Intraspecies polymorphisms of Kluyveromyces marxianus strains from Yaghnob valley

Intraspecies polymorphisms of Kluyveromyces marxianus strains from Yaghnob valley Abstract In this study, 29 strains of Kluyveromyces marxianus with peculiar genetic and phenotypic traits previously isolated from a fermented goat milk of Yaghnob valley were investigated for chromosome length polymorphism (CLP) by PFGE, adhesion properties and carbon usage by Biolog analysis. Obtained data showed that strains differed in terms of number and size of chromosome bands. The number of bands ranged from 5 to 7, suggesting a probable genome size from 1.4 to 2.6 Mb. Strains showed a certain level of cell surface hydrophobicity ranging from 32% to 77.7%. Strains were also tested for their ability to form a biofilm on polystyrene plates: planktonic cells ranged from 6.3 cfu/mL to 7.95 cfu/mL, while sessile from 7.11 cfu/mL to 8.6 cfu/mL. The strains able to adhere to polystyrene plates were also able to form a mature MAT. Biolog analysis revealed that almost all strains were able to use putrescine, malic acid, α-D lactose, phenylethylamine, β-methyl D-gucoside and xylose; 5 strains were able to grow on cellobiose and 3 were able to catabolise α-ketobutyric. The obtained data highlighted a number of interesting features underlying the peculiar capacities of these strains for industrial applications. Kluyveromyces marxianus, Biolog, fermented goat milk, PFGE, adhesion properties, MAT INTRODUCTION Kluyveromyces marxianus is an emerging and promising non-Saccharomyces yeast for biotechnological applications and for some aspects could compete with Saccharomyces cerevisiae (Fonseca et al.2008; Lane and Morrissey 2010). Kluyveromycesmarxianus has been found suitable for biotechnological applications, based on its ability to ferment lactose, its rapid growth, thermotolerance, production of enzymes, bioingredients and aroma compounds (Lane et al.2011; Arrizon et al.2012; Morrissey et al.2015). Because of its strong association with foods, milk and dairy products, it obtained Qualified Presumption of Safety and Generally Regarded as Safe status from the European Food Safety Authority. Although these traits could be considered characteristics of the species, many strains of K. marxianus possess peculiar properties that differentiate them (Lane and Morrissey 2010). Several studies highlighted rich genetic intraspecific polymorphism and different phenotypic features even in strains having the same origin (Belloch et al.1998; Tofalo et al.2014; Fasoli et al.2015). Despite the diverse industrial applications of K. marxianus, there is little systematic comparison of strains, nor of traits in the species (Belloch et al.1998; Lane et al.2011; Tofalo et al.2014; Fasoli et al.2015, 2016; Qvirist et al.2016), and there is a need to have a better understanding of the physiological and genetic variation within this species. Recently, Qvirist et al. (2016) reported that K. marxianus strains from fermented goat milk of Yaghnob valley (an unexplored region of Tajikistan with a low impact from anthropogenic activities and pressures), showed high variability and peculiar genetic (nucleotide variation in the internal transcribed spacer (ITS) region) and phenotypic traits in terms of pH, ethanol and organic acid concentration tolerance, phytate utilization, resistance toward high temperatures, oxidative stress and antifungal tolerance. These features are highly required for biotechnological applications. For instance, traits such as phytate activity, ethanol and lactic acid tolerance can suggest their use in food fermentation especially where yeast and lactic acid bacteria co-fermentation occurs (Di Cagno et al.2014). The analysis of these strains provides unique opportunity to study undisturbed natural population genetics and metabolism suggesting new potential biotechnological roles. In this study, we propose a polyphasic characterization of natural K. marxianus strains. After evaluating intraspecific diversity and metabolic versatility using pulsed field gel electrophoresis (PFGE) and phenotype microarray, respectively, strains were tested for membrane hydrophobicity and adhesion properties. MATERIAL AND METHODS Yeast strains A total of 29 Kluyveromyces marxianus strains, belonging to the Department of Agricultural Sciences (University of Bologna, Italy), (AL1, AL2, AL3, AL4, AL5, BL1, BL3, BL4, BL5, BL6, BL7, BL8, BL12, BL13, BL14, CL5, CL6, DL2, DL4, DL5, DL6, DL10a, DL10b, DL11, DL12, TJY-52, TJY-54, TJY-59 and TJY-60) isolated from fermented goat milk of the Yaghnob valley were studied. The upper valley of the Yaghnob river is located between 2200 and 2600 m above sea level in the Upper-Zarafshan area (North-West Tajikistan). The residual population of Yaghnobis in this area is estimated in less than 500 individuals that rely on an economy of subsistence funded on farming and agriculture (Panaino, Gariboldi and Ognibene 2013). The strains were stored at –80°C in YPD broth (yeast extract 1% w v−1, peptone 2% w v−1, glucose 2% w v−1; Oxoid, Milan, Italy) supplemented with glycerol (20% v v−1; Sigma-Aldrich Srl, Milan, Italy). Pulsed field gel electrophoresis analysis DNA was prepared in agarose plugs as described by Carle and Olson (1985). Chromosome length polymorphism (CLP) was performed by CHEF-DR III electrophoresis unit (Bio-Rad, Milan, Italy) as previously described (Fasoli et al.2015), using DNA-PFGE marker Hansenula wingei (Bio-Rad) to determine the chromosomal sizes. Gels were stained with ethidium bromide 0.5 μg mL−1 for 30 min, washed with deionized water and visualized with UV transillumination using a Gel Doc 2000 EQ System (Bio-Rad). PFGE profiles were grouped using the software Fingerprinting II InformatixTM (Bio-Rad) and similarities between profiles were calculated on the basis of the Pearson's r correlation matrix with the unweighted pair group method using arithmetic averages (UPGMA). Biolog analysis In this study, Biolog ecoplates (Biolog Inc., Hayward, CA, USA) were used to evaluate the catabolic profile of a K. marxianus strains according to the manufacturer's instructions. Cells were initially grown in YPD and then inoculated in each well adjusting the optical density (OD)600 nm at 0.1. Ecoplates were incubated at 30°C for 72 h in the omnilog machine (Biolog Inc.). The omnilog reader photographs the plates at intervals of 15 min to measure dye conversion. The pixel intensity in each well is then converted to a signal value reflecting cell growth. After completion of the run, the signal data are compiled and exported from the Biolog software and compiled using Microsoft Excel. Negative control measurement was obtained from a well containing water. Membrane hydrophobicity and adhesion properties Cell surface hydrophobicity assay Cell surface hydrophobicity (CSH) was assessed according to Bellon-Fontaine, Rault and Van Oss (1996). Briefly, yeast cells grown overnight at 30°C were harvested and washed twice and suspended in PBS to an OD600nm of 0.8 (A0). Three milliliter of this yeast suspension was added to 1 mL of n-hexadecane (Sigma-Aldrich). After 10-min incubation at room temperature, the two-phase system was mixed by vortexing for 5 min at 1400 rpm. After 15-min incubation at room temperature, the aqueous phase was removed and its OD600 nm was measured (A1). The percentage of hydrophobicity was calculated as follows: hydrophobicity (%) = [1 − (A1/A0)] × 100. All assays are performed in triplicate. Adhesion to polystyrene plates and MATS structure formation Adhesion to 96-well polystyrene plate (flat bottom; Nunc, Roskilde, Denmark) was performed as previously described (Perpetuini et al.2018). After incubation, non-adherent cells were removed by washing three times with 0.85% (w v−1) NaCl, while adherent cells were dispersed by pipetting up and down 10 times. Both planktonic and sessile cells were serially diluted, and plated on YPD medium for cell count. The ability to form an elaborate pattern of multicellular growth (MAT formation) was tested according to Reynolds and Fink (2001). Strains were inoculated in the center of YPD plates containing 0.3% w v−1 agar with a toothpick and incubated at 25°C for 5 days. Plates containing 2% w v−1 agar were used as negative controls. All analyses were performed in triplicate. Plates were photographed at the end of incubation. RESULTS Chromosome length polymorphism CLP of 29 Kluyveromyces marxianus strains was assessed by PFGE. At the beginning, the reproducibility was checked performing 3 separate trials starting from DNA extracted from 3 separated cultures of the same strain and no changes in the electrophoretic patterns were observed. Results showed a certain degree of chromosome polymorphisms in terms of size, number and intensity of the bands. Some assumptions are necessary to interpret these results. According to Belloch et al. (1998), we assumed that individual bands correspond to intact chromosomes, some factors such as differences in chromosomal ploidy, can affect the intensity of the stained bands in the gels. Therefore, faint bands correspond to single chromosomes, and the thicker and brighter bands represent several chromosomes of similar or same size, respectively. However, some exceptions can be detected because the intensity of the bands decreases as the chromosomal size increases, due to a diffusing effect suffered by the heaviest bands (Doi et al.1992). For instance, the band between 1.37 Mb and 1.66 Mb showed significantly increased fluorescence intensity compared with the other bands, suggesting that it results from co-migration of chromosomes. Karyotype profiles were compared by cluster analysis and applying a similarity level of 80% 5 clusters were identified (from I to V; Fig. 1). Two main groups were obtained (I and V) containing 12 and 10 strains, respectively, while the others were made up of 2 (II and III) or 3 strains (IV). The number of bands ranged from 5 to 7, with the majority of strains (18 out of 29) having 6 bands and only 1 strain (TJY-59) having 7 bands. Their sizes ranged from 1 to 2 Mb suggesting a probable genome size from 7.7 to 12.6 Mb. Figure 1. View largeDownload slide Karyotype profiles of K. marxianus strains and dendrogram tree showing the clustering based on CLP evaluated by UPGMA. Size in base pairs (bp) of one fragment of ITS1-4 region after restriction digestion with HinfI enzyme: 240, 185, 120, 80 usual K. marxianus profile (Esteve-Zarzoso et al.1999), 240, 185, 140, 80 atypical profile (Qvirist et al.2016). Figure 1. View largeDownload slide Karyotype profiles of K. marxianus strains and dendrogram tree showing the clustering based on CLP evaluated by UPGMA. Size in base pairs (bp) of one fragment of ITS1-4 region after restriction digestion with HinfI enzyme: 240, 185, 120, 80 usual K. marxianus profile (Esteve-Zarzoso et al.1999), 240, 185, 140, 80 atypical profile (Qvirist et al.2016). Biolog analysis Biolog analysis was performed to evaluate the metabolic versatility of tested strains. Almost all strains were able to use putrescine, malic acid, α-D lactose, phenylethylamine, β-methyl D-gucoside and xylose (Fig. 2A). Five strains (AL5, BL6, DL4, AL2 and DL12) were able to grow on cellobiose, and 4 (AL5, BL6, DL4 and AL2) were able to catabolise α-ketobutyric acid that is the precursor of sotolon (3-hydroxy-4,5-dimethyl-2(5H)-furanone), an aroma compound responsible of burnt, sugar and curry flavor in dairy products (Carunchiawhetstine et al.2003). Regarding the growth kinetics in lactose, four different profiles were identified (Fig. 2B). The majority of strains belonged to profile II reaching omnilog values of 200 after 72 h (BL3, DL5, TJY-52, DL10a, DL4, BL8, BL12, BL13, BL14, CL5, DL2, DL6, DL10b, DL11, TJY-54 and TJY-60). Two strains (TJY-52 and DL12) showed a low efficiency of lactose usage, while 3 (BL6, AL2, AL5) were characterized by a strong catabolic activity (profile I). The other 8 strains belonged to profile III showing intermediate values compared to other profiles. Figure 2. View largeDownload slide (A) Heat map showing the metabolic differences among strains with green referring to high catabolic activity and red to low activity. Data were expressed as logR/R0 with R referring to omnilog values obtained after 72 h and R0 to the initial values (T0). (B) Different growth kinetics of strains in presence of lactose. Figure 2. View largeDownload slide (A) Heat map showing the metabolic differences among strains with green referring to high catabolic activity and red to low activity. Data were expressed as logR/R0 with R referring to omnilog values obtained after 72 h and R0 to the initial values (T0). (B) Different growth kinetics of strains in presence of lactose. Membrane hydrophobicity and adhesion properties In order to investigate the relationship between CSH and adhesion properties, the ability of tested strains to form biofilms on abiotic surfaces and MATS structures was analyzed. All 29 strains showed a certain level of CSH with values varying over a wide range, from 32% (AL4) to 77.7% (AL3; Fig. 3A). A certain relation between CSH and CLP was found: 38% of the strains with CSH values higher than 60% belonged to cluster V with the only exception of TJY-59 that clustered together with BL14 and BL13 in cluster IV. Figure 3. View largeDownload slide (A) CSH and strains adhesion to polystyrene plates determined by plate count. (B) MAT structures formed by K. marxianus strains on YPD added 0.3% (w/v) agar. Figure 3. View largeDownload slide (A) CSH and strains adhesion to polystyrene plates determined by plate count. (B) MAT structures formed by K. marxianus strains on YPD added 0.3% (w/v) agar. To be able to assess the ability to form biofilm, strains were grown on polystyrene 96-well plates, and the number of viable planktonic and sessile cells was determined (Fig. 3A). All strains showed a stable, dimorphic growth pattern on polystyrene with both planktonic and biofilm-forming cells. The average of non-attached cells ranged from 6.3 cfu mL−1 (DL10a) to 7.95 cfu mL−1 (BL13), while cells in biofilms from 7.11 cfu mL−1 (BL4) to 8.6 cfu mL−1 (BL13). Sessile life style was predominant in 7 strains (AL3, AL5, BL13, DL10a, DL11, TJY-54 and TJY-60). The ability to form biofilms on the polystyrene surfaces did not reflect the CSH, suggesting that the adhesive strains were not hydrophobic. However, strains preferring a sessile life style were the only ones able to form a mature MAT, composed of a central hub and a leading edge (or rim) that is smooth in appearance with more or less jagged edges (Fig. 3B). DISCUSSION Yaghnob valley is a remote area of Tajikistan and its inhabitants—the Yaghnobis—could be considered as an ethno-linguistic minority living completely isolated from other flanking regions. It is a high-altitude area, partially isolated and virtually inaccessible for several months of the year, due to considerable snowfall and the absence of roads between most of the villages present in the valley (Cilli et al.2011). As other isolated zone, it could represent a precious microbial reservoir since it is poorly contaminated. In this study, K. marxianus strains from Tajikistan isolated by Qvirist et al. (2016) were investigated to deliver advanced knowledge at species level and provide a good basis for K. marxianus exploitation in industrial food or feed processes. CLP data agreed with other authors who observed a species-specific pattern constituted by eight chromosomes resolved into six electrophoretic bands (Belloch et al.1998; Fasoli et al.2015). Fasoli et al. (2015) analyzed K. marxianus strains from different Italian cheeses and observed that 71% of strains isolated from Pecorino di Farindola displayed a similar banding pattern to our strains. Probably, the similarity in the karyotypes could be due to similar sources of isolation (dairy products) or concordances with some of the old species assignations (Kluyveromyces bulgaricus, Kluyveromyces cicerisporus, Kluyveromyces fragilis and Kluyveromyces wickenii; Belloch et al.1998). However, some Yaghnob strains showed some variations of this arrangement with 5 or 7 bands. The predominance of strains with 5 chromosomal bands was also reported by Crafack et al. (2013) who reported this specific karyotype in K. marxianus strains isolated from cocoa beans. In general, the presence of different chromosome arrangements could be a result of the reproductive isolation of tested strains and could reflect a wide haplotype diversity as observed in other K. marxianus strains of different origin (Belloch et al.1997; Suzzi et al.2000). The polymorphism in chromosome number and subsequently in the genome size, suggests a certain degree of chromosomal plasticity within this species that could be exploited in food and industrial biotechnology, similarly to S. cerevisiae (Mattenberger et al.2017). This genetic diversity suggested possible differences at phenotypic level; therefore, strains were tested for metabolic versatility and adhesion properties. To acquire some issues about the metabolic capabilities of tested strains, Biolog analysis was performed. Several phenotypic differences based on their ability to utilize ecoplate substrates were noted. However, it was not possible to establish a direct correlation between the CLP profiles and the metabolic activity. Several recent studies have revealed notable limitations in explaining genotype–metabolic phenotype relations, and the ability to predict the dependence of the metabolic flux distribution on the gene status (presence/absence) has been found to be poor (Jouhten et al.2016; Pereira, Nielsen and Rocha 2016). Of particular interest was the ability of strains to catabolize putrescine, a biogenic amine generally present in several fermented foods such as cheese (Schirone et al.2012). This feature has been firstly proposed by Corpillo et al. (2003), who purified and characterized an amine oxidase (KMAO) from this yeast that belongs to the class of copper-containing amine oxidases (E.C. 1.4.3.6) and is induced by putrescine and, very strongly, by copper (II). The ability of K. marxianus to use lactose and xylose, carbohydrates that are not readily fermented by S. cerevisiae (Kurtzman, Fell and Boekhout 2011) is of great interest, since they are waste products coming from dairy industries (lactose) and forestry (xylose) (Zhang et al.2017). Some differences can be noted in the growth kinetics in presence of lactose (Fig. 3B) in agreement with other studies which reported that this trait is strain dependent (Grba et al.2002; Lane et al.2011). Recently, Varela et al. (2017) demonstrated that the main gene responsible of lactose uptake in K. marxianus is LAC12, which is present in multiple copy in the genome. Strains that failed to transport lactose showed variation in 13 amino acids in the Lac12p protein, making the protein non-functional for lactose transport. Therefore, the slow growth kinetic observed for BL6 and DL4 could be related to the presence of non-functional copies of LAC12. Moreover, 7 strains were able to grow on cellobiose catalyzing the hydrolysis of β 1-4 glycosidic bonds found in cellulose and likely facilitate growth on this substrate. The natural capacity to produce ethanol from lignocellulosic substrates (second-generation ethanol) is of great significance for bioethanol technology. Yeast adhesion and biofilm formation on cheese surfaces are a crucial prerequisite for their establishment and growth on the surface and contribution to the final product quality (Mortensen et al.2005; Gori et al.2011). The studied strains showed differences in terms of biofilm formation and hydrophobicity. Generally, it was not possible to establish a clear relationship between CLP and adhesion properties; however, strains showing the highest values of CSH grouped all together in cluster V with few exceptions confirming the difficulty to find a correlation between phenotype and genotype (Jouhten et al.2016; Bòdi et al.2017; Chari and Church 2017). Biofilm-forming capacity on polystyrene plates was not correlated with CSH: strains showing adhesive characteristics were not hydrophobic under our conditions. This result has been also found in other yeast species such as Candida albicans (Raut, Rathod and Karuppayil 2010), Trichosporon asahii (Ichikawa et al.2017) or Pichia manshurica (Perpetuini et al.2018) suggesting that hydrophobicity alone is not responsible for adhesion and cannot be used as predictor of biofilm formation. However, it is interesting to note that strains with a preference for the sessile life style formed a mature MAT. This phenomenon has been widely investigated in S. cerevisiae. Hope and Dunham (2014) demonstrated that biofilm-related phenotypes, such as complex colony morphology, complex MAT formation, flocculation, agar invasion, polystyrene adhesion and psuedohyphal growth are uncorrelated or weakly correlated except for complex colony and MAT formation, also showing that the phenotypic strength varies significantly depending on ploidy. Biofilm formation in S. cerevisiae is normally related to the expression of FLO11 gene (Reynolds and Fink 2001; Zara et al.2005), even though recent studies demonstrated that additional genes like FLO1, TUP1, ACE2 and FLO9 can play a fundamental role in biofilm formation (Hope et al.2017). The analysis of K. marxianus genome sequence revealed the absence of FLO11 orthologues, suggesting the possible presence of specific genes involved in the regulation of this phenotype. CONCLUSION This study revealed the presence of interesting features in K. marxianus population from the Yaghnob valley. The ability to metabolize putrescine together with the ability to ferment lactose, xylose and cellobiose could represent an important feature for its industrial application. In addition, as suggested by recent evidence, phenotypic heterogeneity of genetically identical cells can generate non-heritable variation in a population facilitating adaptation to adverse conditions in the wild (Bòdi et al.2017). The study of phenotypic traits and specific adaptations of yeast strains for exploitation in industrial niches and their correlation with genetic diversity have only begun to be clarified recently (Gallone et al.2018). The ecological background, the origin of the strains and the data analysis are essential to improve the understanding of genotype–environment–phenotype relationships (Jouhten et al.2016). Phenotypic variability can be considered a sort of evolving trait in population facing a strong selective pressure as probably happens in this remote area. 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Intraspecies polymorphisms of Kluyveromyces marxianus strains from Yaghnob valley

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Abstract

Abstract In this study, 29 strains of Kluyveromyces marxianus with peculiar genetic and phenotypic traits previously isolated from a fermented goat milk of Yaghnob valley were investigated for chromosome length polymorphism (CLP) by PFGE, adhesion properties and carbon usage by Biolog analysis. Obtained data showed that strains differed in terms of number and size of chromosome bands. The number of bands ranged from 5 to 7, suggesting a probable genome size from 1.4 to 2.6 Mb. Strains showed a certain level of cell surface hydrophobicity ranging from 32% to 77.7%. Strains were also tested for their ability to form a biofilm on polystyrene plates: planktonic cells ranged from 6.3 cfu/mL to 7.95 cfu/mL, while sessile from 7.11 cfu/mL to 8.6 cfu/mL. The strains able to adhere to polystyrene plates were also able to form a mature MAT. Biolog analysis revealed that almost all strains were able to use putrescine, malic acid, α-D lactose, phenylethylamine, β-methyl D-gucoside and xylose; 5 strains were able to grow on cellobiose and 3 were able to catabolise α-ketobutyric. The obtained data highlighted a number of interesting features underlying the peculiar capacities of these strains for industrial applications. Kluyveromyces marxianus, Biolog, fermented goat milk, PFGE, adhesion properties, MAT INTRODUCTION Kluyveromyces marxianus is an emerging and promising non-Saccharomyces yeast for biotechnological applications and for some aspects could compete with Saccharomyces cerevisiae (Fonseca et al.2008; Lane and Morrissey 2010). Kluyveromycesmarxianus has been found suitable for biotechnological applications, based on its ability to ferment lactose, its rapid growth, thermotolerance, production of enzymes, bioingredients and aroma compounds (Lane et al.2011; Arrizon et al.2012; Morrissey et al.2015). Because of its strong association with foods, milk and dairy products, it obtained Qualified Presumption of Safety and Generally Regarded as Safe status from the European Food Safety Authority. Although these traits could be considered characteristics of the species, many strains of K. marxianus possess peculiar properties that differentiate them (Lane and Morrissey 2010). Several studies highlighted rich genetic intraspecific polymorphism and different phenotypic features even in strains having the same origin (Belloch et al.1998; Tofalo et al.2014; Fasoli et al.2015). Despite the diverse industrial applications of K. marxianus, there is little systematic comparison of strains, nor of traits in the species (Belloch et al.1998; Lane et al.2011; Tofalo et al.2014; Fasoli et al.2015, 2016; Qvirist et al.2016), and there is a need to have a better understanding of the physiological and genetic variation within this species. Recently, Qvirist et al. (2016) reported that K. marxianus strains from fermented goat milk of Yaghnob valley (an unexplored region of Tajikistan with a low impact from anthropogenic activities and pressures), showed high variability and peculiar genetic (nucleotide variation in the internal transcribed spacer (ITS) region) and phenotypic traits in terms of pH, ethanol and organic acid concentration tolerance, phytate utilization, resistance toward high temperatures, oxidative stress and antifungal tolerance. These features are highly required for biotechnological applications. For instance, traits such as phytate activity, ethanol and lactic acid tolerance can suggest their use in food fermentation especially where yeast and lactic acid bacteria co-fermentation occurs (Di Cagno et al.2014). The analysis of these strains provides unique opportunity to study undisturbed natural population genetics and metabolism suggesting new potential biotechnological roles. In this study, we propose a polyphasic characterization of natural K. marxianus strains. After evaluating intraspecific diversity and metabolic versatility using pulsed field gel electrophoresis (PFGE) and phenotype microarray, respectively, strains were tested for membrane hydrophobicity and adhesion properties. MATERIAL AND METHODS Yeast strains A total of 29 Kluyveromyces marxianus strains, belonging to the Department of Agricultural Sciences (University of Bologna, Italy), (AL1, AL2, AL3, AL4, AL5, BL1, BL3, BL4, BL5, BL6, BL7, BL8, BL12, BL13, BL14, CL5, CL6, DL2, DL4, DL5, DL6, DL10a, DL10b, DL11, DL12, TJY-52, TJY-54, TJY-59 and TJY-60) isolated from fermented goat milk of the Yaghnob valley were studied. The upper valley of the Yaghnob river is located between 2200 and 2600 m above sea level in the Upper-Zarafshan area (North-West Tajikistan). The residual population of Yaghnobis in this area is estimated in less than 500 individuals that rely on an economy of subsistence funded on farming and agriculture (Panaino, Gariboldi and Ognibene 2013). The strains were stored at –80°C in YPD broth (yeast extract 1% w v−1, peptone 2% w v−1, glucose 2% w v−1; Oxoid, Milan, Italy) supplemented with glycerol (20% v v−1; Sigma-Aldrich Srl, Milan, Italy). Pulsed field gel electrophoresis analysis DNA was prepared in agarose plugs as described by Carle and Olson (1985). Chromosome length polymorphism (CLP) was performed by CHEF-DR III electrophoresis unit (Bio-Rad, Milan, Italy) as previously described (Fasoli et al.2015), using DNA-PFGE marker Hansenula wingei (Bio-Rad) to determine the chromosomal sizes. Gels were stained with ethidium bromide 0.5 μg mL−1 for 30 min, washed with deionized water and visualized with UV transillumination using a Gel Doc 2000 EQ System (Bio-Rad). PFGE profiles were grouped using the software Fingerprinting II InformatixTM (Bio-Rad) and similarities between profiles were calculated on the basis of the Pearson's r correlation matrix with the unweighted pair group method using arithmetic averages (UPGMA). Biolog analysis In this study, Biolog ecoplates (Biolog Inc., Hayward, CA, USA) were used to evaluate the catabolic profile of a K. marxianus strains according to the manufacturer's instructions. Cells were initially grown in YPD and then inoculated in each well adjusting the optical density (OD)600 nm at 0.1. Ecoplates were incubated at 30°C for 72 h in the omnilog machine (Biolog Inc.). The omnilog reader photographs the plates at intervals of 15 min to measure dye conversion. The pixel intensity in each well is then converted to a signal value reflecting cell growth. After completion of the run, the signal data are compiled and exported from the Biolog software and compiled using Microsoft Excel. Negative control measurement was obtained from a well containing water. Membrane hydrophobicity and adhesion properties Cell surface hydrophobicity assay Cell surface hydrophobicity (CSH) was assessed according to Bellon-Fontaine, Rault and Van Oss (1996). Briefly, yeast cells grown overnight at 30°C were harvested and washed twice and suspended in PBS to an OD600nm of 0.8 (A0). Three milliliter of this yeast suspension was added to 1 mL of n-hexadecane (Sigma-Aldrich). After 10-min incubation at room temperature, the two-phase system was mixed by vortexing for 5 min at 1400 rpm. After 15-min incubation at room temperature, the aqueous phase was removed and its OD600 nm was measured (A1). The percentage of hydrophobicity was calculated as follows: hydrophobicity (%) = [1 − (A1/A0)] × 100. All assays are performed in triplicate. Adhesion to polystyrene plates and MATS structure formation Adhesion to 96-well polystyrene plate (flat bottom; Nunc, Roskilde, Denmark) was performed as previously described (Perpetuini et al.2018). After incubation, non-adherent cells were removed by washing three times with 0.85% (w v−1) NaCl, while adherent cells were dispersed by pipetting up and down 10 times. Both planktonic and sessile cells were serially diluted, and plated on YPD medium for cell count. The ability to form an elaborate pattern of multicellular growth (MAT formation) was tested according to Reynolds and Fink (2001). Strains were inoculated in the center of YPD plates containing 0.3% w v−1 agar with a toothpick and incubated at 25°C for 5 days. Plates containing 2% w v−1 agar were used as negative controls. All analyses were performed in triplicate. Plates were photographed at the end of incubation. RESULTS Chromosome length polymorphism CLP of 29 Kluyveromyces marxianus strains was assessed by PFGE. At the beginning, the reproducibility was checked performing 3 separate trials starting from DNA extracted from 3 separated cultures of the same strain and no changes in the electrophoretic patterns were observed. Results showed a certain degree of chromosome polymorphisms in terms of size, number and intensity of the bands. Some assumptions are necessary to interpret these results. According to Belloch et al. (1998), we assumed that individual bands correspond to intact chromosomes, some factors such as differences in chromosomal ploidy, can affect the intensity of the stained bands in the gels. Therefore, faint bands correspond to single chromosomes, and the thicker and brighter bands represent several chromosomes of similar or same size, respectively. However, some exceptions can be detected because the intensity of the bands decreases as the chromosomal size increases, due to a diffusing effect suffered by the heaviest bands (Doi et al.1992). For instance, the band between 1.37 Mb and 1.66 Mb showed significantly increased fluorescence intensity compared with the other bands, suggesting that it results from co-migration of chromosomes. Karyotype profiles were compared by cluster analysis and applying a similarity level of 80% 5 clusters were identified (from I to V; Fig. 1). Two main groups were obtained (I and V) containing 12 and 10 strains, respectively, while the others were made up of 2 (II and III) or 3 strains (IV). The number of bands ranged from 5 to 7, with the majority of strains (18 out of 29) having 6 bands and only 1 strain (TJY-59) having 7 bands. Their sizes ranged from 1 to 2 Mb suggesting a probable genome size from 7.7 to 12.6 Mb. Figure 1. View largeDownload slide Karyotype profiles of K. marxianus strains and dendrogram tree showing the clustering based on CLP evaluated by UPGMA. Size in base pairs (bp) of one fragment of ITS1-4 region after restriction digestion with HinfI enzyme: 240, 185, 120, 80 usual K. marxianus profile (Esteve-Zarzoso et al.1999), 240, 185, 140, 80 atypical profile (Qvirist et al.2016). Figure 1. View largeDownload slide Karyotype profiles of K. marxianus strains and dendrogram tree showing the clustering based on CLP evaluated by UPGMA. Size in base pairs (bp) of one fragment of ITS1-4 region after restriction digestion with HinfI enzyme: 240, 185, 120, 80 usual K. marxianus profile (Esteve-Zarzoso et al.1999), 240, 185, 140, 80 atypical profile (Qvirist et al.2016). Biolog analysis Biolog analysis was performed to evaluate the metabolic versatility of tested strains. Almost all strains were able to use putrescine, malic acid, α-D lactose, phenylethylamine, β-methyl D-gucoside and xylose (Fig. 2A). Five strains (AL5, BL6, DL4, AL2 and DL12) were able to grow on cellobiose, and 4 (AL5, BL6, DL4 and AL2) were able to catabolise α-ketobutyric acid that is the precursor of sotolon (3-hydroxy-4,5-dimethyl-2(5H)-furanone), an aroma compound responsible of burnt, sugar and curry flavor in dairy products (Carunchiawhetstine et al.2003). Regarding the growth kinetics in lactose, four different profiles were identified (Fig. 2B). The majority of strains belonged to profile II reaching omnilog values of 200 after 72 h (BL3, DL5, TJY-52, DL10a, DL4, BL8, BL12, BL13, BL14, CL5, DL2, DL6, DL10b, DL11, TJY-54 and TJY-60). Two strains (TJY-52 and DL12) showed a low efficiency of lactose usage, while 3 (BL6, AL2, AL5) were characterized by a strong catabolic activity (profile I). The other 8 strains belonged to profile III showing intermediate values compared to other profiles. Figure 2. View largeDownload slide (A) Heat map showing the metabolic differences among strains with green referring to high catabolic activity and red to low activity. Data were expressed as logR/R0 with R referring to omnilog values obtained after 72 h and R0 to the initial values (T0). (B) Different growth kinetics of strains in presence of lactose. Figure 2. View largeDownload slide (A) Heat map showing the metabolic differences among strains with green referring to high catabolic activity and red to low activity. Data were expressed as logR/R0 with R referring to omnilog values obtained after 72 h and R0 to the initial values (T0). (B) Different growth kinetics of strains in presence of lactose. Membrane hydrophobicity and adhesion properties In order to investigate the relationship between CSH and adhesion properties, the ability of tested strains to form biofilms on abiotic surfaces and MATS structures was analyzed. All 29 strains showed a certain level of CSH with values varying over a wide range, from 32% (AL4) to 77.7% (AL3; Fig. 3A). A certain relation between CSH and CLP was found: 38% of the strains with CSH values higher than 60% belonged to cluster V with the only exception of TJY-59 that clustered together with BL14 and BL13 in cluster IV. Figure 3. View largeDownload slide (A) CSH and strains adhesion to polystyrene plates determined by plate count. (B) MAT structures formed by K. marxianus strains on YPD added 0.3% (w/v) agar. Figure 3. View largeDownload slide (A) CSH and strains adhesion to polystyrene plates determined by plate count. (B) MAT structures formed by K. marxianus strains on YPD added 0.3% (w/v) agar. To be able to assess the ability to form biofilm, strains were grown on polystyrene 96-well plates, and the number of viable planktonic and sessile cells was determined (Fig. 3A). All strains showed a stable, dimorphic growth pattern on polystyrene with both planktonic and biofilm-forming cells. The average of non-attached cells ranged from 6.3 cfu mL−1 (DL10a) to 7.95 cfu mL−1 (BL13), while cells in biofilms from 7.11 cfu mL−1 (BL4) to 8.6 cfu mL−1 (BL13). Sessile life style was predominant in 7 strains (AL3, AL5, BL13, DL10a, DL11, TJY-54 and TJY-60). The ability to form biofilms on the polystyrene surfaces did not reflect the CSH, suggesting that the adhesive strains were not hydrophobic. However, strains preferring a sessile life style were the only ones able to form a mature MAT, composed of a central hub and a leading edge (or rim) that is smooth in appearance with more or less jagged edges (Fig. 3B). DISCUSSION Yaghnob valley is a remote area of Tajikistan and its inhabitants—the Yaghnobis—could be considered as an ethno-linguistic minority living completely isolated from other flanking regions. It is a high-altitude area, partially isolated and virtually inaccessible for several months of the year, due to considerable snowfall and the absence of roads between most of the villages present in the valley (Cilli et al.2011). As other isolated zone, it could represent a precious microbial reservoir since it is poorly contaminated. In this study, K. marxianus strains from Tajikistan isolated by Qvirist et al. (2016) were investigated to deliver advanced knowledge at species level and provide a good basis for K. marxianus exploitation in industrial food or feed processes. CLP data agreed with other authors who observed a species-specific pattern constituted by eight chromosomes resolved into six electrophoretic bands (Belloch et al.1998; Fasoli et al.2015). Fasoli et al. (2015) analyzed K. marxianus strains from different Italian cheeses and observed that 71% of strains isolated from Pecorino di Farindola displayed a similar banding pattern to our strains. Probably, the similarity in the karyotypes could be due to similar sources of isolation (dairy products) or concordances with some of the old species assignations (Kluyveromyces bulgaricus, Kluyveromyces cicerisporus, Kluyveromyces fragilis and Kluyveromyces wickenii; Belloch et al.1998). However, some Yaghnob strains showed some variations of this arrangement with 5 or 7 bands. The predominance of strains with 5 chromosomal bands was also reported by Crafack et al. (2013) who reported this specific karyotype in K. marxianus strains isolated from cocoa beans. In general, the presence of different chromosome arrangements could be a result of the reproductive isolation of tested strains and could reflect a wide haplotype diversity as observed in other K. marxianus strains of different origin (Belloch et al.1997; Suzzi et al.2000). The polymorphism in chromosome number and subsequently in the genome size, suggests a certain degree of chromosomal plasticity within this species that could be exploited in food and industrial biotechnology, similarly to S. cerevisiae (Mattenberger et al.2017). This genetic diversity suggested possible differences at phenotypic level; therefore, strains were tested for metabolic versatility and adhesion properties. To acquire some issues about the metabolic capabilities of tested strains, Biolog analysis was performed. Several phenotypic differences based on their ability to utilize ecoplate substrates were noted. However, it was not possible to establish a direct correlation between the CLP profiles and the metabolic activity. Several recent studies have revealed notable limitations in explaining genotype–metabolic phenotype relations, and the ability to predict the dependence of the metabolic flux distribution on the gene status (presence/absence) has been found to be poor (Jouhten et al.2016; Pereira, Nielsen and Rocha 2016). Of particular interest was the ability of strains to catabolize putrescine, a biogenic amine generally present in several fermented foods such as cheese (Schirone et al.2012). This feature has been firstly proposed by Corpillo et al. (2003), who purified and characterized an amine oxidase (KMAO) from this yeast that belongs to the class of copper-containing amine oxidases (E.C. 1.4.3.6) and is induced by putrescine and, very strongly, by copper (II). The ability of K. marxianus to use lactose and xylose, carbohydrates that are not readily fermented by S. cerevisiae (Kurtzman, Fell and Boekhout 2011) is of great interest, since they are waste products coming from dairy industries (lactose) and forestry (xylose) (Zhang et al.2017). Some differences can be noted in the growth kinetics in presence of lactose (Fig. 3B) in agreement with other studies which reported that this trait is strain dependent (Grba et al.2002; Lane et al.2011). Recently, Varela et al. (2017) demonstrated that the main gene responsible of lactose uptake in K. marxianus is LAC12, which is present in multiple copy in the genome. Strains that failed to transport lactose showed variation in 13 amino acids in the Lac12p protein, making the protein non-functional for lactose transport. Therefore, the slow growth kinetic observed for BL6 and DL4 could be related to the presence of non-functional copies of LAC12. Moreover, 7 strains were able to grow on cellobiose catalyzing the hydrolysis of β 1-4 glycosidic bonds found in cellulose and likely facilitate growth on this substrate. The natural capacity to produce ethanol from lignocellulosic substrates (second-generation ethanol) is of great significance for bioethanol technology. Yeast adhesion and biofilm formation on cheese surfaces are a crucial prerequisite for their establishment and growth on the surface and contribution to the final product quality (Mortensen et al.2005; Gori et al.2011). The studied strains showed differences in terms of biofilm formation and hydrophobicity. Generally, it was not possible to establish a clear relationship between CLP and adhesion properties; however, strains showing the highest values of CSH grouped all together in cluster V with few exceptions confirming the difficulty to find a correlation between phenotype and genotype (Jouhten et al.2016; Bòdi et al.2017; Chari and Church 2017). Biofilm-forming capacity on polystyrene plates was not correlated with CSH: strains showing adhesive characteristics were not hydrophobic under our conditions. This result has been also found in other yeast species such as Candida albicans (Raut, Rathod and Karuppayil 2010), Trichosporon asahii (Ichikawa et al.2017) or Pichia manshurica (Perpetuini et al.2018) suggesting that hydrophobicity alone is not responsible for adhesion and cannot be used as predictor of biofilm formation. However, it is interesting to note that strains with a preference for the sessile life style formed a mature MAT. This phenomenon has been widely investigated in S. cerevisiae. Hope and Dunham (2014) demonstrated that biofilm-related phenotypes, such as complex colony morphology, complex MAT formation, flocculation, agar invasion, polystyrene adhesion and psuedohyphal growth are uncorrelated or weakly correlated except for complex colony and MAT formation, also showing that the phenotypic strength varies significantly depending on ploidy. Biofilm formation in S. cerevisiae is normally related to the expression of FLO11 gene (Reynolds and Fink 2001; Zara et al.2005), even though recent studies demonstrated that additional genes like FLO1, TUP1, ACE2 and FLO9 can play a fundamental role in biofilm formation (Hope et al.2017). The analysis of K. marxianus genome sequence revealed the absence of FLO11 orthologues, suggesting the possible presence of specific genes involved in the regulation of this phenotype. CONCLUSION This study revealed the presence of interesting features in K. marxianus population from the Yaghnob valley. The ability to metabolize putrescine together with the ability to ferment lactose, xylose and cellobiose could represent an important feature for its industrial application. In addition, as suggested by recent evidence, phenotypic heterogeneity of genetically identical cells can generate non-heritable variation in a population facilitating adaptation to adverse conditions in the wild (Bòdi et al.2017). The study of phenotypic traits and specific adaptations of yeast strains for exploitation in industrial niches and their correlation with genetic diversity have only begun to be clarified recently (Gallone et al.2018). The ecological background, the origin of the strains and the data analysis are essential to improve the understanding of genotype–environment–phenotype relationships (Jouhten et al.2016). Phenotypic variability can be considered a sort of evolving trait in population facing a strong selective pressure as probably happens in this remote area. 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FEMS Microbiology LettersOxford University Press

Published: Mar 1, 2018

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