Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis

Abstract

Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm 2 relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses.T he foodborne bacterial pathogen Listeria monocytogenes continues to be a significant issue in the food supply chain, causing repeated recalls and outbreaks of foodborne illness. L. monocytogenes is ubiquitous in nature but is transmitted to humans predominantly through contaminated food, with the majority of listeriosis outbreaks being associated with ready-to-eat (RTE) products, such as deli meats, soft cheeses, and fresh produce (1, 2). Listeriosis is a very serious disease with mortality rates of 20 to 40% (3). Populations which are more susceptible to listeriosis include the elderly, pregnant women, newborns, and immunocompromised individuals.Several foodborne outbreaks of listeriosis have been traced back to the processing facilities in which the products were made (4-8). Recent literature also suggests that contamination at the retail level may be a significant cause of listeriosis (9-11). In both environments, L. monocytogenes has been isolated from food contact surfaces and non-food contact surfaces, such as floors and drains (12). Once introduced into a processing environment, L. monocytogenes can persist for years, despite the harsh environme...

Genotypes Associated with Listeria monocytogenes Isolates Displaying Impaired or Enhanced Tolerances to Cold, Salt, Acid, or Desiccation Stress

“…The plates were then stored for 3 days at 20°C in desiccators (SICCO, Bohlender, Germany) pre-conditioned to 33% relative humidity (RH) using a saturated solution of MgCl 2 (protocol adapted from Hingston et al, 2015). The RH of the chambers was monitored throughout the desiccation periods using data loggers (included with desiccators).…”

Transcriptome Sequencing of Listeria monocytogenes Reveals Major Gene Expression Changes in Response to Lactic Acid Stress Exposure but a Less Pronounced Response to Oxidative Stress

“…Also unique to the 6719 hydrogen peroxide response was the upregulation of two oxidative stress protection genes: sodA (LM6179_2184), a superoxide dismutase upregulated by a log2fold change of 0.79, and LM6179_1300, annotated as a glutathione peroxidase and upregulated by a log2fold change of 0.56 (Chatterjee et al, 2006;Hingston et al, 2015). The importance of the superoxide dismutase in L. monocytogenes stress response has been documented before (Soni et al, 2011;Bucur et al, 2018), and upregulation of the EGD-e homolog of LM6179_1300, lmo0983, was observed following exposure to chlorine dioxide (Pleitner et al, 2014).…”

Xerotolerant bacteria: surviving through a dry spell

“…X14-1, which was isolated from desert soil, encodes proteins for the utilisation of a wide array of carbon substrates, as well as key enzymes for CO 2 fixation that are absent from Pontibacter species isolated from non-xeric environments4 .Many energy-consuming processes, such as flagellar motility and chemotactic machinery26,27,78,85 , are down-regulated in cells under desiccating conditions. Similarly, flagellar motility mutants of the food-borne pathogen L. monocytogenes tolerated desiccation better than wild-type cells90 . Given the energy requirements of flagellar motility, it may become a dispensable function under desiccating conditions, allowing ATP to be redirected to more important metabolic processes90 .…”

“…Similarly, flagellar motility mutants of the food-borne pathogen L. monocytogenes tolerated desiccation better than wild-type cells90 . Given the energy requirements of flagellar motility, it may become a dispensable function under desiccating conditions, allowing ATP to be redirected to more important metabolic processes90 .…”