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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

Colony morphology and motility of constitutive polyP deficient Pseudomonas sp. B4 cell. (A) Standard LB plates and (B) motility assay plates of control and polyP deficient Pseudomonas sp. B4 cells.
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Figure 4: Colony morphology and motility of constitutive polyP deficient Pseudomonas sp. B4 cell. (A) Standard LB plates and (B) motility assay plates of control and polyP deficient Pseudomonas sp. B4 cells.

Mentions: Among many functional and structural problems, P. aeruginosa PAO1 ppk1 knockout mutants failed to develop biofilms and were impaired in all forms of motility (swimming, swarming, and twitching) [9-11,17]. To validate our approach and check whether our transformants resembled the functional deficiencies of ppk1 mutants, we performed motility and biofilm assays in our strains with depleted polyP levels. Wild type Pseudomonas sp. B4 is a highly motile rod with a single polar flagellum and forms widely spread colonies in LB plates [16]. However, the form and size of the colonies varied notoriously in constitutive polyP(-) cells, suggesting a motility defect (Figure 4A). This was confirmed by using semisolid agar plates where cells were able to swim through water-filled channels to create concentric chemotactic rings (Figure 4B). As reported for the P. aeruginosa PAO1 ppk1 mutant, our constitutive polyP(-) cells were impaired in swimming motility in semisolid agarose plates (Figure 4B) despite possessing an apparently normal flagellum (Figure 3 for flagellum detail). Curiously, the phenotype change seen in Figure 4 is as drastic as that seen in a nonmotile strain containing a knockout mutation in a flagellin structural gene fliC and more severe than that observed in a ppk1 mutant [9]. This suggests that the degree of lack of motility is related to the extent of polyP removed from the cell.


Constitutive and regulated expression vectors to construct polyphosphate deficient bacteria.

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

Colony morphology and motility of constitutive polyP deficient Pseudomonas sp. B4 cell. (A) Standard LB plates and (B) motility assay plates of control and polyP deficient Pseudomonas sp. B4 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Colony morphology and motility of constitutive polyP deficient Pseudomonas sp. B4 cell. (A) Standard LB plates and (B) motility assay plates of control and polyP deficient Pseudomonas sp. B4 cells.
Mentions: Among many functional and structural problems, P. aeruginosa PAO1 ppk1 knockout mutants failed to develop biofilms and were impaired in all forms of motility (swimming, swarming, and twitching) [9-11,17]. To validate our approach and check whether our transformants resembled the functional deficiencies of ppk1 mutants, we performed motility and biofilm assays in our strains with depleted polyP levels. Wild type Pseudomonas sp. B4 is a highly motile rod with a single polar flagellum and forms widely spread colonies in LB plates [16]. However, the form and size of the colonies varied notoriously in constitutive polyP(-) cells, suggesting a motility defect (Figure 4A). This was confirmed by using semisolid agar plates where cells were able to swim through water-filled channels to create concentric chemotactic rings (Figure 4B). As reported for the P. aeruginosa PAO1 ppk1 mutant, our constitutive polyP(-) cells were impaired in swimming motility in semisolid agarose plates (Figure 4B) despite possessing an apparently normal flagellum (Figure 3 for flagellum detail). Curiously, the phenotype change seen in Figure 4 is as drastic as that seen in a nonmotile strain containing a knockout mutation in a flagellin structural gene fliC and more severe than that observed in a ppk1 mutant [9]. This suggests that the degree of lack of motility is related to the extent of polyP removed from the cell.

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