Differential protein expression during growth on model and commercial mixtures of naphthenic acids in Pseudomonas fluorescens Pf-5

Abstract

Naphthenic acids (NAs) are carboxylic acids with the formula (C_n H_2n+Z O₂ ) and are among the most toxic, persistent constituents of oil sands process-affected waters (OSPW), produced during oil sands extraction. Currently, the proteins and mechanisms involved in NA biodegradation are unknown. Using LC-MS/MS shotgun proteomics, we identified proteins overexpressed during the growth of Pseudomonas fluorescens Pf-5 on a model NA (4'-n-butylphenyl)-4-butanoic acid (n-BPBA) and commercial NA mixture (Acros). By day 11, >95% of n-BPBA was degraded. With Acros, a 17% reduction in intensity occurred with 10-18 carbon compounds of the Z family -2 to -14 (major NA species in this mixture). A total of 554 proteins (n-BPBA) and 631 proteins (Acros) were overexpressed during growth on NAs, including several transporters (e.g., ABC transporters), suggesting a cellular protective response from NA toxicity. Several proteins associated with fatty acid, lipid, and amino acid metabolism were also overexpressed, including acyl-CoA dehydrogenase and acyl-CoA thioesterase II, which catalyze part of the fatty acid beta-oxidation pathway. Indeed, multiple enzymes involved in the fatty acid oxidation pathway were upregulated. Given the presumed structural similarity between alkyl-carboxylic acid side chains and fatty acids, we postulate that P. fluorescens Pf-5 was using existing fatty acid catabolic pathways (among others) during NA degradation.

Keywords: Pseudomonas fluorescens; naphthenic acids; oil sands process-affected water; proteomics; tailing ponds; toxicity.

FIGURE 1

Structure of (4′‐n‐butylphenyl)‐4‐butanoic acid (n‐BPBA) (a) and (4′‐n‐butylphenyl)ethanoic acid (n‐BPEA) (b)

FIGURE 2

Degradation of n‐BPBA and production of (4′‐n‐butylphenyl)ethanoic acid (n‐BPEA) metabolite by Pseudomonas fluorescens Pf‐5 (a) compared to abiotic controls (b) following 11 days of incubation

FIGURE 3

Heat map of the percentage change for Acros commercial NA species, plotted by carbon number and Z, following 11 days of incubation. Percent changes are calculated relative to killed control following 11 days of incubation

FIGURE 4

Overview of the proteomic analysis of P. fluorescens Pf‐5 growing on naphthenic acids (NAs) compared with pyruvate controls. Venn diagram comparing the common and unique proteins detected and identified during growth on the three substrates. (a) PCA analysis highlighting highly similar replicate proteomes that differ significantly with growth substrate (b), Volcano plots of normalized LC‐MS/MS spectral counts comparing P. fluorescens Pf‐5 during growth on NAs compared with pyruvate controls (c and d). Red points: proteins where p < 0.05 and above two‐fold difference; the green point above horizontal dotted line: proteins where p < 0.05 but below two‐fold difference; green points below horizontal dotted line: proteins above two‐fold difference but not statistically significant; blue points: proteins below two‐fold differential expression and not statistically significant; horizontal dashed line: p = 0.05; vertical dashed lines: two‐fold difference. Thus, all red points represent proteins that are above two‐fold difference (vertical dashed lines) and statistically significant (horizontal dashed line, p = 0.05)

FIGURE 5

Mean spectral count n‐BPBA, (black bars), Acros commercial NA mixture (gray bars), and control (white bars) for metabolism functional COG categories

FIGURE 6

Partial enzymatic pathway diagram for fatty acid degradation in Pseudomonas fluorescens Pf‐5

FIGURE A1

Growth of Pseudomonas fluorescens Pf‐5 in minimal salts medium supplemented with either n‐BPBA or Acros commercial NA mixture (a) or pyruvate control (b)

FIGURE A2

Distribution of NA compounds fitting the molecular formula C_nH_2n+zO₂ from C₇ to C₂₂ faceted by hydrogen deficiency (Z). NAs were acid‐extracted from Pseudomonas fluorescens Pf‐5 cultures containing a commercial NA mixture (Acros) following 11 days incubation (Live) or in killed controls (Killed). Peak areas are normalized to the internal standard. Note differences in y‐scale among faceted plots

FIGURE A3

KEGG pathway diagram for fatty acid degradation in Pseudomonas fluorescens Pf‐5. Enzymes 1.3.8.1 (acyl‐CoA dehydrogenase, Q4KC62) and 2.3.1.9 (acetyl‐CoA C‐acetyltransferase, Q4KEA5) were upregulated by 27 to 30‐fold and 7 to 9‐fold on the different NA mixtures. Enzymes 1.1.1.35 (3‐hydroxyacyl‐CoA dehydrogenase, Q4KC60) and 4.2.1.17 (enoyl‐CoA hydratase, Q4KC63) were upregulated by between 3 to 4‐fold and 3.5‐fold, respectively. Upregulated enzymes are highlighted in red boxes

FIGURE A4

KEGG pathway diagram for amino acid degradation in Pseudomonas fluorescens Pf‐5. Upregulated enzymes 1.3.8.1 (acyl‐CoA dehydrogenase, Q4KC62) and 6.4.1.4 (acetyl/propionyl/methylcrotonyl‐CoA carboxylase subunit alpha, Q4K9P4) are highlighted in red boxes. Enzyme 1.1.1.35 (3‐hydroxyacyl‐CoA dehydrogenase, Q4KC60) and enzyme 2.3.1.9 (acetyl‐CoA C‐acetyltransferase, Q4KEA5) are also shown