Tradeoffs between phage resistance and nitrogen fixation drive the evolution of genes essential for cyanobacterial heterocyst functionality

Abstract

Harmful blooms caused by diazotrophic (nitrogen-fixing) Cyanobacteria are becoming increasingly frequent and negatively impact aquatic environments worldwide. Cyanophages (viruses infecting Cyanobacteria) can potentially regulate cyanobacterial blooms, yet Cyanobacteria can rapidly acquire mutations that provide protection against phage infection. Here, we provide novel insights into cyanophage:Cyanobacteria interactions by characterizing the resistance to phages in two species of diazotrophic Cyanobacteria: Nostoc sp. and Cylindrospermopsis raciborskii. Our results demonstrate that phage resistance is associated with a fitness tradeoff by which resistant Cyanobacteria have reduced ability to fix nitrogen and/or to survive nitrogen starvation. Furthermore, we use whole-genome sequence analysis of 58 Nostoc-resistant strains to identify several mutations associated with phage resistance, including in cell surface-related genes and regulatory genes involved in the development and function of heterocysts (cells specialized in nitrogen fixation). Finally, we employ phylogenetic analyses to show that most of these resistance genes are accessory genes whose evolution is impacted by lateral gene transfer events. Together, these results further our understanding of the interplay between diazotrophic Cyanobacteria and their phages and suggest that a tradeoff between phage resistance and nitrogen fixation affects the evolution of cell surface-related genes and of genes involved in heterocyst differentiation and nitrogen fixation.

Keywords: Cyanobacteria; cost of resistance; genome evolution; nitrogen fixation; phage; resistance; tradeoff.

Figure 1

Phenotypes of the resistant substrains of Nostoc 7120; cells width (A) of the resistant substrains and their susceptible ancestors (wild type, WT); data shown are average +/− standard deviation of 44–139 cells per substrain; (B) bright field images of resistant substrains and their susceptible controls; (C) percentage of the adsorbed A-4L phage particles to four resistant substrains of Nostoc 7120 relative to the adsorption of the phage to their susceptible paired controls (WT); data shown are average +/− standard deviation of three to four biological replicates. ^**P < .01 ^***P < .001.

Figure 2

Growth cost in Nostoc 7120; growth dynamics of resistant substrains of Nostoc 7120 and their susceptible controls in nitrogen replete (A) and in nitrogen deplete conditions; (B) the growth of the resistant substrains in nitrogen-rich medium was significantly lower than that of the susceptible wild type for RD1, RD2, and RE1 (P < .001); the resistant substrains RD2, RE1, and RE2 had a significantly greater cost under nitrogen starvation than in nitrogen replete medium (P < .001); data shown are average +/− standard deviation of five biological replicates; relative cell density is estimated by chlorophyll a autofluorescence; A.U: arbitrary units.

Figure 3

Nitrogen fixation-related cost in Nostoc 7120; (A) nitrogenase activity, relative to the susceptible wild type (WT), of the resistant substrains 48 h after nitrogen stepdown; (B) percentage of filaments with heterocysts of the resistant substrains, relative to the susceptible wild type (WT) 48 h after nitrogen stepdown; (C) expression of nifH gene in the resistant substrains relative to the susceptible ancestor (WT) 48 h after nitrogen stepdown; the transcript levels of nifH values are normalized to the transcript levels of rnpB; data shown are average +/− standard deviation of three to five biological replicates; ^*P < .05, ^**P < .01, ^***P < .001; (D) bright field images (upper panels) and the corresponding fluorescence images (lower panels; red color represents auto fluorescence of chlorophyll, which is absent from mature functional heterocyst cells) of Nostoc 7120 substrains, 48 h after nitrogen stepdown; arrows indicate heterocyst cells; scale equals 10 μm.

Figure 4

Core and accessory genome of Nostoc 7120; (A) pangenome analysis and ANI for 19 Cyanobacteria strains belonging to the Nostocales order (full names and accession numbers are listed in Supplementary Table S4); the inner tree illustrates the relationships between 23 526 gene clusters identified across these Cyanobacteria genomes; each layer of the circle phylogram represents a different organism, and the presence of a gene cluster for each organism is depicted by the darkness of the respective layer; the layers representing Nostoc 7120 and C. raciborskii are highlighted; the outermost partial layer signifies gene clusters found in at least 18 out of the 19 analyzed genomes, representing the “soft core” genome; within this collection of gene clusters, there are two distinct subgroups; the larger subgroup, situated on the left, comprises gene clusters that are universally present in all 19 genomes, thus categorizing them as “core” gene clusters; the locations of 42 gene clusters harboring genes that were found to be affected by mutations associated with resistance are indicated outside the circle phylogram; these genes are distinguished between those that underwent mutation in substrains with a single mutation, and the remaining genes (resistant specific); the ANI percentages are presented as a heatmap; the hierarchical arrangement of the tree above the heatmap is structured based on the ANI percentage values; (B) the distribution of core genes, soft core genes, and noncore genes within the mutants exhibiting a single mutation, across all the mutants, and throughout the entire genome of Nostoc 7120; statistical analysis revealed no significant difference between the observed gene distribution in the resistant mutants and the predicted values across the entire genome.

Figure 5

Genome evolution of Nostoc 7120; (A) heatmap of orthologs of genes involved in resistance to phages and/or in nitrogen fixation in Nostoc 7120 presented as average % identity of amino acid sequences; the heatmap includes protein products of hetF (alr3546) and 16S rRNA (uracil(1498)-N(3))-methyltransferase (alr1349) as references. The hierarchy of bacteria used for the analysis is ordered using maximum likelihood phylogenetic tree of alr1349; (B) a schematic representation of mutations along the chromosome of Nostoc 7120, in the susceptible wild types (control), in resistant mutants with a single mutation, and in resistant mutants with multiple mutations (resistant specific); each position along the horizontal axis corresponds to a different gene within the genome of Nostoc 7120, and the vertical axis provides information about the number of mutations that independently occurred in each gene; (C) genomic neighborhood of a cell surface-related gene cluster (alr4485-alr4494), in which mutations were found in 35 of the resistant strains; the genomic composition (at the amino acid level) within this gene cluster and its flanking regions were compared to the corresponding regions in close relatives of Nostoc 7120; markers represent mutant loci within this cluster, as in (B).

Figure 6

Cost of resistance in C. raciborskii; growth dynamics of resistant substrains and their susceptible controls in nitrogen replete (A) and nitrogen deplete conditions (B); the difference in growth over time for all substrains grown in nitrogen starvation conditions was significantly lower than that of the susceptible wild type (P < .01 for R3C2 and R3C4, and P < .001 for all the rest);however, when grown in nitrogen-rich medium, the resistant substrains had no significant reduction in growth with respect to the susceptible wild type; all resistant substrains had a significantly greater cost under nitrogen starvation than in nitrogen replete medium (P < .05 for R3C2 and R3C4, P < .01 for R23C1 and R3C3, and P < .001 for R3C5). Data shown are average +/− standard deviation of three biological replicates; relative cell density is estimated by chlorophyll a autofluorescence; A.U: arbitrary units; (C) percentage of filaments with heterocysts of the resistant substrains, relative to the susceptible wild type (gray), 6 days after nitrogen stepdown; data shown are average and standard deviation of four biological replicates; ^*P < .05, ^**P < .01, ^***P < .001.