Chlamydomonas reinhardtii secretes compounds that mimic bacterial signals and interfere with quorum sensing regulation in bacteria

Abstract

The unicellular soil-freshwater alga Chlamydomonas reinhardtii was found to secrete substances that mimic the activity of the N-acyl-L-homoserine lactone (AHL) signal molecules used by many bacteria for quorum sensing regulation of gene expression. More than a dozen chemically separable but unidentified substances capable of specifically stimulating the LasR or CepR but not the LuxR, AhyR, or CviR AHL bacterial quorum sensing reporter strains were detected in ethyl acetate extracts of C. reinhardtii culture filtrates. Colonies of C. reinhardtii and Chlorella spp. stimulated quorum sensing-dependent luminescence in Vibrio harveyi, indicating that these algae may produce compounds that affect the AI-2 furanosyl borate diester-mediated quorum sensing system of Vibrio spp. Treatment of the soil bacterium Sinorhizobium meliloti with a partially purified LasR mimic from C. reinhardtii affected the accumulation of 16 of the 25 proteins that were altered in response to the bacterium's own AHL signals, providing evidence that the algal mimic affected quorum sensing-regulated functions in this wild-type bacterium. Peptide mass fingerprinting identified 32 proteins affected by the bacterium's AHLs or the purified algal mimic, including GroEL chaperonins, the nitrogen regulatory protein PII, and a GTP-binding protein. The algal mimic was able to cancel the stimulatory effects of bacterial AHLs on the accumulation of seven of these proteins, providing evidence that the secretion of AHL mimics by the alga could be effective in disruption of quorum sensing in naturally encountered bacteria.

Figure 1.

Effects of C. reinhardtii and Chlorella sp. colonies on quorum sensing responses in bacterial reporters. Streaks of the algae were overlayed with soft agar containing a quorum sensing reporter, then later examined for induced luminescence with a Hamamatsu C2400 intensified CCD camera (Hamamatsu Photonic Systems, Bridgewater, NJ). False color images were superimposed on the digital images of the algal streaks. Increasing luminescence intensity is indicated by red > yellow > green > blue. A, Stimulation of the AI-2-dependent luminescence in V. harveyi BB170 after 9 h. The three Chlorella spp. luminesced strongly at 7 h (not shown) but show dark areas corresponding to inhibited background luminescence at 9 h. B, Inhibition of LasRI'::luxCDABE reporter responses to its cognate AHL, 3-oxo-C12-HSL, by C. reinhardtii colonies after 5 h.

Figure 2.

LasR stimulatory substances from C. reinhardtii culture filtrates. Samples of HPLC fractions equivalent to approximately 22 mL of algal culture filtrate from cultures harvested after 4, 8, and 12 d were assayed with the LasR AHL reporter as described in “Materials and Methods.” Responses are reported as percentage of the maximum response obtained with the reporter to saturating levels of the cognate AHL, 3-oxo-C12-HSL. Results are from a single experiment representative of three independent trials.

Figure 3.

CepR stimulatory substances from C. reinhardtii culture filtrates. Samples of HPLC fractions equivalent to approximately 33 mL of algal culture filtrate from cultures harvested after 4, 8, and 12 d were assayed with the CepR AHL reporter as described in “Materials and Methods.” Responses are reported as percentage of the maximum response obtained with the reporter to saturating levels of the cognate AHL, C8-HSL. Results are from a single experiment representative of at least three independent trials.

Figure 4.

Purification of a LasR stimulatory mimic from C. reinhardtii. Culture filtrate from approximately 10 L of 7-d-old HS algal cultures were extracted, purified, and assayed with the LasR reporter as described in “Materials and Methods.” Relative activity responses to samples of HPLC fractions equivalent to about 22 mL of the original culture are given as x-fold over reporter-only controls. A, Initial purification. B, Repurification of fractions 51 to 54 from the initial HPLC fractionation. The gradient of increasing acetonitrile used to elute the column is indicated by a solid thin line.