Isolation and Characterization of Unsaturated Fatty Acid Auxotrophs of Streptococcus pneumoniae and Streptococcus mutans

Bacterial strains, plasmids, and growth conditions.The strains and plasmids used in this study are listed in Table 1. S. pneumoniae 708 and its derivatives were grown in AGCHYE medium (11) or on brain heart infusion (BHI) agar with 5% sheep blood. AGCHYE medium was prepared with fatty acid-free bovine serum albumin for all experiments unless indicated otherwise. Antibiotics were added at the following concentrations: chloramphenicol (Cm), 3 μg ml⁻¹; and spectinomycin (Sp), 500 μg ml⁻¹. S. mutans UA159 and its derivatives were maintained on BHI (Difco) agar plates or in Todd-Hewitt broth with 0.3% yeast extract (THYE) (Difco) at 37°C. The antibiotic concentrations used for S. mutans strains were 50 μg ml⁻¹ Sp and 1 mg ml⁻¹ kanamycin (Km). Overnight cultures were inoculated from −70°C glycerol stocks. The following day fresh cultures were started by washing and diluting the overnight cultures 1:10. E. coli strains were grown in Luria-Bertani medium supplemented when needed with 50 μg ml⁻¹ Sp and 100 μg ml⁻¹ ampicillin. UFAs neutralized with KOH were added to the medium at the concentrations indicated below. All chemicals were purchased from Sigma Chemical Co.

Construction of mutant strains.Plasmid pLB1, used to disrupt the fabM gene in S. pneumoniae (Table 1), was constructed as follows. Two sets of primers were designed to amplify the predicted FabM coding sequence plus additional flanking regions from the S. pneumoniae genome. The 710-bp upstream fragment containing the 5′ region of the fabM gene was amplified by PCR with primers L1 (5′-TGCATCCGATACCATTTTGGTTAAAGTAGGCAGCA-3′) and L2 (5′-TTGAAGAGATTTCGAAGAGGATCCACTGCTTAT-3′; BamHI site underlined). The 870-bp downstream fragment containing the 3′ region of the fabM gene was amplified by PCR with primers 5′-ACTGAACCTACAGAATCGCATGCTCAAACAGAGGATTTCA-3′ (SphI site underlined) and 5′-CGAGCAGCTGTAGAGGGCCCCATCGAATCTGGAGTA-3′ (ApaI site underlined). The PCR products were cloned in the pGEMT Easy (Promega Life Science) vector, yielding pSL1 and pSL2, respectively, and sequenced. The syn Cm resistance cassette (7) was excised from plasmid pUC18SCATS and inserted downstream of the insert between the BamHI and SphI sites of pSL1, yielding plasmid pSL3. Then the insert of pSL2 was located downstream of the cat gene by insertion within the ApaI and SphI sites of pSL3. The resulting plasmid, pLB1, was transformed in E. coli, and transformants were selected for Cm resistance. Plasmid DNA was prepared using the Wizard DNA purification system (Promega Life Science), linearized, and transferred to S. pneumoniae strain 708 by transformation as described by Lacks and Greenberg (11). The transformed cells were mixed with BHI-blood agar with 3 μg ml⁻¹ of Cm, plated, and incubated at 37°C. As a result of the expected double crossover, the Cm resistance cassette replaced part of the chromosomal copy of the target gene, thereby creating a gene knockout (Fig. 1). Gene replacement in mutant clones was confirmed by PCR using primers that annealed outside the DNA region used for mutant construction. The fabM mutant of S. mutans was constructed by allelic replacement mutagenesis using the Km resistance cassette method described by Song et al. (17). Briefly a Km resistance cassette (904 bp) from Staphylococcus aureus ATCC 43300 was amplified with primers Kan-F (5′-AACAGTGAATTGGAGTTCGTCTTGTTATA-3) and Kan-R (5′-GCTTTTTAGACATCTAAATCTAGGTA-3). Two pairs of gene-specific primers, L-F/L-R and R-F/R-R, were used to amplify the left and right flanking regions of the target gene, generating PCR products that were 500 bp long. Primers L-R and R-F each consisted of 21 nucleotides (5-GACGAACTCCAATTCACTGTT-3 and 5-AGATTTAGATGTCTAAAAAGC-3, respectively) identical to a segment of the promoter region and the 3′ end of the Km^r gene plus 29 nucleotides of the fabM sequence. PCR DNA amplifications were performed as described by Song et al. (17), and the linear fused product was cloned in the pGEMT vector, yielding plasmid pLB2, and sequenced. Transformation and gene disruption of S. mutans were performed as described by Ahn et al. (3). To this end, overnight cultures were diluted 20-fold in prewarmed THYE supplemented with 5% heat-inactivated horse serum and incubated at 37°C until the optical density at 600 nm was approximately 0.2. Competence of the cultures was induced by addition of 1 μg of synthetic competence-stimulating peptide (3) and incubation for 20 min. Then transformation was performed by incubating the cultures for 30 min with linearized plasmid pLB2 (Sp^r Km^r). To allow expression of the antibiotic resistance marker before plating, cultures were incubated for 2 h after addition of 0.5 ml of fresh THYE supplemented with 5% heat-inactivated horse serum. Transformants were selected by plating cultures on BHI-blood agar supplemented with 1 mg ml⁻¹ Km and incubating the preparations at 37°C for 48 h. Gene replacement in mutant clones was confirmed by PCR. Genomic DNAs of mutant and wild-type strains were used as templates for PCR amplification with primers L-F and R-R to verify correct incorporation of the fused construct into the mutant genome.

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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.

Fatty acid analysis.For measurement of fatty acid biosynthesis cells were grown in AGCHYE medium to mid-exponential phase and labeled for one generation time with 2 μCi of [1-¹⁴C]acetate. Lipids were extracted, converted to methyl esters, and separated into unsaturated and saturated fractions by chromatography on 10% silver nitrate-impregnated Silica Gel G plates (thickness, 0.5 mm; Analtech) developed with toluene at −20°C (1). Radioactivity on the plates was visualized using a Typhoon 9200 PhosphorImager screen and was quantified using ImageQuant software (version 5.2). Lipid species were identified by comigration with standards. To analyze the fatty acid profiles, streptococci were grown to mid-log phase in AGCHYE medium containing 0.8% sucrose at 37°C. Total cellular fatty acids were prepared by the method of Bligh and Dyer (6). The fatty acid methyl esters were prepared by transesterification of glycerolipids with 0.5 M sodium methoxide-methanol and then analyzed with a Perkin Elmer Turbo Mass gas chromatograph-mass spectrometer using a capillary column (30 m by 0.25 mm [inside diameter]) containing 100% dimethylpolysiloxane (PE-1; Perkin Elmer Co). Helium at a flow rate of 1 ml min⁻¹ was used as the carrier gas, and the column temperature was programmed to increase at a rate of 4°C min⁻¹ from 140 to 240°C. The spectra were recorded in the electron impact mode at 70 eV, using 1-s scans of m/z 40 to 400 (5). The SFAs and UFAs used as reference compounds were obtained from Sigma Chemical Co.

cis-trans isomerase FabM is essential in S. pneumoniae and S. mutans.The fabM gene of S. pneumoniae codes for the FabM enzyme proposed to be essential for UFA biosynthesis based on its vitro enzymatic activity (15). To directly test the role of this enzyme in the physiology of S. pneumoniae, we disrupted the fabM gene with a Cm resistance cassette. The resulting strain, designated SH9, did not exhibit a colony phenotype on BHI-blood agar plates, and it autolysed faster than the parental strain. More importantly, fabM mutants completely failed to grow in AGHYE complex medium unless it was supplemented with UFAs (Fig. 2). The growth defect of SH9 could be overcome by supplementing the medium with oleate or linoleate (Fig. 2A). On the other hand, SFAs were unable to support the growth of SH9 (data not shown), indicating that this strain is a UFA auxotroph.

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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.

To confirm the UFA auxotrophy of SH9, this strain was grown in AGCHYE medium supplemented with 0.1 mM oleic acid, and its fatty acid composition was determined by gas chromatography-mass spectrometry (Table 2). The membrane of wild-type strain 708 contained 89% UFAs, including three C_16:1 fatty acids with double bonds at positions Δ7, Δ9, and Δ11 and three C_18:1 fatty acids with double bonds at positions Δ9, Δ11, and Δ13 (Table 2), while the UFA content of the mutant strain membrane was 84% and the membrane was composed almost exclusively of oleic acid. In addition, strain SH9 produced a larger amount of shorter-chain C_12:0 and C_14:0 saturated fatty acids than the parental strain (6% versus 1%). We verified that the UFA composition changes arose from alterations in de novo fatty acid synthesis by labeling strains 708 and SH9 with [1-¹⁴C]acetate. The lipids were extracted, and the distribution of label in the fatty acyl methyl esters was determined by argentation chromatography. As shown in Fig. 3, the parent strain (lanes 1 and 4) incorporated radioactivity into both SFAs and UFAs, whereas SH9 (lanes 2 and 5) incorporated radioactivity only into SFAs. These results provided a direct demonstration that SH9 failed to synthesize UFAs.

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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 ⁺).

We confirmed that the deficiency in UFA synthesis of strain SH9 was due to the absence of a functional pneumococcal FabM protein by introducing into strain SH9 a plasmid that expressed the wild-type fabM gene. Expression of fabM in strain SH9 (strain SH11) completely eliminated its growth deficiency in AGCHYE medium (Fig. 2B) and reestablished the synthesis of UFAs (Fig. 3, lane 3). As shown in Table 2 and Fig. 3, strain SH11 synthesized a smaller amount of UFAs than wild-type strain 708. This was likely due to the fact that the His-tagged version of FabM expressed by strain SH11 (Table 1) was less active than native FabM. In any case, we concluded from these data that fabM is an essential gene in S. pneumoniae and that the absence of FabM was the sole cause for the biochemical and growth phenotypes exhibited by strain SH9.

It has recently been reported that a fabM mutant of S. mutans is viable, although it exhibited an increased doubling time compared to the wild-type parent strain (10). This result could indicate that S. mutans has the ability to synthesize other fatty acids that partially overcome the deficiency of UFAs in the absence of the fabM gene product. To test this possibility, we constructed the S. mutans SA20 strain, which is a fabM insertional null mutant derivative of the UA159 streptococcal strain (see details in Materials and Methods). Mutant strain SA20 was still able to generate small colonies on BHI-blood agar, but we found that it required supplementation with UFAs for growth in AGCHYE complex medium (data not shown). SFAs did not cure the growth defect of SA20 in AGCHYE medium, indicating that this strain is an UFA auxotroph. The inability of the fabM strain to synthesize UFAs was verified by in vivo labeling of the fatty acids with [1-¹⁴C]acetate, followed by argentation chromatography. While SA20 synthesized only SFAs (Fig. 4, lane 2), the control strain synthesized both SFAs and UFAs (Fig. 4, lane 1). We also examined the membrane lipid composition of the S. mutans strains by gas chromatography-mass spectrometry analysis (Table 3). We found that like S. pneumoniae, S. mutans UA159 synthesized straight-chain saturated and monounsaturated fatty acids, but the carbon atom chain lengths of the UFAs were predominately 18 and 20 (Table 3). As shown in Table 3, when SA20 was grown in AGCHYE medium supplemented with oleate, the UFA content of the mutant strain was 19% oleate and 14% C_20:1, the elongation product of oleic acid. Although these data are fully consistent with the conclusion that fabM is an essential gene in S. mutans, they are not in agreement with recent results of Fozo and Quivey (10), who reported that a fabM S. mutans strain, UR117, is able to grow in complex medium without a UFA supplement. Since these experiments were performed in TY rich medium, we reexamined whether the UFA auxotrophy of SA20 could be relieved in this medium. However, this was not the case, since in TY medium SA20 was still unable to grow without addition of exogenous UFAs (data not shown). We cannot readily explain why our results differ from those of Fozo and Quivey (10). A possible explanation is that the growth medium used in the experiment of Fozo and Quivey contained contaminant fatty acids that partially met the UFA requirement of UR117. In agreement with this possibility, it was reported that UR117 contains almost 40% nonidentified fatty acids in its membranes (10). These unknown fatty acids could in some way partially support the growth of UR117.

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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 ⁺).

The isolation of UFA growth-dependent fabM null mutants of S. pneumoniae demonstrated that FabM is essential for growth and cell viability of this bacterium and that the role of UFAs cannot be filled by other fatty acids synthesized by this organism. In addition, under the conditions used in this work we found that fabM is also essential in S. mutans, indicating that FabM is the only enzyme involved in the control of membrane fluidity in streptococci.

Functional replacement of S. pneumoniae FabM by E. faecalis FabN.The gram-positive bacterium E. faecalis has a fatty acid composition very similar to that of E. coli, but it lacks FabA, FabB, and FabM homologues (18). However, it contains a protein, FabN, which is a bifunctional dehydratase/isomerase capable of introducing a double bond into a growing acyl chain (18). It has been demonstrated that FabN can produce UFAs in E. coli fabA cells, but the low level of UFAs produced was unable to support growth (18). Here we investigated whether expression of the FabN enzyme corrected the FabM deficiency of S. pneumoniae SH9. To do this, we transformed S. pneumoniae SH9 with plasmid pDLfabN containing the fabN coding sequences from E. faecalis. The resulting transformant, strain SH13 (Table 1), was selected on a BHI-blood agar plate containing the appropriate antibiotics (see Materials and Methods) and tested for growth in AGCHYE medium in the absence of a UFA supplement. As shown in Fig. 2B, fabN was able to complement the growth defect of the SH9 fabM mutant. Analysis of the de novo synthesis of [1-¹⁴C]acetate-labeled fatty acids by argentation thin-layer chromatography and quantification of the spots revealed that strain SH13 produced about 22% UFAs. The UFAs synthesized by SH13 were identified as C_16:1Δ9 and C_18:1Δ11 (Fig. 3, lane 6), which are identical to the UFAs produced by E. faecalis (18). These data indicate that FabN functionally replaces the S. pneumoniae FabM protein. Our experiments also demonstrate that in S. pneumoniae FabM-deficient cells expressing FabN, the synthesis of UFAs was not strongly decreased by competition for trans-2-decenoyl-ACP between FabN and the host enoyl-ACP reductase FabK. This was not the case in E. coli, where FabN was unable to complement the UFA auxotrophy of a fabA mutant strain because the FabI enoyl reductase efficiently competed with FabN for trans-2-decenoyl-ACP (14). It should be noted that the fabN-complemented fabM SH13 strain synthesized about 20% of the UFAs synthesized by the wild-type strain (Fig. 3). This appears to indicate that S. pneumonie requires smaller amounts of UFAs for growth than E. coli. Therefore, this observation could also explain the finding that fabN functionally complements the growth of S. pneumonie but not the growth of E. coli. In any case, it is clear from our work that UFA auxotrophs of S. pneumoniae could be helpful in the identification of novel genes involved in the anaerobic synthesis of UFAs in organisms that lack recognizable homologues of FabA or FabM genes in their genomes.