Volume 27, Issue 17, Pages 2579-2588.e6 (September 2017) Transposon Sequencing Uncovers an Essential Regulatory Function of Phosphoribulokinase for Methylotrophy  Andrea M. Ochsner, Matthias Christen, Lucas Hemmerle, Rémi Peyraud, Beat Christen, Julia A. Vorholt  Current Biology  Volume 27, Issue 17, Pages 2579-2588.e6 (September 2017) DOI: 10.1016/j.cub.2017.07.025 Copyright © 2017 Elsevier Ltd Terms and Conditions

Current Biology 2017 27, 2579-2588.e6DOI: (10.1016/j.cub.2017.07.025) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Experimental Setup of Transposon Sequencing Colonies containing random transposon insertions formed on methanol or succinate agar plates are washed off, DNA is extracted, and insertion sites are amplified using a semi-arbitrary nested PCR. The products are then sequenced using paired-end Illumina sequencing, the reads are mapped to the genome, and every gene is rated as “essential” (or “fitness relevant”) or “non-essential” on methanol as well as on succinate, here exemplarily shown for a hypothetic gene. Current Biology 2017 27, 2579-2588.e6DOI: (10.1016/j.cub.2017.07.025) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Genome-wide Identification of Methylotrophy Genes (A) The Z score, a measure to compare insertion densities between the two conditions, is plotted for every gene in the genome. Genes that have fewer insertions on methanol have a positive value, whereas genes that have fewer insertions on succinate have a negative value. Only the section between −10 and 100 is shown to highlight the methylotrophy genes; for the entire figure, refer to Figure S2. The color of the dot represents the assigned class: non-essential under both conditions (light gray); required under both conditions or only on succinate (dark gray); and required on methanol only (pink). Known genes and clusters (marked with boxes) are shown in black, whereas some newly identified genes and clusters are shown in blue. All labeled genes are listed with full names in Data S1. (B) Insertions detected in selected clusters highlighted in (A). Insertions on methanol are shown in blue and insertions on succinate in gray. Arrow length corresponds to gene length and arrow color to essentiality data as determined in the screen. Genes that are only essential or fitness relevant on methanol are shown in pink (dark and light), genes that are required under both conditions are shown in dark gray, and genes that are not required under either condition are shown in white. See also Figures S1 and S2. Current Biology 2017 27, 2579-2588.e6DOI: (10.1016/j.cub.2017.07.025) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Functional Categorization of Shared and Specific Required Genes The number of genes belonging to each functional category assigned by the MicroScope platform (see Key Resources Table) is shown. “Total” shows the distribution of all ORFs in the genome, “shared” shows the distribution of all genes that are either essential or fitness relevant on both substrates, whereas “succinate” and “methanol” show the distributions of genes only essential or fitness relevant on succinate or methanol, respectively. For the methanol-specific genes, the known genes are shown as hatched parts. See also Data S1. Current Biology 2017 27, 2579-2588.e6DOI: (10.1016/j.cub.2017.07.025) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Integration of Conditional Gene Essentiality Using the Genome-Scale Model The central part of the genome-scale model is shown. Reversible reactions are shown as double arrows, reactions that show gene redundancy in the model are shown with parallel arrows, and reactions that have no gene assigned are shown as hatched arrows. Gene requirement is indicated by colors: gray for reactions not required under either conditions; black for reactions required (essential or fitness relevant) under both conditions; and pink or green for genes required only on methanol or succinate, respectively. Lipoic acid (lipAB) biosynthesis is not in the model and was added manually. See also Figure S3 and Data S3. Current Biology 2017 27, 2579-2588.e6DOI: (10.1016/j.cub.2017.07.025) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Analysis of Phosphoribulokinase (A) Transposon insertions into the phosphoribulokinase gene (prk) and the neighboring genes encoding for the serine cycle regulator (qscR) and fructose-1,6-bisphosphate phosphatase (fbp). (B) Growth of wild-type (wt), prk knockout (Δprk), Δprk suppressor (Δprk_sup), and complementation of Δprk with prk from P. denitrificans (Δprk+prk [Pden]), endogenous prk (Δprk+prk), or endogenous prk carrying an active-site mutation (Δprk+prk D42A) on a methanol plate. (C) Extracted ion chromatogram of ribulose-1,5-bisphosphate (RuBP) in the wild-type strain grown on methanol or succinate. (D) Investigation of serine glyoxylate aminotransferase (sga) expression level using fluorescence quantification of a reporter construct of Psga with mCherry on succinate (green) and methanol (pink) in different backgrounds: wt; knockouts in prk (Δprk) and qscR (ΔqscR); and the reconstructed Δprk suppressor strain that carries a mutation in qscR (Δprk_sup). Δprk and ΔqscR were only measured on succinate because they show no growth on methanol. Mean ± SD is shown (n = 8). (E) Protein abundance of three serine cycle proteins in the Δprk strain compared to the Δprk suppressor strain grown on succinate: serine glyoxylate aminotransferase (Sga); malate thiokinase subunit A (MtkA); and glycerate kinase (Gck). Mean ± SD is shown (n = 4). (F) Model for the function of Prk in methylotrophy. See also Figure S4, Table S1, and Data S4. Current Biology 2017 27, 2579-2588.e6DOI: (10.1016/j.cub.2017.07.025) Copyright © 2017 Elsevier Ltd Terms and Conditions