Isolation and characterization of unsaturated fatty acid auxotrophs of Streptococcus pneumoniae and Streptococcus mutans

Abstract

Unsaturated fatty acid (UFA) biosynthesis is essential for the maintenance of membrane structure and function in many groups of anaerobic bacteria. Like Escherichia coli, the human pathogen Streptococcus pneumoniae produces straight-chain saturated fatty acids (SFA) and monounsaturated fatty acids. In E. coli UFA synthesis requires the action of two gene products, the essential isomerase/dehydratase encoded by fabA and an elongation condensing enzyme encoded by fabB. S. pneumoniae lacks both genes and instead employs a single enzyme with only an isomerase function encoded by the fabM gene. In this paper we report the construction and characterization of an S. pneumoniae 708 fabM mutant. This mutant failed to grow in complex medium, and the defect was overcome by addition of UFAs to the growth medium. S. pneumoniae fabM mutants did not produce detectable levels of monounsaturated fatty acids as determined by gas chromatography-mass spectrometry and thin-layer chromatography analysis of the radiolabeled phospholipids. We also demonstrate that a fabM null mutant of the cariogenic organism Streptococcus mutants is a UFA auxotroph, indicating that FabM is the only enzyme involved in the control of membrane fluidity in streptococci. Finally we report that the fabN gene of Enterococcus faecalis, coding for a dehydratase/isomerase, complements the growth of S. pneumoniae fabM mutants. Taken together, these results suggest that FabM is a potential target for chemotherapeutic agents against streptococci and that S. pneumoniae UFA auxotrophs could help identify novel genes encoding enzymes involved in UFA biosynthesis.

FIG. 1.

Construction and structure of the FabM mutant. The fabM gene was disrupted with a Cm resistance cassette as outlined in Materials and Methods, and the structure of the chromosomal DNA of strain SH9 is diagrammed.

FIG. 2.

Essentiality and growth phenotype of S. pneumoniae fabM mutant. (A) Cells of S. pneumoniae SH9 (fabM) were grown as described in the text. Cells were harvested, washed, and resuspended in fresh AGCHYE medium that was not supplemented (▪) or was supplemented with 0.1 mM oleate (○) or 0.1 mM linoleic acid (▴). S. pneumoniae 708 (fabM⁺) was grown in the same medium without supplements (•). (B) Growth curves for strains grown in AGCHYE medium without supplementation, including S. pneumoniae 708 (fabM⁺) (•), SH9 (fabM/pDL278) (▪), SH11 (fabM/pfabM⁺) (▴), and SH13 (fabM/pfabN⁺) (○). OD, optical density.

FIG. 3.

[1-¹⁴C]acetate labeling profiles of S. pneumoniae strains. Cultures were grown in AGCHYE medium with [1-¹⁴C]acetate, lipids were extracted, and fatty acid methyl esters were prepared and separated by thin-layer chromatography on silver nitrate-impregnated silica plates. Lanes 1 and 4, strain 708 (fabM⁺); lanes 2 and 5, strain SH9 (fabM); lane 3, strain SH11 (fabM/pfabM⁺); lane 6, strain SH13 (fabM/pfabN⁺).

FIG. 4.

[1-¹⁴C]acetate labeling profiles of S. mutans strains. Labeling of fatty acids and chromatography on silver nitrate-impregnated silica plates were performed as described in the legend to Fig. 3. Lane 1, strain U159 (fabM⁺); lane 2, strain SA20 (fabM); lane 3, strain SA21 (fabM/pfabM⁺).