Bacillus subtilis homologs of MviN (MurJ), the putative Escherichia coli lipid II flippase, are not essential for growth - PubMed (original) (raw)

Bacillus subtilis homologs of MviN (MurJ), the putative Escherichia coli lipid II flippase, are not essential for growth

Allison Fay et al. J Bacteriol. 2009 Oct.

Abstract

Although peptidoglycan synthesis is one of the best-studied metabolic pathways in bacteria, the mechanism underlying the membrane translocation of lipid II, the undecaprenyl-disaccharide pentapeptide peptidoglycan precursor, remains mysterious. Recently, it was proposed that the essential Escherichia coli mviN gene encodes the lipid II flippase. Bacillus subtilis contains four proteins that are putatively homologous to MviN, including SpoVB, previously reported to be necessary for spore cortex peptidoglycan synthesis during sporulation. MviN complemented the sporulation defect of a DeltaspoVB mutation, and SpoVB and another of the B. subtilis homologs, YtgP, complemented the growth defect of an E. coli strain depleted for MviN. Thus, these B. subtilis proteins are likely to be MviN homologs. However, B. subtilis strains lacking these four proteins have no defects in growth, indicating that they likely do not serve as lipid II flippases in this organism.

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Figures

FIG. 1.

FIG. 1.

Alignment of B. subtilis MviN homologs. A ClustalW alignment of E. coli MviN and B. subtilis SpoVD, YtgP, YabM, and YkvU is shown. The alignment was generated after a BLASTp query of the B. subtilis genome with the E. coli MviN sequence followed by a second BLASTp query using YtgP, the highest-scoring sequence.

FIG. 2.

FIG. 2.

Growth of B. subtilis strains carrying mutations in putative MviN homologs. (A) Strains carrying the single mutation Δ_ytgP_::mls (JDB2327), Δ_ykvU_::mls (JDB2330), Δ_yabM_::cm (JDB2386), or _spoVB_Δ::tet (JDB1097) or all four mutations (JDB2361) were grown in LB at 37°C following dilution to an optical density at 600 nm (OD600) of 0.04 from an overnight culture grown in LB. OD600 was measured at a series of time points for each of the strains. (B) B. subtilis strains carrying the mutations in single MviN homologs or mutations in all four homologs were grown in LB to an OD600 of 0.9. After collection, cells were resuspended in phosphate-buffered saline with 1 mg/ml FM4-64 and imaged. Bar, 5 mm.

FIG. 3.

FIG. 3.

Localization of GFP fusions. (A) A strain expressing YtgP-GFP (JDB2356) was grown in CH growth medium, cells were collected at an OD600 of 0.7, and the membranes were stained with FM4-64. (B) Strains expressing SpoVB-GFP (JDB2329) and YkvU-GFP (JDB2358) were sporulated by resuspension in A+B medium, cells were collected at T3 of sporulation, and membranes were stained with FM4-64.

FIG. 4.

FIG. 4.

SpoVB-GFP and YkvU-GFP targeting requires additional sporulation-specific proteins. (A) SpoVB-GFP-expressing strains JDB2329 (wt), JDB2416 (spoIIIAG-H::kan), and JDB2418 (spoVE::tet) were sporulated by resuspension, and images were taken at 3 h. (B) YkvU-GFP-expressing strains JDB2358 (wt), JDB2377 (spoIIIAG-H::kan), and JDB2378 (spoVE::tet) were sporulated by resuspension, and images were taken at T3. Recruitment ratios are the ratios of the average maximum forespore fluorescence to the average maximum mother cell fluorescence.

FIG. 5.

FIG. 5.

SpoVB-GFP and YtgP-GFP rescue the mviN depletion mutant. JDE1148 (pMMB207) (▪), JDE1140 (pAF374) (+), and JDE1147 (pAF375) (•) were depleted for mviN by growth in LB in the absence (dashed) or presence (solid) of the inducer arabinose. The presence of pMMB207 did not affect mviN depletion, as the strain required arabinose for growth and IPTG had no effect. Leaky expression of YtgP-GFP or SpoVB-GFP rescued the mviN mutation, as seen by growth without arabinose.

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