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Appl Environ Microbiol. Jan 2011; 77(1): 281–290.
Published online Oct 29, 2010. doi:  10.1128/AEM.01403-10
PMCID: PMC3019715

Ethanolamine Utilization Contributes to Proliferation of Salmonella enterica Serovar Typhimurium in Food and in Nematodes[down-pointing small open triangle]

Abstract

Only three pathogenic bacterial species, Salmonella enterica, Clostridium perfringens, and Listeria monocytogenes, are able to utilize both ethanolamine and 1,2-propanediol as a sole carbon source. Degradation of these substrates, abundant in food and the gut, depends on cobalamin, which is synthesized de novo only under anaerobic conditions. Although the eut, pdu, and cob-cbi gene clusters comprise 40 kb, the conditions under which they confer a selection advantage on these food-borne pathogens remain largely unknown. Here we used the luciferase reporter system to determine the response of the Salmonella enterica serovar Typhimurium promoters PeutS, PpocR, PpduF, and PpduA to a set of carbon sources, to egg yolk, to whole milk, and to milk protein or fat fractions. Depending on the supplements, specific inductions up to 3 orders of magnitude were observed for PeutS and PpduA, which drive the expression of most eut and pdu genes. To correlate these significant expression data with growth properties, nonpolar deletions of pocR, regulating the pdu and cob-cbi genes, and of eutR, involved in eut gene activation, were constructed in S. Typhimurium strain 14028. During exponential growth of the mutants 14028ΔpocR and 14028ΔeutR, 2- to 3-fold-reduced proliferation in milk and egg yolk was observed. Using the Caenorhabditis elegans infection model, we could also demonstrate that the proliferation of S. Typhimurium in the nematode is supported by an active ethanolamine degradation pathway. Taking these findings together, this study quantifies the differential expression of eut and pdu genes under distinct conditions and provides experimental evidence that the ethanolamine utilization pathway allows salmonellae to occupy specific metabolic niches within food environments and within their host organisms.

An estimated 26% of all food-borne infections in the United States are caused by Salmonella enterica (39). Consumption of contaminated foods, such as poultry, eggs, raw milk, seafood, and fresh produce, has been the major cause of salmonellosis (18). Eggs and egg products are the food vehicles most often identified in S. enterica outbreaks (10). Dairy farms are well-known reservoirs of S. enterica (21, 41). Between 1992 and 2000, 52% of the food-borne outbreaks in England and a similar proportion in France were attributed to raw milk (16, 21). In the United States, the sale of contaminated raw milk to the public has led to the outbreak of multidrug-resistant salmonellosis in California and Washington (45). Several studies report that S. enterica is the most predominant organism in raw bulk tank milk; Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Newport are the major serotypes identified here (23, 24, 41).

A variety of gut and environmental genera, including Salmonella, Escherichia, Enterococcus, and Arthrobacter, can utilize ethanolamine as the sole source of carbon, nitrogen, and energy (6, 12, 17, 48; reviewed in reference 19). The precursor molecule is phosphatidylethanolamine, an abundant phospholipid in bacterial and mammalian cell membranes that is broken down to glycerol and ethanolamine by phophodiesterases (33, 44, 48). The enzymes responsible for the degradation of ethanolamine are encoded mainly by 17 clustered genes whose functions have been characterized in great detail (29, 55). The ethanolamine lyase EutBC degrades ethanolamine to acetaldehyde and ammonia within a multiprotein complex termed a carboxysome (48, 49). This microcompartment is assumed to prevent the loss of acetaldehyde and to protect the cell from its potential toxic effects (11, 42). The ammonia serves as the cellular supply of reduced nitrogen, while acetaldehyde is converted to acetyl coenzyme A (acetyl-CoA) by the eutE-encoded enzyme aldehyde oxidoreductase (48, 49). Acetyl-CoA is subsequently absorbed in various metabolic cycles, such as the tricarboxylic acid (TCA) cycle, the glyoxylate cycle, or lipid biosynthesis. Under aerobic conditions, the activity of EutBC depends on the exogenous supply of cobalamin (vitamin B12), which is synthesized de novo in the presence of the cob-cbi operon under anaerobic conditions only (25, 49, 50). In many genomes, the cob-cbi operon is clustered with the pdu genes, which are responsible for cobalamin-dependent propanediol degradation, and both operons are transcriptionally activated by the common regulator pocR when Salmonella grows on poor carbon sources (7, 8, 13, 46, 61). The electron acceptor tetrathionate is essential for the anaerobic growth of Salmonella on ethanolamine and 1,2-propanediol (43). Products of the pdu operon have been found in high abundance by proteome analysis of intracellularly replicating S. Typhimurium strain 14028, and its genes are upregulated under in vivo-mimicking conditions (2, 52). Three pathogenic bacteria are able to use both ethanolamine and 1,2-propanediol: the food-borne pathogens S. enterica, Clostridium perfringens, and Listeria monocytogenes (30). Recently, we were able to demonstrate that cobalamin synthesis and propanediol degradation are required for the intracellular replication of S. Typhimurium, as suggested previously with in vivo expression techniques (22, 28). Evidence has also been provided that pdu mutations are responsible for attenuation of the virulence of Salmonella (14) and that the global Salmonella virulence regulators CsrA and Fis influence the expression of the eut gene (27, 34). Moreover, a lack of EutBC activity in L. monocytogenes results in attenuated proliferation of this food-borne pathogen in epithelial cells (26), and all listerial eut genes are upregulated in the intestine in a mouse infection model (58).

However, much less is known about nonmammalian environments in which the eut and pdu genes provide a selection advantage for salmonellae. Using luciferase-based reporter assays, we investigated the expression of the eut and pdu operons in minimal medium supplemented with a set of carbon sources, as well as in milk and egg yolk. eutR and pocR deletion mutants of S. Typhimurium were tested for their fitness under the same conditions and during proliferation in Caenorhabditis elegans. Our data suggest that ethanolamine degradation constitutes a neglected but crucial metabolic determinant of Salmonella-associated food poisoning and that it also contributes to S. Typhimurium proliferation in vivo.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

The bacterial strains and plasmids used in this study are listed in Table Table1.1. S. enterica strains were grown in Luria-Bertani (LB) broth (10 g/liter tryptone, 5 g/liter yeast extract, 5 g/liter NaCl) or in minimal medium (MM or M9) supplemented with 2 mM MgSO4, 0.1 mM CaCl2 and 27.8 mM (0.5% [wt/vol]) glucose. If appropriate, glucose was replaced by 66 mM (0.5% [wt/vol]) 1,2-propanediol or 82 mM (0.5% [wt/vol]) ethanolamine. To produce growth on the latter two substrates under aerobic growth conditions, the media contained cyanocobalamin (Sigma-Aldrich, Taufkirchen, Germany) at a final concentration of 200 nM. For solid media, 1.5% agar (wt/vol) was added. If appropriate, sodium tetrathionate was added as the final electron acceptor to a final concentration of 40 mM. MacConkey agar (Becton Dickinson, Heidelberg, Germany) with ethanolamine was also used as an indicator medium for acid production. Where necessary, the medium contained the antibiotic ampicillin (150 μg/ml), kanamycin (50 μg/ml), or chloramphenicol (25 μg/ml). For all growth and promoter probe experiments, bacterial strains were grown in LB medium overnight at 37°C, washed twice with phosphate-buffered saline (PBS), and then adjusted to an optical density at 600 nm (OD600) of 0.001 in the desired liquid growth medium or in MM. Growth curves were obtained from bacterial cultures incubated at 37°C in sterile 25-ml bottles with 25 ml of MM containing the appropriate carbon source. The OD600 was measured at appropriate time intervals.

TABLE 1.
Bacterial strains and plasmids used in this study

Standard procedures.

DNA manipulations and isolation of chromosomal or plasmid DNA were performed according to standard protocols (51) by following the manufacturers' instructions. GeneRuler DNA Ladder Mix (Fermentas, St. Leon-Rot, Germany) was used as a marker for DNA analysis. Plasmid DNA was transformed via electroporation by using a Bio-Rad Gene pulser II as recommended by the manufacturer and as described previously (28). PCRs were carried out with Taq polymerase (Fermentas). As a template for PCR, chromosomal DNA, plasmid DNA, or an aliquot of a single colony resuspended in 100 μl H2O was used. The oligonucleotides used for PCRs are listed in Table Table2.2. S. Typhimurium gene numbers refer to LT2 annotation (NC 003197). The home pages for NCBI and Microbes Online were used to determinate the distribution of the pdu and eut clusters in different serotypes of S. enterica. Promoter sequences located upstream of the genes identified were predicted with BPROM (Softberry).

TABLE 2.
Primers used in this study

Construction of deletion mutants.

STM2036 (pocR), STM2454 (eutR), and STM4048-STM4049 (rhaS-rhaR) were deleted in frame in S. Typhimurium strain 14028 by the one-step deletion protocol based on the λ Red recombinase system (15). Briefly, PCR products comprising the kanamycin resistance cassette of plasmid pKD4, including the FLP recombination target (FRT) sites, were generated using pairs of 70-nucleotide primers that included 20-nucleotide priming sequences for pKD4 as template DNA (Table (Table2).2). Homology extensions of 50 bp overlapped 18 nucleotides of the 5′ end and 36 nucleotides of the 3′ end of the target gene (38). The fragment DNA was transferred to 14028 cells harboring plasmid pKD46. Allelic replacement of the kanamycin resistance gene cassette was confirmed by PCR, and nonpolar deletion mutants were obtained following transformation of pCP20. Gene deletions were verified by PCR analysis and DNA sequencing with the primers listed in Table Table22.

Cloning of promoter fusions.

The promoter regions of approximately 250 bp upstream of the start codons of STM2036 (pocR), STM2037 (pduF), STM2038 (pduA), and STM2470 (eutS) were amplified from the chromosomal DNA of 14028 by PCR using the primers listed in Table Table2.2. The fragments were then cloned via EcoRI and BamHI into pDEW201. After transformation into Escherichia coli DH5α, plasmids were verified by PCR analysis and sequencing. The correct plasmids were transformed into strain 14028 cells by electroporation.

Preparation of milk and food fractions.

Pasteurized milk and poultry eggs used in this assay were obtained from the local markets and were used for analysis under sterile conditions. Milk was taken directly for analysis. The yolk was separated from the poultry egg and was mixed with a 1% NaCl solution to a final concentration of 45%.

Quantification of promoter activities.

Raw milk and egg yolk preparations were taken directly for the assay. The milk fractions were prepared to a concentration of 1% in MM, stirred constantly for dissolution, and then used for the assay. The ampicillin-resistant reporter strains carrying the corresponding plasmid were grown overnight in LB broth, washed twice in phosphate-buffered saline, and inoculated into the corresponding food material to an OD600 of 0.001. Bioluminescence measurements were performed in microtiter plates. At appropriate time points, 200 μl of each sample was transferred to a 96-well plate, and the OD600 and the emitted bioluminescence, measured as relative light units (RLU), were recorded in a Wallac Victor3 1420 multilabel counter (Perkin-Elmer Life Sciences, Turku, Finland). At the same time points, aliquots were plated onto LB plates containing the antibiotic ampicillin. The resulting bacterial counts were used to determine the corresponding OD600 values from a curve of the OD600 value versus the CFU per milliliter, prepared earlier from a Salmonella culture in LB with ampicillin. These OD600 values were used to calculate the respective RLU/OD600 values.

Growth assays of strain 14028 and mutant 14028ΔeutR in food.

Strains 14028 and 14028ΔeutR were initially starved by growing the bacterium overnight in MM with glucose. Bacteria were washed twice in PBS, inoculated into 50 ml of the particular food sample to an OD600 of 0.001, and incubated at 37°C without shaking. Aliquots of the spiked food samples were taken at regular intervals and were plated onto LB broth-based agar plates at appropriate dilutions for determination of the counts.

C. elegans infection and quantification of intestinal bacterial cells.

Maintenance of wild-type C. elegans N2 (var. Bristol), including feeding, transfer, and synchronization, was performed according to standard procedures (54). Briefly, an E. coli OP50 overnight culture was seeded on nematode growth medium (NGM) agar plates containing 3.0 g NaCl, 2.5 g peptone, 1.0 ml of 1 M CaCl2, 1.0 ml cholesterol (5-mg/ml stock prepared in 95% ethanol), 25.0 ml of 1 M KPO4 buffer (108.3 g/liter KH2PO4 and 35.6 g/liter K2HPO4; pH 6.0), 1.0 ml of 1 M MgSO4, and 17.0 g high-strength Bacto agar per liter (36). Plates with E. coli OP50 were incubated overnight at room temperature and were stored at 4°C. N2 worms were cultivated at 22°C and were transferred to new plates every 2 to 3 days. A 50-μl volume of an overnight culture of bacterial strains was spread onto NGM agar plates (diameter, 8.5 cm), which were then incubated overnight at the appropriate temperature. Plates were equilibrated to room temperature (22°C) before use. C. elegans larvae at larval stage 4 (L4) were individually transferred to the bacterial lawn. The infection assay was performed at 22°C for 5 h. The infected worms were then transferred back to NGM with E. coli OP50 until the end of the experiment.

For quantification, nematodes were transferred at appropriate time points to 600 μl ice-cold lysis buffer (M9 buffer with 0.1% Triton X-100) and were shaken for 10 min at 400 rpm to release bacteria from the cuticula (53). Nematodes were sedimented by centrifugation for 2 min at 430 × g, washed twice with M9 buffer, and resuspended in 1 ml M9 buffer. After the addition of 500 μl of 1.0-mm-diameter zirconia silica beads (BioSpec, Bartlesville, OK), nematodes were disrupted in a FastPrep-24 instrument (MP Biomedicals, Solon, OH) for 20 s at maximal speed, and the suspension was placed on ice. This step was repeated twice. Dilutions were made in LB medium, and aliquots of the suspensions were plated on MacConkey agar containing 1% lactose. The colorless colonies of Salmonella were then enumerated.

RESULTS

Distribution of the cob, pdu, and eut gene clusters in salmonellae.

The genetic organization and distribution of the cob, pdu, and eut operons of the S. Typhimurium LT2 genome were compared with those of different Salmonella serotypes using amino acid homology BLAST programs (4). Within the S. Typhimurium LT2 genome, 20 genes responsible for cobalamin biosynthesis (cob operon) and 23 genes responsible for 1,2-propanediol utilization (pdu operon) are clustered in a genomic island from STM2016 to STM2058 (GEI2016/2058) (Fig. (Fig.1).1). The eut operon comprises 17 genes encoding proteins contributing to ethanolamine degradation. In Salmonella enterica subsp. enterica serovar Paratyphi B strain SPB7, Salmonella enterica subsp. enterica serovar Choleraesuis strain SC-B67, S. enterica subsp. enterica serovar Paratyphi strain AKU_12601, S. enterica subsp. enterica serovar Agona strain SL483, S. enterica serovar Typhi strain CT18, S. enterica subsp. enterica serovar Paratyphi A strain ATCC 9150, S. enterica subsp. enterica serovar Weltevreden strain HI_NO5-537, S. enterica subsp. enterica serovar Virchow strain SL491, S. enterica subsp. enterica serovar Newport strain SL317, and S. enterica subsp. arizonae strain RSK2980, the genetic organization of the eut, pdu, and cob gene cluster is essentially similar to that of the clusters in the reference strain S. Typhimurium LT2, and the encoded amino acid sequences are homologous with at least 95% identity to the LT2 counterparts. However, minor aberrations among those serovars with respect to absence and replacements of single genes within the sequenced clusters were observed. S. Choleraesuis strain SC-B67 contains a hypothetical protein between pduT and pduU. S. Newport strain SL317 lacks pduP, encoding a propionaldehyde dehydrogenase. Within the S. Newport strain SL317 genome, pduG, encoding a 1,2-propanediol dehydratase reactivation protein, pduU, encoding a polyhedral body protein, and pduW, encoding a propionate kinase, are replaced by a glycerol dehydrogenase reactivation protein, a putative ethanolamine utilization protein, and an acetate kinase, respectively. The two latter replacements were also found in the S. Virchow strain SL491 genome. Here, pduP, encoding a propionaldehyde dehydrogenase, is replaced with eutE, encoding a putative aldehyde oxidoreductase. A major island reduction was observed in the Salmonella enterica subsp. enterica serovar Gallinarum strain 287/91 genome, which lacks pocR, pduO, pduG, cbiO, cbiK, cbiD, and cbiC (Fig. (Fig.1).1). The eut operon is conserved within all the tested Salmonella serovars mentioned above except S. Choleraesuis strain SC-B67, S. Newport strain SL317, and S. Virchow strain SL491. S. Choleraesuis strain SC-B67 lacks eutK, eutC, and eutA (Fig. (Fig.1);1); S. Virchow strain SL491 lacks eutM and eutN; and S. Newport strain SL317 lacks eutM. Lack of EutM and EutN in Salmonella Typhimurium does not inhibit the bacterium from utilizing ethanolamine as the carbon source (11, 29).

FIG. 1.
Genetic organization of the eut, pdu, and cob-cbi genes in S. Typhimurium LT2. The genomes of the S. enterica serovars tested (Table (Table1)1) carry intact operons with most of the genes homologous to each other (filled arrows). The cob and ...

Correlation of genotype and growth properties.

For the study of genotypes and growth properties, we deleted pocR, encoding the pdu transcriptional regulator (7), and eutR, encoding the positive regulator of the eut operon, resulting in the nonpolar deletion mutants 14028ΔpocR and 14028ΔeutR. Both genes appeared to be essential for acid production on MacConkey agar base or MM supplemented with 1,2-propanediol or ethanolamine (Fig. 2A and B), thus confirming the literature (29, 47, 49). The S. enterica subsp. arizonae and S. enterica serovar Paratyphi B, Choleraesuis, Newport, Virchow, and Gallinarum strains were streaked onto MacConkey agar plates containing 1,2-propanediol or ethanolamine and cyanocobalamin as the sole carbon source (Table (Table1).1). All but one serovar tested showed pink colonies due to a decrease in the pH upon 1,2-propanediol or ethanolamine utilization (35). S. Gallinarum strain 287/91 utilizes ethanolamine, but not propanediol, irrespective of the presence or absence of cyanocobalamin, due to an incomplete pdu operon (Fig. (Fig.11 and 2C and D). While S. Choleraesuis strain SC-B67 lacks the eutK, eutC, and eutA, these genes are present in the ATCC strain used here, as demonstrated by PCR (data not shown). The phenotypes of all serovars and mutants mentioned above were further confirmed in growth assays using MM supplemented with cyanocobalamin and 1,2-propanediol or ethanolamine (data not shown).

FIG. 2.
Growth on MacConkey agar base supplemented with 1% propanediol or ethanolamine with or without cyanocobalamin. (A) Colonies of strain 14028 appear dark pink on MacConkey agar containing propanediol or ethanolamine. (B) 14028ΔpocR on propanediol-containing ...

Differential expression of genes involved in propanediol and ethanolamine utilization.

According to the literature, the pdu operon has a single transcription start site upstream of pduA, and pduF and pocR are controlled by their own promoters (13); the eut operon has a main promoter located upstream of eutS and a minor promoter upstream of eutR (29) (Fig. (Fig.1).1). Fragments 250 bp upstream of pduA, pduF, pocR, and eutS, which were computationally confirmed to possess promoter regions, were cloned upstream of the luxCDABE cassette in plasmid pDEW201, and the resulting constructs were transformed into 14028. Strains with plasmids carrying the argS promoter or an intergenic fragment of STM0047 (‵STM0047′) without promoter homology served as the positive and negative controls. The probes were then inoculated into growth medium or food, and promoter expression was determined as RLU/OD600.

All promoters except the positive control were transcriptionally inactive in MM containing glucose. Therefore, the fold induction of promoters tested in the presence of other carbon sources was correlated to the light emission values measured in glucose-containing medium. The induction of PpduA was 241-fold higher during growth with 1,2-propanediol than the threshold level in medium with glucose, thus quantifying former qualitative data (7). The addition of rhamnose and fucose induced the pdu operon 27-fold and 14-fold, respectively (Table (Table3).3). When tested with arabinose and sucrose in MM, the induction of PpduA was only slightly above the threshold (data not shown). To determine whether rhamnose or its fermentation products act as PpduA inducers, rhamnose metabolism was interrupted by the deletion of rhaS and rhaR. PpduA did not show significant transcriptional activity in mutant 14028ΔrhaSR cultivated in M9 containing 1% rhamnose and 0.1% yeast extract (data not shown).

TABLE 3.
Quantification of promoter activities in 1,2-propanediol, rhamnose, and fucose

The promoters of pduF and pocR, whose products are involved in propanediol transport and in the regulation of the pdu operon, were found to be upregulated 3-fold and 2-fold, respectively, in medium with 1,2-propanediol. The latter finding probably reflects the pocR autoinduction (7). No significant induction of PpduF and PpocR in the presence of rhamnose and fucose was observed. Growth in MM with 1,2-propanediol led to a 21-fold induction of the transcriptional activity of the main promoter for the eut operon, PeutS, possibly due to CsrA-dependent regulation of both pathways (3). In MM with glucose, no signal above the threshold set by the negative control was observed for PeutS, while the signal with the positive control, PargS, was approximately 140-fold upregulated (Table (Table4).4). In MM containing ethanolamine, high transcriptional activity of PeutS during the growth of strain 14028 was observed in the late-logarithmic phase in comparison to that for the control fragment ‵STM0047′ and PargS. Standardization to the data of the control experiment with glucose revealed that these values correspond to a 2,118-fold induction of PeutS, but only to 4.6-fold- and 2.4-fold-enhanced transcriptional activities of PargS and ‵STM0047′, respectively (Table (Table44).

TABLE 4.
Quantification of promoter activities in the presence of MM with glucose or ethanolamine

14028ΔeutR neither transcriptionally activates the eut operon and nor grows with ethanolamine. As a control, plasmid pDEW-PeutS was therefore transformed into 14028ΔeutR and investigated for promoter induction in ethanolamine-containing MM. The RLU/OD600 values of 6.4 × 102 were similar to the light emission by the negative control, thus confirming the role of EutR as an essential inductor of the eut operon.

The data presented above show the specific induction of the cloned promoters in response to carbon sources added to the medium. They also quantify the response of PpduA and PeutS to the presence of 1,2-propanediol or ethanolamine in the absence of glucose, resulting in an increase in pdu or eut gene transcription by as much as 2 to 3 orders of magnitude at the end of the logarithmic phase.

Induction of the eut operon in milk and egg yolk.

The impacts of raw milk and egg on the expression of the eut operon were then tested using plasmids pDEW-PeutS, pDEW-PargS, and pDEW-‵STM0047′. To this end, the strains harboring the promoter fusions were directly inoculated into raw milk, and the bioluminescence of the three constructs was measured. During the growth of S. Typhimurium in milk, the activity of PeutS reached a maximum of 1.5 × 106 RLU/OD600 (Fig. (Fig.3A).3A). This is approximately two times higher than the bioluminescence observed with PargS controlling a housekeeping gene and 428 times higher than the background expression from the negative-control sequence without promoter homology (‵STM0047′). To determine which factor in milk specifically induced the operon, the constructs were further tested in the presence of three milk fractions, namely a lipid-rich milk fat globule fraction, a protein-rich casein macropeptide (CMP) fraction, and an ultrafiltrate permeate fraction rich in carbohydrates. We observed that the lipid-rich milk fat fraction (1% in MM) upregulated the eut operon to 11-fold-higher activity of PeutS in comparison to ‵STM0047′ (Table (Table5).5). In contrast, only a background emission level of PeutS, similar to that of the negative control, was detected when S. Typhimurium was grown in MM with 1% of a protein-rich CMP fraction, or with an ultrafiltrate permeate fraction lacking CMP and the milk fat globule membrane material (data not shown). The strain with pDEW-PeutS was also inoculated into milk with different concentrations of fat. While the milk rich in fat (3.5%) induced the eut operon 428-fold with respect to the negative control, the milk with less fat—0.1 or 1.5%—activated this operon by a factor of 62 or 46, respectively (Table (Table55).

FIG. 3.
Luciferase-based expression profiling of the eut and pdu operons in milk and egg yolk. (A) The transcriptional activity of PeutS in milk was 428-fold higher than the background expression of the negative control (‵STM0047′) and 2-fold ...
TABLE 5.
Quantification of promoter activities in the presence of MM with CMP or the fat fraction of milk

This induction of the eut operon in the presence of lipid-rich food fractions was further investigated using pure egg yolk containing 80% lipids, of which as much as 30% is phospholipids (31). Under these conditions, a 139-fold induction of PeutS with respect to the control fragment ‵STM0047′ was determined (Fig. (Fig.3B).3B). During growth in both milk and egg yolk, the induction of the eut operon started approximately 4 h after the inoculation of 14028 into the food medium, and the promoter reached its maximal activity approximately 5 h later (Fig. 3A and B). The pdu operon did not show any upregulation in the different milk fractions. The specific induction of PeutS during S. Typhimurium growth in milk and egg yolk was also monitored with a Xenogen IVIS Lumina station (Fig. (Fig.3C3C).

Growth deficiency of mutant 14028ΔeutR in milk and egg yolk.

The high and specific induction of PeutS in the presence of milk and egg yolk suggests that the eut operon contributes to the replication of S. Typhimurium under these conditions. We therefore constructed the mutant 14028ΔeutR, which is unable to grow in a minimal medium with ethanolamine as the only source of carbon and energy (data not shown).

The bacterial proliferation of strains 14028 and 14028ΔeutR spiked into sterile milk was monitored over a period of 24 h at 37°C by enumerating the bacteria at several time points. Four and 6 h after inoculation, 2 × 106 and 3.7 × 107 CFU/ml of mutant 14028ΔeutR, but 6.6 × 106 and 1.1 × 108 CFU/ml of strain 14028, were counted (Fig. (Fig.4A4A ). A significantly reduced cell number of mutant 14028ΔeutR was still observed after 8 h, but not after 10 h or later, when cells reached the stationary phase.

FIG. 4.
Contribution of ethanolamine degradation to S. Typhimurium proliferation in food. (A) Replication of strains 14028 and 14028ΔeutR in milk. 14028ΔeutR exhibited a 3-fold lower cell number than 14028 after 4 to 6 h, a pattern that parallels ...

A correlation between ethanolamine operon induction and the proliferation of S. Typhimurium in food was further examined using egg yolk as the growth medium. The data obtained were similar to those from the milk experiment. Four and 8 h after inoculation, mutant 14028ΔeutR exhibited 2.1 × 107 and 3.5 × 108 CFU/ml, in comparison to 4.7 × 107 and 6.5 × 108 CFU/ml obtained with strain 14028 (Fig. (Fig.4B).4B). This 2-fold reduction in the mutant cell counts obliterated gradually to an equal cell number of the two strains 10 h postinfection. The reduced proliferation of 14028ΔeutR both in milk and in egg yolk fits well with the pattern of PeutS expression, showing that the ethanolamine operon is induced 4 to 6 h after the entry of the bacterium into the food and that this induction lasts approximately 10 h. These results, in combination with the expression data, demonstrate that the ethanolamine utilization pathway not only is stimulated during the initial hours of proliferation within milk and egg yolk but also contributes to the proliferation of S. Typhimurium in food.

Impact of ethanolamine utilization for proliferation of S. Typhimurium within C. elegans.

The relevance of PocR and EutR, and thus of ethanolamine and propanediol utilization, in vivo was tested by comparing the bacterial proliferation of the respective mutants with that of strain 14028 following C. elegans infection. Both mutants 14028ΔeutR and 14028ΔpocR exhibited a compromised ability to proliferate within nematodes compared with that of strain 14028 (Fig. (Fig.5).5). Twelve hours postinfection, 1.9 × 103 CFU/worm of strain 14028, but only 1.1 × 103 CFU/worm and 1.2 × 103 CFU/worm of 14028ΔeutR and 14928ΔpocR, respectively, were obtained, corresponding to a 1.7- and 1.6-fold reduction in the 14028ΔeutR and 14028ΔpocR counts in comparison with those of strain 14028. This growth attenuation increased considerably during the next hours of infection. Twenty-four hours after C. elegans worms were fed on Salmonella strains, a 2.4-fold-lower number of viable 14028ΔeutR cells than of viable strain 14028 cells was determined. 14028ΔpocR even showed 8.6-fold-weaker growth than strain 14028. The measurements after 36 and 48 h revealed 1.8-fold and 2.3-fold reductions of 14028ΔeutR and 14028ΔpocR cell numbers from those of the control. Thus, both PocR and EutR are required for wild-type-like growth of S. Typhimurium during infection of C. elegans.

FIG. 5.
Bacterial proliferation of strains 14028, 14028ΔpocR, and 14028ΔeutR in C. elegans. Strain 14028 showed 2.4-fold-increased proliferation relative to that of 14028ΔeutR and a cell number 8.6-fold higher than that of 14028Δ ...

DISCUSSION

S. enterica is widely known to utilize 1,2-propanediol and ethanolamine as the sole sources of carbon and energy, but very little is known about the impact of these metabolic pathways on the replication of the pathogen under natural conditions. 1,2-Propanediol and, indirectly, sugars such as rhamnose and fucose, known as sources of 1,2-propanediol (5), were demonstrated here to upregulate the pdu operon significantly and specifically. The highest induction, by more than 3 orders of magnitude, was measured for PeutS in the presence of ethanolamine.

About 95% of cases of human salmonellosis are attributed to contaminated food products such as meat, poultry, eggs, and milk (18). Interestingly, raw milk and egg yolk were shown here to strongly upregulate the eut operon, but not the pdu operon. On average, bovine milk contains about 33 g of total lipid per liter. This includes a variety of phospholipids derived from the mammary plasma membrane surrounding each milk fat globule (20). Phosphatidylethanolamine is the second most abundant phospholipid in mammalian cell membranes, constituting 20% to 50% of the total phospholipids (59). Further investigation of eut induction in a casein macropepetide (CMP) fraction and the fat fraction of milk revealed that the fat fraction, with the higher phospholipid content, but not the CMP fraction, induced the eut operon. A 139-fold induction in egg yolk confirms that eut operon induction in Salmonella is specifically high in food materials rich in lipids. Indeed, in both raw milk and egg yolk, the proliferation of the mutant 14028ΔeutR, which is unable to utilize ethanolamine, was significantly retarded up to 10 h after inoculation. This is in line with the observation that cells preadapted by overnight growth in MM with ethanolamine exhibited PeutS induction as early as 2 h postinoculation (data not shown). This provides evidence that the utilization of phosphatidylethanolamine from phospholipids plays a neglected role during the proliferation of S. Typhimurium in foods moderately rich in fat.

C. elegans infection is a feasible model for the study of Salmonella pathogenesis (56). Different strains of S. Typhimurium, including SL1344 and 14028, had similar proliferation patterns within C. elegans, and the bacterium killed the worms with a TD50 (time for 50% of the nematodes to die) of 5.1 ± 0.7 days (1). The C. elegans model was used here to evaluate the importance of ethanolamine and propanediol utilization during the proliferation of Salmonella within the nematode. Our experiments demonstrated that 14028ΔeutR and 14028ΔpocR are significantly growth deficient within the worm compared with strain 14028. The lysis of E. coli OP50 cells by the nematode probably results in the accumulation of bacterial phospholipids and polysaccharides in the C. elegans intestine. These substrates and their degradation products might induce the ethanolamine and/or propanediol pathways of S. enterica strains that have successfully colonized the nematode, allowing this pathogen to proliferate further within the worm. Mutants incapable of utilizing those substrates have a significant growth disadvantage during proliferation within nematodes. Interestingly, an increasing number of recent reports provide further evidence that the functionally coupled eut, pdu, and cob operons contribute to the replication of pathogenic bacteria in eukaryotic hosts, as reviewed recently (19). Examples, in addition to data from mouse infection or the cell culture assays mentioned above, include the induction of the Photorhabdus luminescens eut genes in insects (40) and the observation that an eut mutant of Enterococcus faecalis is attenuated in the killing of C. elegans (19). Moreover, tetrathionate, which acts as a terminal electron acceptor during the anaerobic degradation of 1,2-propanediol, is formed in the inflamed gut upon infection (62).

Since the cob and pdu operons constitute a single regulon controlled by PocR, the possibility cannot be excluded that the proliferation phenotype of 14028ΔpocR is caused by the lack of de novo cobalamin synthesis, thus reflecting the attenuated growth of 14028ΔeutR, which is unable to use ethanolamine in a cobalamin-dependent manner. However, nematode growth medium (NGM) harbors traces of vitamin B12. Furthermore, it has been shown previously that pdu genes are essential for the replication of Salmonella in macrophages (28), suggesting a role for ethanolamine and propanediol degradation in vivo.

Taken together, these findings point to an overlooked contribution of ethanolamine and propanediol utilization to the proliferation of S. Typhimurium in soil organisms, in food, and in different mammalian compartments. Thus, both metabolic traits provide a selection advantage for the food-borne pathogen not only in a single step of infection, but over all stages of the infection chain, and might therefore constitute a promising new set of possible targets for food formulas that suppress bacterial growth (9, 37).

Acknowledgments

We thank Siegfried Scherer for financial support. The Bayerische Forschungsstiftung is gratefully acknowledged for a postdoctoral stipendium to S.S.

We thank Ulrich Kulozik for helpful discussions, Nadja Siegert for providing CMP and milk fat fractions, Patrick Schiwek for technical assistance, and Nicholas Thomson for supplying S. Gallinarum strain 287/91.

Footnotes

[down-pointing small open triangle]Published ahead of print on 29 October 2010.

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