Nonenzymatic turnover of an Erwinia carotovora quorum-sensing signaling molecule

Abstract

The production of virulence factors and carbapenem antibiotic in the phytopathogen Erwinia carotovora is under the control of quorum sensing. The quorum-sensing signaling molecule, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), accumulates in log-phase culture supernatants of E. carotovora but diminishes in concentration during the stationary phase. In this study, we show that the diminution in OHHL was not due to sequestration of the ligand by the cells, although some partitioning did occur. Rather, it was caused by degradation of the molecule. The rate of stationary-phase degradation of OHHL was as rapid as the rate of log-phase accumulation of the ligand, but it was nonenzymatic and led to a decrease in the expression of selected genes known to be under the control of quorum sensing. The degradation of OHHL was dependent on the pH of the supernatant, which increased as the growth curve progressed in cultures grown in Luria-Bertani medium from pH 7 to approximately 8.5. OHHL became unstable over a narrow pH range (pH 7 to 8). Instability was increased at high temperatures even at neutral pH but could be prevented at the growth temperature (30 degrees C) by buffering the samples at pH 6.8. These results may provide a rationale for the observation that an early response of plants which are under attack by Erwinia is to activate a proton pump which alkalizes the site of infection to a pH of >8.2.

FIG. 1.

Carbapenem and OHHL production through the growth curve of a wild-type E. carotovora subsp. carotovora culture. Aliquots of an MS1 culture were withdrawn every hour through the growth curve for the measurement of cell density (•) and the carbapenem (□) (A) or OHHL concentration (○) (B). Panel B also shows the growth curve (▴) and the OHHL concentration (▵) of a culture of GB7.

FIG. 2.

Sequestration of OHHL by wild-type E. carotovora subsp. carotovora cells. The amount of OHHL in the supernatant (•) and in the cells (○) from a culture of MS1 was measured throughout the growth curve. The high initial value in the supernatant is due to carryover from the sample used to inoculate the culture.

FIG. 3.

(A) Degradation of synthetic OHHL by a carI mutant. Synthetic OHHL (4.7 μM) was added to a culture of the carI mutant, jbC1. Aliquots of the culture were withdrawn every hour throughout the growth curve (•), and the OHHL content of the supernatant was measured (○). (B) OHHL stability in conditioned medium. Cell-free supernatant was prepared from throughout the growth curve (•) of a jbC1 culture. Some of this supernatant was retained for the measurement of endogenous OHHL (◊). Synthetic OHHL (2 μM) was added to the remaining samples, which were then divided into two portions. The OHHL content of one set of these portions was measured immediately (○), while the other was boiled for 10 min before being assayed (□).

FIG. 4.

pH changes in the culture supernatants throughout the growth curve. The growth curves (circles) and supernatant pH values (triangles) of an MS1 culture (solid symbols) and of the jbC1 culture in Fig. 5 (open symbols) are shown.

FIG. 5.

Degradation of OHHL in conditioned medium. Synthetic OHHL (2 μM) and chloramphenicol (50 μg/ml) were added to jbC1 culture supernatants which had been harvested throughout the growth curve. The mixtures were incubated for 18 h at 30°C before measurement of the OHHL concentration (▴) and pH (▵) of the samples. Error bars represent the standard error of the mean from two independent cultures.

FIG. 6.

pH-dependent degradation of OHHL. Solutions of OHHL (2 μM) were prepared in 100 mM potassium phosphate buffer at the indicated pH and incubated at 30°C for 7 h. The samples were then diluted 100-fold in LB medium and assayed for OHHL content.

FIG. 7.

(A) Buffering of growing cultures alters OHHL turnover. Cultures of MS1 were grown in LB medium buffered with 100 mM potassium phosphate at pH 6.7 (circles) or at pH 7.7 (squares). The OHHL levels (solid symbols) and pH values (open symbols) at each time point are shown. (B) pH-dependent degradation of OHHL in conditioned medium. Supernatants taken throughout the growth curve of GB7 were buffered with 200 mM potassium phosphate at pH 6.8 (filled symbols) or pH 8.3 (open symbols) either before (circles) or after (triangles) an 18 h of incubation at 30°C. The residual OHHL concentration in each set of supernatants is shown. Error bars represent the standard error of the mean from two independent cultures.

FIG. 8.

OHHL does not decline in stationary-phase anaerobic cultures. A culture of MS1 was grown in LB medium in anaerobic conditions as described in Materials and Methods. Symbols: •, cell density; ○, OHHL level. (Inset) pH of the culture supernatant through the growth curve.