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. 13 C 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. (28), and contamination of food is therefore common. Listeriosis occurs mainly in immunocompromised individuals, such as pregnant women, AIDS patients, and transplant recipients, and is approximately 25% fatal (19). L. monocytogenes has been shown to be tolerant to levels of osmotic stress as high as that afforded by 10% NaCl and grows at temperatures as low as Ϫ0.1°C (5). Thus, it is resistant to two major forms of food preservation. Listeria monocytogenes is a gram-positive, food-borne pathogen (15). It is widely distributed in the environmentA common response to hyperosmotic stress, after an initial loss of cellular water, is the intracellular accumulation of compatible solutes in order to restore osmotic pressure and turgor (29). The presence of the compatible solute glycine betaine or carnitine in the medium therefore confers a degree of osmotolerance (3,13,14,22). We have also found that L. monocytogenes accumulates glycine betaine and carnitine in response to chill stress and that the presence of these compatible solutes in the medium confers cryotolerance (11,13,22). These osmolytes are not synthesized by L. monocytogenes and must be transported from the environment. The rate and the extent of their transport can be controlled by regulation of the synthesis of the transport systems and by their biochemical activation by osmotic gradients, temperature, or other factors.In earlier work, we demon...

The Type II Protein Secretion System of Legionella pneumophila Promotes Growth at Low Temperatures

“…Indeed, in the only previous study on L. pneumophila physiology at low temperature, Mauchline et al found increases in the amount of unsaturated fatty acids in bacteria grown at 24°C (52). Newly recognized forms of cold adaptation are increases in trehalose and palmitoylated lipid A within E. coli (41,87) and solute (e.g., glycine, betaine) uptake in Listeria monocytogenes (3,49). The ability of wild-type L. pneumophila to stimulate, in trans, low-temperature growth of lsp mutants suggests that low-temperature-in- VOL.…”

Listeria monocytogenesσBRegulates Stress Response and Virulence Functions

“…Interestingly, both the L. monocytogenes and the B. subtilis B regulons include multiple genes that have been shown experimentally to contribute to protection against osmotic stress, including genes encoding several solute transporters. The ABC transporter OpuC contributes to osmotic stress resistance by facilitating uptake of the osmoprotectants choline and glycine betaine in both L. monocytogenes and B. subtilis (1,35,36,60), and expression of the encoding operon is B dependent in L. monocytogenes. Similarly, lmo1421 and the B. subtilis homologue opuB, each of which is a member of the B regulon in its species, both encode a putative choline transporter which may also contribute to osmotic stress resistance (22,36).…”

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

“…Adaptation and response to chill stress have been studied in detail in Bacillus subtilis (9,15,45,46) and in Listeria monocytogenes (3,22,39). In B. subtilis, global responses at the level of the transcriptome and its contribution to low-temperature adaptation (7, 9) have been described, as well as the response at the level of fatty acid synthesis (21,46).…”

“…Physiological responses and adaptation phenomena to chill stress have been reported for a number of prokaryotes, e.g., Bacillus subtilis, Listeria monocytogenes, and cyanobacteria (3,9,12,18,19,22,25,39,46); related systems have also been studied in plants and in humans (17,44).…”