A Bacillus subtilis gene induced by cold shock encodes a membrane phospholipid desaturase

Abstract

Bacillus subtilis grown at 37 degrees C synthesizes saturated fatty acids with only traces of unsaturated fatty acids (UFAs). However, when cultures growing at 37 degrees C are transferred to 20 degrees C, UFA synthesis is induced. We report the identification and characterization of the gene encoding the fatty acid desaturase of B. subtilis. This gene, called des, was isolated by complementation of Escherichia coli strains with mutations in either of two different genes of UFA synthesis. The des gene encodes a polypeptide of 352 amino acid residues containing the three conserved histidine cluster motifs and two putative membrane-spanning domains characteristic of the membrane-bound desaturases of plants and cyanobacteria. Expression of the des gene in E. coli resulted in desaturation of palmitic acid moieties of the membrane phospholipids to give the novel mono-UFA cis-5-hexadecenoic acid, indicating that the B. subtilis des gene product is a delta5 acyl-lipid desaturase. The des gene was disrupted, and the resulting null mutant strains were unable to synthesize UFAs upon a shift to low growth temperatures. The des null mutant strain grew as well as its congenic parent at 20 or 37 degrees C but showed severely reduced survival during stationary phase. Analysis of operon fusions in which the des promoter directed the synthesis of a lacZ reporter gene showed that des expression is repressed at 37 degrees C, but a shift of cultures from 37 to 20 degrees C resulted in a 10- to 15-fold increase in transcription. This is the first report of a membrane phospholipid desaturase in a nonphotosynthetic organism and the first direct evidence for cold induction of a desaturase.

FIG. 1

Biosynthesis of UFAs in E. coli. β-Hydroxydecanoyl-ACP dehydrase (HDD) catalyzes the key step in UFA production, whereas β-ketoacyl-ACP synthase I (KAS I) is required for the elongation of these unsaturated acyl-ACP intermediates. β-Ketoacyl-ACP synthase II (KAS II) is capable of participating in SFA synthesis and in the elongation of 16:1 Δ9 palmitoleoyl-ACP but is unable to replace KAS I in the elongation of cis-3-decenoyl-ACP.

FIG. 2

Hydropathy profiles and conserved histidine clusters of the putative desaturase of B. subtilis. The sequence we obtained has been reported to GenBank (accession no. AF037430), and the identical sequence is found as ORF yocE in the genomic sequence of B. subtilis (; Genbank accession no. Z99114), which was reported since the completion of our work. Hydropathy indices for the B. subtilis, Synechocystis sp. (GenBank accession no. 488509), Anabaena variabilis (GeneBank accession no. 628916), Arabidopsis thaliana (GenBank accession no. 1169601), and Glycine max (GenBank accession no. 1345979) desaturases were calculated by the algorithm of Kyte and Doolittle (18) with a window size of 11 residues. Histidine residues in conserved clusters are indicated by H’s in shaded circles and by the black boxes at the bottom. Shaded boxes indicate hydrophobic domains containing greater than 40 amino acid residues (capable of spanning the membrane twice). Note that the hydropathy profiles are aligned by the histidine clusters, and thus, the N termini of the desaturases are not coincident.

FIG. 3

Effects of the expression of the B. subtilis des gene in E. coli. (A) Autoradiogram of the products of [1-¹⁴C]palmitate labeling of E. coli strains harboring either vector plasmid pHP13 or des plasmid pDM10. Lane 1 contains a culture of strain AK7/pHP13 that was grown to exponential phase at 30°C. Cells were washed twice with RB medium to remove the oleate, and a 2-ml sample was exposed to 2 μCi of radioactive palmitate for 90 min at the same temperature. Lane 2 contains fatty acids synthesized by B. subtilis JH642 labeled with [1-¹⁴C]acetate after a shift from 37 to 20°C (see Materials and Methods). Lanes 3 to 5 contain strain AK7/pDM10 grown to exponential phase at 30°C. A 2-ml sample of each culture was exposed to 2 μCi of radioactive palmitate and then incubated for 90 min at 30°C (lane 3), 37°C (lane 4), or 42°C (lane 5). The lipids were analyzed as described in Materials and Methods. The UFA and SFA migration positions are indicated on the left. (B) Desaturation of [1-¹⁴C]palmitic acid at different growth temperatures. The radioactive unsaturated methyl esters shown in lanes 3 to 5 of panel A were quantified by scintillation counting, and the results are expressed as percentages of the total methyl esters recovered at the indicated growth temperatures. (C) Autoradiogram of the products of [1-¹⁴C]acetate labeling of E. coli strains harboring vector plasmid pHP13 or des plasmid pDM10. Cultures of strains HC71/pHP13 (lane 1), AK7/pDM10 (lanes 2 to 4), and AK7/pHP13 (lane 5) were grown at 30°C to exponential phase. A 2-ml sample of each culture was exposed to 10 μCi of [1-¹⁴C]acetate and then incubated for 90 min at 30°C (lanes 1, 2, and 5), 37°C (lane 3), or 42°C (lane 4). After incubation, the lipids were extracted and chromatographed as in Fig. 2A. The UFAs synthesized by strain HC71/pHP13 are palmitoleic acid (16:1) and cis-vaccenic acid (18:1). Neither UFA is synthesized by AK7/pDM10 or AK7/pHP13 due to the fabB null mutation of the host strain. The final UFAs and SFA positions are indicated on the left. (D) Percentages of UFA synthesis at different growth temperatures. The total radioactive methyl esters were separated into unsaturated and saturated fractions as shown in panel C. The radioactivity contents of lanes 2 to 4 were quantitated as described in Materials and Methods. Results are expressed as percentages of the total methyl esters recovered at the indicated growth temperatures.

FIG. 4

Analysis of the double bond position of dimethyl disulfide adducts of 16:1 fatty acid methyl esters of strain AK7/pDM10. Strain AK7/pDM10 was grown at 30°C to exponential phase. Lipids were extracted, and the dimethyl disulfide adducts of the fatty acid methyl esters were prepared as described in Materials and Methods. GC-MS analysis of the adducts was carried out on an SPB-1 capillary column (60 m by 0.25 mm) in a Shimadzu QP-5000 gas chromatograph-mass spectrometer.

FIG. 5

Fatty acids synthesized by des mutant cultures. Cultures of the B. subtilis JH642 wild type (lane 1), the JH642 des null mutant (lane 2), and the JH642 des null mutant carrying pDM10 (lane 3) were grown to exponential phase at 37°C. A 2-ml sample of each culture was shifted to 20°C and exposed to 10 μCi of [1-¹⁴C]acetate for 12 h. The lipids were then extracted and transesterified, and the resulting methyl esters were chromatographed as described in the legend to Fig. 3. The sample in lane 1 contained 10,000 and 1,400 cpm of radioactivity in the SFA and UFA fractions, respectively. The sample in lane 2 contained 8,000 cpm of radioactivity in the SFA fraction, while the UFA fraction contained only background levels of radioactivity. The sample in lane 3 contained 10,000 and 1,750 cpm of radioactivity in the SFA and UFA fractions, respectively. des plasmid pDM10 was introduced into the des null mutant by transformation of competent cells, followed by selection for resistance to erythromycin (10 μg/ml).

FIG. 6

Pattern of des-lacZ expression after a temperature shift. B. subtilis AKP2 cells harboring a des-lacZ transcriptional fusion located in the amyE locus were grown at 37°C to an optical density (O.D.) of 0.35 (at 525 nm) and then divided into two samples. One sample was transferred to 20°C (□, ▪), and the second was kept at 37°C (○, •). Optical densities (□, ○) and β-galactosidase specific activities (▪, •) were determined at the indicated time intervals.