Mutualistic Interactions Between Vitamin B12-Dependent Algae and Heterotrophic Bacteria

bs_bs_banner Environmental Microbiology (2012) 14(6), 1466–1476 doi:10.1111/j.1462-2920.2012.02733.x Mutualistic interactions between vitamin B12-dependent algae and heterotrophic bacteria exhibit regulationemi_2733 1466..1476 Elena Kazamia,1 Hjördis Czesnick,1 results in reduction of METE expression, demon- Thi Thanh Van Nguyen,1 Martin Tom Croft,1 strating that the bacterium can deliver the vitamin to 1 1 Emma Sherwood, Severin Sasso, this B12-independent alga. We discuss the implica- Sarah James Hodson,2 Martin James Warren2 and tions of this for the widespread distribution of cobal- Alison Gail Smith1* amin auxotrophy in the algal kingdom. 1Department of Plant Sciences, University of Cambridge, Cambridge, UK. Introduction 2Department of Biosciences, University of Kent, Canterbury, UK. Despite their photosynthetic lifestyle, an estimated half of all microalgal species are auxotrophs for the organic micronutrient vitamin B (Croft et al., 2005). Vitamin B Summary 12 12 (cobalamin) is a water-soluble vitamin that is required Many algae are auxotrophs for vitamin B12 by many eukaryotic organisms, but is synthesized by (cobalamin), which they need as a cofactor for B12- prokaryotes only (Warren et al., 2002). It is a complex dependent methionine synthase (METH). Because Co2+-containing modified tetrapyrrole that acts as a cofac- only prokaryotes can synthesize the cobalamin, they tor for enzymes involved in C1 metabolism and certain must be the ultimate source of the vitamin. In the radical reactions. In prokaryotes, there are over 20 laboratory, a direct interaction between algae and enzymes that have a cobalamin cofactor (Marsh, 1999), heterotrophic bacteria has been shown, with bacteria while in animals there are just two B12-dependent supplying cobalamin in exchange for fixed carbon. enzymes, methylmalonyl-CoA mutase, involved in odd- Here we establish a system to study this interaction chain-fatty-acid metabolism in the mitochondria, and at the molecular level. In a culture of a B12-dependent methionine synthase (METH), which catalyses the C1 green alga Chlamydomonas nivalis, we found a con- transfer from methylhydrofolate to homocysteine to make taminating bacterium, identified by 16S rRNA analy- methionine (Drummond et al., 1993). Vitamin B12 is not sis as Mesorhizobium sp. Using the sequenced found in, or required by, land plants or fungi, because they strain of M. loti (MAFF303099), we found that it contain no cobalamin-dependent enzymes (Eichel et al., was able to support the growth of B12-dependent 1995), and instead encode a B12-independent form of Lobomonas rostrata, another green alga, in return methionine synthase (METE). In contrast, the vitamin for fixed carbon. The two organisms form a stable is widespread throughout the algal kingdom, and algae equilibrium in terms of population numbers, which such as the seaweed Porphyra yezoensis (nori), are an is maintained over many generations in semi- extremely rich dietary source of cobalamin (Watanabe continuous culture, indicating a degree of regulation. et al., 2002). However, algae do not synthesize cobal- However, addition of either vitamin B12 or a carbon amin, and so must acquire it from the environment; those source for the bacteria perturbs the equilibrium, algae that are non-requirers do not synthesize vitamin B12, demonstrating that the symbiosis is mutualistic but some can use it if it is available. We have surveyed and facultative. Chlamydomonas reinhardtii does not several complete algal genome sequences, and together require B12 for growth because it encodes a B12- with growth assays and expression studies, have found independent methionine synthase, METE, the gene that vitamin B12-independent species all encode a func- for which is suppressed by addition of exogenous tional METE, whereas this is absent from auxotrophic B12. Co-culturing C. reinhardtii with M. loti also species (Helliwell et al., 2011). Interestingly in two of the latter, Volvox carteri and Gonium pectorale METE pseudogenes were present, providing evidence of the Received 5 November, 2011; revised 28 February, 2012; accepted 3 March, 2012. *For correspondence. E-mail [email protected]; Tel. evolution of B12-dependence in action. Some algae, such (+44) 1223 333952; Fax (+44) 1223 333953. as Chlamydomonas reinhardtii, possess both isoforms of © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd Algal–bacterial interactions for delivery of vitamin B12 1467 the enzyme, and can use vitamin B12 if it is available, in late host (Wagner-Döbler et al., 2010). Numerous other which case the METE gene is repressed, a situation symbiotic interactions between bacteria and algae have analogous to that in many eubacteria, including Escheri- been reported in recent years. Uptake of Fe by algae has chia coli (Whitfield et al., 1970). It is important to note been demonstrated to be enhanced by the presence of that methionine synthase is involved not just in synthesis oligotrophic bacteria (Keshtacher-Liebson et al., 1995). of the amino acid methionine, but also in C1 metabolism, Several clades of Marinobacter were found in close asso- in particular in folate cycling. Thus, B12-dependent algae ciation with dinoflagellates and coccolithophores, produc- can grow if supplemented with both methionine and ing a specific siderophore that promotes algal assimilation folate, but not methionine alone (Croft et al., 2005), due of iron, which they did not synthesize as free-living bac- to the phenomenon of folate-trapping, a characteristic teria. In return, the bacteria use organic carbon from the also seen in humans with B12 deficiency (Scott, 1999). algae (Amin et al., 2009). Co-inoculation of presumed Because vitamin B12 auxotrophy is so widespread in the bacterial contaminants isolated from a culture of Chlorella algal kingdom, and with no phylogenetic relationship ellipsoidea with the alga resulted in 0.5–3 times greater between auxotrophs (Croft et al., 2005), the loss of algal growth than that of C. ellipsoidea alone (Park et al., METE must have happened multiple times throughout 2008). Finally, many green algae cannot develop normally evolution. This implies that there is a readily available when grown under axenic conditions. Monostroma supply of cobalamin in the environment to allow so many oxyspermum, for example, proliferates unicellularly in an algal species to thrive. aseptic culture, but develops into a normal foliaceous The source of the vitamin remains the subject of gametophyte in the presence of select marine bacteria debate. In axenic cultures in the laboratory, growth of (Matsuo et al., 2003). vitamin B12-dependent algae is proportional to the amount Algal–bacterial symbiosis centred on vitamin B12 provi- of vitamin available (Carlucci and Silbernagel, 1969; Car- sion is one of simplest such examples, as it appears to be lucci and Cuhel, 1977; Graneli and Risinger, 1994). A driven by loss of a single gene, METE (Helliwell et al., minimum of 7 pM (10 ng l-1) is required for growth in 2011), and thus can be readily studied at the molecular culture of most species (Croft et al., 2005), but measure- level. To this end we wanted to establish a laboratory ments of free cobalamin in solution in natural habitats model of a co-culture system that is genetically tractable rarely find levels above this concentration. For example, and lends itself to easy molecular and physiological in seawater vitamin B12 levels typically vary between 0 manipulations. In this paper we report on such a system and 3 pM (Provasoli and Carlucci, 1974; Panzeca et al., by describing new symbiotic associations between the 2009). Furthermore, enrichment experiments show that green alga Lobomonas rostrata and the soil bacterium B-vitamins can have a significant effect on phytoplankton Mesorhizobium loti. We investigate the stability of the dynamics, as addition of B12 was shown to stimulate phy- co-cultures, and have started to probe the nature of the toplankton growth in both temperate coastal waters symbiotic interaction between these organisms. (Sañudo-Wilhelmy et al., 2006; Gobler et al., 2007), in the Southern Ocean’s Gerlache Straight (Panzeca et al., 2006), and in the Ross Sea (Bertrand et al., 2007). That Results half of all the algal species have an absolute requirement Identification of algal and bacterial partners for vitamin B12 suggests that these species are able to persist and thrive alongside non-requirers, especially Chlamydomonas reinhardtii does not require vitamin B12 during such times as algal blooms, in an environment that for growth, because it has both isoforms of methionine appears to contain only trace amounts of the free bioavail- synthase, METE and METH (Croft et al., 2005; Helliwell able cofactor in solution. et al., 2011). In contrast, the related Chlamydomonas Because only prokaryotes can synthesize cobalamin, nivalis (CCAP 11/128) was reported to have an obligate they must be the ultimate source of the micronutrient. We requirement for vitamin B12 (Provasoli and Carlucci, have shown that, in the laboratory, B12-producing het- 1974). A culture of C. nivalis was obtained from the erotrophic bacteria are able to supply cobalamin to algae Culture Collection of Algae and Protozoa (CCAP), Oban, in co-cultures, in return for photosynthate (Croft et al., UK, but it was able to grow without addition of vitamin B12 2005). A marine bacterium, Halomonas sp., was isolated to the medium. Examination of the culture revealed the from cultures of the dinoflagellate Amphidinium carterae presence of bacterial cells. The 16S rRNA gene from the and from blades of Porphyra miniata (a red alga), and bacterium was amplified by PCR and sequenced, and could support the growth of several marine algae in the bacterium identified as closely related to Mesorhizo- minimal media (Croft et al., 2005). Similarly, the symbiotic bium loti. Mesorhizobium loti is a soil bacterium known to bacterium Dinoroseobacter shibae was shown to deliver be able to synthesize vitamin B12. This was confirmed by both vitamin B12 and vitamin B1 (thiamine) to its dinoflagel- inspection of the genome sequence of M.

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