High Levels of CO2 Induce Spoilage by Leuconostoc mesenteroides by Upregulating Dextran Synthesis Genes | Applied and Environmental Microbiology
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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?0.05 for 1% sucrose and P?0.01 for 5% to 20% sucrose) (Fig. 2). No exudate was seen in minimal medium that lacked sucrose (0%) and contained 2% glucose as the carbon source. Open in new tab.Download powerpoint.FIG. 2 The volume of exudate produced by L. mesenteroides is associated with sucrose concentration and growth atmosphere. The volumes of exudate were determined after incubating for 4?days at 20°C under aerated and 100% CO2 conditions (n?=?6). Error bars represent standard deviations. , P?0.05; , P?0.01 between treatments at each time point.To further examine the relationship between exudate formation, growth medium, and growth atmosphere, we tested the volume of the exudate and the content of dextran following the growth of L. mesenteroides in 5% sucrose or in carrot medium under ambient air or a 100% CO2 environment. The exudate produced on each plate (8 plates per treatment) was collected into 15-ml Falcon tubes, and the total volume was determined per treatment. The collected exudates were later dried and used for dextran weight measurements. Exudate production and dextran synthesis were both higher in the sucrose medium and were significantly increased under 100% CO2 versus aerated conditions (Student's t test, P?0.05) (Fig. 3). Open in new tab.Download powerpoint.FIG. 3 One hundred percent CO2 conditions induce higher exudate volume and dextran amounts in L. mesenteroides YL48. (A) Total exudate volume. (B) Total dextran (dry weight). L. mesenteroides (100?μl of 5?×?107 CFU/ml) was inoculated on 5% sucrose and carrot agar plates and incubated for 7?days at 30°C. Data are averages (n?=?8 plates) and error bars represent standard deviations. , P?0.05 between environments (CO2 and air).These findings imply interactions between the high-CO2 environment and sucrose concentration, exudate volume, and dextran content. Therefore, our next step was to identify possible mechanisms governing these interactions.High CO 2 levels do not increase bacterial growth rate. To determine whether exudate volume and dextran content were elevated in the high-CO2 environment simply due to differences in L. mesenteroides growth under the different atmospheric conditions, we examined the growth curves of L. mesenteroides on de Man, Rogosa, and Sharpe (MRS) medium under both aerated and 100% CO2 conditions, both at 30°C. Overall, L. mesenteroides grew at a slightly lower rate in CO2 than in aerated conditions during the first 6?h of growth (Fig. 4). The growth rates and doubling times were 1.58 and 46 min, respectively, in the aerated environment and 1.55 and 48 min, respectively, in the CO2 environment; under both conditions, the bacteria reached the stationary phase after 8 to 10?h, with no significant difference (P?0.05) between environments (CO2 and air) (Fig. 4). Open in new tab.Download powerpoint.FIG. 4 CO2 environment slightly inhibits bacterial growth during the exponential phase. L. mesenteroides YL48 was grown on optimal growth medium (MRS) for 10?h under either air or 100% CO2 conditions at 30°C (κ?=?1.58 and 1.55, r = 0.0148 and 0.144 for air and CO2 environments, respectively). Growth curves were fitted to logistic model with sigma values of 0.041 and 0.038 for air and CO2, respectively.Dextransucrase activity correlates with the exudate formation on carrots. The abundant dextransucrases found in the exudate produced by L. mesenteroides in the 100% CO2 environment led us to examine whether these enzymes might be correlated with the exudate observed on carrots. We inoculated carrot medium plates with commercial purified dextransucrase (derived from L. mesenteroides BF-512) and with L. mesenteroides YL48 exudate-derived protein extract. The plates were incubated for 24?h at 30°C, and the exudate produced on the plates was collected, extracted, and analyzed using HPLC-RID to determine sugar composition. The HPLC-RID analysis demonstrated that purified dextransucrase enzyme and L. mesenteroides -derived extract of exudate proteins are able to synthesize dextran from the ingredients present in the carrot agar medium (Fig. 5). Moreover, the treated plates looked similar, with a large number of drops seen over the plates (Fig. 5). The control plates inoculated with sterile acetate buffer without commercial enzyme or MRS agar plates did not show fluid production and remained dry (not shown). These findings suggest a role for the enzyme dextransucrase in the production of exudate on carrots by L. mesenteroides . Open in new tab.Download powerpoint.FIG. 5 Commercial purified dextransucrases produce exudate that is similar to that produced by L. mesenteroides exudate-derived protein extract. HPLC-RID analysis and binocular imaging (insets) of exudate produced by purified dextransucrase (A) and by protein extract of L. mesenteroides YL48 (B). Dextran peaks are marked as 1. Glucose (2) and fructose (3) were additionally detected in the commercial dextransucrase-derived exudate.Differential dextransucrase gene expression in aerated versus CO 2 environments. Differences in the levels of dextransucrases and the amounts of dextran present in the L. mesenteroides -mediated exudate may result from differences in the expression of the dextransucrase genes responsible for dextran synthesis. On the basis of the draft genome of YL48, we identified two putative dextransucrase genes, named YL4825 and YL4874, which showed homology to the genes dsrD and dsrT, respectively (see Table S1). A BLAST analysis of the sequences of the three dextransucrase proteins found in the exudate against the YL48 genome further confirmed the matching presence of these two genes (see Table S2). Full sequencing of the genome enabled us to design specific primers for the two genes and perform a quantitative real-time PCR (qPCR) analysis to explore the expression profiles of those genes under aerated and high-CO2 conditions. Bacterial total RNA was isolated from L. mesenteroides cells grown on carrot medium plates after 2, 4, 8, and 24?h in each environment, namely, 100% CO2 and ambient air. The qPCR analysis of the dextransucrase genes showed that dsrD transcript levels were 35-fold to 25-fold greater after 2 to 8?h of growth under 100% CO2 conditions, with only a 10- to 15-fold difference under aerated conditions (Fig. 6A). However, no differences in transcript levels appeared after 24?h of incubation (Fig. 6A). On the other hand, the dsrT expression profile under 100% CO2 exhibited only a modest gradual increase after 2 to 4?h compared to that in the aerated environment, with transcript levels only 1.5-fold to 3-fold greater in the high-CO2 environment (Fig. 6B). After 8?h of growth, there was no difference in transcript levels in 100% CO2 versus that in ambient air environments. Open in new tab.Download powerpoint.FIG. 6 CO2 environment induces upregulation of dextransucrase-encoding genes. Expression of dsrD (A) and dsrT (B) over time compared to time zero. Bacterial total RNA was isolated from independent carrot medium plates after 2, 4, 8, and 24?h in each environment, 100% CO2 or air. , P?0.05 between air and CO2 conditions; NS, not significant.Overall, these findings suggest that the observed increases in exudate production and dextran synthesis under high CO2 levels are regulated at the transcriptional level, mainly by the upregulation of dsrD.Since we showed that dsrD expression is highly upregulated by CO2, it was of interest to assess whether the gene found in L. mesenteroides YL48 is related to other known dextransucrase genes present in other L. mesenteroides strains. A BLASTn analysis yielded 26 homologous sequences of dsrD present in different L. mesenteroides strains (see Table S3). A phylogenetic tree was generated on the basis of these homologous sequences, and it demonstrated that YL48 dsrD is indeed highly similar to other dsr genes harbored by different L. mesenteroides strains (Fig. 7). Open in new tab.Download powerpoint.FIG. 7 Rooted phylogenetic tree of homologous dsrD-encoding genes, relative to L. mesenteroides YL48 dsrD gene (presented in bold font). Streptococcus downei (AB476746.1) served as an outgroup.DISCUSSION.Dextransucrase and dextran are the main components of L. mesenteroides exudates on carrots. L. mesenteroides is known to inhabit different and diverse habitats, including plants, meat and dairy products, soil, and even the guts of different animals (12, 20, 27–31). However, under certain circumstances, such as high CO2 levels, we previously found that it dominates the product microflora and damages it via several mechanisms, mainly by secreting a slimy exudate. A high-N2 atmosphere had no visible effect on carrot exudate (10). Here, we further characterized L. mesenteroides exudates on carrots and found that the exudates contain large quantities of dextransucrase enzymes and the EPS dextran (Table 1; Fig. 1; see also Fig. S1 in the supplemental material). Dextran is a glucan, a homopolysaccharide of glucose that features a substantial number of α-1,6 linkages in its major chain, along with different kinds of branched linkages (32). L. mesenteroides produces the exudate following or during growth on carrot tissue, which is rich in simple sugars such as sucrose, glucose, and fructose (33). The abundance of dextransucrases and its sugar products in the exudate led us to examine whether they might be involved in its production. We indeed found that purified dextransucrase can mimic L. mesenteroides -mediated exudate, supporting the involvement of L. mesenteroides dextransucrase in the exudate phenomenon (Fig. 5). Still, the purified enzyme produces other by-products, such as glucose and fructose, that were missing in the exudate activity, suggesting other enzymatic activity downstream of dextransucrase.High levels of CO 2 correlate with upregulation of dextransucrase-synthesizing genes. L. mesenteroides strain YL48 carries two dextransucrase-encoding genes, dsrD and dsrT, that are upregulated upon exposure to a high-CO2 environment (Fig. 6); the expression of dsrD is more pronounced than that of dsrT.The observed increase in the transcription of dextran-synthesizing genes under high CO2 levels may potentially explain the increased exudate volume and dextran amounts produced in the carrot and sucrose media (Fig. 2 and 3). The underlying role of CO2 as a factor regulating bacterial growth and activity has been demonstrated previously, mainly with human pathogens (34). A high CO2 level can activate and induce virulence of different bacterial species. For example, in Bordetella species, high CO2 causes increased transcription of genes encoding adenylate cyclase toxin (ACT), filamentous hemagglutinin, pertactin, fimbriae, and the type III secretion system, along with increased cytotoxicity and adherence ability (35). Similarly, CO2 has been reported to regulate the expression of several plasmid and chromosomal virulence genes of Bacillus anthracis , the causative agent of anthrax (36).Herein, a high CO2 level is thus implicated in the increased spoilage caused by L. mesenteroides and in the upregulation of dextran synthesis enzymes. Our findings demonstrate the ability of L. mesenteroides , generally regarded as a facultative or aerotolerant anaerobe, to sense its environment and, under certain conditions, respond with dextran synthesis. Others have shown that the exposure of L. mesenteroides BD3749 to O2 leads to an increased synthesis of insoluble EPSs and an upregulation of a novel glucansucrase, Gsy (37). The authors explained this phenomenon as a defense mechanism by which the facultative anaerobic bacteria buffer themselves from the aerobic environment using an EPS appendage. This notion may not explain our finding regarding enhanced dextran synthesis in a high-CO2 environment, suggesting different dextransucrase regulation or strain-specific differences. One potential explanation for this observed phenomenon is related to motility. EPS has been reported to play a role in the motility of the benthic bacterium Pseudoalteromonas (38). It might be that L. mesenteroides uses the secreted fluid as a sliding mechanism that enables it to migrate and drift over the host surface. Another explanation might be related to the carbon dioxide that is produced during hexose metabolism in LAB (39). However, the fact that we observed upregulation of dextransucrase genes, dextran production, and exudate under aerated conditions (Fig. 3 and 6) implies the involvement of other, currently unknown factors in the synthesis of dextran.The pattern that we found, in which high CO2 levels increased the produced dextran by upregulating dextransucrase transcription (Fig. 6), might also have practical and industrial implications. First, our isolated strain YL48 might be used as a dextran-producing agent because of its ability to synthesize large amounts of dextran under high CO2 levels. The fact that the extracted dextran molecules structurally resembled commercial dextran (derived from L. mesenteroides B-512F) with some modifications in the branching distribution (see Table S4) provides more support for the commercial potential of L. mesenteroides YL48. Second, some of the strains in our homology analysis have been researched in the context of commercial and novel dextran synthesis, for instance, L. mesenteroides CMG713 (40) and L. mesenteroides URE-13 (41). If the synthesis of biotechnologically important dextrans can be facilitated by combining high CO2 levels in the large-scale fermentation process, this would have beneficial implications for a variety of biotechnological industries.We were able to demonstrate one potential mechanism in which high CO2 upregulates dextran synthesis enzyme transcription, resulting in an increased production of slimy exudate over raw carrots. The exact ecological benefit to the bacterium of expressing more EPS-synthesizing genes and increasing its EPS production under high CO2 levels, rather than in an aerated less-optimal environment, warrants future investigation. These and our previous data (10) suggested that a high CO2 level, rather than a lack of O2, induces exudate secretion and the consequent development of a brownish color following air ventilation. We believe that future studies should deal with the identification of the threshold CO2 concentration that influences the L. mesenteroides response.MATERIALS AND METHODS.Bacterial strain and inoculum preparation. L. mesenteroides YL48 was isolated from minimally processed carrots grown in the northern Negev, Israel (10). The genomic DNA of this strain was sequenced at the Center for Genomic Technologies (The Hebrew University of Jerusalem, Israel).Overnight cultures were started by picking a fresh colony from an MRS agar plate (Sigma-Aldrich, Rehovot, Israel), using it to inoculate 10?ml of MRS broth medium, and incubating at 30°C with shaking at 170?rpm. Overnight cultures were diluted 1:10 in 10?ml of MRS broth medium and grown for 3?h to an optical density at 600?nm (OD600) of 0.6.Culture medium preparation. Bacteria were grown in three different growth media. The first one was raw carrot discs, cut from whole washed carrots, washed again with tap water, and sterilized in sodium hypochlorite (1% NaClO) for 2?min. The sterile carrot discs were washed with sterile double-distilled water to remove excess NaClO and cut into 2-mm discs using a sterile scalpel blade. The discs were placed on sterile 20-mm Whatman paper no. 2 (Sigma-Aldrich) in 20-mm petri dishes.Carrot agar medium was made as previously described (10), by using raw carrots as the starting material. Sucrose and glucose media were prepared with concentrations of 1% to 20% sucrose or 2% glucose as the sole carbon source, as detailed elsewhere (42). Petri plates (20?mm) containing 10?ml of agar medium (or carrot discs) were inoculated with 100?μl of 5?×?107 CFU/ml bacterial inoculum, as described above in “Bacterial strain and inoculum preparation.” The plates were placed inside 2-liter glass chambers, either filled with 100% CO2 or air ventilated, and incubated for 4?days at 20°C.Proteomic analysis. L. mesenteroides YL48 was grown for 4?days on carrot medium under 100% CO2 conditions, and the exudate secreted on the plate was collected and centrifuged (2?min at 12,000?×?g). The supernatant was subjected to tryptic digestion. The samples were first subjected to buffer exchange to 50?mM ammonium bicarbonate on 10-kDa-molecular-weight-cutoff centrifugal concentrators (Sartorius; Fisher Scientific, Rockford, IL, USA). The proteins were reduced by incubation with dithiothreitol (5?mM; Sigma, Rehovot, Israel) for 60?min at room temperature and alkylated with 10?mM iodoacetamide (Sigma) in the dark for 30?min at room temperature. Trypsin (Promega, Madison, WI, USA) was added at a 1:50 trypsin/protein ratio (wt/wt) and incubated overnight at 37°C, followed by a second addition of trypsin for 4?h at 37°C. Digestion was stopped by the addition of trifluoroacetic acid (1% [vol/vol] final concentration). After the digestion, the peptides were desalted using the Oasis HLB μElution format (Waters, Milford, MA, USA), vacuum dried, and stored at ?80°C until further analysis.Liquid chromatography (LC) and mass spectrometry (MS) were conducted as detailed elsewhere (43) with minor modifications. Each sample was loaded using splitless nanoscale ultraperformance liquid chromatography (nanoUPLC) (10,000 lb/in2, nanoAcquity; Waters, Milford, MA, USA). The mobile phase consisted of solutions A (H2O plus 0.1% formic acid) and B (acetonitrile plus 0.1% formic acid). Desalting of the samples was performed online using a reversed-phase Symmetry C18 trapping column (180-?m internal diameter, 20-mm length, 5-?m particle size; Waters). The peptides were then separated using a T3 HSS nano-column (75-?m internal diameter, 250-mm length, 1.8-?m particle size; Waters) at 0.35 ?l/min. The peptides were eluted from the column into the mass spectrometer using the following gradient: 4% to 30% solution B for 105?min, 30% to 90% solution A for 5?min, maintained at 90% for 5?min, and then back to initial conditions. The nanoUPLC was coupled online through a nanoscale electrospray ionization (nanoESI) emitter (10-μm tip; New Objective, Woburn, MA, USA) to a quadrupole Orbitrap mass spectrometer (Q Exactive Plus; Thermo Scientific) using a FlexIon nanospray apparatus (Proxeon). Data were acquired in data-dependent acquisition (DDA) mode, using a Top20 method (53). MS1 resolution was set to 70,000 (at 400?m/z), mass range of 300 to 1,650?m/z, and automatic gain control (AGC) of 3e6, and the maximum injection time was set to 20?ms. MS2 resolution was set to 17,500, quadrupole isolation 1.7?m/z, AGC of 1e6, dynamic exclusion of 60?s, and a maximum injection time of 60?ms.Raw data were processed using Proteome Discoverer v1.4.1. Tandem mass spectrometry (MS/MS) spectra were searched using Mascot v2.4 (Matrix Sciences) and Sequest HT. Data were searched against the carrot (Daucus carota ) and L. mesenteroides protein sequences as downloaded from UniProt (http://www.uniprot.org/), appended with common laboratory contaminant proteins. A fixed modification was set to carbamidomethylation of cysteines. Variable modifications were set to methionine oxidation and asparagine and glutamine deamidation. The proteins were then grouped on the basis of shared peptides, and the identifications were filtered such that the global false discovery rate was a maximum of 1%. The Mascot score for each protein identification represents the statistical confidence score in protein identification. It is the sum of the Mascot score of each peptide identified from the given protein, where the score of each peptide is reported as ?10?log10(P), where P is the absolute probability of peptide identification. Mascot scores of >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. 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