Isolation and pure culturing are required to determine the ability of bacteria to produce AHLs. Due to the heterogeneity of the AHL synthetase (I) genes, it has not been possible to develop PCR protocols or rRNA probes allowing a direct determination of AHL-producing capability at the molecular level in single cells. Therefore, preliminary screening for AHLs was done by preparing sterile filtered supernatants from cultures grown for 1 1/2 to 2 weeks at 15°C and testing the samples in three AHL monitor systems using Agrobacterium tumefaciens
) and Chromobacterium violaceum
) as described by Ravn et al. (31
) and in the E. coli
pSB403 LuxR assay (8
) as described by Gram et al. (15
). Since AHLs are not stable at high pH (above 8), all cultures were also grown in MB in which pH was adjusted to 6.2. In none of these outgrown cultures did the pH increase above 7.5. All cultures eliciting an AHL response were positive both when grown in MB at normal pH (7.8) and when grown at pH 6.2. For the monitor assays, A. tumefaciens
strain NT1(pZLR4) was grown with 20 μg of gentamicin ml−1
in Luria-Bertani broth (2
) with 5 g of NaCl liter−1
(LB5) for 24 h and inoculated into 50 ml of ABt broth with 0.5% glucose and 0.5% Casamino Acids (4
). The outgrown culture was mixed with 100 ml of melted, 45°C ABt agar containing 50 μg of X-Gal (5-bromo-4-chloro-3-indolyl-β-d
-galactopyranoside) (Promega 9683801 L) ml−1
and poured into petri dishes. C. violaceum
) was grown in LB5 with 20 μg of kanamycin ml−1
for 24 h, inoculated in 50 ml of LB5, and incubated overnight. Plates were poured after the outgrown culture was mixed with 100 ml of 45°C LB5 agar. Wells of 6 mm in diameter were punched in the solidified agars, and samples of 60 μl were pipetted into the wells. Plates with A. tumefaciens
or C. violaceum
were incubated for 2 days and 1 day, respectively, at 25°C and read for zones of blue color due to AHL-induced β-galactosidase activity or zones of purple pigment due to AHL-induced violacin formation in the agar. E. coli
pSB403 was grown in LB5 with 10 μg of tetracycline ml−1
overnight and diluted to an optical density at 450 nm of 0.8. One hundred microliters of this culture was mixed with 100 μl of sterile filtered supernatant from marine bacteria, and luminescence was measured with a MicroBeta 1450 TriLux scintillation and luminescence counter (Wallac).
Sterile filtered supernatants from 4 strains (HP12, HP30, HP32, and HP36) out of 43 tested elicited a reproducible response in the A. tumefaciens
monitor system (Fig. ), indicating the presence of AHLs. No responses were elicited in the C. violaceum
or E. coli
pSB403 system from any of the 43 strains, while the standard N
-hexanoyl-homoserine lactone (C6-HSL) caused induction of a purple zone in the CV026 assay. In principle, it is possible that some of the 38 strains that did not elicit AHL responses in the monitor systems do produce AHLs, since they may cause a reaction in other AHL receptors. However, several authors have reported that the TraR system is broad (31
), and it is therefore a good screening system.
FIG. 1. Presence of acylated homoserine lactones in supernatants on bacteria isolated from marine snow particles. AHLs were determined by the sensor strain A. tumefaciens NT1(pZLR4). Plates 1 and 2, twofold dilutions of N-3-oxo-hexanoyl-homoserine lactone. Plate (more ...)
Three strains producing AHLs were closely related and clustered in the Roseobacter
subgroup of the α-Proteobacteria
. This is, to our knowledge, the first report of AHLs in this group (7
). However, AHLs have been detected in other members of the α-Proteobacteria
, such as Rhodobacter sphaeroides
), and A. tumefaciens
). We tested four Roseobacter
strains isolated from similar marine environments and found two, isolated from diatom aggregates in the North Sea, also to be AHL positive. Thus, several but not all marine Roseobacter
strains produce AHLs. Many genera of the γ-Proteobacteria
) are common in marine particles (23
), and although these organisms are common producers of AHLs (7
), we detected AHLs only from one Marinobacter
strain in this bacterial group (Table ).
species are important members of the aquatic microbial community (39
), and 20 to 30% of bacterial small-subunit ribosomal DNAs isolated from the upper 50 m of Monterey Bay belong to this clade (42
). The exact ecological role of Roseobacter
species is not known, but they metabolize dimethyl-sulfonio-propionate and other organic sulfur compounds (M. A. Moran, J. M. Gonzáles, R. P. Kiene, R. Simó, and C. Pedrós-Alió, talk presented at the American Society of Limnology and Oceanography, 2001). They display chemotactic behavior towards S and C compounds and are likely to be of major importance for cycling of these compounds in the ocean.
All AHL-positive Roseobacter
strains were isolated from living marine diatoms (S. costatum
and T. rotula
). While AHLs clearly are involved in bacterium-bacterium interactions allowing up-regulation of production of hydrolytic enzymes or antibiotics, these regulatory systems may also be a key to understanding specific bacterium-eucaryote interactions. Thus, the marine alga Delisea pulchra
produces a range of halogenated furanones which specifically interfere with the bacterial AHL regulatory systems and eliminate or reduce bacterial surface colonization (13
). The possible involvement of AHL regulation in Roseobacter
metabolism can have major implications for turnover of organic material in the ocean as well as their colonization of and growth on phytoplankton or organic aggregates.
Three Roseobacter strains (HP12, HP30, and HP32) which were positive in the initial screening were inoculated in MB at approximately 105 CFU ml−1 and incubated at 25°C. Samples were withdrawn regularly for 1 week, and sterile filtered supernatants were tested in the AHL assay. The strains grew well in MB at 25°C, and AHL-inducing zones were seen from sterile filtered culture supernatants only when counts exceeded 5 × 107 to 108 CFU ml−1 (Fig. ). Thus, AHL production in Roseobacter species appears to be similar to that in other organisms in being a phenomenon related to high bacterial densities. AHL production clearly occurs at the bacterial densities typically found in marine snow.
Growth of three Roseobacter strains isolated from marine snow in MB at 25°C. Closed symbols, AHLs were not detected in sterile filter supernatants; filled symbols, AHLs were detected in sterile filtered supernatants.