scholarly journals Di-Adenosine Tetraphosphate (Ap4A) Metabolism Impacts Biofilm Formation by Pseudomonas fluorescens via Modulation of c-di-GMP-Dependent Pathways

2010 ◽  
Vol 192 (12) ◽  
pp. 3011-3023 ◽  
Author(s):  
Russell D. Monds ◽  
Peter D. Newell ◽  
Jeffrey C. Wagner ◽  
Julia A. Schwartzman ◽  
Wenyun Lu ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Biofilm Formation ◽  
Inorganic Phosphate ◽  
De Novo ◽  
Cellular Level ◽  
Pho Regulon ◽  
Biosynthetic Pathways ◽  
Gene Encoding ◽  
Dependent Inhibition ◽  
Significant Point

ABSTRACT Dinucleoside tetraphosphates are common constituents of the cell and are thought to play diverse biological roles in organisms ranging from bacteria to humans. In this study we characterized two independent mechanisms by which di-adenosine tetraphosphate (Ap4A) metabolism impacts biofilm formation by Pseudomonas fluorescens. Null mutations in apaH, the gene encoding nucleoside tetraphosphate hydrolase, resulted in a marked increase in the cellular level of Ap4A. Concomitant with this increase, Pho regulon activation in low-inorganic-phosphate (Pi) conditions was severely compromised. As a consequence, an apaH mutant was not sensitive to Pho regulon-dependent inhibition of biofilm formation. In addition, we characterized a Pho-independent role for Ap4A metabolism in regulation of biofilm formation. In Pi-replete conditions Ap4A metabolism was found to impact expression and localization of LapA, the major adhesin regulating surface commitment by P. fluorescens. Increases in the level of c-di-GMP in the apaH mutant provided a likely explanation for increased localization of LapA to the outer membrane in response to elevated Ap4A concentrations. Increased levels of c-di-GMP in the apaH mutant were associated with increases in the level of GTP, suggesting that elevated levels of Ap4A may promote de novo purine biosynthesis. In support of this suggestion, supplementation with adenine could partially suppress the biofilm and c-di-GMP phenotypes of the apaH mutant. We hypothesize that changes in the substrate (GTP) concentration mediated by altered flux through nucleotide biosynthetic pathways may be a significant point of regulation for c-di-GMP biosynthesis and regulation of biofilm formation.

scholarly journals Disruption of de novo purine biosynthesis in Pseudomonas fluorescens Pf0-1 leads to reduced biofilm formation and a reduction in cell size of surface-attached but not planktonic cells

2015 ◽  
Author(s):  
Shiro Yoshioka ◽  
Peter D Newell
Keyword(s):  
Pseudomonas Fluorescens ◽  
Cell Size ◽  
Biofilm Formation ◽  
De Novo ◽  
Purine Biosynthesis ◽  
Model Organisms ◽  
Purine Nucleotides ◽  
Surface Attachment ◽  
Nucleotide Biosynthesis ◽  
Mutant Cells

Pseudomonas fluorescens Pf0-1 is one of the model organisms for biofilm research. Our previous transposon mutagenesis study suggested a requirement for the de novo purine nucleotide biosynthesis pathway for biofilm formation by this organism. This study was performed to verify that observation and investigate the basis for the defects in biofilm formation shown by purine biosynthesis mutants. Constructing deletion mutations in 8 genes in this pathway, we found that they all showed reductions in biofilm formation that could be partly or completely restored by nucleotide supplementation or genetic complementation. We demonstrated that, despite a reduction in biofilm formation, more viable mutant cells were recovered from the surface-attached population than from the planktonic phase under conditions of purine deprivation. Analyses using scanning electron microscopy revealed that the surface-attached mutant cells were 25~30% shorter in length than WT, which partly explains the reduced biomass in the mutant biofilms. The laser diffraction particle analyses confirmed this finding, and further indicated that the WT biofilm cells were smaller than their planktonic counterparts. The defects in biofilm formation and reductions in cell size shown by the mutants were fully recovered upon adenine or hypoxanthine supplementation, indicating that the purine shortages caused reductions in cell size. Our results are consistent with surface attachment serving as a survival strategy during nutrient deprivation, and indicate that changes in the cell size may be a natural response of P. fluorescens to growth on a surface. Finally, cell sizes in WT biofilms became slightly smaller in the presence of exogenous adenine than in its absence. Our findings suggest that purine nucleotides or related metabolites may influence the regulation of cell size in this bacterium.

scholarly journals Escherichia coliO157:H7 responds to phosphate starvation by modifying LPS involved in biofilm formation

2019 ◽  
Author(s):  
Philippe Vogeleer ◽  
Antony T. Vincent ◽  
Samuel M. Chekabab ◽  
Steve J. Charette ◽  
Alexey Novikov ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Inorganic Phosphate ◽  
Type Strain ◽  
Wild Type Strain ◽  
Pho Regulon ◽  
Wild Type ◽  
E Coli ◽  
Pi Starvation ◽  
Pst System

ABSTRACTIn open environments such as water, enterohemorrhagicEscherichia coliO157:H7 responds to inorganic phosphate (Pi) starvation by inducing the Pho regulon controlled by PhoB. The phosphate-specific transport (Pst) system is the high-affinity Pi transporter. In the Δpstmutant, PhoB is constitutively activated and regulates the expression of genes from the Pho regulon. InE. coliO157:H7, the Δpstmutant, biofilm, and autoagglutination were increased. In the double-deletion mutant ΔpstΔphoB, biofilm and autoagglutination were similar to the wild-type strain, suggesting that PhoB is involved. We investigated the relationship between PhoB activation and enhanced biofilm formation by screening a transposon mutant library derived from Δpstmutant for decreased autoagglutination and biofilms mutants. Lipopolysaccharide (LPS) genes involved in the synthesis of the LPS core were identified. Transcriptomic studies indicate the influence of Pi-starvation andpstmutation on LPS biosynthetic gene expression. LPS analysis indicated that the O-antigen was deficient in the Δpstmutant. Interestingly,waaH, encoding a glycosyltransferase associated with LPS modifications inE. coliK-12, was highly expressed in the Δpstmutant ofE. coliO157:H7. Deletion ofwaaHfrom the Δpstmutant and from the wild-type strain grown in Pi-starvation conditions decreased the biofilm formation but without affecting LPS. Our findings suggest that LPS core is involved in the autoagglutination and biofilm phenotypes of the Δpstmutant and that WaaH plays a role in biofilm in response to Pi-starvation. This study highlights the importance of Pi-starvation in biofilm formation of E. coli O157:H7, which may affect its transmission and persistence.IMPORTANCEEnterohemorrhagicEscherichia coliO157:H7 is a human pathogen responsible for bloody diarrhea and renal failures. In the environment, O157:H7 can survive for prolonged periods of time under nutrient-deprived conditions. Biofilms are thought to participate in this environmental lifestyle. Previous reports have shown that the availability of extracellular inorganic phosphate (Pi) affected bacterial biofilm formation; however, nothing was known about O157:H7 biofilm formation. Our results show that O157:H7 membrane undergoes modifications upon PhoB activation leading to increased biofilm formation. A mutation in the Pst system results in reduced amount of the smooth type LPS and that this could influence the biofilm composition. This demonstrates how theE. coliO157:H7 adapts to Pi starvation increasing its ability to occupy different ecological niches.

Cellular mechanisms in proximal tubular reabsorption of inorganic phosphate

1991 ◽  
Vol 260 (5) ◽  
pp. C885-C899 ◽  
Author(s):  
H. Murer ◽  
A. Werner ◽  
S. Reshkin ◽  
F. Wuarin ◽  
J. Biber
Keyword(s):  
Inorganic Phosphate ◽  
De Novo ◽  
Basolateral Membrane ◽  
Transport Systems ◽  
Xenopus Laevis Oocytes ◽  
Cellular Mechanisms ◽  
Regulatory Cascade ◽  
Igf I ◽  
Dependent Inhibition ◽  
Pi Deprivation

Filtered inorganic phosphate (Pi) is largely reabsorbed in the proximal tubule. Na-Pi cotransport, with a stoichiometry of at least 2:1, mediates uphill transport at the apical membrane; at the basolateral membrane different types of transport systems can be involved in efflux and uptake of Pi from the interstitium. Regulation of transcellular Pi flux involves alteration of the apical Na-Pi cotransport; at least three different cellular control/sensing systems seem to participate in this regulation and are exemplified by parathyroid hormone (PTH)-dependent inhibition, Pi deprivation-dependent increase, and insulin-like growth factor I (IGF-I)-dependent increase in Na-Pi cotransport. For PTH inhibition, recent evidence suggests a role of the phospholipase C/protein kinase C-dependent regulatory cascade in inhibition of Na-Pi cotransport, at least at low PTH concentrations. In addition, an endocytic mechanism seems to be involved in this PTH action. Little is known of the cellular mechanisms in Pi deprivation-dependent and/or IGF-I-dependent increases in Na-Pi cotransport; they are dependent on de novo protein synthesis. Recent experiments involving an expression in Xenopus laevis oocytes led to the identification of an approximately 50 kDa membrane protein that is a good candidate for being involved in brush-border membrane Na-Pi cotransport activity.

scholarly journals Disruption of de novo purine biosynthesis in Pseudomonas fluorescens Pf0-1 leads to reduced biofilm formation and a reduction in cell size of surface-attached but not planktonic cells

2015 ◽  
Author(s):  
Shiro Yoshioka ◽  
Peter D Newell
Keyword(s):  
Pseudomonas Fluorescens ◽  
Cell Size ◽  
Biofilm Formation ◽  
De Novo ◽  
Purine Biosynthesis ◽  
Model Organisms ◽  
Purine Nucleotides ◽  
Surface Attachment ◽  
Nucleotide Biosynthesis ◽  
Mutant Cells

Pseudomonas fluorescens Pf0-1 is one of the model organisms for biofilm research. Our previous transposon mutagenesis study suggested a requirement for the de novo purine nucleotide biosynthesis pathway for biofilm formation by this organism. This study was performed to verify that observation and investigate the basis for the defects in biofilm formation shown by purine biosynthesis mutants. Constructing deletion mutations in 8 genes in this pathway, we found that they all showed reductions in biofilm formation that could be partly or completely restored by nucleotide supplementation or genetic complementation. We demonstrated that, despite a reduction in biofilm formation, more viable mutant cells were recovered from the surface-attached population than from the planktonic phase under conditions of purine deprivation. Analyses using scanning electron microscopy revealed that the surface-attached mutant cells were 25~30% shorter in length than WT, which partly explains the reduced biomass in the mutant biofilms. The laser diffraction particle analyses confirmed this finding, and further indicated that the WT biofilm cells were smaller than their planktonic counterparts. The defects in biofilm formation and reductions in cell size shown by the mutants were fully recovered upon adenine or hypoxanthine supplementation, indicating that the purine shortages caused reductions in cell size. Our results are consistent with surface attachment serving as a survival strategy during nutrient deprivation, and indicate that changes in the cell size may be a natural response of P. fluorescens to growth on a surface. Finally, cell sizes in WT biofilms became slightly smaller in the presence of exogenous adenine than in its absence. Our findings suggest that purine nucleotides or related metabolites may influence the regulation of cell size in this bacterium.

scholarly journals Disruption of de novo purine biosynthesis in Pseudomonas fluorescens Pf0-1 leads to reduced biofilm formation and a reduction in cell size of surface-attached but not planktonic cells

2015 ◽  
Author(s):  
Shiro Yoshioka ◽  
Peter D Newell
Keyword(s):  
Pseudomonas Fluorescens ◽  
Cell Size ◽  
Biofilm Formation ◽  
De Novo ◽  
Purine Biosynthesis ◽  
Model Organisms ◽  
Purine Nucleotides ◽  
Surface Attachment ◽  
Nucleotide Biosynthesis ◽  
Mutant Cells

Pseudomonas fluorescens Pf0-1 is one of the model organisms for biofilm research. Our previous transposon mutagenesis study suggested a requirement for the de novo purine nucleotide biosynthesis pathway for biofilm formation by this organism. This study was performed to verify that observation and investigate the basis for the defects in biofilm formation shown by purine biosynthesis mutants. Constructing deletion mutations in 8 genes in this pathway, we found that they all showed reductions in biofilm formation that could be partly or completely restored by nucleotide supplementation or genetic complementation. We demonstrated that, despite a reduction in biofilm formation, more viable mutant cells were recovered from the surface-attached population than from the planktonic phase under conditions of purine deprivation. Analyses using scanning electron microscopy revealed that the surface-attached mutant cells were 25~30% shorter in length than WT, which partly explains the reduced biomass in the mutant biofilms. The laser diffraction particle analyses confirmed this finding, and further indicated that the WT biofilm cells were smaller than their planktonic counterparts. The defects in biofilm formation and reductions in cell size shown by the mutants were fully recovered upon adenine or hypoxanthine supplementation, indicating that the purine shortages caused reductions in cell size. Our results are consistent with surface attachment serving as a survival strategy during nutrient deprivation, and indicate that changes in the cell size may be a natural response of P. fluorescens to growth on a surface. Finally, cell sizes in WT biofilms became slightly smaller in the presence of exogenous adenine than in its absence. Our findings suggest that purine nucleotides or related metabolites may influence the regulation of cell size in this bacterium.

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