Roles of Four Putative DEAD-Box RNA Helicase Genes in Growth of Listeria monocytogenes EGD-e under Heat, pH, Osmotic, Ethanol, and Oxidative Stress Conditions

Abstract

To examine the role of the four putative DEAD-box RNA helicase genes of Listeria monocytogenes EGD-e in stress tolerance, the growth of the Δlmo0866, Δlmo1246, Δlmo1450, and Δlmo1722 deletion mutant strains at 42.5°C, at pH 5.6 or pH 9.4, in 6% NaCl, in 3.5% ethanol, and in 5 mM H₂O₂ was studied. Restricted growth of the Δlmo0866 deletion mutant strain in 3.5% ethanol suggests that Lmo0866 contributes to ethanol stress tolerance of L. monocytogenes EGD-e. The Δlmo1450 mutant strain showed negligible growth at 42.5°C, at pH 9.4, and in 5 mM H₂O₂ and a lower maximum growth temperature than the wild-type EGD-e, suggesting that Lmo1450 is involved in the tolerance of L. monocytogenes EGD-e to heat, alkali, and oxidative stresses. The altered stress tolerance of the Δlmo0866 and Δlmo1450 deletion mutant strains did not correlate with changes in relative expression levels of lmo0866 and lmo1450 genes under corresponding stresses, suggesting that Lmo0866- and Lmo1450-dependent tolerance to heat, alkali, ethanol, or oxidative stress is not regulated at the transcriptional level. Growth of the Δlmo1246 and Δlmo1722 deletion mutant strains did not differ from that of the wild-type EGD-e under any of the conditions tested, suggesting that Lmo1246 and Lmo1722 have no roles in the growth of L. monocytogenes EGD-e under heat, pH, osmotic, ethanol, or oxidative stress. This study shows that the putative DEAD-box RNA helicase genes lmo0866 and lmo1450 play important roles in tolerance of L. monocytogenes EGD-e to ethanol, heat, alkali, and oxidative stresses.

INTRODUCTION

Listeria monocytogenes is a widespread food-borne pathogen found in nature. It causes a rare but severe infection in susceptible people, with an incidence of 3 cases per million persons in the United States (8) and 0 to 8 cases per million persons in Europe (14, 36), with a fatality rate as high as 20% (Centers for Disease Control and Prevention [http://www.cdc.gov/listeria/surveillance.html]) (22, 36).

L. monocytogenes is frequently found in raw and processed foods and in food-processing environments (3, 16, 27, 32, 39, 56, 57). In the food chain, the bacterium is exposed to various stresses through sanitation, disinfection, heating, cooling, and the addition of food additives and preservatives. L. monocytogenes tolerates a wide variety of stressful conditions and is able to grow at temperatures as low as −1.5°C (24), in the pH range of 4.3 to 9.6 (17, 51), in water activity down to 0.90 (40), and under anaerobic atmospheres (51).

Adaptation to various stresses results in great changes in the gene expression of L. monocytogenes. Whole-genome microarray studies have shown 714, 411, and 355 genes of L. monocytogenes to be differentially transcribed under heat (58), cold (10), and alkali stresses (20), respectively, compared with control growth conditions. Mutational analyses have confirmed the role of alternative sigma factors in the bacterium's tolerance to all these, as well as osmotic, acid, oxidative, and ethanol stresses (9, 12, 18, 19, 41, 47–49, 60). In addition, the involvement of approximately 30 other factors in the tolerance of L. monocytogenes to these stresses has been reported (54).

DEAD-box proteins are conserved RNA helicases with 12 characteristic sequence motifs and are found in most living organisms (15, 34). They are associated with RNA metabolism and have roles in many cellular events (13, 42, 53). Despite intensive research on DEAD-box RNA helicases during the past decade, only a few studies have clarified the roles of these proteins in abiotic stress tolerance in bacteria. Recently, DEAD-box proteins were linked with cold stress tolerance in L. monocytogenes (4, 37), Bacillus subtilis (25), Bacillus cereus (44), Escherichia coli (11), and Yersinia enterocolitica (43) by mutational analyses. DEAD-box RNA helicases also contribute to heat, alkali, and oxidative stress tolerance in B. cereus (45), whereas in the anaerobic Clostridium perfringens, a DEAD-box protein was associated with decreased tolerance to oxidative stress (7). In addition, exposure to salt and light resulted in altered transcription of the DEAD-box RNA helicase gene in the cyanobacterium Synechocystis sp. (30, 59).

L. monocytogenes EGD-e has four putative DEAD-box RNA helicase-encoding genes (21). Three of these have significant roles in the cold tolerance of L. monocytogenes EGD-e (37). In the present study, the role of all four putative DEAD-box RNA helicase genes in the tolerance of L. monocytogenes EGD-e to heat, pH, osmotic, ethanol, and oxidative stresses, to which the bacterium may be exposed in processed foods or food-processing environments, was examined.

MATERIALS AND METHODS

Strains.

The sequenced L. monocytogenes EGD-e strain (21) and the Δlmo0866, Δlmo1246, Δlmo1450, and Δlmo1722 DEAD-box RNA helicase gene deletion mutant strains (37), the respective Δlmo0866c and Δlmo1450c complementation strains (37) containing a wild-type copy of each deleted gene in the pPL2 backbone (31), and the EGD-epPL2, Δlmo0866pPL2, and Δlmo1450pPL2 (37) vector controls, containing only the vector pPL2, were used. Deletion mutants without an associated antibiotic resistance gene were constructed by deleting the entire coding sequence of lmo0866, lmo1246, lmo1450, or lmo1722 using a temperature-sensitive shuttle vector, pMAD, for allelic replacement (2). Complementation of the lmo0866 and lmo1450 deletions was performed according to the methods of Lauer et al. (31) by restoring the wild-type copy of each deleted coding sequence with approximately 500-bp and 200-bp upstream regions, including the putative promoters of lmo0866 and lmo1450, respectively, into the respective mutant strain using the site-specific integration vector pPL2 (received from Martin Loessner, Swiss Federal Institute of Technology, Zurich, Switzerland). Correspondence between the optical density at 600 nm (OD₆₀₀) and viable cell numbers examined by plating indicate similar cell sizes for the wild-type EGD-e and all four deletion mutant strains at 37°C (37).

Growth conditions.

Before each experiment, the bacterial strains were grown on blood agar plates at 37°C. For growth curve and gene expression analyses, growth of the strains in brain heart infusion (BHI) broth (Becton Dickinson [BD], Franklin Lakes, NJ) at 37°C was used as a control. To study the growth curves and gene expression under heat stress, the strains were grown in BHI broth at 42.5°C. Acid and alkali stresses on the strains were studied in BHI broth adjusted to pH 5.6 with 1 M HCl (37%) or pH 9.4 with 1 M NaOH, respectively. To study osmotic, ethanol, or oxidative stress tolerance, BHI broth supplemented with 6% NaCl, 3.5% ethanol (vol/vol) (99.5%), or 5 mM H₂O₂ (30%), respectively, was used. The maximum growth temperatures were examined in tryptic soy agar (TSA) (BD) containing 25 g agar per liter.

Growth curve analyses.

Ten-milliliter aliquots of BHI broth were inoculated with single colonies of L. monocytogenes EGD-e and each deletion mutant strain in five replicates. After a 20-h incubation at 37°C with shaking, the strains were diluted 1:100 in test or control growth medium. The strains were grown in the Bioscreen C microbiology reader (Growth Curves Ltd, Helsinki, Finland) at 37°C or 42.5°C according to the methods of Markkula et al. (37). The OD₆₀₀ was monitored at 15-min intervals within 48 h. To obtain the maximum growth rate for each strain, the OD₆₀₀ data were fitted to growth curves using DMFit software version 2.1 (Computational Microbiology Research Group, Institute of Food Research, Colney, Norwich, United Kingdom) (5).

Maximum growth temperatures.

The mean maximum growth temperatures of the five replicate cultures of the wild-type EGD-e and each deletion mutant were examined according to the methods of Hinderink et al. (23), with a few modifications. The strains grown for 20 h in BHI broth at 37°C with shaking and diluted 1:1,000 in the same medium were plated by stamping them as parallel lines onto TSA containing 25 g agar per liter. The strains were grown for 48 h in a Gradiplate W10 temperature gradient incubator (BCDE Group Waste Management Ltd., Helsinki, Finland) using a temperature gradient from 39.1°C to 45.6°C over the growth lines. The maximum growth boundaries were observed with a stereomicroscope.

Phenotypic characterization of the complementation strains.

To verify that the altered phenotypes observed for the Δlmo0866 and Δlmo1450 deletion mutant strains under heat and ethanol and heat, alkali, and oxidative stresses, respectively, were specifically due to gene deletions, the maximum growth temperatures and the growth of three replicate cultures of the Δlmo0866c and Δlmo1450c complementation strains in 3.5% ethanol and at 42.5°C, pH 9.4, or in 5 mM H₂O₂, respectively, were examined using the protocols described above. The maximum growth temperatures and growth curves were compared with those of the EGD-epPL2 and Δlmo0866pPL2 or Δlmo1450pPL2 vector controls.

RNA extraction, reverse transcription, and quantitative real-time PCR.

To study the transcription levels of the DEAD-box RNA helicase-encoding genes lmo0866, lmo1246, lmo1450, and lmo1722 under stress conditions in relation to their levels under a control growth condition, total RNA was extracted from three replicate cultures of L. monocytogenes EGD-e grown to the midlogarithmic growth phase at 42.5°C, pH 5.6 or pH 9.4, in 6% NaCl, 3.5% ethanol, 5 mM H₂O₂, and BHI broth at 37°C according to the methods of Mattila et al. (38). In brief, bacterial cells from 5-ml cultures were lysed in Tris-EDTA buffer (Fluka Biochemica, Buchs, Switzerland) containing 25 mg/ml lysozyme (Sigma-Aldrich, St. Louis, MO) and 250 U/ml mutanolysin (Sigma-Aldrich). Total RNA isolated with the RNeasy midi-kit (Qiagen, Valencia, CA) was treated with the RNase-Free DNase set (Qiagen) and finally with the DNA-free kit (Ambion, Austin, TX). The RNA yield was determined by using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), and RNA integrity was verified with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). A total of 400 ng of each RNA sample was used to synthesize cDNA by using the DyNAmo cDNA synthesis kit (Finnzymes, Espoo, Finland) in duplicate. In addition, single reactions without reverse transcriptase enzyme were established. Real-time PCR with two replicate reactions for each cDNA sample diluted 1:5,000 was performed using previously published primers (37) and the SYBR green chemistry (DyNAmo Flash SYBR green quantitative PCR kit; Finnzymes) in a Rotor-Gene 3000 device (Corbett Research, Sydney, Australia). The relative expression levels of lmo0866, lmo1246, lmo1450, and lmo1722 normalized to those of the 16S rRNA, the most stably expressed housekeeping gene of L. monocytogenes (55), were calculated using the 2^−ΔΔCt method (50). The amplification efficiencies of the four target genes and the reference gene ranged from 0.98 to 1.07.

Statistical analyses.

The significance of differences in the growth rates and maximum growth temperatures between the deletion mutant and wild-type EGD-e strains were tested using Student's t test. Differences in the expression levels of lmo0866, lmo1246, lmo1450, and lmo1722 in the wild-type EGD-e between each stress condition and the control growth condition were tested by the paired t test.

RESULTS

Growth of DEAD-box RNA helicase gene deletion mutants under different stress conditions.

In 3.5% ethanol, the mean growth rate of the Δlmo0866 DEAD-box RNA helicase gene deletion mutant strain was decreased by 75% compared to that of the wild-type EGD-e (P < 0.001) (Fig. 1; Table 1). In 6% NaCl and under the control growth condition, the mean growth rates of the Δlmo0866 deletion mutant strain were 14% and 17% lower, respectively, than those of the wild-type EGD-e (P < 0.001), whereas at 42.5°C, the mean growth rate of the Δlmo0866 deletion mutant strain was increased by 22% (P < 0.001).

Fig 1.

Growth of Listeria monocytogenes EGD-e and the Δlmo0866, Δlmo1246, Δlmo1450, and Δlmo1722 DEAD-box RNA helicase gene deletion mutant strains in BHI broth at 42.5°C (B), at pH 5.6 (C), at pH 9.4 (D), in 6% NaCl (E), in 3.5% ethanol (F), and in 5 mM H₂O₂ (G). (A) Growth of the five strains in BHI broth at 37°C is included as a control. (Reprinted from reference 37 with permission of the publisher.) The optical density at 600 nm (OD₆₀₀) was monitored at 15-min intervals for 48 h. The data shown represent the median OD₆₀₀ values of four (F) and five (A to E, G) independent cultures. The error bars indicate the variations in the replicate cultures.

Table 1.

Average maximum growth rates of Listeria monocytogenes EGD-e and the Δlmo0866, Δlmo1246, Δlmo1450, and Δlmo1722 DEAD-box RNA helicase gene deletion mutant strains under different growth conditions

Growth condition	Growth rate ± SD (OD600 units/h)a
	EGD-e	Δlmo0866 mutant	Δlmo1246 mutant	Δlmo1450 mutant	Δlmo1722 mutant
37°C, controlb	0.29 ± 0.004	0.24 ± 0.006*	0.28 ± 0.007	0.17 ± 0.004*	0.26 ± 0.006*
42.5°C	0.09 ± 0.004	0.11 ± 0.003*	0.10 ± 0.003	0.01 ± 0.001*	0.11 ± 0.003*
pH 5.6	0.05 ± 0.002	0.05 ± 0.005	0.05 ± 0.003	0.04 ± 0.005*	0.05 ± 0.002
pH 9.4	0.07 ± 0.014	0.05 ± 0.006	0.06 ± 0.006	0.02 ± 0.023	0.05 ± 0.006
6% NaCl	0.07 ± 0.002	0.06 ± 0.003*	0.07 ± 0.001	0.05 ± 0.004*	0.07 ± 0.001
3.5% ethanol	0.04 ± 0.002	0.01 ± 0.004*	0.04 ± 0.003	0.02 ± 0.001*	0.05 ± 0.001*
5 mM H2O2	0.21 ± 0.041	0.17 ± 0.015	0.21 ± 0.015	0.02 ± 0.019*	0.22 ± 0.013

^a*, significant difference (Student's t test, P < 0.001) compared with the corresponding value of the EGD-e strain.

^bData for control reprinted from Environmental Microbiology (37) with permission from the publisher.

Growth of the Δlmo1450 deletion mutant strain was totally impaired at 42.5°C (Fig. 1; Table 1). At pH 9.4 and in 5 mM H₂O₂, growth of the Δlmo1450 deletion mutant strain was restricted, mainly due to the extended lag phase (Fig. 1; Table 1). At pH 5.6, in 6% NaCl, in 3.5% ethanol, and under the control growth condition, the mean growth rates of the Δlmo1450 deletion mutant strain were decreased by 20%, 29%, 50%, and 41%, respectively, compared to those of the wild-type EGD-e (P < 0.001) (Table 1).

The deletion of lmo1722 resulted in 22% and 25% increases and a 10% decrease in the mean growth rates of L. monocytogenes EGD-e at 42.5°C, in 3.5% ethanol, and under the control growth condition, respectively (P < 0.001) (Table 1). Growth of the Δlmo1246 deletion mutant strain was similar to that of the wild-type EGD-e under all conditions tested (Fig. 1; Table 1).

Impact of DEAD-box RNA helicase-encoding genes on maximum growth temperatures of L. monocytogenes EGD-e.

The maximum growth temperatures of the Δlmo0866 and Δlmo1450 deletion mutant strains were 0.6°C higher and 0.6°C lower, respectively, than the maximum growth temperature of the wild-type EGD-e (P < 0.05) (Fig. 2). The maximum growth temperatures of the Δlmo1246 and Δlmo1722 deletion mutant strains did not differ from that of the wild-type EGD-e.

Fig 2.

Growth of Listeria monocytogenes EGD-e and the DEAD-box RNA helicase gene Δlmo0866, Δlmo1246, Δlmo1450, and Δlmo1722 deletion mutant strains in tryptic soy broth solidified with 2.5% agar within 48 h in a Gradiplate W10 temperature gradient incubator at a temperature range from 39.1°C to 45.6°C.

Phenotypes of complementation strains.

Growth of the Δlmo0866c complementation strain in 3.5% ethanol (Fig. 3A) and the Δlmo1450c complementation strain at 42.5°C, at pH 9.4, and in 5 mM H₂O₂ (Fig. 3B to D) and the maximum growth temperatures of both strains (Fig. 4) were similar to those of the EGD-epPL2 control strain.

Fig 3.

Growth of Listeria monocytogenes EGD-e containing the unmodified pPL2 vector (EGD-epPL2), the DEAD-box RNA helicase gene Δlmo0866 deletion mutant strain containing the pPL2 vector (Δlmo0866pPL2), and the complemented Δlmo0866 deletion mutant strain containing a wild-type copy of lmo0866 in the pPL2 backbone (Δlmo0866c) in 3.5% ethanol (A) and EGD-epPL2, the DEAD-box RNA helicase gene Δlmo1450 deletion mutant strain containing the pPL2 vector (Δlmo1450pPL2), and the complemented Δlmo1450 containing a wild-type copy of lmo1450 in the pPL2 backbone (Δlmo1450c) at 42.5°C (B), at pH 9.4 (C), and in 5 mM H₂O₂ (D). The optical density at 600 nm (OD₆₀₀) was monitored at 15-min intervals for 48 h. The data shown represent median OD₆₀₀ values of three independent cultures. The error bars indicate the variations in the replicate cultures.

Fig 4.

Growth of Listeria monocytogenes EGD-e containing the unmodified pPL2 vector (EGD-epPL2), the DEAD-box RNA helicase gene Δlmo0866 and Δlmo1450 deletion mutant strains containing the pPL2 vector (Δlmo0866pPL2 and Δlmo1450pPL2), the complemented Δlmo0866 deletion mutant strain containing a wild-type copy of lmo0866 in the pPL2 backbone (Δlmo0866c), and the Δlmo1450 deletion mutant strain containing a wild-type copy of lmo1450 in the pPL2 backbone (Δlmo1450c) in tryptic soy broth solidified with 2.5% agar within 48 h in a Gradiplate W10 temperature gradient incubator at a temperature range from 39.1°C to 45.6°C.

Expression of DEAD-box RNA helicase-encoding genes of L. monocytogenes exposed to different stresses.

The relative expression levels of the DEAD-box RNA helicase-encoding genes lmo0866 and lmo1246 were significantly lower in 6% NaCl than under the control growth condition (P < 0.05) (Fig. 5), while the relative expression levels of lmo1450 and lmo1722 were not affected. In 3.5% ethanol, the transcription levels of lmo1246, lmo1450, and lmo1722 were significantly higher than those under the control condition (P < 0.05). The relative expression level of lmo0866 was unaffected by the presence of 3.5% ethanol. At 42.5°C, at pH 5.6 and pH 9.4, and in 5 mM H₂O₂, the transcription levels of the DEAD-box RNA helicase-encoding genes did not differ from those under control conditions.

Fig 5.

Relative expression of the DEAD-box RNA helicase genes lmo0866, lmo1246, lmo1450, and lmo1722 in Listeria monocytogenes EGD-e during midlogarithmic growth at 42.5°C, at pH 5.6 or pH 9.4, in 6% NaCl, in 3.5% ethanol, and in 5 mM H₂O₂ in relation to midlogarithmic growth at 37°C. Error bars represent the standard deviations of three independent replicates. Significant differences (paired t test, P < 0.05,) in relative expression levels are indicated by asterisks.

DISCUSSION

L. monocytogenes is able to survive and multiply in raw and processed foods and food-processing environments despite the conventional preservation agents and sanitizing practices (3, 16, 27, 39, 56, 57). Understanding the mechanisms behind tolerance to stresses caused by food processing, additives, preservatives, sanitizers, and disinfectants may provide new insights into the control of L. monocytogenes in the food chain. In this study, the role of DEAD-box RNA helicase-encoding genes, so far linked mainly to cold stress tolerance of bacteria (4, 7, 11, 37, 43–45, 59), in tolerance of L. monocytogenes to various stress conditions the bacterium may meet in the food chain was examined. Single deletions of the putative DEAD-box RNA helicase-encoding genes lmo0866 and lmo1450 were found to significantly alter the tolerance of L. monocytogenes EGD-e to heat, alkali, ethanol, and oxidative stresses. The remaining two DEAD-box RNA helicase-encoding genes, lmo1246 and lmo1722, had no role in tolerance of L. monocytogenes EGD-e to the stress conditions examined.

The restricted growth of the lmo0866 deletion mutant in 3.5% ethanol suggests that Lmo0866 contributes to the ethanol stress tolerance of EGD-e. The finding was confirmed by complementation of the mutation, which restored the growth of the Δlmo0866 deletion mutant strain in 3.5% ethanol to the wild-type level. To our knowledge, this is the first report on the role of a DEAD-box RNA helicase gene in tolerance to ethanol stress in prokaryotes. Ethanol increases membrane leakage and affects the properties of all biological molecules in bacteria (6, 26). In B. subtilis, ethanol induces recruitment of the molecular chaperones DnaK and GroEL to the cell membrane (52). Interestingly, the DEAD-box RNA helicase CrhR of the cyanobacterium Synechocystis sp. regulates the expression of GroEL, at least under low temperature (46). Whether Lmo0866 regulates the expression of GroEL or some other protein under ethanol stress is not known.

Enhanced tolerance of the Δlmo0866 deletion mutant strain to heat stress, albeit representing a minimal effect, suggests that Lmo0866 represses the growth of the wild-type EGD-e at high temperatures. Successful complementation of the lmo0866 deletion, restoring the maximum growth temperature of the Δlmo0866 deletion mutant strain to the wild-type level, confirmed our finding. Previously, a DEAD-box protein was associated with repression of oxidative stress tolerance in the anaerobe Clostridium perfringens (7). In other studies, bacterial DEAD-box RNA helicases either increased or had no role in tolerance to temperature, pH, and oxidative stresses (11, 25, 37, 43, 44–46). The slightly restricted growth of the Δlmo0866 deletion mutant strain compared to that of the wild type EGD-e detected at pH 9.4 and in 6% NaCl, but also under the control growth condition, suggests no specific role for Lmo0866 under alkali and osmotic stresses.

The observed growth characteristics of the Δlmo1450 deletion mutant strain under temperature, pH, and H₂O₂ stresses indicate an important role for Lmo1450 in heat, alkali, and oxidative stress conditions. This was confirmed by successful complementation of the lmo1450 deletion, restoring the wild-type phenotype in the Δlmo1450c complementation strain. DEAD-box RNA helicase-dependent heat and oxidative stress tolerances are further supported by a study by Pandiani et al. (45), which showed that cshA, cshB, and cshC, orthologous with lmo0866, lmo1450, and lmo1722, respectively (35), are associated with heat and oxidative stress tolerance in B. cereus. The differences between the growth rates of the Δlmo1450 deletion mutant strain and the wild-type EGD-e at pH 5.6, in 6% NaCl, and in 3.5% ethanol were similar to those under control conditions, indicating that the Δlmo1450 deletion mutant strain has no specific role in acid, osmotic, and ethanol stresses.

Slightly increased growth of the Δlmo1722 deletion mutant strain was observed both at 42.5°C and in 3.5% ethanol. However, the increased growth rate of the Δlmo1722 deletion mutant strain at 42.5°C was not supported by any altered maximum growth temperature or induced transcription of lmo1722 at 42.5°C. Moreover, repeated growth of the wild-type and deletion mutant strains in ethanol stress (data not shown) suggests that the location in the outermost wells of the growth plate increased the growth rate of the Δlmo1722 deletion mutant strain in this study, presumably because of more potent evaporation of ethanol. Thus, we assume that growth of the Δlmo1722 deletion mutant strain was similar to that of the wild-type EGD-e under heat and ethanol stresses. Based on our study and previous reports by us (37) and Azizoglu and Kathariou (4), Lmo1722 has a role in the tolerance of L. monocytogenes to cold stress and is not associated with heat, pH, osmotic, ethanol, or oxidative stress tolerance. This differentiates Lmo1722 from Lmo0866 and Lmo1450, as well as from the stress-associated DEAD-box RNA helicases of B. cereus, which all play roles in tolerance to multiple stress conditions (45).

The identical growth of the Δlmo1246 deletion mutant strain and the wild-type EGD-e under all the conditions tested indicates that Lmo1246 has no role in the tolerance of L. monocytogenes EGD-e to the stress conditions examined in this study. Moreover, lmo1246 showed only a negligible role in the cold stress tolerance of L. monocytogenes EGD-e (37). Accordingly, the DEAD-box RNA helicases CshD (containing the conserved C-terminal domain DpbA present also in Lmo1246 and not found in the other DEAD-box helicases of L. monocytogenes EGD-e or B. cereus) and CshE (with no ortholog in L. monocytogenes EGD-e) (35) have no role in temperature, pH, or oxidative stress tolerance of B. cereus (44, 45). Based on previous studies, showing that the paralogous DEAD-box proteins do not usually cross-complement each other (33, 35), Lmo1246 is not expected to compensate for the other DEAD-box helicases of L. monocytogenes EGD-e. Further studies are warranted to reveal if Lmo1246 has a role in some cellular reaction other than abiotic stress tolerance.

Quantitative real-time PCR analysis revealed alterations solely in the relative expression of lmo0866 and lmo1246 under osmotic stress and lmo1246, lmo1450, and lmo1722 under ethanol stress. The transcription analysis with DNA microarrays by Giotis et al. (20), showing no significant changes in the expression of alkali-stressed DEAD-box RNA helicase-encoding genes of L. monocytogenes 10403S, and that of van der Veen et al. (58), showing a decreased transcription level of lmo0866 3 min after heat shock but no change in expression of any DEAD-box RNA helicase genes of L. monocytogenes EGD-e later, support our findings. Thus, Lmo0866- or Lmo1450-associated tolerance to heat, alkali, ethanol, or oxidative stress is likely controlled through mechanisms other than those behind tolerance to low temperature (37), which appears to be regulated at least at the transcriptional level. The Lmo0866 and Lmo1450 activity may be controlled at a translational or posttranslational level, a well-known phenomenon in the regulation of RNA helicase activity in eukaryotes but yet unknown in bacteria (42).

The distinct behavior of the Δlmo0866, Δlmo1246, Δlmo1450, and Δlmo1722 deletion mutant strains under various stresses may result from different cellular functions of Lmo0866, Lmo1246, Lmo1450, and Lmo1722. Functional divergence of paralogous DEAD-box proteins, resulting from structural variability in the C-terminal domains, has been reported in B. subtilis, whose four DEAD-box protein-encoding genes are orthologous to those in L. monocytogenes EGD-e (1, 13, 25, 28, 29, 33, 35). An Lmo0866 ortholog, CshA, is involved with ribosome assembly. Moreover, it seems to be the only DEAD-box protein in B. subtilis that confers interaction with a membrane-bound degradosome, a large multiprotein complex responsible for RNA turnover, and thus controls the steady-state concentration of any given RNA (1, 33). An Lmo1450 ortholog, CshB, has an mRNA-dependent role in rescuing misfolded mRNA molecules and maintaining proper initiation of translation at low temperatures together with the cold shock protein CspB (25). The C-terminal domain of an Lmo1246 ortholog, DpbA (previously YxiN or DeaD), specifically binds 23S rRNA and has a role in ribosome assembly (28, 29). Whether the DEAD-box helicases of L. monocytogenes possess the same physiological functions as their orthologs in B. subtilis needs to be elucidated.

In conclusion, the DEAD-box RNA helicases Lmo0866 and Lmo1450 played important roles in the tolerance of L. monocytogenes EGD-e to stress conditions that this pathogen may be exposed to in the food-processing chains. While Lmo0866 appeared to increase ethanol stress tolerance, it was associated with decreased heat stress tolerance. Lmo1450 contributed to tolerance to heat, alkali, and oxidative stresses. Lmo1246 and Lmo1722 played no role in the tolerance of L. monocytogenes EGD-e to heat, acid, alkali, osmotic, ethanol, or oxidative stresses.