Bacterial microcompartments linked to the flavin-based extracellular electron transfer drives anaerobic ethanolamine utilization inListeria monocytogenes

Abstract

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.

Anaerobic growth ofListeria monocytogeneson rhamnose is stimulated by Vitamin B12 and bacterial microcompartment dependent 1,2-propanediol utilization

“…Additional end product analysis at 72 h shows accumulation of approximately 6.7 mM of 1,2-propanediol in pdu non-induced condition and nearly zero production of propionate and 1propanol. In pdu induced condition a significant lower amount of 1,2-propanediol is found, about 1.4 mM, and higher levels of approximately 3.4 mM propionate and 3.6 mM 1-propanol are produced at 72 h, in line with the expected 1:1 molar stoichiometry of L. monocytogenes BMC-dependent pdu [9]. Enhanced rhamnose metabolism in pdu-induced cells is also evident from production of acetate and lactate.…”

“…Thin sections (<100 nm) of polymerized resin samples were obtained with microtomes. After staining with 2% (w/v) aqueous uranyl acetate, the samples were analyzed with a Jeol 1400 plus TEM with 120 kV setting [8,9].…”

“…Samples were prepared according to the filter assisted sample preparation protocol (FASP) with the following steps: reduction with 15 mM dithiothreitol, alkylation with 20 mM acrylamide, and digestion with sequencing grade trypsin overnight [22]. Each prepared peptide sample was analyzed by injecting (18 μl) into a nanoLC-MS/MS (Thermo nLC1000 connected to a LTQ-Orbitrap XL) as described previously [8,9]. LCMS data with all MS/MS spectra were analyzed with the MaxQuant quantitative proteomics software package as described before [8,9,23].…”

“…Each prepared peptide sample was analyzed by injecting (18 μl) into a nanoLC-MS/MS (Thermo nLC1000 connected to a LTQ-Orbitrap XL) as described previously [8,9]. LCMS data with all MS/MS spectra were analyzed with the MaxQuant quantitative proteomics software package as described before [8,9,23]. A protein database with the protein sequences of L. monocytogenes EGDe (ID: UP000000817) was downloaded from UniProt.…”

“…Recent studies on anaerobic growth of L. monocytogenes have provided evidence that it has the capacity to form proteinaceous organelles so-called bacterial microcompartments (BMCs) that enable extension of its metabolic repertoire by supporting the utilization of 1,2-propanediol and ethanolamine [7][8][9]. BMCs are selfassembling organelles that consist of an enzymatic core that is encapsulated by a semi-permeable protein shell [7,10,11].…”