Biogeochemical niche of phosphorus sequestrating magnetotactic bacteria in Lake Pavin, a freshwater feruginous environment.

<p>Phosphorus (P) is essential to life but a limiting nutrient in many ecosystems. Understanding the role of microorganisms in P cycling, especially the processes of P uptake and storage, is a major environmental issue.  Only few models are known to highly sequestrate phosphorus and mostly in marine environments. We thus need to improve our knowledge about other model of sequestration and especially in freshwater environments.</p><p>Freshwater magnetotactic bacteria (MTB) affiliated to the Magnetococcaceae family have been identified within the water column of Lake Pavin in France [3]. Similarly, to the marine Thiomarguarita and Beggiatoa [1, 2], they accumulate intracellular polyphosphates (PolyP) to a uniquely high extent, up to 90% of their cell volume. In contradiction with the marine Thiomarguarita and Beggiatoa, the Magnetococcaceae accumulate PolyP in anoxic conditions. They represent the major population of MTB and are located right under the oxic-anoxic interface in a zone of strong chemical and redox gradients. These gradients allow the study of the impact of varying chemical conditions on microbial physiology.</p><p>We aim at characterizing Magnetococcaceae distribution as a function of depth and therefore of different chemical parameters, but also at determining the drivers of PolyP accumulation. </p><p>Here, we combine a variety of methods to analyse these MTB and their potential appartenance to a specific ecological niche in the water column. We measured the physico-chemical parameters of the water column (O<sub>2</sub>, pH, redox, conductivity, FDOM, turbidity, etc.). We used a new sampling system that allowed us to reach a better spatial resolution [4], from 1 m to 20 cm. We were therefore able to better estimate the impact of the chemical parameters on the MTB. We then sampled the water to measure the geochemical parameters using ICP-OES and to characterize MTB via optical and electronic microscopy. Optical microscopy helped identify the main populations of MTB and their concentrations, while electronic microscopy permitted the characterization of the different magnetosome organisation and PolyP accumulation capacities. Multivariate statistics were finally performed on all data.</p><p>Multivariate statistics identified several parameters positively and significantly correlated to the Magnetococcaceae. These parameters are different from the ones correlated to other MTB of the water column. We therefore show that the Magnetococcaceae live into a specific niche with specific biogeochemical parameters. These correlated parameters include dissolved lithium concentration, mass percentage of nitrogen, magnesium and particulate P. Phosporus  and magnesium are linked to the formation of PolyP, lithium represent a cofactor for phosphate transport [5] and nitrogen might be linked to nitrate transportation by the MTB [6].</p><p>Genomic analyses will be done in the future to allow further comprehension on molecular mecanisms and PolyP formation.</p><p> </p><p>[1] Brock J, Schulz-Vogt HN. (2011) ISME Journal <strong>5</strong>, 497-506. [2] Mubmann M et al. (2007) PLoS Biology <strong>5</strong>(9), e230. [3] Rivas-Lamelo S et al. (2017) Geochem. Persp. Let. <strong>5</strong>, 35–41. [4] Busigny et al., submitted to Environmental Microbiology. [5] Jakobsson E et al. (2017) J. Membr. Biol. <strong>250</strong>,587-604. [6] Li et al. (2020) Geophys. Res. Biogeosciences.</p>

Inorganic Polyphosphate Accumulation inEscherichia coliIs Regulated by DksA but Not by (p)ppGpp

ABSTRACTProduction of inorganic polyphosphate (polyP) by bacteria is triggered by a variety of different stress conditions. polyP is required for stress survival and virulence in diverse pathogenic microbes. Previous studies have hypothesized a model for regulation of polyP synthesis in which production of the stringent-response second messenger (p)ppGpp directly stimulates polyP accumulation. In this work, I have now shown that this model is incorrect, and (p)ppGpp is not required for polyP synthesis inEscherichia coli. However, stringent mutations of RNA polymerase that frequently arise spontaneously in strains defective in (p)ppGpp synthesis and null mutations of the stringent-response-associated transcription factor DksA both strongly inhibit polyP accumulation. The loss of polyP synthesis in a mutant lacking DksA was reversed by deletion of the transcription elongation factor GreA, suggesting that competition between these proteins for binding to the secondary channel of RNA polymerase plays an important role in controlling polyP activation. These results provide new insights into the poorly understood regulation of polyP synthesis in bacteria and indicate that the relationship between polyP and the stringent response is more complex than previously suspected.IMPORTANCEProduction of polyP in bacteria is required for virulence and stress response, but little is known about how bacteria regulate polyP levels in response to changes in their environments. Understanding this regulation is important for understanding how pathogenic microbes resist killing by disinfectants, antibiotics, and the immune system. In this work, I have clarified the connections between polyP regulation and the stringent response to starvation stress inEscherichia coliand demonstrated an important and previously unknown role for the transcription factor DksA in controlling polyP levels.

Mutations inEscherichia coliPolyphosphate Kinase That Lead to Dramatically IncreasedIn VivoPolyphosphate Levels

ABSTRACTBacteria synthesize inorganic polyphosphate (polyP) in response to a wide variety of stresses, and production of polyP is essential for stress response and survival in many important pathogens and bacteria used in biotechnological processes. However, surprisingly little is known about the molecular mechanisms that control polyP synthesis. We have therefore developed a novel genetic screen that specifically links growth ofEscherichia colito polyP synthesis, allowing us to isolate mutations leading to enhanced polyP production. Using this system, we have identified mutations in the polyP-synthesizing enzyme polyP kinase (PPK) that lead to dramatic increases inin vivopolyP synthesis but do not substantially affect the rate of polyP synthesis by PPKin vitro. These mutations are distant from the PPK active site and found in interfaces between monomers of the PPK tetramer. We have also shown that high levels of polyP lead to intracellular magnesium starvation. Our results provide new insights into the control of bacterial polyP accumulation and suggest a simple, novel strategy for engineering bacteria with increased polyP contents.IMPORTANCEPolyP is an ancient, universally conserved biomolecule and is important for stress response, energy metabolism, and virulence in a remarkably broad range of microorganisms. PolyP accumulation by bacteria is also important in biotechnology applications. For example, it is critical to enhanced biological phosphate removal (EBPR) from wastewater. Understanding how bacteria control polyP synthesis is therefore of broad importance in both the fields of bacterial pathogenesis and biological engineering. UsingEscherichia colias a model organism, we have identified the first known mutations in polyP kinase that lead to increases in cellular polyP content.

phoUInactivation in Pseudomonas aeruginosa Enhances Accumulation of ppGpp and Polyphosphate

ABSTRACTInorganic polyphosphate (polyP) is a linear polymer composed of several molecules of orthophosphate (Pi) linked by energy-rich phosphoanhydride bonds. InPseudomonas aeruginosa, Piis taken up by the ABC transporter Pst, encoded by an operon consisting of five genes. The first four genes encode proteins involved in the transport of Piand the last gene of the operon,phoU, codes for a protein which exact function is unknown. We show here that the inactivation ofphoUinP. aeruginosaenhanced Piremoval from the medium and polyP accumulation. ThephoUmutant also accumulated high levels of the alarmone guanosine tetraphosphate (ppGpp), which in turn increased the buildup of polyP. In addition,phoUinactivation had several pleiotropic effects, such as reduced growth rate and yield and increased sensitivity to antibiotics and stresses. However, biofilm formation was not affected by thephoUmutation.

Accumulation of Inorganic Polyphosphate in phoU Mutants of Escherichia coli and Synechocystis sp. Strain PCC6803

ABSTRACT The biological process for phosphate (Pi) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the Pi regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more Pi from the medium than the wild-type strain removed.

Inorganic Polyphosphate in Escherichia coli: the Phosphate Regulon and the Stringent Response

ABSTRACT Escherichia coli transiently accumulates large amounts of inorganic polyphosphate (polyP), up to 20 mM in phosphate residues (Pi), in media deficient in both Pi and amino acids. This transient accumulation is preceded by the appearance of nucleotides ppGpp and pppGpp, generated in response to nutritional stresses. Mutants which lack PhoB, the response regulator of the phosphate regulon, do not accumulate polyP even though they develop wild-type levels of (p)ppGpp when subjected to amino acid starvation. When complemented with a phoB-containing plasmid,phoB mutants regain the ability to accumulate polyP. PolyP accumulation requires high levels of (p)ppGpp independent of whether they are generated by RelA (active during the stringent response) or SpoT (expressed during Pi starvation). Hence, accumulation of polyP requires a functional phoB gene and elevated levels of (p)ppGpp. A rapid assay of polyP depends on its adsorption to an anion-exchange disk on which it is hydrolyzed by a yeast exopolyphosphatase.

Novel Assay Reveals Multiple Pathways Regulating Stress-Induced Accumulations of Inorganic Polyphosphate in Escherichia coli

ABSTRACT A major impediment to understanding the biological roles of inorganic polyphosphate (polyP) has been the lack of sensitive definitive methods to extract and quantitate cellular polyP. We show that polyP recovered in extracts from cells lysed with guanidinium isothiocynate can be bound to silicate glass and quantitatively measured by a two-enzyme assay: polyP is first converted to ATP by polyP kinase, and the ATP is hydrolyzed by luciferase to generate light. This nonradioactive method can detect picomolar amounts of phosphate residues in polyP per milligram of extracted protein. A simplified procedure for preparing polyP synthesized by polyP kinase is also described. Using the new assay, we found that bacteria subjected to nutritional or osmotic stress in a rich medium or to nitrogen exhaustion had large and dynamic accumulations of polyP. By contrast, carbon exhaustion, changes in pH, temperature upshifts, and oxidative stress had no effect on polyP levels. Analysis of Escherichia coli mutants revealed that polyP accumulation depends on several regulatory genes, glnD (NtrC), rpoS,relA, and phoB.