Characterisation of the growth behaviour of Listeria monocytogenes in Listeria synthetic media

Abstract The foodborne pathogen Listeria monocytogenes can grow in a wide range of environmental conditions. For the study of the physiology of this organism, several chemically defined media have been developed over the past decades. Here, we examined the ability of L. monocytogenes wildtype strains EGD‐e and 10403S to grow under salt and pH stress in Listeria synthetic medium (LSM). Furthermore, we determined that a wide range of carbon sources could support the growth of both wildtype strains in LSM. However, for hexose phosphate sugars such as glucose‐1‐phosphate, both L. monocytogenes strains need to be pre‐grown under conditions, where the major virulence regulator PrfA is active. In addition, growth of both L. monocytogenes strains was observed when LSM was supplemented with the amino acid sugar N‐acetylmannosamine (ManNAc). We were able to show that some of the proteins encoded in the operon lmo2795‐nanE, such as the ManNAc‐6‐phosphate epimerase NanE, are required for growth in the presence of ManNAc. The first gene of the operon, lmo2795, encodes a transcriptional regulator of the RpiR family. Using electrophoretic mobility shift assays and quantitative real‐time PCR analysis, we were able to show that Lmo2795 binds to the promoter region of the operon lmo2795‐nanE and activates its expression.


INTRODUCTION
The foodborne pathogen Listeria monocytogenes is a Gram-positive, facultative anaerobic, rod-shaped bacterium, which is ubiquitously found in nature.It can survive as a saprophyte on decaying plant material, in soil or wastewater.L. monocytogenes and other Listeria species can also enter food processing facilities and food chains, for instance, due to contaminated water or raw materials (Lourenco et al., 2022).Several recent outbreaks of L. monocytogenes were associated with contaminated fruits and vegetables, such as melons, apples or mushrooms or contaminated meat products (Lachmann et al., 2021;Matle et al., 2020;Townsend et al., 2021).The intracellular pathogen L. monocytogenes is the causative agent of the disease listeriosis.In healthy individuals, the intake of L. monocytogenes via the consumption of contaminated food products mostly leads to mild symptoms, such as fever and self-limiting gastroenteritis.However, for immunocompromised individuals, the elderly, newborns and pregnant women, the infection with L. monocytogenes can cause more severe symptoms, such as muscle aches, vomiting, diarrhoea, encephalitis or meningitis.For pregnant women, it can also lead to premature or still birth (Radoshevich & Cossart, 2018).This is achieved by its ability to cross all human barriers, namely the intestinal, placental and blood-brain barriers (Lecuit, 2005).
The survival of L. monocytogenes in food processing facilities and within the host is enabled due to its ability to grow under diverse stress conditions (reviewed in (Gaballa et al., 2019;Osek et al., 2022;Wiktorczyk-Kapischke et al., 2021)).Previous studies reported that L. monocytogenes can grow at temperatures between 1 and 45 C, in a pH range from 4.0 to 9.5 or in the presence of up to 10% salt (Arizcun et al., 1998;Patchett et al., 1992;Vasseur et al., 2001).L. monocytogenes is also able to use a variety of carbon sources ranging from simple monosaccharides (glucose and fructose) to more complex sugars (maltodextrin) and polyols (glycerol, D-arabitol and D-xylitol) (Deutscher et al., 2014;Gopal et al., 2010;Kentache et al., 2016).The ability to take up such a wide range of carbon sources is facilitated by a diverse set of transporters, which are encoded in the genome of L. monocytogenes.Most of the carbon sources are imported into the cell via the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS).The genome of L. monocytogenes wildtype strain EGD-e contains 86 PTS genes, which encode for 29 complete and 12 incomplete PTS complexes (Glaser et al., 2001;Stoll & Goebel, 2010).These PTS transporters are involved in the transport of diverse carbon sources such as glucose, fructose, D-arabitol and D-xylitol (Deutscher et al., 2014;Kentache et al., 2016).Some of these PTS transporters also have overlapping substrate specificities.For instance, the two PTS systems ManLMN (MptACD) and MpoABCD, encoded by lmo0096-0098 and lmo0784-0781, respectively, are both involved in the transport of glucose and mannose (Aké et al., 2011;Stoll & Goebel, 2010).Inactivation of all PTS systems, e.g. by deleting the PTS enzyme I encoding gene ptsI, did not abolish fermentation of some sugars such as glucose, fructose or mannose, suggesting that L. monocytogenes possesses also other sugar transporters (Deutscher et al., 2014).Indeed, L. monocytogenes encodes three GlcU-like non-PTS permeases (Lmo0169, Lmo0176 and Lmo0424), which could be involved in the import of these sugars (Aké et al., 2011;Deutscher et al., 2014).The import of L-rhamnose is accomplished by the major facilitator superfamily protein Lmo2850, which uses a proton symport mechanism (Deutscher et al., 2014;Fieseler et al., 2012;Zeng et al., 2021).Within the host, L. monocytogenes uses mainly glucose-1-phosphate, glucose-6-phosphate and glycerol as carbon sources (Grubmüller et al., 2014;Ripio et al., 1997).The transporter Hpt, also named UhpT, takes up hexose phosphates from the host cell cytosol (Chico-Calero et al., 2002), while the glycerol uptake facilitator GlpF facilitates the import of glycerol (Joseph et al., 2008).The expression of hpt thereby depends on PrfA, the main virulence regulator of L. monocytogenes (Chico-Calero et al., 2002;Ripio et al., 1997).L. monocytogenes possesses eight predicted carbohydrate ATP-binding cassette (ABC) transporters.One of these transporters, which is encoded by lmo2123-2125, is required for the import of maltose and maltodextrin (Gopal et al., 2010).The remaining seven ABC transporters have not yet been studied.
For many studies on the physiology and stress tolerance of L. monocytogenes, bacteria were cultured in a complex medium such as brain heart infusion (BHI) or tryptic soy broth (TSB).In their natural habitat or within their host, L. monocytogenes likely encounters more limiting growth conditions.Within the last decades, several minimal media have been developed which can be used to grow L. monocytogenes (Jarvis et al., 2016;Phan-Thanh & Gormon, 1997;Premaratne et al., 1991;Tsai & Hodgson, 2003;Welshimer, 1963;Whiteley et al., 2017).In this study, we aimed to characterise the growth of the two widely used L. monocytogenes wildtype strains EGD-e and 10403S, which belong to serotype 1/2a, in the recently developed Listeria synthetic medium (LSM) (Whiteley et al., 2017).L. monocytogenes strains belonging to serotype 1/2a are often recovered from food, environmental and clinical samples (Orsi et al., 2011;Ward et al., 2008) and thus encounter diverse environmental conditions.We thus determined the growth behaviour of EGD-e and 10403S in LSM under stress conditions, which they encounter in food or the environment or within the host during infection, namely high salt and low pH stress.To our knowledge, the growth of L. monocytogenes in LSM was so far only assessed with glucose as a sole carbon source.We therefore determined, which other carbon sources can facilitate the growth of L. monocytogenes EGD-e and 10403S in LSM.While 95% of the open reading frames are conserved between EGD-e and 10403S, the genomes differ by over 30,000 single nucleotide polymorphisms (Bécavin et al., 2014;Glaser et al., 2001).It is, thus, not surprising that we could observe differences in the growth behaviour between these two strains.

Bacterial strains and growth conditions
All strains and plasmids used in this study are listed in Table 1.Escherichia coli strains were grown in Luria-Bertani (LB) medium and Listeria monocytogenes strains in BHI medium or LSM at 37 C unless otherwise stated.Where required, antibiotics and supplements were added to the medium at the following concentrations: for E. coli cultures, ampicillin (Amp) at 100 μg mL À1 and kanamycin (Kan) at 50 μg mL À1 , and for L. monocytogenes cultures, chloramphenicol (Cam) at 7.5 μg mL À1 and Kan at 50 μg mL À1 .
A previous study showed that the expression of prfA is induced in the presence of low levels of branchedchain amino acids (Lobel et al., 2015).For the growth of L. monocytogenes under PrfA-activating conditions, the concentration of branched-chain amino acids was 10-fold reduced in freshly prepared LSM yielding 80 μM L-isoleucine, 80 μM L-leucine and 90 μM Lvaline.

Growth assays
Overnight cultures of the indicated L. monocytogenes strains were diluted in fresh BHI medium to an OD 600 of 0.1 and incubated at 37 C until an OD 600 of 0.3 was reached.Cells of 2 mL culture were collected per condition, washed twice with 1 mL of the corresponding LSM and re-suspended in 1 mL LSM.The cultures were adjusted to an OD 600 of 0.2 and 100 μL of the cell suspension were transferred into wells of a 96-well plate containing 100 μL of the corresponding LSM.The plate was incubated at 37 C with orbital shaking in the Bio-Tek Epoch 2 microplate reader and the OD 600 was measured every 15 min for at least 36 h.Averages and standard deviations of at least three independent growth assays were plotted.
To test whether the growth of L. monocytogenes was possible in LSM Glc-1-P and Glc-6-P after pregrowth of the indicated strains under PrfA-activating conditions, overnight cultures of the indicated L. monocytogenes strains were diluted in LSM glucose with low concentrations of BCAA to an OD 600 of 0.1 and incubated at 37 C until an OD 600 of 0.3 was reached.Cells were collected, washed and used to inoculate a 96-well plate containing the corresponding LSM with low concentrations of BCAA as described above.

Strain and plasmid construction
All primers used in this study are listed in Table 3.For markerless deletion of lmo2795, 1-kb fragments upand downstream of lmo2795 were amplified by PCR using primer pairs LMS346/LMS347 and LMS348/ LMS349, respectively.The resulting PCR fragments were fused by PCR using primers LMS346/LMS349.The deletion fragment was subsequently digested with XbaI and SacI and ligated into plasmid pKSV7 that had Note: The recipe of LSM was adapted from Whiteley et al. (2017).All stock solutions were sterile filtrated prior to use and stored at 4 C, except for Phosphate, which is stored at room temperature.LSM is prepared by combining the stock solutions in accordance with their dilution factor and in the order they are listed in the table.
The final LSM medium is again sterile filtrated.These ingredients are usually freshly added to LSM according to Whiteley et al. (2017); however, a stock solution could be prepared and is stable for at least 4 weeks.
For the purification of Lmo2795 with an N-terminal His-tag, lmo2795 was amplified using primer pair AK004/AK005.The resulting PCR product was digested with BamHI and SalI and ligated into pWH844.Plasmid pWH844-lmo2795 was recovered in E. coli DH5α yielding strain EJR131.

RNA extraction
RNA of the indicated L. monocytogenes strains was isolated following a previously published method with minor modifications (Hauf et al., 2019).Briefly, a single colony was used to inoculate 10 mL LSM medium with 1% glucose as the sole carbon source and cultures were grown overnight at 37 C.The next day, cultures were used to inoculate 30 mL of fresh LSM medium with 1% glucose to an OD 600 of 0.1.When the cultures reached an OD 600 of 0.5 ± 0.05, 25 mL of the cell suspensions were harvested by centrifugation for 15 min at 4000 rpm and 4 C and the pellet was snap frozen in liquid nitrogen and stored at À80 C. To isolate the RNA, pellets were re-suspended in 1 mL killing buffer (20 mM Tris, pH 7.5, 5 mM MgCl 2 , 20 mM NaN 3 ), transferred to 1.5 mL microtubes and centrifuged at 13,000 rpm for 60 sec.Cells were then resuspended in 1 mL lysis buffer I (25% sucrose; 20 mM Tris-HCl pH 8, 0.25 mM EDTA) and 2 μL lysozyme (100 mg mL À1 ) and incubated for 5 min on ice, followed by centrifugation at 5000 rpm and 4 C for 5 min.Pellets were then re-suspended in 300 μL lysis buffer II (3 mM EDTA; 200 mM NaCl) and added to pre-heated (95 C) lysis Buffer III (3 mM EDTA; 200 mM NaCl; 1% SDS).Samples were incubated for exactly 5 min at 95 C and 600 μL phenol/chloroform/isoamyl alcohol (25:24:1) (PCI) was added.After shaking the samples at 700 rpm for 5 min, the two phases were separated by centrifugation at 13,000 rpm for 5 min and the upper aqueous phase was transferred to a new 1.5 mL microtube containing 600 μL PCI.The extraction was repeated with 600 μL chloroform/ isoamyl alcohol (25:1).Finally, the upper phase was transferred into an RNAse-free tube and RNA was precipitated by the addition of 0.1 x volume 3 M sodium acetate (pH 5.2) and 1.5 volumes 96% ethanol and incubated at 20 C overnight.To precipitate the RNA, samples were centrifuged at 13,000 rpm for 15 min, washed in 70% ethanol and dried under the fume hood.Finally, samples were re-suspended in 25 μL RNAse-free H 2 O.
To avoid DNA contamination, 5 μg isolated RNA were digested with 5 μL DNase I (1 U μl À1 , Thermo Scientific) for 40 min at 37 C.The reaction was stopped by the addition of 2.5 μL 25 mM EDTA and incubation at 65 C for 10 min.To verify that the isolated RNA is free of DNA, a check PCR was performed using primers LMS169 and LMS170.Genomic DNA from L. monocytogenes EGD-e was used as a control.

Quantitative real-time PCR (qRT-PCR)
qRT-PCR was carried out using the One-Step reverse transcription PCR kit, the Bio-Rad iCycler and the Bio-Rad iQ5 software (Bio-Rad, Munich, Germany).Three biological replicates were performed.Primer pairs LMS375/LMS376 and LMS169/170 were used to determine the transcript amounts of rpID and gyrB, respectively, which were used as internal controls.Transcript amounts for lmo2796 and nanE were monitored using primer pairs LMS384/LMS385 and LMS369/LMS377, respectively.The average of the cycle threshold (C T ) values of rpID and gyrB were used to normalise the C T values obtained for lmo2796 and nanE.For each strain, the fold changes in lmo2796 and nanE expression were calculated using the ΔΔC T method.

Purification of His-Lmo2795
For the overexpression of His-Lmo2795, plasmid pWH844 was used, which contains the lacI gene, enabling the strictly IPTG-dependent overproduction of His-tagged proteins (Schirmer et al., 1997).pWH844-lmo2795 was transformed into E. coli BL21 and grown in 2x LB.When cultures reached an OD 600 of 0.6-0.8,expression was induced by the addition of IPTG at a final concentration of 1 mM, and the cells were grown for 2 h at 37 C. Cells were collected by centrifugation, washed once with 1x ZAP buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl) and the cell pellet stored at À20 C. The next day, cells were re-suspended in 1x ZAP (50 mM Tris-HCl, pH 7.5, 200 mM NaCl) and lysed by three passages (18,000 lb/in2) through an HTU DIGI-F press (G.Heinemann, Germany).The resulting crude extract was centrifuged at 46,400x g for 60 min followed by protein purification using a Ni 2+ nitrilotriacetic acid column (IBA, Göttingen, Germany).His-Lmo2795 was eluted using imidazole and elution fractions analysed using SDS-PAGE.Selected elution fractions were subsequently subjected to dialysis against 1x ZAP buffer at 4 C overnight.The protein concentration was determined according to the method of Bradford (Bradford, 1976) using the Bio-Rad protein assay dye reagent concentrate.Bovine serum albumin was used as standard.The protein samples were stored at À80 C until further use.

Electrophoretic mobility shift assay (EMSA)
EMSA was performed as previously described with some modifications (Dhiman et al., 2014).Briefly, the upstream region of the lmo2795-nanE operon containing a putative promoter site was amplified using primer pair LMS430/LMS431.A 204 bp region within the operon, which served as an unspecific control, was amplified using primers LMS428/LMS429.Binding reactions were performed at 25 C for 20 min in 20 μL binding buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10% glycerol, 5 mM EDTA, 20 mM DTT and varying amounts of His-Lmo2795 (3.5-14 μg), as well as 250 pmol of DNA.To ease the loading of the samples into the wells, 1 μL DNA loading dye was added to each sample.A 60-min pre-run was carried out at 70 V prior to sample loading.The samples were separated on an 8% polyacrylamide native gel in 0.5x TBE buffer (50 mM Tris-HCl, pH 10, 50 mM boric acid, 1 mM Na 2 EDTA) at 4 C for 2.5 h at 50 V.The gel was stained in 50 mL 0.5x TBE buffer containing 5 μL HDGreen™ Plus DNA dye (INTAS, Göttingen, Germany) for 3 min, followed by a 5 min washing step in 0.5x TBE buffer, three 20 sec washing steps in H 2 O and an additional 30 min washing step in H 2 O.DNA bands were visualised using a GelDoc™ XR+ (Bio-Rad, Munich, Germany).

Growth of L. monocytogenes wildtype strains under salt and pH stress
For most physiological studies, which aimed at the characterisation of salt and pH resistance of L. monocytogenes, bacteria were grown in complex media such as BHI or TSB.In nature as well as within the host, L. monocytogenes likely encounters limited access to nutrients, such as carbon and nitrogen sources or vitamins, which likely affects their ability to adapt to environmental stress conditions.We, therefore, assessed the ability of the two widely used L. monocytogenes wildtype strains EGD-e and 10403S to grow under high salt and low pH stress in the chemically defined medium LSM.Growth of EGD-e was barely affected in the presence of 1% NaCl.In the presence of 2, 3 and 4% NaCl, the growth of EGD-e was delayed as compared to LSM.At a concentration of 5% salt, EGD-e was still able to grow but only reached an optical density of around 0.3 after 36 h.The L. monocytogenes wildtype strain 10403S showed a lower salt resistance as compared to EGD-e.Growth of 10403S was already impaired in LSM containing 3% salt.In the presence of 4% and 5% salt, 10403S could only reach an optical density of around 0.2 after 36 h (Figure 1A).In contrast, L. monocytogenes is able to grow in the presence of up to 10% salt, when a complex medium is used (Patchett et al., 1992;Vasseur et al., 2001).To assess the pH resistance, we compared the growth of L. monocytogenes strains grown in standard LSM, which has a pH of 8.7, and LSM adjusted to a pH of 8, 7, 6 and 5.5.Growth of the L. monocytogenes wildtype strain EGD-e was similar in LSM with a pH of 7 as compared to standard LSM, while the growth of 10403S was slightly reduced.Both strains had a severe growth defect in LSM with a pH of 6 and were unable to grow in LSM with a pH of 5.5 (Figure 1B).Overall, we observed that the L. monocytogenes wildtype strain 10403S was less tolerant to salt and pH stress than EGD-e (Figure 1).Interestingly, 10403S grew better in the BHI medium, which was adjusted to a low pH, than EGD-e (Cheng et al., 2015).Resistance of L. monocytogenes to pH stress is mainly conferred by the activity of the glutamate decarboxylase activity (GAD) system, a process, which depends on the availability of extracellular glutamate (Cotter et al., 2001;Feehily et al., 2014).The two L. monocytogenes wildtype strains EGD-e and 10403S seem to use two distinct GAD systems, which confer a different degree of acid resistance.The GAD system used by 10403S was shown to give a higher acid tolerance (Cheng et al., 2015;Feehily et al., 2014).However, complex medium such as BHI likely contains higher glutamate levels than LSM, which can be used by the GAD system.Thus, the GAD system of 10403S might be less efficient when the cells are grown in LSM and are therefore less resistant to acid stress.

Growth of L. monocytogenes in LSM with different carbon sources
Over the last decades, several chemically defined media have been developed that support growth of L. monocytogenes (Jarvis et al., 2016;Phan-Thanh & Gormon, 1997;Pine et al., 1989;Premaratne et al., 1991;Tsai & Hodgson, 2003;Whiteley et al., 2017).Most of these media contain glucose as the sole carbon source.Other carbon sources such as fructose and mannose were shown to support the growth of L. monocytogenes in Hsiang-Ning Tsai medium (HTM) or modified Welshimer's Broth (MWB) (Premaratne et al., 1991;Tsai & Hodgson, 2003).Glucose could also be replaced by the amino sugars GlcNAc and N-acetylmuramic acid in MWB (Premaratne et al., 1991).Interestingly, lactose and rhamnose could support the growth of L. monocytogenes in ACES-buffered chemically defined (ABCD) medium but not in HTM and/or MWB (Table 4) (Pine et al., 1989;Premaratne et al., 1991;Tsai & Hodgson, 2003).Similarly, glycerol was shown to support the growth of L. monocytogenes in HTM medium, while only weak growth could be observed for MWB with glycerol as the sole carbon source (Table 4) (Premaratne et al., 1991;Tsai & Hodgson, 2003).To our knowledge, no information is available on the carbon sources that support the growth of L. monocytogenes in LSM.We, therefore, performed growth experiments with L. monocytogenes wildtype strains EGD-e and 10403S in LSM containing 1% of selected carbon sources.This analysis revealed that growth of both strains was similar in LSM containing GlcNAc, GlcN, mannose, cellobiose or trehalose as the sole carbon source compared to glucose (Figure 2, Table 4).Both strains were also able to use glycerol as carbon sources, while only weak growth was obtained for maltose, rhamnose and succinate (Figure 2, Table 4).No growth was observed for both strains, when glucose was replaced with galactose, sucrose, glucose-1-phosphate (Glc-1-P), glucose-6-phosphate (Glc-6-P) or mannitol (Figure 3A, Table 4).The hexose phosphate transporter Hpt, which is required for the import of Glc-1-P and Glc-6-P, is expressed in a PrfAdependent manner.The expression and activity of PrfA is controlled on transcriptional, translational and posttranslational level (reviewed in (Gaballa et al., 2019;Xayarath & Freitag, 2012)).One factor involved in the control of prfA expression is the global regulator CodY, which senses the presence of branched-chain amino acids (BCAAs).In the presence of high levels of BCAAs, CodY acts as a prfA repressor, while expression of prfA is induced when BCAA levels drop, which occurs upon invasion of host cells (Lobel et al., 2015).We, therefore, tested whether reduction of the BCAA concentration in LSM could lead to the growth of F I G U R E 1 Salt and pH resistance of L. monocytogenes wildtype strains.(A) L. monocytogenes strains EGD-e and 10403S were grown in LSM containing 1% glucose and increasing concentrations of NaCl as described in the methods section.(B) L. monocytogenes strains EGD-e and 10403S were grown in LSM containing 1% glucose (pH 8.7).The pH of LSM was adjusted to the indicated pH values using HCl.The average values and standard deviations of three independent experiments were plotted.
L. monocytogenes wildtype strains EGD-e and 10403S on Glc-1-P and Glc-6-P as the sole carbon source.While both strains grew fine in LSM with low concentrations of BCAA and glucose as the sole carbon source, no growth was observed in LSM with Glc-1-P or Glc-6-P (Figure 3A).For growth analysis, L. monocytogenes strains were pre-grown in a BHI complex medium, in which PrfA is inactive (Renzoni et al., 1997).We, thus, wondered whether the strains have simply not sufficient time to activate PrfA and by this also do not induce the expression of hpt, encoding the Glc-1-P and Glc-6-P importer, when they are transferred into LSM containing the hexose phosphate sugars.To test this, we pre-grew L. monocytogenes strains EGD-e and 10403S in LSM glucose with low levels of BCAA and subsequently transferred them either into the same medium as a control, or into LSM with low levels of BCAA and containing Glc-1-P or Glc-6-P as sole carbon source.A strain lacking the virulence regulator PrfA was used as a control.Indeed, EGD-e and 10403S were now able to metabolise Glc-1-P and Glc-6-P, while the prfA mutant was unable to grow (Figure 3B).In contrast, the absence of PrfA results in a growth advantage in LSM with glucose as the sole carbon source, suggesting that the expression and/or activity of PrfA is a burden for L. monocytogenes, which has previously been shown (Friedman et al., 2017;Vasanthakrishnan et al., 2015).Therefore, characterisation of growth and the general physiology of L. monocytogenes in the presence of Glc-1-P and Glc-6-P as sole carbon source, requires a pre-growth under PrfA-activating conditions, for instance, in a chemically defined medium or BHI medium containing activated charcoal, Chelex or Amberlite XAD (Chico-Calero et al., 2002;Gaballa et al., 2021;Portman et al., 2017).
was able to support the growth of L. monocytogenes strains EGD-e and 10403S (Figure 2A, Table 4).Man-NAc is the precursor of the sialic acid Nacetylneuraminic acid (Neu5Ac), which can serve as a carbon source for Staphylococcus aureus inside the host (Angata & Varki, 2002;Olson et al., 2013;Vimr et al., 2004).However, Neu5Ac could not support the growth of L. monocytogenes, suggesting that this organism does not contain the necessary catabolic pathway (Table 4).

The lmo2795-nanE operon is required for ManNAc catabolism
In E. coli, ManNAc is imported into the cell by the PTS transporter ManXYZ yielding ManNAc-6-phosphate.ManNAc-6-phosphate is subsequently converted into GlcNAc-6-phosphate by the ManNAc-6-phosphate epimerase NanE.The genome of L. monocytogenes strain EGD-e contains one homologue of NanE encoded by lmo2801 (37% identity and 58% similarity).Based on the presence of the NanE homologue, it was already previously proposed that the lmo2795-nanE operon might be involved in the transport of ManNAc (Deutscher et al., 2014).In addition to NanE, the lmo2795-nanE operon encodes a RpiR-type regulator, a member of the repressor, ORF, kinase (ROK) family, the EIIA and EIIBC components of a PTS system, a putative HAD hydrolase and a putative oxidoreductase (Figure 4A).To test whether these proteins are required for the catabolism of ManNAc, single deletion mutants and a mutant lacking both PTS components, Lmo2797 and Lmo2799, were constructed in the EGD-e wildtype background and their growth was analysed.All deletion mutants were able to grow in LSM containing glucose; however, growth was enhanced for L. monocytogenes strains Δlmo2799 and Δlmo2797 Δlmo2799, lacking one or two of the PTS components and the nanE deletion mutant (Figure 4B, C).In contrast, growth on Man-NAc as the sole carbon source was reduced for the lmo2795 and lmo2798 deletion strains lacking the RpiR transcriptional regulator and the putative HAD hydrolase, respectively, and was nearly abolished for the strain lacking NanE (Figure 4D,E).Surprisingly, deletion strains Δlmo2799 and Δlmo2797 Δlmo2799 showed better growth in LSM with ManNAc, similar to what was observed for the glucose-containing LSM (Figure 4C-E), indicating that this PTS system is either not involved in the import of ManNAc or that ManNAc can also be imported by other PTS systems of L. monocytogenes.The absence of individual PTS systems can also lead to the overexpression of another PTS system (Stoll & Goebel, 2010), which could explain why strains lacking the EIIB/C component Lmo2799 grew better than the wildtype in LSM with glucose or ManNAc.

Expression of the lmo2795-nanE operon is controlled by Lmo2795
RpiR-type transcriptional regulators are often involved in the control of sugar metabolic pathways in Grampositive and Gram-negative bacteria (Aleksandrzak-Piekarczyk et al., 2019;Jaeger & Mayer, 2008;Li et al., 2017;Sørensen & Hove-Jensen, 1996;Yamamoto et al., 2001).We, thus, wondered whether Lmo2795 regulates the expression of the lmo2795-nanE operon.First, we conducted EMSA experiments to assess whether Lmo2795 binds to the promoter region of the operon.For this purpose, increasing concentrations of purified His-Lmo2795 (Figure 5A) were incubated with the DNA fragment containing the putative promoter of the lmo2795-nanE operon.The migration of the DNA fragment was retarded in the presence of His-Lmo2795 as compared to the DNA sample that did not contain the protein, suggesting that a protein-DNA complex was formed.In addition, a so-called supershift could also be observed, whose intensity increases with increasing protein concentrations.In contrast, no retardation of the unspecific DNA control was observed in the presence of His-Lmo2795 (Figure 5B).To test whether Lmo2795 activates or represses the expression of the lmo2795-nanE, we quantified the expression of lmo2796 and nanE via quantitative real-time PCR.To allow for a better comparison of the gene expression, we decided to isolate RNA of the L. monocytogenes wildtype strain EGD-e and the lmo2795 mutant that were grown in LSM with glucose as the sole carbon source due to the observed growth deficit of the lmo2795 mutant in ManNAccontaining LSM.This analysis revealed that the expression of both genes, lmo2796 and nanE, was reduced in the lmo2795 mutant as compared to the wildtype strain (Figure 5C), suggesting that Lmo2795 acts as a transcriptional activator under the tested condition.Based on the observation that a strain lacking NanE had a growth deficit in LSM with ManNAc as the sole carbon source, we assume that Lmo2795 also activates the expression of the lmo2795-nanE under this condition as we would expect at least wildtype-like growth of the nanE mutant if Lmo2795 acts as a repressor.Further analysis is required to support this hypothesis.

CONCLUSION
The human pathogen L. monocytogenes is able to withstand and adapt to a variety of environmental stress F I G U R E 5 Lmo2795 regulates the expression of the lmo2795-nanE operon.(A) SDS-PAGE of elution fraction 3 containing purified His-Lmo2795.The expected molecular weight of His-Lmo2795 is 31.5 kDa.(B) EMSA assay.Increasing concentrations of His-Lmo2795 were incubated with the DNA fragment containing the predicted promoter region of the lmo2795-nanE operon.The DNA-protein complexes were separated on an 8% polyacrylamide gel, which was subsequently stained using HDGreen™.A DNA fragment containing a short region from within the operon was used as the unspecific control.Reactions without protein were used as negative controls (À).(C) Analysis of lmo2796 and nanE expression by qRT-PCR.RNA was isolated from L. monocytogenes strains EGD-e (wt) and Δlmo2795 grown in LSM containing 1% glucose as the sole carbon source as described in the methods section.Expression of lmo2796 and nanE was normalised to the expression of gyrB and rplD and fold changes were calculated using the ΔΔC T method.Averages and standard deviations of three independent RNA extractions were plotted.For statistical analysis, a one-way ANOVA coupled with Dunnett's multiple comparison test was performed (**p ≤ 0.01; ***p ≤ 0.001).
conditions.Most studies on the physiology and stress tolerance of this organism were performed in complex media without any nutrient limitation; however, this condition is rarely found in their natural habitat or within the host during infection.Our study focused on the characterisation of the growth of two widely used L. monocytogenes wildtype strains in the newly developed LSM.We were able to show that both strains can utilise a variety of carbon sources when supplied as the sole carbon source.To our knowledge, this is the first time that the catabolism of ManNAc has been investigated for L. monocytogenes.We were able to show that the growth of this pathogen depends on the presence of NanE, which is encoded in the lmo2795-nanE operon.The expression of the lmo2795-nanE operon is regulated by the RpiR-type regulator Lmo2795, which acts as a transcriptional activator under the tested conditions.
Our detailed characterisation of the growth of L. monocytogenes in LSM in the presence of diverse stress conditions or carbon sources can serve as a starting point for future studies focusing on the adaptation of this important human pathogen under changing environmental conditions.

a
Adjust pH of MOPS stock solution to pH 7.5 with 10 N NaOH.b Dissolve ingredients in boiling water followed by filter sterilisation.c Dissolve amino acids in hot 1 N NaOH followed by filter sterilisation.d Dissolve adenine in 20 mL 0.2 M HCl and fill up with H 2 O to 250 mL.e

b
Pre-growth in LSM glucose with low concentrations of BCAA, a PrfA-activating condition, is required.GROWTH OF L. MONOCYTOGENES IN LISTERIA SYNTHETIC MEDIUM F I G U R E 2 Growth in LSM media with different carbon sources.(A, B) L. monocytogenes strains EGD-e and 10403S were grown in Listeria synthetic medium (LSM) containing 1% of the indicated carbon sources as described in the methods section.The average values and standard deviations of three independent experiments were plotted.GlcNAc, N-acetylglucosamine; ManNAc, N-acetylmannosamine; GlcN, glucosamine.

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I G U R E 4 The lmo2795-nanE operon is required for ManNAc utilisation.(A) Gene arrangement of the lmo2795-nanE operon of L. monocytogenes.Predicted protein functions are indicated above the genes.ROK, member of the repressor, ORF, kinase family; PTS-EIIA and PTS-EIIB/C, components of a phosphotransferase system; HAD, member of the haloacid dehydrogenase-like hydrolase superfamily.(B-E) Growth curves.The indicated L. monocytogenes strains were grown in LSM containing (B, C) 1% glucose, (D, E) 1% ManNAc as the sole carbon source.The average values and standard deviations of three independent experiments were plotted.
Bacterial strains used in this study.
T A B L E 1 T A B L E 2 Composition of the Listeria synthetic medium (LSM).