The fabM gene product of Streptococcus mutans is responsible for the synthesis of monounsaturated fatty acids and is necessary for survival at low pH

Abstract

Previously, it has been demonstrated that the membrane fatty acid composition of Streptococcus mutans is affected by growth pH (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929-936, 2004; R. G. Quivey, Jr., R. Faustoferri, K. Monahan, and R. Marquis, FEMS Microbiol. Lett. 189:89-92, 2000). Specifically, the proportion of monounsaturated fatty acids increases when the organism is grown in acidic environments; if the shift to increased monounsaturated fatty acids is blocked by the addition of a fatty acid biosynthesis inhibitor, the organism is rendered more acid sensitive (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929-936, 2004). Recently, work with Streptococcus pneumoniae has identified a novel enzyme, FabM, responsible for the production of monounsaturated fatty acids (H. Marrakchi, K. H. Choi, and C. O. Rock, J. Biol. Chem. 277:44809-44816, 2002). Using the published S. pneumoniae sequence, a putative FabM was identified in the S. mutans strain UA159. We generated a fabM strain that does not produce unsaturated fatty acids as determined by gas chromatography of fatty acid methyl esters. The mutant strain was extremely sensitive to low pH in comparison to the wild type; however, the acid-sensitive phenotype was relieved by growth in the presence of long-chain monounsaturated fatty acids or through genetic complementation. The strain exhibited reduced glycolytic capability and altered glucose-PTS activity. In addition, the altered membrane composition was more impermeable to protons and did not maintain a normal DeltapH. The results suggest that altered membrane composition can significantly affect the acid survival capabilities, as well as several enzymatic activities, of S. mutans.

FIG. 1.

(A) ClustalW alignment of predicted sequence of S. mutans UR159 PhaB/FabM with the sequence of S. pneumoniae FabM. (B) Genomic organization of the fatty acid biosynthesis gene cluster of S. mutans. The website used to generate the map was http://www.oralgen.lanl.gov/. The insertion site of the erythromycin resistance cassette into the fabM coding region is indicated. The numbers refer to the nucleotides in the coding region of each gene.

FIG. 2.

fabM mutant strain UR117 does not survive extreme acidification of its environment. Cultures were grown as described in Materials and Methods and treated as described. Shown are averages ± standard deviations of two separate cultures, each performed in duplicate. ⧫, wild-type S. mutans UA159; ▪, S. mutans fabM mutant UR117; ▴, UR117 with 10 μg of cis-vaccenic acid ml⁻¹; •, UR117 with 10 μg of cis-eicosenoic acid ml⁻¹; X, S. mutans UR119.

FIG. 3.

Glycolytic profiles of UA159, UR117, UR119, and nutritionally supplemented UR117. Glycolytic pH drops were performed as stated in Materials and Methods. Each strain was grown three separate times, and each overnight culture was assayed in triplicate. The averages ± standard deviations of an overnight culture read in triplicate are shown. ⧫, S. mutans UA159; ▪, S. mutans fabM mutant UR117; ▴, UR117 with 10 μg of cis-vaccenic acid ml⁻¹; •, UR117 with 10 μg of cis-eicosenoic acid ml⁻¹; X, S. mutans UR119.

FIG. 4.

Different proton permeabilities between the fabM mutant strain and the wild type. Proton permeability assays were performed as stated in Materials and Methods. At 50 min, butanol was added to a final concentration of 10% to permeablize the cells and allow for pH to reach equilibrium. The average values ± standard deviations are shown from two independent cultures assayed in duplicate. ⧫, wild-type S. mutans UA159; ▪, fabM S. mutans UR117; ▴, UR117 with 10 μg of cis-vaccenic acid ml⁻¹; •, UR117 with 10 μg of cis-eicosenoic acid ml⁻¹; X, S. mutans UR119.