Impairment of O-antigen production confers resistance to grazing in a model amoeba–cyanobacterium predator–prey system

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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...

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confidence: 99%

An Amoebal Grazer of Cyanobacteria Requires Cobalamin Produced by Heterotrophic Bacteria

Beld

Brahamsha

2017

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“…For example, cyclic di-GMP has been shown to play a role in biofilm formation and cell buoyancy (32), and spurious activation of pathways that lead to high cytosolic di-GMP also lead to increased sedimentation in the cyanobacterium Fremyella diplosiphon (33). Similarly, genetic knockout of factors that regulate biofilm formation or extracellular lipopolysaccharide composition leads to increased aggregation and sedimentation in S. elongatus (34,35). It is possible that combinatorial control over regulatory pathways for both cell morphology and biofilm development might provide additive effects on the rate of cell sedimentation relative to relying on one approach alone.…”

Section: Discussionmentioning

confidence: 99%

Engineering Cyanobacterial Cell Morphology for Enhanced Recovery and Processing of Biomass

Jordan

Chandler

MacCready

et al. 2017

Cyanobacteria are emerging as alternative crop species for the production of fuels, chemicals, and biomass. Yet, the success of these microbes depends on the development of cost-effective technologies that permit scaled cultivation and cell harvesting. Here, we investigate the feasibility of engineering cell morphology to improve biomass recovery and decrease energetic costs associated with lysing cyanobacterial cells. Specifically, we modify the levels of Min system proteins in Synechococcus elongatus PCC 7942. The Min system has established functions in controlling cell division by regulating the assembly of FtsZ, a tubulin-like protein required for defining the bacterial division plane. We show that altering the expression of two FtsZ-regulatory proteins, MinC and Cdv3, enables control over cell morphology by disrupting FtsZ localization and cell division without preventing continued cell growth. By varying the expression of these proteins, we can tune the lengths of cyanobacterial cells across a broad dynamic range, anywhere from an ϳ20% increased length (relative to the wild type) to near-millimeter lengths. Highly elongated cells exhibit increased rates of sedimentation under low centrifugal forces or by gravity-assisted settling. Furthermore, hyperelongated cells are also more susceptible to lysis through the application of mild physical stress. Collectively, these results demonstrate a novel approach toward decreasing harvesting and processing costs associated with mass cyanobacterial cultivation by altering morphology at the cellular level. IMPORTANCEWe show that the cell length of a model cyanobacterial species can be programmed by rationally manipulating the expression of protein factors that suppress cell division. In some instances, we can increase the size of these cells to near-millimeter lengths with this approach. The resulting elongated cells have favorable properties with regard to cell harvesting and lysis. Furthermore, cells treated in this manner continue to grow rapidly at time scales similar to those of uninduced controls. To our knowledge, this is the first reported example of engineering the cell morphology of cyanobacteria or algae to make them more compatible with downstream processing steps that present economic barriers to their use as alternative crop species. Therefore, our results are a promising proof-of-principle for the use of morphology engineering to increase the cost-effectiveness of the mass cultivation of cyanobacteria for various sustainability initiatives.KEYWORDS cyanobacteria, cell morphology, biomass recovery, biotechnology, cell division, Min system W hile cyanobacteria and algae offer many potential benefits relative to traditional land plants, the commercialization of such photosynthetic crop species has been limited due to the technical problems of scaled cultivation. Cyanobacteria exhibit rapid

“…All vectors and strains used in this study are listed in Table 1. To generate novel constitutive expression vectors for complementation studies, open reading frames (ORFs) were PCR amplified from WT S. elongatus cells using Q5 DNA polymerase (NEB) and TOPO cloned into the pSyn_1/D-TOPO expression vector (Life Technologies) as previously reported (21). WT-like antibiotic-resistant strains (29), transposon-insertion knockout mutants (27,28), and expression clones were derived from a WT strain of S. elongatus PCC 7942 (lab collection accession no.…”

Section: Methodsmentioning

confidence: 99%

“…We previously identified and characterized, through mutational studies, four genes in S. elongatus PCC 7942 involved in a Wzm/Wzt-like O-antigen synthesis and transport pathway homologous to that of E. coli serotypes O8 and O9 (21). Prior to that study, only a single publication on phageresistant mutants in Anabaena sp.…”

Section: Ipopolysaccharide (Lps) Serves As a Critical Interface Betmentioning

confidence: 99%

“…We recently demonstrated that removal of the O antigen from the LPS of S. elongatus PCC 7942 confers resistance to predation by a heterolobosean amoebal grazer, HGG1 (21). Strains that lack the O antigen display a visibly identifiable "rough" phenotype, which enabled the identification of new resistant mutants through random mutagenesis.…”

Section: Ipopolysaccharide (Lps) Serves As a Critical Interface Betmentioning

confidence: 99%

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Mutations in Novel Lipopolysaccharide Biogenesis Genes Confer Resistance to Amoebal Grazing in Synechococcus elongatus

Simkovsky

Effner

Iglesias-Sánchez

et al. 2016

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In natural and artificial aquatic environments, population structures and dynamics of photosynthetic microbes are heavily influenced by the grazing activity of protistan predators. Understanding the molecular factors that affect predation is critical for controlling toxic cyanobacterial blooms and maintaining cyanobacterial biomass production ponds for generating biofuels and other bioproducts. We previously demonstrated that impairment of the synthesis or transport of the O-antigen component of lipopolysaccharide (LPS) enables resistance to amoebal grazing in the model predator-prey system consisting of the heterolobosean amoeba HGG1 and the cyanobacterium Synechococcus elongatus PCC 7942 (R. S. Simkovsky et al., Proc Natl Acad Sci U S A 109:16678 -16683, 2012, http://dx.doi.org/10.1073/pnas.1214904109). In this study, we used this model system to identify additional gene products involved in the synthesis of O antigen, the ligation of O antigen to the lipid A-core conjugated molecule (including a novel ligase gene), the generation of GDP-fucose, and the incorporation of sugars into the lipid A core oligosaccharide of S. elongatus. Knockout of any of these genes enables resistance to HGG1, and of these, only disruption of the genes involved in synthesis or incorporation of GDP-fucose into the lipid A-core molecule impairs growth. Because these LPS synthesis genes are well conserved across the diverse range of cyanobacteria, they enable a broader understanding of the structure and synthesis of cyanobacterial LPS and represent mutational targets for generating resistance to amoebal grazers in novel biomass production strains. L ipopolysaccharide (LPS) serves as a critical interface between aGram-negative bacterium's internal physiology and its abiotic and biotic environments, protecting the cell from specific antimicrobials, evading the innate immune and complement systems, enabling symbiosis with eukaryotic hosts, acting as a recognition factor for phage infection, and protecting bacteria against digestion by predatory amoebae (1-5). Alterations to any of the three primary components of the LPS structure (the endotoxin lipid A, the core oligosaccharide, or the O-antigen polysaccharide), through changes in sugar content or sequence, acylation, phosphorylation, or other molecular modifications, can alter these interactions, modulate the toxicity of the endotoxin, or prevent cellular behaviors such as motility (6, 7).While much is known about the structure and synthesis of LPS in enteric and pathogenic proteobacteria, little is known about either aspect of LPS in photosynthetic cyanobacteria and how the cyanobacterial LPS affects interactions with the environment (8-11). To date, only three cyanobacterial LPS structures have been reported: the partial structural determination of the filamentous freshwater cyanobacterium Oscillatoria planktothrix LPS (12) and the complete structural determination of the LPSs of the unicellular marine Synechococcus strains WH8102 and CC9311 (9). Beyond these structures, the remaining...