Identification of Listeria monocytogenes Genes Expressed in Response to Growth at Low Temperature

Listeria monocytogenes is a food-borne bacterial pathogen that is able to grow at refrigeration temperatures. To investigate microbial gene expression associated with cold acclimation, we used a differential cDNA cloning procedure known as selective capture of transcribed sequences (SCOTS) to identify bacterial RNAs that were expressed at elevated levels in bacteria grown at 10°C compared to those grown at 37°C. A total of 24 different cDNA clones corresponding to open reading frames in the L. monocytogenes strain EGD-e genome were obtained by SCOTS. These included cDNAs for L. monocytogenes genes involved in previously described cold-adaptive responses (flaA and flp), regulatory adaptive responses (rpoN, lhkA, yycJ, bglG, adaB, and psr), general microbial stress responses (groEL, clpP, clpB, flp, and trxB), amino acid metabolism (hisJ, trpG, cysS, and aroA), cell surface alterations (fbp, psr, and flaA), and degradative metabolism (eutB, celD, and mleA). Four additional cDNAs were obtained corresponding to genes potentially unique to L. monocytogenes and showing no significant similarity to any other previously described genes. Northern blot analyses confirmed increased steady-state levels of RNA for all members of a subset of genes examined during growth at a low temperature. These results indicated that L. monocytogenes acclimation to growth at 10°C likely involves amino acid starvation, oxidative stress, aberrant protein synthesis, cell surface remodeling, alterations in degradative metabolism, and induction of global regulatory responses.     

Response regulator DegU of Listeria monocytogenes regulates the expression of flagella-specific genes

An isogenic mutant of Listeria monocytogenes EGD with a deletion of the response regulator gene degU showed a lack of motility due to the absence of flagella. In the present study, we used two-dimensional gel electrophoresis, mass-spectrometry and microarray analyses to identify the listerial genes that depend on DegU for expression. We found that the two L. monocytogenes operons encoding flagella-specific genes and the monocistronically transcribed flaA gene are positively regulated by DegU at 24 degrees C, but are not expressed at 37 degrees C.     

Control of Listeria monocytogenes in a biofilm by competitive-exclusion microorganisms

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37 degrees C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10(3) CFU of L. monocytogenes/ml and 10(5) CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37 degrees C for 24 h, 15 degrees C for 14 days, 8 degrees C for 21 days, and 4 degrees C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37 degrees C, two at 15 and 8 degrees C, and three at 4 degrees C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4 degrees C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log(10) CFU of L. monocytogenes/cm(2)). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37 degrees C.     

Electron paramagnetic resonance studies of the membrane fluidity of the foodborne pathogenic psychrotroph Listeria monocytogenes

Listeria monocytogenes is a foodborne psychrotrophic pathogen that grows at refrigeration temperatures. Previous studies of fatty acid profiles of wild-type and cold-sensitive, branched-chain fatty acid deficient mutants of L. monocytogenes suggest that the fatty acid 12-methyltetradecanoic (anteiso-C(15:0)) plays a critical role in low-temperature growth of L. monocytogenes, presumably by maintaining membrane fluidity. The fluidity of isolated cytoplasmic membranes of wild-type (SLCC53 and 10403S), and a cold-sensitive mutant (cld-1) of L. monocytogenes, grown with and without the supplementation of 2-methylbutyric acid, has been studied using a panel of hydrocarbon-based nitroxides (2N10, 3N10, 4N10, and 5N10) and spectral deconvolution and simulation methods to obtain directly the Lorentzian line widths and hence rotational correlation times (tau(c)) and motional anisotropies of the nitroxides in the fast motional region. tau(c) values over the temperature range of -7 degrees C to 50 degrees C were similar for the membranes of strains SLCC53 and 10403S grown at 10 degrees C and 30 degrees C, and for strain cld-1 grown with 2-methylbutyric acid supplementation (which restores branched-chain fatty acids) at 30 degrees C. However, strain cld-1 exhibited a threefold higher tau(c) when grown without 2-methylbutyric acid supplementation (deficient in branched-chain fatty acids) compared to strains SLCC53, 10403S, and supplemented cld-1. No evidence was seen for a clear lipid phase transition in any sample. We conclude that the fatty acid anteiso-C(15:0) imparts an essential fluidity to the L. monocytogenes membrane that permits growth at refrigeration temperatures.     

Thermotolerance of Listeria monocytogenes and Salmonella typhimurium after sublethal heat shock.

The effect of prior heat shock on thermotolerance of Listeria monocytogenes and Salmonella typhimurium in broth culture was determined. Bacteria were grown at the permissive temperature of 35 degrees C, sublethally heated at 35 (control), 42, 48, and 52 degrees C (nonpermissive control) for various times, and inactivated at either 57.8 or 52 degrees C. The induction of increased thermotolerance by heat shock, although consistent within each experiment, was generally not significant for L. monocytogenes; the increase was significant for S. typhimurium. Temperature shift experiments with L. monocytogenes suggested that induced thermotolerance was not long lived unless the shock temperature was maintained.     

Effects of several factors on the heat-shock-induced thermotolerance of Listeria monocytogenes.

The influence of the temperature at which Listeria monocytogenes had been grown (4 or 37 degrees C) on the response to heat shocks of different durations at different temperatures was investigated. For cells grown at 4 degrees C, the effect of storage, prior to and after heat shock, on the induced thermotolerance was also studied. Death kinetics of heat-shocked cells is also discussed. For L. monocytogenes grown at 37 degrees C, the greatest response to heat shock was a fourfold increase in thermotolerance. For L. monocytogenes grown at 4 degrees C, the greatest response to heat shock was a sevenfold increase in thermotolerance. The only survival curves of cells to have shoulders were those for cells that had been heat shocked. A 3% concentration of sodium chloride added to the recovery medium made these shoulders disappear and decreased decimal reduction times. The percentage of cells for which thermotolerance increased after a heat shock was smaller the milder the heat shock and the longer the prior storage.     

Influence of the natural microbial flora on the acid tolerance response of Listeria monocytogenes in a model system of fresh meat decontamination fluids

Depending on its composition and metabolic activity, the natural flora that may be established in a meat plant environment can affect the survival, growth, and acid tolerance response (ATR) of bacterial pathogens present in the same niche. To investigate this hypothesis, changes in populations and ATR of inoculated (10(5) CFU/ml) Listeria monocytogenes were evaluated at 35 degrees C in water (10 or 85 degrees C) or acidic (2% lactic or acetic acid) washings of beef with or without prior filter sterilization. The model experiments were performed at 35 degrees C rather than lower (8.0 log CFU/ml) by day 1. The pH of inoculated water washings decreased or increased depending on absence or presence of natural flora, respectively. These microbial and pH changes modulated the ATR of L. monocytogenes at 35 degrees C. In filter-sterilized water washings, inoculated L. monocytogenes increased its ATR by at least 1.0 log CFU/ml from days 1 to 8, while in unfiltered water washings the pathogen was acid tolerant at day 1 (0.3 to 1.4 log CFU/ml reduction) and became acid sensitive (3.0 to >5.0 log CFU/ml reduction) at day 8. These results suggest that the predominant gram-negative flora of an aerobic fresh meat plant environment may sensitize bacterial pathogens to acid.     

Chlamydomonas reinhardtii mutants abnormal in their responses to phosphorus deprivation.

P-starved plants scavenge inorganic phosphate (Pi) by developing elevated rates of Pi uptake, synthesizing extracellular phosphatases, and secreting organic acids. To elucidate mechanisms controlling these acclimation responses in photosynthetic organisms, we characterized the responses of the green alga Chlamydomonas reinhardtii to P starvation and developed screens for isolating mutants (designated psr [phosphorus-stress response]) abnormal in their responses to environmental levels of Pi. The psr1-1 mutant was identified in a selection for cells that survived exposure to high concentrations of radioactive Pi. psr1-2 and psr2 were isolated as strains with aberrant levels of extracellular phosphatase activity during P-deficient or nutrient-replete growth. The psr1-1 and psr1-2 mutants were phenotypically similar, and the lesions in these strains were recessive and allelic. They exhibited no increase in extracellular phosphatase activity or Pi uptake upon starvation. Furthermore, when placed in medium devoid of P, the psr1 strains lost photosynthetic O2 evolution and stopped growing more rapidly than wild-type cells; they may not be as efficient as wild-type cells at scavenging/accessing P stores. In contrast, psr2 showed elevated extracellular phosphatase activity during growth in nutrient-replete medium, and the mutation was dominant. The mutant phenotypes and the roles of Psr1 and Psr2 in P-limitation responses are discussed.     

Detection of low levels of Listeria monocytogenes cells by using a fiber-optic immunosensor

Biosensor technology has a great potential to meet the need for sensitive and nearly real-time microbial detection from foods. An antibody-based fiber-optic biosensor to detect low levels of Listeria monocytogenes cells following an enrichment step was developed. The principle of the sensor is a sandwich immunoassay where a rabbit polyclonal antibody was first immobilized on polystyrene fiber waveguides through a biotin-streptavidin reaction to capture Listeria cells on the fiber. Capture of cells on the fibers was confirmed by scanning electron microscopy. A cyanine 5-labeled murine monoclonal antibody, C11E9, was used to generate a specific fluorescent signal, which was acquired by launching a 635-nm laser light from an Analyte 2000 and collected by a photodetector at 670 to 710 nm. This immunosensor was specific for L. monocytogenes and showed a significantly higher signal strength than for other Listeria species or other microorganisms, including Escherichia coli, Enterococcus faecalis, Salmonella enterica, Lactobacillus plantarum, Carnobacterium gallinarum, Hafnia alvei, Corynebacterium glutamicum, Enterobacter aerogenes, Pseudomonas aeruginosa, and Serratia marcescens, in pure or in mixed-culture setup. Fiber-optic results could be obtained within 2.5 h of sampling. The sensitivity threshold was about 4.3 x 10(3) CFU/ml for a pure culture of L. monocytogenes grown at 37 degrees C. When L. monocytogenes was mixed with lactic acid bacteria or grown at 10 degrees C with 3.5% NaCl, the detection threshold was 4.1 x 10(4) or 2.8 x 10(7) CFU/ml, respectively. In less than 24 h, this method could detect L. monocytogenes in hot dog or bologna naturally contaminated or artificially inoculated with 10 to 1,000 CFU/g after enrichment in buffered Listeria enrichment broth.     

Pathogenicity of Listeria monocytogenes grown on crabmeat

The pathogenicity of Listeria monocytogenes as influenced by growth on crabmeat at 5 and 10 degrees C was studied. Crabmeat was inoculated with L. monocytogenes V7 (ca. 10(4) CFU/g) and incubated for up to 14 days at 5 and 10 degrees C. At selected incubation times, L. monocytogenes was removed from crabmeat by washing with 0.1 M potassium phosphate buffer (pH 7.0), and populations were determined by surface plating on LiCl-phenylethanol-moxalactam agar. Buffered suspensions were then centrifuged, and the resulting pellets were suspended in phosphate buffer containing 10% glycerol and stored at -18 degrees C. Thawed, diluted suspensions of cells were tested for pathogenicity by intraperitoneal injection into immunocompromised and nonimmunocompromised mice. L. monocytogenes cells recovered from crabmeat and then recultured in tryptose phosphate broth (TPB), as well as cells which had not been passed through crabmeat but had been cultured in TPB, were likewise harvested, suspended in buffered 10% glycerol, frozen, thawed, diluted, and tested for pathogenicity by intraperitoneal injection. Growth on crabmeat at 5 and 10 degrees C did not have a significant effect on pathogenicity. The population of L. monocytogenes necessary to kill about 50% of the immunocompromised mice in each test set within 7 days was about 10(4) CFU, and this result was not significantly affected by storage temperature of the crabmeat or type of substrate, i.e., crabmeat or TPB, on which it had grown.     

Pathogenicity of Listeria monocytogenes grown on crabmeat.

The pathogenicity of Listeria monocytogenes as influenced by growth on crabmeat at 5 and 10 degrees C was studied. Crabmeat was inoculated with L. monocytogenes V7 (ca. 10(4) CFU/g) and incubated for up to 14 days at 5 and 10 degrees C. At selected incubation times, L. monocytogenes was removed from crabmeat by washing with 0.1 M potassium phosphate buffer (pH 7.0), and populations were determined by surface plating on LiCl-phenylethanol-moxalactam agar. Buffered suspensions were then centrifuged, and the resulting pellets were suspended in phosphate buffer containing 10% glycerol and stored at -18 degrees C. Thawed, diluted suspensions of cells were tested for pathogenicity by intraperitoneal injection into immunocompromised and nonimmunocompromised mice. L. monocytogenes cells recovered from crabmeat and then recultured in tryptose phosphate broth (TPB), as well as cells which had not been passed through crabmeat but had been cultured in TPB, were likewise harvested, suspended in buffered 10% glycerol, frozen, thawed, diluted, and tested for pathogenicity by intraperitoneal injection. Growth on crabmeat at 5 and 10 degrees C did not have a significant effect on pathogenicity. The population of L. monocytogenes necessary to kill about 50% of the immunocompromised mice in each test set within 7 days was about 10(4) CFU, and this result was not significantly affected by storage temperature of the crabmeat or type of substrate, i.e., crabmeat or TPB, on which it had grown.     

Survey of gene expression in winter rye during changes in growth temperature,irradiance or excitation pressure

Previous comparisons of winter rye plants (Secale cereale L. cv. Musketeer) grown in a combination of specific temperature (degrees C)/irradiance (micromol m(-2) s(-1)) regimes (20/50; 20/250; 20/800; 5/50; 5/250) revealed (1) that photosynthetic acclimation to low temperature mimics photosynthetic acclimation to high light because both conditions result in comparable reduction states of photosystem II (PSII), that is, comparable PSII excitation pressure; (2) that the relative redox state of PSII also appears to regulate a specific cold acclimation gene, Wcs19. In order to identify additional genes regulated differentially by either low temperature, irradiance or excitation pressure, we initiated a detailed analysis of gene expression. We identified and characterized 42 differentially expressed genes from wheat and rye. Based on their patterns of regulation under the five growth conditions employed, 37 of the cDNAs could be classified into four groups: genes regulated by PSII excitation pressure, low temperature, growth irradiance and interaction between growth temperature and irradiance. Partial sequence analyses revealed that several of these genes encode known chloroplastic proteins such as ELIPs, transketolase, carbonic anhydrase and Mg-chelatase. However, five of the genes could not be classified unambiguously into any one of these four categories. The implications of these results and the limitations of the experimental design are discussed in terms of larger-scale genomic studies designed to understand the interactions of multiple abiotic stresses to which a plant may be exposed when examining regulation of gene expression.