Aerobic adaptation and metabolic dynamics of Propionibacterium freudenreichii DSM 20271: insights from comparative transcriptomics and surfaceome analysis

Abstract

Propionibacterium freudenreichii (PFR) DSM 20271^T is a bacterium known for its ability to thrive in diverse environments and to produce vitamin B12. Despite its anaerobic preference, recent studies have elucidated its ability to prosper in the presence of oxygen, prompting a deeper exploration of its physiology under aerobic conditions. Here, we investigated the response of DSM 20271^T to aerobic growth by employing comparative transcriptomic and surfaceome analyses alongside metabolite profiling. Cultivation under controlled partial pressure of oxygen (pO₂) conditions revealed significant increases in biomass formation and altered metabolite production, notably of vitamin B12, pseudovitamin-B12, propionate, and acetate, under aerobic conditions. Transcriptomic analysis identified differential expression of genes involved in lactate metabolism, tricarboxylic acid cycle, and electron transport chain, suggesting metabolic adjustments to aerobic environments. Moreover, surfaceome analysis unveiled growth environment-dependent changes in surface protein abundance, with implications for adaptation to atmospheric conditions. Supplementation experiments with key compounds highlighted the potential for enhancing aerobic growth, emphasizing the importance of iron and α-ketoglutarate availability. Furthermore, in liquid culture, FeSO₄ supplementation led to increased heme production and reduced vitamin B12 production, highlighting the impact of oxygen and iron availability on the metabolic pathways. These findings deepen our understanding of PFR's physiological responses to oxygen availability and offer insights for optimizing its growth in industrial applications.

Importance: The study of the response of Propionibacterium freudenreichii to aerobic growth is crucial for understanding how this bacterium adapts to different environments and produces essential compounds like vitamin B12. By investigating its physiological changes under aerobic conditions, we can gain insights into its metabolic adjustments and potential for enhanced growth. These findings not only deepen our understanding of P. freudenreichii's responses to oxygen availability but also offer valuable information for optimizing its growth in industrial applications. This research sheds light on the adaptive mechanisms of this bacterium, providing a foundation for further exploration and potential applications in various fields.

Keywords: Propionibacterium; aerobic; anaerobic; cobamides; surfaceomics; transcriptomics; vitamin B12.

Fig 1

(A) A workflow illustrating the sequence of experiments, including bioreactor cultivations with studied parameters and follow-up growth experiments conducted in media supplemented with aspartate (Asp), succinate (SA), and α-ketoglutarate (α-KG). (B) Cell density (dotted line) and viable cell counts (solid line) in bioreactor-conducted experiments under aerobic (green) and anaerobic (orange) growth conditions. Sampling points for OD₆₀₀ measurements and colony-forming unit determinations are marked with squares (aerobic) and circles (anaerobic), respectively. Key metabolite production dynamics and transcriptomic analyses were performed over four growth stages: mid-logarithmic (I), late logarithmic (II), early stationary (III), and late stationary (IV) phases. RNA sequencing was conducted on samples I and III. Red arrows indicate the specific time points/growth stages at which the cells were sampled for transcriptomic (T) and surfaceomic (S) analyses.

Fig 2

Lactate utilization (A), excreted propionate (B), acetate (C), pyruvate (D), succinate (E), and intracellular vitamin B12 and pseudo-B12 (F; vitamin B12 – solid line, pseudo-B12 – dashed line) of PFR DSM 20271 during cultivation under aerobic (green) and anaerobic (orange) fermentations performed in bioreactors. N = 3, except pseudo-B12 samples III and IV: n = 2. No pseudo-B12 was detected under aerobic growth conditions.

Fig 3

Differential expression of genes in PFR DSM 20271 across two sampling points under aerobic versus anaerobic growth conditions. (A) A total of 1,375 genes exhibited significant differential expression (adjusted P-value < 0.05), with 713 genes downregulated and 662 upregulated at sampling point I, and 906 genes were differentially expressed at sampling point III, including 388 downregulated and 518 upregulated. (B) Venn diagram illustrating the overlap of differentially expressed genes (DEGs) at the two sampling points. Sampling point I coincides with the logarithmic growth phase, while sampling point III corresponds to the stationary phase. Of the 662 upregulated DEGs at sampling point I, 300 remained upregulated and 19 were downregulated at sampling point III. Additionally, 277 of the 713 downregulated DEGs at sampling point I remained downregulated at sampling point III, and 11 were upregulated.

Fig 4

Schematic view of metabolism during aerobic and anaerobic growth. The presentation of the Wood–Werkman and TCA cycles was adapted from (50–52). Differentially expressed genes (≥ twofold difference, adjusted P-value < 0.05) are shown in green (increased aerobically) and orange (increased anaerobically). The pathways with the most differentially expressed genes, the TCA cycle and the C5 pathway for porphyrin synthesis, are highlighted with a green background. A tentative diagram of the aerobic electron transport chain, consisting of NADH dehydrogenase/complex I (NDH-I), succinate dehydrogenase/fumarate reductase (SDH/FDR), cytochrome bd complex (CytBD), and ATP synthase (ATPase), is shown, as well as the predicted transmembrane transport systems for iron and iron siderophore with detected with higher abundance under aerobic condition RNA-seq and surfaceome analyses. Putative membrane spanning structures of nitrate and DMSO reductases possibly involved in anaerobic respiration are shown. Differentially abundant proteins (FDR 0.05) detected in surfaceome analysis are indicated using green/orange fonts.

Fig 5

Genes associated with transport, import, or export that are upregulated (adjusted P-value ≤ 0.05) under aerobic conditions at Sampling Point I (SP_I) and/or Sampling Point III (SP_III) compared to anaerobic conditions. Genes exhibiting an upregulation fold change of ≥2.0 (log2FoldChange ≥ 1) are marked with an asterisk.

Fig 6

Proteomic analysis of PFR DSM 20271^T surfaceomes. (A) Venn diagram indicates uniquely and intersectively identified proteins in aerobic and anaerobic growth environments. (B) Subcellular localization of the identified surfaceomes (predicted by PSORTb v. 3.0.3). (C) The principal component analysis (PCA) plot of the LFQ intensities of the individual samples with three replicates. PCA identifies a correlation between the protein abundance profiles between aerobically (green) and anaerobically (orange) cultured bacterial cells. (D) Functional categorization of proteins that were either uniquely identified from aerobic (green) or anaerobic (orange) culture or differentially abundant between the two growth environments (predicted by eggNOG 5.0.0).

Fig 7

Colony formation of DSM 4902 (1) and DSM 20271 (2) under anaerobic (-O2) and aerobic (+O2) conditions on YEL agar plates with and without supplementation of FeSO₄ (1 mM), aspartic acid (10 mM), α-ketoglutaric acid (1 mM), or succinic acid (1 mM) after 4 (anaerobic) or 6 (aerobic) days of incubation at 30°C. The images are representative of experiments repeated at least three times for each condition. YEL, yeast extract–lactate medium; a-KG, a-ketoglutarate; Asp, aspartic acid; SA, succinic acid.

Fig 8

Effect of FeSO₄ (1 mM) supplementation on the biosynthesis of heme (A), vitamin B12 (B), and accumulation of organic acids (C) when grown under microaerobic and anaerobic conditions in yeast extract–lactate medium supplemented with CoCl₂ (5 mg/L). The values are means and standard deviations of three biological replicates. A significant difference between the treatments (P < 0.05) is indicated by an asterisk.