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Applied and Environmental Microbiology, November 2008, p. 6848-6858, Vol. 74, No. 22
0099-2240/08/$08.00+0     doi:10.1128/AEM.00442-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification of Components of the Sigma B Regulon in Listeria monocytogenes That Contribute to Acid and Salt Tolerance

F. Abram,¹ E. Starr,¹ K. A. G. Karatzas,¹ K. Matlawska-Wasowska,¹ A. Boyd,¹ M. Wiedmann,² K. J. Boor,² D. Connally,¹ and C. P. O'Byrne^1*

Department of Microbiology, National University of Ireland—Galway, Galway, Ireland,¹ Department of Food Science, 412 Stocking Hall, Cornell University, Ithaca, New York 14853²

Received 22 February 2008/ Accepted 10 September 2008

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Sigma B (^B) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the sigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The sigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that ^B contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the sigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of ^B in L. monocytogenes, we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the sigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of ^B. It also demonstrated clear roles for ^B in both osmotic and low-pH stress tolerance and identified specific components of the ^B regulon that contribute to the responses observed.

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Listeria monocytogenes is a gram-positive bacterium that has a remarkable ability to grow and survive in very diverse environments. It is almost ubiquitous in nature and can readily be isolated from soil, water, and decaying vegetation. It has been found associated with a wide range of different food products (21, 30, 39, 48), which can be problematic since it causes life-threatening illness in humans, particularly individuals who are immunocompromised. It can grow at temperatures as low as 0°C (17, 57) and can survive in the presence of wide ranges of salt concentrations (17) and pH values (56). In animals L. monocytogenes is a facultative intracellular pathogen that is capable of invading and growing within epithelial cells and macrophages. It can also grow and persist within the gall bladder (28), displaying a remarkable tolerance for bile salts.

The tolerance of L. monocytogenes to a variety of harsh environmental conditions is at least partly attributed to genes under the control of the alternative sigma factor, sigma B (^B). This sigma factor is conserved in closely related gram-positive bacteria, where it coordinates the transcription of genes required for the general stress response (11, 12, 14, 26, 32, 43, 44, 50, 60). Mutants of L. monocytogenes that lack the sigB gene have a variety of stress-sensitive phenotypes. They are sensitive to low pH (24, 55, 59), osmotic stress (5, 25, 55), bile salts (8), bacteriocins (7), and high hydrostatic pressure (58). They survive poorly during prolonged carbon limitation (16, 23, 29), and they grow poorly at low temperatures (5, 6). In addition, mutants lacking sigB have a reduced ability to invade epithelial cells, a finding that is explained by the low levels of internalin expression in such mutants (35, 38). While some of the stress-sensitive phenotypes can be partially explained at the molecular level, it is clear that full elucidation of the ^B regulon is required to understand all of the phenotypes observed in sigB mutant strains.

A number of studies have sought to identify components of the ^B regulon in L. monocytogenes, either using gene array technology (34, 45) or using proteomic approaches (1, 58). Collectively, these studies have identified about 150 L. monocytogenes genes that are expressed in a ^B-dependent manner. The functional categories represented by these genes include stress tolerance, carbon metabolism, transport, cell envelope-related functions, and virulence. In each of these studies L. monocytogenes was cultured in brain heart infusion (BHI) medium, a complex growth medium with an undefined composition. In our recent proteomic study we found that ^B-dependent differences in expression could not be detected in the exponential phase of growth in BHI medium, suggesting that cells experience little stress under these growth conditions. This medium also does not allow clear analysis of osmotolerance since it potentially contains an undefined mixture of different compatible solutes. Moreover, the complexity of this growth medium makes it impossible to study the utilization of individual nutrients by wild-type or sigB mutant strains. For these reasons in the present study we used a chemically defined growth medium (DM) to compare protein expression in wild-type and sigB strains of L. monocytogenes.

Overall, this study highlighted two new phenotypes associated with the loss of ^B. Mutants lacking ^B have a rapid-growth phenotype in DM with added NaCl, and they also exhibit an unusual pattern of Gram staining. The present study also revealed clear roles for ^B in both osmotic and low-pH stress tolerance and identified specific components of the ^B regulon that contribute to these characteristics.

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Bacterial strains and growth conditions.
Bacterial strains and plasmids used in this study are listed in Table 1. All the L. monocytogenes strains were cultured either in BHI broth or in the DM described previously (2) supplemented with either 0.04% (wt/vol) glucose, 0.4% (wt/vol) glucose, 0.4% (wt/vol) mannose, 0.4% (wt/vol) fructose, 0.4% (wt/vol) trehalose, or 0.4% (wt/vol) cellobiose in the presence or absence of 0.5 M NaCl with continuous shaking at 37°C. L. monocytogenes strains containing the shuttle vector pKSV7 or derivatives of this vector were cultured in BHI broth supplemented with 12.5 µg ml^–1 chloramphenicol. Escherichia coli strains were routinely grown in Luria-Bertani broth (1% [wt/vol] tryptone peptone, 1% [wt/vol] NaCl, 0.5% [wt/vol] yeast extract) with continuous shaking at 37°C. E. coli strains containing the shuttle vector pKSV7 or derivatives of this vector were cultured in Luria-Bertani broth containing 50 µg ml^–1 ampicillin. When required, growth was monitored using 96-well plates. The plates were incubated at 37°C in a 96-well plate reader (Genios, Tecan) and shaken every 30 min for 30 s. Optical densities at 595 nm (OD₅₉₅) were automatically recorded in triplicate for each well for 48 h. All cultures were inoculated to obtain a starting OD₆₀₀ of 0.05 using 16-h overnight cultures as inocula. Two-dimensional gel electrophoresis (2-DGE) experiments were carried out with cells grown in DM either to the exponential phase (OD₆₀₀, 0.6) or following 8 h of growth to stationary phase.

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TABLE 1. Bacterial strains and plasmids used in this study

2-DGE.
Proteins were extracted by sonication and subsequently separated by 2-DGE using a modified version of the O'Farrell method (42), as previously described (1). Briefly, the first dimension consisted of isoelectric focusing using 11-cm IPG strips with linear pH gradients (pH 4 to 7; Amersham). The second-dimension gels contained 12% acrylamide and were run in pairs along with molecular mass markers with a range of 10 to 225 kDa (Broad Range protein molecular markers supplied by Promega). Gels were stained overnight in GelCode Blue staining reagent (Pierce) and then destained in deionized, distilled water for several hours. For each growth condition and strain investigated six gels were run, representing samples extracted from two independent cultures and three technical replicates. Gels were analyzed with PDQuest-Advanced software, version 8.0 (Bio-Rad), and data were normalized using the local regression model. Protein expression differences greater than twofold that were obtained for all six pairs of replicates were considered significant. The proteins were identified using a combination of tryptic digestion and matrix-assisted laser desorption ionization-time of flight mass spectrometry, as previously described (10).

Construction of in-frame deletion mutants and complementation.
We constructed four individual in-frame deletion mutants of L. monocytogenes using the splicing by overlap extension PCR technique, followed by allelic replacement (33). This procedure relies on two recombination events: homologous recombination into the L. monocytogenes chromosome of a truncated copy of the gene of interest carried on the suicide shuttle vector pKSV7 (52), followed by a second recombination event leading to the loss of the wild-type copy of the gene of interest along with the suicide vector. Nonpolar lmo0796, lmo0913, lmo2391, and lmo2748 mutants with 492-, 1,407-, 516-, and 399-bp internal deletions, respectively, were created. Primers were designed for each gene (Table 2) to amplify two fragments (AB and CD) of similar size flanking the portion of the gene to be deleted. The amplified flanking regions were mixed at a 1:1 ratio and reamplified using outer flanking primers A and D (Table 2). The resulting AD fragments were digested with EcoRI and cloned into pKSV7. Each plasmid carrying the truncated copy of the gene of interest was transformed into E. coli TOP10 competent cells (Invitrogen), purified, and subsequently electroporated into L. monocytogenes 10403S. Transformants were selected by growth for 48 h at 30°C on BHI agar plates containing chloramphenicol (12.5 µg ml^–1). Subsequently, plasmid integration was forced by growing the organisms at a nonpermissive temperature (42°C) on BHI agar plates in the presence of chloramphenicol. Plasmid excision was achieved by continuous passage of cells growing at 30°C in BHI medium containing no antibiotics with shaking and spreading at intervals onto BHI agar plates. Replica plating on BHI agar plates with and without 12.5 µg ml^–1 chloramphenicol allowed screening for vector loss. Homologous recombination was confirmed by PCR using outside primers (for and rev primers [Table 2]) with chloramphenicol-sensitive colonies.

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TABLE 2. Primers used in this study

Complementation studies were carried out by transforming each of the deletion mutants with a vector carrying the wild-type version of the deleted gene. In short, each gene was amplified by PCR, resulting in an amplicon containing the wild-type version of the gene, including its native promoter region. The corresponding A and D primers used for construction of the deletion cassettes were also used for amplification of the lmo0796, lmo2391, and lmo2748 genes for cloning, while primers lmo0913-compF and lmo0913-compR were used for cloning lmo0913 (Table 2). Subsequently, each of the amplicons carrying the lmo0913 (2,196 bp), lmo2391 (1,511 bp), or lmo2748 (1,165 bp) gene was cloned into pCR-XL-TOPO (Invitrogen Life Technologies) and then subcloned in pKSV7, resulting in plasmids pKAK0913, pKAK2391, and pKAK2748, respectively (Table 1); it was not possible to clone the lmo0796 gene amplicon (1,278 bp) into either pCR-XL-TOPO or pKSV7. Cloning in pKSV7 was performed by digestion of the vector and the amplicons with EcoRI for lmo0796 and lmo2391 and with BamHI for lmo0913 and lmo2748, followed by ligation with T4 ligase. The newly constructed plasmids were electroporated into E. coli One-Shot TOP10 electrocompetent cells purchased from Invitrogen Life Technologies (200 , 25 µF, and 2.4 kV for 0.2-cm cuvettes) and subsequently into both the L. monocytogenes wild-type strain and the corresponding mutant strains (400 , 25 µF, and 2 kV for 0.2-cm cuvettes). All strains were also transformed with the empty vector pKSV7 as a control. The resulting transformants were used for the complementation studies.

Acid resistance.
L. monocytogenes strains were grown for 16 h in BHI broth containing 0.5 M NaCl (BHIS) with shaking at 37°C. Two 1-ml aliquots were centrifuged at 13,000 x g for 2 min. For each culture, one of the cell pellets was resuspended in 1 ml of fresh BHIS in order to determine the initial count for the bacterial population. The other cell pellet was resuspended in 1 ml of fresh BHIS previously acidified to pH 2.5 with HCl. Assay tubes were incubated at room temperature, and 20-µl aliquots were removed at regular intervals, serially diluted, and plated in triplicate on BHI agar plates, which were incubated at 37°C for 48 h before enumeration of CFU.

Caco-2 invasion assays.
The gentamicin protection assay was performed with the wild-type strain and the mutants derived from it, as described previously by Elsinghorst (20), with minor modifications. Two days before the invasion assays were performed, 1.5 x 10⁵ Caco-2 human colon adenocarcinoma cells (European Collection of Cell Cultures number 86010202) were seeded in 24-well plates in Dulbecco's modified Eagle's medium containing 2 mM glutamine, 1% (wt/vol) nonessential amino acids, and 20% (vol/vol) fetal bovine serum supplemented with 100 U ml^–1 penicillin/streptomycin (Sigma). Thirty minutes before coincubation, the medium in each well was replaced with prewarmed fresh medium without antibiotics. The OD₆₀₀ of stationary-phase bacterial cultures grown in BHI broth overnight at 37°C were determined, all cultures were washed twice with sterile phosphate-buffered saline (PBS), and the concentrations were adjusted to obtain similar OD₆₀₀ values. We previously confirmed that there was a good correlation between OD₆₀₀ and the number of cells for each strain, as assessed by comparing numbers of CFU and OD₆₀₀ values. Coincubation was performed with approximately 10⁸ CFU of stationary-phase bacteria for 45 min at 37°C. Subsequently, Caco-2 cells were washed twice with PBS and suspended in Dulbecco's modified Eagle's medium containing 150 mg liter^–1 gentamicin. After 45 min of incubation at 37°C, cells were washed twice with sterile PBS and lysed with 2 ml Triton X-100 (1% vol/vol) in PBS. Following incubation for 5 min at 37°C, cell lysates were serially diluted and plated on BHI agar to determine the number of intracellular bacteria. The invasion efficiency (expressed as a percentage) was calculated by using the ratio of the number of bacterial cells that survived the gentamicin assay to the total number of bacterial cells added initially to each well. A statistical analysis was performed using the Mann-Whitney nonparametric test, which was appropriate for the number of observations conducted (8 < n < 20). P values less than 0.05 were considered statistically significant.

Gram staining.
Gram staining of L. monocytogenes strains was performed with colonies from BHI agar plates. Single colonies were suspended in sterilized distilled water, and a drop of a suspension was placed on a glass microscope slide and fixed by heating. The heat-fixed cells were flooded with crystal violet for 1 min. Then an iodine solution was added for 3 min, and decolorization with alcohol was performed for 20 s, which was followed by counterstaining by addition of safranin for 2 min. Stained bacterial cells were visualized with a Nikon Eclipse E600 microscope, and the images were captured using Q Capture Pro software, version 5.1 (QImaging).

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L. monocytogenes sigB mutant grows faster than the wild type in DMS.
The growth of L. monocytogenes 10403S and the growth of the corresponding sigB mutant were monitored in DM supplemented with 0.4% (wt/vol) glucose both in the presence of 0.5 M NaCl (DMS) and in the absence of 0.5 M NaCl (DM). In DM the two strains exhibited very similar patterns of growth, and during exponential phase they grew with a doubling time of approximately 100 min (Fig. 1). The presence of 0.5 M NaCl caused both strains to grow more slowly, but surprisingly, the sigB deletion mutant was found to grow significantly faster than the wild-type strain. The growth rate of the sigB mutant under these conditions was approximately 60% higher than that of the parent strain (Fig. 1, inset). This result was quite unexpected since ^B is known to play a positive role in osmoregulation, at least in media containing compatible solutes. The presence of the sigB allele in mutant cultures was confirmed by performing PCRs with samples taken from each culture (data not shown).

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FIG. 1. Rapid-growth phenotype of an L. monocytogenes sigB mutant in the presence of 0.5 M NaCl. L. monocytogenes wild-type strain 10403S (squares) and the sigB mutant (triangles) were grown in defined media supplemented with 0.4% (wt/vol) glucose at 37°C with shaking in the absence (DM) (filled symbols) and in the presence (DMS) (open symbols) of 0.5 M NaCl. Representative growth curves for the conditions investigated are shown; all experiments were performed in triplicate. The inset shows the specific growth rates (SGR) during exponential growth derived from the curves for the wild type (open bars) and the sigB mutant (shaded bars) in DM and DMS. The error bars indicate the standard deviations from the means of triplicate measurements.

We investigated the possibility that the higher growth rate of the sigB mutant in DMS might have been due to an ability of this strain to utilize glucose more efficiently than the parent strain. If this phenomenon was specific for glucose, then it should not have been observed if alternative carbon sources were provided in the growth media. Therefore, the growth rates of the two strains were compared in DM and DMS without glucose and supplemented with either mannose, fructose, trehalose, or cellobiose. These growth experiments were performed in a temperature-controlled 96-well plate reader, where the growth rate of L. monocytogenes is typically about 50% of that observed in conical flasks, presumably due to differences in culture aeration. Under these growth conditions with 0.4% (wt/vol) glucose, the wild-type strain and sigB mutant were again found to grow at the same rate in DM (Fig. 2A), while the rapid-growth phenotype of the sigB mutant in DMS was confirmed (Fig. 2B). The rapid-growth phenotype of the sigB mutant was also observed after addition of 0.5 M NaCl when the growth medium was supplemented with either 0.4% (wt/vol) fructose, 0.4% (wt/vol) mannose, 0.4% (wt/vol) trehalose, 0.4% (wt/vol) cellobiose, or a limiting concentration of glucose (0.04%, wt/vol) (Fig. 2B). The effect was less dramatic for fructose than for the other carbon sources tested. In all cases, in the absence of salt (Fig. 2A), the wild type and the sigB deletion strain grew at similar rates. In each case addition of salt to the medium resulted in both strains growing at a lower rate, and the decrease was more dramatic for the wild-type strain (Fig. 2). These data indicate that the rapid-growth phenotype of the sigB mutant in DMS is not dependent on the carbon source provided in the medium.

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FIG. 2. sigB mutant exhibits greater specific growth rates than the wild-type strain in DM supplemented with different carbon sources in the presence of 0.5 M NaCl. Specific growth rates (SGR) of L. monocytogenes 10403S (open bars) and the corresponding sigB deletion mutant (shaded bars) in defined media supplemented with 0.04 or 0.4% (wt/vol) glucose (glu), 0.4% (wt/vol) mannose (man), 0.4% (wt/vol) fructose (fru), 0.4% (wt/vol) trehalose (tre), or 0.4% (wt/vol) cellobiose (cel) were determined. Cells were grown at 37°C in 96-well plates in the absence (A) and presence (B) of 0.5 M NaCl. The errors bars indicate the standard deviations from the means of triplicate measurements.

^B-Dependent protein expression.
We performed a proteomic comparison of L. monocytogenes wild-type strain 10403S and the corresponding sigB mutant. Protein expression in both DM and DMS was investigated in both the exponential and stationary phases of growth. Overall, 11 proteins were found to be differentially expressed in the sigB background under all conditions tested (Fig. 3 and Table 3). The expression pattern of each of these 11 proteins is summarized in Table 3. Ten proteins were found to be under positive control of ^B, while the level of expression of one protein, LmaA, was higher in the sigB mutant than in the wild-type strain. Eight proteins (Lmo0539, Lmo0783, Lmo0796, Lmo1830, Lmo2391, Lmo2696, Lmo0913, and Lmo2748) (Fig. 3A) were always expressed in a ^B-dependent manner when they were detectable. The levels of two of these proteins, Lmo0796 and Lmo0539, were lower in the sigB mutant strain, while the other six proteins were undetectable in extracts prepared from the sigB mutant but were clearly detectable in extracts of the wild-type strain (Fig. 3A).

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FIG. 3. Eleven proteins were found to be expressed in a B-dependent manner. The gel images show representative sections of 2-DGE profiles for proteins extracted from either L. monocytogenes wild-type strain 10403S or sigB cells grown to exponential phase or stationary phase in DM. Proteins were extracted from cells growing either in the presence (+NaCl) or absence (–NaCl) of 0.5 M NaCl. The arrows indicate the locations of the proteins showing altered expression patterns in the sigB background. (A) Proteins showing B-dependent expression in stationary phase (the same proteins showed B-dependent expression in exponential phase [data not shown]). The asterisk indicates the Lmo0913 protein referred to as Lmo0913a. (B) Proteins showing B-dependent expression under specific conditions. Extracts of exponential-phase cells were used for Lmo2425, while extracts of stationary-phase cells were used for Lmo2829 and LmaA.

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TABLE 3. Proteins identified in extracts of DM and DMS cultures

Three other proteins (Lmo2425, Lmo2829, and LmaA) (Fig. 3B and Table 3) were found to be expressed in a ^B-dependent manner only under specific growth conditions. Lmo2425 was under ^B control only in the presence of salt in exponential phase (Fig. 3B). Under all other conditions this protein was detectable but was not found to be differentially expressed (data not shown). Lmo2829 was expressed in a ^B-dependent manner in both phases of growth, but only in the presence of salt (Fig. 3B; data not shown for exponential phase). In the absence of NaCl, similar levels of Lmo2829 were detected in both genetic backgrounds in stationary phase (Fig. 3B) and in exponential phase (data not shown). LmaA was the only protein that showed increased expression in the sigB mutant compared to the wild type. This protein was detectable only in stationary phase and was found to be differentially expressed only when salt was present in the medium (Fig. 3B).

Deletion of ^B-dependent genes lmo0796, lmo0913, lmo2391, and lmo2748.
We attempted to obtain new insights into the ^B regulon by constructing deletion mutants with mutations in targeted ^B-dependent genes. Five genes were selected for this deletion analysis, lmo0796, lmo0913, lmo1830, lmo2391, and lmo2748. These genes were selected because they were ^B dependent under every condition that we tested and they are also known to be ^B dependent when cells are grown in BHI medium (or BHIS) to stationary phase (1). In addition, we previously confirmed that all of these genes are transcribed in a ^B-dependent manner by reverse transcription-PCR (1). Indeed, for three of them (lmo0796, lmo1830, and lmo2391) ^B promoters have been mapped (1). Deletions were constructed using the splicing by overlap extension PCR method (33), resulting in in-frame deletion of 492, 1,407, 516, and 399 bp for lmo0796, lmo0913, lmo2391, and lmo2748, respectively. The presence of the correct deletion constructs in the chromosome of L. monocytogenes was confirmed by PCR analysis and by DNA sequencing (data not shown). Several attempts to delete the lmo1830 gene were unsuccessful. Heterozygous recombinants (lmo1830⁺ and lmo1830) were obtained, but repeated attempts to obtain lmo1830 secondary recombinants failed to yield the required mutant.

To test the possibility that reduced expression of lmo0796, lmo0913, lmo2391, or lmo2748 might contribute to the rapid-growth phenotype of the sigB mutant, we measured the growth of each deletion mutant in DM and in DMS. The mutants all grew at the same rate and reached the same final optical densities as the wild-type strain, and only the sigB mutant grew faster in DMS (data not shown). This result suggested that none of these four ^B-regulated genes is responsible for the higher growth rate observed for the sigB strain in DMS. It also showed that these genes are not essential for growth in a chemically defined medium.

Growth of the sigB and lmo2748 mutants in complex media is impaired in the presence of 1.75 M NaCl.
To further characterize the lmo0796, lmo0913, lmo2391, and lmo2748 mutants, we investigated whether any of them displayed reduced osmotolerance in complex growth media. In BHI broth with an NaCl concentration of 1 M or less, there were no detectable differences in the growth rate between the wild type and either the sigB mutant or the four newly constructed deletion mutants (data not shown). However, when the organisms were grown in BHI broth supplemented with 1.75 M NaCl, the growth of the sigB mutant was clearly impaired compared to the growth of the wild-type strain (0.016 h^–1 versus 0.076 h^–1) (Fig. 4). The lmo0796, lmo0913, and lmo2391 mutants did not show any growth defect in the presence of 1.75 M NaCl. However, the lmo2748 mutant was reproducibly found to grow at a lower rate than the parent strain (0.053 h^–1 versus 0.076 h^–1) under these conditions (Fig. 4). These data suggest that the loss of Lmo2748 expression in the sigB mutant may contribute to the osmotic sensitivity of this strain.

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FIG. 4. lmo2748 and sigB mutants have a slow-growth phenotype in BHI medium supplemented with 1.75 M NaCl. L. monocytogenes wild-type strain 10403S (black squares), lmo0796 mutant (open squares), lmo0913 mutant (gray triangles), lmo2391 mutant (open circles), lmo2748 mutant (black triangles), and sigB mutant (asterisks) cells were grown in BHI medium supplemented with 1.75 M NaCl at 37°C with shaking. Representative growth curves for the conditions investigated are shown; all experiments were performed in triplicate. The inset shows the specific growth rates (SGR) derived from the curves. The error bars indicate the standard deviations from the means of triplicate measurements. wt, wild type.

Lmo0796, Lmo0913, and Lmo2391 contribute to acid tolerance.
The sigB mutant had a clear disadvantage for survival under acidic conditions (pH 2.5) in BHI medium (Fig. 5), a result that confirmed previous findings (24, 55, 59). A 4-log reduction in the size of the sigB population after 2.25 h was observed, compared with a 3-log reduction in the size of the wild-type population. After 3.75 h of incubation at pH 2.5 in BHI medium, the size of the sigB mutant population had decreased by a total of 6 logs, while the size of the wild-type population had decreased by only 3 logs. The lmo0796 and lmo0913 mutants both displayed an acid-sensitive profile that was very similar to that of the sigB mutant (Fig. 5). The level of survival of the lmo2391 strain was reduced compared to that of the wild-type strain, but the difference was not as marked as it was in the case of the sigB strain; after 3 h of incubation at pH 2.5, the number of lmo2391 mutant cells was reduced by 4 logs, whereas the number of wild-type cells was reduced by only 3 logs. In contrast, the lmo2748 mutant did not show any defect in survival under acid conditions (Fig. 5). These data suggest that lmo0796, lmo0913, and lmo2391 (albeit to a lesser extent) contribute to acid tolerance in L. monocytogenes and that decreased levels of the corresponding proteins in the sigB mutant might contribute to the acid sensitivity of this strain.

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FIG. 5. lmo0796, lmo0913, lmo2391, and sigB mutants have a survival defect in BHIS at pH 2.5. L. monocytogenes wild-type strain 10403S (black squares), lmo0796 mutant (open squares), lmo0913 mutant (gray triangles), lmo2391 mutant (open circles), lmo2748 mutant (black triangles), and sigB mutant (asterisks) cells were grown to stationary phase in BHIS at 37°C with shaking and were subsequently subjected to pH 2.5 in fresh BHIS at room temperature. Survival under the acid conditions was monitored in triplicate, and the errors bars indicate the standard deviations from the means.

Genetic complementation of deletion mutants.
In order to confirm the roles of the lmo0796, lmo0913, lmo2391, and lmo2748 genes in the newly identified phenotypes associated with the deletions, attempts were made to genetically complement each mutant. Three of the genes, lmo0913, lmo2391, and lmo2748, were successfully amplified by PCR and cloned into the shuttle vector pKSV7, generating pKAK0913, pKAK2391, and pKAK2748, respectively. These genes were cloned together with their native promoter and regulatory elements. It was not possible to clone the lmo0796 gene despite repeated attempts, suggesting that the presence of multiple copies of this gene may be deleterious to cell growth. It was therefore not possible to complement the acid-sensitive phenotype associated with the lmo0796 deletion. When plasmid pKAK0913 or pKAK2391 was present, there was a significant increase in survival under acid conditions (28- and 310-fold, respectively) (data not shown) compared with the survival of the corresponding deletion strains carrying the empty cloning vector, pKSV7, confirming the role of the lmo0913 and lmo2391 genes in acid tolerance. When plasmid pKAK0913 was present, there was a smaller increase in acid tolerance than when plasmid pKAK2391 was present, but this might be attributed to the presence of a missense mutation in the cloned allele of the lmo0913 gene. Sequencing of the clone revealed the presence of a missense mutation (A to C at position 697), which is predicted to change codon 232 from Thr to Ala in the translated protein, a change that could conceivably influence the activity of the protein. Complementing the salt-sensitive phenotype of the lmo2748 mutant proved to be difficult because the presence of the empty vector was found to have a significant effect on the growth rate when a high NaCl level (1.6 M) was present in the growth medium. However, it was observed that the wild-type strain carrying pKAK2748 grew significantly faster (0.026 ± 0.003 h^–1) than an isogenic strain carrying the empty vector (0.012 ± 0.001 h^–1) when 1.6 M NaCl was present in the growth medium, suggesting that the lmo2748 product may confer some protection against osmotic stress.

Invasion is not impaired by the lmo0796, lmo0913, lmo2391, and lmo2748 mutations.
The invasiveness of the deletion strains was investigated using the Caco-2 human epithelial cell line. The wild-type bacteria were found to invade Caco-2 cells with an invasion efficiency of approximately 0.075%, while the sigB mutant had a significantly lower invasion efficiency (0.025%) (Table 4), which was consistent with previous reports (35, 36). None of the four deletion mutants showed a decrease in invasion compared to the wild-type bacteria, suggesting that none of the genes is required for efficient host cell invasion. The lmo0913 mutant was found to invade Caco-2 cells at a higher rate than the wild-type strain, and the difference appeared to be significant (Table 4). The reason for this difference is not clear at present.

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TABLE 4. Invasion of Caco-2 epithelial cells

sigB and lmo2748 mutants have an altered pattern of Gram staining.
In order to investigate if the deletion mutants had a defect in cell morphology, microscopic observation following Gram staining was performed. The lmo0796, lmo0913, and lmo2391 strains appeared to be identical to the wild type (Fig. 6). They were all found to be dark purple, as expected for gram-positive bacteria, while both the sigB and lmo2748 mutant strains stained red (Fig. 6). Similar results were obtained regardless of whether the microscope slides were prepared directly from colonies growing on BHI agar or from liquid cultures grown in DMS or BHIS (data not shown). These results suggest that ^B might contribute to the integrity of the cell wall and that the absence of Lmo2748 expression in the sigB mutant background could be involved in this phenotype.

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FIG. 6. Unusual pattern of Gram staining of the lmo2748 and sigB mutants. L. monocytogenes wild-type strain 10403S (wt), lmo0796 mutant, lmo0913 mutant, lmo2391 mutant, lmo2748 mutant, and sigB mutant cells were observed with the microscope after Gram staining.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study we investigated the role of ^B in L. monocytogenes cells growing in a chemically defined medium (DM), using a combination of proteomic and genetic approaches. Overall, 11 proteins were found to be differentially expressed in the sigB background compared to the parent. Eight of these proteins were ^B dependent under all conditions under which they were detectable (Fig. 3A). The remaining three proteins (Lmo2425, Lmo2829, and LmaA) showed ^B-dependent expression only under specific conditions. For example, Lmo2829 was found to be ^B dependent only when NaCl was present in the growth medium. In the absence of NaCl this protein was expressed at detectable levels, but the sigB deletion had no effect on its expression level. Seven of the proteins identified here (Lmo0539, Lmo0796, Lmo1830, Lmo2391, Lmo2696, Lmo0913, and Lmo2748) were previously described as proteins that were under ^B control when L. monocytogenes was grown in complex medium (1). Four of these proteins have putative functions related to carbon utilization (Lmo0539, Lmo0913, Lmo1830, and Lmo2696), while three are similar to proteins that have unknown functions but are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). Thus, this study identified four new members of the ^B regulon in L. monocytogenes, namely, Lmo0783, Lmo2425, Lmo2829, and LmaA. Lmo0783 is related to component IIB of the mannose-specific phosphotransferase system (Table 3). Despite the reduced levels of this protein in the sigB background, no effect on the ability of the Lmo0783 mutant to grow was observed when mannose was the sole carbon source in the medium (Fig. 2A). However, there are at least two other genes in the L. monocytogenes genome encoding proteins that also have similarity to component IIB of this transport system (Lmo0096 and Lmo2002), suggesting that there may be functional redundancy that allows mannose transport. Lmo2425 is similar to a subunit of the glycine cleavage system, protein H (Table 3), which is involved in balancing the requirements of the cells for glycine and single carbon units. Lmo2829 is related to nitroreductase, which may have a role in membrane bioenergetics. LmaA is the only protein that has been found to be induced in the sigB mutant compared to the parent, suggesting that ^B has an indirect negative effect on its expression. LmaA is a surface-expressed antigen that induces cell-mediated responses in mice (27). Moreover, lmaA appears to be absent in nonpathogenic species of Listeria and in some L. monocytogenes strains that are attenuated for virulence (47).

Surprisingly, the loss of ^B was found to result in a higher growth rate in DM in the presence of 0.5 M NaCl (Fig. 1), regardless of the carbon source in the medium (Fig. 2B). This result was particularly unexpected since ^B is known to play a central role in osmoregulation (5, 25, 55). Indeed, the present study revealed that ^B is required for osmotolerance in a complex medium with 1.75 M NaCl added (Fig. 4). Neither the proteomic data nor the genetic analysis performed here provided any obvious insight into this unusual phenotype. However, this phenotype is not without precedent in other bacteria; Bacillus subtilis mutants lacking ^B have a selective growth advantage in glucose-limited continuous cultures (49). A similar phenomenon has been described for E. coli, continuous glucose-limited cultures of which are known to accumulate rpoS mutations that confer a growth advantage (40). The nature of the nutrient limitation in E. coli was not the determining factor in selecting for the emergence of these mutants (37, 40). For E. coli these observations have been explained by proposing that sigma factors compete for an RNA polymerase core enzyme whose availability is limited (22). In this model ^s provides protection against adverse conditions to the bacterium, but at the cost of diverting resources away from growth-related functions; in the absence of ^s faster growth can therefore be achieved (40). Nyström (41) suggested that environmental conditions can influence the trade-off between stress resistance and growth by altering the availability of RNA polymerase and competing sigma factors. Further study is necessary to determine if the same phenomenon exists in L. monocytogenes.

This study also identified two other novel phenotypes associated with the loss of ^B. Strains lacking ^B are known to be defective for the uptake of compatible solutes under osmotic stress conditions (25, 34, 54). This is explained by the known role of ^B in the transcription of the opuC and gbu operons, which are involved in carnitine and betaine uptake, respectively (13, 15, 25, 54). Despite this, no obvious growth defect has been reported for sigB mutants growing in complex medium in the presence of additional NaCl. Here we show that at a very high salt concentration (1.75 M NaCl) the growth of the sigB mutant was extremely poor compared to the growth of the parent strain (Fig. 4). Microscopic examination of the sigB mutant after Gram staining suggested that ^B may contribute to the maintenance of cell wall integrity (Fig. 6). In a recent study we found that the expression of DapE is under control of ^B (1). This enzyme is involved in biosynthesis of diaminopimelate, a precursor that is required for assembly of the peptidoglycan cell wall, and its absence could contribute to the staining defect seen in the sigB mutant.

Of the 11 proteins identified by proteomics, 5 were targeted for further genetic analysis (Lmo0796, Lmo0913, Lmo1830, Lmo2391, and Lmo2748). Four deletion mutants were successfully constructed; however, despite repeated attempts we were unable to generate an lmo1830 deletion mutant. This might indicate that lmo1830 is an essential gene, at least under the conditions used during mutant construction. Using the deletion mutants, the contributions of lmo0796, lmo0913, lmo2391, and lmo2748 to osmotolerance were investigated using complex medium with a high salt concentration (1.75 M NaCl). As discussed above, the sigB mutant grows very poorly under these conditions. It is clear that lmo2748 contributes to this phenotype since the growth of the lmo2748 strain was slower than the growth of the wild-type strain under these conditions (Fig. 4). This apparent function of Lmo2748 correlates well with the finding that this protein is expressed only in the presence of NaCl both in DM and in BHI medium (1). Interestingly, a gene encoding a homologue of Lmo2748 in B. subtilis, designated ydaG, is known to be expressed in a ^B-dependent manner and is also induced in response to osmotic shock (43). Although the function of Lmo2748 remains unknown at present, this protein may be involved in maintaining the integrity of the cell wall since, like the sigB mutant, the lmo2748 strain has an unusual color after Gram staining (Fig. 6). Motif searching revealed that Lmo2748 possesses a potential pyridoxamine 5'-phosphate oxidase motif, which might indicate that it is involved in pyridoxal phosphate biosynthesis. Pyridoxal phosphate is a cofactor for many enzymatic reactions in the cell, and it is possible that one or more of these reactions could account for the phenotypes observed for the lmo2748 mutant.

^B is known to contribute to acid tolerance in L. monocytogenes (24, 55, 58, 59). Three of the deletion mutants tested here were found to have acid-sensitive phenotypes; two were as sensitive as the sigB mutant (lmo0913 and lmo0796), while one (lmo2391) had an intermediate phenotype (Fig. 5). Interestingly, both lmo0796 and lmo2391 have been shown to be induced in exponentially growing L. monocytogenes cells exposed to pH 5.0 (46). Lmo0796 has a conserved potential lipid/isoprenoid binding motif and is homologous (44% identity and 61% similarity) to YceI from E. coli, which is a periplasmic protein that is induced in response to basic pH stress but whose function remains unknown (53). Lmo2391 is homologous to YhfK (46% identity and 61% similarity) from B. subtilis, a general stress protein that is induced in response to osmotic shock (31), heat shock, and ethanol stress (40).

Lmo0913 is homologous (46% identity and 61% similarity) to succinate semialdehyde dehydrogenase from B. subtilis, which is proposed to be involved in the metabolism of -aminobutyrate (GABA) to succinate (19). GABA is the decarboxylated product of glutamate, and its production plays a central role in acid tolerance in L. monocytogenes (18, 19). During the response to low pH, intracellular GABA is thought to be exchanged via an antiporter for extracellular glutamate, which is then decarboxylated to produce another GABA molecule, thereby establishing a cycle (4, 51). It might be that an inability to metabolize intracellular GABA increases the susceptibility of cells to low pH, perhaps through the known toxic effects of this metabolite (3, 9). Although the mutation in none of the four mutants tested reduced the ability of L. monocytogenes to invade human epithelial cells (Table 4), the lmo0913 mutant appeared to be significantly more invasive (seven- to eightfold) than the parent strain. The underlying reason for this difference is not clear at present.

In summary, this study identified two new phenotypes associated with the loss of ^B in L. monocytogenes. It demonstrated clear roles for ^B in both osmotic and low-pH stress tolerance and identified specific components of the ^B regulon that contribute to these responses.

 
We are grateful to members of the Bacterial Stress Response Group at National University of Ireland—Galway, Galway, Ireland, for useful discussions and for providing critical comments on the manuscript. We thank Cyril Carroll for providing unrestricted access to the 2-DGE apparatus and also Gavin Collins for his assistance with microscopy. We are grateful to Eoin Cosgrave for help with some preliminary experiments.

This work was supported by National Institutes of Health award RO1-AI052151-01A1 (to K.J.B.), by the Science Foundation Ireland Research Frontiers Programme, and by a Framework 6 Marie Curie Transfer of Knowledge grant.

 
* Corresponding author. Mailing address: Department of Microbiology, National University of Ireland—Galway, Galway, Ireland. Phone: 35391512342. Fax: 35391494598. E-mail: conor.obyrne{at}nuigalway.ie

Published ahead of print on 19 September 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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