multiple biotic (microbial degrada- transported in coolers to the laboratory and processed tion) and abiotic (oxidation, photodegradation) factors. immediately. Pelagic bacteria were used immediately Since the allelochemical activity of Myriophyllum in enrichment cultures. Epiphytic bacteria were de- spicatum depends on the presence of tellimagrandin II tached by ultrasonication at maximum output (Labo- and other hydrolysable polyphenols, a better knowl- son 200 ultrasonic bath, Bender & Hobein) from 3 to edge of the role of microorganisms in the degradation 20 Myriophylllum spicatum leaves in 2 ml sodium of these polyphenols is needed. In polyphenols, the pyrophosphate (0.1 M Na4P2O7 × 10 H2O) for 2 × 30 s ester bonds between the sugar (often glucose) and gal- with a 10 s break. lic or ellagic acid are cleaved by tannase (tannin acyl Isolation and enrichment of polyphenol-degrading hydrolase, EC 3.1.1.20), an enzyme frequently found in bacteria. Water samples (2 ml) or detached epiphytic fungi or bacteria (Chowdhury et al. 2004, Li et al. 2006) bacteria (2 ml) were incubated under sterile conditions and even in plants (Niehaus & Gross 1997). The micro- in 250 ml Erlenmeyer flasks with 100 ml modified bial degradation of gallic acid is possible by several Medium B (Hempel 2004: 5 mM NH4Cl, 0.5 mM distinct mechanisms (Li et al. 2006). Under aerobic MgSO4 × 7 H2O, 14 mM KCl, 7.2 mM NaCl; instead of conditions, gallic acid can be degraded either (1) by 10 mM HEPES, 10 mM TRIS were used as buffer, following the β-ketoadipate pathway starting with a medium adjusted to pH 7). Directly before use, 1 ml of NADH-dependent reduction (Armstrong & Patel 1000-fold concentrated trace element solution SL10 1992); or (2) by conversion into pyruvate and oxal- (Widdel et al. 1983), 0.1 ml of 100 mM Ca2Cl and 0.3 ml acetate feeding into the citrate cycle, initiated by the 0.15 mM Na+/K+-hydrogenphosphate solution (pH 7) action of gallate dioxygenase (Kasai et al. 2005, were added. Instead of 0.05% tryptone and 0.0005% Nogales et al. 2005). The anaerobic degradation starts yeast extract, we offered 294 μM tannic acid as the sole with gallate decarboxylase to yield pyrogallol, which is source of carbon and energy. Cultures were incubated further degraded to acetate (Zeida et al. 1998). In in the dark at 16°C and 200 rpm on an orbital shaker aquatic systems, only a few strains (e.g. Pelobacter and growth was followed by measuring optical density acidigallici from aquatic sediments: Schink et al. 2000) (OD) at 600 nm. Growing cultures were diluted after have been investigated for their capacity to degrade 3 to 7 d in a series from 10–1 to 10–6 and plated on phenolic compounds, including gallic acid. Aerobic Medium B agar plates solidified with 1.5% agar, con- degradation is most plausible for epiphytic bacteria or taining 0.05% tryptone and 0.0005% yeast extract, but bacteria living in the water surrounding M. spicatum. no tannic acid. This change in carbon source was used The aims of the present study were (1) to investigate to allow a faster growth of colonies. The plates were whether bacteria capable of growing with tannic acid cultivated at 16°C in the dark. After 4 d, colonies were or hydrolysable polyphenols can be isolated from the picked and transferred to new plates. This procedure environment of Myriophyllum spicatum; (2) to then was repeated 3 times to single out colonies. identify bacterial strains that are capable of growing Each isolate was again tested for growth on tannic solely on tannic or gallic acid; and (3) to investigate acid as sole carbon and energy source. Five microlitres how these strains degrade tannic acid, gallic acid and of an actively growing culture of each strain were tellimagrandin II. diluted in 295 μl Medium B with 294 μM tannic acid in 96-well microtitre plates. Growth was recorded in a microplate reader at 595 nm (Genios, Tecan). Only MATERIALS AND METHODS strains exhibiting significant growth (OD595 > 0.09) were used further. Origin of bacterial isolates and aquatic plants. Bac- Identification of polyphenol-degrading strains. terial samples were collected from Myriophyllum spi- Selected strains were identified by DNA extraction, catum stands in mesocosms at the Limnological Insti- PCR amplification of the 16S rDNA gene, sequenc- tute, University of Konstanz (9.192° N, 47.694° E) ing and BLAST searches using standard protocols. during winter 2004/2005. The mesocosms are 2 × 2 × Growing isolates were harvested by centrifugation at 1 m (depth) concrete basins, filled with a 7 to 10 cm 12 000 rpm (14 800 × g) and 4°C for 15 min. The pellets layer of lake sediment, flushed constantly with water were washed twice with sterile ultrapure water to from Lake Constance, and planted with M. spicatum, break the cells by hypoosmotic shock. The final pellet which is wintergreen in these basins. was shock-frozen in liquid nitrogen and ultrasonicated Pelagic bacteria were sampled in the vicinity of plant at maximum output for 2 × 30 s with a 10 s break. Cell stands in the mesocosms. Epiphytic bacteria were debris was removed after resuspension of the pellet in retrieved from the upper 20 cm of plant shoots (mea- 100 μl sterile ultrapure water and centrifugation. The sured from the apex) and placed directly in poly- supernatant contained the DNA. 85 One PCR reaction to amplify the 16S-rDNA gene (>80%) isolated from apical shoots of M. spicatum. contained 42.8 μl PCR buffer (Taq buffer, Eppendorf), Plant material was extracted with aqueous acetone (1:1 5 μl dNTPMix (500 mM, Eppendorf), 0.5 μl forward [v/v]; 2 × 2 h, 14°C, constant stirring) followed by SPE primer at 50 pmol μl–1, 27f 5’AGAGTTTGATCCTG on C18 cartridges (Phenomenex; 2 g sorbens) using a GCTCAG-3’, 0.5 μl reverse primer at 50 pmol μl–1, step-gradient elution with increasing methanol con- 1492r 5’-TACGG(CT)TACC TTACGACTT-3’, 1 μl of centrations in water (Leu et al. 2002). The fraction DNA-template, and finally 1 U Taq polymerase (0.2 μl). containing tellimagrandin II eluted with 25% [v/v] We used a Thermocycler T-Gradient (Biometra) and methanol. the following protocol: (1) 3 min at 94°C; (2) 30 s at Growth experiments. Matsuebacter sp. FB25 was 55°C; (3) 1 min at 72°C; (4) 30 s at 94°C; (5) 30 s at 55°C; pregrown on Medium B with 294 μM tannic acid. This (6) 1 min at 72°C; repeat Steps 4 to 6 33×; (7) 7 min at strain was selected because of its high growth rate on 72°C.