Growth and gene expression of Pseudomonas nitroreducens TX1 on octylphenol polyethoxylate surfactants

Abstract

Pseudomonas nitroreducens ATCC PTA-6168 (also named TX1) efficiently grows on non-ionic surfactants as the sole source of carbon and energy aerobically. The global gene expression and bacterial stress responses during degradation remain further investigated. This study compared the growth, viability, and transcriptomic profiles of strain TX1 during the log phase when grown on minimal salts basal medium supplemented with 0.5% octylphenol polyethoxylates (OPEO_n, commercial name Triton^® X-100) vs. in 0.5% succinate. Differentially expressed genes were identified, with 219 upregulated and 22 downregulated. Gene ontology analysis revealed upregulation of oxidoreductase activity (53%), electrotransfer activity (11%), heme/FAD/cofactor binding (11/19/27%), and membrane (42%)-related functions. In ethanol oxidation, adh18 and adh19 (for two pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases), aldh1 and aldh2 (for two aldehyde dehydrogenases), c₅₅₀ (for cytochrome c₅₅₀), and pqqABCDEF (for PQQ biosynthesis proteins) and aceA (for isocitrate lyase) and aceB (for malate synthase) acting in the glyoxylate cycle were upregulated. In addition to the above genes, the expression level of the representative genes from fatty acid degradation and chemotaxis was verified by RT-qPCR. The global gene expression of P. nitroreducens TX1 provides candidate genes involved in the bacteria grown on nonionic surfactants. KEY POINTS: • Growth characteristics of Pseudomonas nitroreducens ATCC PTA-6168 on surfactants • Differentially expressed genes related to growth on surfactants were identified • The ethanol oxidation system was upregulated when grown on surfactants.

Keywords: Pseudomonas nitroreducens; Alcohol dehydrogenase; Differentially expressed genes; Ethanol oxidation system; Octylphenol polyethoxylates; Transcriptomic analysis.

Fig. 1

Growth of P. nitroreducens TX1 in MSB medium containing 0.5% succinate or 0.5% OPEO_n. Bacterial growth was determined by measuring a the optical density (OD) and by b viable cell counting (colony-forming units per milliliter, CFU/mL). The green arrows indicate the harvesting times for RNA-seq analysis. Succinate or OPEO_n designates 0.5% succinate or 0.5% OPEO_n in the MSB. Generation times (hours) were calculated based on c OD and d CFU values during the logarithmic growth phase (2- to 6-h period for succinate and 4- to 12-h period for OPEO_n) with regression lines. e The generation time values are composited and compared from two media and by OD and CFU. All data represent the mean ± SEM of three biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant

Fig. 2

Gene expression profile of P. nitroreducens TX1 cultured in MSB/OPEO_n vs. MSB/succinate. Transcriptomes of TX1 wild-type strains were harvested in the log phase at four hours or ten hours on succinate and OPEO_n medium, respectively. a Correlation matrix of each comparison pair labeled with Spearman’s correlaztion coefficient. b Heatmap of hierarchical cluster of all genes. c Principal-component analysis (PCA) of the transcriptomes from the comparison set. S, MSB/S and O, MSB/O. S1, S2, and S3 are three replicate experiments on the MSB/S medium. O1, O2, and O3 are three replicate experiments on the MSB/OPEO_n medium

Fig. 3

Transcriptomic analysis of P. nitroreducens TX1 cultured in MSB/OPEO_n vs. succinate. a Volcano plot shows the DEGs. The red dots indicate the significantly upregulated genes, and the blue dots represent the significantly downregulated genes (absolute log₂ fold change > 2 and adjusted p < 0.05); grey dots correspond to genes that are not significantly differentially expressed. b Chord graph of the ontology enrichment analysis result of the DEGs. On the right side of the diagram, the enriched GO biological processes are presented, while on the left side, the DEGs contributing to this enrichment are displayed. Within the left portion of the circular diagram, individual DEGs are symbolized by rectangles, with each rectangle’s color corresponding to the log₂FC value. Specifically, upregulated genes are denoted in red, while downregulated genes are represented in blue. Chords within the diagram connect gene names with their respective biological process GO term groups, delineating each GO term by a colored line. CC, cellular components; MF, molecular functions. c KEGG pathway enrichment analysis of DEGs. The y-axis shows the significantly enriched pathways with an adjusted p < 0.1, the x-axis indicates the number of DEGs enriched in the pathway. The color gradient from blue to red indicates a correlation with the level of significance of the enrichment. d COG function classification of the annotated DEGs. The x-axis represents different function classes, while the y-axis shows the number of genes in each function class

Fig. 4

Upregulation of ethanol oxidation system in P. nitroreducens TX1 when grown on MSB/OPEO_n vs. succinate. a Genetic map of the DNA region containing NAD(P)-independent ADH cluster. Arrows indicate gene position and orientation of transcription of the gene. The mRNA expression level is described in log₂ fold change, and the bold number indicates the upregulated mRNA. Color intensity indicates the expression level of the corresponding genes. b Organization of the genes coding for ethanol oxidation in P. nitroreducens TX1, P. aeruginosa PAO1, and P. putida KT2440. Arrows indicate the direction of gene transcription and the relative size of each open reading frame (ORF); the same color represents the same function; the percentages indicate sequence identity between encoded proteins from each strain. ▭, coenzyme PQQ synthesis proteins

Fig. 5

Validation of transcriptomic analysis by qRT-PCR. The log₂ fold changes in expression levels of selected genes when P. nitroreducens TX1 was grown on OPEO_n vs. succinate using RNA-seq and qRT-PCR. Upregulated genes include the following: glyoxylate and dicarboxylate metabolism (aceA, aceB, aldh2, fadA1, glcD, and glcF), ethanol oxidation system (adh18, adh19, aldh1, aldh2, c₅₅₀, pqqB, and pqqE), glycolysis/gluconeogenesis (acs2, adh18, adh19, and aldh1), fatty acid degradation (fadA1, fadA2, fadB1, and fadB2), and chemotaxis pathway (cheA and tar). Downregulated genes include hcp2 and mqo2. The encoded products of the above genes are as follows: aceA, isocitrate lyase; aceB, malate synthase; glcD and glcF, glycolate oxidase; adh18, quinoprotein alcohol dehydrogenase; adh19, quinohemoprotein alcohol dehydrogenase; aldh1 and aldh2, aldehyde dehydrogenases; c₅₅₀, cytochrome c₅₅₀; pqqB, coenzyme PQQ synthesis protein B; pqqE, coenzyme PQQ synthesis protein E; acs2, acetyl-coenzyme A synthetase; fadA1 and fadA2, 3-ketoacyl-CoA thiolases; fadB1 and fadB2, 3-hydroxyacyl-CoA dehydrogenases; cheA, chemotaxis protein histidine kinase CheA; tar, methyl-accepting chemotaxis protein; hcp2, type VI protein secretion system component Hcp; and mqo2, malate: quinone oxidoreductase. All data represent the mean ± SEM of three replicates

Fig. 6

Summary of metabolic pathways regulated when strain TX1 is grown on MSB/0.5% OPEO_n vs. 0.5% succinate. TCA and glyoxylate cycle: aceA, isocitrate lyase; aceB, malate synthase; glcDEF, glycolate oxidase. Ethanol oxidation system: adh18 and adh19, quino(hemo)protein alcohol dehydrogenases; adh1, NAD(P)-dependent ADH; aldh1, aldh2, aldh3, and aldh4, aldehyde dehydrogenases; acs1 and acs2, acetyl-CoA synthetases; c₅₅₀, cytochrome c₅₅₀; pqqABCDEF, PQQ synthesis gene cluster; PQQ, pyrroloquinoline quinone. Two-component system: SK, sensor kinase (a histidine kinase); H, histidine residue; P, phosphate group