The Two Acyl Carrier Proteins of Enterococcus faecalis Have Nonredundant Functions

Abstract

Enterococcus faecalis encodes two proteins, AcpA and AcpB, having the characteristics of acyl carrier proteins (ACPs). We report that the acpA gene located in the fatty acid synthesis operon is essential for fatty acid synthesis and the ΔacpA strain requires unsaturated fatty acids for growth. The ΔacpA strain could be complemented by a plasmid carrying a wild-type acpA gene, but not by a plasmid carrying a wild-type acpB gene. Substitution of four AcpA residues for those of AcpB resulted in a protein that modestly complemented the ΔacpA strain and restored fatty acid synthesis, although the acyl chains synthesized were unusually short. IMPORTANCE Enterococcus faecalis, as well as related species, has two genes-acpA and acpB-encoding putative acyl carrier proteins (ACPs). It has been assumed that AcpA is essential for fatty acid synthesis whereas AcpB is involved utilization of environmental fatty acids. We report here the first experimental test of the essentiality of acpA and show that it is indeed an essential gene that cannot be replaced by acpB.

Keywords: acyl carrier proteins; fatty acid synthesis; phospholipids.

FIG 1

Strategy for construction of the E. faecalis ΔacpA strain and its characterization by PCR. The genomic locations of the fatty acid synthesis genes are given above the scheme.

FIG 2

Growth phenotypes of the ΔacpA strain with either 5% serum or different fatty acids.

FIG 3

Growth phenotypes and fatty acid synthesis by [¹⁴C]acetate labeling of the ΔacpA strain and the complemented derivative strains. The wild-type acpA gene was expressed from the strong P32 promoter (1) in the high-copy-number plasmid pBM02. The number above each lane is the incorporation relative to the wild-type strain. Note that the label at the origin is from lipoproteins that extract into chloroform. One of the lipoprotein fatty acids is in amide linkage which is not cleaved under the basic conditions we use. Others have estimated 60 to 80 different lipoproteins in E. faecalis. The ester-linked lipoprotein fatty acids are derived from phospholipids.

FIG 4

Growth phenotypes of the ΔacpA strain complemented with the wild-type acpA gene under agmatine control. Growth with agmatine induction at various concentrations was tested.

FIG 5

Fatty acid synthesis of the agmatine-induced complemented strains of Fig. 4 by [¹⁴C]acetate labeling. The number above each lane is the incorporation relative to the wild-type strain.

FIG 6

(Upper panel) Sequence alignments of ACPs of various bacteria with the helix II sequences in a bracket. (Lower panel) Helix II sequences from the upper panel and sites where AcpA residues were substituted in place of AcpB residues. The ACPs are from various organisms as follows: Ec, E. coli; Bs, Bacillus subtilis; Ll, Lactococcus lactis; and Ef, E. faecalis.

FIG 7

Complementation of the ΔacpA strain by acpB and substitution derivatives in complementation of the ΔacpA strain. 2S and 4S denote the residue substitutions given in Fig. 6. The acpB genes were expressed from the strong P32 promoter (10) in the high-copy-number plasmid pBM02.

FIG 8

Fatty acid synthesis by [¹⁴C]acetate labeling of the ΔacpA strain complemented by plasmids encoding the substituted acpB derivatives. The two unsaturates in the right-hand lane are 14:1 (probably cis-7-tetradecenoic acid) and palmitoleic acid (cis-9-hexadecenoic acid) (Table 1). The number above each lane is the incorporation relative to the wild-type strain.

FIG 9

Fatty acid uptake by [¹⁴C]oleic acid labeling of the ΔacpA strain complemented by plasmids encoding the substituted acpB derivatives. The compounds running at the top of the plate are cyclopropane fatty acid derived from [¹⁴C]oleic acid. The number above each lane gives the incorporation relative to the wild-type strain.