Bacterial microcompartments (BMCs) are proteinaceous organelles that optimize specific metabolic pathways referred to as metabolosomes involving transient production of toxic volatile metabolites such as aldehydes. Previous bioinformatics analysis predicted the presence of BMCs in 23 bacterial phyla including foodborne pathogens and a link with gene clusters for the utilization of host-derived substrates such as 1,2-propanediol utilization, i.e., the Pdu cluster. Although, transcriptional regulation of the Pdu cluster and its role in Listeria monocytogenes virulence in animal models have recently been reported, the experimental identification and the physiological role of BMCs in L. monocytogenes is still unexplored. Here, we ask whether BMCs could enable utilization of 1,2-propanediol (Pd) in L. monocytogenes under anaerobic conditions. Using L. monocytogenes EGDe as a model strain, we could demonstrate efficient utilization of Pd with concomitant production of 1-propanol and propionate after 24 h of anaerobic growth, while the utilization was significantly reduced in aerobic conditions. In line with this, expression of genes encoding predicted shell proteins and the signature enzyme propanediol dehydratase is upregulated more than 20-fold in cells anaerobically grown in Pdu-induced versus non-induced control conditions. Additional proteomics analysis confirmed the presence of BMC shell proteins and Pdu enzymes in cells that show active degradation of Pd. Furthermore, using transmission electron microscopy, BMC structures have been detected in these cells linking gene expression, protein composition, and BMCs to activation of the Pdu cluster in anaerobic growth of L. monocytogenes. Studies in defined minimal medium with Pd as an energy source showed a significant increase in cell numbers, indicating that Pdu and the predicted generation of ATP in the conversion of propionyl-phosphate to the end product propionate can support anaerobic growth of L. monocytogenes. Our findings may suggest a role for BMC-dependent utilization of Pd in L. monocytogenes growth, transmission, and interaction with the human host.

Ethanolamine (EA) is a valuable microbial carbon and nitrogen source derived from cell membranes. EA catabolism is suggested to occur in a cellular metabolic subsystem called a bacterial microcompartment (BMC), and the activation of EA utilization (eut) genes is linked to bacterial pathogenesis. Despite reports showing that the activation of eut is regulated by a vitamin B12-binding riboswitch and that upregulation of eut genes occurs in mice, it remains unknown whether EA catabolism is BMC dependent in Listeria monocytogenes. Here, we provide evidence for BMC-dependent anaerobic EA utilization via metabolic analysis, proteomics, and electron microscopy. First, we show vitamin B12-induced activation of the eut operon in L. monocytogenes coupled to the utilization of EA, thereby enabling growth. Next, we demonstrate BMC formation connected with EA catabolism with the production of acetate and ethanol in a molar ratio of 2:1. Flux via the ATP-generating acetate branch causes an apparent redox imbalance due to the reduced regeneration of NAD+ in the ethanol branch resulting in a surplus of NADH. We hypothesize that the redox imbalance is compensated by linking eut BMCs to anaerobic flavin-based extracellular electron transfer (EET). Using L. monocytogenes wild-type, BMC mutant, and EET mutant strains, we demonstrate an interaction between BMCs and EET and provide evidence for a role of Fe3+ as an electron acceptor. Taken together, our results suggest an important role of BMC-dependent EA catabolism in L. monocytogenes growth in anaerobic environments like the human gastrointestinal tract, with a crucial role for the flavin-based EET system in redox balancing. IMPORTANCE Listeria monocytogenes is a foodborne pathogen causing severe illness, and as such, it is crucial to understand the molecular mechanisms contributing to pathogenicity. One carbon source that allows L. monocytogenes to grow in humans is ethanolamine (EA), which is derived from phospholipids present in eukaryotic cell membranes. It is hypothesized that EA utilization occurs in bacterial microcompartments (BMCs), self-assembling subcellular proteinaceous structures and analogs of eukaryotic organelles. Here, we demonstrate that BMC-driven utilization of EA in L. monocytogenes results in increased energy production essential for anaerobic growth. However, exploiting BMCs and the encapsulated metabolic pathways also requires the balancing of oxidative and reductive pathways. We now provide evidence that L. monocytogenes copes with this by linking BMC activity to flavin-based extracellular electron transfer (EET) using iron as an electron acceptor. Our results shed new light on an important molecular mechanism that enables L. monocytogenes to grow using host-derived phospholipid degradation products.

Ethanolamine (EA) is a valuable microbial carbon and nitrogen source derived from phospholipids present in cell membranes. EA catabolism is suggested to occur in so-called bacterial microcompartments (BMCs) and activation of EA utilization (eut) genes is linked to bacterial pathogenesis. Despite reports showing that activation of eut in Listeria monocytogenes is regulated by a vitamin B12-binding riboswitch and that upregulation of eut genes occurs in mice, it remains unknown whether EA catabolism is BMC dependent. Here, we provide evidence for BMC-dependent anaerobic EA utilization via metabolic analysis, proteomics and electron microscopy. First, we show B12-induced activation of the eut operon in L. monocytogenes coupled to uptake and utilization of EA thereby enabling growth. Next, we demonstrate BMC formation in conjunction to EA catabolism with the production of acetate and ethanol in a molar ratio of 2:1. Flux via the ATP generating acetate branch causes an apparent redox imbalance due to reduced regeneration of NAD+ in the ethanol branch resulting in a surplus of NADH. We hypothesize that the redox imbalance is compensated by linking eut BMC to anaerobic flavin-based extracellular electron transfer (EET). Using L. monocytogenes wild type, a BMC mutant and a EET mutant, we demonstrate an interaction between BMC and EET and provide evidence for a role of Fe3+ as an electron acceptor. Taken together, our results suggest an important role of anaerobic BMC-dependent EA catabolism in the physiology of L. monocytogenes, with a crucial role for the flavin-based EET system in redox balancing.

Bacterial microcompartments (BMCs) are proteinaceous prokaryotic organelles that enable the utilization of substrates such as 1,2-propanediol and ethanolamine. BMCs are mostly linked to the survival of particular pathogenic bacteria by providing a growth advantage through utilization of 1,2-propanediol and ethanolamine which are abundantly present in the human gut. Although a 1,2-propanediol utilization cluster was found in the probiotic bacterium Propionibacterium freudenreichii, BMC-mediated metabolism of 1,2-propanediol has not been demonstrated experimentally in P. freudenreichii. In this study we show that P. freudenreichii DSM 20271 metabolizes 1,2-propanediol in anaerobic conditions to propionate and 1-propanol. Furthermore, 1,2-propanediol induced the formation of BMCs, which were visualized by transmission electron microscopy and resembled BMCs found in other bacteria. Proteomic analysis of 1,2-propanediol grown cells compared to L-lactate grown cells showed significant upregulation of proteins involved in propanediol-utilization (pdu-cluster), DNA repair mechanisms and BMC shell proteins while proteins involved in oxidative phosphorylation were down-regulated. 1,2-Propanediol utilizing cells actively produced vitamin B12 (cobalamin) in similar amounts as cells growing on L-lactate. The ability to metabolize 1,2-propanediol may have implications for human gut colonization and modulation, and can potentially aid in delivering propionate and vitamin B12in situ.

L. monocytogenes has evolved various mechanisms for carbon source utilization, stress adaptation and virulence factors that allow for transmission from the food environment to the human GI tract. The mechanisms of carbon sources utilization and stress adaptation discovered in this bacterium include numerous catabolic enzymes, transporter systems and gene expression regulating proteins [1, 6, 12, 22, 23]. Activation of L. monocytogenes virulence factors is primarily regulated via transcription regulator PrfA (positive regulatory factor A) [1, 3, 24]. The activity of PrfA is modulated by selected environmental signals at the transcriptional and post-transcriptional level, including temperature and the availability of efficiently metabolizable carbon sources [1, 24]. For example glycerol and lactose, resulted in differential activation of SigB and stress resistance in L. monocytogenes [25].PrfA expression is also controlled through stress response regulatory proteins such as Sigma B [24, 26]. Therefore, the impact of carbon source utilization on stress resistance activation and virulence of L. monocytogenes deserves more detailed analysis.L. monocytogenes is a well-studied model for intracellular infection [1, 3]. L. monocytogenes binds to epithelial host cells and promotes invasion in a process mediated by InlA (Internalin A) [1,[27][28][29] and InlB (Internalin B) [26, 28, 29], but also other factors have been reported to contribute to the process, including Listeria Adhesion Protein (LAP) [1, 30, 31] and other host cell surface modulation Shell Proteins of BMCsBMCs are typically about 40-200 nm in diameter and are made of three types of shell proteins: hexamers (BMC-H), pseudohexamers (BMC-T), and pentamers (BMC-P) [52-54]. Hexamers and pseudohexamers are formed by the classical BMC shell proteins containing the Pf00936 domain, while pentamers are formed by the non-classical BMC shell proteins containing the Pf03319 domain [50, 55].A0A0B6X1Q4 Carboxysome shell protein Acidobacteria Pyrinomonas methylaliphatogenes 5.80E-24 A0A0B6X4B3 Carboxysome shell protein Acidobacteria Pyrinomonas methylaliphatogenes 2.60E-23 A0A143PG95 Ethanolamine utilization protein EutM Acidobacteria Luteitalea pratensis 1.70E-22 A0A0B6X296 Carboxysome shell protein Acidobacteria Pyrinomonas methylaliphatogenes 2.60E-21 A0A143PYA9 Ethanolamine utilization protein EutM Acidobacteria Luteitalea pratensis 2.60E-20 Q02C23 Microcompartments protein Acidobacteria Solibacter usitatus (strain Ellin6076) 3.40E-20 A0A143PPE2 Propanediol utilization protein PduA Acidobacteria Luteitalea pratensis 5.30E-20 Q02C83 Microcompartments protein Acidobacteria Solibacter usitatus (strain Ellin6076) 7.40E-20 Q029G1 Microcompartments protein Acidobacteria Solibacter usitatus (strain Ellin6076) 4.40E-18 A8AEM1 Uncharacterized protein Proteobacteria Citrobacter koseri ATCC BAA-895 2.10E-40 D2TPS2 Propanediol utilization protein PduT Proteobacteria Citrobacter rodentium (strain ICC168) 3.60E-40 A0A1T4W4Z8 Shell CcmL/EutN Proteobacteria Desulfovibrio bizertensis...