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Abstract

Due to their particular properties, detergents are widely used in household cleaning products, cosmetics, pharmaceuticals, and in agriculture as adjuvants tailoring the features of pesticides or other crop protection agents. The continuously growing use of these various products means that water soluble detergents have become one of the most problematic groups of pollutants for the aquatic and terrestrial environments. Thus it is important to identify bacteria having the ability to survive in the presence of large quantities of detergent and efficiently decompose it to non-surface active compounds. In this study, we used peaty soil sampled from a surface flow constructed wetland in a wastewater treatment plant to isolate bacteria that degrade sodium dodecyl sulfate (SDS). We identified and initially characterized 36 Pseudomonas spp. strains that varied significantly in their ability to use SDS as their sole carbon source. Five isolates having the closest taxonomic relationship to the Pseudomonas jessenii subgroup appeared to be the most efficient SDS degraders, decomposing from 80 to 100% of the SDS present in an initial concentration 1 g/L in less than 24 h. These isolates exhibited significant differences in degree of SDS degradation, their resistance to high detergent concentration (ranging from 2.5 g/L up to 10 g/L or higher), and in chemotaxis toward SDS on a plate test. Mass spectrometry revealed several SDS degradation products, 1-dodecanol being dominant; however, traces of dodecanal, 2-dodecanol, and 3-dodecanol were also observed, but no dodecanoic acid. Native polyacrylamide gel electrophoresis zymography revealed that all of the selected isolates possessed alkylsulfatase-like activity. Three isolates, AP3_10, AP3_20, and AP3_22, showed a single band on native PAGE zymography, that could be the result of alkylsulfatase activity, whereas for isolates AP3_16 and AP3_19 two bands were observed. Moreover, the AP3_22 strain exhibited a band in presence of both glucose and SDS, whereas in other isolates, the band was visible solely in presence of detergent in the culture medium. This suggests that these microorganisms isolated from peaty soil exhibit exceptional capabilities to survive in, and break down SDS, and they should be considered as a valuable source of biotechnological tools for future bioremediation and industrial applications.

Abstract

Due to their particular properties, detergents are widely used in household cleaning products, cosmetics, pharmaceuticals, and in agriculture as adjuvants tailoring the features of pesticides or other crop protection agents. The continuously growing use of these various products means that water soluble detergents have become one of the most problematic groups of pollutants for the aquatic and terrestrial environments. Thus it is important to identify bacteria having the ability to survive in the presence of large quantities of detergent and efficiently decompose it to non-surface active compounds. In this study, we used peaty soil sampled from a surface flow constructed wetland in a wastewater treatment plant to isolate bacteria that degrade sodium dodecyl sulfate (SDS). We identified and initially characterized 36 Pseudomonas spp. strains that varied significantly in their ability to use SDS as their sole carbon source. Five isolates having the closest taxonomic relationship to the Pseudomonas jessenii subgroup appeared to be the most efficient SDS degraders, decomposing from 80 to 100% of the SDS present in an initial concentration 1 g/L in less than 24 h. These isolates exhibited significant differences in degree of SDS degradation, their resistance to high detergent concentration (ranging from 2.5 g/L up to 10 g/L or higher), and in chemotaxis toward SDS on a plate test. Mass spectrometry revealed several SDS degradation products, 1-dodecanol being dominant; however, traces of dodecanal, 2-dodecanol, and 3-dodecanol were also observed, but no dodecanoic acid. Native polyacrylamide gel electrophoresis zymography revealed that all of the selected isolates possessed alkylsulfatase-like activity. Three isolates, AP3_10, AP3_20, and AP3_22, showed a single band on native PAGE zymography, that could be the result of alkylsulfatase activity, whereas for isolates AP3_16 and AP3_19 two bands were observed. Moreover, the AP3_22 strain exhibited a band in presence of both glucose and SDS, whereas in other isolates, the band was visible solely in presence of detergent in the culture medium. This suggests that these microorganisms isolated from peaty soil exhibit exceptional capabilities to survive in, and break down SDS, and they should be considered as a valuable source of biotechnological tools for future bioremediation and industrial applications.

Abstract

The aim of this work was to study the chemical composition of Allium obliquum L., A. senescens L. subsp. montanum (Fries) Holub, and A. schoenoprasum L. subsp. schoenoprasum. Sulphur-containing compounds analysis was performed by an LC-MS method, the identification and quantification of polyphenolic compounds through a HPLC-UV-MS method, and the presence of five sterols was simultaneously assessed by HPLC-MS-MS. Alliin was identified only in A. obliquum and A. senescens subsp. montanum extracts, whilst allicin was present in all extracts, with higher amounts in A. schoenoprasum and A. obliquum. The pattern of phenol carboxylic acids shows the presence of p-coumaric and ferulic acids in all species. Isoquercitrin was identified in A. obliquum and A. schoenoprasum, and rutin in A. senescens subsp. montanum and A. schoenoprasum. Luteolin and apigenin were identified only in A. obliquum. All three species contain glycosides of kaempferol and quercetol. β-Sitosterol and campesterol were identified in all species. The results obtained showed significant differences in the composition of the three Allium species.

2.1. The Analysis of Polyphenols

Luteolin and apigenin were determined only in A. obliquum, before (172.35 μg/100 g, and 149.03 μg/100 g, respectively), and after acid hydrolysis (280.74 μg/100 g, and 277.06 μg/100 g, respectively). Kaempferol and quercetol were present in both non-hydrolysed and hydrolysed sample of A. schoenoprasum, and only in hydrolysed extracts of A. obliquum and A. senescens subsp. montanum. The richest species in quercetol derivatives was A. senescens (993.90 μg/100 g), and in kaempferol derivatives was A. schoenoprasum (1563.46 μg/100 g).

Abstract

Specialized pro‐resolving mediators (SPMs), such as resolvin D5 (7S,17S‐dihydroxydocosahexaenoic acid, 7S,17S‐DiHDHA; RvD5) and protectin DX (10S,17S‐DiHDHA; PDX), are critical in resolving inflammation in humans. In this study, a unique double‐oxygenating 15S‐lipoxygenase (15S‐LOX) from the alga Chlamydomonas incerta was identified and characterized for its ability to simultaneously produce RvD5 and PDX from docosahexaenoic acid (DHA). Recombinant Escherichia coli expressing the C. incerta 15S‐LOX demonstrated enhanced RvD5 and PDX production under the following optimized reaction conditions: pH 8.0, 25°C, 0.5 g dry cells/L, 7.0 mM DHA, 2.0% (w/v) PVP, 2.0% (v/v) DMSO, and 200 mM cysteine used as a reductant. This one‐step biocatalytic process produced 2.91 mM (1.05 g/L) RvD5 and 2.18 mM (0.78 g/L) PDX in 90 min, with a total of 5.09 mM (1.83 g/L) and a total conversion yield of 79.6% (w/w). Compared to previously reported two‐step biocatalytic processes, this one‐step process significantly enhanced the production of particular PDX with improved productivity and simplicity. Structural analysis identified residues Phe667, Ile705, and Leu713 as regioselectivity modulators for the second oxygenation step. This study demonstrates the efficiency and industrial potential of the double‐oxygenating LOX as a biocatalyst for simultaneously producing RvD5 and PDX.

2.2. EBC Was Attractive to Non-Viruliferous N. Virescens, while 2-Heptanol Was Repellent to Viruliferous N. Virescens

Behavioral assays revealed that 2-heptanol was not attractive or repellent to non-viruliferous N. virescens at any of the three dosages tested (C, 0.01 μg μL−1, χ2 = 4.832, P = 0.185, n = 38; 0.1 μg μL−1, χ2 = 0.270, P = 0.966, n = 40; 1 μg μL−1, χ2 = 1.215, P = 0.749, n = 41). However, 2-heptanol was significantly repellent to viruliferous N. virescens at the three dosages tested (D, 0.01 μg μL−1, χ2 = 58.304, p < 0.001, n = 36; 0.1 μg μL−1, χ2 = 44.367, p < 0.001, n = 36; 1 μg μL−1, χ2 = 82.395, p < 0.001, n = 37). The walking paths of N. virescens are shown in (2-heptanol, 0.1 μg μL−1).

2.2. EBC Was Attractive to Non-Viruliferous N. Virescens, while 2-Heptanol Was Repellent to Viruliferous N. Virescens

Behavioral assays revealed that 2-heptanol was not attractive or repellent to non-viruliferous N. virescens at any of the three dosages tested (C, 0.01 μg μL−1, χ2 = 4.832, P = 0.185, n = 38; 0.1 μg μL−1, χ2 = 0.270, P = 0.966, n = 40; 1 μg μL−1, χ2 = 1.215, P = 0.749, n = 41). However, 2-heptanol was significantly repellent to viruliferous N. virescens at the three dosages tested (D, 0.01 μg μL−1, χ2 = 58.304, p < 0.001, n = 36; 0.1 μg μL−1, χ2 = 44.367, p < 0.001, n = 36; 1 μg μL−1, χ2 = 82.395, p < 0.001, n = 37). The walking paths of N. virescens are shown in (2-heptanol, 0.1 μg μL−1).

Abstract

Patients with rheumatoid arthritis have subnormal vitamin B6 status, both quantitatively and functionally. Abnormal vitamin B6 status in rheumatoid arthritis has been associated with spontaneous tumor necrosis factor (TNF)-α production and markers of inflammation, including C-reactive protein and erythrocyte sedimentation rate. Impaired vitamin B6 status could be a result of inflammation, and these patients may have higher demand for vitamin B6. The aim of this study was to determine if daily supplementation with 50 mg of pyridoxine for 30 days can correct the static and/or the functional abnormalities of vitamin B6 status seen in patients with rheumatoid arthritis, and further investigate if pyridoxine supplementation has any effects on the pro-inflammatory cytokine TNF-α or IL-6 production of arthritis. This was a double-blinded, placebo-controlled study involving patients with rheumatoid arthritis with plasma pyridoxal 5'-phosphate below the 25th percentile of the Framingham Heart Cohort Study. Vitamin B6 status was assessed via plasma and erythrocyte pyridoxal 5'-phosphate concentrations, the erythrocyte aspartate aminotransferase activity coefficient (αEAST), net homocysteine increase in response to a methionine load test (ΔtHcy), and 24 h urinary xanthurenic acid (XA) excretion in response to a tryptophan load test. Urinary 4-pyridoxic acid (4-PA) was measured to examine the impact of pyridoxine treatment on vitamin B6 excretion in these patients. Pro-inflammatory cytokine (TNF-α and IL-6) production, C-reactive protein levels and the erythrocyte sedimentation rate before and after supplementation were also examined. Pyridoxine supplementation significantly improved plasma and erythrocyte pyridoxal 5'-phosphate concentrations, erythrocyte αEAST, urinary 4-PA, and XA excretion. These improvements were apparent regardless of baseline B6 levels. Pyridoxine supplementation also showed a trend (p < 0.09) towards a reduction in post-methionine load ΔtHcy. Supplementation did not affect pro-inflammatory cytokine production. Although pyridoxine supplementation did not suppress pro-inflammatory cytokine production in patients with rheumatoid arthritis, the suboptimal vitamin B6 status seen in rheumatoid arthritis can be corrected by 50 mg pyridoxine supplementation for 30 days. Data from the present study suggest that patients with rheumatoid arthritis may have higher requirements for vitamin B6 than those in a normal healthy population.

Study interventions

Qualified subjects started taking the study treatment within one week of plasma pyridoxal 5'-phosphate analysis. These subjects were randomly assigned through the NEMC pharmacy to receive either active vitamin B6 (B6 group) or placebo (placebo group) tablets in double-blinded fashion for 30 days. To minimize the potential confounding effect of methotrexate and prednisone treatment on the functional tests, subjects were stratified by prednisone and methotrexate treatment, and then the subjects in each group were randomized to receive either active or placebo treatment. The randomization procedure was under guidance of a statistician and performed by registered pharmacists not directly involved in the present study.

Study interventions

The placebo tablet, made specifically for the study, was identical in appearance as well as ingredients to the active tablet, except that the active tablet (Nutro Laboratories, South Plainfield, NJ, USA) contained 50 mg of pyridoxine hydrochloride and the placebo did not (Tishcon Corp., Westbury, NY, USA). Both tablets contained microcrystalline cellulose, croscarmellose sodium, calcium phosphate, stearic acid, and magnesium stearate, ingredients commonly found in over-the-counter vitamin B6 supplements. Each phase 2 participant was asked to take one assigned tablet daily throughout the 30 day period. To assure compliance with the treatment regimen, each subject was given a personal study calendar with the 30 supplement days highlighted. The subject was asked to record the time of ingestion of each tablet on the calendar. In addition, the study coordinator made phone calls to remind each subject to take the tablets during the 30 day supplement period. The subjects were asked to return the bottle for a tablet count at the end of the 30 day treatment. To test the efficacy of the vitamin B6 supplementation, each subject went through the same 2 day testing procedure described above at the end of the 30 day supplementation period.

Results

Indicators of vitamin B6 status before and after treatment are shown in Table . All markers of vitamin B6 status improved significantly in the B6 group after supplementation, except for net increase in total homocysteine concentration, which only showed a modest trend towards improvement. None of the vitamin B6 status parameters showed significant improvements after treatment in the placebo group. Analysis of co-variance further demonstrated that initial levels of plasma pyridoxal 5'-phosphate, ΔtHcy, post-load urinary XA, αEAST (Table ) and CRP and erythrocyte sedimentation rate (ESR) (Table ) in phase 1 were strong predictors of those indicators after treatment in phase 2. After adjusting for the initial levels in (before treatment), the vitamin B6 supplementation significantly improved plasma and erythrocyte pyridoxal 5'-phosphate concentrations, αEAST, post-load XA and 24 h 4-PA excretion. We found a trend for normalization of plasma ΔtHcy in the vitamin B6 treatment versus placebo group (p = 0.086, ANCOVA). In patients who had abnormal ΔtHcy (above 15 μmol/l) before treatment (n = 22/28), the effect of vitamin B6 treatment was significant (p < 0.02). Plasma pyridoxal 5'-phosphate and ΔtHcy levels were related to CRP in patients with rheumatoid arthritis [], thus CRP could be a potential targets for vitamin B6 supplementation. The correlations between CRP and plasma pyridoxal 5'-phosphate and ΔtHcy disappeared in the B6 group after supplementation, whereas the relationships remained in the placebo group after the 30 day treatment (CRP versus plasma pyridoxal 5'-phosphate, r = -0.61, p = 0.02; CRP versus ΔtHcy, r = 0.48, p = 0.098) (n = 14). We found that vitamin B6 supplementation had no effect on inflammatory cytokines, plasma CRP, ESR, or rheumatoid factor levels in these patients (Table ).

Cell culture & proliferation assays

Ishikawa cells were obtained from the European Collection of Cell Cultures (Cat. No. 99040201) [] and were grown in DMEM/F-12 Ham medium (Cat. No. D6421, Sigma-Aldrich, UK) supplemented with L-glutamine and 10% fetal calf serum in 96-well plates (proliferation assays), 6-well plates (FACS analysis, luciferase reporter assays) or cell culture flasks (RNA extraction, tumour explant preparation). Cells were cultured at 37°C and 5%CO2 with varying doses of fenofibrate (Cat. No. F6020), 9-cis retinoic acid (9-cRA) (Cat. No. R4643) or all-trans retinoic acid (ATRA) (Cat. No. R6265, all Sigma-Aldrich, UK) dissolved in DMSO (Sigma-Aldrich). Control cells were treated with DMSO and the concentration of DMSO was kept the same throughout each experiment, to a maximum of 1% v/v. A minimum of five replicates per dose was performed for automated assays (BrdU and MTS) and all other cell culture experiments were performed independently in duplicate or triplicate. Cell proliferation was measured by the uptake of 5-bromo-2'-deoxyuridine (BrdU) using the BrdU Labelling and Detection Kit (Cat. No. 1444611, Roche, UK) according to the manufacturer's instructions. Relative cell abundance was measured either by cell counting, or using an MTS assay (CellTiter 96 Aqueous Cell Proliferation Assay, Cat. No. TB169, Promega, UK). Absorbance values for each well were measured using a microtiter plate reader (Anthos Labtech Instruments, Salzburg, Austria)

Objectives:

We investigated whether lowering of fasting homocysteine concentrations, either with folic acid or with betaine supplementation, differentially affects vascular function, a surrogate marker for risk of cardiovascular disease, in healthy volunteers. As yet, it remains uncertain whether a high concentration of homocysteine itself or whether a low folate status—its main determinant—is involved in the pathogenesis of cardiovascular disease. To shed light on this issue, we performed this study.

Design:

This was a randomized, placebo-controlled, double-blind, crossover study.

Participants:

Participants were 39 apparently healthy men and women, aged 50–70 y.

Interventions:

Participants ingested 0.8 mg/d of folic acid, 6 g/d of betaine, and placebo for 6 wk each, with 6-wk washout in between.

Results:

Folic acid supplementation lowered fasting homocysteine by 20% (−2.0 μmol/l, 95% confidence interval [CI]: −2.3; −1.6), and betaine supplementation lowered fasting plasma homocysteine by 12% (−1.2 μmol/l; −1.6; −0.8) relative to placebo. Mean (± SD) FMD after placebo supplementation was 2.8 (± 1.8) FMD%. Supplementation with betaine or folic acid did not affect FMD relative to placebo; differences relative to placebo were −0.4 FMD% (95%CI, −1.2; 0.4) and −0.1 FMD% (−0.9; 0.7), respectively.

Conclusions:

Folic acid and betaine supplementation both did not improve vascular function in healthy volunteers, despite evident homocysteine lowering. This is in agreement with other studies in healthy participants, the majority of which also fail to find improved vascular function upon folic acid treatment. However, homocysteine or folate might of course affect cardiovascular disease risk through other mechanisms.

Methodology/Principal Findings

Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein) and quencher (dabcyl) and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71±0.07 indicating that it is a sensitive assay for the identification of inhibitors.

Introduction

To kinetically characterise Dam and its substrates or inhibitors, it was essential to develop an assay that reported the initial rate of reaction. To do this, we have used a hemimethylated break light oligonucleotide which upon turnover yields the fully methylated product. This is cleaved in situ by the restriction enzyme DpnI, resulting in an increase in fluorescence. Having established a direct relationship between the observed fluorescence increase and the methylation activity of Dam with this hemimethylated substrate, we have used the assay to determine the K M and V max of Y. pestis Dam for S-adenosylmethionine and the oligonucleotide substrate. To demonstrate the convenience of this assay, we have used it to determine the K i of the known Dam inhibitor, S-adenosylhomocysteine with Y. pestis Dam.

Measurement of K i of S-adenosylhomocysteine for Y. pestis Dam

The effect of a known Dam inhibitor, S-adenosylhomocysteine on Y. pestis Dam was investigated using the break light assay. From the data shown in , the K i was calculated to be 6.93±2.01 µM and is lower than the reported value for the E. coli enzyme (K i = 41.6±10.4 µM). These experiments demonstrate the application of the assay to the detailed kinetic analysis of a single inhibitor.

Background

The human gut harbors a diverse community of microorganisms which serve numerous important functions for the host wellbeing. Functional foods are commonly used to modulate the composition of the gut microbiota contributing to the maintenance of the host health or prevention of disease. In the present study, we characterized the impact of one month intake of a synbiotic food, containing fructooligosaccharides and the probiotic strains Lactobacillus helveticus Bar13 and Bifidobacterium longum Bar33, on the gut microbiota composition and metabolic profiles of 20 healthy subjects.

Results

The synbiotic food did not modify the overall structure of the gut microbiome, as indicated by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The ability of the probiotic L. helveticus and B. longum strains to pass through the gastrointestinal tract was hypothesized on the basis of real-time PCR data. In spite of a stable microbiota, the intake of the synbiotic food resulted in a shift of the fecal metabolic profiles, highlighted by the Gas Chromatography Mass Spectrometry Solid Phase Micro-Extraction (GC-MS/SPME) analysis. The extent of short chain fatty acids (SCFA), ketones, carbon disulfide and methyl acetate was significantly affected by the synbiotic food consumption. Furthermore, the Canonical discriminant Analysis of Principal coordinates (CAP) of GC-MS/SPME profiles allowed a separation of the stool samples recovered before and after the consumption of the functional food.

Effects of the synbiotic food on composition of the gut microbiota

PCR-DGGE analysis with universal primers targeting the V2-V3 region of the 16S rRNA gene was used to monitor the impact of the synbiotic food intake on the predominant bacterial population (Figure ). Population fingerprint profiles were compared and numerically analyzed by FPQuest Software. DGGE band profiles (mean of bands: 15.3) were stable for each subject over a month of feeding with the functional food. Only a slight difference in band richness was found between the time points of the study (T0, mean of bands: 15.8; T1, mean of bands: 14.8). DGGE bands were subjected to Mann-Whitney U-test in order to search for significant differences in the intensities between T0 and T1. No band showed a significant variation, indicating that the consumption of the synbiotic food did not alter the concentration of any major species of intestinal microbiota. Pearson correlation was used to calculate the similarity index (SI) between DGGE band profiles related to the time points T0 and T1 for each healthy volunteer (Table ). The high median value of SI (67.1%) revealed that the dominant bacterial composition remained constant over the treatment. Only 3 subjects presented SIs lower than 50% (subjects 8, 12 and 20). No subject showed significant variations between DGGE band profiles related to T0 and T1, as evaluated using the Pearson correlation analysis (P > 0.05).

Rgc1 and Rgc2 serve a redundant role in the regulation of glycerol efflux through the Fps1 glycerol channel

To understand the cause of the cell wall stress in the rgc1/2Δ mutant, we conducted a dosage suppressor screen for high-copy number plasmids that conferred growth at 37°C. A single class of strong suppressor was identified as the FPS1 gene (). FPS1 encodes an aquaglyceroporin that is the major facilitator of glycerol uptake and efflux in yeast ,. This plasma membrane channel protein also mediates uptake of toxic metalloids, such as arsenite and antimonite ,. One interpretation of the suppression result is that the rgc1/2Δ mutant experiences abnormally high turgor pressure from accumulation of glycerol, which yeast cells use as a compatible solute for osmoregulation. Measurement of intracellular glycerol concentration confirmed that the rgc1/2Δ mutant has a 5.9-fold higher glycerol level than wild-type cells under normal growth conditions, a value that is approximately half that of an fps1Δ mutant and approximately equal to that of wild-type cells exposed to hyper-osmotic shock (). To determine if excess intracellular glycerol is responsible for the phenotypic defects of this mutant, we blocked glycerol biosynthesis at the first committed and rate limiting step, glycerol-3-phosphate dehydrogenase (GPD) –. GPD is encoded by the paralogous genes GPD1 and GPD2. Deletion of either GPD1 or GPD2 alone did not suppress the lysis defect of the rgc1/2Δ mutant, but blocking glycerol biosynthesis completely by deletion of both GPD1 and GPD2 allowed growth at 37°C (), confirming that glycerol accumulation is responsible for the cell lysis defect. This also provides an explanation for the fortified cell wall of the rgc1/2Δ mutant as a response to the stress of abnormally high turgor pressure. Consistent with this interpretation, the gpd1/2Δ mutations relieved the cell wall stress signaling observed in the rgc1/2Δ mutant (). Finally, the gpd1/2Δ mutations relieved the caspofungin sensitivity of the rgc1/2Δ mutant ().

Rgc1 and Rgc2 serve a redundant role in the regulation of glycerol efflux through the Fps1 glycerol channel

We considered two possible explanations for the hyper-accumulation of glycerol in the rgc1/2Δ mutant – the mutant either produces excess glycerol, or it is impaired for glycerol efflux through Fps1. These hypotheses generate different predictions for the impact of the rgc1/2Δ mutations on the phenotype of an fps1Δ mutant. If the rgc1/2Δ mutant produces excess glycerol, this should exacerbate the lysis defect of an fps1Δ mutant, which is blocked for glycerol efflux. By contrast, if the rgc1/2Δ mutant is blocked for glycerol efflux through Fps1, loss of the glycerol channel should not result in an additive defect. shows that an fps1Δ mutant displays a temperature-sensitive growth defect that is slightly more severe than that of the rgc1/2Δ mutant, with a semi-permissive growth temperature of 34.5°C. The fps1Δ mutant growth defect at elevated temperature is also the result of cell lysis (data not shown). Significantly, the rgc1/2Δ fps1Δ triple mutant behaves identically to the fps1Δ mutant (), supporting the hypothesis that the rgc1/2Δ mutant is impaired for glycerol efflux through Fps1. To test this directly, we measured export of glycerol from cells exposed to a hypo-osmotic shock, a condition that would induce glycerol efflux through Fps1. We found that glycerol was released from wild-type cells, but not from the rgc1/2Δ mutant or the fps1Δ mutant (), supporting the conclusion that Rgc1 and Rgc2 regulate glycerol efflux through Fps1. The varied initial content of 14C-labeled glycerol among these mutants is a consequence of differential glycerol loading, reflecting the importance of Fps1 for glycerol influx as well as efflux. Finally, strong overexpression of RGC2 failed to suppress the temperature-sensitivity of the fps1Δ mutant (data not shown), thus establishing an epistatic relationship that places RGC1 and RGC2 above FPS1 in a common pathway for glycerol efflux.

Rgc1 and Rgc2 serve a redundant role in the regulation of glycerol efflux through the Fps1 glycerol channel

To determine the cause of the increased steady-state level of Fps1 in the rgc1/2Δ mutant, we conducted a test of Fps1 stability. Fps1 levels were followed in cells in which FPS1 transcription was shut down with the simultaneous inhibition of protein synthesis. We found that Fps1 was stabilized in the rgc1/2Δ mutant relative to wild-type cells (). Therefore, we conclude that increased intracellular glycerol in the rgc1/2Δ mutant, which is caused by a deficiency in Fps1 function, induces an increase in the level of weakly functional Fps1 through protein stabilization.