Gary M. Smith

Role of the Glycine Betaine and Carnitine Transporters in Adaptation of Listeria monocytogenes to Chill Stress in Defined Medium

ABSTRACT The food-borne pathogen Listeria monocytogenes proliferates at refrigeration temperatures, rendering refrigeration ineffective in the preservation of Listeria-contaminated foods. The uptake and intracellular accumulation of the potent compatible solutes glycine betaine and carnitine has been shown to be a key mediator of the pathogens cold-tolerant phenotype. To date, three compatible solute systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and the carnitine transporter OpuC. We investigated the specificity of each transporter towards each compatible solute at 4°C by examining mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state compatible solute accumulation data together with growth rate experiments demonstrated that under cold stress glycine betaine transport is primarily mediated by Gbu and that Gbu-mediated betaine uptake results in significant growth stimulation of chill-stressed cells. BetL and OpuC can serve as minor porters for the uptake of betaine, and their action is capable of providing a small degree of cryotolerance. Under cold stress, carnitine transport occurs primarily through OpuC and results in a high level of cryoprotection. Weak carnitine transport occurs via Gbu and BetL, conferring correspondingly weak cryoprotection. No other transporter in L. monocytogenes 10403S appears to be involved in transport of either compatible solute at 4°C, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown at that temperature.

Three Transporters Mediate Uptake of Glycine Betaine and Carnitine by Listeria monocytogenes in Response to Hyperosmotic Stress

ABSTRACT The uptake and accumulation of the potent osmolytes glycine betaine and carnitine enable the food-borne pathogen Listeria monocytogenes to proliferate in environments of elevated osmotic stress, often rendering salt-based food preservation inadequate. To date, three osmolyte transport systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and a carnitine transporter OpuC. We investigated the specificity of each transporter towards each osmolyte by creating mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state osmolyte accumulation data together with growth rate experiments demonstrated that osmotically activated glycine betaine transport is readily and effectively mediated by Gbu and BetL and to a lesser extent by OpuC. Osmotically stimulated carnitine transport was demonstrated for OpuC and Gbu regardless of the nature of stressing salt. BetL can mediate weak carnitine uptake in response to NaCl stress but not KCl stress. No other transporter in L. monocytogenes 10403S appears to be involved in osmotically stimulated transport of either osmolyte, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown under elevated osmotic stress.

Osmotic and Chill Activation of Glycine Betaine Porter II in Listeria monocytogenes Membrane Vesicles

Listeria monocytogenes is a foodborne pathogen known for its tolerance to conditions of osmotic and chill stress. Accumulation of glycine betaine has been found to be important in the organisms tolerance to both of these stresses. A procedure was developed for the purification of membranes from L. monocytogenes cells in which the putative ATP-driven glycine betaine permease glycine betaine porter II (Gbu) is functional. As is the case for the L. monocytogenes sodium-driven glycine betaine uptake system (glycine betaine porter I), uptake in this vesicle system was dependent on energization by ascorbate-phenazine methosulfate. Vesicles lacking the gbu gene product had no uptake activity. Transport by this porter did not require sodium ion and could be driven only weakly by artificial gradients. Uptake rates could be manipulated under conditions not affecting secondary transport but known to affect ATPase activity. The system was shown to be both osmotically activated and cryoactivated. Under conditions of osmotic activation, the system exhibited Arrhenius-type behavior although the uptake rates were profoundly affected by the physical state of the membrane, with breaks in Arrhenius curves at approximately 10 and 18 degrees C. In the absence of osmotic activation, the permease could be activated by decreasing temperature within the range of 15 to 4 degrees C. Kinetic analyses of the permease at 30 degrees C revealed K(m) values for glycine betaine of 1.2 and 2.9 microM with V(max) values of 2,200 and 3,700 pmol/min. mg of protein under conditions of optimal osmotic activation as mediated by KCl and sucrose, respectively.

Mechanism of osmotically regulated N-acetylglutaminylglutamine amide production inRhizobium meliloti

Rhizobium meliloti adapts to environments of high osmolarity by accumulating glutamate, trehalose, and the dipeptide N-acetylglutaminylglutamine amide (NAGGN) intracellularly. In this study, the mechanism of NAGGN production and accumulation was examined. NAGGN was produced in osmotically shocked cultures after a lag period of more than one hour, and NAGGN was undetectable in cultures treated with chloramphenicol, indicating that genetic induction is required for NAGGN accumulation.In vitro radiolabeling experiments demonstrated that the peptide synthesis step in NAGGN production did not occur ribosomally. Rather, it was catalyzed by an ATP-dependent enzyme that appeared to be both induced by high osmolarity and activated by K+. Also, a mutant analysis suggested that NAGGN may be partly responsible for the osmotic tolerance observed inR. meliloti. 36% of mutants that were characterized as osmotically sensitive compared to the parent strain, were also found to contain reduced levels of NAGGN. The phenomenon of osmolyte accumulation as it relates to adaptation to other environmental stresses is discussed.

Identification of OpuC as a Chill-Activated and Osmotically Activated Carnitine Transporter in Listeria monocytogenes

ABSTRACT The food-borne pathogen Listeria monocytogenes is notable for its ability to grow under osmotic stress and at low temperatures. It is known to accumulate the compatible solutes glycine betaine and carnitine from the medium in response to osmotic or chill stress, and this accumulation confers tolerance to these stresses. Two permeases that transport glycine betaine have been identified, both of which are activated by hyperosmotic stress and one of which is activated by low temperature. An osmotically activated transporter for carnitine, OpuC, has also been identified. We have isolated a Tn917-LTV3 insertional mutant that could not be rescued from hyperosmotic stress by exogenous carnitine. The mutant, LTS4a, grew indistinguishably from a control strain (DP-L1044) in the absence of stress or in the absence of carnitine, but DP-L1044 grew substantially faster under osmotic or chill stress in the presence of carnitine. LTS4a was found to be strongly impaired in KCl-activated as well as chill-activated carnitine transport. 13C nuclear magnetic resonance spectroscopy of perchloric acid extracts showed that accumulation of carnitine by LTS4a was negligible under all conditions tested. Direct sequencing of LTS4a genomic DNA with a primer based on Tn917-LTV3 yielded a 487-bp sequence, which allowed us to determine that the opuC operon had been interrupted by the transposon. It can be concluded that opuC encodes a carnitine transporter that can be activated by either hyperosmotic stress or chill and that the transport system plays a significant role in the tolerance of L. monocytogenes to both forms of environmental stress.

Rapid determination of total cholesterol in egg yolk using commercial diagnostic cholesterol reagent

Abstract A rapid and accurate method for cholesterol determination using a commercial diagnostic cholesterol reagent (‘enzyme method’) was developed and evaluated with four different yolk preparations. The cholesterol content of The National Institute of Standards and Technology (NIST) Standard Reference Material in whole egg powder, fresh, frozen, and dried egg yolk was determined using gas chromatography and the enzyme method. All samples were subjected to direct saponification and solubilization prior to analysis. No treatment was applied to control samples, which were analyzed by the enzyme method. Solubilization of samples was performed at 0.85, 2 and 5% (w/v) NaCl concentrations. Solubilization measured in terms of cholesterol value was best at 2 and 5% NaCl levels. For egg powder standard and fresh or frozen egg, the cholesterol values obtained by the enzyme method from saponified or solubilized samples were not significantly different from values obtained by gas chromatography. For solubilized dried egg yolk the enzyme method gave results indistinguishable from chromatography for saponified yolk. However, the results obtained by the enzyme method and gas chromatography from saponified dried egg yolk were significantly different. The simple and rapid procedure developed for sample preparation (solubilization) eliminates the need for saponification prior to cholesterol determination using the enzyme method, and provides an alternative to an expensive and time-consuming gas chromatographic method.

Isolation and fatty acid analysis of neutral and polar lipids of the food bacterium Listeria monocytogenes

Abstract Listeria monocytogenes is a Gram-positive bacterium that causes meningitis, septicemia and death in humans. Found in low-acid cheeses, vegetables and meat, L. monocytogenes is resistant to osmotic and chill stress. Food handling practices that suppress microbial competitors can therefore promote its growth. In response to hyperosmotic or chill stress, L. monocytogenes accumulates the potent protectant glycine betaine from the medium, which decreases the lag time and increases the growth rate of the organism. The molecular basis for activation of glycine betaine transport by chill (7 °C), despite the expected membrane lipid phase transition, may reside in the lipid composition. The present research identified the lipids of L. monocytogenes. Extraction of total lipids yielded 7 ± 1 mg ml−1 wet cells, with a 5–6% phosphorus content. Polar lipids represented 64% of total lipids. There was a clear difference in the relative complexity of the fatty acids: neutral lipids were more varied and unsaturated fatty acids represented 19% of the total. Polar lipid fatty acids were primarily 15:anteiso (50%) and 17:anteiso (25%).

Elevated carnitine accumulation by Listeria monocytogenes impaired in glycine betaine transport is insufficient to restore wild-type cryotolerance in milk whey.

Listeria monocytogenes accumulates low molecular weight compounds (osmolytes, or compatible solutes) in response to chill stress. This response has been shown to be responsible, in part, for the chill tolerance of the species. Among the osmolytes tested to date, glycine betaine, gamma-butyrobetaine and carnitine display the strongest cryoprotective effect. These osmolytes are not synthesized in the cell and must be transported from the medium. In this study, the compatible solute accumulation profile of the food-borne pathogen L. monocytogenes was determined in balanced growth and stationary phase cultures grown in milk whey at 7 and 30 degrees C. In balanced growth cultures at 7 degrees C, glycine betaine (720 nmol/10(10) cfu) and carnitine (130 nmol/10(10) cfu) were the major osmolytes accumulated by wild-type L. monocytogenes 10403S, whereas carnitine (490 nmol/10(10) cfu) was the dominant osmolyte and glycine betaine was present in smaller amounts (270 nmol/10(10) cfu) in a mutant (L. monocytogenes LTG59) blocked in the major glycine betaine uptake system, glycine betaine porter II. In strain 10403S, glycine betaine and carnitine were present in eightfold and twofold excess at 7 degrees C compared to 30 degrees C; the respective ratios for strain LTG59 were 6 and 8. The intracellular concentration of osmolytes in stationary phase cultures at 7 degrees C was markedly reduced compared to that during balanced growth. Furthermore, at 4 degrees C, small but highly significant differences in growth were observed between strains. Strain LTG59 grew with a lag phase that was significantly longer, a generation time that was significantly greater and reached a final cell yield that was significantly lower than that of strain 10403S. The elevated accumulation of carnitine in the absence of glycine betaine porter II was insufficient to confer the magnitude of the cryoprotective effect displayed by the wild type.

Diversity of the polar lipids of the food-borne pathogenListeria monocytogenes

Listeria monocytogenes is a Gram-positive bacterium that can adapt to high salinity and cold. Because the membrane lipids may play a role in its survival and adaptation, we have examined the polar lipids ofL. monocytogenes. Extraction of total lipids fromL. monocytogenes yielded 7±1 mg/mL wet cells. Polar lipids represented 64% of total lipids and contained 9% lipid-phosphorus. Polar lipids were separated into 14 components by two-dimensional thin-layer chromatography. Eight components (88% of polar lipids) contained lipid-phosphorus; among these was one major component (34% of polar lipids). Two other phospholipids were ninhydrin-positive components and accounted for 15% of the polar lipids. Orcinol staining revealed two glyco- or sulfo-lipids accounting for 9% of polar lipids. Five components (4% of polar lipids) were amino components free of phosphorus. The major component contained 46% of its fatty acids as 15:0anteiso, 24% as 17:0anteiso, and 11% as 15:0iso. The fatty acid profile of the remaining polar lipids was variable, consisting primarily of 16:0, 18:0, 15:0anteiso, and 17:0anteiso. Their unsaturation level was ≤20%; however, the major phosphoaminolipid component was 46% unsaturated. The ratios of 15:0anteiso/17:0anteiso and 15:0anteiso/15:0iso were similar in all classes, averaging 1.5 and 4.5, respectively. Since the adaptation process to stressful environments involves activation of a membrane transport system for the protectant glycine betaine, the membrane lipids may play a role in enabling transport.

Effect of Hg(II) on d(GCGCATATGCGC)2 conformation: UV absorption and circular dichroism studies

Exposing the palindromic dodecamer d(GCGCATATGCGC)2, dissolved in the neutral-salt solvent 0.1 M (identical to low salt) or 3.0 M (identical to high salt) NaC1O4, to increasing amounts of Hg(C1O4)2 (identical to Hg(II)) produces major changes in its chirality in the low-salt medium but not high-salt. Let r identical to [Hg(C1O4)2]added/[DNA-P]. At low salt, the conformational changes are noted as follows: in absence in Hg(II), the near-conservative CD spectrum of the dodecamer is that of a B-type DNA. In the presence of Hg(II), the long-wavelength positive Cotton band (central location near 280 nm) undergoes inversion at r > 0.2 and reaches maximum negative extension at r = 0.5-0.6. A new minor positive CD peak appears around 315 nm. The long-wavelength negative Cotton band (central location near 250 nm) experiences a slight red-shift by about 10 nm but remains otherwise rather unaffected by Hg(II). Major CD changes are also noted in the short-wavelength region near 220 nm. The inverted CD spectrum is in general more complex in structure than the spectrum of the untreated oligomer. By contrast, the CD changes seen in the high-salt medium are not as complex, and the CD scripture of a right-handed B-type helix is retained. It is stressed that all CD changes are fully reversible: both the low-salt and high-salt CD spectra of the untreated dodecamer are regained subsequent to the removal of Hg(II) with the help of a complexing agent such as sodium cyanide. The (ordinary) UV absorption spectra of the dodecamer are altered by Hg(II) in less spectacular fashion although differences in progression in the two-salt media are noted. We interpret the CD changes as signaling (right-handed) B<-->(left-handed) non-B transitions of the oligodeoxyribonucleotide at low-salt levels but not at high-ionic strength. The presence of isodichroic (isosbestic) points in the various spectra shows that the Hg(II)-induced conformational B<-->Z transitions is unclear at present.