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Constitutive and regulated expression vectors to construct polyphosphate deficient bacteria.

Chávez FP, Mauriaca C, Jerez CA - BMC Res Notes (2009)

Bottom Line: This was achieved by the overexpression of yeast exopolyphosphatase (PPX1).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Microbiology and Biotechnology & Millennium Institute for Advanced Studies in Cell Dinamics and Biotechnology (ICDB), Department of Biology, Faculty of Sciences, University of Chile, Las Palmeras 3425, Nuñoa, Santiago, Chile. fpchavez@uchile.cl

ABSTRACT

Background: 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. 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.

Results: As 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.

Conclusion: To 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.

No MeSH data available.


Related in: MedlinePlus

PolyP content in recombinant Pseudomonas sp. B4 cells. PolyP removal was checked by quantification of cellular polyP (A) and by Transmission Electron Microscopy (TEM) of unstained cells (B). Arrows indicate the cellular flagellum. Error bars are the average of three determinations in three biological replicates.
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Figure 3: PolyP content in recombinant Pseudomonas sp. B4 cells. PolyP removal was checked by quantification of cellular polyP (A) and by Transmission Electron Microscopy (TEM) of unstained cells (B). Arrows indicate the cellular flagellum. Error bars are the average of three determinations in three biological replicates.

Mentions: Previous studies from our laboratory have shown that Pseudomonas sp. B4 is a polyP-accumulating bacteria and in certain conditions polyP can be stored and observed as electron dense granules by transmission electron microscopy (TEM) of unstained cells [15,16]. To determine whether the overproduction of yeast PPX1 (Figures 1 and 2) affected the accumulation of polyP in the bacterium, we monitored the levels of this biopolymer by observing the presence of electron dense granules by TEM. Figure 3A shows the results of quantification of the isolated polyP from recombinant Pseudomonas sp. B4 cells in LB medium. PolyP from arabinose induced regulated polyP(-) (pBADPPX1) and constitutive polyP(-) (pS7PPX1) cells was removed to barely detectable levels (around 2 pmoles Pi/mg protein). The reduction of polyP levels (>96%) due to PPX1 overproduction could be prevented in regulated polyP(-) (pBADPPX1) cells to levels similar to those of the control cells (pMLBAD and pMLS7) when growing in the presence of 1% glucose.


Constitutive and regulated expression vectors to construct polyphosphate deficient bacteria.

Chávez FP, Mauriaca C, Jerez CA - BMC Res Notes (2009)

PolyP content in recombinant Pseudomonas sp. B4 cells. PolyP removal was checked by quantification of cellular polyP (A) and by Transmission Electron Microscopy (TEM) of unstained cells (B). Arrows indicate the cellular flagellum. Error bars are the average of three determinations in three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667530&req=5

Figure 3: PolyP content in recombinant Pseudomonas sp. B4 cells. PolyP removal was checked by quantification of cellular polyP (A) and by Transmission Electron Microscopy (TEM) of unstained cells (B). Arrows indicate the cellular flagellum. Error bars are the average of three determinations in three biological replicates.
Mentions: Previous studies from our laboratory have shown that Pseudomonas sp. B4 is a polyP-accumulating bacteria and in certain conditions polyP can be stored and observed as electron dense granules by transmission electron microscopy (TEM) of unstained cells [15,16]. To determine whether the overproduction of yeast PPX1 (Figures 1 and 2) affected the accumulation of polyP in the bacterium, we monitored the levels of this biopolymer by observing the presence of electron dense granules by TEM. Figure 3A shows the results of quantification of the isolated polyP from recombinant Pseudomonas sp. B4 cells in LB medium. PolyP from arabinose induced regulated polyP(-) (pBADPPX1) and constitutive polyP(-) (pS7PPX1) cells was removed to barely detectable levels (around 2 pmoles Pi/mg protein). The reduction of polyP levels (>96%) due to PPX1 overproduction could be prevented in regulated polyP(-) (pBADPPX1) cells to levels similar to those of the control cells (pMLBAD and pMLS7) when growing in the presence of 1% glucose.

Bottom Line: This was achieved by the overexpression of yeast exopolyphosphatase (PPX1).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Microbiology and Biotechnology & Millennium Institute for Advanced Studies in Cell Dinamics and Biotechnology (ICDB), Department of Biology, Faculty of Sciences, University of Chile, Las Palmeras 3425, Nuñoa, Santiago, Chile. fpchavez@uchile.cl

ABSTRACT

Background: 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. 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.

Results: As 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.

Conclusion: To 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.

No MeSH data available.


Related in: MedlinePlus