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 pathogen's 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.The animal and human pathogen Listeria monocytogenes is the causative agent of listeriosis, a food-borne disease primarily affecting immunocompromised individuals with a 20 to 30% mortality rate (28, 36). L. monocytogenes has long been recognized as an organism capable of proliferating at refrigeration temperatures (46). During the last decade, several aspects of the L. monocytogenes physiology have been identified that are linked to the ability of this otherwise mesophilic pathogen to adapt to environments of low temperature, including the expression of cold shock proteins, the retailoring of the membrane lipid composition, and the accumulation of compatible solutes.Cold-stress (cold-shock and cold acclimation) proteins whose synthesis is increased after temperature downshifts have been isolated, but for the most part their identities and functions remain uncertain (6,16,32,47). Analogous proteins have been studied for Escherichia coli and Bacillus subtilis and have been postulated to function as RNA chaperons, transcription antiterminators, or transcription activators (9,13,14,19). A recent study of gene expression in response to growth of L. monocytogenes at 10°C showed that the pathogen's acclimation involves amino acid starvation, oxidative stress, aberrant protein synthesis, cell surface remodeling, alterations in degradative metabolism, and...

The complete genome sequence of the meat-borne lactic acid bacterium Lactobacillus sakei 23K

“…It is likely that accumulation of these compounds is driven by three ABC uptake systems (LSA0616 to LSA0619, LSA1694 to LSA1696, LSA1869-LSA1870), together with a Na + -dependent symporter (LSA0264). This genetic repertoire is similar to that found in Listeria monocytogenes, an organism also known for salt and cold resistance 20,21 . Similarly, L. sakei has more putative cold stress proteins (LSA0768, LSA0946, LSA1152, LSA1561) than other lactobacilli, but as many as L. monocytogenes.…”

Crystal Structure of the Substrate-Binding Domain from Listeria monocytogenes Bile-Resistance Determinant BilE

“…There are many transporters known to mediate bacterial stress responses and virulence through the uptake of compatible solutes, low molecular weight osmolytes that can be accumulated to high concentrations without interfering with cellular processes [19]. In Listeria monocytogenes, the ABC transporters Gbu and OpuC, which are homologous to BilE, as well as the functionally related secondary transporter BetL, contribute to osmo-, bile-and chill-tolerance by importing glycine betaine and carnitine [16,[20][21][22]. Although it was initially hypothesized that BilE (then called OpuD) was an additional compatible solute transporter, it has since been demonstrated that it is unable to mediate transport of glycine betaine, carnitine, or choline (a metabolic precursor of glycine betaine), and plays no role in osmo-or chill-tolerance [18].…”

Chill Activation of Compatible Solute Transporters in Corynebacterium glutamicum at the Level of Transport Activity

“…The third response may also involve regulation at the level of protein activity, at least as far as uptake of compatible solutes is concerned. These phenomena have been studied in great detail in L. monocytogenes (4,5,48). To date, three uptake systems for compatible solutes are known in this bacterium, both a primary (Gbu) and a secondary (BetL) betaine carrier as well as the primary carnitine transporter OpuC (4).…”

“…Chill stress response in bacteria includes at least three different mechanisms, synthesis of so-called cold shock proteins (6,7), synthesis of branched-chain and unsaturated fatty acids (2,21,26,45,47), and uptake and synthesis of compatible solutes (4,23). The first two types of response necessarily involve regulation at the level of expression and have been studied in detail in Escherichia coli, Bacillus subtilis, and Listeria monocytogenes (1,2,11,15,18,19,20,32,33,41,45,46,50).…”