Abstract
The genome of Pseudomonas aeruginosa contains three open reading frames, PA2966, PA1869, and PA3334, which encode putative acyl carrier proteins, AcpP1, AcpP2, and AcpP3, respectively. In this study, we found that, although these apo-ACPs were successfully phosphopantetheinylated by P. aeruginosa phosphopantetheinyl transferase (PcpS) and all holo-forms of these proteins could be acylated by Vibrio harveyi acyl-ACP synthetase (AasS), only AcpP1 could be used as a substrate for the synthesis of fatty acids, catalyzed by P. aeruginosa cell free extracts in vitro, and only acpP1 gene could restore growth in the Escherichia coli acpP mutant strain CY1877. And P. aeruginosa acpP1 could not be deleted, while disruption of acpP2 or acpP3 in the P. aeruginosa genome allowed mutant strains to grow as well as the wild type strain. These findings confirmed that only P. aeruginosa AcpP1 functions in fatty acid biosynthesis, and that acpP2 and acpP3 do not play roles in the fatty acid synthetic pathway. Moreover, disruption of acpP2 and acpP3 did not affect the ability of P. aeruginosa to produce N-acylhomoserine lactones (AHL), but replacement of P. aeruginosa acpP1 with E. coli acpP caused P. aeruginosa to reduce the production of AHL molecules, which indicated that neither P. aeruginosa AcpP2 nor AcpP3 can act as a substrate for synthesis of AHL molecules in vivo. Furthermore, replacement of acpP1 with E. coli acpP reduced the ability of P. aeruginosa to produce some exo-products and abolished swarming motility in P. aeruginosa.
Highlights
IntroductionMembers of the superfamily of 4 -phosphopantetheine (Ppant)-dependent carrier proteins play central roles in fatty acid synthesis, polyketide synthesis and non-ribosomal peptide synthesis (Lai et al, 2006; Smith, 2006; Byers and Gong, 2007; Chan et al, 2009)
In order to test whether P. aeruginosa acpP1, acpP2, and acpP3 function in fatty acid biosynthesis, these genes were inserted into the IPTG-inducible vector pTac85 (Marsh, 1986) to yield plasmids plasmid encoding acpP1 (pCD1), pCD2, and pCD13, respectively
E. coli CY1877 carrying the plasmid encoding acpP1 grew on Luria– Bertani (LB) medium in the presence of IPTG and the absence of arabinose, whereas derivatives of strain CY1877 carrying empty vector, or plasmids encoding acpP2 or acpP3 failed to grow under this condition (Figure 1A)
Summary
Members of the superfamily of 4 -phosphopantetheine (Ppant)-dependent carrier proteins play central roles in fatty acid synthesis, polyketide synthesis and non-ribosomal peptide synthesis (Lai et al, 2006; Smith, 2006; Byers and Gong, 2007; Chan et al, 2009). The remaining two ACP homologs, SMc01553 and SMb20651, have been identified as ACP-like proteins, but their functions need further investigation (Ramos-Vega et al, 2009; Davila-Martinez et al, 2010)
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