Gene-specific random mutagenesis of Escherichia coli in vivo: isolation of temperature-sensitive mutations in the acyl carrier protein of fatty acid synthesis

Abstract

Acyl carrier proteins (ACPs) are very small acidic proteins that play a key role in fatty acid and complex lipid synthesis. Moreover, recent data indicate that the acyl carrier protein of Escherichia coli has a large protein interaction network that extends beyond lipid synthesis. Despite extensive efforts over many years, no temperature-sensitive mutants with mutations in the structural gene (acpP) that encodes ACP have been isolated. We report the isolation of three such mutants by a new approach that utilizes error-prone PCR mutagenesis, overlap extension PCR, and phage lambda Red-mediated homologous recombination and that should be generally applicable. These mutants plus other experiments demonstrate that ACP function is essential for the growth of E. coli. Each of the mutants was efficiently modified with the phosphopantetheinyl moiety essential for the function of ACP in lipid synthesis, and thus lack of function at the nonpermissive temperature cannot be attributed to a lack of prosthetic group attachment. All of the mutant proteins were largely stable at the nonpermissive temperature except the A68T/N73D mutant protein. Fatty acid synthesis in strains that carried the D38V or A68T/N73D mutations was inhibited upon a shift to the nonpermissive temperature and in the latter case declined to a small percentage of the rate of the wild-type strain.

FIG. 1.

PCR mutagenesis coupled to λ Red recombinase-mediated allele replacement. (A) The acpP gene and the downstream fabF::cat gene were amplified to produce fragments that overlapped by 20 bp. The acpP genes were then mutagenized using 12 or 35 rounds of mutagenic PCR. The mutagenized acpP genes were then combined with the fabF::cat PCR product, and overlapping extension PCR was performed. (B) The resulting population of mutagenized acpP genes linked to fabF::cat was then transformed into strain MC1061 induced for λ Red recombinase. After recovery, the cells were plated at 30°C with selection for chloramphenicol resistance. The colonies that formed after 48 h were patched onto three plates, each of which was incubated at a different temperature (30, 37, or 42°C) to screen for colonies showing temperature-sensitive growth. TS, temperature-sensitive.

FIG. 2.

Complementation analysis of the temperature-sensitive mutants and demonstration of the essentiality of acpP. (A) The three temperature-sensitive mutants isolated by mutagenesis were tested for the ability to grow at 42°C. Each of these mutant strains was then transformed with a plasmid expressing wild-type E. coli AcpP under the control of the arabinose-inducible paraBAD promoter. The resulting strains were streaked onto RB medium containing either arabinose (induction conditions) or glucose (repression conditions), and the plates were incubated at 42°C. (B) The plasmids encoding wild-type, A68T, N73D, or A68T N73D AcpP species were tested for the ability to allow the growth of the acpP(Ts) mutant strain NRD29 at 42°C. (C) Strains DY330 and NRD62, a derivative of DY330 harboring an acpP deletion, carrying a wild-type acpP allele under the control of an araBAD promoter (pNRD25) were tested for the ability to grow in the presence of arabinose or glucose. Note that the growth of the wild-type strain MC1061 containing the wild-type AcpP-encoding plasmid was inhibited by arabinose but that a similar derivative of the other wild-type strain, DY330, was not. Inhibition of growth by overproduction of ACP is due to the accumulation of apo-ACP (the unmodified form), which inhibits phospholipid synthesis (22). We attribute the differential effects seen to the inability of strain MC1061 to metabolize arabinose, which increases the extent of induction compared to that of arabinose-catabolizing strains such as DY330 (13, 27). WT, wild type.

FIG. 3.

Analysis of growth and fatty acid synthesis in two acpP(Ts) mutants and the parental strain. Strains NRD28 (encoding AcpP A68T/N73D), NRD29 (encoding AcpP D38V), and NRD52 (encoding wild-type AcpP) were grown at 30°C to an OD₆₀₀ of ∼0.2, and the culture was then split into two flasks. One flask was incubated at 30°C, and the second flask was incubated at 44°C. The turbidities of the cultures were then measured at various time points. (A) Growth curves of strains NRD52 (squares), NRD28 (triangles), and NRD29 (circles) at 30°C. (B) Growth curves of the same strains after a shift of the exponentially growing cultures from 30°C to 44°C. The symbols are the same as those used for panel A. (C) Rates of fatty acid synthesis (labeled acetate incorporation [incorp.]) at various time points after shifting of the cultures of the three strains to 44°C. Fatty acid synthesis rates were determined as described in Materials and Methods. The symbols are the same as those used for panel A. Note that the y axes are log scales.

FIG. 4.

Analysis of 4′-PP attachment to the temperature-sensitive ACPs (with the mutations D38V, E49G, and A68T) at 30°C and 42°C. The protein encoded by the plasmid is shown above the lane (in the lane marked “None,” the strain carried the empty vector). The ΔpanD strain NRD44 harboring plasmids that encoded the various mutant AcpPs expressed from the araBAD promoter was first starved for β-alanine at 30°C on minimal E medium-glucose plates. The cells were then resuspended in minimal E liquid medium. Arabinose and β-alanine were then added to final concentrations of 0.2% and 0.5 μM, respectively. One culture of each strain was incubated at 30°C and the other at 42°C for 3 h. The cells were then harvested by centrifugation, resuspended in 100 mM MES (pH 6.1), and sonicated. The cell debris was pelleted, and the soluble cell extract was fractionated on a 20% native polyacrylamide gel. The gel was then fixed, treated with fluorography solution, dried, and exposed to film. The autofluorogram is shown. WT, wild type.

FIG. 5.

Stabilities of the mutant ACPs. Western blot analysis of the in vivo turnover of wild-type (WT) E. coli AcpP and the three temperature-sensitive AcpP species. Derivatives of strain CY321 cells harboring plasmids expressing wild-type, D38V, E49G, A68T, or A68T N73D AcpP from the E. coli araBAD promoter were grown in RB-ticarcillin-clavulanate medium to an OD₆₀₀ of ∼0.4. Expression of the ACPs was induced for 1 h with arabinose (0.2%). The cells were washed once with RB-ticarcillin-clavulanate-glucose (0.2%) medium and then suspended in this medium. A sample of culture was then removed. The cell culture was then divided, and half the culture was incubated at 30°C and the other half was incubated at 42°C. Additional samples were removed from both cultures after 30 min, 60 min, 120 min, and 240 min of incubation. The soluble fraction of the cell extracts was then fractionated on a 20% native polyacrylamide gel, and AcpP species were detected by Western blotting as described in Materials and Methods. Note that the apparently decreased intensities of the E49G protein bands are due to a shorter exposure of the film in that experiment. The initial band intensities were comparable to those of the wild-type strain in that experiment. Strain CY321 encodes a functional mutant ACP that has an unusually fast migration on native polyacrylamide gels (23) and thus allows resolution of the chromosomally and plasmid-encoded ACPs.

FIG. 6.

Modeling of the amino acid substitutions present in the AcpP(Ts) proteins onto the E. coli butyryl-ACP crystal structure (39). Segments of the crystal structures of wild-type AcpP (A, C, and E) are compared to the corresponding segments containing the following amino acid substitutions present in the AcpP(Ts) proteins: D38V (B), E49G (D), and A68T (F). (A-D) The hydrophobic residues are shown in brown. (E and F) The measurements are in angstroms.