Anaerobic growth of Listeria monocytogenes on rhamnose is stimulated by Vitamin B12 and bacterial microcompartment dependent 1,2-propanediol utilization

The food-borne pathogen Listeria monocytogenes is able to form proteinaceous organelles called bacterial microcompartments (BMCs) that optimize the utilization of substrates, such as 1,2-propanediol, and confer an anaerobic growth advantage. Rhamnose is a deoxyhexose sugar abundant in a range of environments including the human intestine, and can be degraded in anaerobic conditions into 1,2-propanediol, next to acetate and lactate. Rhamnose-derived 1,2-propanediol has been found to link with BMCs in a limited number of commensal human colonic species and some human pathogens such as Salmonella enterica, but the involvement of BMCs in rhamnose metabolism and potential physiological effects on L. monocytogenes are still unknown. In this study, we firstly test the effect of rhamnose uptake and utilization on anaerobic growth of L. monocytogenes EGDe without and with added vitamin B12, followed by metabolic analysis. We unveil that the vitamin B12-dependent activation of pdu stimulates metabolism and anaerobic growth of L. monocytogenes EGDe on rhamnose via 1,2-propanediol degradation into 1-propanol and propionate. Transmission electron microscopy of pdu-induced cells shows that BMCs are formed and additional proteomics experiments confirm expression of pdu BMC shell proteins and enzymes. Finally, we discuss physiological effects and energy efficiency of L. monocytogenes pdu BMC-driven anaerobic rhamnose metabolism and impact on competitive fitness in environments such as the human intestine.


Introduction
Listeria monocytogenes is a Gram-positive facultative anaerobe and a food-borne pathogen which causes a severe human infection called listeriosis [1,2]. The pathogen continues to cause food-borne illness outbreaks characterised by high mortality ranging from 20 to 30% [1,3]. L. monocytogenes is found ubiquitously in natural environments and it can survive a variety of stress conditions leading to the colonization of different niches including a range of food processing environments [1, 3,4]. To survive in such a variety of niches, L. monocytogenes should be able to adapt to environmental stresses and to use a range of nutrients for growth in aerobic and anaerobic conditions [1, 5,6].
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]. The separation of the encapsulated enzymes from the cytosol is thought to protect the cell from toxic metabolic intermediates such as aldehydes, and prevent unwanted side reactions [7,10,11]. In our previous studies, we showed that the L. monocytogenes 1,2-propanediol utilization gene cluster (pdu) is activated in the presence of 1,2-propanediol and vitamin B12, resulting in stimulation of growth in anaerobic conditions [8]. Vitamin B12 is required for activation of the pdu cluster in L. monocytogenes [8,12] and to act as a cofactor of 1,2-propoanediol reductase [13]. Activation of BMC-dependent pdu supports degradation of 1,2-propanediol via the toxic intermediate propionaldehyde into 1-propanol and propionate via respective reductive and oxidative branches, with the latter resulting in extra ATP generation leading to enhanced anaerobic growth of L. monocytogenes [8]. Notably, 1,2-propanediol is a major end product from the anaerobic degradation of mucus-derived rhamnose by human intestinal microbiota and it is thought to be an important energy source supporting intestinal growth of selected pathogens such as Salmonella spp. and L. monocytogenes [7,[14][15][16].
Rhamnose is a naturally occurring deoxyhexose sugar abundant in glycans on surfaces of mammalian and bacterial cells and in cell walls of many plant and insect species [14,17]. Anaerobic metabolism of rhamnose has been studied previously in a range of bacteria including E. coli , and rhamnose is parallelly metabolized into lactaldehyde and dihydroxyacetone phosphate (DHAP) [18,19]. DHAP is converted in the glycolytic pathway leading to a variety of fermentation products, while lactaldehyde is converted to 1,2-propanediol that is subsequently secreted [18,19]. Notably, for example in Salmonella spp. and Clostridium phytofermentans, rhamnose-derived 1,2-propanediol can be converted to 1-propanol and propionate via BMC-dependent pdu [14,16]. Although rhamnose-derived 1,2-propanediol was found to be metabolised via a pduD-dependent pathway in Listeria innocua [20], the possible activation and contribution of BMC-dependent pdu to anaerobic metabolism and growth of L. monocytogenes on rhamnose remains to be investigated.
In this study, we firstly quantified the effect of rhamnose as sole carbon source on anaerobic growth and metabolism of L. monocytogenes in absence and presence of vitamin B12 (cobalamine), an essential co-factor of 1,2-propoanediol reductase, the signature enzyme of BMC-dependent pdu [13] Next, we analysed rhamnose utilization and end product formation, and combined with Transmission Electron Microscopy and proteomics, we provide evidence for a B12-dependent pdu-induced metabolic shift. We summarize our findings in a model integrating BMC-dependent pdu with rhamnose metabolism, and discuss impact on growth and survival of L.
monocytogenes in anaerobic environments such as the human intestine.

Strains, Culture Conditions, and Growth Measurements
All experiments in this study were carried out with L. monocytogenes EGDe anaerobically grown at 30°C in defined medium MWB [21]. Overnight grown cells in Luria Broth (LB) were washed three times in PBS before inoculation into MWB. MWB was supplemented with 20mM L-rhamnose as sole carbon source with or without addition of 20nM vitamin B12. Anaerobic conditions were achieved by Anoxomat Anaerobic Culture System with a gas mixture composed of 10% CO2, 5% H2, 85% N2. MWB with 20 mM rhamnose and 20 nM vitamin B12 was defined as rhamnose pdu-induced, while MWB with 20 mM rhamnose was defined as rhamnose pdu noninduced condition. OD600 measurements in MWB were performed every 12 h for 3 days. Plate counting in MWB to quantity Colony Forming Units (CFUs) was performed every 24 h for 3 days.

Analysis of metabolites for Rhamnose metabolism using High Pressure Liquid Chromatography (HPLC)
Samples were taken from the cultures at 0, 24, 48, and 72 h. After centrifugation, the supernatant was collected for the HPLC measurements of rhamnose, acetate, lactate, 1,2-propanediol, 1-propanol and propionate. The experiment was performed with three biological replicates. Additionally, the standard curves of all the metabolites were measured in the concentration range of 0.1, 1, 5, 10, and 50 mM. HPLC was performed using an Ultimate 3000 HPLC (Dionex) equipped with an RI-101 refractive index detector (Shodex, Kawasaki, Japan), an autosampler and an ion-exclusion Aminex HPX-87H column (7.8 mm × 300 mm) with a guard column (Bio-Rad, Hercules, CA). As the mobile phase 5 mM H2SO4 was used at a flow rate of 0.6 ml/min, the column was kept at 40°C. The total run time was 30 min and the injection volume was 10 μl.

Transmission Electron Microscopy (TEM)
L. monocytogenes EGDe cultures were grown anaerobically at 30°C rhamnose pdu-induced and rhamnose pdu non-induced condition. Samples were collected at 48 h of incubation. About 10 μg dry cells were fixed for 2 h in 2.5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). After rinsing in the same buffer, a postfixation was done in 1% (w/v) OsO4 for 1 h at room temperature. The samples were dehydrated by ethanol and were then embedded in resin (Spurr HM20) 8 h at 70°C. 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].

Proteomics
L. monocytogenes cultures were anaerobically grown at 30°C in rhamnose pdu-induced and rhamnose pdu noninduced condition. Samples were collected at 48 h of incubation and then washed twice with 100 mM Tris (pH 8). About 10 mg wet weight cells in 100 μl 100 mM Tris was sonicated for 30 s twice to lyse the cells. 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]. A protein database with the protein sequences of L. monocytogenes EGDe (ID: UP000000817) was downloaded from UniProt. Filtering and further bioinformatics and statistical analysis of the MaxQuant ProteinGroups file were performed with Perseus [24]. Reverse hits and contaminants were filtered out. Protein groups were filtered to contain minimally two peptides for protein identification of which at least one is unique and at least one is unmodified. The volcano plot was prepared based on the Student's t-test difference of Pduinduced/non-induced control.

Bioinformatics and Statistical Analysis
Pathview R package [25] to visualize the proteomics data: The UniProt protein IDs from Supplementary Table 1 were collected and retrieved to Entre IDs. A list of Entrez IDs, protein expression indicated by LFQ intensity (Supplementary Table 2

Activation of pdu stimulates anaerobic growth of L. monocytogenes EGDe on rhamnose
We first examined if rhamnose can function as a sole carbon source to support anaerobic growth of L.
monocytogenes EGDe in MWB defined medium without and with added vitamin B12 (cobalamin) (Figure 1). In MWB defined medium supplied with 20mM rhamnose OD600 reaches a maximum of about 0.37 after 48 h, while in MWB supplied with 20mM rhamnose and 20nM B12 OD600 continues to increase after 48 h, reaching a significant higher OD600 of 0.51 at 72 h. Enhanced growth on MWB supplied with rhamnose and B12 compared to MWB plus rhamnose, is also evident from plate counts which increase from 6.5 to 8.2 log10 CFU/ml and from 6.5 to 7.2 log10 CFU/ml, respectively (figure 1B). There is no significant difference in growth performance of L. monocytogenes EGDe on MWB supplied with 20mM glucose and MWB supplied with 20mM glucose and 20 nM B12, and at 48 h final levels of 8.8 log10 CFU/ml were reached (Supplementary Figure 1). These results suggest that in B12 stimulated anaerobic growth of L. monocytogenes EGDe on MWB medium with rhamnose as sole carbon source is linked to activation of pdu.

Activation of pdu supports 1,2-propanediol degradation and stimulates rhamnose metabolism
To confirm possible activation of pdu, metabolic analysis via HPLC was conducted to quantify substrate consumption and product formation following anaerobic growth of L. monocytogenes EGDe on MWB plus 20mM rhamnose and MWB plus 20mM rhamnose and 20nM B12. As shown in Figure 2A

Visualization of BMCs and expression analysis of BMC shell proteins
To answer the question if BMCs are formed to support the utilization of rhamnose-derived 1,2-propanediol, Transmission Electron Microscopy (TEM) was performed to observe BMCs structures, and proteomics was applied to measure the expression of BMC shell proteins ( Figure 3A). The pdu induced cells clearly contain BMC-like structures with an approximate diameter of 50-80 nm, which are absent in pdu non-induced cells.
Notably, the identified structures strongly resemble TEM pictures of previously reported pdu BMCs in L. monocytogenes [8,9] and in S. enterica and E. coli [13,26].  Figure 3B), which indicates that the activation of pdu BMC does not affect the expression of these enzymes.

Proteomics-based pathway visualization of propanoate metabolism and vitamin B12 metabolism
To visualize the metabolism from 1,2-propanediol to propanoate (propionate) and 1-propanol, the identified proteins and expression levels presented in Supplementary Table 1, are mapped to propanoate metabolic pathways of L. monocytogenes EGDe. As shown in Figure 4A, the enzymes involved in degradation of rhamnose-derived 1,2-propanediol into propanoate (propionate) and 1-propanol are all significantly upregulated in pdu induced condition compared to pdu non-induced condition. The propanediol dehydratase (EC 4.2.1.28) is an enzyme with three subunits encoded by pduC, pduD and pduE, which converts 1,2propanediol into propanal (propionaldehyde). Propionaldehyde is metabolized to 1-propanol by propanol dehydrogenase PduQ and propanol-CoA by propionaldehyde dehydrogenase PduP (EC 1.2.1.87). Propanol-CoA is converted to propanoyl-phosphate by phosphate propanoyltransferase PduL (EC 2.3.1.222), with propanoylphosphate subsequently converted to propanoate by propionate kinase PduW (EC 2.7.2.1). We found that the vitamin B12 biosynthesis pathway that is grouped in porphyrin and chlorophyll metabolism, is significantly downregulated in pdu induced condition compared to pdu non-induced condition ( Figure 4B), which suggests that supplementation of 20nM B12 represses the expression of proteins required for B12 biosynthesis. This also includes the three enzymes mediating the final steps in B12 biosynthesis, CobU, CobS and CobC, encoded by the respective genes located in the pdu cluster ( Figure 4B) [8,[27][28][29]. Apparently, B12 accumulation from the medium supports activation of pdu BMCs, whereas despite expression of B12 biosynthesis enzymes, production of B12 and levels reached are not sufficient to induce pdu in L. monocytogenes EGDe grown in MWB without added B12.

Discussion
The presented model of 1,2-propanediol BMCs in rhamnose metabolism is based on growth phenotypes, metabolic analysis, proteomics, TEM visualization and our understanding of 1,2-propanediol BMCs in anaerobic growth of L. monocytogenes EGDe. As illustrated in Figure 5, the rhamnose catabolism gene cluster (rha) in L.
monocytogenes EGDe is composed of lmo2846-lmo2851 [30]. lmo2850 encodes a secondary transporter which has high similarity with L-rhamnose permease RhaT in E. coli [31][32][33], and is conceivably acting as the transporter of α-L-rhamnose. L-rhamnose mutarotase RhaM mediates the conversion of α-L-rhamnose into β-Lrhamnose (also called L-rhamnopyranose) [30,34]. β-L-rhamnose is converted to L-rhamnulose by L-rhamnose isomerase RhaA [30,35]. L-rhamnose is then phosphorylated to L-rhamnulose 1-phosphate by rhamnulokinase RhaB with one ATP consumption [30,35]. L-rhamnulose 1-phosphate is split into (S)-lactaldehyde and dihydroxyacetone phosphate (DHAP) by rhamnulose-1-phosphate aldolase RhaD [30,35]. DHAP can be metabolized to glyceraldehyde 3-phosphate via Triosephosphate isomerase 1 TpiA1 and via the glycolytic pathway [14,36] and the GABA (γ-aminobutyric acid) shunt in the incomplete TCA cycle in L. monocytogenes [37], to the end products acetate and lactate, as confirmed in our metabolic analysis. The observed production of 1,2-propanediol in pdu non-induced conditions confirms the predicted anaerobic conversion of lactaldehyde to 1,2-propanediol in L. monocytogenes EGDe. The activity of lactaldehyde reductase has not been described in L. monocytogenes [38], but protein similarity alignment with lactaldehyde reductase FucO of Escherichia coli [38], suggests four putative candidates annotated as alcohol dehydrogenase in L. monocytogenes EGDe including lmo1166, lmo1171, lmo1634 and lmo1737, detected in the proteomes of both pdu non-induced and pdu-induced cells (for details see Supplementary File 1). Since the discovery of the role of pdu BMCs dehydratase in rhamnose (and fucose) utilization, two pathway scenarios have been proposed, one with and one without lactaldehyde reductase encapsulated inside BMCs [30,34]. In line with previously reported comparative genomic analysis [30,34], our data now provide evidence for the latter model to be active in L. Our data provide evidence for another extension of the BMC dependent metabolic repertoire of L. monocytogenes in anaerobic conditions, that now includes BMC-dependent ethanolamine utilization (eut) [9], BMC pdu [8], and BMC pdu-stimulated rhamnose metabolism. The indicated substrates can be found in a wide range of environments including foods and human gastrointestinal tract. In the latter case, competitive fitness of L. monocytogenes in the human intestine may be enhanced by supply of these compounds by enzymatic activity including release of ethanolamine following membrane phospholipid degradation and release of rhamnose following mucus glycan hydrolysis activity of for example Bacteriodes spp, and propanediol as a fermentation product [15]. Notably, despite the presence of a complete vitamin B12 synthesis cluster, we found that eut [9], pdu [8], and pdu-stimulated rhamnose utilization in L. monocytogenes in the current study, requires supplementation of B12 to the medium. This points to an important role of B12 in activation of L. monocytogenes BMC-mediated metabolic pathways containing B12-dependent signature aldehyde reductases.
Vitamin B12 can be found in foods including meat and dairy products [28,39] and is also found in human intestine, where part of the B12 is derived from gut microbiota that have the capacity to produce B12 [12,28].
The fact that we observed in the current study induction of the B12 synthesis pathway in cells grown in MWB plus rhamnose but no activation of B12-dependent pdu, while activation was found with B12 added to the medium, points to an intricate regulation of the B12 synthesis pathway and its connection to BMCs activation.
In addition to earlier studies on transcriptional and translational control of BMC eut and pdu in L.
monocytogenes [12,28,40], studies are required to assess for example impact of extracellular and intracellular B12 concentrations on BMC pathway activation and their role in L. monocytogenes ecophysiology and virulence.