Aminophosphonate mineralisation is a major step in the global oceanic phosphorus redox 1 cycle 2

21 The planktonic synthesis of reduced organophosphorus molecules, such as 22 alkylphosphonates and aminophosphonates, represents one half of a vast global oceanic 23 phosphorus redox cycle. Whilst alkylphosphonates tend to accumulate in recalcitrant 24 dissolved organic matter, aminophosphonates do not. Thus, we hypothesised unknown 25 pathways for the uptake of aminophosphonates must exist in seawater. Here, we identify 26 three novel bacterial 2-aminoethylphosphonate (2AEP) transporters, named AepXVW, AepP 27 and AepSTU, whose expression is independent of phosphate concentrations (phosphate- 28 insensitive). AepXVW, is found in diverse marine heterotrophs and is ubiquitously distributed 29 in mesopelagic and epipelagic waters. Unlike the archetypal phosphate-regulated 30 phosphonate binding protein, PhnD, the newly identified AepX is heavily transcribed (~100- 31 fold>PhnD) in the global ocean independently of phosphate and nitrogen concentrations. 32 Collectively, our data identifies a mechanism responsible for the oxidative step in the marine 33 phosphorus redox cycle and suggests 2AEP may be an important source of regenerated 34 phosphate, which is required for oceanic primary production.


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Phosphonates are reduced organic phosphorus (P) molecules with a carbon (C)-P 38 bond, as opposed to the more common C-oxygen (O)-P ester bonds found in many other 39 organic P molecules 1 . Phosphonates are synthesised as both primary and secondary 40 metabolites in various bacterial, archaeal and eukaryotic organisms 1-7 where they are 41 incorporated into lipids (phosphonolipids) and glycans (phosphonoglycans) 4,8 . A significant 2AEP degradation was the remineralisation and release of labile Pi 22 , due to the greater 69 cellular demand for N over P and the 1:1 N:P stoichiometry of 2AEP. 70 To date, only two 2-AEP transport systems have been identified. Both are ATP-binding PhnWAY phosphonatases, which is surprising, given the fact 2AEP is a charged molecule and 80 ubiquitous in marine and terrestrial ecosystems 1,20 .

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Here, we sought to identify transporters, which provide superb molecular tools for 82 investigating the in situ cycling of specific environmental metabolites 28,33-35 , required for 2AEP 83 catabolism in environmental bacteria. Through combining laboratory-based molecular and 84 genetic analyses with environmental meta-omics, we identified three novel transporters 85 which have a role in 2AEP uptake and revealed Pi-insensitive 2AEP catabolism is widespread 86 in the global ocean, likely representing a major step in the marine phosphorus redox cycle.

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Pseudomonas putida BIRD-1 possesses a novel Pi-sensitive 2-aminoethylphosphonate ABC 90 transporter, AepXVW 91 We recently identified several candidate 2-AEP transporters (ABC-type) in 92 Pseudomonas rhizobacteria that contain the PhnWX phosphonatase but lack both the 93 archetypal PhnCDE transporter and PhnSTUV 36 (Fig 1A). In Pseudomonas putida BIRD-1 94 (hereafter BIRD-1), a periplasmic substrate binding protein associated with one of these 95 putative transporters (PPUBIRD1_4925), which we hereafter refer to as AepX, was induced 96 under Pi-deplete growth conditions in a PhoBR-dependent manner 36 . AepX belongs to the 97 same family (pfam13343) as PhnS, iron and sulphate SBPs but is clearly distinct (Coverage = 98 40%, Identity = 25.09%, 1.1e -05 ) ( Fig 1B). 99 BIRD-1 was capable of growth on 1.5 mM 2AEP as either a sole N, P, or N and P source, 100 the latter resulting in mineralisation of Pi which was subsequently exported from the cell (Fig   101   1C, Fig S1). Mutagenesis of phnWX confirmed this phosphonatase was essential for 2AEP 102 catabolism under both growth conditions in this bacterium (Fig S2A and S2B). Next, we 103 investigated if AepX and its corresponding ABC transporter components, the ATP binding 104 domain protein (AepV), and the permease domain protein (AepW) were essential for its 105 growth on 2AEP. Surprisingly, deletion of aepXVW BIRD had no effect on growth as a sole N 106 source ( Fig 1C). However, the mutant (ΔaepXVW BIRD ::Gm) had significantly (P <0.0001) 107 attenuated growth on 2AEP as a sole P source (Fig 1C). The growth defect observed during 108 growth on 2AEP as a sole P source was largely restored by complementing the mutant with a 109 plasmid-encoded native homolog (Fig. 1C). These data suggest that whilst the aepXVW 110 transporter is not essential, it is involved in 2AEP uptake as a sole P source but is not involved 111 6 in its growth as an N source. Therefore, another 2AEP transport system must also exist in this 112 bacterium.

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AepP shares conserved residues essential for binding Pi and the phosphate moiety of G3P 39-122 42 with GlpT, whereas residues that impact the binding affinity to the glycerol moiety of G3P 123 but not Pi are not conserved 41 (Fig S4). Mutation of aepP in either the wild type parental strain 124 (ΔaepP, Fig1D) or the aepXVW mutant (ΔaepXVW:Gm:aepP, Fig 1E) led to an inability to grow 125 on 2AEP as sole N source. Subsequent complementation of ΔaepP with its native homolog 126 (ΔaepP +pBB:aepP) restored growth on 2AEP as a sole N source ( Fig 1D). Interestingly, delayed 127 but significant growth on 2AEP as a P source still occurred in the ΔaepXVW:Gm:aepP double 128 mutant, revealing the presence of a third 2AEP transporter in this bacterium.

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To identify the unknown 2AEP transporter, we reanalysed our proteomics data (Fig   130   S3). Another substrate binding protein (PPUBIRD1_3891) containing the same pfam domain 131 (pfam13343) as AepX (Fig 1B), hereafter named AepS, was constitutively expressed in all 132 growth conditions. In order to uncover the role of AepS in the utilisation of 2AEP as a sole P 133 source, a triple mutant ΔaepXVW:Gm;aepP:aepSTU was generated in BIRD-1 (Fig 1E). This 134 7 triple knockout mutant was unable to grow on 2AEP as a sole P source (Fig 1E), suggesting 135 AepSTU is a functional 2AEP transporter. However, generation of a single aepSTU knockout 136 mutant did not affect Pi-sensitive growth compared to the wild type (Fig S2C), suggesting 137 AepXVW is the major transporter involved in Pi-sensitive 2AEP uptake and AepSTU only has AepSTU having an auxiliary role, whereas AepP is essential for Pi-insensitive 2AEP growth.  (Fig 2A). We also found an 153 orthologous ORF in the model rhizosphere alphaproteobacterium Sinorhizobium meliloti 154 strain 1021 that is capable of 2AEP catabolism via a phosphonatase 43 (Fig 2A). In all cases, 155 ORFs encoding AepXVW were located adjacent to ORFs encoding the phosphonatases, 156 PhnWAY or PhnWX, strongly suggesting a role in 2AEP transport. S. stellulata DSM 5886, A.  (Table S1). 159 Indeed, both Aliiroseovarius strains lack other characterised 2AEP transport and degradation 160 systems (Table S2). As previously reported, Pi was exported from cells and accumulated in the 161 medium during growth on 2AEP as a sole N source (Fig S5A).

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To confirm that Stappia AepXVW can take up 2AEP, we complemented the BIRD-1 null 163 2AEP transporter mutant (ΔaepXVW:aepP:aepSTU:gm) with this transporter fused with the 164 aepXVW BIRD-1 promoter ( Fig 2B). This duly restored growth of the triple mutant confirming S. 165 stellulata AepXVW is also a functional 2AEP transporter. In BIRD-1, AepXVW was only involved in Pi-sensitive growth whilst AepP was induced 169 during Pi-insensitive metabolism (Fig 1). However, S. stellulata lacks AepP but is still capable 170 of Pi-insensitive growth and Pi export ( Fig S5A). In addition to aepXVW and genes encoding 171 the 2AEP phosphonatase (phnWAY), S. stellulata also possesses genes (phnCDEFGHIJKLMN) 172 encoding the P-regulated C-P lyase operon and we also confirmed this strain grew on several 173 other alkylphosphonates as sole P source ( Fig S5B). Therefore, to determine which transport 174 and degradation systems were upregulated during growth on 2AEP as either a sole N or P 175 source, we performed comparative proteomics. Unlike BIRD-1, AepX was abundantly 176 expressed during growth on 2AEP as either a sole N or P source, as was the phosphonatase 177 (PhnWAY), whilst the C-P lyase operon was not (Fig 3). Importantly, whilst we detected 178 several general nitrogen stress-response proteins induced under Pi-insensitive growth, we 179 9 didn't identify any other potential 2AEP transporters (Table S3). Therefore, unlike in BIRD-1, 180 AepXVW likely represents the major route for 2AEP uptake in this bacterium. Deltaproteobacteria, as well as marine Vibrio spp. AepX was also found in terrestrial 189 Betaproteobacteria, Firmicutes, and other gram-positive bacteria (Fig 4). AepX was 190 partitioned into several subclades, with AepX Stappia and AepX BIRD well separated (Fig 4). Many 191 taxonomically divergent AepX ORFs were co-localised with ORFs encoding the various 192 phosphonatase systems or the C-P lyase, supporting a role in 2AEP transport (Fig 4). 193 AepP was also found in a wide range of phylogenetically divergent taxa, such as   The majority of aepX sequences were related to the cosmopolitan Alphaproteobacteria and 215 Deltaproteobacteria (Fig 4). We confirmed that these abundant environmental sequences 216 were also co-localised with phosphonate degradation genes (Fig 4). In broad agreement with 217 aepX, the cumulative transcription of the two phosphonatase markers phnA and phnX is 218 significantly greater than the C-P lyase marker phnJ (Kruskal-wallis Χ 2 = 206.6, p<0.001) 219 strengthening this observation that 2AEP mineralisation is a major oceanic process.