High cellular organization of pyoverdine biosynthesis in Pseudomonas aeruginosa: clustering of PvdA at the old cell pole

Abstract

Pyoverdine I (PVDI) is the major siderophore produced by Pseudomonas aeruginosa PAO1 to import iron. Its biosynthesis requires the coordinated action of cytoplasmic, periplasmic and membrane proteins. The individual enzymatic activities of these proteins are well known. However, their subcellular distribution in particular areas of the cytoplasm, periplasm, or within the membrane has never been investigated. We used chromosomal replacement to generate P.aeruginosa strains producing fluorescent fusions with PvdA, one of the initial enzymes in the biosynthetic pathway of PVDI in the cytoplasm, and PvdQ, involved in the maturation of PVDI in the periplasm. Cellular fractionation indicated that a substantial amount of PvdA-YFP was located in the membrane fraction. Epifluorescence microscopy imaging showed that PvdA-YFP was mainly clustered at the old cell pole of bacteria, indicating a polar segregation of the protein. Epifluorescence and TIRF imaging on cells expressing labelled PvdQ showed that this enzyme was uniformly distributed in the periplasm, in contrast with PvdA-YFP. The description of the intracellular distribution of these enzymes contributes to the understanding of the PVDI biosynthetic pathway.

Cellular organization of siderophore biosynthesis in Pseudomonas aeruginosa: Evidence for siderosomes

“…The first investigations of the cellular organization of PVDI biosynthesis considered the cytoplasmic enzyme PvdA and the periplasmic enzyme PvdQ [43]. These two proteins are involved in two very distant steps in the PVDI biosynthetic pathway ( Figure 2).…”

“…PvdQ acts in a much later step in PVDI biosynthesis: once the cytoplasmic PVDI precursor has been exported across the inner membrane by the ABC transporter PvdE, the myristic or myristoleic acid chain is removed from the siderophore precursor by the periplasmic PvdQ. Our group [43] has used chromosomal replacement to generate P. aeruginosa strains producing fluorescent PvdA or PvdQ fusion proteins, cell fractionation and fluorescent microscopy and found that these two enzymes had completely different distributions within the cell. PvdQ was uniformly distributed throughout the periplasm whereas the PvdA distribution was much more heterogeneous and complex [43].…”

“…Our group [43] has used chromosomal replacement to generate P. aeruginosa strains producing fluorescent PvdA or PvdQ fusion proteins, cell fractionation and fluorescent microscopy and found that these two enzymes had completely different distributions within the cell. PvdQ was uniformly distributed throughout the periplasm whereas the PvdA distribution was much more heterogeneous and complex [43].…”

Sensor Kinase PA4398 Modulates Swarming Motility and Biofilm Formation in Pseudomonas aeruginosa PA14

“…In particular, microarray data analysis revealed downregulation by 44-fold and 28-fold, respectively, of genes coding for PvdA, one of the initial enzymes in the biosynthetic pathway of pyoverdine in the cytoplasm, and PvdQ, which is involved in pyoverdine maturation in the periplasm (43). Additionally, PvdQ, which exhibits an N-acyl homoserine lactone (HSL) acylase activity and has been shown to degrade the quorum-sensing signal 3-oxododecanoyl-HSL (3OC 12 -HSL), is known for its role in biofilm formation, swarming motility, and virulence (16,44).…”

Acyl peptidic siderophores: structures, biosyntheses and post-assembly modifications

“…It has been hypothesized that the acylated pyoverdine precursor prevents the siderophore from diffusing through the cytoplasm and periplasm. The acyl chain is thus proposed to hold the siderophore in the proper cellular location until biosynthesis is complete (Hannauer et al 2012;Guillon et al 2012). …”