INTRODUCTION

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Survival of the food-borne pathogen Listeria monocytogenes in hypersaline environments is attributed mainly to the accumulation of organic compounds termed osmolytes (57). Osmolytes, often referred to as compatible solutes (9) owing to their compatibility with cellular metabolism at high internal concentrations, can be either transported into the cell or synthesized de novo and act by counterbalancing the external osmotic strength, thus preventing water loss and plasmolysis (14, 15).

Beumer et al. (8) identified three principal compatible solutes in Listeria: proline, betaine, and carnitine. While much information is available regarding uptake of these osmolytes from the external environment (8, 40, 53), detailed analysis of osmolyte synthesis systems in Listeria has remained largely unexplored. Unlike the recently identified transport systems BetL (47, 48), OpuC (19; R. D. Sleator, unpublished) and GbuABC (21, 30), osmolyte synthesis is not restricted by the availability of external osmolytes, a factor which may represent an important limitation for growth in hostile environments of elevated osmolarity such as the macrophage phagosome. Optimal growth of L. monocytogenes in low a_w environments may thus depend on osmolyte synthesis in combination with uptake.

Perhaps the best-characterised bacterial osmolyte synthesis system is that of proline (5, 24, 32). For the majority of bacteria, proline is synthesized from glutamate by a four-step reaction catalyzed by gamma-glutamyl kinase (GK; proB product; EC 2.7.2.11), -glutamyl phosphate reductase (GPR; proA product; EC 1.2.1.41), and ¹-pyrroline-5-carboxylate (P5C) reductase (proC product; EC 1.5.1.2). The remaining step, third in the sequence, occurs spontaneously (12). In other genera, the proB and proA genes generally constitute an operon, which is distant from the proC gene on the chromosome. In addition to this pathway, a number of bacteria have been shown to synthesize proline via offshoots of the arginine biosynthetic pathway (5).

As well as its role as an osmoprotectant, recent evidence suggests that proline may function as a virulence factor for certain pathogenic bacteria (6, 16, 46). Marquis et al. (35), using an uncharacterized listerial proline auxotroph obtained following transposon mutagenesis, concluded that while proline auxotrophy had no effect on virulence following intravenous inoculation, the possibility of reduced virulence during the intestinal phase of natural infection could not be ruled out. In this communication we describe the isolation, characterization, and disruption of the listerial proBA operon and investigate the role of this genetic element in contributing to the growth and survival of L. monocytogenes in environments of elevated osmolarity and during subsequent infection (both intraperitoneal and peroral) of a murine model.

	MATERIALS AND METHODS

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Media, chemicals, and growth conditions. Bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were grown at 37°C in either Luria-Bertani (LB) medium (34) or M9 minimal medium (Gibco-BRL, Eggenstein, Federal Republic of Germany [FRG]) containing appropriate additional requirements. L. monocytogenes strains were grown either in brain heart infusion (BHI) broth (Oxoid, Unipath Ltd., Basingstoke, United Kingdom) or in chemically defined minimal medium (DM) (43). Blood agar plates consisted of blood agar base (Lab M) to which 5% sheep blood was added following autoclaving. All experiments involving the selection of proline-prototrophic (Pro⁺) derivatives of proline-auxotrophic (Pro) strains were carried out using proline-deficient minimal media supplemented with 0.2 mM arginine to eliminate spontaneous Pro⁺ phenotypic revertants carrying suppressor mutations, which may allow the arginine biosynthetic pathway to function in proline biosynthesis (7). Where necessary, proline (Sigma) and 4-nitropyridine 1-oxide (Merk-Schuchardt, Hohenbrunn, FRG) were added to the growth medium at the appropriate concentration, as filter-sterilized solutions. Radiolabeled L-[2,3,4,5-³H]proline (100 Ci/mmol) was purchased from American Radiolabeled Chemicals Inc., St. Louis, Mo. Ampicillin (AMP), carbenicillin (CAR), chloramphenicol (CHL), kanamycin (KAN), and rifampin (RIF) were made up as described by Maniatis et al. (34) as concentrated stocks and added to media at the required levels. Where indicated, medium osmolarity was adjusted by the addition of NaCl.

DNA manipulations and sequence analysis. Restriction enzymes, RNase, shrimp alkaline phosphatase, and T4 DNA ligase were obtained from Boehringer GmbH (Mannheim, FRG) and used according to the manufacturer's instructions. Genomic DNA was isolated from L. monocytogenes as described by Hoffman and Winston (25). Plasmid DNA was isolated using the Qiagen QIAprep Spin miniprep kit (Qiagen, Hilden, FRG). E. coli was transformed using standard methods (34), while electrotransformation of L. monocytogenes was achieved using the protocol outlined by Park and Stewart (38). Restriction fragments were isolated using the Qiaex II gel extraction Kit (Qiagen). PCR reagents (Taq polymerase and deoxynucleoside triphosphates) were purchased from Boehringer and used according to the manufacturer's instructions with a Hybaid (Middlesex, United Kingdom) PCR express system. Unless otherwise stated, PCR was carried out following lysis of cells with Igepal CA-630 (Sigma). PCR products were purified using the QIAquick PCR purification kit. Oligonucleotide primers (listed in Table 2) used for PCR and sequence purposes were synthesized on a Beckman Oligo 1000M DNA synthesizer (Beckman Instruments Inc., San Diego, Calif.). Nucleotide sequence determination was performed on an ABI 373A automated sequencer using the dye terminator sequence kit (Applied Biosystems, Warrington, U.K.). Nucleotide and protein sequence analysis were done using Lasergene (Dnastar Ltd., London, United Kingdom). Protein secondary structure analysis was determined by using the PredictProtein program (EMBL, Heidelberg, FRG) (44). Homology searches were performed with the Blast program (2).

Isolation of proBA from L. monocytogenes. A DNA library consisting of genomic DNA from L. monocytogenes LO28, partially digested with Sau3A and ligated to plasmid pUC18 DNA, digested with BamHI and dephosphorylated with shrimp alkaline phosphatase, was constructed as described previously (47). Plasmids were isolated and transformed into the proline synthesis mutant E. coli CSH26; transformants were then plated on minimal medium containing no added proline to select proline prototrophs. Plasmids isolated from complementing clones were tested for recomplementation of the proline auxotrophy and analyzed by agarose gel electrophoresis. Restriction deletion analysis (using enzymes whose recognition sites constitute the multiple cloning site of plasmid pUC18 [54]) followed by recomplementation experiments was used to isolate those plasmids with the smallest complementing insert.

Tn1000 mutagenesis. Tn1000 mutagenesis was carried out essentially as described by Strathmann et al. (51), using E. coli DPWC as the Tn1000-containing host strain and E. coli BW26 as the F recipient. The cloned insert on the smallest complementing plasmid (pCPL8) was amplified by PCR using primers 5EF2EcoRI and 5ER2EcoRI, digested with EcoRI, and ligated to similarly digested pMOB. The resulting construct, designated pCPL10, was isolated by functional complementation of E. coli CSH26, selected on proline-deficient minimal medium. Plasmid pCPL10 was then transformed into E. coli DPWC, which carries Tn1000 on the F factor. Following transformation, mobilization of the transposon into pCPL10 occurred in E. coli DPWC, the transposition transiently fusing the F factor and pCPL10 in a cointegrated structure that was subsequently transferred to E. coli BW26 by bacterial mating. Following conjugation, resolution of the cointegrate in E. coli BW26 resulted in a single copy of Tn1000 placed randomly within pCPL10. Since E. coli BW26 is KAN resistant and pCPL10 codes for CAR resistance (pMOB carries the -lactamase gene for AMP and CAR resistance), plating onto medium with both antibiotics selected E. coli BW26 cells harboring pCPL10 mutated randomly with Tn1000. These cells were pooled and grown for 2 h in LB medium containing CAR (50 µg/ml). Plasmid DNA was extracted and used to transform E. coli CSH26, selected on LB medium containing 10 mM proline and AMP (50 µg/ml). Clones in which the complementing insert was functionally inactivated were isolated by replica plating, based on their lack of growth on proline-deficient minimal medium. Since the presence of the transposon places known sequencing primer sites adjacent to unknown, unsequenced regions of the target DNA, isolation of a set of clones in which Tn1000 is situated 100 to 500 nucleotides (nt) apart allowed the operon sequence to be assembled from overlapping DNA sequences generated using the Tn1000-specific primers G186 and G187 in combination with the Pharmacia (Uppsala, Sweden) universal and reverse primers

Transport assays. Radiolabeled proline uptake was measured essentially as described by Culham et al. (16).

Generation of an L. monocytogenes proBA mutant. The splicing by overlap extension (SOE) PCR procedure described by Horton et al. (26) was used to create PSOE, a mutant with an internal 1,394-bp deletion in proBA. SOE PCR primers were designed to amplify two ~300-bp DNA fragments, one comprising the 5' end of proBA (nt 76 to 384), amplified by primers SOEA (proBA) and SOEB (proBA) (Table 2), and the other comprising the 3' end of the operon (nt 1779 to 2082), amplified by primers SOEC (proBA) and SOED (proBA) (Table 2). The resulting products were gel extracted, mixed in a 1:1 ratio, and reamplified using the SOEA (proBA) and SOED (proBA) primers. The amplified 613-bp product was digested with EcoRI and XbaI and cloned into the temperature-sensitive shuttle vector pKSV7 (49) before being transformed into E. coli DH5. The resultant plasmid, designated pCPL11, was electroporated into L. monocytogenes LO28, and transformants were selected on BHI agar plates containing CHL (10 µg/ml). Forced chromosomal integration of pCPL11 at 42°C, followed by sequential passaging in BHI at 30°C in the absence of CHL, facilitated allelic exchange between the intact proBA operon and the 613-bp insert on pCPL11. The successful mutation event was confirmed by PCR using the SOEX (proBA) and SOED (proBA) primers (Table 2).

Virulence assays. Bacterial virulence was determined by intraperitoneal and peroral inoculation of 8- to 12-week old BALB/c mice. Intraperitoneal inoculations were carried out as described previously (48), using overnight cultures of mutant and wild-type cells (3 × 10⁶ cells) suspended in 0.2 ml of phosphate-buffered saline. For peroral inoculations, mutant and wild-type strains suspended in buffered saline with gelatin were mixed at a 1:1 ratio of LO28(Rif) to PSOE. Mice were infected with approximately 10⁹ cells (total) using a micropipette tip placed immediately behind the incisors. Three days postinfection, mice were euthanized, and listerial numbers were determined by spread plating homogenized samples onto BHI (for liver and spleen) and blood agar (for Peyer's patches and small intestine wall and contents) with and without RIF (50 µg/ml).

Nucleotide sequence accession number. The nucleotide sequence data reported in this paper have been submitted to GenBank and assigned accession no. AF282880.

	RESULTS

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Complementation of E. coli CSH26. The proBA mutant E. coli CSH26 is unable to synthesize proline, rendering it incapable of growth in proline-deficient minimal medium. A pUC18::LO28 genome library (see Materials and Methods) was transformed into CSH26, and transformants were selected on minimal medium containing no added proline. While no transformants were obtained with pUC18 alone, transformation efficiencies of approximately 50 CFU/µg of DNA were achieved from the plasmid bank, colonies appearing after 24 h at 37°C. Plasmids isolated from five random transformants were retransformed into CSH26 to confirm complementation. Following analysis by gel electrophoresis, all five clones were shown to contain the same ~8.5-kb insert. A representative plasmid, designated pCPL7, was chosen for further characterization.

Functional expression of a listerial proline synthesis system is the basis for complementation of E. coli CSH26. To confirm that complementation of E. coli CSH26 with pCPL8 was the result of proline biosynthesis, a number of growth experiments were performed. Both E. coli CSH26(pCPL8) and E. coli CSH26(pUC18) were inoculated into minimal medium with and without 10 mM proline. While growth of CSH26(pCPL8) was observed in both the presence and absence of proline, the control strain CSH26(pUC18) only grew in the medium containing added proline. To further characterize the insert on pCPL8, the plasmid was introduced into E. coli strains RC711 (proA), J5-3 (proB), JM240 (proC), and MKH13 (putPA proP proU) with well-characterized mutations in various proline biosynthetic and uptake genes. For strains with mutations in proline biosynthetic genes, the results indicated that the plasmid contained sufficient genetic information to restore the proline prototrophy in the proA and proB but not proC mutants. The plasmid was unable to complement the proline uptake deficiency in MKH13, and as expected, no measurable L-[2,3,4,5-³H]proline uptake was observed for MKH13 containing pCPL8 (data not shown),

Sequence analysis of complementing insert. Tn1000 mutagenesis (33, 51) facilitated rapid localization and sequence determination of the complementing genes on pCPL10. Following transposon insertion, replica plating based on functional inactivation of the Pro⁺ phenotype led to the isolation of approximately 50 Pro mutants. In each Pro mutant tested, the site of the Tn1000 insertion mapped to a ~2-kb portion of the insert. Based on this analysis, 2,707 bp of DNA sequence was generated by bidirectional sequencing from a set of five clones (Table 1) in which Tn1000 insertions were positioned at approximately 300-bp intervals (Fig. 1).

View larger version (31K): [in this window] [in a new window]   FIG. 1.   Random Tn1000 insertion within the pCPL10 plasmid of E. coli CSH26 clones A to E. The oligonucleotide combination used for PCR was the transposon-specific primer G186 and the M13 forward primer. Creation of the proBA deletion mutant PSOE is also illustrated. Part of the coding region of proBA was eliminated by using the SOE procedure (see Materials and Methods) and confirmed by PCR.

Creation of an L. monocytogenes proBA mutant. In order to properly evaluate the role of proBA in contributing to the growth and survival of L. monocytogenes, we used allelic exchange mutagenesis to create a 1,394-bp deletion in the proBA operon, designed to inactivate both genes (Fig. 1). The resulting mutant, designated PSOE, exhibited complete proline auxotrophy, requiring upwards of 10 mM proline to restore growth to a level comparable to that of the parent strain in DM. As expected, this mutation could be complemented by the introduction of pCPL9, a plasmid constructed by cloning the proBA operon into the lactococcal vector pCI372 (23), which is capable of replication in Listeria. In the absence of added salt, the growth rate of the PSOE mutant in complex media such as BHI was unaffected (Fig. 2), presumably due to high levels of both free proline (~5 mM) and proline-containing peptides in this environment (3). A peculiar feature of proBA mutants of E. coli is their increased resistance to the compound 4-nitropyridine 1-oxide (28). However this phenotype (the biochemical basis of which is unknown) appears not to extend to the corresponding Listeria mutant (data not shown).

View larger version (11K): [in this window] [in a new window]   FIG. 2.   Growth rates of LO28 () and PSOE () as a function of NaCl added to the medium. Overnight cultures of L. monocytogenes LO28 and PSOE (proBA) were cultured in the appropriate medium, and the specific growth rates (µ) were determined during exponential growth. Each point represents the mean value of three independent experiments.

View larger version (11K): [in this window] [in a new window]   FIG. 3.   Analysis of the effects of proline and glycine betaine on the growth of the Listeria proline auxotroph PSOE. L. monocytogenes PSOE grown overnight in BHI was washed twice in sterile Ringer's solution before being inoculated (at 2%) into DM at various proline concentrations in both the presence () and absence () of 1 mM glycine betaine. The optical density at 600 nm (OD600) after 60 h of growth at 37°C for each sample was determined in triplicate.

Virulence studies. The effects of deleting proBA on the virulence of L. monocytogenes were analyzed by both intraperitoneal and peroral inoculation of BALB/c mice. Consistent with the findings of Marquis et al. (35), the proline auxotroph PSOE showed no obvious reduction in virulence when administered by the peritoneal route. Mutant and wild-type strains were recovered at approximately equal levels from both livers and spleens of infected animals 3 days postinoculation (Table 3). Given that the small intestine may represent a more osmotically stressful environment than the peritoneal cavity (the osmolarity of the gastrointestinal tract is equivalent to 0.3 M NaCl, as opposed to 0.15 M NaCl for the bloodstream [10]), we analyzed the role of proBA in contributing to the intestinal phase of natural infection. The use of bacterial coinfection by the peroral route allowed direct comparison between mutant and parent strains in individual mice. Similar to the results obtained for intraperitoneal inoculation, mutating ProBA failed to inhibit colonization of the upper small intestine or to disrupt the subsequent invasion and spread to internal organs (Table 3). In addition, osmotically stressing the cells prior to inoculation failed to affect virulence (data not shown). Thus, we can conclude that neither proline nor proline-containing peptides are limiting during murine infection (35), nor is proline biosynthesis likely to function as a source of compatible solutes.

	DISCUSSION

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The ubiquitous nature of the food-borne pathogen L. monocytogenes means that the organism is frequently exposed to a variety of environmental insults, both in foods prior to ingestion (1) and subsequently within the infected host, where it is exposed, among other stresses, to the osmotic challenge of the gastrointestinal tract (10) and macrophage phagosomes (20). Survival of Listeria both at high salt concentrations and in low-temperature environments is attributed mainly to the accumulation of compatible solutes (9). Beumer et al. (8) identified the three principal compatible solutes in Listeria as proline, betaine, and carnitine. The isolation of genes encoding BetL (47) and OpuC (19; Sleator, unpublished), osmolyte transport systems dedicated to the uptake of glycine betaine and carnitine, has previouly been reported. However, to date, no equivalent system has been described for the accumulation of proline in Listeria.

Here we report the identification and disruption of the listerial proBA operon encoding homologues of the GK and GPR complex of the E. coli proline biosynthetic pathway. The pathway from glutamate via glutamate--semialdehyde (GSA) and its spontaneous cyclization product P5C to proline was first proposed in 1952 (55) and has since been described in other prokaryotes, both gram-positive and gram-negative (5, 32). However, not until 1955 was the effectiveness of proline as a compatible solute realized by Christian (11), who observed that accumulation of the osmolyte could relieve bacterial growth inhibition by osmotic stress.

Sequence analysis revealed that the physical organization of the listerial proBA homologue is similar to that of E. coli (17), in which proB (coding for GK) and proA (coding for GPR) constitute an operon with a single ^A consensus promoter proximal to proB. While exhibiting a high degree of sequence similarity and functional compatibility, the two systems differ in a number of respects. First, while the E. coli genes are separated by a 14-nt intergenic region, the listerial proB and proA genes overlap by 17 nt. This, together with the reduced size of the listerial proB gene (273 bp shorter than the equivalent gene in E. coli), leads to the formation of a tighter genetic domain, a feature which may reflect a degree of evolutionary divergence between the two systems. This is particularly relevant given that Hu et al. (27) proposed that the evolutionary origins of the bifunctional plant enzyme P5C synthetase might be linked to a genetic fusion of proB and proA.

The predicted secondary structure composition of ProBA (as determined with the PHD E-mail server and emotif search programs) is a mixed class of -helix, -sheet, and loop structures. Conserved sequences at amino acids (aa) 103 to 110 and aa 321 to 342 of ProA correspond to the [AG]-X4-G-K [ST] sequence fingerprint of an ATP/GTP binding site and -glutamyl phosphate signature sequence, respectively (44). As with A. thaliana (45), a secondary structure near the carboxy-terminal domain of ProA (~aa 210 to 290) may function as a noncovalent NAD(P)H binding domain. The existence of both ATP and putative NAD(P)H binding sites on ProA alone supports the formation of a GK-GPR enzyme complex (as previously described in E. coli [32]) for the catalysis of the ATP- and NAD(P)H-dependent first and second steps, respectively, of proline biosynthesis. Since prokaryotic proline synthesis is known to be regulated by proline-mediated inhibition of GK activity, we used multiple sequence alignments in combination with emotif searches to identify possible allosteric binding domains and adjacent or overlapping enzyme active sites. Two conserved regions of ProB (identified by emotif searches) extended from aa 77 to 109 and 120 to 144. These domains map closely to ProB mutations in E. coli (aa 107, Asp to Asn) (13) and S. marcesens (aa 117, Ala to Val) (37), which result in proline overproduction due to reduced feedback repression, and thus may represent the allosteric binding domain of the enzyme.

The phenotypic consequences of eliminating the ProBA complex provided a unique opportunity to study proline transport in the absence of endogenous proline synthesis. Perhaps the most detailed knowledge of proline transport in gram-positive bacteria involves the halotolerant food-borne pathogen Staphylococcus aureus. Proline uptake in S. aureus is mediated by two transport systems, a specific high-affinity system (corresponding to PutP in E. coli [56]) and an osmotically inducible low-affinity system, which is also dedicated to the uptake of glycine betaine (4, 41, 4, 52) and thus resembles ProP and ProU of E. coli (14). The relatively high concentration of proline (10 mM) required to complement PSOE suggests that Listeria may lack the scavenging capacity of a high-affinity proline transporter, a hypothesis previously suggested by the findings of Patchett et al. (39). The relatively high proline concentrations (>10 mM) demonstrated by Beumer et al. (8) to be osmotically significant may be attributed to the presence of a low-affinity uptake system, the activity of which we have shown to be inhibited by glycine betaine. Proline uptake in Listeria thus may resemble the situation in Lactococcus lactis, in which proline appears to be transported by a single low-affinity system which also transports glycine betaine (36). Alternatively, the system may be specific for proline but inhibited by preaccumulated glycine betaine, as was described previously for proline transport in S. aureus (42). Since regulation of osmolyte uptake by feedback inhibition due to preaccumulated solute has previously been described for both glycine betaine and carnitine uptake in Listeria (53), it is possible that this process may also extend to the accumulation of proline.

While the role of proline as an effective compatible solute is well documented (14, 15), recent evidence suggests that accumulation of the osmolyte may also be linked to the virulence potential of certain pathogenic bacteria (6, 16, 46). Mutating the osmotically sensitive proline transporter ProP resulted in a 100-fold reduction in the colonization of the murine urinary tract by uropathogenic E. coli (16), while putP mutants of S. aureus have been shown to demonstrate reduced virulence in both wound and murine abscess infection models, as well as in experimental endocarditis (a prototypical model of invasive S. aureus infection) (6, 46). We conducted mouse virulence assays to investigate the effects of mutating ProBA (proline auxotrophy) on the colonization of the murine gastrointestinal tract and subsequent growth within the intracellular milieu of the internal organs (liver and spleen). Consistent with previous observations by other workers (35) we observed from intraperitoneal inoculations that proline auxotrophy has no significant effect on the growth of Listeria within the seemingly nutrient-rich environment of the macrophage cytoplasm (35). The elevated osmolarity (0.3 M NaCl [10]) and otherwise limiting environment of the gastrointestinal tract also failed to inhibit growth and survival of PSOE, notwithstanding suggestions that proline biosynthesis may be important during the intestinal phase of natural infection (35). From these results it can be concluded that reduced proline synthesis has no significant effect on Listeria pathogenesis. Given the observed role of ProP and PutP in the virulence of uropathogenic E. coli and S. aureus, respectively, a detailed analysis of proline transport may be required to fully appreciate the importance (if any) of proline in contributing to the virulence potential of L. monocytogenes.