A polyphosphate kinase 1 ( ppk1 ) mutant of Pseudomonas aeruginosa exhibits multiple ultrastructural and functional defects

Abstract

Pseudomonas aeruginosa, of medical, environmental, and industrial importance, depends on inorganic polyphosphate (poly P) for a wide range of functions, especially survival. Mutants of PAO1 lacking poly P kinase 1, PPK1, the enzyme responsible for most poly P synthesis in Escherichia coli and other bacteria, are defective in motility, quorum sensing, biofilm formation, and virulence. We describe here multiple defects in the ppk1 mutant PAOM5, including a striking compaction of the nucleoid, distortion of the cell envelope, lack of planktonic motility and exopolymer production, and susceptibility to the ␤-lactam antibiotic carbenicillin as well as desiccation. We propose that P. aeruginosa with reduced poly P levels undergoes ultrastructural changes that contribute to profound deficiencies in cellular functions.carbenicillin ͉ exopolymer ͉ motility ͉ desiccation ͉ nucleoid P olyphosphate (poly P), a linear chain of phosphate residues linked by phosphoanhydride bonds, is present in all cells and was likely present throughout evolution (1). Poly P is synthesized in prokaryotic cells from ATP by poly P kinases (PPKs) for which two families, PPK1 and PPK2, have been identified. PPK1 is responsible principally for the synthesis of poly P in Escherichia coli, Salmonella enterica serovar Typhimurium, Shigella flexneri, Vibrio cholerae, Helicobacter pylori, Bacillus cereus, Myxococcus xanthus, and, as described here, P. aeruginosa. Homologous PPK1 amino acid sequences are in the databases of Ͼ40 organisms, including many bacterial pathogens (2). Knockout mutants of ppk1 in several pathogens demonstrate phenotypes including responses to stresses, motility, and virulence (3-7) as well as developmental defects in other species (8, 9). The only apparent eukaryotic homolog is in the social slime mold Dictyostelium discoideum (2), the mutant of which exhibits growth and developmental phenotypes (10).We describe here the construction and biochemical characterization of a P. aeruginosa ppk1 knockout mutant, PAOM5, for which the motility, biofilm formation, burned-mouse, and ocular virulence phenotypes were reported in refs. 3, 6, and 11 and which, unlike the WT, is susceptible to predation by D. discoideum (10). Electron microscopy studies reveal significant differences in ultrastructure between the WT and mutant, including compaction of the nucleoid, withdrawal of the cytoplasm from the inner membrane, abnormal envelope structure, as well as a failure to produce exopolymer. Video microscopy studies show that the mutant is almost immotile in liquid medium, despite having about the same number of polar flagella. We also demonstrate that the ppk1 mutant exhibits much reduced viability after exposure to a ␤-lactam antibiotic, carbenicillin, or the hyperosmolarity of desiccation. Results PPK1 Activity and poly P Levels Are Reduced but Not Abolished in the ppk1Mutant. P. aeruginosa PAO1 (WT) grown in rich (LB) medium exhibits high levels of PPK1 activity during midexponential phase growth, almost wholly associated with the membrane f...

Defects in Phosphate Acquisition and Storage Influence Virulence of Cryptococcus neoformans

“…The polyphosphate kinase in bacteria is also essential for biofilm formation in Pseudomonas aeruginosa, Vibrio cholerae, and Escherichia coli (74)(75)(76), while deletion of the kinase gene in Campylobacter jejuni increased biofilm formation (72). Virulence during macrophage interaction or during infection of the host was also reduced for polyphosphate metabolism mutants of P. aeruginosa, Helicobacter pylori, C. jejuni, Salmonella enterica, Mycobacterium tuberculosis, and E. coli (72)(73)(74)(76)(77)(78)(79)(80)(81)(82)(83).…”

Accumulation and enhanced cycling of polyphosphate by Sargasso Sea plankton in response to low phosphorus

“…Therefore, predominant strategies for reducing cellular P probably involve DNA (e.g., "genome streamlining") (24), RNA (e.g., slower growth) (25), or other P-containing biochemicals whereas polyP cannot be spared beyond a certain extent. Indeed, polyP is intimately linked to primary metabolism in both eukaryotes and prokaryotes such that the inability to synthesize polyP results in a diverse range of cellular defects (26,27).…”

Role of Polyphosphate in Thermophilic Synechococcus sp. from Microbial Mats

“…MreB and ParM, for example, both engage in the formation of actin-like filaments that are indispensable in the maintenance of cell shape and plasmid segregation (55,56). Poly P is known to have a role in maintaining the extended shape of the nucleoid and the cell envelope so that in Pseudomonas aeruginosa ppk1 mutant cells, the nucleoid is compacted, and the cytoplasm is detached in many places from the cell poles and borders (44). Other proteins like CTPase (57,58) have also been reported to make filaments inside the cell.…”

“…The shape and size of WT cells appear to be affected by growth conditions (Devaki Bhaya, unpublished observations); thus, without a more detailed analysis, the exact cause of the smaller ppk cells is not clear. However, the finding that PPK can form tubular structures suggests a potential role of PPK in the maintenance of cell shape (27,44) (see the Discussion; also see Fig. S2 in the supplemental material).…”