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Constitutive and regulated expression vectors to construct polyphosphate deficient bacteria.

Francisco P Chávez1, Cecilia Mauriaca, Carlos A Jerez

  • 1Laboratory 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

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Summary

Researchers developed a new method to create polyphosphate (polyP)-deficient bacteria by overexpressing yeast exopolyphosphatase (PPX1) using broad-host-range vectors. This approach efficiently reduces polyP levels, impacting bacterial functions like motility and biofilm formation, offering an alternative to gene deletion methods.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • Inorganic polyphosphate (polyP) is a vital polymer found in all organisms, synthesized by polyphosphate kinases (PPK1, PPK2) and degraded by exopolyphosphatase (PPX).
  • PolyP deficiency in bacteria impairs critical functions including motility, quorum sensing, biofilm formation, and virulence.
  • Current methods for generating polyP-deficient bacteria often involve knockout mutants of the ppk1 gene.

Purpose of the Study:

  • To develop an alternative and efficient method for depleting cellular polyphosphate (polyP) content in bacteria.
  • To investigate the functional consequences of polyP depletion using a novel expression vector system.
  • To establish a versatile tool for studying polyP roles in diverse bacterial species.

Main Methods:

  • Construction of constitutive and regulated broad-host-range expression vectors.
  • Overexpression of yeast exopolyphosphatase (PPX1) to deplete intracellular polyP.
  • Application of the method in polyphosphate accumulating bacteria (Pseudomonas sp. B4) and validation in other species (Escherichia coli, Burkholderia, Salmonella).

Main Results:

  • Achieved significant depletion of cellular polyP (>95%) in Pseudomonas sp. B4 via PPX1 overexpression.
  • Observed functional defects in motility and biofilm formation, mirroring those in ppk1 mutants of Pseudomonas aeruginosa PAO1.
  • Demonstrated successful plasmid replication and polyP depletion capability across multiple bacterial genera.

Conclusions:

  • Broad-host-range expression vectors overexpressing PPX1 offer a more efficient alternative to ppk gene deletion for depleting bacterial polyP.
  • This method facilitates the study of polyP's role in essential cellular processes.
  • The developed system is valuable for investigating polyP in bacteria with unsequenced genomes or unidentified ppk orthologs.