Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Wed, 09 Jan 2019 09:17:25 Microbiology (2000), 146, 839–849 Printed in Great Britain Expression and purification of four different rhizobial acyl carrier proteins Isabel M. Lo  pez-Lara† and Otto Geiger† Author for correspondence : Otto Geiger. Tel : 52 73 131697. Fax: 52 73 175581. e-mail : Ottocifn.unam.mx Institute of Biotechnology, Technical University of Berlin, Seestrasse 13, D-13353 Berlin, Germany In rhizobia, besides the constitutive acyl carrier protein (AcpP) involved in the biosynthesis and transfer of common fatty acids, there are at least three specialized acyl carrier proteins (ACPs) : (1) the flavonoid-inducible nodulation protein NodF ; (2) the RkpF protein, which is required for the biosynthesis of rhizobial capsular polysaccharides ; and (3) AcpXL, which transfers 27- hydroxyoctacosanoic acid to a sugar backbone during lipid A biosynthesis. Whereas the nucleotide sequences encoding the three specialized ACPs are known, only the amino acid sequence of the AcpP of Sinorhizobium meliloti was available. In this study, using reverse genetics, the genes for the constitutive AcpPs of S. meliloti and of Rhizobium leguminosarum were cloned and sequenced. Previously, it had been shown that NodF and RkpF can be overproduced in Escherichia coli using the T7 polymerase expression system. Using the same system, the constitutive AcpPs of S. meliloti and of R. leguminosarum, together with the specialized ACP AcpXL, were overproduced and purified. All the known ACPs of rhizobia can be labelled in vivo during expression in E. coli with radioactive β-alanine added to the growth medium due to their modification with a 4’-phosphopantetheine prosthetic group. The availability of all functionally different ACPs should help to unravel how different fatty acids are targeted towards different biosynthetic pathways in one organism. Keywords : Rhizobium, acyl carrier protein, acpP, acpXL INTRODUCTION Biosynthesis and transfer of fatty acids is of major importance during the formation of biological mem- branes, storage lipids, or certain amphiphilic signal molecules [i.e. Nod factors in rhizobia (De  narie  et al., 1996) or autoinducers in Gram-negative bacteria (Fuqua et al., 1996)]. In higher animals, fatty acid biosynthesis occurs on a multienzyme complex whereas in bacteria, fatty acids are formed by the catalytic activity of a number of monofunctional enzymes. In the latter organisms, a small acidic protein carries the growing fatty acyl chains via a thioester linkage of a 4- phosphopantetheine prosthetic group, which itself is ................................................................................................................................................. † Present address : CIFN – UNAM, Av. Universidad s/n, Colonia Chamilpa, Apdo postal 565-A, CP 62210, Cuernavaca, Morelos, Mexico. Abbreviations : ACP, acyl carrier protein; iPCR, inverse PCR. The GenBank accession numbers for the nucleotide sequences reported in this paper are AF159244 and AF159243 for the acpP-containing regions of Sinorhizobium meliloti 1021 and of Rhizobium leguminosarum LPR5045, respectively. attached to a conserved serine residue. This protein is called acyl carrier protein (ACP). In bacteria, ACPs are involved not only in the synthesis of fatty acyl chains, but also in their transfer during phospholipid, lipid A or α-haemolysin biosynthesis (Issartel et al., 1991; Jackowski et al., 1991). In Escherichia coli, all these functions seem to be realized by a single and absolutely essential ACP which is produced constitutively from the acpP gene (Jackowski et al., 1991 ; Rawlings & Cronan, 1992) and which is now named AcpP. Some bacteria have additional ACPs which are involved in the biosynthesis of special β-ketides or fatty acids (Bibb et al., 1989 ; Shearman et al., 1986). In rhizobial species infecting legume plants of the Trifolieae, Vicieae, and Galegeae tribes, one specialized ACP, the nodu- lation protein NodF, is involved, together with NodE, in the synthesis of polyunsaturated fatty acids which are host-specific substituents of nodulation (Nod) factors (Yang et al., 1999). In addition to AcpP and NodF, there are at least two additional ACPs in rhizobia. The first was identified as 0002-3775  2000 SGM 839