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High Levels of CO2 Induce Spoilage by Leuconostoc mesenteroides by Upregulating Dextran Synthesis Genes | Applied and Environmental Microbiology
ABSTRACT.During nonventilated storage of carrots, CO2 gradually accumulates to high levels and causes modifications in the carrot's microbiome toward dominance of Lactobacillales and Enterobacteriales. The lactic acid bacterium Leuconostoc mesenteroides secretes a slimy exudate over the surface of the carrots. The objective of this study was to characterize the slime components and the potential cause for its secretion under high CO2 levels. A proteomic analysis of the exudate revealed bacterial glucosyltransferases as the main proteins, specifically, dextransucrase. A chemical analysis of the exudate revealed high levels of dextran and several simple sugars. The exudate volume and dextran amount were significantly higher when L. mesenteroides was incubated under high CO2 levels than when incubated in an aerated environment. The treatment of carrot medium plates with commercial dextransucrase or exudate protein extract resulted in similar sugar profiles and dextran production. Transcriptome analysis demonstrated that dextran production is related to the upregulation of the L. mesenteroides dextransucrase-encoding genes dsrD and dsrT during the first 4 to 8?h of exposure to high CO2 levels compared to aerated conditions. A phylogenetic analysis of L. mesenteroides YL48 dsrD revealed a high similarity to other dsr genes harbored by different Leuconostoc species. The ecological benefit of dextran production under elevated CO2 requires further investigation. However, this study implies an overlooked role of CO2 in the physiology and fitness of L. mesenteroides in stored carrots, and perhaps in other food items, during storage under nonventilated conditions.IMPORTANCE The bacterium Leuconostoc mesenteroides is known to cause spoilage of different types of foods by secreting a slimy fluid that damages the quality and appearance of the produce. Here, we identified a potential mechanism by which high levels of CO2 affect the spoilage caused by this bacterium by upregulating dextran synthesis genes. These results have broader implications for the study of the physiology, degradation ability, and potential biotechnological applications of Leuconostoc.INTRODUCTION.The long-term storage and transport of postharvest carrots (Daucus carota L.) require a low-temperature, high-relative-humidity environment, usually with low ventilation (1, 2). Following long-term storage, rots often appear on the carrots, leading to severe spoilage. The postharvest soft rot of stored carrot roots is attributed to well-known pathogens, such as Pectobacterium carotovorum (3–5), the fungus Sclerotinia sclerotiorum (6–8), and other microorganisms (9). We recently discovered an additional bacterium, Leuconostoc mesenteroides , responsible for deterioration of stored carrots (10). L. mesenteroides causes the formation of a slimy exudate on the carrots' surfaces, termed “oozing,” under high CO2 levels. When exudate carrots are exposed to air, they undergo browning, and soft rot develops by unidentified phytopathogenic fungi (10). The finding that L. mesenteroides -mediated exudate occurs only under high CO2 levels led us to examine the potential mechanisms by which CO2 may contribute to this phenomenon.L. mesenteroides belongs to the Leuconostocaceae, a bacterial family mainly consisting of organisms generally recognized as safe that are best known for their use as starter cultures in the food and dairy industries (11). In addition, these bacteria are used for the production of various biomolecules, such as vitamins, lactic acid, bacteriocins, and different exopolysaccharides (EPSs) (12, 13). These EPSs, such as dextran, mutan, and alternan, are synthesized mainly during sucrose metabolism (14) and are considered to play a central role in biofilm formation (15, 16).Despite the beneficial role of different Leuconostoc species and strains in the industry, several Leuconostoc species have also been found as spoilage agents in a variety of food products, from meat and dairy to fruit and vegetables (17). The spoilage characteristics include off-odors and flavors, gas formation, discoloration, and slime formation. It appears that slime or exudate formation can occur under different conditions and environments. L. mesenteroides has been documented to spoil sugar beet storage piles (18, 19), fresh tomatoes (20), sugarcane (21), and packaged refrigerated foods, particularly meat and dairy products and ready-to-eat salads (22, 23). An understanding of the factors leading to the growth and dominance of Leuconostoc species in stored food products, and eventually to spoilage, is essential for preventing food losses.Since we previously demonstrated the role of high CO2 in disposing L. mesenteroides to exudate secretion in stored carrots, the aim of this study was to determine the mechanism of induction by high levels of CO2.RESULTS.Dextransucrases and dextran are the main components in high-CO 2 -derived exudate. To identify the bacterial factors involved in Leuconostoc-mediated exudate, we analyzed the protein content of exudate formed on carrots during storage under 100% CO2 conditions. A proteomic analysis of the fluid revealed a predominance of dextransucrases and levansucrases (Table 1), two groups of extracellular enzymes responsible for converting sucrose to dextran and levan, respectively (13, 24, 25). The three most abundant proteins found in the exudate were dextransucrases, belonging to glycoside hydrolase family 70 (GH70). This family accommodates different glucansucrases, which convert sucrose into several α-glucan polysaccharides and are found in lactic acid bacteria (LAB), most Bacilli, and Enterobacteriaceae (26). In addition to these enzymes, other functional proteins were also found in the exudate, including proteins responsible for metabolism, protein synthesis, and folding, as well as membrane proteins. However, their relative abundance was less pronounced than those of dextransucrase and levansucrase (Table 1). Following the identification of abundant dextransucrases, we conducted a high-performance liquid chromatography-refractive index detector (HPLC-RID) analysis to determine the presence of polysaccharides and sugars in the exudate. We found dextran, along with a variety of other simple sugars, such as sucrose, fructose, glucose, and probably other soluble sugars (see Fig. S1 in the supplemental material). To further confirm the presence of dextran in the exudate, we used a proton nuclear magnetic resonance (1H-NMR) analysis of the EPSs present in the exudate formed by the bacteria in both carrot and 10% sucrose growth media in a 100% CO2 environment. This analysis explicitly revealed the presence of dextran in both media, raising its potential involvement in carrot deterioration (Fig. 1).View this table: View inline.View popup.TABLE 1 Proteomic analysis of exudate induced by high levels of CO2 Open in new tab.Download powerpoint.FIG. 1 L. mesenteroides YL48 produces dextran-rich exudate when incubated on carrot medium. 1H-NMR spectrum of dextran synthesized by L. mesenteroides YL48 in 100% CO2 atmosphere (top). The proton number is annotated on each proton signal in the spectrum and related to the positions in the dextran biochemical structure (bottom).Exudate level is affected by both sucrose and CO 2 levels. Previously, we reported that a CO2-rich environment enhances exudate on carrots, produced by L. mesenteroides (10). To examine the interplay between sucrose concentrations and CO2, we incubated L. mesenteroides YL48 with different sucrose concentrations over 4?days under both aerated and 100% CO2 environments (n?=?6 per treatment). After 4?days, the exudate produced by the bacteria was collected with sterile tips and put in 15-ml Falcon tubes, and the resulting collected exudate volume was determined using a 200-?l pipette. There was a significant increase in the exudate volume produced by L. mesenteroides in 5% versus 1% sucrose medium, regardless of the gaseous conditions, with a 7-fold increase and 4.5-fold increase under CO2 and atmospheric air conditions, respectively (Fig. 2). However, at the higher sucrose concentrations of 10% and 20%, there was no significant change under either aerated or CO2 conditions. The exudate volume was significantly higher in the 100% CO2 environment than in ambient air for all sucrose concentrations used (Student's t test, P?50 were included as significant.Extraction and identification of sugars. After extraction, the sugars in the exudate were identified, with additional filtration of the supernatant through a 0.2-μm membrane filter (Millex-GV filter unit; Merck Millipore, Tullagreen, Ireland). The filtrate was used for dextran, sucrose, glucose, and fructose analyses. Samples were separated on an ion-exchange column (6.5?nm by 300?nm) (Sugar-Pak I; Waters) using an HPLC system (LC-10A UFLC series; Shimadzu, Japan) equipped with an RID (SPD-20A). The column temperature was set to 80°C, and the mobile phase (ultrapurified deionized water; Bio Lab, Jerusalem, Israel) was eluted through the system for 30?min at a flow rate of 0.5?ml/min. The chromatographic peak corresponding to each sugar was identified by comparing the retention time of each peak, at its maximum height, with that of a standard. A calibration curve was prepared using standards (Sigma-Aldrich) to determine the relationship between peak area and concentration.Carrot sugars were identified and quantified using the same procedure, with the following extraction method. Every 2?weeks after harvest, carrot roots stored at 1°C were extracted for sugar identification and quantification. One gram of carrot tissue was excised from the roots using a scalpel. The tissues were incubated three times in 80% ethanol at 80°C, for 30?min each time. The solution was then dried for 7?h using a speed vacuum (Centrivap concentrator; Labconco, Kansas City, MO, USA) and passed through a 0.2-μm membrane filter (Millex-GV filter unit; Merck Millipore). The filtrate was used for sucrose, glucose, and fructose analyses.EPS extraction. After 4?days of incubation at 20°C under aerated or 100% CO2 conditions, the exudate produced over the sucrose, glucose, or carrot medium was collected from each plate into individual 15-ml sterile Falcon tubes (Thermo Fisher Scientific, San Jose, CA, USA) using edged-cut 200- and 1,000-?l sterile tips. The total collected volume of the exudate was determined using a 200-?l pipette for each condition. Sterile double-distilled water was added at the same volume as the resulting collected exudate to each 15-ml Falcon tube, and bacterial cells were removed from the exudate by centrifugation (10?min at 8,000?×?g at room temperature). After this step, EPSs were extracted and purified by ethanol precipitation as previously described (44). The resultant purified EPSs were lyophilized for 5?h, ground to a powder using a sterile mortar and pestle, and weighed on an analytical balance.Structural analysis of dextran by 1 H-NMR. Samples of 10?mg dextran from YL48 grown on carrot medium under aerated or 100% CO2 conditions were dissolved in 0.5?ml of pure D2O and then placed in 5-mm NMR tubes. NMR spectra were recorded on a Bruker Avance III spectrometer at 500.13?MHz for 1H. The spectra were recorded using standard Bruker pulse sequences. NMR chemical shifts were referenced to external 3-trimethylsilyltetradeutero-propionic acid sodium salt (TSP; δH?=?0?ppm). The 1H-NMR data collection consisted of 256 acquisitions per sample. The resulting NMR profile was compared with known 1H-NMR dextran profiles (45).Determining bacterial growth rates. L. mesenteroides was grown on MRS agar plates at 30°C. Individual colonies were used to inoculate 10?ml of MRS broth and grown overnight at 30°C. A 1-ml aliquot of this culture was used to inoculate 10?ml of fresh MRS broth. Cultures were shaken at 170?rpm and 30°C for 10?h in an aerated or 100% CO2 environment, and the OD600 was recorded every 2?h. Model fit, growth rate, and doubling time calculations were conducted using the R package growthcurver, based on the optical density (OD600) of the sample (46).RNA extraction and cDNA synthesis. L. mesenteroides overnight broth stock (as described above in “Bacterial strain and inoculum preparation”) was diluted 1:10 and grown for 3 h at 30°C. This stock was used to inoculate carrot medium plates (300??l), placed inside 2-liter glass chambers, and incubated for up to 96?h under aerated or 100% CO2 atmospheres. For each predefined sampling time point, three plates for each environment were separately swabbed, and the swabs were washed inside sterile 1.5-ml Eppendorf tubes filled with sterile 0.9% saline. After centrifuging (10?min at 600?rpm), total RNA was extracted from the samples using the GeneJet RNA purification kit (Thermo Fisher Scientific). The procedures were performed according to the manufacturer's protocol with the following modifications: the lysozyme concentration was 15?mg/ml and the incubation time was extended to 60?min. The RNA concentrations were measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Agarose gel electrophoresis was then used to determine the integrity and quality of the RNA. RNA samples were digested with an RNase-free DNase kit (Thermo Fisher Scientific) and were then used to synthesize cDNA using the Verso cDNA synthesis kit (Thermo Fisher Scientific).Quantitative real-time PCR of dextransucrase genes. We used qPCR to determine differential transcript expression. The gene-specific primers are described in Table 2. Each PCR mixture (20?μl) contained SYBR green mix, cDNA samples, and forward and reverse gene-specific primers. All qPCRs were performed in a StepOne Plus real-time PCR system (Applied Biosystems, Foster City, CA, USA). The thermocycling conditions were 95°C for 30?s and 40 cycles of 95°C for 30?s and 60°C for 30?s. Differential gene expression levels were normalized to the level of the gyrA transcript. This gene was previously shown to be expressed constitutively in several bacterial species (47, 48). The expression profiles at the start of the experiment (time zero) were used as a reference, and the relative transcription level under each environment was determined by fold change relative to that condition. Three independent experiments were performed, and the results were averaged.View this table: View inline.View popup.TABLE 2 Primers used for quantitative real-time PCR analysisSequence alignment and phylogenetic tree construction. The target sequence was annotated using BLASTn to reveal homologous gene sequences. Those sequences, together with the target sequence, were aligned using the L-INS-I option of the web-based version of MAFFT version 7 (49). After the alignment, a phylogenetic tree was constructed with the web-based platform PhyML 3.0 using the SH-like aLRT (Shimodaira–Hasegawa-like approximate likelihood ratio test) method (50). The tree was graphically designed using FigTree version 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).Statistical analysis. All experiments were performed at least in duplicate. The means, standard errors, and standard deviations were calculated and analyzed using Microsoft Excel 2010. The different statistical tests were conducted with R, using RStudio version 1.0.44 (51). Bacterial growth curves were calculated, compared, and fitted using the R package growthcurver (52).Accession number(s). The whole-genome shotgun project was deposited at DDBJ/EMBL/GenBank under accession no. MUXD00000000.ACKNOWLEDGMENTS.This research was funded by a grant from the Chief Scientist of the Ministry of Agriculture and Rural Development of Israel (no. 430-0522-16). The article is a contribution of the Agricultural Research Organization (ARO), the Volcani Center, Rishon LeZion, Israel, no. 810/18. S. Sela was partially supported by BARD-NIFA grant no. NB-8316-17, the EU COST Action FA1202 (A European Network for Mitigating Bacterial Colonisation and Persistence on Foods and Food Processing Environments; http://www.bacfoodnet.org/), and the EU COST Action CA16110 entitled Control of Human Pathogenic Micro-organisms in Plant Production Systems.FOOTNOTES.Received 26 February 2018..Accepted 10 October 2018..Accepted manuscript posted online 26 October 2018..Supplemental material for this article may be found at https://doi.org/10.1128/AEM.00473-18.Copyright ? 2018 American Society for Microbiology..All Rights Reserved.REFERENCES.1. ?Phan C , .Hsu H , .Sarkar S .. 1973 . Physical and chemical changes occurring in the carrot root during storage . Can J Plant Sci 53 :635 –641 . doi: 10.4141/cjps73-124 .OpenUrl CrossRef 2. ?Selj?sen R , .Bengtsson GB , .Hoftun H , .Vogt G .. 2001 . Sensory and chemical changes in five varieties of carrot ( Daucus carota L) in response to mechanical stress at harvest and post‐harvest . 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