Construction of Listeria monocytogenes Mutants with In-Frame Deletions in the Phosphotransferase Transport System (PTS) and Analysis of Their Growth under Stress Conditions Yanhong Liu, Marina Ceruso, Yuji Jiang, Atin R. Datta, Laurenda Carter, Errol Strain, Tiziana Pepe, Aniello Anastasi, and Pina Fratamico
Listeria monocytogenes is a foodborne pathogen that is difficult to eliminate due to its ability to survive under different stress conditions such as low pH and high salt. To better control this pathogen in food, it is important to understand its survival mechanisms under these stress conditions. LMOf2365_0442, 0443, and 0444 encode for phosphotransferase transport system (PTS) permease (fructose-specific IIABC components) that is responsible for sugar transport. LMOf2365_0445 encodes for glycosyl hydrolase. These genes were induced by high pressure and inhibited under salt treatments; therefore, we hypothesized that genes encoding these PTS proteins may be involved in general stress responses. To study the function of these genes, deletion mutants of the PTS genes (LMOf2365_0442, LMOf2365_0443, and LMOf2365_0444) and the downstream gene LMOf2365_0445 were created in L. monocytogenes strain F2365. These deletion mutants were tested under different stress conditions. The growth of LMOf2365_0445 was increased under nisin (125 μg/mL) treatments compared to the wild-type (P < 0.01). The growth of LMOf2365_0442 in salt (brain–heart infusion medium with 5% NaCl) was significantly increased (P < 0.01), and LMOf2365_0442 showed increased growth under acidic conditions (pH 5.0) compared to the wild-type (P < 0.01). The results from phenotypic arrays demonstrated that some of these mutants showed slightly slower growth under different carbon sources and basic conditions. The results indicate that deletion mutants LMOf2365_0442 and LMOf2365_0445 were more resistant to multiple stress conditions compared to the wild-type, suggesting that they may contribute to the general stress response in L. monocytogenes. An understanding of the growth of these mutants under multiple stress conditions may assist in the development of intervention strategies to control L. monocytogenes in food.
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Keywords: Listeria monocytogenes, phosphotransferase transport system (PTS), stress response
Introduction Listeria monocytogenes is a foodborne pathogen that causes human listeriosis, which is responsible for 28% of annual deaths attributable to known foodborne pathogens (Scallan and others 2011). Listeriosis affects primarily the fetus in pregnant women, newborns, the elderly, and immune-compromised individuals. Transmission of L. monocytogenes is generally through consumption of contaminated food, such as dairy products made from unpasteurized milk and ready-to-eat fruits, vegetables, meat, and fish products (Gandhi and Chikindas 2007). In addition, L. monocytogenes has been found in a variety of raw foods, as well as in processed foods that become contaminated after processing, such as soft cheeses and cold cuts at the deli counter (Cordano and MS 20130246 Submitted 2/21/2013, Accepted 5/9/2013. Authors Liu and Fratamico are with the Molecular Characterization of Foodborne Pathogens Research Unit, Eastern Regional Research Center, Agricultural Research Service, U.S. Dept. of Agriculture, 600 East Mermaid Lane, Wyndmoor PA 19038, U.S.A. Authors Ceruso, Pepe, and Anastasi are with the Dipto. di Scienze Zootecniche e Ispezione degli Alimenti, Sezione Ispezione, Facolt`a di Medicina Veterinaria, Univ. degli Studi di Napoli Federico II, Via Delpino 1, Naples 80137. Author Jiang is with the College of Food Science, Fujian Agriculture and Forestry Univ., Fuzhou, Fujian 3, China 50002. Authors Datta, Carter, and Strain are with the Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Laurel MD 20708, U.S.A. Direct inquiries to author Liu (E-mail: [email protected]
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Rocourt 2001). Potential sources of L. monocytogenes contamination in foods include the incoming product, food handlers, consumers, and environmental sources (Lianou and Sofos 2007). L. monocytogenes is also widely distributed in the environment. Biofilms form readily in places such as food processing plants. L. monocytogenes also grows under refrigeration conditions and is relatively resistant to acid and high-salt concentrations. Therefore, it is difficult to eliminate L. monocytogenes from foods and/or foodprocessing plants. The genome sequence of L. monocytogenes serotype 1/2a strain EGDe (Glaser and others 2001) was published in 2001, followed by the complete sequence of the epidemic L. monocytogenes isolate (serotype 4b strain F2365) in 2004 (Nelson and others 2004). More recently, a bank of 46 human, animal, food, and environmental L. monocytogenes isolates were sequenced, and the sequences are available in the public domain (http://www.ncbi.nlm.nih.gov/genome/159). The available sequences should provide an invaluable source of information for identification of genetic determinants involved in stress responses and the environmental biology of the organism. In response to changes in the natural environment, bacteria undergo a complex program of differential gene expression. A number of transcriptional regulators important for stress-response gene expression have been identified in L. monocytogenes (Hanawa and others 2000; Nair and others 2000; Stack and others 2008; R C 2013 Institute of Food Technologists
doi: 10.1111/1750-3841.12181 Further reproduction without permission is prohibited
L. moncytogenes growth under stress . . .
Materials and Methods Bacteria and growth conditions L. monocytogenes strain F2365, isolated from Mexican-style soft cheese that was implicated in an outbreak of listeriosis in California in 1985 (Linnan and others 1988), was used in this study since its genome is fully sequenced and annotated (Nelson and others 2004). Glycerol stock cultures of L. monocytogenes F2365 and isogenic mutants of this parent strain (Table 1) stored at –80 ◦ C were streaked onto brain–heart infusion (BHI; SigmaAldrich St. Louis, Mo., U.S.A.) agar plates and grown at 37 ◦ C prior to each experiment. Construction of in-frame deletion mutants for LMOf2365_0442, 0443, 0444, and 0445 in L. monocytogenes F2365 Gene deletion fragments were constructed by splice overlap extension (SOE) polymerase chain reaction (PCR) (Horton and others 1990) to generate two 400-bp fragments: 1 upstream
including the ATG codon (AB product) and 1 downstream beginning from the stop codon (CD product). The positions and sequences of PCR primers are listed in Table 1. The 5’ end of the SOEB primers was complementary to the SOEC primers. The initial PCR products (AB and CD products) were used as templates in 2nd rounds of PCR with the SOEA and SOED primers to generate 800-bp products (PCR product AD). The 2nd round PCR products were cloned into pKSV7 vector (a gift from S. Kathariou, North Carolina State Univ.) and transformed into E. coli competent cells (C4040–03, Invitrogen, Carlsbad, Calif., U.S.A.), according to the manufacturer’s instructions. The plasmids containing PCR products ADs were electroporated into L. monocytogenes F2365 competent cells as described (Park and Stewart 1990). Chloramphenicol sensitive colonies were screened by colony PCR using SOEA and SOED primers to identify isolates with the mutant alleles. The sizes of the fragments in the deletion mutants were about 800 bp while the sizes in the wildtype were 800 bp plus the size of the deleted genes (Liu and others 2012). Each of the genes (LMOf2365_0442, 0443, 0444, and 0445) was deleted individually.
Growth assays of L. monocytogenes F2365 wild-type and LMOf2365_0442, 0443, 0444, and 0445 under different stress conditions L. monocytogenes F2365 (wild-type), and the LMOf2365_0442 to 0445 mutants were used for growth assays. To make a log-phase culture, 1 L. monocytogenes colony was inoculated into 5 mL BHI and grown at 37 ◦ C with agitation at 200 rpm overnight. A 50-μL aliquot of overnight culture was added into 5 mL BHI and grown at 37 ◦ C with agitation at 180 rpm for 3 h until the O.D.600 was close to 0.4. Growth assays were performed in a 100-well plate format using the log-phase bacteria (O.D.600 approximately 0.4). For the nisin inhibition assay, nisin (containing 2.5% pure nisin, sodium chloride, and denatured milk solids, activity of 1 × 106 IU/g, according to the manufacturer) from Lactococcus lactis was purchased from Sigma-Aldrich (N5764). Nisin at a concentration of 125 μg/mL in BHI was used for growth studies, and BHI medium was used as a negative control. For the salt tolerance assays, the growth of log-phase cells was tested in BHI containing 5% NaCl, and BHI medium was used as a negative control. BHI at pH 5 was used for the acid tolerance assays, and BHI at pH 7 was used as a control. The plates were placed into a Bioscreen C Analyzer (Oy Growth Curves AB Ltd., Helsinki, Finland) at 37 ◦ C, with O.D. readings at λ = 600 nm taken every hour for 25 h. All of the growth experiments were repeated 5 times. Analysis of variance (ANOVA) was used to compare the difference in the means of the growth of bacteria as measured by O.D.600 among different strains/mutants of L. monocytogenes. The statistical analysis was conducted using SAS (Version 9.2, Cary, N.C., U.S.A.). If significant differences in O.D.600 were observed (P < 0.05), the Fisher’s least significant difference test procedure was used to compare and group the means of O.D.600 among the L. monocytogenes mutants. For growth at pH 5, all O.D.600 data were used for analysis. For the nisin experiments, O.D.600 values after 7 h were analyzed. For the 5% NaCl treatment, O.D.600 values after 10 h were analyzed. Phenotypic arrays. Phenotypic assessment of the mutants was performed using Phenotype MicroArrays (PM) as described previously by Bochner (2009). The PM consists of about 2000 assays in 20 (PM1 to PM20) 96-well culture plates. The technology measures growth/respiration through a NADH coupled reduction of tetrazolium dye. For our purpose, we utilized PM1 and PM2A assay plates, which evaluate the growth potential in 190 Vol. 78, Nr. 9, 2013 r Journal of Food Science M1393
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Chaturongakul and others 2011), and the pathogen has developed efficient strategies for survival under stress conditions, such as starvation, wide variations in temperature, pH, and osmolarity (Gandhi and Chikindas 2007). The regulation of gene expression in response to environmental stress conditions is essential for bacterial survival (Akbar and others 1997). The phosphotransferase transport system (PTS) serves as a complex system for sugar phosphorylation and transport in bacteria. A typical PTS contains 2 enzymes, enzyme I (EI) and enzyme II (EII), and a heat-stable protein (HPr). HPr and EI are cytoplasmic whereas the location of EII is more variable, and it is often composed of 3 domains. EIIA is cytoplasmic and soluble. EIIB is hydrophilic and bound to EIIC, a hydrophobic membrane protein. EI and HPr are common whereas EII is sugar-specific, and it usually consists of 3 domains (IIA, IIB, and IIC) (Deutscher and others 2006). There are more than 30 copies of different PTSs present in the genome of L. monocytogenes (Barabote and Saier 2005). In addition to sugar transport, some PTSs have been associated with hydrogen peroxide resistance (Stevens and others 2010), biofilm formation, virulence (Wu and others 2012), and bacteriocins (Kjos and others 2010). For example, mannose-specific PTS was regulated by the σ 54 factor encoded by rpoN (Gravesen and others 2002; Dalet and others 2003; Arous and others 2004; Vadyvaloo and others 2004). The gene that is upstream of the LMOf2365_0442 has been shown to be responsible for growth under cold, osmotic, and acid stress conditions (Michel and others 2011). LMOf2365_0442 (encoding for PTS system, fructose-specific, IIA component), LMOf2365_0443 (encoding for PTS system, fructose-specific, IIB component), LMOf2365_0444 (encoding for PTS system, fructose-specific, IIC component), and LMOf2365_0445 (encoding for glycosyl hydrolase, family 38) were highly induced under high-pressure treatment (Liu and others 2011); however, these genes were inhibited under salt stress (Bae and others 2011, 2012). Since these genes were induced or inhibited under stress conditions, we hypothesized that these genes may be involved in general stress responses. Therefore, in-frame deletion mutants of LMOf236_0442 to 0445 were constructed, and the growth of the mutants were tested under different foodrelated stress conditions including exposure to nisin, acid, and high salt.
L. moncytogenes growth under stress . . . Table 1–Strains, plasmids, and primers used in this study. Strains/plasmids/primers
M: Food Microbiology & Safety
E. coli strains TOP10 DH5α Plasmids pKSV7 L. monocytogenes strains F2365 LMOf2365_0442 LMOf2365_0443 LMOf2365_0444 LMOf2365_0445 Primers F2365_0442SOEA F2365_0442SOEB F2365_0442SOEC F2365_0442SOED F2365_0443SOEA F2365_0443SOEB F2365_0443SOEC F2365_0443SOED F2365_0444SOEA F2365_0444SOEB F2365_0444SOEC F2365_0444SOED F2365_0445SOEA F2365_0445SOEB F2365_0445SOEC F2365_0445SOED
Source or reference
Competent cells Competent cells
Temperature-sensitive integration vector; Cmr
Gift from S. Kathariou
Wild-type serotype 4b strain, genome sequenced LMOf2365_0442deletion LMOf2365_0443deletion LMOf2365_0444 deletion LMOf2365_0445 deletion
Nelson and others 2004 This study This study This study This study
5’GGGGTACCTTGCGAAGCTAGATGTTGC3’ 5’GCGATAATTTTACGTTTCATATCATTACATCCCATCCTTTAAC3’ 5’ATATGAAACGTAAAATTATCGC3’ 5’ GCTCTAGACAACCCGAGCAATCGCCAT3’ 5’ GGGGTACCCAAAAGAATTAGTCGTC 3’ 5’ CTGAACATGGTAGATCCTCCTTATTTCATATCTCTACTACGCC 3’ 5’ TAAGGAGGATCTACCATGTTCAG 3’ 5’ GCTCTAGATACAATCGAAGCCGCCGC 3’ 5’ GGGGTACCATACAAGAGACGAAGAAG3’ 5’ CCATAGTTACCTCCGTTTTTTACATGGTAGATCCTCCTTA3’ 5’ TAAAAAACGGAGGTAACTATGG3’ 5’ GCTCTAGACCCACGCCAGAAAATCAC3’ 5’ GGGGTACCACTTGGTCTTGGTTTAGC3’ 5’ CTTTGAGCAAGTTTTCTTTCTACATAGTTACCTCCGTTTTTTA3’ 5’ TAGAAAGAAAACTTGCTCAAAG3’ 5’ GCTCTAGAGCGGTTGTAACTCCTAGTG3’
This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study
Restriction sites (KpnI and XbaI) are highlighted in bold. Regions overlapping complementary to SOEC primers are underlined.
alternative carbon sources, effects of ions and osmolytes (PM9), and low- and high-pH (PM10). The protocol for the assays was as described by the manufacturer (Biolog Inc. Hayward, Calif., U.S.A.) except that the cultures were grown overnight on BHI agar at 37 ◦ C and used to prepare the cell suspension inoculums. The assay plates were incubated at 37 ◦ C for 48 h in the Omnilog chamber, and the reduction of the tetrazolium dye from colorless to violet color was captured by the proprietary software. For this work, the phenotypes in each well were compared on the basis of the total area under each graph generated by the manufacturer’s software. Statistical analysis was performed using R (www.r-project.org) version 2.13. Briefly, after 48 h incubation at 37 ◦ C, the Biolog total area readings from plates PM1, PM2A, PM9, and PM10 for the 4 mutant strains (LMOf2365_0442, 0443, 0444, and 0445) were compared with the parent strain LMOf2365. To avoid differences in samples processed on different days, comparisons were made only between test (mutant) strains and the parent strain that were processed on the same day. Four mutant strains were divided into 2 groups of 2. Each run consisted of 3 strains (2 mutant test strains and the parent strain) with 3 replicates per strain. Two-sample t-tests were used to compare the area values for the test strain compared with the wild-type. Corrections for multiple tests were made using a stepwise Bonferroni procedure (Holm 1979) with α = 0.05.
Results In this paper, in-frame deletions of the PTS operon (LMOf2365_0442 to 0444) and LMOf2365_0445 were constructed, and the growth of these deletion mutants was tested under food-related stress conditions. The effect of nisin was investigated because it has antimicrobial activity and can be used as a food preservative (G´alvez and others 2007). The effect of salt was studied because salt is often used as a general preservative and food M1394 Journal of Food Science r Vol. 78, Nr. 9, 2013
additive to enhance the flavor and shelf life of food (Ruusunen and Puolanne 2005). pH 5 was selected for the acid tolerance assay because human gastric pH is between 3.0 and 5.0 during food digestion, and L. monocytogenes is generally consumed with contaminated food (Cotter and Hill 2003). Furthermore, the pH of cheese that is often implicated in listeriosis outbreaks is usually pH 5 (Faleiro and others 2003), and L. monocytogenes mounts an acid tolerance response after exposure to this pH (Gahan and others 1996). LMOf2365_0442 to 0445 showed over a 3-fold induction using a DNA microarray assay and over 5-fold induction by real-time quantitative PCR assays under pressure treatment (Liu and others 2011). The current work extends our previous studies through the construction of the knockout mutants to determine a phenotype. Although LMOf2365_0442 to 0445 was highly induced under high-pressure treatment, only the knockout mutants of LMOf2365_0442 and LMOf2365_0445 displayed a growth difference under nisin, salt and acid treatments.
Genetic organization of the PTS operon and in silico analysis of LMOf2365_0442, 0443, 0444, and 0445 The whole genome of L. monocytogenes F2365 has been sequenced (Nelson and others 2004), and DNA sequence analysis indicates that the PTS operon is comprised of 4 genes: LMOf2365_0442, LMOf2365_0443, and LMOf2365_0444. LMOf2365_0445 is located downstream of the PTS operon and encodes for glycosyl hydrolase, family 38. Blast searches from NCBI databases indicated that the protein encoded by LMOf2365_0444 (encoding for IIC) is predicted to contain 8 transmembrane domains whereas LMOf2365_0443 (encoding for IIB) is predicted to have only 1 transmembrane domain. Our results are consistent with the previous finding that IIC is usually
L. moncytogenes growth under stress . . .
125ug/ml Nisin 0.8 0.7
0.6 0443 0.5 0444
0.2 LMOf 2365
0.1 0 442
membrane bound whereas IIA and IIB are mainly cytosolic proteins (Deutscher and others 2006). The in-frame deletion mutants for LMOf2365_0442 to 0445 were constructed using SOE primers (Table 1). The mutants were generated by an in-frame deletion of the entire gene with only the 2nd or 3rd amino acid and the stop codon remaining. LMOf2365_0442 had amino acids 2 to 153 removed, and LMOf2365_0443 had amino acids 3 to 106 removed. LMOf2365_0444 had amino acids 2 to 368 removed, and LMOf2365_0445 had amino acids 2 to 860 removed. These inframe deletions were verified by PCR (Figure 1) and confirmed by sequencing.
Mutant growth in nisin, acid, salt (NaCl), and carbon source Growth phenotypes of the LMOf2365 parental strain and its isogenic LMOf2365_0442, 0443, 0444, and 0445 mutants were tested under different types of stress (nisin, salt, and acid) potentially experienced by this pathogen in food-associated environments. As shown in Figure 2(A), with the nisin (125 μg/mL) treatment, the O.D.600 values of LMOf2365_0445 were significantly higher (P < 0.01) than that of the wild-type and other mutants (LMOf2365_0442, 0443, and 0444) between 0 and 25 h at 37 ◦ C. The fact that LMOf2365_0445 grew faster compared to the wild-type at the concentration of 125 μg/mL nisin indicates that mutants were more resistant to nisin. The resistance
Figure 2–Growth curves of L. monocytogenes F2365, LMOf2365_0442 to 0445 at 37 ◦ C with (A) nisin (250 μg/mL in BHI), (B) acid (BHI acidified to pH 5 with HCl), and (C) salt (5% NaCl in BHI). Cell growth was measured spectrophotometrically by monitoring the O.D.600 at 1-h intervals for 25 h at 37 ◦ C. Data presented here are averages of 5 independent experiments with standard deviations.
to nisin in LMOf2365_0445 was abolished as the nisin concentrations increased to 250 and 500 μg/mL (data not shown). At pH 5, the growth rate (slope) of LMOf2365_0442 was higher than that of the wild-type and other mutants, and the difference in growth between the mutants and the wild-type was maximum at 13 h (Figure 2B). Under salt (5% NaCl in BHI) treatment, the growth rate of LMOf2365_0442 (slope) was higher than that of the wild-type and other mutants after 9 h. The difference in growth of LMOf2365_0442 became apparent at 11 h and reached a maximum at 20 h (Figure 2C). The growth of the mutants in the presence of carbon sources such as maltose, glucose, fructose, and mannose was also tested; however, the mutants grew similarly as compared to the wild-type under these conditions (data not shown). Phenotypic arrays of the deletion mutants. Phenotypic arrays were performed to screen for potential phenotypic differences in the mutants under different growth conditions. The PM1 and PM2A plates measured growth/metabolism in different carbon sources whereas PM9 and PM10 measured the effects of various pH levels and osmolytes, respectively. As shown in Table 2, LMOf2365_0442 to 0445 showed slightly reduced growth with certain carbon sources and different pH conditions. When these growth conditions were tested at 37 ◦ C, the differences shown in phenotypic arrays were not observed (data not shown). The results obtained from growth studies (Figure 2) do not correlate well with the phenotypic array results (Table 2). The use of different assay systems in the growth studies and phenotypic arrays may explain the different results obtained from the 2 assays. It is interesting to note that both LMOf2365_0043 and LMOf2365_ 0045 showed increased sensitivity to pH 9 and 10, as well as to the presence of 6% NaCl.
Discussion In this study, in-frame deletions of the PTS operon (LMOf2365_0442 to 0444) and LMOf2365_0445 were constructed, and the growth of these deletion mutants was tested under food-related stress conditions. The effect of nisin was Vol. 78, Nr. 9, 2013 r Journal of Food Science M1395
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Figure 1–Schematic diagram of the PTS (LMOf2365_0442 to 0444) operon and LMOf2365_0445 in the L. monocytogenes F2365 genome (A) and generation of chromosomal deletion mutants (B). (A) LMOf2365_0442 to 0444 encoded fructose-specific, IIA-C components of the PTS system. LMOf2365_0445 encodes for glycosyl hydrolase, family 38. (B) PCR products of the wild-type genes (LMOf2365_0442 to 0445) and the genes from the respective deletion mutants (LMOf2365_0442 to 0445) amplified using specific primers (SOEA and SOED) flanking each gene. Lane 1, 1kb Plus DNA ladder. Lane 2, 100-bp ladder. Lane 3, LMOf2365_0442 gene and flanking region of the wild-type. Lane 4, flanking region of LMOf2365_0442. Lane 5, LMOf2365_0443 gene and flanking region of the wild-type. Lane 6, flanking region of LMOf2365_0443. Lane 7, LMOf2365_0444 gene and flanking region of the wild-type. Lane 8, flanking region of LMOf2365_0444. Lane 9, LMOf2365_0445 gene and flanking region of the wild-type. Lane 10, flanking region of LMOf2365_0445. The predicted sizes are as follows: flanking regions of LMOf2365_0442, 1318 bp; LMOf2365_0442, 863 bp; LMOf2365_0443, 1214 bp; LMOf2365_0443, 803 bp; LMOf2365_0444, 1968 bp; LMOf2365_0444, 868 bp; LMOf2365_0445, 3378 bp; LMOf2365_0445, 799 bp.
L. moncytogenes growth under stress . . . Table 2–Results from the comparative global phenotypic array analysis of the LMOf2365_0442, 0443, 0444, and 0445 and the L. monocytogenes F2365 parental strain.
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LMOf2365_0442/PM1 C06/L-Rhamnose LMOf2365_0442/PM1 F12/Inosine LMOf2365_0442/PM2A A06/Dextrin LMOf2365_0442/PM2A C04/D-Melezitose LMOf2365_0443/PM1 A11/D-Mannose LMOf2365_0443/PM1 C10/Maltose LMOf2365_0443/PM1 C12/Thymidine LMOf2365_0443/PM1 D12/Uridine LMOf2365_0443/PM10 A10 pH 9 LMOf2365_0443/PM10 A11/pH 9.5 LMOf2365_0443/PM10 A12/pH 10 LMOf2365_0443/PM10 C11/pH 4.5 + L-Homoarginine LMOf2365_0443/PM10 E11/pH 9.5 + L-Leucine LMOf2365_0443/PM10 E12/pH 9.5 + L-Lysine LMOf2365_0443/PM2A A06/Dextrin LMOf2365_0443/ PM2A B12/3-0-β-D-Galactopyranosyl-D-Arabinose LMOf2365_0443/PM2A C10 α-Methyl-D-Mannoside LMOf2365_0443/PM9 A11 9%/NaCl LMOf2365_0443/PM9 B11/6% NaCl + Creatinine LMOf2365_0443 PM9 B12/6% NaCl + L-Carnitine LMOf2365_0443/PM9 C11/6% NaCl + Octopine LMOf2365_0443/ PM9 D12/20% Ethylene Glycol LMOf2365_0443/PM9 H02/20-mM Sodium Nitrate LMOf2365_0444/PM1 C06 L-Rhamnose LMOf2365_0444/PM2A C06/α-Methyl-D-Galactoside LMOf2365_0445/PM1 A10/D-Trehalose LMOf2365_0445/PM1 A11/D-Mannose LMOf2365_0445/PM1 C10/Maltose LMOf2365_0445/PM1 C12/Thymidine LMOf2365_0445/PM1 D12/Uridine LMOf2365_0445/PM10 A11/pH 9.5 LMOf2365_0445/PM10 A12/pH 10 LMOf2365_0445/PM10 E11/pH 9.5 + L-Leucine LMOf2365_0445/PM10 E12/pH 9.5 + L-Lysine LMOf2365_0445/PM10 F12/pH 9.5 + L-Homoserine LMOf2365_0445/PM2A C10/α-Methyl-D-Mannoside LMOf2365_0445/PM9 B11/6% NaCl + Creatinine LMOf2365_0445 PM9 B12 6% NaCl + L-Carnitine LMOf2365_0445 PM9 C11 6% NaCl + Octopine
− 7.972 − 8.065 − 15.264 − 7.56 − 10.354 − 7.305 − 5.087 − 9.781 − 5.471 − 11.82 − 14.549 − 13.184 − 7.546 − 10.183 − 7.436 − 5.6 − 11.302 − 5.189 − 6.435 − 5.025 − 5.954 − 5.657 − 5.593 − 5.027 − 6.426 − 5.892 − 11.037 − 6.071 − 5.122 − 7.173 − 7.643 − 9.314 − 6.166 − 6.428 − 5.751 − 9.458 − 5.245 − 5.522 − 6.273
0.642 0.512 0.424 0.337 0.587 0.755 0.736 0.723 0.816 0.633 0.598 0.664 0.807 0.741 0.711 0.723 0.793 0.738 0.756 0.762 0.71 0.839 0.845 0.762 0.709 0.779 0.572 0.799 0.805 0.75 0.625 0.619 0.696 0.728 0.66 0.81 0.777 0.751 0.721
Statistically significant results from the comparative phenotypic array analysis of the deletion mutants (LMOf2365_0442 to 0445) and the parent strain is shown. T-statistic value. Fold of changes indicate the ratios of total areas of readings between L. monocytogenes deletion mutants (LMOf2365_0442 to 0445) and the parent strain (LMOf2365) grown under different treatments.
b The c
investigated because it has antimicrobial activity and can be used as a food preservative (G´alvez and others 2007). The effect of salt was studied because salt is often used as a general preservative and food additive to enhance the flavor and shelf life of food (Ruusunen and Puolanne 2005). pH 5 was selected for the acid tolerance assay because human gastric pH is between 3.0 and 5.0 during food digestion, and L. monocytogenes is generally consumed with contaminated food (Cotter and Hill 2003. Furthermore, the pH of cheese that is often implicated in listeriosis outbreaks is usually about pH 5 (Faleiro and others 2003), and L. monocytogenes mounts an acid tolerance response after exposure to this pH (Gahan and others 1996). LMOf2365_0442 to 0445 showed over a 3-fold induction using a DNA microarray assay and over 5-fold induction by real-time quantitative PCR assays under pressure treatment (Liu and others 2011). The current work extends our previous studies through the construction of the knockout mutants to determine a phenotype. Although LMOf2365_0442 to 0445 were highly induced under high-pressure treatment, only the knockout mutants of LMOf2365_0442 and LMOf2365_0445 displayed a growth difference under nisin, salt, and acid treatments.
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Although the mutants grew differently than the wild-type under stress conditions such as nisin, salt, and acid treatments in laboratory medium, they may behave differently in real-food matrices. Studying bacterial growth in food matrices is much more complex than in culture media. Food matrices may provide different factors to regulate gene expression, and further studies will continue to explore growth of the L. monocytogenes mutants in food matrices. A number of L. monocytogenes strains have been sequenced; however, mutants from only 1 strain (LMOf2365) were studied in this paper. Deletion mutants from other strains/serotypes/lineages may behave differently. It was puzzling that the growth of the deletion mutants (LMOf2365_0442 to 0444) in media supplemented with fructose was the same as that of the wild-type (data not shown), although the fructose-specific permease components (FruABC) were deleted in the chromosome, Our results are consistent with a previous study in which inactivation of FruBCA in Steptococcus mutans had no impact on growth with 0.5% fructose as sole carbon source (Wen and others 2001). Growth with different carbon sources including mannose, fructose, raffinose, sorbitol, sucrose, xylose, and glucose did not alter mutant growth compared to the
wild-type (data not shown). Although the phenotypic microarray results (Table 2) identified small differences with several carbon sources, differences in separate growth studies could not be confirmed. This could be due to the fact that dye measurement in the PM assay is based on respiration, which may or may not be coupled with growth. Additionally, the marginal effect on growth with different carbon sources could be due to the fact that the PTS operon is dispensable since there are several copies on the chromosome, and deleting one will not affect growth (Stoll and Goebel 2010). Alternatively, the PTS operon may not be named correctly based on its function. It may be related to the transport of other sugars, not fructose. The increased sensitivity of LMO2365_0043 and LMO2365_0045 to high osmolarity in presence of carnitine, creatinine, and octopine appears to indicate a similar mechanism in both these mutants (Table 2). It is well established that bacterial growth suppression at high osmolarity can be alleviated by compatible solutes, also known as osmoprotectants such as glycerol, glycine-betaine, proline, and carnitine (Stack and others 2008). It is possible that the enzymatic defects in these mutants may lead to transport defects of carnitine, creatinine, and octopine leading to enhanced growth suppression although there is no evidence in literature that creatinine and octopine can act as osmoprotectants. Similarly both LMO2365_0043 and 0045 showed increased sensitivity to high pH. Alkaline pH homeostasis in bacteria can be achieved by a variety of mechanisms including induction of amino acid-deaminases (Padan and others 2005). Increased sensitivity of LMO2365_0043 and LMO2365_0045 in the presence of leucine, lysine, and homoserine may indicate that these mutations affected the deamination process resulting in increased high-pH sensitivity. Although the deletion mutants showed some resistance to nisin, acid, and salt, the differences compared to the wild-type were not large. The lack of identifiable phenotypes may possibly be due to the fact that activity of the genes can be replaced by other similar genes in the cell since there are other PTS systems present in the genome. This notion is supported by the fact that deletion of other PTS systems in Streptococcus pneumonia also resulted in no phenotypic change (Bidossi and others 2012). This may suggest that L. monocytogenes is highly adaptive in response to inactivation of these genes or, alternatively, that other PTS systems that have not yet been identified are also involved in sensitivity to acid and salt in L. monocytogenes. PTS systems are located on cell membranes that are usually targets for the action of nisin. Our findings also suggest that putative PTS systems have the potential to be used as targets for the development of new antimicrobials against L. monocytogenes.
Acknowledgments We thank Amy Ream for performing the real-time PCR assays, and appreciate the assistance of Dr. Lihan Huang for the statistical analyses. We are also grateful to Dr. James Smith for critical reading of the manuscript.
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