Downloaded from www.microbiologyresearch.org by IP: 54.144.193.201 On: Thu, 18 Jan 2018 02:48:23 Genetic characterization of pcpS, encoding the multifunctional phosphopantetheinyl transferase of Pseudomonas aeruginosa Nazir Barekzi,3 Swati Joshi, Scott Irwin, Todd Ontl and Herbert P. Schweizer Correspondence Herbert P. Schweizer Herbert.Schweizer@colostate.edu Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1658, USA Received 8 October 2003 Revised 2 December 2003 Accepted 9 December 2003 Fatty acid synthases (primary metabolism), non-ribosomal peptide synthases and polyketide synthases (secondary metabolism) contain phosphopantetheinyl (Ppant)-dependent carrier proteins that must be made functionally active by transfer of the 49-Ppant moiety from coenzyme A. These reactions are usually catalysed by dedicated Ppant transferases. Although rich in Ppant-dependent carrier proteins, it was previously shown that Pseudomonas aeruginosa possesses only one Ppant transferase, encoded by pcpS, which functions in both primary and secondary metabolism. Consistent with this notion are our findings that pcpS can genetically complement mutations in the Escherichia coli acpS and entD genes, encoding the apo-acyl carrier protein (ACP) synthase of fatty acid synthesis and a Ppant transferase of enterobactin synthesis, respectively. It also complements a Bacillus subtilis sfp mutation affecting a gene encoding a Ppant transferase essential for surfactin synthesis. A pcpS insertion mutant could only be constructed in a strain carrying the E. coli acpS gene on a chromosomally integrated element in trans, implying that the in vitro essentiality of pcpS is due to its requirement for activation of apo-ACP of fatty acid synthesis. The conditional pcpS mutant is non-fluorescent, does not produce pyoverdine and pyochelin, and does not grow in the presence of iron chelators. The data presented here for the first time confirm that PcpS plays an essential role in both fatty acid and siderophore metabolism. INTRODUCTION Members of the superfamily of 49-phosphopantetheine (Ppant)-dependent carrier proteins play central roles in fatty acid synthesis, polyketide synthesis and non-ribosomal peptide synthesis. This family includes the acyl carrier proteins (ACP), aryl carrier proteins and peptidyl carrier proteins, which can exist as distinct proteins or be part of larger polypeptide chains (Lambalot et al., 1996; Walsh et al., 1997). All carrier proteins are synthesized in the inactive apo form and must be converted to the active holo form. This is achieved by enzymic transfer of the Ppant moiety from co- enzyme A to a conserved serine residue in the carrier protein by a usually dedicated Ppant transferase (Flugel et al., 2000). Most bacteria use more than one Ppant transferase pathway. The conversion of ACPs of primary metabolism (fatty acid synthesis) is catalysed by AcpS enzymes. Ppant transferases of this group are ~120 aa in size, act as homotrimers and exhibit a narrow substrate specificity in that they only modify ACPs of fatty acid synthesis and type II polyketide synthesis (Flugel et al., 2000; Mootz et al., 2001). Other Ppant transferases are involved in the synthesis of second- ary metabolites. Escherichia coli and Bacillus subtilis, for example, produce the catecholic siderophore enterobactin and the cyclic lipopeptide surfactin via non-ribosomal peptide synthesis pathways, using the associated Sfp-type Ppant transferases EntD and Sfp, respectively (Coderre & Earhardt, 1989; Nakano et al., 1992). EntD cannot substitute for AcpS in primary metabolism (Flugel et al., 2000) but B. subtilis strains from which acpS has been deleted can sustain fatty acid synthesis, presumably because Sfp can accept ACPs of fatty acid synthesis pathways (Mootz et al., 2001). Sfp is about twice the size of AcpS, exhibits a very broad substrate specificity and functions as a monomeric enzyme (Mootz et al., 2001; Reuter et al., 1999). P. aeruginosa is unique in that it apparently contains only one Ppant transferase enzyme, PcpS, which can modify the carrier proteins of both primary and secondary metabolism 3Present address: Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA. The GenBank accession number for the sequence reported in this paper is AAG04554. Abbreviations: ACP, acyl carrier protein; CAS, chrome azurol S; PcpS, P. aeruginosa carrier protein synthase; Ppant, 49-phosphopantetheine. 0002-6823 G 2004 SGM Printed in Great Britain 795 Microbiology (2004), 150, 795–803 DOI 10.1099/mic.0.26823-0