Trade-offs of lipid remodeling in a marine predator-prey interaction in response to phosphorus limitation.

Phosphorus (P) is a key nutrient limiting bacterial growth and primary production in the oceans. Unsurprisingly, marine microbes have evolved sophisticated strategies to adapt to P limitation, one of which involves the remodeling of membrane lipids by replacing phospholipids with non-P-containing surrogate lipids. This strategy is adopted by both cosmopolitan marine phytoplankton and heterotrophic bacteria and serves to reduce the cellular P quota. However, little, if anything, is known of the biological consequences of lipid remodeling. Here, using the marine bacterium Phaeobacter sp. MED193 and the ciliate Uronema marinum as a model, we sought to assess the effect of remodeling on bacteria-protist interactions. We discovered an important trade-off between either escape from ingestion or resistance to digestion. Thus, Phaeobacter grown under P-replete conditions was readily ingested by Uronema, but not easily digested, supporting only limited predator growth. In contrast, following membrane lipid remodeling in response to P depletion, Phaeobacter was less likely to be captured by Uronema, thanks to the reduced expression of mannosylated glycoconjugates. However, once ingested, membrane-remodeled cells were unable to prevent phagosome acidification, became more susceptible to digestion, and, as such, allowed rapid growth of the ciliate predator. This trade-off between adapting to a P-limited environment and susceptibility to protist grazing suggests the more efficient removal of low-P prey that potentially has important implications for the functioning of the marine microbial food web in terms of trophic energy transfer and nutrient export efficiency.

2-Aminoethylphosphonate utilization in Pseudomonas putida BIRD-1 is controlled by multiple master regulators.

Bacteria possess various regulatory mechanisms to detect and coordinate a response to elemental nutrient limitation. In pseudomonads, the two-component system regulators CbrAB, NtrBC and PhoBR, are responsible for regulating cellular response to carbon (C), nitrogen (N) and phosphorus (P) respectively. Phosphonates are reduced organophosphorus compounds produced by a broad range of biota and typified by a direct C-P bond. Numerous pseudomonads can use the environmentally abundant phosphonate species 2-aminoethylphosphonate (2AEP) as a source of C, N, or P, but only PhoBR has been shown to play a role in 2AEP utilization. On the other hand, utilization of 2AEP as a C and N source is considered substrate inducible. Here, using the plant-growth-promoting rhizobacterium Pseudomonas putida BIRD-1 we present evidence that 2AEP utilization is under dual regulation and only occurs upon depletion of C, N, or P, controlled by CbrAB, NtrBC, or PhoBR respectively. However, the presence of 2AEP was necessary for full gene expression, i.e. expression was substrate inducible. Mutation of a LysR-type regulator, termed AepR, upstream of the 2AEP transaminase-phosphonatase system (PhnWX), confirmed this dual regulatory mechanism. To our knowledge, this is the first study identifying coordination between global stress response and substrate-specific regulators in phosphonate metabolism.

A widely distributed phosphate-insensitive phosphatase presents a route for rapid organophosphorus remineralization in the biosphere.

The regeneration of bioavailable phosphate from immobilized organophosphorus represents a key process in the global phosphorus cycle and is facilitated by enzymes known as phosphatases. Most bacteria possess at least one of three phosphatases with broad substrate specificity, known as PhoA, PhoX, and PhoD, whose activity is optimal under alkaline conditions. The production and activity of these phosphatases is repressed by phosphate availability. Therefore, they are only fully functional when bacteria experience phosphorus-limiting growth conditions. Here, we reveal a previously overlooked phosphate-insensitive phosphatase, PafA, prevalent in Bacteroidetes, which is highly abundant in nature and represents a major route for the regeneration of environmental phosphate. Using the enzyme from Flavobacterium johnsoniae, we show that PafA is highly active toward phosphomonoesters, is fully functional in the presence of excess phosphate, and is essential for growth on phosphorylated carbohydrates as a sole carbon source. These distinct properties of PafA may expand the metabolic niche of Bacteroidetes by enabling the utilization of abundant organophosphorus substrates as C and P sources, providing a competitive advantage when inhabiting zones of high microbial activity and nutrient demand. PafA, which is constitutively synthesized by soil and marine flavobacteria, rapidly remineralizes phosphomonoesters releasing bioavailable phosphate that can be acquired by neighboring cells. The pafA gene is highly diverse in plant rhizospheres and is abundant in the global ocean, where it is expressed independently of phosphate availability. PafA therefore represents an important enzyme in the context of global biogeochemical cycling and has potential applications in sustainable agriculture.

Transporter characterisation reveals aminoethylphosphonate mineralisation as a key step in the marine phosphorus redox cycle.

The planktonic synthesis of reduced organophosphorus molecules, such as alkylphosphonates and aminophosphonates, represents one half of a vast global oceanic phosphorus redox cycle. Whilst alkylphosphonates tend to accumulate in recalcitrant dissolved organic matter, aminophosphonates do not. Here, we identify three bacterial 2-aminoethylphosphonate (2AEP) transporters, named AepXVW, AepP and AepSTU, whose synthesis is independent of phosphate concentrations (phosphate-insensitive). AepXVW is found in diverse marine heterotrophs and is ubiquitously distributed in mesopelagic and epipelagic waters. Unlike the archetypal phosphonate binding protein, PhnD, AepX has high affinity and high specificity for 2AEP (Stappia stellulata AepX Kd 23 ± 4 nM; methylphosphonate Kd 3.4 ± 0.3 mM). In the global ocean, aepX is heavily transcribed (~100-fold>phnD) independently of phosphate and nitrogen concentrations. Collectively, our data identifies a mechanism responsible for a major oxidation process in the marine phosphorus redox cycle and suggests 2AEP may be an important source of regenerated phosphate and ammonium, which are required for oceanic primary production.

Niche-adaptation in plant-associated Bacteroidetes favours specialisation in organic phosphorus mineralisation.

Bacteroidetes are abundant pathogen-suppressing members of the plant microbiome that contribute prominently to rhizosphere phosphorus mobilisation, a frequent growth-limiting nutrient in this niche. However, the genetic traits underpinning their success in this niche remain largely unknown, particularly regarding their phosphorus acquisition strategies. By combining cultivation, multi-layered omics and biochemical analyses we first discovered that all plant-associated Bacteroidetes express constitutive phosphatase activity, linked to the ubiquitous possession of a unique phosphatase, PafA. For the first time, we also reveal a subset of Bacteroidetes outer membrane SusCD-like complexes, typically associated with carbon acquisition, and several TonB-dependent transporters, are induced during Pi-depletion. Furthermore, in response to phosphate depletion, the plant-associated Flavobacterium used in this study expressed many previously characterised and novel proteins targeting organic phosphorus. Collectively, these enzymes exhibited superior phosphatase activity compared to plant-associated Pseudomonas spp. Importantly, several of the novel low-Pi-inducible phosphatases and transporters, belong to the Bacteroidetes auxiliary genome and are an adaptive genomic signature of plant-associated strains. In conclusion, niche adaptation to the plant microbiome thus appears to have resulted in the acquisition of unique phosphorus scavenging loci in Bacteroidetes, enhancing their phosphorus acquisition capabilities. These traits may enable their success in the rhizosphere and also present exciting avenues to develop sustainable agriculture.

Identification of extracellular glycerophosphodiesterases in Pseudomonas and their role in soil organic phosphorus remineralisation.

In soils, phosphorus (P) exists in numerous organic and inorganic forms. However, plants can only acquire inorganic orthophosphate (Pi), meaning global crop production is frequently limited by P availability. To overcome this problem, rock phosphate fertilisers are heavily applied, often with negative environmental and socio-economic consequences. The organic P fraction of soil contains phospholipids that are rapidly degraded resulting in the release of bioavailable Pi. However, the mechanisms behind this process remain unknown. We identified and experimentally confirmed the function of two secreted glycerolphosphodiesterases, GlpQI and GlpQII, found in Pseudomonas stutzeri DSM4166 and Pseudomonas fluorescens SBW25, respectively. A series of co-cultivation experiments revealed that in these Pseudomonas strains, cleavage of glycerolphosphorylcholine and its breakdown product G3P occurs extracellularly allowing other bacteria to benefit from this metabolism. Analyses of metagenomic and metatranscriptomic datasets revealed that this trait is widespread among soil bacteria with Actinobacteria and Proteobacteria, specifically Betaproteobacteria and Gammaproteobacteria, the likely major players.

The 'known' genetic potential for microbial communities to degrade organic phosphorus is reduced in low-pH soils.

In soil, bioavailable inorganic orthophosphate is found at low concentrations and thus limits biological growth. To overcome this phosphorus scarcity, plants and bacteria secrete numerous enzymes, namely acid and alkaline phosphatases, which cleave orthophosphate from various organic phosphorus substrates. Using profile hidden Markov modeling approaches, we investigated the abundance of various non specific phosphatases, both acid and alkaline, in metagenomes retrieved from soils with contrasting pH regimes. This analysis uncovered a marked reduction in the abundance and diversity of various alkaline phosphatases in low-pH soils that was not counterbalanced by an increase in acid phosphatases. Furthermore, it was also discovered that only half of the bacterial strains from different phyla deposited in the Integrated Microbial Genomes database harbor alkaline phosphatases. Taken together, our data suggests that these 'phosphatase lacking' isolates likely increase in low-pH soils and future research should ascertain how these bacteria overcome phosphorus scarcity.

Comparative genomic, proteomic and exoproteomic analyses of three Pseudomonas strains reveals novel insights into the phosphorus scavenging capabilities of soil bacteria.

Bacteria that inhabit the rhizosphere of agricultural crops can have a beneficial effect on crop growth. One such mechanism is the microbial-driven solubilization and remineralization of complex forms of phosphorus (P). It is known that bacteria secrete various phosphatases in response to low P conditions. However, our understanding of their global proteomic response to P stress is limited. Here, exoproteomic analysis of Pseudomonas putida BIRD-1 (BIRD-1), Pseudomonas fluorescens SBW25 and Pseudomonas stutzeri DSM4166 was performed in unison with whole-cell proteomic analysis of BIRD-1 grown under phosphate (Pi) replete and Pi deplete conditions. Comparative exoproteomics revealed marked heterogeneity in the exoproteomes of each Pseudomonas strain in response to Pi depletion. In addition to well-characterized members of the PHO regulon such as alkaline phosphatases, several proteins, previously not associated with the response to Pi depletion, were also identified. These included putative nucleases, phosphotriesterases, putative phosphonate transporters and outer membrane proteins. Moreover, in BIRD-1, mutagenesis of the master regulator, phoBR, led us to confirm the addition of several novel PHO-dependent proteins. Our data expands knowledge of the Pseudomonas PHO regulon, including species that are frequently used as bioinoculants, opening up the potential for more efficient and complete use of soil complexed P.