Utilization of phosphonic acid compounds by marine bacteria of the genera Phaeobacter, Ruegeria, and Thalassospira (α-Proteobacteria)

Abstract

Phosphonic acid (phosphonate) that possesses a carbon-phosphours bond is a chemically stable form of organic phosphorus. Various phosphonic acids are widely distributed in oceanic waters; in particular, methylphosphonic acid (namely methylphosphonate) is believed to be responsible for global methane production. To discuss the microbial degradation of phosphonic acids, we investigated the utilization of phosphonic acid compounds by cultures of marine bacteria, Phaeobacter sp., Ruegeria sp. (Rhodobacterales), and Thalassospira sp. (Rhodospirillales). These bacterial cultures were able to grow on methylphosphonic acid as well as on the tested alkyl-, carboxy-, aminoalkyl-, and hydroxyalkyl-phosphonic acid compounds. Cell yields and growth rates of Ruegeria and Thalassospira cultures grown on methyl-, ethyl-, propyl-, and butyl-phosphonic acid compounds tended to decrease with increasing alkyl chain length. In contrast, Phaeobacter sp. grew well on such alkyl-phosphonic acids. Our results suggest that these marine bacteria, which exhibit varied utilization, are involved in microbial degradation of various phosphonic acid compounds.

bacteria, methylphosphonate, phosphonate, phosphonic acid, phosphorus, utilization

Introduction

Phosphorus (P) is essential for cellular processes such as energy transfer and biosynthesis of nucleic acids, phospholipids, and so on. Orthophosphate, which is an important P source for microorganisms, is present in seawaters, but sometimes occurs at concentrations below the detection limits of current analytical methods. Dissolved organic P (DOP) concentrations in surface seawaters often exceed orthophosphate concentrations, sometimes by an order of magnitude; this is especially true in temperate and tropical regions (Orrett and Karl 1987, Duhamel et al. 2021). DOP in the regions could potentially support a fraction of the microbial community P demand, as mentioned by Duhamel et al. (2021). The chemical forms of DOP are generally considered to comprise phosphate esters.

Clark et al. (1998) examined the P components of high-molecular-weight DOP compounds in the collected seawaters using ³¹P-nucleic magnetic resonance, thereby identifying the specific signals of phosphonic acid (phosphonate). Phosphonic acid compounds comprise of ∼25% P compounds (Clark et al. 1998); and are widely and vertically distributed in the oceans (Kolowith et al. 2001, Sannigrahi et al. 2006). Repeta et al. (2016) reported that phosphonoacetic and hydroxyalkylphosphonic acids, as well as methylphosphonic acid (CH₃PO₃: methylphosphonate), were present in polysaccharide esters extracted from oceanic waters. Furthermore, Karl et al. (2008) indicated that methylphosphonic acid is a source of methane in surface oceans. Therefore, to reveal the marine P cycle and global climate change, it is essential to understand the marine microbial degradation of phosphonic acids.

Repeta et al. (2016) showed that marine microbes in surface waters of A Long-term Oligotrophic Habitat Assessment (ALOHA) stations produced methane and ethylene gases by degrading methylphosphonic and ethylphosphonic acids, respectively. Martínez et al. (2013) examined bacterial composition profiles in seawaters into which methylphosphonic acid was provided artificially together with carbon and nitrogen sources. They found a large relative increase in the number of bacteria (Rhodobacterales) after 48 h of incubation. Culture experiments conducted by Martinez et al. (2010) support the conclusion that Ruegeria pomeroyi, which belongs to the order Rhodobacterales, can utilize methylphosphonic acid. Sosa et al. (2017) revealed that Sulfitobacter sp. HI0054 (of the order Rhodobacterales) appears to catabolize phosphonic acid compounds such as methyl-, ethyl-, and hydroxyethyl-phosphonic acids. Among the bacterial species within SAR11 clade (α-Proteobacteria), Pelagibacterales sp. strain HTCC7211 can degrade methylphosphonic acid, whereas Candidatus Pelagibacter ubique strain HTCC1062 cannot (Carini et al. 2014).

Bacteria are likely to uptake phosphonic acids through transporters, and subsequently degrade them via some degradation pathways (Villarreal-Chiu et al. 2012, Horsman and Zechel 2017, Murphy et al. 2021). Several phn genes coding related proteins are present in marine microbial genomes. Many bacteria appear to possess phnJ, a key phn gene in phosphonic acid degradation (Martinez et al. 2010, Seweryn et al. 2015). Pelagibacterales sp. strain HTCC7211 possesses phn series and strongly expresses some of them (e.g. phnCDE) in phosphate-depleted conditions (Carini et al. 2014).

α-Proteobacteria species are both numerous and diverse. Furthermore, a variety of phosphonic acid compounds are present in natural seawaters, e.g. methylphosphonic, phosphonoacetic, 2-hydroxyoethylphosphonic, hydroxymethylphosphonic, and hydroxypropylphosphonic acids (Repeta et al. 2016). However, little is known about the degradation and utilization of phosphonic acid compounds by marine bacteria, regarding Rhodobacterales and the close orders of α-Proteobacteria. To discuss the marine microbial degradation of phosphonic acids, we investigated the utilization of phosphonic acid compounds by Phaeobacter, Ruegeria (Rhodobacterales), and Thalassospira (Rhodospirillales) isolated from the coastal waters of Japan.

Materials and methods

Marine bacterial cultures of Phaeobacter sp. URN3, Ruegeria sp. URN111, and Thalassospira sp. URN109 were used; these bacteria were isolated from the coastal waters of Japan (Yamaguchi et al. 2016). These stock cultures were maintained at 20°C in seawater-based media containing 0.5 g l⁻¹ tryptone (Nacalai Tesque Inc.) and 0.05 g l⁻¹ dried extract yeast (Nacalai Tesque Inc.).

To cultivate the marine bacteria and evaluate their utilization of phosphonic acid, we modified the medium components of Widdel and Pfennig (1981), as shown in Table 1. The modified medium, in which carbon, nitrogen, vitamin, and P sources were not yet added, was prepared in a screw-capped glass test tube with a flat bottom (16 mm × 100 mm, Fisher Scientific). Tris and metal sources were added into seawater, following which the pH was adjusted to 7.5. The seawater was then autoclaved at 121°C. Carbon, nitrogen, vitamins (Table 1), and P sources (Fig. 1) were added through a 0.2 µm-pore size syringe filter (Minisart^® NML, Sartorius). The prepared media, containing either various P sources or no additional P source, were used as test media.

Figure 1.

Chemical structures and abbreviations (bold font) of the tested P compounds. CAS numbers are shown in parentheses.

Table 1.

Components of modified medium of Widdel and Pfennig (1981). Reagents were added to 1 l of aged surface seawater collected from offshore Tosa Bay.

Reagent	Chemical formula	Final concentration
Tris(hydroxymethyl) aminomethane	(HOCH2)3CNH2	3.34 mM
Metal mixturea		0.1% (v/v)
Selenite–tungstate solutionb		0.1% (v/v)
Vitamin mixturec,*		0.1% (v/v)
Glucose*	C6H12O6	5.17 mM
Urea*	NH2CONH2	0.39 mM
Ammonium chloride*	NH4Cl2	0.39 mM
Phosphorus*	See Fig. 1	50 µM

a

Metal mixture contains 7.5 mM Fe(III)-EDTA, 0.5 mM H₃BO₃, 0.5 mM MnCl, 0.8 mM CoCl₂, 0.1 mM NiCl₂, 0.01 mM CuCl₂, 0.5 mM ZnSO₄, 0.15 mM NaMoO₄, and 0.1 M HCl.

b

Selenite–tungstate solution contains 0.4 mM NaOH, 5.3 µM NaSeO₃, and 6.6 µM Na₂WO₄.

c

Vitamin mixture contains 29.2 µM p-aminobenzoic acid, 4.1 µM D(+)-biotin, 81.2 µM nicotinic acid, 10.2 µM D(+)-calcium pantothenate, 88.7 µM pyridoxine hydrochloride, 29.7 µM thiamine hydrochloride, and 3.7 µM cyanocobalamin.

*

Reagents were added into the autoclaved media through a 0.2-µm filter.

A small portion of each bacterial culture (pregrown on the test media containing only glycerophosphate as the additional P source) was inoculated into the test medium with a platinum loop. Cultures were then incubated in triplicate and/or quadruplicate at 20°C, in the dark. During the cultivation period, the optical densities (OD₆₆₀) of bacterial cultures were monitored using a mini photo 518R (Titeck). Cultivation was terminated when no temporal increase in OD₆₆₀ was detected. The highest OD₆₆₀ obtained during the incubation period represented the maximum cell yield. Growth rate (ΔOD₆₆₀ day⁻¹) was calculated using OD₆₆₀ data (n = 3 or 4) from the exponential portion of the growth curve; it was determined by least-squares regression of the OD₆₆₀ and the number of days. By averaging the independent estimates of growth rates and cell yields obtained for each regime, the average cell yields and growth rates of their bacterial cultures were calculated. We tested for significant differences in maximum cell yields and maximum growth rates among P compounds using the Bonferroni method (α = 0.05).

Results and discussion

We found that the tested marine bacterial cultures were able to significantly utilize various compounds of alkyl-, carboxy-, aminoalkyl-, and hydroxyalkyl-phosphonic acids as the P source (Fig. 2; Figures A1–A3). For Phaeobacter sp., cell yields and growth rates were significantly higher for cultures using phosphonic acid compounds than for P-depleted cultures, except for phosphonoformic acid (P < .05; Fig. 2A). Meanwhile, Ruegeria sp. and Thalassospira sp. cultures did not grow well when utilizing butylphosphonic acid (Fig. 2); the former also showed a low cell yield when was cultured on media containing phosphonoacetic acid. The cell yields and growth rates of Ruegeria sp. and Thalassospira sp. cultures decreased significantly with increasing carbon numbers for alkylphosphonic acid compounds [r ≤ −0.694 (P < .01, n ≥ 15), data not shown]. None of the bacterial cultures grew on phosphonoformic acid (also known as a DNA polymerase inhibitor, foscarnet).

Figure 2.

Maximum OD₆₆₀ (A), (C), and (E) and growth rate (B), (D), and (F) of Phaeobacter sp. (A) and (B), Ruegeria sp. (C) and (D), and Thalassospira sp. (E) and (F) cultures on P sources (as shown by their abbreviations, see Fig. 1). Mean values that did not differ significantly (Bonferroni test, P > .05) are indicated by the same letter. Error bars indicate standard deviation (n = 4); asterisks indicate n = 3.

Marine bacteria belonging to the order Rhodobacterales (α-Proteobacteria) showed a relatively significant increase in the marine bacterial communities when glucose, nitrate, and methylphosphonic acid are provided artificially (Martínez et al. 2013). In support of this phenomenon, Sosa et al. (2017) found that methylphosphonic-, ethylphosphonic-, and 2-hydroxyethylphosphonic-acids were degraded by Sulfitobacter sp. HI0054 of the order Rhodobacterales. Phaeobacter and Ruegeria, which belong to the order Rhodobacterales, are found worldwide in oceanic waters (Pommier et al. 2005, Gram et al. 2010); species of the genus Thalassospira (Rhodospirillales) are also widespread in the oceans (Liu et al. 2007, Hütz et al. 2011). In the present study, we show that these bacterial strains are able to utilize phosphonic acids. Together with the fact that oceanic waters contain phosphonic acids (Clark et al. 1998, Kolowith et al. 2001, Sannigrahi et al. 2006), we conclude that the widespread bacteria of the orders Rhodobacterales and Rhodospirillales are responsible for the degradation of phosphonic acids in oceanic waters.

Little is known about the varieties of phosphonic acid compounds that marine bacteria can utilize. The microbial degradation of 2-aminoethylphosphonic, phosphonoacetic, and methylphosphonic acids have been studied well. Murphy et al. (2021) demonstrated that Stappia stellulata (Hyphomicrobiales, α-Proteobacteria) is likely to utilize not only methylphosphonic acid but also vinylphosphonic and dimethylthiophosphonic acids. Taken together with our data, we suggest that various phosphonic acid compounds can be P sources of marine bacteria.

Phaeobacter sp. utilizes various phosphonic acid compounds, whereas Ruegeria sp. and Thalassospira sp. cannot completely utilize alkylphosphonic acids, such as butylphosphonic acid. Alkylphosphonic acids with a long alkyl chain possess both hydrophilic PO₃ and hydrophobic C_nH_2n+1 (n ≥ 1). As per the chemical properties of those compounds (as obtained from SciFinderⁿ), the partition coefficients (log P-values) of methyl-, ethyl-, propyl-, and butyl-phosphonic acids are −1.246, −0.736, −0.227, and 0.283, respectively. Long-chain alkyl-phosphonic acid is of hydrophobic, and may have a low microbial degradability and/or accessibility at high concentrations. The degradability and accessibility of phosphonic acids may possibly be divergent among bacterial species and alkylphosphonic acids. Future research is needed to clarify accurate components of alkylphosphonic acids in seawaters.

Seweryn et al. (2015) determined the crystal structure of the 240 kDa Esherichia coli C–P lyase core complex (PhnGHIJ) and showed that PhnK binds to a conserved insertion domain of PhnJ. In addition to phnCDE [which codes an adenosine triphosphate (ATP)-binding cassette transporter] phnGHIJ likely plays a central role in the degradation and utilization of phosphonic acid compounds by microbes. Indeed, phnCDE and phnGHIJ are present in the genome of Pelagibacterales sp. strain HTCC7211, which can utilize methylphosphonic and 2-aminoethylphosphonic acids as P sources (Carini et al. 2014). Expression of phnD and phnJ clearly appeared in extractions of Pelagibacterales sp. strain HTCC7211 (Carini et al. 2014) and field-collected cyanobacteria, Trichodesmium erythraeum, which is known to utilize methylphosphonic acid (Dyhrman et al. 2006). Our preliminary search found that Phaeobacter gallaeciensis, Ruegeriamobilis(Tritonibacter mobilis), and Thalassospira profundimaris also possess phnCDE, phnGHIJ, and other phn series in their genome sequences (Table A1); as shown in our research paper, these bacteria are the same as and/or closely related with Phaeobacter sp., Ruegeria sp., and Thalassospira sp., respectively (Yamaguchi et al. 2016). This observation may partly support, at the genome level, the significant utilization of phosphonic acid compounds by species of the genera Phaeobacter, Ruegeria, and Thalassospira. Recently, bacterial 2-aminoethylphophonate transporters, namely AepXVW, AepP, and AepSTU, were identified in the genome of S. stellulata DSM5886 belonging to Hyphomicrobiales (Murphy et al. 2021). These characteristics may possibly be responsible for the varied utilization of phosphonic acid compounds among marine bacteria. Our future research will aim to search not only phnCDE and phnGHIJ, but also other phn genes in marine bacteria genomes.

This study provides new insights into the microbial degradation of phosphonic acids in seawater. To fully understand how the marine P cycle is associated with the microbial degradation of phosphonic acids, future studies should investigate the utilization of phosphonic acid compounds by various species of α-Proteobacteria using bioassays, chemical analysis, and molecular approaches.

Authors’ contributions

S.U., H.Y., and N.N. conceived the original idea and designed the study; S.U. and H.Y. carried out the culture experiments; S.U., N.Y., H.Y., and H.Y. prepared samples and reagents; Y.H. and M.A. proposed the genetic analyses in discussion with S.U., Y.K., and H.Y. S.U., Y.K., and H.Y. conducted genome informatics; S.U., N.N., Y.K., and H.Y. drafted and revised the manuscript and designed/prepared the tables and figures with support from M.A. Y.H., N.Y., H.Y., and N.N. verified the analytical methods and calculations; and H.Y. and N.N. supervised the projects. All authors discussed the results and contributed to the final manuscript.

ACKNOWLEDGEMENTS

We thank Dr Eiji Onodera (Kochi University) for the technical assistance.

Author notes

Present address: Nomura Micro Science Co., Ltd., Okata, Atsugi, Kanagawa 243-0021, Japan.