Transcriptome analysis shows activation of the arginine deiminase pathway in Lactococcus lactis as a response to ethanol stress

Accepted Manuscript Transcriptome analysis shows activation of the arginine deiminase pathway in Lactococcus lactis as a response to ethanol stress

Lorena Díez, Ana Solopova, Rocío Fernández, Miriam González, Carmen Tenorio, Oscar P. Kuipers, Fernanda Ruiz-Larrea PII: DOI: Reference:

S0168-1605(17)30219-2 doi: 10.1016/j.ijfoodmicro.2017.05.017 FOOD 7590

To appear in:

International Journal of Food Microbiology

Received date: Revised date: Accepted date:

18 November 2016 8 May 2017 21 May 2017

Please cite this article as: Lorena Díez, Ana Solopova, Rocío Fernández, Miriam González, Carmen Tenorio, Oscar P. Kuipers, Fernanda Ruiz-Larrea , Transcriptome analysis shows activation of the arginine deiminase pathway in Lactococcus lactis as a response to ethanol stress, International Journal of Food Microbiology (2017), doi: 10.1016/j.ijfoodmicro.2017.05.017

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Transcriptome analysis shows activation of the arginine deiminase pathway in

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Lactococcus lactis as a response to ethanol stress

Lorena Díez1, Ana Solopova2, Rocío Fernández1, Miriam González 1, Carmen Tenorio1,

University of La Rioja, Instituto de Ciencias de la Vid y del Vino (CSIC, Universidad

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Oscar P. Kuipers2 and Fernanda Ruiz-Larrea1

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de La Rioja, Gobierno de La Rioja), Av. Madre de Dios 51, 26006 Logroño, Spain.

Department of Molecular Genetics, University of Groningen, Groningen Biomolecular

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Sciences and Biotechnology Institute, Nijenborgh 7, 9747 AG Groningen, The

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Netherlands

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Running title: Response of Lactococcus lactis to ethanol stress

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ABSTRACT This paper describes the molecular response of Lactococcus lactis NZ9700 to ethanol. This strain is a well-known nisin producer and a lactic acid bacteria (LAB) model strain. Global transcriptome profiling using DNA microarrays demonstrated a

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bacterial adaptive response to the presence of 2 % ethanol in the culture broth and

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differential expression of 67 genes. The highest up-regulation was detected for those genes involved in arginine degradation through the arginine deiminase (ADI) pathway

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(20-40 fold up-regulation). The metabolic responses to ethanol of wild type L. lactis

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strains were studied and compared to those of regulator-deletion mutants MGargR and MGahrC. The results showed that in the presence of 2 % ethanol those strains with an

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active ADI pathway reached higher growth rates when arginine was available in the culture broth than in absence of arginine. In a chemically defined medium strains with

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an active ADI pathway consumed arginine and produced ornithine in the presence of 2

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% ethanol, hence corroborating that arginine catabolism is involved in the bacterial response to ethanol. This is the first study of the L. lactis response to ethanol stress to

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demonstrate the relevance of arginine catabolism for bacterial adaptation and survival in

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an ethanol containing medium.

Key words: Lactococcus lactis; ethanol; stress response; ADI pathway; arginine

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INTRODUCTION Lactic acid bacteria (LAB) play an essential role in the process of fermentation of numerous foods and beverages (Bourdichon et al., 2012) giving rise to dairy products, meat and cereal-based foods (Kabak and Dobson, 2011), fermented vegetables (Hurtado

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et al., 2012; Settanni and Corsetti, 2008) and wine (Mathews et al., 2004; Mills et al.,

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2005). Lactococcus lactis has been associated with food production and preservation for centuries and it is by far the best studied of the food-related LAB, which is largely due

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to its major industrial importance as a starter in the manufacture of cheese. Its main

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activity during milk fermentation is the conversion of lactose to lactic acid, which results in the lowering of the pH in the product. Moreover, the capacity for lactate and

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bacteriocin production of L. lactis is beneficial for food preservation. During these food- and beverage related industrial processes, LAB can be exposed to a number of

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environmental stresses, among which low and high temperatures, oxidative stress, high

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osmotic pressure, acidity, nutrient starvation and the presence of ethanol are included. Growth performance and robustness to withstand environmental stresses are key

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properties for good starters. Bacterial mechanisms of stress resistance are based on bacterial adaptive responses and cross protection to those external factors. Advances in

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the genome, transcriptome and proteome research of L. lactis have turned this economically important LAB also into a widely used Gram-positive model organism (Pinto et al., 2001). L. lactis stress responses have been studied over the last years (Papadimitriou et al., 2016) and reports can be found on the response of L. lactis to osmotic stress (Sanders et al., 1998; Zhang et al., 2010), oxidative stress (Larsen et al., 2016; Miyoshi et al., 2003; Sheng et al., 2016), to both oxidative and acidic conditions (Cretenet et al., 2011), to acid stress (Budin-Verneuil et al., 2007; Carvalho et al., 2011;

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Carvalho et al., 2013; Hartke et al., 1996; Rallu et al., 1996; Sanders et al., 1995; Zhang et al., 2007), to heat- (Kim and Batt, 1993) and cold- (Panoff et al., 1994; Wouters et al, 2001) shocks, to starvation (Dressaire et al., 2011; Price et al., 2012) and to the presence of antibiotics (Dorrian et al., 2011). Cross-protective responses and interactive pathways

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have been demonstrated in a number of such responses of L. lactis to oxidative stress (Dijkstra et al., 2014; Duwat et al., 2000), osmotic, acid and thermal stress (Abdullah-

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al-Mahin et al., 2010; van de Guchte et al., 2002; Zhang et al., 2014). Cross-protection

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induced by the expression of an adaptive response to one stress agent can be advantageous for bacterial tolerance to subsequent stress conditions; it increases the

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fitness of a bacterial culture to harsh conditions and will allow an optimal performance

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of a fermentative process carried out by this culture. Ethanol is a well-known antimicrobial agent, and tolerance to ethanol may be considered an indicator of bacterial

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robustness and might become a criterion for starter selection.

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Arginine, a non-essential amino acid in L. lactis, can be synthesized de novo from glutamate in eight enzymatic steps, and is completely degraded into ornithine,

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ammonium and carbon dioxide via the arginine deiminase pathway (ADI pathway), which takes place in three enzymatic steps catalysed by the enzymes arginine deiminase

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(ArcA), ornithine carbamoyltransferase (ArcB) and carbamate kinase (ArcC). Arginine metabolism in L. lactis has been shown to be regulated by two transcriptional regulators named ArgR and AhrC (Larsen et al., 2004); both transcriptional regulators are required for repression of arginine biosynthesis in presence of the amino acid, and AhrC is an anti-repressor required to activate the ADI pathway of arginine degradation (Larsen et al., 2005). This study aimed to identify the global adaptive response of L. lactis during growth in

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the presence of ethanol, which is a notorious stress factor for bacterial growth. Additionally, the bacteriocin nisin produced by some L. lactis strains had been previously reported to exert an inhibitory effect upon LAB strains isolated from wines and responsible for wine spoilage (Rojo-Bezares et al., 2007). The putative usage of a

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nisin-producer for wine preservation was an additional issue of interest for our study. Under these oenological conditions, ethanol exposure of wine LAB strains is a

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continuous and concentration-increasing exposure. We chose the model strain L. lactis

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subsp. cremoris NZ9700, which is a well-known nisin producer, its full genome had been sequenced and had been extensively studied (de Ruyter et al., 1996; Mu et al.,

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2015), nevertheless, no reports can be found on its response to ethanol. In this work we

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studied the molecular response of L. lactis NZ9700 to 2 % ethanol exposure by wholegenome transcription profiling. To confirm and extend the obtained results, we then studied the arginine metabolism of the plasmid-free model strain L. lactis subsp.

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cremoris MG1363 and its single deletion mutants MGΔargR and MGΔahrC, whose

MATERIALS AND METHODS

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ADI pathways of arginine degradation are either expressed or repressed.

Bacterial strains and media. L. lactis strains used in this study are listed in Table 1. L. lactis was grown at 30º C in M17 broth (Terzaghi and Sandine, 1975) with 0.5 % glucose as the carbon source (GM17). A chemically defined medium (CDM) was prepared as described by Larsen et al. (2004); CDM buffer containing 15 free amino acids (CDM15) was prepared as previously described (Larsen et al., 2004). Arginine (Merck-VWR, Llinars del Vallès, Spain) stock solution was made in distilled water;

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pH was set to 7.0 with HCl. Growth and cell density were determined by measurement of the optical density at 600 nm (OD600) of the culture using a spectrophotometer (Ultraspec 2000, Pharmacia Biotech, Cambridge, UK). Transcriptome analysis using L. lactis DNA microarrays. RNA was isolated from

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cells grown to mid-exponential (OD600= 0.4) and stationary phase (OD600= 1) in GM17

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with 0 % and 2 % ethanol. Cells were harvested by centrifugation at 12,000 x g for 2 min at 4 ºC. Supernatants were discarded and cell pellets were immediately frozen in

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liquid nitrogen and stored at -80 ºC. Pellets were resuspended in 400 µl of T10E1 buffer

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(10 mM Tris-HCl pH 8.0, 1 mM Na2-EDTA), and 50 µl 10 % SDS (w/v), 500 µl phenol/chloroform: isoamyl alcohol (24/24:1) (Sigma-Aldrich Chemie, Zwijndrecht,

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Netherlands), 500 mg glass beads (50-105 µm of diameter, Fischer Scientific BV, Den Bosch, the Netherlands), and 175 µl Macaloid suspension (Bentone MA, Elementis

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Specialities Inc, Hightstown, NJ) was added. The Macaloid suspension was made as

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follows: 2 g macaloid was boiled for 5 min in 100 ml T10E1, cooled to room temperature, sonicated by bursts until a gel was formed, centrifuged and resuspended in

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50 ml T10E1. Cells were disrupted by shaking twice for 45 s in a Biospec Mini-bead Beater-8 (Biospec, OK, USA). The cell lysate was cleared by centrifugation and 500 µl

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supernatant was extracted with 500 µl chloroform:isoamyl alcohol (24:1). Total RNA was isolated from the water phase using the High Pure RNA Isolation Kit (Roche Applied Science, Mannheim, Germany) according the manufacturer´s instructions. All reagents used for RNA work were treated with diethylpyrocarbonate (DEPC) (SigmaAldrich, St. Louis, MO). RNA quantity was determined spectrophotometrically and RNA quality was verified on an Agilent Bioanalyzer 2100 using RNA 6000 LabChips (Agilent Technologies Netherlands BV, Amstelveen, the Netherlands). 20 µg total RNA

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was used for the synthesis of aminoallyl-dUTP-labelled copy DNA (cDNA) using SuperScript III Reverse Transcriptase (Life Technologies, Carlsbad, California, US). Aminoallyl-dUTPs-containing cDNA was subsequently labelled using CyDye-NHSesters Cy3 and Cy5 (Amersham Biosciences Europe GmbH). Labelled DNA was

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purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, GmbH&Co. KG, Germany). Hybridisation (16 h at 45ºC) of Cy-labeled cDNA was performed in

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Ambion Slidehyb 1 hybridisation buffer (Ambion Europe Ltd., Huntington, UK) on

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full-genome L. lactis NZ9000 DNA Microarray slides (Kuipers et al., 2002) supplemented with probes for the nisin biosynthesis-cluster genes. Slides were scanned

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using a GenePix Autoloader 4200AL scanner (Molecular Devices Corporation,

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Sunnyvale, CA).

DNA microarray data analysis. Slide images were analysed using ArrayPro 4.5

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(Media Cybernetics Inc., Silver Spring, MD) and the data processed and normalized

provided

by

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using MicroPrep software (van Hijum et al., 2003) and following standard routines GENOME2D

software

available

at

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http://genome2d.molgenrug.nl/index.php/analysis-pipeline. For each DNA microarray experiment, at least three independent biological replicates and two technical replicates

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(dye-swaps) were performed to discard possible differences due to variations in Cy3/Cy5 hybridisation. Expression ratios were calculated and a gene was considered differentially expressed when a p value of at least <0.05 was obtained and the expression fold-change was at least > 1.8. Nisin production. The production of nisin by L. lactis NZ9700 grown in presence of ethanol was determined by calculating the minimal inhibitory concentration (MIC) in

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the microtiter dilution assay (Rojo-Bezares et al, 2006) of cell free supernatants. For these experiments fresh inocula of L. lactis NZ9700 (105 cells/ml) were incubated for up to 30 h at 25 ºC in 100 ml of culture broth containing 0 %, 2 %, 4 %, 6 %, 8 % and 10 % (vol/vol) ethanol (Panreac, S.A., Barcelona, Spain) in GM17 with its

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concentration adjusted to account for the addition of ethanol. Bacterial growth was monitored by OD600. Aliquots were taken at different incubation times and centrifuged

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at 12,000 x g for 5 min at 4 ºC. Cell free supernatants were boiled in a water bath for 10

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min. Samples were tested for their antimicrobial activity by the microtiter dilution method, using serial double dilutions against the indicator strain Pediococcus

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pentosaceus FBB63 and incubation in microtiter plates at 30 ºC for 48 h following the

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method described by Rojo-Bezares et al. (2006) to determine the corresponding MIC values. Positive and negative controls were included in all the assays.

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Ethanol stress. To confirm and further investigate the results of ethanol resistance

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revealed by the transcriptome analysis, the wild type NZ9700 and MG1363 L. lactis subsp. cremoris strains and the mutant strains MGΔargR and MGΔahrC were grown at

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25º C with the following concentrations of ethanol (Panreac, S.A., Barcelona, Spain) in the culture broth: 2 %, 4 %, 6 % and 10 % (vol/vol). Growth and cell density were

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determined by measurement of the OD600 of the culture. All experiments were carried out in triplicates. Culture conditions in the CDM for strain growth were: either absence or presence of arginine (Merck, Darmsladt, Germany) (2.5 mg/ml) and either absence or presence of ethanol (Panreac, Barcelona, Spain) (2 % vol/vol) in the culture broth. Strains were incubated at 25 ºC without agitation. We determined growth curves by measuring OD600. Samples were taken at the initial moment of inoculation and after 10

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h incubation, which corresponded to the stationary growth phase. These samples were stored at -80 ºC until HPLC processing.

HPLC Analysis. Cell free supernatants obtained at stationary phase (OD600= 1) of

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growth in CDM (10 h incubation) containing either 0.25 g/L arginine or no arginine,

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with either 0 % or 2 % ethanol as described above, were analyzed by HPLC. Samples were centrifuged at 12,000 x g for 5 min at 4 ºC and were boiled in a water bath for 10

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min. Analyses were performed on a modular Agilent 1200 Series liquid chromatograph

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(Agilent Technologies, Waldbronn, Germany) equipped with one G1311A quaternary pump, an on-line G1322A degasser, a G1316A column oven, a G2913A automatic

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injector and a G1315B photodiodearray detector (DAD) controlled by the Chemstation Agilent software. Chromatographic separation was performed in an ACE HPLC column

et

al.

(2007)

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Gomez-Alonso

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(5 C18-HL) particle size 5 mm (250 mm, 4.6 mm) following the method described by with

the

derivatisation

reagent

diethyl

ethoxymethylenemalonate (Sigma-Aldrich Chemie, Steinhein, Germany). Amino acids

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were identified on the basis of the aminoenone derivative retention times of the corresponding standards (Merck, Darmstadt, Germany) and quantified using the internal

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standard method. Statistical analysis of data was performed using SPSS 12 statistical software (SPSS Inc., Chicago, IL). Analysis of variance (ANOVA) was applied for the hplc data, which showed normal distribution and homogeneous variances. IBM-SPSS Statistics 19.0 software for Windows (IBM-SPSS Inc., Chicago, IL, USA) was used for data processing.

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RESULTS L. lactis NZ9700 growth and nisin production in the presence of ethanol. To determine the effect of ethanol on cell growth and nisin production of our model strain L. lactis NZ9700, it was incubated in GM17 containing from 0 % to 10 % (vol/vol)

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ethanol as indicated in Methods section, and the results of cell growth and nisin activity

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are shown in Fig. 1. L. lactis NZ9700 was not able to grow in the presence of 8 % and 10 % ethanol. Remarkably, it was able to grow in the presence of 2 % ethanol with a

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growth rate of about half of that of control samples without ethanol in the culture broth.

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The strain still produced nisin, but only 25 % when compared to control conditions in the absence of ethanol, as shown in Fig. 1. Culture broth pH values were lower (pH

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4.88) for cultures in the absence of ethanol after 24 h incubation, than for cultures containing 2 % - 6 % ethanol (pH 5.08 - 5.15), which correlated with the higher cell

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density of ethanol-free cultures. Given these results, we chose 2 % ethanol in the culture

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broth for studying the L. lactis NZ9700 transcriptome response with DNA microarrays . Transcriptome profile of L. lactis NZ9700 grown in the presence of 2% ethanol.

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Results of the transcriptome analysis of L. lactis NZ9700 grown in GM17 containing 2

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% ethanol are shown in the supplementary material. Comparison of transcriptomes of L. lactis NZ9700 grown in the presence of 2 % ethanol with those of L. lactis NZ9700 grown under reference ethanol-free conditions showed differential expression of 67 genes in the mid-exponential growth phase. This response to ethanol involved upregulation of the expression of the following genes: 

genes of the deiminase pathway of arginine degradation (ADI pathway) (up to 42.7 fold activation) (Fig. 2)

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genes of the alcohol dehydrogenase pathway (up to 4.9 fold activation)

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genes of ABC-type multidrug resistance transporters (up to 4.3 fold activation)

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genes involved in sugar transport and metabolism (up to 4.0 fold activation)

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genes encoding stress proteins: cspC and cspD (up to 2.0 fold activation)

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gene involved in synthesis of fatty acids (1.9 fold activation)

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

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The up-regulation of expression of the majority of genes was maintained till the

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stationary phase of bacterial growth. Additionally, in the stationary phase up-regulation of cell wall protein-encoding genes was detected and the expression of two cold shock

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protein genes increased even more (Table S1 of the supplementary material).

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In contrast, down-regulation of the expression of genes during the mid-exponential growth phase (Table S2 of the supplementary material) included the following genes: genes involved in pyrimidine and purine biosynthesis (down 16.4- fold),

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iron transport genes (down 4.2-fold)

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genes of the nisin operon: nisB, nisC, nisE, nisF, nisG, nisI, nisK, nisP and

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

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nisT (down 4.3-fold).

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According to these results, the strongest response to ethanol was the activation of expression of genes encoding proteins of the ADI pathway for arginine degradation shown in Fig. 2. In the section below the results of the metabolic study carried out with wild type NZ9700 and MG1363 L. lactis strains and the deletion mutants MGΔargR and MGΔahrC are shown. Mutant MGΔargR is the deletion mutant of strain MG1363 that lacks the transcriptional regulator ArgR, which represses the expression of the ADI genes, and in combination with arginine and the transcriptional regulator AhrC

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completely repressess arginine biosynthetic pathways (Larsen et al., 2008). MGΔahrC is the deletion mutant of the transcriptional regulator AhrC, the anti-repressor that allows expression of the ADI pathway genes in presence of arginine (Larsen et al., 2004).

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Effect of ethanol on arginine metabolism. Fig. 3 shows the growth curves of

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strains L. lactis NZ9700, MG1363, MGΔargR, and MGΔahrC when grown in the chemically defined medium in the presence of 0 % and 2 % ethanol, w/o arginine (2.5

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mg/ml) in the culture broth. Fig. 3A and 3B show that both wild type strains NZ9700

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and MG1363 grew at slower rates when grown in 2 % ethanol in the absence of arginine when compared to conditions of presence of arginine in the culture broth, which

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indicates that arginine availability positively contributed to bacterial growth in the ethanol containing broth. In contrast, L. lactis mutant MGΔargR, expressing both fully

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active ADI pathway and arginine biosynthetic pathway, showed higher growth rate

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during growth in 2 % ethanol in the absence of arginine (Fig. 3C) than the wild type. Finally, Fig. 3D shows the growth curves of L. lactis mutant MGΔahrC, which does not

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express its ADI pathway. This mutant, in the presence of ethanol, showed reduced growth rates either with arginine (2.5 mg/ml) or without arginine, as it was not able to

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degrade this amino acid for obtaining energy; its growth rates in the presence of 2 % ethanol were half of the values reached in the absence of ethanol (Fig 3D). Fig. 4 shows arginine and ornithine concentrations in the bacterial culture supernatants at stationary growth phase after 10 h incubation of the L. lactis strains in the presence of either 0 % or 2 % ethanol, and either 0 mg/ml or 2.5 mg/ml arginine in the chemically defined culture broth. It is shown that ornithine was produced (>20 mg/L

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in the culture supernatant) only when arginine was available in the culture broth and only in those samples of strains expressing an active ADI pathway (wild type MG1363 and mutant MGΔargR, Fig. 4A and Fig. 4B respectively), and the highest production of ornithine (122.1 mg/L) was shown for mutant MGΔargR, with an active ADI pathway

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and no repression of its arginine biosynthetic pathways, growing in the presence of 2 % ethanol and arginine (Fig. 4B). Thus, the deletion mutant MGΔahrC (Fig. 4C), not

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expressing the ADI pathway for arginine degradation, was unable to generate ornithine

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(less than 20 mg/L threshold limit). Moreover, it is shown that more arginine was consumed in those samples grown in the presence of 2 % ethanol than in the

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corresponding samples grown in absence of ethanol.

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DISCUSSION

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We have studied the molecular responses of L. lactis subsp. cremoris to the stress due to the presence of ethanol in the bacterial growth medium. Ethanol is known to be a

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potent antimicrobial agent and to act at the lipid-water interface, altering the stability and integrity of bacterial cell membranes (Weber and de Bont, 1996). The effect of

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ethanol on microorganisms related to industrial alcoholic fermentations, such as Saccharomyces cerevisiae, Oenococcus oeni or Clostridium thermocellum (Cafaro et al., 2014; Ma and Liu, 2010; Voigt et al., 2013; Yang et al., 2012), and on some model microorganisms (Chong et al., 2013; Seydlová et al., 2012) has been reported before. L. lactis subsp. cremoris NZ9700 is a strain obtained from a fermented dairy starter and a well-known nisin producer, which is distributed in laboratories and collections around the world and has become a prototype for genetic and physiological studies in LAB.

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Nevertheless, this is to our knowledge the first transcriptome and metabolic study of the response of L. lactis to ethanol, which is an antimicrobial agent that may well be considered a good indicator of bacterial robustness. Our transcriptome analyses identified down-regulation of genes related to purine and

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pyrimidine biosynthesis, which implies the inhibition of bacterial growth and is in

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accordance with the observed reduced growth of L. lactis in presence of ethanol (Fig 1). These results were as expected for a potent bactericidal agent such as ethanol. Down-

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regulation was also observed for genes related to nisin production, which is encoded by

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the nisin operon nisABTCIP that contains the structural gene nisA and the necessary biosynthetic and immunity genes for its expression (Kuipers et al., 1993), and two

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contiguous operons, i.e. nisRK, encoding regulation by a two-component regulatory system, and nisFEG, also involved in immunity. Our results showed that with the

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exception of the structural gene nisA and the regulatory nisR, all the other nisin-related

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genes were down regulated at the mid-exponential growth phase in presence of ethanol. This down-regulation at the transcriptional level was corroborated by the results of Fig.

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1 that shows a progressive decrease of nisin production as ethanol concentration in the culture broth increased. It should be taken into account that nisin production implies a

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fitness cost, and its inhibition at transcriptional level could favor cell survival and adaption to harsh conditions. In contrast to our results, some L. lactis engineered strains with enhanced nisin production showed increased tolerance to low pHs during fermentation (Zhang et al., 2016). Regarding the activation of genes related to cold shock stress (cspA, cspC and cspD), the proteins encoded by these genes act as chaperons to protect peptide synthesis

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from cold and a variety of other stress factors (Wouters et al., 2001; Yu et al., 2009), and similarly Lactobacillus plantarum (van Bokhorst et al., 2011) was also reported to down regulate the transcriptional factor ctsR in the presence of 8 % ethanol and thus to activate expression of genes coding for chaperon proteins. Oenococcus oeni, a LAB of

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relevance in wine making, was also reported to utilize the ctsR gene product to regulate stress responses through the major molecular chaperones, which include Hsp, Csp

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(Grandvalet et al., 2005) and the small heat shock protein Lo18 (Maitre et al., 2014),

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expressed in L. lactis (Weidmann et al., 2016).

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which seems to improve tolerance not only to heat, but also to acid conditions when

Our transcriptome analyses also revealed that L. lactis subsp. cremoris requires

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energy in the presence of ethanol and activated pathways for sugar transport and metabolism, as well as ABC-type multidrug pumps and alcohol dehydrogenase

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activities to expel and degrade the toxic agent ethanol. Similarly, O. oeni (Bourdineaud

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et al., 2004) was shown to over-express the ABC-transporter gene omrA as response toand protection from ethanol, and L. plantarum (van Bokhorst et al., 2011) was reported

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to transcriptionally activate its citrate metabolism (citCDEF operon) and utilize citrate from the medium to obtain energy to overcome ethanol stress. Similarly to our results, a

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study by NMR of L. lactis subsp. cremoris MG1363 (Carvalho et al., 2013) also reported activation of glucose uptake by a cellobiose-specific PTS system and genes coding for glycolytic enzymes as a response to acid stress. Up-regulation of transcription of ABC transporter permeases and cellobiose-transport PTS genes has been associated with strain robustness under heat and oxidative stress (Dijkstra et al., 2014) and in this regard, our results show the same type of response, suggesting that

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ethanol tolerance could also be considered a robustness characteristic for L. lactis strains. Our results showed that the transcriptional response of L. lactis subsp. cremoris to ethanol also includes activation of genes related to fatty acid synthesis at the stationary

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growth phase. The Lb. plantarum transcriptional response to ethanol (van Bokhorst et

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al., 2011) was also reported to differentially express genes encoding cell-surface lipoproteins and teichoic acid biosynthesis enzymes. These results imply changes in the

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bacteria cell wall and membrane, which are obviously needed to overcome the effects of

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ethanol at the lipid-water interface.

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The core response to ethanol of L. lactis subsp. cremoris was transcriptional upregulation (up to 42.7 fold) of the arcABC1C2TD1D2 operon (Fig. 2), encoding proteins of the ADI pathway for arginine degradation (Fig. 5). The final products of

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arginine degradation through this pathway are ornithine, ammonia and CO2. This pathway renders one molecule of ATP (Fig. 5) and consumes two protons, which contributes to the internal pH homeostasis and opposes external acid stress. Activation

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of this pathway has been shown to be a major protection mechanism of LAB species

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against acidic environments such as the oral cavity (Curran et al., 1995), lactic fermentation processes (Pessione et al., 2010), wine (Arena and Manca de Nadra, 2005), and salt- and temperature stresses (Vrancken et al., 2009). Our results of transcriptional activation of L. lactis ADI pathway for arginine consumption in presence of ethanol, corroborated by the metabolic study with the wild type and mutant strains, indicated that the ADI pathway provides useful energy for cell survival and tolerance to ethanol. Although the pleiotropic transcriptional regulator CodY has been described as the

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overall regulator of nitrogen metabolism in L. lactis (Guédon et al., 2005), in the presence of glucose the arc operon is actually regulated by the carbon catabolite control protein (CcpA), which represses arc expression and constitutes a link between regulation of carbon metabolism and regulation of nitrogen metabolism in L. lactis

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MG1363 (Zomer et al., 2007). As glucose is being consumed, both transcriptional factors ArgR and AhrC take the lead and regulate arc expression. When the L. lactis

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strains of our study were grown in chemically defined culture broth, the presence of

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glucose initially could have triggered repression of the ADI pathway through CcpA. Our results show that when glucose is partially consumed (middle of exponential

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growth phase and stationary phase) and in the presence of arginine in the culture broth,

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the transcriptional regulators ArgR and AhrC exert their action and the ADI pathway of arginine degradation contributes to bacterial growth in the presence of ethanol in those

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strains expressing the arc operon. Fig. 3A and 3B show that at the end of the

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exponential growth phase (8 - 10 h culture) and in the presence of 2 % ethanol, samples with active ADI pathways and with available arginine showed the largest differences in cell population when compared with control cultures without arginine. This mechanism

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of resistance through the ADI pathway has been described at the proteomic level for L.

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lactis subsp. cremoris against oxidative stress (H2O2), to which this species is subjected during cheese manufacturing (Rochat et al., 2012). Other studies related to the production of cheese flavour also reported arginine degradation as the source of energy for L. lactis subsp. lactis to survive under starvation stress when cells were deprived of sugar (Brandsma et al., 2012), and more recently it has been suggested that the capability of L. lactis to persist in a viable not culturable state could be due to the fact

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that L. lactis cells could switch from glycolysis to nitrogen catabolism for obtaining the required energy to survive (Ruggirello et al., 2016). In the wine industry LAB play an important role in the secondary fermentation that takes place after alcoholic fermentation, named the malolactic fermentation (MLF) that

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is a requisite for the sensory properties of premium red wines. Wine heterofermentative

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LAB are able to degrade arginine, and do so through the ADI pathway (Liu and Pilone, 1998). Although many LAB species are present in the initial grape must, O. oeni

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becomes the dominant species and finally triggers and conducts MLF, and this is mainly

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due to the excellent adaptation of this species to the aggressive ecological medium that wine is for bacterial growth. O. oeni possesses the arc genes (Araque et al., 2009) and

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therefore, arginine would stimulate O. oeni growth and resistance in wine, which was reported by Bourdineaud (2006). Nevertheless, some other studies reported that arginine

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does not stimulate growth of O. oeni in wine under standard MLF conditions (Terrade

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and Mira de Orduña, 2009). Our results support that arginine and glucose enhance bacterial growth and resistance to ethanol by utilizing the sugar and the ADI pathway

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for arginine degradation to obtain energy and overcome the stress due to the presence of

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ethanol in their growth medium. Summarizing, our study provides deeper insight into the stress tolerance mechanisms against ethanol that L. lactis, and other ADI pathway-possessing LAB strains of industrial relevance utilize when they become exposed to ethanol.

Acknowledgments

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This work was supported by grant AGL2010-15466 of the Ministry of Research and Science of Spain and FEDER of the European Community. Lorena Diez was a contractual technician supported by the grant AGL2010-15466. Ana Solopova was supported by a Stichting Technische Wetenschappen grant in the scope of Project 10619

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“Understanding Preculture- Dependent Growth and Acidification Rates of Lactococcus lactis as the Result of Population Heterogeneity”. Rocío Fenández was a Predoctoral

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Researcher of the Regional Autonomous Government of La Rioja.

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Fig. 1: Growth curves and nisin production of L. lactis NZ9700 in GM17 with 0 %, 2 %, 4 % and 6 % (vol/vol) ethanol Antimicrobial activity

Growth curve

Antimicrobial activity

Growth curve

Antimicrobial activity

Growth curve

Antimicrobial activity

in absence of ethanol in presence of 2 % ethanol in presence of 4 % ethanol in presence of 6 % ethanol

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Growth curve

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Fig. 2. The ADI pathway genes up-regulated in presence of 2 % ethanol.

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Fig. 3: Growth curves of L. lactis strains in CDM in presence of 0% and 2% ethanol, with (2.5 mg/ml) and without arginine in the culture broth.

A: L. lactis NZ9700 B: L. lactis MG1363 C: L. lactis MGΔargR D: L. lactis MGΔahrC

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0 % ethanol with 2.5 mg/ml arginine

0 % ethanol without arginine

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2 % ethanol without arginine

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Fig. 4: Arginine and ornithine concentrations in the bacterial culture broth: at initial time, with Arg in the culture broth at stationary growth phase (10 h incubation) under the following conditions: with 2.5 mg/mL Arg and without ethanol without Arg and without ethanol with 2.5 mg/mL Arg and with 2 % ethanol

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without Arg and with 2 % ethanol The dotted line indicates the detection threshold. L. lactis subs. cremoris strains: A) wild type MG1363; B) mutant MGΔargR; C) mutant MGΔahrC.

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Fig. 5. Scheme of the metabolic pathways of arginine in L. lactis

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Table 1.- Bacterial strains

MGΔargR

Source of reference Kuipers et al., 1993

plasmid free strain

Gasson 1983

active ADI pathway

Larsen et al., 2004

ADI pathway not expressed

Larsen et al., 2004

Supplementary material

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MGΔahrC

Characteristics nisin producer

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MG1363

Descriptions Lactococcus lactis subsp. cremoris; Lactococcus lactis subsp. cremoris deletion mutant argR of L. lactis subsp. cremoris MG1363 deletion mutant ahrC of L. lactis subsp. cremoris MG1363

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Strains NZ9700

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Table S1.- Up-regulated genes1 in presence of ethanol at middle exponential growth phase (stage 1) and expression at the beginning of the stationary phase (stage 2).

llmg_2306 llmg_2306 -MG2015 llmg_2307 -arcD2MG2014 llmg_2308 -arcTMG2013 llmg_2309 -arcC2MG2011 llmg_2310 -arcC1MG2010 llmg_2311 -arcD1-

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Via ADI

pa

Fold 2.48

1

St age2 fold si g 1.93 0

llmg_230 6

8.54E -07

Bayes pb 5.14E -13

arcD2

9.78E -05

1.18E -08

3.69

1

3.59

0

arginine/ornithine antiporter

arcT

2.90E -05

9.90E -11

11.6 6

1

2.06

0

aminotransferase

arcC2

2.11E -06

5.34E -12

28.8 7

1

-1.92

1

carbamate kinase

arcC1

8.54E -07

5.14E -13

38.7 0

1

-3.27

1

carbamate kinase

arcD1

4.90E -05

8.61E -11

26.0 5

1

-1.98

0

arginine:ornithine antiporter

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gene

Stage 1

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sig c

Description hypothetical protein

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7.44E -12

28.0 8

1

-2.01

1

arcA

7.19E -06

8.21E -12

42.7 1

1

-1.81

0

arcA

1.61E -05

1.78E -11

23.0 7

1

-2.17

1

llmg_251 3

2.19E -04

8.66E -09

4.39

1

-4.98

1

llmg_251 4

6.80E -04

1.61E -07

2.85

1

-4.15

llmg_251 5

3.79E -03

4.40E -06

2.56

llmg_251 6

1.36E -05

9.15E -08

llmg_251 6

2.81E -03

llmg_251 7

8.48E -04

ABC-Type Multidrug Resistance Transporter LmrCDI hypothetical protein

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1

-3.81

0

2.42

1

1.34

0

hypothetical: universal stress protein A hypothetical protein

4.01E -06

2.54

1

-1.15

0

hypothetical protein

1.20E -07

3.98

1

-3.08

1

hypothetical protein

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ornithine carbamoyltransferas e ornithine carbamoyltransferas e arginine deiminase

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1.03E -05

adhA

1.45E -04

4.05E -09

1.96

1

3.56

1

alcohol dehydrogenase

adhE

4.32E -04

2.92E -08

3.93

1

5.32

1

bifunctional acetaldehydeCoA/alcohol dehydrogenase bifunctional acetaldehydeCoA/alcohol dehydrogenase carbon starvation protein A

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ADH

llmg_2514 llmg_2514 -mg7577 llmg_2515 llmg_2515 llmg_2516 llmg_2516 -mg7579 llmg_2516 llmg_2516 -MG98 llmg_2517 llmg_2517 -MG96 llmg_1991 -adhAMG813 llmg_2432 -adhEMG265

arcB

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MG2009 llmg_2312 -arcBMG2008 llmg_2313 -arcAmg12120s llmg_2313 -arcAMG2007 llmg_2513 llmg_2513 -mg7439

adhE

1.45E -04

4.05E -09

4.98

1

6.04

1

llmg_0430 -cstAMG2027 llmg_0430 -cstAMG752 Fatty acids llmg_0627 -fadDMG2358 llmg_0784 Ribose -rbsRMG2451

cstA

4.04E -04

8.42E -08

3.15

1

1.89

0

cstA

2.26E -05

2.43E -08

3.08

1

1.42

0

carbon starvation protein A

fadD

6.61E -03

3.03E -05

1.86

1

2.35

1

long-chain acylCoA synthetase

rbsR

2.46E -04

1.65E -07

2.26

1

2.26

1

ribose operon repressor

Carbon starvatio n

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llmg_2432 -adhEMG266

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3.95E -08

2.86

1

1.64

0

ribokinase

rbsD

8.29E -05

2.25E -08

3.19

1

2.23

0

D-ribose pyranase

rbsD

4.46E -05

7.82E -09

3.97

1

2.54

1

llmg_0788 -rbsCMG2447 llmg_0789 -rbsBMG2446

rbsC

8.39E -04

1.18E -07

3.60

1

3.07

1

rbsB

8.29E -05

2.25E -08

3.07

1

2.34

llmg_0437 -ptcBmg5538 llmg_0438 -ptcAmg13888s llmg_0439 -llmg_043

ptcB

1.05E -03

8.51E -07

2.78

1

ptcA

5.24E -05

9.97E -09

2.84

llmg_043 9

4.20E -03

1.10E -05

llmg_1847 -cspAmg8166 llmg_1662 -uspAMG1212 llmg_1238 -cspD2MG15008 4 llmg_1253 llmg_1253 llmg_1254 llmg_1254 llmg_1255 -cspCMG15031 3 llmg_1256 -cspDMG15008 8 llmg_1126 llmg_1126 llmg_1127 llmg_1127 llmg_1680 -

cspA

2.66E -01

rbsD ribose ABC transporter permease protein RbsD ribose transport system permease protein RbsC ribose ABC transporter substrate binding protein RbsB cellobiose-specific PTS system IIB component cellobiose-specific PTS system IIA component LacI family transcription regulator cold shock-like protein CspA

uspA

2.31E -01

AC Cell wall

PT

2.02E -05

RI

1

SC

6.59

1

30.6 7

1

3.36

1

10.5 7

1

8.22E -02

1.25

0

2.34

1

3.45E -04

1.30

0

2.50

1

universal stress protein A

6.36E -02

2.81E -03

1.65

0

3.53

1

cold shock protein CspD

1.46E -02

1.31E -04

1.85

1

-1.01

0

hypothetical protein

llmg_125 4

2.60E -02

8.03E -04

2.14

1

1.99

0

hypothetical protein

cspC

4.05E -02

8.24E -04

1.82

1

2.66

1

cold shock protein CspC

cspD

3.36E -02

4.70E -04

2.02

1

2.76

0

cold shock protein CspD

llmg_112 6

1.71E -02

7.46E -04

1.39

0

2.15

1

hypotetical protein

5.82E -01

5.19E -01

7.62

1

2.41E -03

8.09E -05

1.93

1

cell wall surface anchor family protein cell division protein

MA

D

cspD2

llmg_125 3

llmg_112 7 llmg_168 0

NU

1

CE

Stress

rbsK

PT E

Sugar

llmg_0785 -rbsKMG2450 llmg_0786 -rbsDmg6892 llmg_0787 -rbsDMG2448

39

1.58 1.45

0 0

ACCEPTED MANUSCRIPT

1

llmg_1680 gene expression showing a p value of at least <0.05, and a fold-change at least > 1.8 at middle

exponential growth phase (stage 1). pa: probability Bayes pb: Bayes probability

AC

CE

PT E

D

MA

NU

SC

RI

PT

sigc: statistical significance, 1, statistically significant; 0, not significant

40

ACCEPTED MANUSCRIPT

Table S2.- Down-regulated genes in presence of ethanol at middle exponential growth phase (stage 1) and expression at the beginning of the stationary phase (stage 2).

feoA

6.21 E-06

2.54 E-09

fhuC

fhuG

misc-nisA

nisA

misc-nisB

nisB

misc-nisC

nisC

misc-nisE

nisE

misc-nisF

nisF

misc-nisG

nisG

misc-nisI

nisI

misc-nisin

nisin

misc-nisK

nisK

misc-nisP

nisP

misc-nisR

nisR

misc-nisT

nisT

misc-nisX

nisX

sig c

0 1 1

2.18 1.64 1.90 1.41 1.14

Description nis A

0

nis B

0

nis C nis E

0

nis F

0

nis G

2.57 1.28

1

nis I

0

nisin

1

nisK

1

nis P

0

nis R

1

nis T

1

2.73 4.21 1.26 2.08 1.43 2.54

0

ferrous iron transport protein B

3.54

1

2.11

1

ferrous iron transport protein A

6.37 E-05

8.90 E-09

3.80

1

3.85

1

ferrichrome ABC transporter FhuC

6.21 E-06

3.22 E-08

3.01

1

1.88

0

ferrichrome ABC transporter permease

fhuD

3.94 E-05

2.54 E-09

4.23

1

2.23

1

pyrR

1.12 E-05

4.76 E-11

1

2.66

0

pyrP

1.38 E-03

1.04 E-07

15.1 1 9.78

ferrichrome ABC transporter substrate binding protein bifunctional pyrimidine regulatory protein PyrR

1

2.29

1

uracil permease (uracil transporter)

pyrB

7.18 E-05

5.20 E-10

8.68

1

2.17

0

9.65

1.72

-

1

2.81

1

aspartate carbamoyltransferase catalytic subunit carbamoyl phosphate

AC

SC

1 1 0

NU

MA

D

feoB

PT E

llmg_0199feoBMG1899 llmg_0200feoAMG1898 llmg_0346fhuCMG696 llmg_0348fhuGMG698 llmg_0349fhuDMG700 Pyrimidin llmg_089 0-pyrRes MG2412 llmg_089 1-pyrPMG2413 llmg_089 3-pyrBMG2414 llmg_089

1

41

RI

0

Ferrus

1

Stage 2 fold si g 1.67 0

CE

Nisin

Fol d 1.14 3.48 2.31 2.82 2.80 3.46 3.97 1.81 2.71 4.35 1.36 2.78 1.14 2.83

PT

5.47 E-01 8.04 E-04 9.92 E-04 2.87 E-03 1.40 E-03 8.22 E-04 1.03 E-03 9.09 E-02 1.67 E-03 9.62 E-04 1.58 E-01 8.16 E-03 4.70 E-02 6.21 E-06

Stage 1 Baye s pb 3.33 E-01 1.08 E-07 6.45 E-07 1.65 E-06 4.37 E-07 1.28 E-07 1.32 E-07 4.94 E-03 5.96 E-07 9.06 E-08 2.34 E-02 1.41 E-05 3.35 E-02 3.22 E-08

pa

gene

1 1 0 1 0

0

ACCEPTED MANUSCRIPT

4.35 E-11

pyrD B

1.42 E-05

2.47 E-11

pyrF

2.91 E-05

7.95 E-11

1.83 E-04

1.19 E-09

7.59 E-03

1.83 E-05

2.22

pyrC

2.14 E-02

9.82 E-05

8.52

pyrE

1.02 E-05

8.40 E-11

purC

7.62 E-04

1.32 E-06

AC

2.32

1

dihydroorotate dehydrogenase,

1

1.94

0

dihydroorotate dehydrogenase 1B

1

2.20

1

orotidine 5'-phosphate decarboxylase

1

4.20

1

hypotetical protein

1

1.26

0

hypotetical protein

5.54

1

dihydroorotase

1

1

2.88

1

orotate phosphoribosyltransferase

1.93

1

16.1 2

1

3.72 E-07 9.73 E-08

3.84 2.62

1

17.8 2 1.21

1

phosphoribosylaminoimida zole-succinocarboxamide synthase PurS

0

hydroxyethylthiazole kinase

MA

9.04

D

thiM

1

PT

2.73 E-06

synthase small subunit

RI

pyrK

1.73 E-03 1.51 E-04

14.3 9 10.7 4 16.4 4 12.4 3 11.9 7

SC

E-11

NU

E-06

1

2.41 E-04

2.40 E-07

2.52

1

1.62

0

phosphomethylpyrimidine kinase

thiE

7.10 E-05

2.22 E-07

2.28

1

1.43

0

thiamine-phosphate pyrophosphorylase

3.00 E-03 1.01 E-03 3.63 E-03

3.29 E-06 8.69 E-07 7.02 E-06

2.27 2.37 2.19

1

1.81 1.81 2.01

1

xanthine/uracil permease

1

xanthine/uracil permease

1

xanthine phosphoribosyltransferase

PT E

thiD1

CE

4-carAMG2415 llmg_110 5-pyrKMG1867 llmg_110 6-pyrDBMG1866 llmg_110 7-pyrFMG1865 llmg_110 8llmg_110 8 llmg_150 7llmg_150 7 llmg_150 8-pyrCMG15 llmg_150 9-pyrEMG1464 llmg_097 Purines 3-purCMG2328 llmg_097 4 llmg_121 6-thiMmg5862 llmg_121 7-thiD1mg5861 llmg_121 8-thiEmg5860 Xanthines llmg_134 5 llmg_134 5-pbuX llmg_134 6-xptMG1501 77 pa: probability

pbuX xpt

1 1

Bayes pb: Bayes probability sigc: statistical significance, 1, statistically significant; 0, not significant

42

ACCEPTED MANUSCRIPT

Highlights 

Transcriptome analysis of the response to ethanol of the model strain L. lactis NZ9700.



Activation of the deiminase (ADI) pathway for arginine degradation as a response to ethanol stress.

PT

Stress tolerance mechanism of LAB strains of industrial relevance that possess the ADI

CE

PT E

D

MA

NU

SC

RI

pathway.

AC



43