Identification and disruption of BetL, a secondary glycine betaine transport system linked to the salt tolerance of Listeria monocytogenes LO28

The trimethylammonium compound glycine betaine (N,N, N-trimethylglycine) can be accumulated to high intracellular concentrations, conferring enhanced osmo- and cryotolerance upon Listeria monocytogenes. We report the identification of betL, a gene encoding a glycine betaine uptake system in L. monocytogenes, isolated by functional complementation of the betaine uptake mutant Escherichia coli MKH13. The betL gene is preceded by a consensus sigmaB-dependent promoter and is predicted to encode a 55-kDa protein (507 amino acid residues) with 12 transmembrane regions. BetL exhibits significant sequence homologies to other glycine betaine transporters, including OpuD from Bacillus subtilis (57% identity) and BetP from Corynebacterium glutamicum (41% identity). These high-affinity secondary transporters form a subset of the trimethylammonium transporter family specific for glycine betaine, whose substrates possess a fully methylated quaternary ammonium group. The observed Km value of 7.9 microM for glycine betaine uptake after heterologous expression of betL in E. coli MKH13 is consistent with values obtained for L. monocytogenes in other studies. In addition, a betL knockout mutant which is significantly affected in its ability to accumulate glycine betaine in the presence or absence of NaCl has been constructed in L. monocytogenes. This mutant is also unable to withstand concentrations of salt as high as can the BetL+ parent, signifying the role of the transporter in Listeria osmotolerance.     

Heterologous expression of BetL, a betaine uptake system, enhances the stress tolerance of Lactobacillus salivarius UCC118

Given the increasing commercial and clinical relevance of probiotic cultures, improving the technological robustness of what are often process-sensitive cultures is an important biological goal. The nisin-controlled expression system was used to direct the heterologous expression of the listerial betaine uptake system BetL in the probiotic strain Lactobacillus salivarius UCC118. Following nisin induction, strains expressing betL exhibited a significant increase in resistance to several stresses, including elevated osmo-, cryo-, baro-, and chill tolerance, as well as increased resistance to spray- and freeze-drying. The ability to confer additional stress tolerance on a probiotic culture may be an important step in delivering viable cultures for maximal efficacy.     

Isolation and Functional Validation of Salinity and Osmotic Stress Inducible Promoter from the Maize Type-II H+-Pyrophosphatase Gene by Deletion Analysis in Transgenic Tobacco Plants

Salinity and drought severely affect both plant growth and productivity, making the isolation and characterization of salinity- or drought-inducible promoters suitable for genetic improvement of crop resistance highly desirable. In this study, a 1468-bp sequence upstream of the translation initiation codon ATG of the promoter for ZmGAPP (maize Type-II H⁺-pyrophosphatase gene) was cloned. Nine 5´ deletion fragments (D1–D9) of different lengths of the ZmGAPP promoter were fused with the GUS reporter and translocated into tobacco. The deletion analysis showed that fragments D1–D8 responded well to NaCl and PEG stresses, whereas fragment D9 and CaMV 35S did not. The D8 segment (219 bp; -219 to -1 bp) exhibited the highest promoter activity of all tissues, with the exception of petals among the D1–D9 transgenic tobacco, which corresponds to about 10% and 25% of CaMV 35S under normal and NaCl or PEG stress conditions, respectively. As such, the D8 segment may confer strong gene expression in a salinity and osmotic stress inducible manner. A 71-bp segment (-219 to -148 bp) was considered as the key region regulating ZmGAPP response to NaCl or PEG stress, as transient transformation assays demonstrated that the 71-bp sequence was sufficient for the salinity or osmotic stress response. These results enhance our understanding of the molecular mechanisms regulating ZmGAPP expression, and that the D8 promoter would be an ideal candidate for moderating expression of drought and salinity response genes in transgenic plants.     

Mapping salinity tolerance during Arabidopsis thaliana germination and seedling growth

To characterize and dissect genetic variation for salinity tolerance, we assessed variation in salinity tolerance during germination and seedling growth for a worldwide sample of Arabidopsis thaliana accessions. By combining QTL mapping, association mapping and expression data, we identified genomic regions involved in salinity response. Among the worldwide sample, we found germination ability within a moderately saline environment (150 mM NaCl) varied considerable, from >90% among the most tolerant lines to complete inability to germinate among the most susceptible. Our results also demonstrated wide variation in salinity tolerance within A. thaliana RIL populations and identified multiple genomic regions that contribute to this variation. These regions contain known candidate genes, but at least four of the regions contain loci not yet associated with salinity tolerance response phenotypes. Our observations suggest A. thaliana natural variation may be an underutilized resource for investigating salinity stress response.     

Salicylic acid negatively affects the response to salt stress in pea plants

We studied the effect of salicylic acid (SA) treatment on the response of pea plants to salinity. Sodium chloride (NaCl)-induced damage to leaves was increased by SA, which was correlated with a reduction in plant growth. The content of reduced ascorbate and glutathione in leaves of salt-treated plants increased in response to SA, although accumulation of the respective oxidised forms occurred. An increase in hydrogen peroxide also occurred in leaves of salt-exposed plants treated with SA. In the absence of NaCl, SA increased ascorbate peroxidase (APX; 100 μm) and glutathione-S transferase (GST; 50 μm) activities and increased catalase (CAT) activity in a concentration-dependent manner. Salinity decreased glutathione reductase (GR) activity, but increased GST and CAT activity. In salt-stressed plants, SA also produced changes in antioxidative enzymes: 100 μm SA decreased APX but increased GST. Finally, a concentration-dependent increase in superoxide dismutase (SOD) activity was induced by SA treatment in salt-stressed plants. Induction of PR-1b was observed in NaCl-stressed plants treated with SA. The treatment with SA, as well as the interaction between salinity and SA treatment, had a significant effect on PsMAPK3 expression. The expression of PsMAPK3 was not altered by 70 mm NaCl, but was statistically higher in the absence than in the presence of SA. Overall, the results show that SA treatment negatively affected the response of pea plants to NaCl, and this response correlated with an imbalance in antioxidant metabolism. The data also show that SA treatment could enhance the resistance of salt-stressed plants to possible opportunistic pathogen attack, as suggested by increased PR-1b gene expression.     

Glutamine synthetase and glutamate dehydrogenase contribute differentially to proline accumulation in leaves of wheat (Triticum aestivum) seedlings exposed to different salinity

To investigate the roles of ammonium-assimilating enzymes in proline synthesis under salinity stress, the activities of glutamine synthetase (GS; EC 6.3.1.2) and NADH-dependent glutamate dehydrogenase (NADH-GDH; EC 1.4.1.2) were determined in leaves of wheat (Triticum aestivum) seedlings exposed to salt stress at 150 and 300 mM NaCl for 5d. At the lower salinity, only GS activity increased markedly. At 300 mM NaCl, however, NADH-GDH activity increased while GS activity decreased. A significant accumulation of proline was found only at high-salinity exposure while glutamate, a proline precursor, increased dramatically under both low and high salinity. These data suggests that GS-catalysis might be the main glutamate synthesis pathway under low salinity. At 300 mM NaCl, glutamate seems to be preferentially produced through the process catalyzed by NADH-GDH. The increase of ammonium in salinity-stressed wheat seedlings might have resulted from increased photorespiration, which is responsible for the higher NADH-GDH activity. The activity of Delta(1)-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) was significantly enhanced at 300 mM NaCl but remained unchanged at 150 mM. Delta(1)-Pyrroline-5-carboxylate synthetase (P5CS) activity did not show a specific response, indicating that P5CR might be the limiting step in proline synthesis from glutamate at high salinity.     

Growth and nitrogen removal characteristics of Halomonas sp. B01 under high salinity

At present, the nitrogen (N) removal efficiency of the microbial treatment in the high-salinity nitrogenous wastewaters is relatively low. Study on the N removal behavior and properties of moderately halophilic bacteria Halomonas under high salinity is of great significance for the microbial treatment of high-salinity nitrogenous wastewater. The response mechanism of Halomonas sp. B01 to high osmotic pressure stress was investigated by measuring the compatible solute ectoine concentration and superoxide dismutase (SOD) activity. The salt tolerance during growth and N removal of the strain was evaluated by measuring the activities of growth-related and N removal–related enzymes and the mRNA expression abundance of ammonia monooxygenase-encoding gene (amoA). The process of simultaneous heterotrophic nitrification and aerobic denitrification (SND) under high salinity was described by measuring the concentration of inorganic N. Halomonas sp. B01 synthesized ectoine under NaCl stress, and the intracellular ectoine concentration increased with increased NaCl concentration in the growth medium. When the NaCl concentration of the medium reached 120 g L−1, the malondialdehyde concentration and SOD activity were significantly increased to 576.1 μg mg−1 and 1.7 U mg−1, respectively. The growth-related and N removal–related enzymes of the strain were active or most active in medium with 30–60 g L−1 NaCl. The amoA of the strain cultured in medium with 60 g L−1 NaCl had the highest mRNA expression abundance. In the N removal medium containing 60 g L−1 NaCl and 2121 mg L−1 NH4+-N, SND by Halomonas sp. B01 was performed over 96 h and the N removal rate reached 98.8%. In addition to the protective mechanism of synthetic compatible solutes, Halomonas sp. B01 had the repair mechanism of SOD for lipid peroxidation. The growth-related and N removal–related enzymes of the strain were most active at a certain salt concentration; amoA also had the highest mRNA expression abundance under high salinity. Halomonas sp. B01 could efficiently perform N removal by SND under high salinity.     

Sugars and acid invertase mediate the physiological response of Schenkia spicata root cultures to salt stress

A heterotrophic model system was established in our studies in order to differentiate the effect of high salt concentrations in external medium on growth and sugar metabolism in roots from the effect of reduced sugar availability resulting from decreased photosynthesis under salinity. Soluble sugar content and the activity of acid invertase in root cultures of salt-tolerant (ST) and salt-sensitive (SS) Schenkia spicata (L.) Mansion genotypes were investigated during exposure to different NaCl concentrations (0-200mM). Their response to severe salinity was characterized by a metabolic adjustment that led to the accumulation of sucrose (Suc) in root tissues. There was clear evidence that cell wall invertase (CW-Inv) is the major contributor to the Suc/hexose ratio in roots during exposure to elevated salinity. The results of CW-Inv activity and immunodetection assays in our study suggest that the regulation of CW-Inv expression is most likely achieved in a salt stress dependent manner. Also, NaCl modulated soluble acid invertase (SA-Inv) expression differentially in SS and ST genotypes of S. spicata. Regardless of the salt treatment, genotype, or the amount of enzyme, SA-Inv activity was generally low, indicating regulation at the posttranslational level. The results suggest no direct role of SA-Inv in the regulation of the root tissue carbohydrate pool and therefore in the control of the availability of glucose and fructose for the primary metabolism and/or osmotic adjustment in the present heterotrophic model system.     

Specific regulation of SOD isoforms by NaCl and osmotic stress in leaves of the C3 halophyte Suaeda salsa L

The halophyte Suaeda salsa L., exposed to different NaCl concentrations (100 and 400 mmol/L) and polyethylene glycol (isoosomotic to 100 mmol/L NaCl) containing nutrient solutions under normal or K+-deficient conditions for 7 days, was used to study effects of NaCl salinity and osmotic stress on chlorophyll content, chlorophyll fluorescence characteristics, malonedialdehyde (MDA) content, and superoxide dismutase (SOD) isoform activities. Photosynthetic capacity was not decreased by NaCl treatment, indicating that S. salsa possesses an effective antioxidative response system for avoiding oxidative damage. Seven SOD activity bands were detected in S. salsa leaf extracts, including an Mn-SOD and several isoforms of Fe-SOD and CuZn-SOD. It turned out that NaCl salinity and osmotic stress lead to a differential regulation of distinct SOD isoenzymes. This differential regulation is suggested to play a major role in stress tolerance of S. salsa.     

Differential sensitivity of stomatal and non-stomatal components to NaCl or Na2SO4 salinity in horsegram, Macrotyloma uniflorum (Lam.)

14CO2 assimilation rate (P), leaf diffusive conductance (gs), photosynthetic electron flow, and activities of enzymes of Calvin cycle were studied in a horsegram [Macrotyloma uniflorum (Lam.)] in response to salinity induced by NaCl or Na2SO4. A significant reduction in P and gs by both salt treatments was registered. Na2SO4 caused a greater reduction in gs than the NaCl salinity. Studies with isolated chloroplasts confirmed a greater sensitivity to NaCl than to Na2SO4. Salinity inhibited the photosynthetic electron transport. The activity of ribulose-1,5-bisphosphate carboxylase (E.C.4.1.1.39) was under salinity inhibited more than the activities of other three enzymes of the Calvin cycle, ribulose-5-phosphate kinase (E.C.2.7.1.19), ribose-5-phosphate isomerase (E.C.5.3.16), and NADP-glyceraldehyde-3-phosphate dehydrogenase (E.C.1.2.13). These inhibitions lead to a reduced capacity for ribulose-1,5-bisphosphate regeneration. Isolated chloroplasts extracted from salt stressed plants and supplemented with the substrates of Calvin cycle could elevate P, but the P was always lower than in the controls. Decreased P in horsegram exposed to high salinity can be attributed to both stomatal and non-stomatal components, however, the sensitivity to the salt source, NaCl or Na2SO4, was different.     

盐度和CO2倍增环境下碱蓬幼苗呼吸酶活性的变化

研究了生长在正常大气CO₂和CO₂倍增环境中的盐生植物碱蓬(Suaedasalsa)幼苗呼吸酶活性对KCl和NaCl的反应。结果表明,在CO₂倍增(700μl·L^-1)和正常大气CO₂(350μl·L^-1)下,300mmol·L^-1KCl和NaCl均能抑制琥珀酸脱氢酶(SDH)和苹果酸脱氢酶(MDH)活性,而异柠檬酸脱氢酶(IDH)活性为NaCl抑制、KCl促进; NaCl和KCl明显抑制细胞色素氧化酶(CO)和光呼吸中乙醇酸氧化酶(GO)、羟基丙酮酸还原酶(HPR)活性; 并指出在KCl胁迫下,CO₂使三羧酸循环(TCAC)的运行变慢,NaCl胁迫下使其加快,TCAC运行限速步骤与MDH无关,CO为盐对呼吸代谢影响的重要位点。另外,K⁺、Na⁺对蛋白表达的影响有差异,CO₂可使盐胁迫下的碱蓬幼苗蛋白表达降低。     

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.     

Salinity effects on growth,photosynthetic parameters,and nitrogenase activity in estuarine planktonic cyanobacteria

Salinity has been suggested as being a controlling factor for blooms of N2-fixing cyanobacteria in estuaries. We tested the effect of salinity on the growth, N2 fixation, and photosynthetic activities of estuarine and freshwater isolates of heterocystous bloom-forming cyanobacteria. Anabaena aphanizomenoides and Anabaenopsis sp. were isolated from the Neuse River Estuary, North Carolina, and Cylindrospermopsis raciborskii from Lakes Dora and Griffin, central Florida. Salinity tolerance of these cyanobacteria was compared with that of two Nodularia strains from the Baltic Sea. We measured growth rates, N2 fixation (nitrogenase activity), and CO2 fixation at salinities between 0 and 20 g L(-1) NaCl. We also examined photosynthesis-irradiance relation-ships in response to salinity. Anabaenopsis maintained similar growth rates in the full range of salinities from 2 to 20 g L(-1) NaCl. Anabaena grew at up to 15 g L-', but the maximum salinity 20 g L(-1) NaCl was inhibitory. The upper limit for salinity tolerance of Cylindrospermopsis was 4 g L(-1) NaCl. Nodularia spp. maintained similar growth rates in the full range of salinities from 0 to 20 g L(-1) . Between 0 and 10 g L(-1), the growth rate of Nodularia spumigena was slower than that of the Neuse Estuary strains. In most strains, the sensitivity of nitrogenase activity and CO2 fixation to salinity appeared similar. Anabaenopsis, Anabaena, and the two Nodularia strains rapidly responded to NaCl by increasing their maximum photosynthetic rates (Pmn). Overall, both Neuse River Estuary and Baltic Sea strains showed an ability to acclimate to salt stress over short-(24 h) and long-term (several days to weeks) exposures. The study suggested that direct effect of salinity (as NaCl in these experiments) on cyanobacterial physiology does not alone explain the low frequency and magnitude of blooms of N2-fixing cyanobacteria in estuaries.