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.

FIG 1

Colony morphology of the Listeria monocytogenes 568 wild type and its DT transposon mutants on BHI agar after 2 days of incubation at 30°C. (a) L. monocytogenes 568; (b) DT01 (lmo0289, yycH); (c and d) DT11 (lmo1371, lpd) plated on BHI after 49 h of osmotic stress (TSB-glu plus 20% NaCl, 15°C) (c) and 4 days of desiccation (43% RH, 15°C) (d).

FIG 2

Putative functions of the interrupted genes in 15 desiccation-tolerant (DT) and 15 desiccation-sensitive (DS) Listeria monocytogenes 568 Himar1 transposon insertion mutants. Note that two DS mutants contained transposons in the same gene, making the total number of affected genes 14.

FIG 3

Locations of the sequenced Himar1 transposon insertion sites in desiccation-tolerant and -sensitive Listeria monocytogenes 568 mutants mapped to the EGD-e chromosome. Each quarter of the chromosome is identified with the approximate base pair, except for bp 1, which is identified by an arrow.