Production of pectate lyases and cellulases by Chryseomonas luteola strain MFCL0 depends on the growth temperature and the nature of the culture medium: evidence for two critical temperatures

Abstract

Several extracellular enzymes that are responsible for plant tissue maceration were detected in culture supernatant of the psychrotrophic bacterium Chryseomonas luteola MFCL0. Isoelectrofocusing experiments showed that pectate lyase (PL) activity resulted from the cumulative action of three major isoenzymes, designated PLI, PLII, and PLIII. Cellulolytic activity was also detected in culture supernatants. These enzymes exhibited different behaviors with respect to growth temperature. PLII was not regulated by temperature, whereas PLI and PLIII were regulated similarly by growth temperature. Maximal levels of PLI and PLIII were produced at 14 degrees C when cells were grown in polygalacturonate-containing synthetic medium and at around 20 to 24 degrees C in nutrient broth. In contrast, thermoregulation of cellulolytic activity production differed from thermoregulation of PL. The level of cellulolytic activity was low in all media at temperatures up to 20 degrees C, and then it increased dramatically until the temperature was 28 degrees C, which is the optimal temperature for growth of C. luteola. Previously, we defined the critical temperature by using the modified Arrhenius equation to characterize bacterial behavior. This approach consists of monitoring changes in the maximal specific growth rate as a function of temperature. Our most striking result was the finding that the temperature at which maximum levels of PLI and PLIII were produced in two different media was the same as the critical temperature for growth observed in these two media.

FIG. 1

Arrhenius plots for growth of C. luteola MFCL0 (ln maximum specific growth rate [k] versus the reciprocal of the absolute temperature [T]). The maximum specific growth rate k is expressed in hours⁻¹, and the temperature is expressed in degrees Kelvin. Each datum point represents a mean based on at least six independent determinations. The lines were drawn by calculating linear regressions for experimental data with the shareware Nonlin (r = 0.999). Symbols: □, PGA medium; ■, NB. The standard deviations were too small to be shown.

FIG. 2

PL production during growth of C. luteola MFCL0. Cells were grown in PGA medium at 8°C (A), 17°C (B), and 28°C (C). PL activity was assayed as described in the text. Each datum point represents a mean based on at least three independent determinations. Symbols: ⧫, growth; ○, PL activity. The maximum standard deviation for any enzymatic determination was 5%, and the maximum standard deviation for growth was 4%. O.D. 580 nm, optical density at 580 nm.

FIG. 3

PL specific activity of C. luteola MFCL0. Cells were grown in PGA medium, NB, and NB+PGA at 8, 11, 14, 17, 20, 24, and 28°C. PL activity was assayed as described in the text. Each datum point represents a mean based on at least six independent determinations. The error bars indicate standard deviations. Symbols: □, PGA medium; ○, NB; ⊕, NB+PGA. OD₅₈₀, optical density at 580 nm.

FIG. 4

Silver-stained SDS-PAGE gel (A) and PL activity-stained pectate agarose overlay (B). The SDS-PAGE gel was loaded with concentrated supernatants from cultures of C. luteola grown in PGA medium at different temperatures. Lane 1, 8°C; lane 2, 11°C; lane 3, 14°C; lane 4, 17°C; lane 5, 20°C; lane 6, 24°C; lane 7, 28°C; lane MM, molecular mass standards.

FIG. 5

Gel filtration of a concentrated culture supernatant from C. luteola MFCL0 on a Sephacryl S-300 HR column. The column was loaded with a concentrated supernatant from a culture grown at 14°C in PGA medium. Fractions (1 ml) were collected and assayed to determine their protein contents (○) and PL activities (⧫) as described in the text. Calibration of the column showed that fraction 30 corresponded to approximately 35 kDa.

FIG. 6

IEF profiles of extracellular PL isoenzymes produced by C. luteola MFCL0 (pH 9 to 11). Concentrated supernatants from C. luteola cultures were prepared from cultures grown in PGA medium (A), NB (B), and NB+PGA (C). Each lane was loaded with 5 mg of proteins. Lane 1, 8°C; lane 2, 11°C; lane 3, 14°C; lane 4, 17°C; lane 5, 20°C; lane 6, 24°C; lane 7, 28°C. PL isoenzymes were activity stained with a pectate agarose overlay following IEF. The pH gradient of the corresponding IEF gel is shown.

FIG. 7

Specific activity of cellulase produced by C. luteola MFCL0. Cells were grown in PGA medium, NB, and NB+PGA at 8, 11, 14, 17, 20, 24, and 28°C. Cellulase activity was assayed as described in the text. Each datum point represents a mean based on at least three independent determinations. The error bars indicate standard deviations. Symbols: □, PGA medium; ○, NB; ⊕, NB+PGA. OD₅₈₀, optical density at 580 nm.