Construction of Listeria monocytogenes Mutants with In‐Frame Deletions in the Phosphotransferase Transport System (PTS) and Analysis of Their Growth under Stress Conditions

Abstract

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.

LMOf2365_0442 Encoding for a Fructose Specific PTS Permease IIA May Be Required for Virulence in L. monocytogenes Strain F2365

“…The expression levels of clpE and sigB were moderately elevated (3.5- and 4.8-folds) in Δ LMOf2365_0444 and Δ LMOf2365_0442 , respectively. The increased levels of stress-related gene expression confirmed our previous observation that these deletion mutants may contribute to general stress response ( Liu et al, 2013 ).…”

“…Interestingly, the gene expression levels of hly, lap, actA , and plcB had little change in Δ LMOf2365_0443. Our previous studies indicated that the deletion mutants were more resistant to multiple stress conditions ( Liu et al, 2013 ), indicating that they may contribute to a general stress response. RT-PCR assays were used to determine the gene expression levels of three stress-related genes ( sigB, clpC , and clpE ).…”

“…It was used in this study since its genome is fully sequenced and annotated ( Nelson et al, 2004 ). Glycerol stock cultures of L. monocytogenes F2365 (serotype 4b), L. innocua (ATCC ® 51742 TM ), and three isogenic deletion mutants Δ LMOf2365_0442-0444 of the parent strain L. monocytogenes F2365 ( Liu et al, 2013 ) stored at -80°C were streaked onto Brain Heart Infusion (BHI) (Sigma–Aldrich St. Louis, MO, United States) agar plates. Single colonies picked from agar plates were grown to logarithmic or stationary phases prior to each experiment.…”

“…LMOf2365_0442 (encoding for the PTS system, fructose-specific, IIA component), LMOf2365_0443 (encoding for the PTS system, fructose-specific, IIB component), and LMOf2365_0444 (encoding for the PTS system, fructose-specific, IIC component) were highly induced under high hydrostatic pressure treatment ( Liu et al, 2011 ) and inhibited under salt stress ( Bae et al, 2012 ) as tested by microarray and real-time PCR assays. In our previous study, mutants with in-frame deletions of these genes had different growth patterns under multiple stress conditions, including acid and salt stresses, indicating that these genes may contribute to the general stress response ( Liu et al, 2013 ). Since a mannose PTS permease has been shown to be related to virulence gene expression in L. monocytogenes ( Vu-Khac and Miller, 2009 ), we hypothesized that the LMOf2365_0442, 0443 , and 0444 encoding for PTS permease might also be involved in virulence in L. monocytogenes .…”

Aurantimycin resistance genes contribute to survival of Listeria monocytogenes during life in the environment

“…For example, 30 complete phosphotransferase systems for the uptake of different sugars are present in the L. monocytogenes reference strain EGD-e (Barabote and Saier, 2005). Likewise, over 30 different ATP-binding cassette (ABC) transporters are encoded in the listerial genome (Liu et al, 2013), serving different cellular functions such as nutrient uptake and antibiotic resistance (Fraser et al, 2000;Gopal et al, 2010).…”