Nutrient Limitation Causes Differential Expression of Transport- and Metabolism Genes in the Compartmentalized Anammox Bacterium Kuenenia stuttgartiensis

Abstract

Anaerobic ammonium-oxidizing (anammox) bacteria, members of the "Candidatus Brocadiaceae" family, play an important role in the nitrogen cycle and are estimated to be responsible for about half of the oceanic nitrogen loss to the atmosphere. Anammox bacteria combine ammonium with nitrite and produce dinitrogen gas via the intermediates nitric oxide and hydrazine (anammox reaction) while nitrate is formed as a by-product. These reactions take place in a specialized, membrane-enclosed compartment called the anammoxosome. Therefore, the substrates ammonium, nitrite and product nitrate have to cross the outer-, cytoplasmic-, and anammoxosome membranes to enter or exit the anammoxosome. The genomes of all anammox species harbor multiple copies of ammonium-, nitrite-, and nitrate transporter genes. Here we investigated how the distinct genes for ammonium-, nitrite-, and nitrate- transport were expressed during substrate limitation in membrane bioreactors. Transcriptome analysis of Kuenenia stuttgartiensis planktonic cells showed that four of the seven ammonium transporter homologs and two of the nine nitrite transporter homologs were significantly upregulated during ammonium-limited growth, while another ammonium transporter- and four nitrite transporter homologs were upregulated in nitrite limited growth conditions. The two nitrate transporters were expressed to similar levels in both conditions. In addition, genes encoding enzymes involved in the anammox reaction were differentially expressed, with those using nitrite as a substrate being upregulated under nitrite limited growth and those using ammonium as a substrate being upregulated during ammonium limitation. Taken together, these results give a first insight in the potential role of the multiple nutrient transporters in regulating transport of substrates and products in and out of the compartmentalized anammox cell.

Keywords: amtB; anammox; anammoxosome; focA; narK; nutrient limitation; planctomycete; transcriptomics.

FIGURE 1

(A) Measured ammonium and nitrite levels in the K. stuttgartiensis MBR1 bioreactor and sampling times for transcriptomic analyses, indicated with vertical lines above the graph. (B) Venn diagram showing the number of genes that were exclusively expressed during either ammonium- (yellow) or nitrite-limitation (purple), and the genes that were expressed in both conditions. (C) Venn diagram showing only the genes significantly upregulated > 1.5-fold in either ammonium- (yellow) or nitrite-limitation (purple). The overlap includes all genes with either (1) very low expression levels (basemean < 4), or (2) expression levels that were within 1.5-fold change between ammonium- and nitrite limited conditions, or (3) with changes in expression levels that were below the significance level (padj > 0.05). Note that the 707 genes not expressed in either condition are not included in the Venn diagrams. (D) Bar graph of P_II regulator genes (nrp1-4), ammonium transporter genes (amt1-4, amtb1-2, and amtB/1), nitrite transporter genes (focA1-5 and nar1), nitrate transporter genes (narK1-2) and their expression levels under both ammonium- (yellow) and nitrite (purple) limited growth. Statistical significance ns: *p = 0.05, **p = 0.01, ****p = 0.0001. RPKM values corresponding to each bar are shown in Supplementary File 2.

FIGURE 2

Schematic representation of K. stuttgartiensis MBR1 genes that are either up- or downregulated in response to ammonium- or nitrite-limiting bioreactor growth conditions. (A) graph showing all genes as dots on vertical line, with the relative expression on the y-axis. Genes indicated in translucent blue were either not significantly expressed, or not significantly different between the two conditions. Genes indicated in purple were expressed at a significantly higher level in response to ammonium limitation compared to nitrite limitation. Genes indicated as red dots were expressed at a significantly higher level in response to nitrite limitation when compared to ammonium limitation. Circles were drawn around genes that are in very close genomic proximity. XX = KSMBR1_3177, KSMBR1_3122, and KSMBR1_3846. (B) Synteny and description of genes that were expressed at a significantly different level between nitrite- and ammonium-limiting conditions. Roman numerals I–V indicate genes that were expressed relatively more during nitrite-limiting conditions, and roman numerals VI–XII indicate genes expressed relatively more during ammonium-limiting conditions.

FIGURE 3

A schematic overview of a K. stuttgartiensis cell and the differential expression of gene products related to metabolism and nutrient transport under nitrite- versus ammonium limited bioreactor growth conditions. Transporters have been drawn on the anammoxosome membrane for comparison but their localization on either of the three anammox membranes awaits experimental validation. Genes up-regulated under nitrite- relative to ammonium limiting conditions are indicated in purple and genes upregulated under ammonium- relative to nitrite-limiting conditions are indicated in orange. Genes that are not affected when grown under either nitrite- or ammonium-limiting conditions are indicated in green. A circle within each transport related gene indicates relative expression level. OM, outer membrane; IM, inner (cytoplasmic) membrane; AM, anammoxosome membrane; HK, histidine kinase domain. Proteins depicted in this image: Ammonium transporters: AmtB/amt1 (KSMBR1_2086), Amt_1 (KSMBR1_2627), Amt_2 (KSMBR1_2630), Amt_3 (KSMBR1_2632), Amtb_2 (KSMBR1_3866), Amtb_1 (KSMBR1_3722), Amt_4 (KSMBR1_3716); Ammonium transport P_II regulators: Nrp1 (KSMBR1_2628), Nrp2 (KSMBR1_2631), Nrp3 (KSMBR1_3717), Nrp4 (KSMBR1_3718); Nitrite transporters: FocA_5 (KSMBR1_3008), Nar1 (focA_6) (KSMBR1_3009), FocA_3 (KSMBR1_1474), FocA_4 (KSMBR1_1475), FocA_2 (KSMBR1_1070), FocA_1 (KSMBR1_0300), HPP (Putative nitrite transporter, KSMBR_1075); Nitrate transporters: NarK1 (KSMBR1_3317), NarK2 (KSMBR1_3299); Anammox key metabolism: NirS (KSMBR1_0452), HZS (hzsA: KSMBR1_2705, 2713, 3603), hzsB: (KSMBR1_2704, 2712 and 3602), and hzsC: (KSMBR1_2703, _2711, and 3601), NXR (A: KSMBR1_1455, B: 1458, and C: 1459), HAO (KSMBR1_2163), HAO r/b3 (KSMBR1_3792), HOX (KSMBR1_2670); Electron transport chain: FDH (KSMBR1_2469, 2470, 2471, 2479), r/b 1 (KSMBR1_1020 – 1021), r/b 3 (KSMBR1_ 3792 – 3797), r/b 2 (KSMBR1_1275 – 1283), ATPase (KSMBR1_0120 – 0130), NADH-DH (KSMBR1_3978 – 3990), ETM (unknown electron transport module).