Abstract
BackgroundInorganic polyphosphate (polyP), a polymer of tens or hundreds of phosphate residues linked by ATP-like bonds, is found in all organisms and performs a wide variety of functions. PolyP is synthesized in bacterial cells by the actions of polyphosphate kinases (PPK1 and PPK2) and degraded by an exopolyphosphatase (PPX). Bacterial cells with polyP deficiencies are impaired in many structural and important cellular functions such as motility, quorum sensing, biofilm formation and virulence. Knockout mutants of the ppk1 gene have been the most frequent strategy employed to generate polyP deficient cells.ResultsAs an alternative method to construct polyP-deficient bacteria we developed constitutive and regulated broad-host-range vectors for depleting the cellular polyP content. This was achieved by the overexpression of yeast exopolyphosphatase (PPX1). Using this approach in a polyphosphate accumulating bacteria (Pseudomonas sp. B4), we were able to eliminate most of the cellular polyP (>95%). Furthermore, the effect of overexpression of PPX1 resembled the functional defects found in motility and biofilm formation in a ppk1 mutant from Pseudomonas aeruginosa PAO1. The plasmids constructed were also successfully replicated in other bacteria such as Escherichia coli, Burkholderia and Salmonella.ConclusionTo deplete polyP contents in bacteria broad-host-range expression vectors can be used as an alternative and more efficient method compared with the deletion of ppk genes. It is of great importance to understand why polyP deficiency affects vital cellular processes in bacteria. The construction reported in this work will be of great relevance to study the role of polyP in microorganisms with non-sequenced genomes or those in which orthologs to ppk genes have not been identified.
Highlights
Inorganic polyphosphate, a polymer of tens or hundreds of phosphate residues linked by ATP-like bonds, is found in all organisms and performs a wide variety of functions
The best-known enzymes involved in the metabolism of polyP in bacteria are the polyphosphate kinases (PPKs) that catalyze the reversible conversion of the terminal phosphate of ATP into polyP and the exopolyphosphatase (PPX) that processively hydrolyzes the terminal residues of polyP to liberate Pi [1,2]
As a tool to remove cellular polyP content, we report here the construction of expression vectors with constitutive and regulated promoters that overexpress in bacteria the yeast PPX1 fused to a hexa histidine-tag (6 Ht)
Summary
Inorganic polyphosphate (polyP), a polymer of tens or hundreds of phosphate residues linked by ATP-like bonds, is found in all organisms and performs a wide variety of functions. PPK1 knockout mutant cells lacking polyP survive poorly during growth in the stationary phase and are less resistant to heat, oxidants, osmotic challenge, antibiotics and UV [48]. Important cellular process such as motility, quorum sensing, biofilm development and virulence are affected [9,10,11]. In contrast to the ATP-dependent polyP synthetic activity of PPK1, PPK2 preferentially catalyses the opposite reaction, i.e. poly P-driven synthesis of GTP from GDP. Orthologs to both proteins have been found in many bacterial genomes. The enzyme in charge of polyP synthesis still remains unknown in several bacteria containing the biopolymer [13]
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