Viability of an Escherichia coli pgsA null mutant lacking detectable phosphatidylglycerol and cardiolipin - PubMed (original) (raw)

Viability of an Escherichia coli pgsA null mutant lacking detectable phosphatidylglycerol and cardiolipin

S Kikuchi et al. J Bacteriol. 2000 Jan.

Abstract

Phosphatidylglycerol, the most abundant acidic phospholipid in Escherichia coli, has been considered to play specific roles in various cellular processes and is believed to be essential for cell viability. It is functionally replaced in some cases by cardiolipin, another abundant acidic phospholipid derived from phosphatidylglycerol. However, we now show that a null pgsA mutant is viable, if the major outer membrane lipoprotein is deficient. The pgsA gene normally encodes phosphatidylglycerophosphate synthase that catalyzes the committed step in the biosynthesis of these acidic phospholipids. In the mutant, the activity of this enzyme and both phosphatidylglycerol and cardiolipin were not detected (less than 0.01% of total phospholipid, both below the detection limit), although phosphatidic acid, an acidic biosynthetic precursor, accumulated (4.0%). Nonetheless, the null mutant grew almost normally in rich media. In low-osmolarity media and minimal media, however, it could not grow. It did not grow at temperatures over 40 degrees C, explaining the previous inability to construct a null pgsA mutant (W. Xia and W. Dowhan, Proc. Natl. Acad. Sci. USA 92:783-787, 1995). Phosphatidylglycerol and cardiolipin are therefore nonessential for cell viability or basic life functions. This notion allows us to formulate a working model that defines the physiological functions of acidic phospholipids in E. coli and explains the suppressing effect of lipoprotein deficiency.

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Figures

FIG. 1

PCR analysis of the pgsA alleles of E. coli pgsA mutants. PCR products amplified with Ex Taq DNA polymerase were subjected to 1.2% agarose gel electrophoresis. Lanes 1, W3110; lanes 2, S301; lanes 3, MDL12; lanes 4 to 6, independent clones of the transductants (S330). Primer pairs FPPG5-ASFPPG1 (a) and FPPG5-ASFPPG3 (b) were used. The design and sequences of the primers are described in Materials and Methods. With the former primer pair, the wild-type pgsA allele and the pgsA::kan allele gave products of 0.71 and 1.9 kbp, respectively. With the latter primer pair, the wild-type pgsA and the pgsA::kan alleles gave products of 0.5 and 1.7 kbp, respectively. A DNA fragment of ca. 1.5 kbp which appeared in MDL12 (panel b, lane 3) may be the product of a false annealing of the antisense primer with a site in _lacZ_′ fused to pgsA (12). The molecular size markers included (two left lanes of each gel) were λ-_Hin_dIII digest (23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.56 kbp) and λ-_Eco_T14 I digest (19.3, 7.7, 6.2, 4.3, 3.5, 2.7, 1.9, 1.5, 0.93, and 0.42 kbp).

FIG. 2

Autoradiograms of 32P-labeled phospholipids of pgsA mutants. Cells of E. coli S301 (wild type) (a), S303 (pgsA3) (b), and S330 (pgsA30::kan) (c) were grown at 37°C in NBY medium supplemented with 1% NaCl in the presence of 7.5 μCi of 32Pi/ml for six generations (to the late-exponential growth phase). The lipids were extracted and separated by two-dimensional thin-layer chromatography (Silica gel 60; Merck) as described previously (34). After the preparation of autoradiograms, the spots were scraped off the plates for measurement of their radioactivities (Table 3). CL, cardiolipin; PE, phosphatidylethanolamine; PA, phosphatidic acid; PG, phosphatidylglycerol.

FIG. 3

Growth characteristics of E. coli pgsA mutants. Cells of E. coli S301 (wild type) (a), S303 (pgsA3) (b), and S330 (pgsA30::kan) (c) were grown in LB medium at 37°C to Klett 5 to 7. They were cultured further at 37°C (○) or transferred to 30 (▵) or 42°C (●), and Klett units were measured every hour.

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Viability of an Escherichia coli pgsA null mutant lacking detectable phosphatidylglycerol and cardiolipin - PubMed (original) (raw)