Genetic and biochemical properties of an alkaline phosphatase PhoX family protein found in many bacteria

Maintenance of cellular phosphate homeostasis is essential for cellular life. The PhoU protein has emerged as a key regulator of this process in bacteria, and it is suggested to modulate phosphate import by PstSCAB and control activation of the phosphate limitation response by the PhoR-PhoB two-component system. However, a proper understanding of PhoU has remained elusive due to numerous complications of mutating phoU, including loss of viability and the genetic instability of the mutants. Here, we developed two sets of strains of Sinorhizobium meliloti that overcame these limitations and allowed a more detailed and comprehensive analysis of the biological and molecular activities of PhoU. The data showed that phoU cannot be deleted in the presence of phosphate unless PstSCAB is inactivated also. However, phoU deletions were readily recovered in phosphatefree media, and characterization of these mutants revealed that addition of phosphate to the environment resulted in toxic levels of PstSCAB-mediated phosphate accumulation. Phosphate uptake experiments indicated that PhoU significantly decreased the PstSCAB transport rate specifically in phosphate-replete cells but not in phosphate-starved cells and that PhoU could rapidly respond to elevated environmental phosphate concentrations and decrease the PstSCAB transport rate. Sitedirected mutagenesis results suggested that the ability of PhoU to respond to phosphate levels was independent of the conformation of the PstSCAB transporter. Additionally, PhoU-PhoU and PhoU-PhoR interactions were detected using a bacterial two-hybrid screen. We propose that PhoU modulates PstSCAB and PhoR-PhoB in response to local, internal fluctuations in phosphate concentrations resulting from PstSCAB-mediated phosphate import.IMPORTANCE Correct maintenance of cellular phosphate homeostasis is critical in all kingdoms of life and in bacteria involves the PhoU protein. This work provides novel insights into the role of the Sinorhizobium meliloti PhoU protein, which plays a key role in rapid adaptation to elevated phosphate concentrations. It is shown that PhoU rapidly responds to elevated phosphate levels by significantly decreasing the phosphate transport of PstSCAB, thereby preventing phosphate toxicity and cell death. Additionally, a new model for phosphate sensing in bacterial species which involves the PhoR-PhoB two-component system is presented. This work provides new insights into the bacterial response to changing environmental conditions and into regulation of the phosphate limitation response that influences numerous bacterial processes, including antibiotic production and virulence.KEYWORDS Pho regulon, PhoU, PstSCAB, Sinorhizobium meliloti, environmental adaptation, modulation of TCS, phosphate homeostasis, phosphate sensing, phosphate transport, transport modulation

show abstract

“…The PhoX alkaline phosphatase is strongly induced in wild-type S. meliloti during P i starvation in a PhoR-PhoB-dependent manner (38) (Fig. 4C).…”

Section: Resultsmentioning

confidence: 99%

PhoU Allows Rapid Adaptation to High Phosphate Concentrations by Modulating PstSCAB Transport Rate in Sinorhizobium meliloti

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…The high working efficacy and stability of a monomeric CmAP can be attributed to the intrinsically tight binding of both catalytic and structural metal ions. Strong metal-enzyme interactions in the AP active site have been reported (Zappa et al 2001;Zaheer et al 2009). Estimates of binding strengths and specificities of metal ions using the CmAP 3D structure model and atomic absorption analysis confirmed two zinc and one magnesium ions and their ligands in the active site (Figs.…”

Section: Discussionmentioning

confidence: 94%

Recombinant Production and Characterization of a Highly Active Alkaline Phosphatase from Marine Bacterium Cobetia marina

et al. 2014

View full text Add to dashboard Cite

The psychrophilic marine bacterium, Cobetia marina, recovered from the mantle tissue of the marine mussel, Crenomytilus grayanus, which contained a gene encoding alkaline phosphatase (AP) with apparent biotechnology advantages. The enzyme was found to be more efficient than its counterparts and showed k cat value 10-to 100-fold higher than those of all known commercial APs. The enzyme did not require the presence of exogenous divalent cations and dimeric state of its molecule for activity. The recombinant enzyme (CmAP) production and purification were optimized with a final recovery of 2 mg of the homogenous protein from 1 L of the transgenic Escherichia coli Rosetta(DE3)/Pho40 cells culture. CmAP displayed a half-life of 16 min at 45°C and 27 min at 40°C in the presence of 2 mM EDTA, thus suggesting its relative thermostability in comparison with the known cold-adapted analogues. A high concentration of EDTA in the incubation mixture did not appreciably inhibit CmAP. The enzyme was stable in a wide range of pH (6.0-11.0). CmAP exhibited its highest activity at the reaction temperature of 40-50°C and pH 9.5-10.3. The structural features of CmAP could be the reason for the increase in its stability and catalytic turnover. We have modeled the CmAP 3D structure on the base of the high-quality experimental structure of the close homologue Vibrio sp. AP (VAP) and mutated essential residues predicted to break Mg 2+ bonds in CmAP. It seems probable that the intrinsically tight binding of catalytic and structural metal ions together with the flexibility of intermolecular and intramolecular links in CmAP could be attributed to the adapted mutualistic lifestyle in oceanic waters.

show abstract

“…2, lanes 1-3, 5, 6). SMc02634 was recently shown to be a PhoX alkaline phosphatase (19), which is found in many bacteria but is distinct from the well-characterized PhoA family. Mutant PHO01907, deficient in smc01907, also degraded zwitterionic phospholipids and formed DGTS (Fig.…”

Section: Smc00171-deficient Mutant Of S Meliloti Is Unable To Degradementioning

confidence: 99%

Sinorhizobium meliloti phospholipase C required for lipid remodeling during phosphorus limitation

Zavaleta-Pastor

Sohlenkamp

Gao

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Rhizobia are Gram-negative soil bacteria able to establish nitrogenfixing root nodules with their respective legume host plants. Besides phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine, rhizobial membranes contain phosphatidylcholine (PC) as a major membrane lipid. Under phosphate-limiting conditions of growth, some bacteria replace their membrane phospholipids with lipids lacking phosphorus. In Sinorhizobium meliloti, these phosphorus-free lipids are sulfoquinovosyl diacylglycerol, ornithinecontaining lipid, and diacylglyceryl trimethylhomoserine (DGTS). Pulse-chase experiments suggest that the zwitterionic phospholipids phosphatidylethanolamine and PC act as biosynthetic precursors of DGTS under phosphorus-limiting conditions. A S. meliloti mutant, deficient in the predicted phosphatase SMc00171 was unable to degrade PC or to form DGTS in a similar way as the wild type. Cell-free extracts of Escherichia coli, in which SMc00171 had been expressed, convert PC to phosphocholine and diacylglycerol, showing that SMc00171 functions as a phospholipase C. Diacylglycerol , in turn, is the lipid anchor from which biosynthesis is initiated during the formation of the phosphorus-free membrane lipid DGTS. Inorganic phosphate can be liberated from phosphocholine. These data suggest that, in S. meliloti under phosphate-limiting conditions, membrane phospholipids provide a pool for metabolizable inorganic phosphate, which can be used for the synthesis of other essential phosphorus-containing biomolecules. This is an example of an intracellular phospholipase C in a bacterial system; however, the ability to degrade endogenous preexisting membrane phospholipids as a source of phosphorus may be a general property of Gram-negative soil bacteria.nimal cells have access to relatively abundant sources of phosphorus for the formation of biomolecules such as membrane phospholipids and nucleic acids. The characteristic lipid composition for a particular animal cell membrane is thought to result from a steady state between formation and turnover of the lipids. In contrast, plants and many environmental microbes often live in environments where available phosphorus is a growth-limiting factor. The strategies employed by organisms to deal with phosphorus limitation include: (i) increased solubilization of phosphorus-containing compounds; (ii) more efficient uptake into cells; and (iii) less phosphorus use when synthesizing their biomolecules (1). The replacement of phospholipids by galacto-and sulfolipids in plant membranes constitutes an important adaptive process for growth on phosphate-limited soils. In Arabidopsis thaliana, several phospholipases D and C (2-5) are induced under phosphate-limiting conditions, and they degrade membrane phospholipids to phosphatidic acid or diacylglycerol (DAG), respectively. DAG then serves as the initial substrate for the formation of galacto-and sulfolipids, which lack phosphorus.In some bacteria, the membrane phospholipids are partially replaced during phosphate limitation by phosphoru...

show abstract

Genetic and biochemical properties of an alkaline phosphatase PhoX family protein found in many bacteria

Cited by 60 publications

References 70 publications

PhoU Allows Rapid Adaptation to High Phosphate Concentrations by Modulating PstSCAB Transport Rate in Sinorhizobium meliloti

PhoU Allows Rapid Adaptation to High Phosphate Concentrations by Modulating PstSCAB Transport Rate in Sinorhizobium meliloti

Recombinant Production and Characterization of a Highly Active Alkaline Phosphatase from Marine Bacterium Cobetia marina

Sinorhizobium meliloti phospholipase C required for lipid remodeling during phosphorus limitation

Contact Info

Product

Resources

About