Limits...
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

Broad-host range constitutive (pS7PPX1) and regulated (pBADPPX1) expression vector maps to obtain polyP deficient bacteria. Source of yeast DNA and primers used in plasmid construction were described in Methods. A detail description of pMLBAD and pMLS7 plasmids can be found in reference [22].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2667530&req=5

Figure 1: Broad-host range constitutive (pS7PPX1) and regulated (pBADPPX1) expression vector maps to obtain polyP deficient bacteria. Source of yeast DNA and primers used in plasmid construction were described in Methods. A detail description of pMLBAD and pMLS7 plasmids can be found in reference [22].

Mentions: Details about the construction of the constitutive and regulated expression vector and the verification of yeast PPX1 overexpression can be found in the Additional File 1 and Figures 1 and 2.


Constitutive and regulated expression vectors to construct polyphosphate deficient bacteria.

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

Broad-host range constitutive (pS7PPX1) and regulated (pBADPPX1) expression vector maps to obtain polyP deficient bacteria. Source of yeast DNA and primers used in plasmid construction were described in Methods. A detail description of pMLBAD and pMLS7 plasmids can be found in reference [22].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Broad-host range constitutive (pS7PPX1) and regulated (pBADPPX1) expression vector maps to obtain polyP deficient bacteria. Source of yeast DNA and primers used in plasmid construction were described in Methods. A detail description of pMLBAD and pMLS7 plasmids can be found in reference [22].
Mentions: Details about the construction of the constitutive and regulated expression vector and the verification of yeast PPX1 overexpression can be found in the Additional File 1 and Figures 1 and 2.

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