Isolation of the Rhizobium leguminosarum NodF nodulation protein: NodF carries a 4'-phosphopantetheine prosthetic group

Abstract

Cell 60: [281][282][283][284][285][286][287][288][289][290][291][292][293][294] 1990). The nodFE region of Rhizobium kguminosarum has been cloned into a multicopy plasmid and has been shown in R. leguminosarum to code for a flavonoid-inducible protein that is effectively labeled by radioactive I-alanine added to the growth medium. After purification, the labeled protein migrates as a single band with an apparent molecular weight of 5,000 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, more rapidly than E. coli ACP. In contrast, in native gels the protein is resolved into two bands, both identified as NodF by analysis of the amino terminus and both migrating more slowly than E. coli ACP. Pulse-chase experiments with labeled ji-alanine suggested that the slower-moving band may be the precursor of the faster band. The NodF protein carries a 4'-phosphopantetheine as a prosthetic group. A NodF fusion protein under the control of the lac promoter is expressed in E. coli and is labeled with Il-alanine, indicating that it is recognized by the ACP synthase of E. coli. The ACP phosphodiesterase of E. coli, which catalyzes the release of phosphopantetheine from E. coli ACP, does not remove phosphopantetheine from NodF.Bacteria of the genus Rhizobium are able to interact symbiotically with specific plant hosts, leading to the formation of nitrogen-fixing root nodules. In the exchange of signals prior to nodulation, flavonoids secreted by the plant cause the induction of rhizobial nodulation (nod) genes. The nodABCIJ genes are termed common nod genes because they are functionally interchangeable among different Rhizobium species. In contrast, genes determining the host range of nodulation are termed host specific. Among them are genes of the nodFEL operon (7,22,37) (Fig. 1). NodF functions together with NodE in the production of a hostspecific rhizobial signal (31).Some indication of the function of NodFEL proteins can be derived from studying their homology, deduced from the gene sequence, to known proteins. The NodL protein appears to be homologous to the acetyl transferases LacA and CysE (9, 44), and NodE shares homology with a group of ,-ketoacyl synthases, such as FabB (the condensing enzyme of fatty acid biosynthesis of Escherichia coli), or those presumed to be involved in the synthesis of ,B-ketide antibiotics in Streptomyces species (2, 34). The NodF sequence was found to resemble that of the acyl carrier protein (ACP) of E. coli in the region near the serine to which the phosphopantetheine is attached, although amino acid identity to the ACP was very low in the rest of the molecule (see Fig. 6A tutive ACP, which presumably does function in the synthesis of essential membrane lipids, has been recently isolated from R. meliloti and studied in this laboratory (27).The periplasmic cyclic P-1,2-glucans of Rhizobium species are also involved in the cell signalling leading to nodulation (13) in a way that is so far not understood. These glucans are very similar to the periplasmic membrane-derived oligosa...

Biochemical Characterization of Acyl Carrier Protein (AcpM) and Malonyl-CoA:AcpM Transacylase (mtFabD), Two Major Components ofMycobacterium tuberculosis Fatty Acid Synthase II

“…2 Until recently, only ACPs expressed constitutively and involved in the biosynthesis of essential fatty acids were known. The discovery of specialized ACPs for the biosynthesis of polyunsaturated fatty acids in Rhizobium meliloti (33) and in Rhizobium leguminosarum (34) were thought to represent unusual cases involved in complex secondary metabolism. Recently, a second ACP was isolated from R. leguminosarum and was shown to be involved in the transfer of 27-hydroxyoctacosanoic acids during lipid A biosynthesis (35).…”

NMR investigations of the structural properties of the nodulation protein, NodF, from Rhizobium leguminosarum and its homology with Escherichia coli acyl carrier protein

“…Like AcpP, the NodF protein can carry this Abstract Heteronuclear NMR methods have been used to elucidate the secondary structure and the general tertiary fold of prosthetic group [9]. However, the sequence homology outside the protein NodF from Rhizobium leguminosarum.…”

Tandemly Duplicated Acyl Carrier Proteins, Which Increase Polyketide Antibiotic Production, Can Apparently Function Either in Parallel or in Series

“…They can also act as acyl donors in the biosynthesis of lipids (2), can carry membranederived oligosaccharides (3), and can be involved in the activation of RTX toxins in Gram-negative bacteria (4). Specialized ACPs may also act as signal factors in rhizobial nodulation (5) as well as substrates and carriers in the synthesis of lipoteichoic acid (6) and polyketide antibiotics (7).…”