In Vivo Characterization of Dimethylsulfoniopropionate Lyase in the Fungus Fusarium lateritium

Abstract

A fungus, Fusarium lateritium, with dimethylsulfoniopropionate (DMSP) lyase activity was isolated from both seawater and a salt marsh due to its ability to grow on DMSP (with the evolution of dimethyl sulfide) as the sole source of carbon. This is the first reported case of DMSP lyase activity in a fungus. Several other common fungal genera tested did not have DMSP lyase activity. DMSP was taken up more rapidly by F. lateritium than it was utilized, leading to its intracellular accumulation. Inhibitor studies with nystatin and cyanide indicated that DMSP uptake was an energy-dependent process. The lyase was inducible by its substrate, DMSP (K(m), 1.2 mM), and by the substrate analogs choline and glycine betaine. During induction, DMSP lyase activity increased with time and then dropped rapidly. This loss of activity could be prevented by spiking the culture with fresh DMSP or choline. The V(max) for DMSP lyase was 34.7 mU . mg of protein. The inhibitory effects of nystatin, and p-chloromercuriphenylsulfonate on DMSP lyase activity suggested that the enzyme is cytosolic. Because plants like Spartina (a marsh grass) and marine algae contain high concentrations of DMSP, we speculate that DMSP-utilizing fungi may be involved in their decay.

FIG. 1

Scanning electron micrograph of F. lateritium macroconidia. Fungal cultures were fixed in 2% glutaraldehyde and postfixed in 1% OsO₄. Magnification, ×1,200.

FIG. 2

Growth of F. lateritium on various carbon sources. (A) Fungal dry weight produced per mole of substrate; (B) fungal dry weight per gram of carbon. Substrate concentrations were as follows: sucrose, 2.5 mM; DMSP and acrylate, 5 mM each. The results show the mean and standard deviation of three replicas.

FIG. 3

DMS emissions by various fungal genera. DMSP (1 mM) was added to fungal cell suspensions containing approximately 40 mg of biomass; the appearance of DMS in the gas phase was monitored by gas chromatography.

FIG. 4

Effect of DMSP concentration on DMSP lyase induction. The kinetics of induction were monitored by assaying washed cells for DMSP lyase activity after 3 h of induction.

FIG. 5

Effect of DMSP and various DMSP analogs on DMSP lyase stability. DMSP (2 mM) was added to four identical fungal cell suspensions at time zero. At the times indicated, an aliquot of cells was removed, filtered, washed, and resuspended with 1 mM DMSP. The cultures were amended after 3 and 6 h, as indicated by the arrows, with 1 mM choline, glycine betaine, or DMSP. The line indicates the course of an unamended culture. Bars show the level of activity after the additions listed.

FIG. 6

Effects of pH and temperature on DMSP lyase activity in F. lateritium. Fungal cultures were induced with 1 mM DMSP; the temperature and pH were adjusted for each aliquot of fungal biomass. DMSP lyase rates were averaged from three separate experiments and are shown with their standard deviations.

FIG. 7

DMSP uptake by F. lateritium as measured by increases in intracellular DMSP concentrations with time. The inset shows the effect of cyanide on DMSP uptake. The 0% inhibition of DMSP uptake represents 1.62 μmol of DMS · mg of protein⁻¹ after 40 min of uptake.

FIG. 8

Effect of nystatin concentrations on DMSP uptake and induction and turnover of DMSP lyase. The 100% DMSP lyase activities for induction and turnover were 0.06 and 0.03 μmol of DMS · min⁻¹ · mg of protein⁻¹, respectively. DMSP uptake in the absence of inhibitor (0% was 1.6 μmol of DMS · mg of protein⁻¹; all uptake measurements were done at 40 min.

FIG. 9

Effect of p-CMBS concentrations on DMSP uptake and induction and turnover of DMSP lyase. The 100% DMSP lyase activities for induction and turnover were 0.037 and 0.031 μmol of DMS · min⁻¹ · mg of protein⁻¹, respectively. DMSP uptake was 1.2 μmol of DMS · mg of protein⁻¹ in the absence of inhibitor (0% inhibition); all uptake measurements were made at 40 min.