An Amoebal Grazer of Cyanobacteria Requires Cobalamin Produced by Heterotrophic Bacteria

Abstract

Amoebae are unicellular eukaryotes that consume microbial prey through phagocytosis, playing a role in shaping microbial food webs. Many amoebal species can be cultivated axenically in rich media or monoxenically with a single bacterial prey species. Here, we characterize heterolobosean amoeba LPG3, a recent natural isolate, which is unable to grow on unicellular cyanobacteria, its primary food source, in the absence of a heterotrophic bacterium, a Pseudomonas species coisolate. To investigate the molecular basis of this requirement for heterotrophic bacteria, we performed a screen using the defined nonredundant transposon library of Vibrio cholerae, which implicated genes in corrinoid uptake and biosynthesis. Furthermore, cobalamin synthase deletion mutations in V. cholerae and the Pseudomonas species coisolate do not support the growth of amoeba LPG3 on cyanobacteria. While cyanobacteria are robust producers of a corrinoid variant called pseudocobalamin, this variant does not support the growth of amoeba LPG3. Instead, we show that it requires cobalamin that is produced by the Pseudomonas species coisolate. The diversity of eukaryotes utilizing corrinoids is poorly understood, and this amoebal corrinoid auxotroph serves as a model for examining predator-prey interactions and micronutrient transfer in bacterivores underpinning microbial food webs.IMPORTANCE Cyanobacteria are important primary producers in aquatic environments, where they are grazed upon by a variety of phagotrophic protists and, hence, have an impact on nutrient flux at the base of microbial food webs. Here, we characterize amoebal isolate LPG3, which consumes cyanobacteria as its primary food source but also requires heterotrophic bacteria as a source of corrinoid vitamins. Amoeba LPG3 specifically requires the corrinoid variant produced by heterotrophic bacteria and cannot grow on cyanobacteria alone, as they produce a different corrinoid variant. This same corrinoid specificity is also exhibited by other eukaryotes, including humans and algae. This amoebal model system allows us to dissect predator-prey interactions to uncover factors that may shape microbial food webs while also providing insight into corrinoid specificity in eukaryotes.KEYWORDS amoeba, corrinoids, microbial interactions, vitamin B 12 U nicellular organisms dominate the eukaryotic phylogenetic tree of life. Although protists make up a significant part of natural microbial communities, they have been largely overlooked and are understudied as simple model eukaryotes (1, 2). While culture-independent metagenomic studies provide insight into the abundance and natural diversity of protists in environmental settings (3), in-depth characterization requires the isolation and cultivation of individual species. A large range of eukaryotic microalgal species have been cultivated and studied, particularly as a source of renewable biofuel compounds in recent years (4, 5); however, besides microalgae, there are only a few protist microbes developed as model systems. Amoebae are...

Specificity of cobamide remodeling, uptake and utilization in Vibrio cholerae

“…Genes for corrinoid biosynthesis are widely distributed in aquatic environments (Doxey et al , ), and both cobalamin and pseudocobalamin were directly detected in surface waters, while only cobalamin was found in deeper water samples (Heal et al , ). Production of cobalamin was attributed to Thaumarchaeota, α‐ and γ‐proteobacteria, while production of pseudocobalamin was attributed to cyanobacteria, consistent with other studies examining cultured cyanobacteria (Helliwell et al , ; Ma et al , ). Other bacteria that produce pseudocobalamin include Clostridium (Hoffmann et al , ), Lactobacillus (Santos et al , ) and Salmonella (Anderson et al , ).…”

“…Many eukaryotes also require a source of cobamides, including algae (Croft et al , ; Bertrand et al , ), amoebae (Ma et al , ), nematodes (Bito et al , ) and humans (Stabler and Allen, ). Since eukaryotes cannot synthesize corrinoids, most organisms acquire cobalamin through dietary means, although there are a few examples of cobalamin production by resident intestinal microbiota (Girard et al , ).…”

“…Some eukaryotic algae have mutualistic relationships with cobalamin‐producing bacteria (Croft et al , ; Xie et al , ; Cooper et al , ), while two species have been shown to remodel pseudocobalamin in the presence of DMB (Helliwell et al , ). In the eukaryotic organisms in which different cobamide variants have been examined, the general trend of specificity for the cobalamin variant has been observed (Stupperich and Nexo, ; Helliwell et al , ; Ma et al , ). In humans, this specificity is mediated through B 12 ‐binding proteins intrinsic factor and transcobalamin which bind cobalamin with higher affinity compared to other variants (Stupperich and Nexo, ; Fedosov et al , ).…”

“…V. cholerae is the etiological agent of the diarrheal disease cholera, and is a well‐studied model organism. Previous work characterizing a natural amoebal isolate showed that amoebal growth could be supported by coculture with V. cholerae and cyanobacteria (Ma et al , ). Mutational analysis of V. cholerae showed that btuB and cobS were required, suggesting that pseudocobalamin from cyanobacteria was imported into V. cholerae cells and remodeled into cobalamin.…”

Cofactor selectivity in methylmalonyl-CoA mutase, a model cobamide-dependent enzyme

“…We and others have proposed the possibility of manipulating microbial communities using cobamides by taking advantage of the differential cobamide-dependent growth of bacteria (Abreu and Taga, 2016;Degnan et al, 2014b;Seth and Taga, 2014;Yan et al, 2018). Cobamides are predicted to mediate microbial interactions that are critical to the assembly of complex communities (Belzer et al, 2017;Degnan et al, 2014a;Heal et al, 2017;Helliwell et al, 2016;Ma et al, 2017;Men et al, 2015;Shelton et al, 2019;Yan et al, 2012), so the ability to selectively inhibit or promote the growth of particular species using corrinoids with various lower ligands could be applied to alter the composition of microbial communities in ways that could promote environmental and human health. This possibility hinges on the ability to predict which cobamides support or inhibit growth of an organism of interest, which requires an understanding of the major biochemical determinants of growth.…”