Enriched KEGG pathways in Phycobacter and A. macleodii.

Left: Scatter plot of the log2 fold-change of differentially expressed (DE) genes present in KEGG pathways listed on the y-axis. Each circle represents the differential expression of a gene in the presence of Aze relative to controls. Genes were considered DE if they had a p-adjusted value of < 0.05 and log2 fold change of ≥ ±0.5. Right: Expression ratios for KEGG pathways of interest were calculated as the total number of DE genes: total number of genes present in the genome in the given pathway. Bar sizes are proportional to the contribution of differentially expressed genes in relation to all genes present in a pathway. Circle and bar colors indicate the strain and time point (red=Phycobacter at 0.5 hours; green=Phycobacter at 8 hours; blue=A. macleodii at 0.5 hours).

Proposed Aze catabolism in Phycobacter based on transcriptomics and isotope labeling.

Left: Log2 fold-change of differentially expressed (DE) genes is shown as circles at 0.5 (left circle) and 8 hours (right circle) after the addition of Aze to Phycobacter cells relative to controls. The Aze transport system (azeTSL) consists of 3 genes, the DE values of which are shown next to the transporter. Intermediate reactions and substrates for the successive liberation of acetyl-coA from azeloyl-coA and pimeloyl-coA are not shown; DE values of the genes involved are shown next to each overall reaction. 13C-labeled metabolites detected in the intracellular metabolome of Phycobacter cells are marked with cyan circles at each labeled carbon atom site. Right: Relative abundance of each detected labeled metabolite after addition of 100 μM 13C-Aze to Phycobacter cells, shown as total ion current (TIC). Box plot values are based on triplicates. NS indicates no significant relative abundance, * denotes p<0.05 and ** denotes p<0.005 based on a Student’s t-test.

Genomic neighborhood structure of azeR and azeTSL in Phycobacter and Pseudomonas nitroreducens DSM9128. Genes predicted in silico to belong to a single operon are marked by blue boxes. Bold-faced gene abbreviations in Phycobacter denote upregulated genes in response to Aze. Operon prediction was carried out on OperonMapper [55]. azeT=substrate-binding protein, azeS=small permease, azeL=large permease, azeR=transcriptional regulator, TR=transcriptional regulator, OR=oxidoreductase, HP=hypothetical protein, GNAT=GNAT family N-acetyltransferase, SGD=succinylglutamate desuccinylase, SBP=substrate-binding protein.

Model of Aze catabolism in Phycobacter and toxic effects in A. macleodii.

Colored boxes and circles represent metabolic pathway expression ratios and differential gene expression (log2 fold-change), respectively, at 0.5 and 8 hours for a, Phycobacter and at 0.5 hours for b, A. macleodii. White circles/boxes indicate non-statistically significant DE genes/silent pathways. Putative transcriptional factor regulation of pathways/genes is shown by purple lines. Log2 fold-change values shown for all transporters represent the mean log2 fold-change value of all genes in the cluster. Pathway expression ratios were the same as in Fig. 1 except the sign of the ratio (indicating up- or downregulation) are shown. Amino acid biosynthesis and metabolism ratios in (b) are outside the range of the pathway expression ratio scale (values ∼1.5) but are colored in dark red to indicate significant downregulation. T2SS=Type II secretion system, AHLs=acyl-homoserine lactones.

Transcriptional coexpression networks of hub transcriptional factors (TFs) azeR and xre, and their putative target genes in Phycobacter and A. macleodii.

Nodes represent genes (circles) and TFs (triangles) connected by edges based on significant co-expression correlation (PCIT; r≥0.95) for a, Phycobacter and b, A. macleodii. Nodes are grouped based on functions and represented by different colors. The size of the node corresponds to the normalized mean expression values in Aze-treated samples, whereas the color of the node border corresponds to differential expression. Edge colors indicate the direction of the correlation between each gene pair. DEG=differentially expressed gene, TF=transcriptional factor.

Bacterial and archaeal diversity in seawater treated with Aze.

a, Alpha-diversity indices of observed OTUs, Chao1, Shannon and Simpson across the Aze-treated and control samples. b, PCoA of Bray-Curtis distances (PERMANOVA: R2 = 0.73; p<0.001) between samples. The two principal components (PCoA1 and PCoA2) explained 72.5 % and 15.1 % variance, respectively. c, Relative abundance of the top 25 microbial families based on 16S rRNA gene amplicon sequencing of Aze-treated samples at T=16 hours and control samples at T=0 and T=16 hours. d, Distribution of the amplicon sequence variants (ASVs) belonging to significantly differentially abundant taxa between the Aze-treated and control samples at T=16 hours, according to their log2 fold-change and p-adjusted values. The bubble size indicates the mean taxonomic proportion of each ASV, calculated as the mean number of reads in an ASV: mean number of reads present in all ASVs of the same taxonomic classification. The bubble color indicates the taxonomic classification of each ASV according to (c).

Effect of Aze on growth of Phycobacter.

a, as a sole carbon source (1 mM) and b, in comparison to a control. For (b) Aze was added at T=0 hours to treatments while controls received an equivalent volume of Milli-Q water. RNA samples were collected at the times marked by arrows (T=0.5 hours and T=8 hours). Error bars represent standard deviation of triplicate cultures.

13C-Aze assimilation by Phycobacter and A. macleodii (reported as δ(13C) [‰, VPDB]) during a two-hour incubation.

Asterisks denote significant differences between the two bacteria (repeated measure ANOVA, n=3, p<0.001). Error bars indicate the standard deviation of the mean.

Transcriptional coexpression networks constructed using the Partial Correlation coefficient with Information Theory algorithm in Phycobacter and A. macleodii.

Initial networks (a, d) consisted of key transcriptional factors (TFs) (identified by regulatory impact factor analysis) and differentially expressed genes. Nodes are depicted either as circles for genes or triangles for TFs. Edges represent significant interactions between nodes. Edge color represents directions of the interaction (red=positive correlation and blue=negative correlation). The size of the node corresponds to the normalized mean expression values in Aze-treated samples. Subnetworks were extracted from initial networks based on significant co-expression correlation (PCIT; r ≥ ±0.95) (b, e) and those containing only hub genes (identified based on RIF scores, differential expression, and the degree centrality) and their connected genes (c, f).

Effect of Aze treatment on the alpha and beta-diversity of soil.

a, Alpha-diversity indices of observed OTUs, Chao1, Shannon and Simpson across the Aze-treated and control samples. b, PCoA of Bray-Curtis distances (PERMANOVA: R2 = 0.73; p<0.001) between samples. The two principal components (PCoA1 and PCoA2) explained 25.1 % and 14.5 % variance, respectively.

Effect of Aze treatment on the alpha and beta-diversity of A. thaliana root microbiome.

a, Alpha-diversity indices of observed OTUs, Chao1, Shannon and Simpson across the Aze-treated and control treatments. b, PCoA of Unweighted Unifrac distances between Aze-treatment and controls (PERMANOVA, p<0.05). The two principal components (PCoA1 and PCoA2) explained 13.2 % and 10.8 % variance, respectively.

Bacterial diversity in the roots of A. thaliana treated with Aze.

a, Relative abundance of the top 25 microbial families based on 16S rRNA gene amplicon sequencing of A. thaliana root-microbiome following Aze infiltration. Plant-free control is untreated soil and MES buffer and suberic acid are treatment controls. b, Distribution of the ASVs belonging to significantly differentially abundant taxa between the Aze-treated and MES buffer samples according to their log2 fold-change and p-adjusted values. The bubble color denotes the species belonging to each genus on the x-axis. c, Distribution of the ASVs belonging to significantly differentially abundant taxa between the Aze-treated and suberic acid samples according to their log2 fold-change and p-adjusted values. Bubble colors denote the species belonging to each genus on the x-axis.