Gbu Glycine Betaine Porter and Carnitine Uptake in Osmotically Stressed Listeria monocytogenes Cells

The food-borne pathogen Listeria monocytogenes grows actively under high-salt conditions by accumulating compatible solutes such as glycine betaine and carnitine from the medium. We report here that the dominant transport system for glycine betaine uptake, the Gbu porter, may act as a secondary uptake system for carnitine, with a K_m of 4 mM for carnitine uptake and measurable uptake at carnitine concentrations as low as 10 μM. This porter has a K_m for glycine betaine uptake of about 6 μM. The dedicated carnitine porter, OpuC, has a K_m for carnitine uptake of 1 to 3 μM and a V_max of approximately 15 nmol/min/mg of protein. Mutants lacking either opuC or gbu were used to study the effects of four carnitine analogs on growth and uptake of osmolytes. In strain DP-L1044, which had OpuC and the two glycine betaine porters Gbu and BetL, triethylglycine was most effective in inhibiting growth in the presence of glycine betaine, but trigonelline was best at inhibiting growth in the presence of carnitine. Carnitine uptake through OpuC was inhibited by γ-butyrobetaine. Dimethylglycine inhibited both glycine betaine and carnitine uptake through the Gbu porter. Carnitine uptake through the Gbu porter was inhibited by triethylglycine. Glycine betaine uptake through the BetL porter was strongly inhibited by trigonelline and triethylglycine. These results suggest that it is possible to reduce the growth of L. monocytogenes under osmotically stressful conditions by inhibiting glycine betaine and carnitine uptake but that to do so, multiple uptake systems must be affected.     

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

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.     

Characterization of Glycine Betaine Porter I from Listeria monocytogenes and Its Roles in Salt and Chill Tolerance

Listeria monocytogenes is a pathogenic bacterium that can grow at low temperatures and elevated osmolarity. The organism survives these stresses by the intracellular accumulation of osmolytes: low-molecular-weight organic compounds which exert a counterbalancing force. The primary osmolyte in L. monocytogenes is glycine betaine, which is accumulated from the environment via two transport systems: glycine betaine porter I, an Na⁺-glycine betaine symporter; and glycine betaine porter II, an ATP-dependent transporter. The biochemical characteristics of glycine betaine porter I were investigated in a mutant strain (LTG59) lacking the ATP-dependent transporter. At 4% NaCl, glycine betaine uptake in LTG59 was about fivefold lower than in strain DP-L1044, which has both transporters, indicating that the ATP-dependent transporter is the primary means by which glycine betaine enters the cell. In the absence of osmotic stress, cold-activated uptake by both transporters was most rapid between 7 and 12°C, but a larger fraction of the total uptake was via the ATP-dependent transporter than was observed under salt-stressed conditions. Twelve glycine betaine analogs were tested for their ability to inhibit glycine betaine uptake and growth of stressed cultures. Carnitine, dimethylglycine, and γ-butyrobetaine appear to inhibit the ATP-dependent transporter, while trigonelline and triethylglycine primarily inhibit glycine betaine porter I. Triethylglycine was also able to retard the growth of osmotically stressed L. monocytogenes grown in the presence of glycine betaine.     

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

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.     

Analysis of the Role of OpuC, an Osmolyte Transport System, in Salt Tolerance and Virulence Potential of Listeria monocytogenes

The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the external environment prior to ingestion and subsequently within the animal host. Growth at high salt concentrations and low temperatures is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We utilized a novel system for generating chromosomal mutations (based on a lactococcal pWVO1-derived Ori⁺ RepA⁻ vector, pORI19) to identify a listerial OpuC homologue. Mutating the operon in two strains of L. monocytogenes revealed significant strain variation in the observed activity of OpuC. Radiolabeled osmolyte uptake studies, together with growth experiments in defined media, linked OpuC to carnitine and glycine betaine uptake in Listeria. We also investigated the role of OpuC in contributing to the growth and survival of Listeria in an animal (murine) model of infection. Altering OpuC resulted in a significant reduction in the ability of Listeria to colonize the upper small intestine and cause subsequent systemic infection following peroral inoculation.     

Identification of an ATP-driven, osmoregulated glycine betaine transport system in Listeria monocytogenes.

The ability of the gram-positive, food-borne pathogen Listeria monocytogenes to tolerate environments of elevated osmolarity and reduced temperature is due in part to the transport and accumulation of the osmolyte glycine betaine. Previously we showed that glycine betaine transport was the result of Na(+)-glycine betaine symport. In this report, we identify a second glycine betaine transporter from L. monocytogenes which is osmotically activated but does not require a high concentration of Na(+) for activity. By using a pool of Tn917-LTV3 mutants, a salt- and chill-sensitive mutant which was also found to be impaired in its ability to transport glycine betaine was isolated. DNA sequence analysis of the region flanking the site of transposon insertion revealed three open reading frames homologous to opuA from Bacillus subtilis and proU from Escherichia coli, both of which encode glycine betaine transport systems that belong to the superfamily of ATP-dependent transporters. The three open reading frames are closely spaced, suggesting that they are arranged in an operon. Moreover, a region upstream from the first reading frame was found to be homologous to the promoter regions of both opuA and proU. One unusual feature not shared with these other two systems is that the start codons for two of the open reading frames in L. monocytogenes appear to be TTG. That glycine betaine uptake is nearly eliminated in the mutant strain when it is assayed in the absence of Na(+) is an indication that only the ATP-dependent transporter and the Na(+)-glycine betaine symporter occur in L. monocytogenes.     

Glycine betaine confers enhanced osmotolerance and cryotolerance on Listeria monocytogenes.

Listeria monocytogenes is a gram-positive food-borne pathogen that is notably resistant to osmotic stress and can grow at refrigerator temperatures. These two characteristics make it an insidious threat to public health. Like several other organisms, L. monocytogenes accumulates glycine betaine, a ubiquitous and effective osmolyte, intracellularly when grown under osmotic stress. However, it also accumulates glycine betaine when grown under chill stress at refrigerator temperatures. Exogenously added glycine betaine enhances the growth rate of stressed but not unstressed cells, i.e., it confers both osmotolerance and cryotolerance. Both salt-stimulated and cold-stimulated accumulation of glycine betaine occur by transport from the medium rather than by biosynthesis. Direct measurement of glycine betaine uptake shows that cells transport betaine 200-fold faster at high salt concentration (4% NaCl) than without added salt and 15-fold faster at 7 than at 30 degrees C. The kinetics of glycine betaine transport suggest that the two transport systems are indistinguishable in terms of affinity for betaine and may be the same. Hyperosmotic shock and cold shock experiments suggest the transport system(s) to be constitutive; activation was not blocked by chloramphenicol. A cold-activated transport system is a novel observation and has intriguing implications concerning the physical state of the cell membrane at low temperature.     

Identification and characterization of an ATP binding cassette L-carnitine transporter in Listeria monocytogenes

We identified an operon in Listeria monocytogenes EGD with high levels of sequence similarity to the operons encoding the OpuC and OpuB compatible solute transporters from Bacillus subtilis, which are members of the ATP binding cassette (ABC) substrate binding protein-dependent transporter superfamily. The operon, designated opuC, consists of four genes which are predicted to encode an ATP binding protein (OpuCA), an extracellular substrate binding protein (OpuCC), and two membrane-associated proteins presumed to form the permease (OpuCB and OpuCD). The operon is preceded by a potential SigB-dependent promoter. An opuC-defective mutant was generated by the insertional inactivation of the opuCA gene. The mutant was impaired for growth at high osmolarity in brain heart infusion broth and failed to grow in a defined medium. Supplementation of the defined medium with peptone restored the growth of the mutant in this medium. The mutant was found to accumulate the compatible solutes glycine betaine and choline to same extent as the parent strain but was defective in the uptake of L-carnitine. We conclude that the opuC operon in L. monocytogenes encodes an ABC compatible solute transporter which is capable of transporting L-carnitine and which plays an important role in osmoregulation in this pathogen.     

单核细胞增生李斯特氏菌PCR-DHPLC检测新技术的建立

应用PCR结合变性高效液相色谱(DHPLC)技术建立食品中单核细胞增生李斯特氏菌fimY的快速检测方法。根据单核细胞增生李斯特氏菌prfA和hlyA基因序列的特点设计特异性引物,PCR扩增的产物经DHPLC技术进行快速检测。以单核细胞增生李斯特氏菌等61株参考菌株做特异性试验;单核细胞增生李斯特氏菌菌株稀释成不同梯度,进行灵敏度试验。试验结果表明该方法具有很好的特异性,灵敏度较高,检测低限可达到为181CFU/ml。可以快速、准确检测单核细胞增生李斯特氏菌,是食品中致病菌快速检测的新技术。     

高效液相色谱法测定葡萄叶中甘氨酸甜菜碱、脯氨酸及葫芦巴碱

甘氨酸甜菜碱、脯氨酸及葫芦巴碱的含量与葡萄的抗逆性能密切相关,应用离子交换树脂提取葡萄叶中的这三种物质,采用Suger-pak1(waters)色谱柱,柱温:85℃;样品测定时间:70min;进样体积:100μL;流速:0.6mL/min;检测波长:195nm;流动相:50mg/LCa-EDTA的HPLC同时分析测定这三种物质,甘氨酸甜菜碱、脯氨酸及葫芦巴碱线性相关系数分别为0.9999,0.9994及0.9994;保留时间分别为20.28,25.66及30.65min;检出限分别为1.35,2.56及1.02ng/mL,回收率为93%~96%。此方法分离效果好,稳定,精确。     

Identification and characterization of an essential gene in Listeria monocytogenes using an inducible gene expression system

The Listeria monocytogenes gene lmo1594 is a homolog of the Bacillus subtilis cell division gene ezrA. EzrA is a negative regulator of FtsZ ring formation, which is required for efficient cell division as it regulates the frequency and position of Z-rings in the cell and prevents aberrant polar cell division. Previously identified as a putative high pressure (HP) resistance mechanism; conferring enhanced barotolerance when heterologously expressed against an Escherichia coli background; the aim of the current study was to investigate whether lmo1594 plays a role in listerial barotolerance. When the creation of a deletion mutant proved unsuccessful, the role of lmo1594 was addressed by creating a conditional knockout mutant which demonstrated that the gene is in fact essential for cell survival and growth in L. monocytogenes. In order to investigate the effect of lmo1594 on barotolerance, the gene was over-expressed. The over-expression of lmo1594 increased survival levels in L. monocytogenes treated at 300 MPa, but survival levels similar to those of the wild-type strain were observed when treated at a higher pressure (≥400 MPa). In conclusion, this study reveals for the first time that lmo1594 is absolutely essential for listerial cell growth and survival, and also plays an important role in listerial barotolerance.     

Betaine and L-carnitine transport by Listeria monocytogenes Scott A in response to osmotic signals.

The naturally occurring compatible solutes betaine and L-carnitine allow the food-borne pathogen Listeria monocytogenes to adjust to environments of high osmotic strength. Previously, it was demonstrated that L. monocytogenes possesses an ATP-dependent L-carnitine transporter (A. Verheul, F. M. Rombouts, R. R. Beumer, and T. Abee, J. Bacteriol. 177:3205-3212, 1995). The present study reveals that betaine and L-carnitine are taken up by separate highly specific transport systems and support a secondary transport mechanism for betaine uptake in L. monocytogenes. The initial uptake rates of betaine and L-carnitine are not influenced by an osmotic upshock, but the duration of transport of both osmolytes is directly related to the osmotic strength of the medium. Regulation of uptake of both betaine and L-carnitine is subject to inhibition by preaccumulated solute. Internal betaine inhibits not only transport of external betaine but also that of L-carnitine and, similarly, internal L-carnitine inhibits transport of both betaine and L-carnitine. The inhibition is alleviated upon osmotic upshock, which suggests that alterations in membrane structure are transmitted to the allosteric binding sites for betaine and L-carnitine of both transporters at the inner surface of the membrane. Upon osmotic downshock, betaine and L-carnitine are rapidly released by L. monocytogenes as a consequence of activation of a channel-like activity. The osmolyte-sensing mechanism described is new and is consistent with various unexplained observations of osmoregulation in other bacteria.     

Incidence of Listeria monocytogenes in an acidified fish product, ceviche

Thirty-two samples of ceriche , a South American acidified fish product, were tested for the presence of listeria. Listeria innocua was isolated from 24 samples (75%) and L. monocytogenes was isolated from three samples (9%) of those tested.     

Role for Glycine Betaine Transport in Vibrio cholerae Osmoadaptation and Biofilm Formation within Microbial Communities

Vibrio cholerae is a halophilic facultative human pathogen found in marine and estuarine environments. Accumulation of compatible solutes is important for growth of V. cholerae at NaCl concentrations greater than 250 mM. We have identified and characterized two compatible solute transporters, OpuD and PutP, that are involved in uptake of glycine betaine and proline by V. cholerae. V. cholerae does not, however, possess the bet genes, suggesting that it is unable to synthesize glycine betaine. In contrast, many Vibrio species are able to synthesize glycine betaine from choline. It has been shown that many bacteria not only synthesize but also secrete glycine betaine. We hypothesized that sharing of compatible solutes might be a mechanism for cooperativity in microbial communities. In fact, we have demonstrated that, in high-osmolarity medium, V. cholerae growth and biofilm development are enhanced by supplementation with either glycine betaine or spent media from other bacterial species. Thus, we propose that compatible solutes provided by other microorganisms may contribute to survival of V. cholerae in the marine environment through facilitation of osmoadaptation and biofilm development.     

A mariner-Based Transposition System for Listeria monocytogenes

In this study, we developed a new mariner-based transposition system for Listeria monocytogenes. The mariner-based system has a high rate of transposition and a low rate of plasmid retention, and transposition is very random, making it an ideal tool for high-throughput transposon mutagenesis in L. monocytogenes.     

Identification of Listeria monocytogenes genes involved in salt and alkaline-pH tolerance

The capacity of Listeria monocytogenes to tolerate salt and alkaline stresses is of particular importance, as this pathogen is often exposed to such environments during food processing and food preservation. We screened a library of Tn917-lacZ insertional mutants in order to identify genes involved in salt and/or alkaline tolerance. We isolated six mutants sensitive to salt stress and 12 mutants sensitive to salt and alkaline stresses. The position of the insertion of the transposon was located in 15 of these mutants. In six mutants the transposon was inserted in intergenic regions, and in nine mutants it was inserted in genes. Most of the genes have unknown functions, but sequence comparisons indicated that they encode putative transporters.     

Identification of a sigma B-dependent small noncoding RNA in Listeria monocytogenes

In Listeria monocytogenes, the alternative sigma factor σ^B plays important roles in stress tolerance and virulence. Here, we present the identification of SbrA, a novel small noncoding RNA that is produced in a σ^B-dependent manner. This finding adds the σ^B regulon to the growing list of stress-induced regulatory circuits that include small noncoding RNAs.