Strain-Dependent Transcriptome Signatures for Robustness in Lactococcus lactis

Abstract

Recently, we demonstrated that fermentation conditions have a strong impact on subsequent survival of Lactococcus lactis strain MG1363 during heat and oxidative stress, two important parameters during spray drying. Moreover, employment of a transcriptome-phenotype matching approach revealed groups of genes associated with robustness towards heat and/or oxidative stress. To investigate if other strains have similar or distinct transcriptome signatures for robustness, we applied an identical transcriptome-robustness phenotype matching approach on the L. lactis strains IL1403, KF147 and SK11, which have previously been demonstrated to display highly diverse robustness phenotypes. These strains were subjected to an identical fermentation regime as was performed earlier for strain MG1363 and consisted of twelve conditions, varying in the level of salt and/or oxygen, as well as fermentation temperature and pH. In the exponential phase of growth, cells were harvested for transcriptome analysis and assessment of heat and oxidative stress survival phenotypes. The variation in fermentation conditions resulted in differences in heat and oxidative stress survival of up to five 10-log units. Effects of the fermentation conditions on stress survival of the L. lactis strains were typically strain-dependent, although the fermentation conditions had mainly similar effects on the growth characteristics of the different strains. By association of the transcriptomes and robustness phenotypes highly strain-specific transcriptome signatures for robustness towards heat and oxidative stress were identified, indicating that multiple mechanisms exist to increase robustness and, as a consequence, robustness of each strain requires individual optimization. However, a relatively small overlap in the transcriptome responses of the strains was also identified and this generic transcriptome signature included genes previously associated with stress (ctsR and lplL) and novel genes, including nanE and genes encoding transport proteins. The transcript levels of these genes can function as indicators of robustness and could aid in selection of fermentation parameters, potentially resulting in more optimal robustness during spray drying.

Introduction

Owing to their spoilage-preventing, texture-improving and flavor-enhancing properties, lactic acid bacteria have a long history of application in food fermentations [1, 2]. One of the most widely used lactic acid bacteria in the food industry is Lactococcus lactis, notably for the production of cheese and butter(milk) [2]. These milk fermentation processes are typically initiated with the addition of starter cultures containing high concentrations of one or multiple L. lactis strains. During the production of these starter cultures prior to application in the food industry, L. lactis strains encounter severe stresses, for example heat and oxidative stress during spray drying [3–5]. Although spray drying is a cost-effective and energy-efficient method for the preservation of starter cultures, it generally results in a relatively large decrease in viability as compared with other preservation methods such as freezing and freeze drying [6]. Viability of starter cultures is essential for an adequate contribution to the fermentation end-product, justifying the industrial interest to better understand and improve robustness [1].

Genes involved in stress responses appear highly conserved among bacteria, nevertheless regulation of these stress genes can differ between organisms [7, 8]. Recently, we demonstrated a large diversity in heat and oxidative stress survival among L. lactis strains, suggesting differential regulation of stress responses [5]. Furthermore, strains with an L. lactis subsp. cremoris phenotype appeared to have a less efficient response as compared with strains with an L. lactis subsp. lactis phenotype when these strains were pre-adapted to a minor dose of acid, bile or freezing stress, prior to exposure to a lethal dose of the same stress [9].

Previously, we demonstrated that for the L. lactis subsp. cremoris strain MG1363 [10], oxygen level and fermentation temperature strongly affect subsequent survival during heat and oxidative stress assays, respectively [11]. Furthermore, by applying a transcriptome-phenotype matching approach, we revealed transcriptome signatures associated with robustness towards heat and oxidative stress, which could function as indicators for robustness. These transcriptome signatures included the metC-cysK operon, of which the transcript levels positively correlated with robustness. The role of this operon was confirmed by demonstrating an increase in robustness towards oxidative stress of MG1363 after growth in medium lacking cysteine, which has been demonstrated to induce the metC-cysK operon [11, 12].

L. lactis strains that are applied in food industry are diverse in subspecies and isolation source. It remains unclear if the correlation of gene expression levels and robustness as found in strain MG1363 are generic and, therefore, can also be employed for other L. lactis strains to predict their robustness. Specific individual gene transcripts that associated with robustness in MG1363 [11] were previously established to be important during heat, acid and osmotic stress in L. lactis subsp. lactis strain IL1403 [13], suggesting at least partially overlapping stress responses in these two L. lactis strains.

To investigate if other strains have transcriptome signatures for robustness towards heat and oxidative stress similar to or distinct from those of strain MG1363, we applied an identical transcriptome-phenotype matching strategy [11] on three other strains. These three strains were the dairy L. lactis subsp. lactis strain IL1403 [14], the non-dairy L. lactis subsp. lactis strain KF147 [15] and the dairy L. lactis subsp. cremoris strain SK11 [16]. Besides the differences in subspecies and origin, we previously revealed highly diverse robustness phenotypes of these strains[5]. The strains were individually grown under the twelve conditions that were previously applied to MG1363 [11] and the effect of these conditions on heat and oxidative stress survival was assessed. Moreover, we determined full genome transcriptome profiles, allowing association of gene expression and stress survival to identify transcriptome signatures for robustness towards heat and oxidative stress in the individual strains.

Materials and Methods

Strains and fermentations

L. lactis strains IL1403 [14], KF147 [15] and SK11 [16] were cultivated in chemically defined medium (CDM) as described previously [11]. Briefly, the strains were fermented under twelve different conditions varying in sodium chloride concentration (0 or 100 mM), initial pH (6.0 or 6.5), temperature (27, 30 or 35°C) and level of oxygen (static in 50 ml Falcon tube or shaken at 100 rpm in 500 ml shake flask with a cotton plug) (Table 1). Fermentations were performed on two separate days (fermentation number 1–6 on day 1, 7–12 on day 2) and, therefore, a replicate of fermentation 6 was added on day 2 (fermentation 13). Biomass formation was determined by measurement of the optical density (OD) at 600 nm. In the exponential phase of growth (OD₆₀₀ between 0.5 and 0.7), cells were harvested for heat and oxidative stress survival assays and RNA isolation.

Table 1. Fermentation conditions, growth characteristics and stress survival.

					μ (h-1)			ODfinal			heat stress survival(%)			oxidative stress survival (%)
fermentation number	salt (mM)	initial pH	temperature (°C)	level of oxygen	IL1403	KF147	SK11	IL1403	KF147	SK11	IL1403	KF147	SK11	IL1403	KF147	SK11
1	0	6.0	27	high	0.37	0.87	0.52	1.59	2.12	1.34	0.23	0.38	1.2	6.5	0.045	0.051
2	100	6.5	27	high	0.43	0.77	0.57	2.43	2.56	1.81	0.75	1.1	0.36	12	0.10	0.55
3	0	6.5	27	low	0.59	0.79	0.67	2.42	2.37	2.43	0.000092	0.012	0.63	0.0011	0.10	0.13
4	100	6.0	27	low	0.30	0.87	0.49	1.48	1.50	1.30	4.5	0.021	0.35	0.75	0.12	0.11
5	0	6.0	30	low	0.59	1.16	0.66	1.56	1.83	1.46	0.000044	0.0077	2.5	0.00046	0.27	0.074
6	100	6.5	30	low	0.73	1.09	0.69	2.27	2.23	2.08	0.0075	0.12	4.0	0.032	0.042	0.047
7	0	6.5	30	high	0.74	0.94	0.63	2.62	2.73	1.98	0.0052	1.7	6.8	0.0065	0.071	0.037
8	100	6.0	30	high	0.39	0.85	0.57	1.53	1.80	1.50	3.2	18	9.4	53	0.46	0.038
9	0	6.0	35	high	0.80	1.12	0.26	2.18	1.99	1.16	5.3	25	13	0.0029	1.7	58
10	100	6.5	35	high	0.77	1.09	0.25	2.42	2.75	1.32	2.7	53	6.4	1.2	0.18	20
11	0	6.5	35	low	1.00	1.22	0.61	2.90	2.58	1.89	0.095	0.79	34	0.040	0.012	0.0084
12	100	6.0	35	low	0.92	1.18	0.50	1.77	1.59	1.26	0.45	0.93	7.4	0.0042	0.00070	0.010
13	100	6.5	30	low	0.76	1.06	0.74	2.34	2.21	2.04	0.011	0.033	3.1	0.0066	0.0023	0.036

Fermentation parameters of the various fermentations and resulting maximum growth rates (μ) and optical densities at the end of fermentation (OD_final) and survival after 60 minutes (IL1403) or 10 minutes (KF147 and SK11) of heat stress and after 30 minutes of oxidative stress of strains IL1403, KF147 and SK11. Survival at the other time point of the stress assays can be found in S1 Table. Survival data represent averages of technical duplicates. Shaken and static fermentations are indicated as a relatively high level of oxygen (“high”) and a relatively low level of oxygen (“low”), respectively.

Heat and oxidative stress survival assays

Stress survival was determined as described previously [11]. Cells were harvested from 5 ml of culture by centrifugation at 1865 × g for 10 minutes and resuspended in 2.5 ml sterile 50 mM sodium phosphate (Merck) buffer pH 7.2. For assessment of heat stress survival, 0.5 ml of the cell suspensions was diluted by adding 0.5 ml of phosphate buffer and were incubated in duplicate in a volume of 0.1 ml at 50°C for 10 and 30 minutes (KF147, SK11) or 30 and 60 minutes (IL1403) in 0.2 ml PCR tubes (Bioplastics BV, Landgraaf, The Netherlands) in a Gene-Amp PCR system 9600 (Applied BioSystems, Foster City, California, USA). For assessment of oxidative stress survival, hydrogen peroxide (Merck) in phosphate buffer was added to 0.25 ml of the cell suspensions in duplicate to a final concentration of 5 mM and an end volume of 0.5 ml, followed by incubation for 30 and 60 minutes at 30°C in a water bath. After incubation, samples were centrifuged at 15,000 × g for 3 minutes and cell pellets were resuspended in 0.5 ml of phosphate buffer. Survival was assessed by spotting serial dilutions in triplicate on M17 agar plates supplemented with 0.5% glucose[17]. Colony forming units (CFU) were determined after incubation of the plates for 72 hours at 30°C.

RNA isolation and DNA microarrays

RNA isolation, subsequent cDNA synthesis and labeling, as well as DNA microarray hybridizations were performed using routine procedures, as described previously for MG1363 [11]. Briefly, aliquots of 5 ml of culture were centrifuged at 4000 × g for 3 minutes at 2°C and cells were resuspended in 0.5 ml cold TE buffer. To this suspension, 500 μl 1:1 phenol/chloroform, 30 μl 10% SDS, 30 μl 3M sodium acetate pH 5.2 and 500 mg 0.1 mm zirconia beads (Biospec Products, Inc., Bartlesville, USA) was added in a 2 ml screw-cap tube and samples were frozen in liquid nitrogen and stored at -80°C. The DNA microarray hybridization scheme contained two connected loops, both containing samples derived on a single day (S1 Fig). A two-dye microarray-based gene expression analysis was performed on a custom-made 60-mer oligonucleotide array (Agilent Technologies, Santa Clara, California, USA, submitted in Gene Expression Omnibus under GEO Series accession number GSE72045) to determine genome-wide gene transcription levels. Co-hybridization of Cy5- and Cy3-labeled cDNA probes was performed on these oligonucleotide arrays at 65°C and 10 rpm for 17 h using GEX HI-RPM buffer (Agilent Technologies). After hybridization, slides were washed and scanned.

Data analysis

Data analysis was performed as previously described for strain MG1363 [11]. The raw expression data were Lowess normalized and scaled to normalized probe expression levels using MicroPreP [18]. Multiple probes were designed for each ORF and the ORF expression level was calculated from the median of its probe signals. Normalized gene expression levels were further analyzed using the R BioConductor packages Biobase and limma (www.bioconductor.org). After 2-log transformation, gene expression levels were plotted against robustness levels and significance of the correlation was assessed by a linear model. We selected the genes with a significant correlation (P < 0.05) at both time points of the stress assay and further analyzed the genes with the most significant correlation by calculating the product of both P-values. To identify a generic transcriptome signature, we used survival at the time point at which the dynamic range of robustness was the largest. As a consequence, the selected time points for heat stress were 60, 10 and 10 minutes for IL1403, KF147 and SK11, respectively, whereas for oxidative stress the selected time point was 30 minutes for all strains. These data were compared with the survival of MG1363 after 30 minutes of heat stress or oxidative stress [11]. Correlation of survival and growth rate or optical density was determined by calculating the Pearson correlation coefficient. Differences in the effect of individual fermentation parameters on growth characteristics and robustness were assessed with a t-test in R (version 3.0.1, www.R-project.org) and differences were considered significant if the P-value was smaller than 0.05.

Results and Discussion

Variations in fermentation conditions impose largely similar effects on the growth characteristics of different L. lactis strains

To compare the effect of fermentation conditions on the growth characteristics, L. lactis strains IL1403, KF147 and SK11 were grown under the twelve different conditions that were previously applied to strain MG1363 [11]. These conditions varied in the level of salt and/or oxygen, as well as fermentation pH and temperature and resulted in variation of growth characteristics (Table 1, S2 Fig). Strain KF147 displayed maximum growth rates (μ_max) in the same range (0.7 h^-1 to 1.2 h^-1) as we previously established for strain MG1363 [11], whereas SK11 had lower growth rates (0.5 h^-1 to 0.7 h^-1 [Table 1]). Strain IL1403 displayed the largest variation in maximum growth rate, ranging from 0.3 h^-1 to 1.0 h^-1 (Table 1).

The effect of fermentation temperature on maximum growth rate of the strains KF147 and IL1403 was similar to what we previously observed for MG1363 [11] (Fig 1). In contrast to the other strains, the maximum growth rate of SK11 was significant lower in fermentations at 35°C as compared with 30°C (Fig 1), which is in line with the fact that SK11 has an L. lactis subsp. cremoris phenotype in contrast to MG1363, IL1403 and KF147, which have an L. lactis subsp. lactis phenotype [19]. One of the characteristics that discriminates these phenotypes is that strains with an L. lactis subsp. cremoris phenotype are incapable of growing at high temperature in contrast to strains with an L. lactis subsp. lactis phenotype [20].

Fig 1. Effect of temperature on growth rate.

Boxplots of maximum growth rate (μ_max in h^-1) of strains MG1363, IL1403, KF147 and SK11 in fermentations at 27, 30 and 35°C.

Both biomass formation (OD_final) and final pH at the end of fermentation were strongly affected by the fermentation conditions and the observed effects were similar for all strains (Table 1). The initial pH of fermentation had the most significant effect on biomass formation. In fermentations with an initial pH of 6.5 a significantly higher biomass formation was reached for all strains as compared to fermentations with an initial pH of 6.0 (Fig 2). The final pH at the end of fermentation was mostly affected by the oxygen level and was significantly lower in fermentations with a relatively low level of oxygen as compared with fermentations with a relatively high level (data not shown). This is in line with an earlier study, which demonstrated that the acidifying ability of L. lactis strain CNRZ 483 decreased as initial oxygen concentration increased [21].

Fig 2. Effect of pH on final OD.

Boxplots of final optical density (OD_final) of strains MG1363, IL1403, KF147 and SK11 in fermentations with an initial pH of 6.0 or 6.5.

With the notable exception of the effect of fermentation temperature on growth rate of SK11, all other applied fermentation parameters had similar effects on the growth characteristics of the L. lactis strains, revealing an overlap in responses towards the applied fermentation conditions.

The effect of fermentation conditions on robustness is strain-dependent

To study the effect of the fermentation conditions on robustness phenotypes, cells were harvested in exponential phase of growth for assessment of heat and oxidative stress survival phenotypes, representing robustness during spray drying [5]. During the stress assays, survival was determined at two time points, similar as for MG1363 [11]. For KF147 and SK11 the time points for heat stress survival measurement were adjusted because these strains displayed a higher sensitivity towards heat stress as compared with MG1363 and IL1403 (see Materials and Methods). Variation in fermentation conditions resulted in differences in both heat and oxidative stress survival of up to five log units (Table 1, S1 Table). Moreover, the various fermentation conditions had a different impact on the stress survival of the various strains. Strain IL1403 displayed the largest variation in robustness towards both heat and oxidative stress, which is in line with our observation that differences in fermentation conditions imposed the largest variation on growth characteristics of this strain as well. The observed differences in robustness towards both heat and oxidative stress of strain SK11 in the various fermentations demonstrate that contrary to earlier observations by Kim et al. [9] also strains with an L. lactis subsp. cremoris phenotype can have an adaptive response to stress.

As was observed before for strain MG1363 [11], no correlation of growth rate and survival towards heat stress was observed for the three strains. Only strain SK11 displayed a correlation of growth rate and oxidative stress survival (Pearson correlation coefficient = 0.79). Overall, this appears to support the study of Dressaire et al., which demonstrated that downregulation of stress genes at increasing growth rates, as observed in yeast [22], does not occur in L. lactis [23]. This implies that fermentation conditions resulting in improved robustness are not necessarily more time-consuming. Moreover, neither for heat stress nor oxidative stress, correlation of final biomass formation and survival was found, indicating that increased robustness can be achieved without the necessity to reduce yield.

To identify the individual fermentation parameters with the most pronounced effect on heat or oxidative stress survival, we compared survival phenotypes in fermentations with one variant of this parameter with survival phenotypes in fermentations with the other variant of this parameter. Similar to what was previously observed for MG1363 [11], survival of KF147 during heat stress significantly increased during fermentation with a high level of oxygen (Fig 3A), whereas for SK11 robustness towards heat stress significantly increased with increasing fermentation temperature (Fig 3B). Contrasting our earlier observations in MG1363 [11], oxidative stress survival of strains IL1403, KF147 and SK11 was not significantly higher in fermentation at 35°C as compared with fermentations at 27°C. Survival of IL1403, which displayed a large variation in robustness phenotypes in the various fermentations, was not significantly altered by any of the specific individual fermentation parameters (S2 Table).

Fig 3. Heat stress survival of KF147 and SK11.

Boxplots of robustness phenotypes towards 10 minutes of heat stress at relatively low and high oxygen levels for strain KF147 (A) and at various fermentation temperatures for strain SK11 (B). Robustness is expressed as the difference of log CFU/ml after stress (N_t) and before stress (N₀).

These experiments demonstrate that fermentation parameters have a substantial impact on subsequent stress survival of L. lactis strains. Irrespective of the strain’s general robustness level [5], survival can be dramatically altered by varying fermentation conditions. Although the fermentation parameters had similar effects on growth characteristics, the effect of specific fermentation parameters on survival is strain-dependent. This indicates that a general fermentation strategy to optimize robustness is difficult to achieve and to accomplish optimal robustness, fermentation conditions should be individually optimized for each L. lactis strain.

Transcriptome-phenotype matching reveals strain-specific associations of gene expression with robustness

We determined the effect of the fermentation parameters on gene expression. As previously demonstrated for strain MG1363 [11], the oxygen level and the fermentation temperature also had the most pronounced effect on gene expression in IL1403, KF147 and SK11 (S3 Fig), which appears to be in line with the observed effect of oxygen level and fermentation temperature on robustness phenotypes of several strains.

Subsequently, we calculated the correlation (according to a linear model) of gene expression levels in the various fermentations with the corresponding robustness phenotypes (S1–S6 Files). Similarly as for MG1363 [11], we selected the genes displaying a significant correlation (P < 0.05) with robustness at both time points of the stress assay. The genes with the most significant correlation at both time points of the stress assay (product of P-values < 5×10⁻⁵) were further analyzed (Table 2). For IL1403, 54 and 32 genes met these criteria for heat and oxidative stress survival, respectively. Only two genes displayed a significant correlation with oxidative stress survival in KF147, whereas 174 genes correlated with heat stress survival in this strain. In SK11, 124 and 63 genes displayed a significant correlation with heat and oxidative stress survival, respectively.

Table 2. Individual correlating gene expressions with robustness towards heat stress (A) or oxidative stress (B).

A
Strain	Locus tag	Gene	Function	Correlation	Slope
IL1403	L133770	rpmH	50S_ribosomal_protein_L34	negative	3.1
	L127611	yveD	hypothetical protein	negative	0.6
	L36850	ps104	prophage_ps1_protein_04	negative	0.1
	L52686	ycfD	hypothetical_protein	negative	1.1
	L52019	gntK	gluconate_kinase	positive	0.4
	L18206	ysdB	ABC transporter ATP binding protein	negative	1.8
	L167426	zitS	zinc ABC transporter substrate binding protein	negative	1.9
	L94973	ycjG	hypothetical protein	negative	3.4
	L14408	nagB	glucosamine-6-P isomerase	negative	3.9
	L72115	yohD	hypothetical protein	negative	2.9
	L154225	ylfD	hypothetical protein	negative	2.4
	L0163	ribG	riboflavin-specific deaminase	positive	0.1
	L145739	floL	flotillin-like protein	negative	5.2
	L39365	yqdA	hypothetical protein	negative	2.8
	L11493	arsC	arsenate reductase	negative	1.7
	L175712	ynhD	hypothetical protein	negative	3.2
	L196779	yfjD	tRNA/rRNA methyltransferase	negative	1.5
	L113377	ps221	prophage ps2 protein 21	negative	0.4
	L77017	ykhJ	hypothetical protein	negative	0.3
	L0397	rpsT	30S ribosomal protein S20	negative	17.7
	L0275	dnaN	DNA polymerase III subunit beta	positive	6.1
	L0063	aroF	phospho-2-dehydro-3-deoxyheptonate aldolase	negative	5.2
	L193734	pdc	phenolic acid decarboxylase	negative	0.3
	L156445	ylfH	N-acetylglucosamine catabolic protein	positive	1.8
	L126998	yveC	hypothetical protein	negative	2.4
	L158972	yjfJ	hypothetical protein	negative	5.7
	L189881	rluC	pseudouridine synthase	negative	1.5
	L109379	yjaJ	transcription regulator	negative	4.8
	L198904	ps304	prophage ps3 protein 04	negative	0.3
	L16848	ysdA	ABC transporter permease protein	negative	2.7
	L193031	yhjA	hypothetical protein	negative	11.4
	L0064	aroH	phospho-2-dehydro-3-deoxyheptonate aldolase	negative	17.4
	L30663	ycdA	hypothetical protein	negative	0.8
	L102317	hslA	HU like DNA-binding protein	negative	11.1
	L0285	dnaD	hypothetical protein	positive	2.7
	L0151	rgrB	GntR family transcription regulator	negative	4.1
	L188392	ybiH	hypothetical protein	positive	0.2
	L192589	pydA	dihydroorotate dehydrogenase 1A	negative	4.8
	L19745	bar	acyltransferase	negative	2.4
	L117821	yxdC	cation-transporting ATPase	negative	0.5
	L67463	yuiB	hypothetical protein	negative	7.9
	L199277	ps305	prophage ps3 protein 05	negative	0.7
	L71486	yohC	transcription regulator	negative	2.0
	L140714	adk	adenylate kinase	negative	2.7
	L43222	recX	recombination regulator RecX	negative	5.3
	L72684	ykhE	hypothetical protein	negative	0.3
	L00096	rpmF	50S ribosomal protein L32	negative	13.5
	L155044	dcdA	dCMP deaminase	negative	1.5
	L122849	ybcG	hypothetical protein	negative	7.5
	L3272	yiaD	putative NADH-flavin reductase	negative	3.9
	L0416	rplT	50S ribosomal protein L20	negative	10.5
	L0217	rlrD	LysR family transcription regulator	negative	1.7
	L148007	ybeM	hypothetical protein	negative	0.9
	L162840	yhgC	transcription regulator	negative	0.1
KF147	LLKF_1804	trxB	thioredoxin reductase	positive	12.6
	LLKF_1758	rarA	ArsR family transcriptional regulator	positive	0.7
	LLKF_0447	yeaA	beta-lactamase superfamily Zn-dependent hydrolase	positive	6.0
	LLKF_2085	ytgB	hypothetical protein	positive	17.7
	LLKF_1563	bglH	beta-glucosidase/ 6-phospho-beta-glucosidase	positive	0.4
	LLKF_1820	yrbB	transglycosylase	positive	26.3
	LLKF_2083		hypothetical protein	positive	15.2
	LLKF_2084	ytgA	hypothetical protein	positive	14.1
	LLKF_1723		excisionase	positive	0.1
	LLKF_2082	ytgH	Gls24 family general stress protein	positive	16.5
	LLKF_0716	glgD	glucose-1-phosphate adenylyltransferase regulatory subunit	negative	2.9
	LLKF_0747	menC	O-succinylbenzoate synthase	positive	5.1
	LLKF_0746	yhdA	1,4-dihydroxy-2-naphthoyl-CoA thioesterase	positive	2.2
	LLKF_0965	yjgC	amino acid ABC transporter substrate-binding protein	positive	7.0
	LLKF_0036	pdhC	pyruvate dehydrogenase complex dihydrolipoamide acetyltransferase	positive	23.9
	LLKF_1210		hypothetical protein	positive	1.5
	LLKF_0039	lplL	lipoate-protein ligase	positive	17.5
	LLKF_1293		AMP-dependent synthetase and ligase family protein	negative	0.6
	LLKF_0381	ydcG	Cro/CI family transcriptional regulator	positive	5.6
	LLKF_1201	nanE	N-acetylmannosamine-6-phosphate 2-epimerase	positive	0.8
	LLKF_0715	glgC	glucose-1-phosphate adenylyltransferase catalytic subunit	negative	1.6
	LLKF_0967	yjgE	amino acid transport, ATP-binding protein	positive	4.9
	LLKF_1852	yrfB	NADH-dependent oxidoreductase	positive	5.8
	LLKF_0684		CHW repeat-/cell adhesion domain-containing transglutaminase-like protease	negative	21.7
	LLKF_1259	ymdE	hypothetical protein	positive	16.9
	LLKF_0384	fhuG	ferrichrome ABC transporter permease FhuG	positive	2.9
	LLKF_1275	trmFO	tRNA (uracil-5-)-methyltransferase Gid	positive	11.6
	LLKF_0110	pmrB	MF superfamily multidrug resistance efflux pump protein	positive	0.9
	LLKF_1417	yngB	fibronectin-binding protein A	positive	1.9
	LLKF_1270	ilvA	threonine dehydratase	negative	2.9
	LLKF_1118	ykjI	hypothetical protein	positive	0.7
	LLKF_1265	ymeB	ABC transporter ATP-binding protein	negative	0.3
	LLKF_0493	pyrG	CTP synthase	positive	7.4
	LLKF_0849	trmU	tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase	positive	10.2
	LLKF_0664	scrK	fructokinase	positive	0.7
	LLKF_0555	yfhA	GNAT family acetyltransferase	positive	0.5
	LLKF_1344	xerD	site-specific tyrosine recombinase XerD	positive	1.5
	LLKF_2234		hypothetical protein	negative	1.5
	LLKF_2318		family 2 glycosyltransferase	negative	0.3
	LLKF_0959	yjfG	hypothetical protein	positive	3.5
	LLKF_0901	hslB	DNA-binding protein HU	positive	1.1
	LLKF_0500	dnaE	DNA polymerase III subunit alpha	positive	2.0
	LLKF_2242		hypothetical protein	negative	1.9
	LLKF_1294		acyl carrier protein	negative	0.3
	LLKF_1209		hypothetical protein	positive	0.8
	LLKF_0382	fhuC	ferrichrome ABC transporter ATP-binding protein FhuC	positive	5.8
	LLKF_0518	cysK	cysteine synthase	positive	1.0
	LLKF_2139	yudI	tRNA-dihydrouridine synthase	positive	8.4
	LLKF_1001	ftsE	cell division ATP-binding protein FtsE	positive	10.8
	LLKF_2231	ardA	conjugative transposon antirestriction protein	negative	0.5
	LLKF_1299	nisK	nisin biosynthesis two-component system, sensor histidine kinase NisK	positive	0.9
	LLKF_0094		ABC transporter ATPase protein	negative	8.1
	LLKF_0853	uvrC	excinuclease ABC subunit C	positive	4.1
	LLKF_0037	pdhB	pyruvate dehydrogenase E1 component subunit beta	positive	16.0
	LLKF_1962	nifU	SUF system FeS assembly protein	positive	11.1
	LLKF_0964	yjgB	gamma-D-glutamyl-meso-diaminopimelate peptidase I, NlpC/P60 family	positive	6.7
	LLKF_2244		FtsK/SpoIIIE family DNA segregation ATPase	negative	2.2
	LLKF_0038	pdhA	pyruvate dehydrogenase E1 component subunit alpha	positive	13.4
	LLKF_1851	yrfA	ArsR family transcriptional regulator	positive	3.1
	LLKF_0577	yfiL	GNAT family acetyltransferase	positive	2.0
	LLKF_1710	uxaC	uronate isomerase	negative	0.1
	LLKF_0098		hypothetical protein	negative	2.1
	LLKF_0441	trxH	thioredoxin	positive	3.0
	LLKF_0047	yahA	HAD superfamily hydrolase	positive	5.6
	LLKF_1812	yraD	hypothetical protein	positive	1.1
	LLKF_1857		ABC transporter ATP-binding protein	negative	0.3
	LLKF_0540	uvrB	excinuclease ABC subunit B	positive	2.4
	LLKF_1295		hypothetical protein	negative	0.5
	LLKF_1966	sufC	SUF system FeS cluster assembly protein ATP-dependent transporter SufC	positive	11.5
	LLKF_0052	cysD	O-acetyl-L-homoserine sulfhydrolase/O-acetyl-L-serine sulfhydrolase	positive	1.8
	LLKF_0904	yjaF	hypothetical protein	positive	5.6
	LLKF_0162	ybhA	5-formyltetrahydrofolate cyclo-ligase	negative	0.7
	LLKF_0035	pdhD	pyruvate dehydrogenase complex dihydrolipoamide acetyltransferase	positive	21.6
	LLKF_1720		hypothetical protein	negative	0.1
	LLKF_1579	ypaE	hypothetical protein	negative	4.6
	LLKF_2241		hypothetical protein	negative	1.9
	LLKF_2238		hypothetical protein	negative	1.7
	LLKF_1856		transcriptional regulator	negative	0.7
	LLKF_2243		replication initiation factor	negative	1.5
	LLKF_2233		CHAP domain family N-acetylmuramoyl-L-alanine amidase	negative	0.5
	LLKF_2236		hypothetical protein	negative	1.1
	LLKF_2246		hypothetical protein	negative	2.7
	LLKF_1948	ysdC	hypothetical protein	negative	0.3
	LLKF_1167	ylfFG	acyl-[acyl-carrier-protein] hydrolase	positive	2.5
	LLKF_1550	coaA	pantothenate kinase	positive	2.4
	LLKF_0668		GFO/IDH/MOCA family oxidoreductase	negative	0.2
	LLKF_0861	choS	glycine betaine ABC transporter permease/substrate-binding protein	positive	2.7
	LLKF_0999	yjjH	calcineurin-like phosphoesterase	positive	1.0
	LLKF_1961	sufB	cysteine desulfurase activator complex subunit SufB	positive	13.9
	LLKF_0443	noxE	NADH oxidase	positive	29.5
	LLKF_0020	tilS	tRNA(Ile)-lysidine synthetase	positive	2.3
	LLKF_0802	cysK	cysteine synthase	positive	2.2
	LLKF_0898	pnuC	nicotinamide mononucleotide transporter/n-ribosylnicotinamide transporter	positive	4.0
	LLKF_1536	pp270	phage protein	positive	0.6
	LLKF_0661	scrR	LacI family sucrose operon repressor	positive	0.8
	LLKF_1521	pp255	phage protein	negative	0.3
	LLKF_0284		transcriptional regulator	positive	2.5
	LLKF_0982	grpE	molecular chaperone GrpE	negative	6.4
	LLKF_1261	leuB	3-isopropylmalate dehydrogenase	negative	0.5
	LLKF_2093	ytgF	2,3-cyclic-nucleotide 2-phosphodiesterase	positive	10.6
	LLKF_0100		short chain dehydrogenase	negative	5.8
	LLKF_1331	ymjE	family 2 glycosyltransferase	positive	2.3
	LLKF_0093		ABC transporter permease	negative	8.2
	LLKF_1359	rnhB	ribonuclease HII	positive	0.9
	LLKF_0165	ybhD	GNAT family acetyltransferase	positive	0.5
	LLKF_1075	pp146	phage protein	positive	2.7
	LLKF_0310		hypothetical protein	negative	0.6
	LLKF_0981	hrcA	Heat-inducible transcription repressor HrcA	negative	5.7
	LLKF_0695		hypothetical protein	positive	7.5
	LLKF_1578	ypaD	hypothetical protein	negative	4.5
	LLKF_1799	aroD	3-dehydroquinate dehydratase	negative	1.9
	LLKF_2229		conjugative transposon Tn5276 integrase	negative	0.8
	LLKF_1872	yrgF	hypothetical protein	negative	0.5
	LLKF_1527	pp261	phage protein	negative	0.1
	LLKF_0029	yafF	hypothetical protein	positive	0.8
	LLKF_2431	gntR	RpiR family transcriptional regulator	negative	2.1
	LLKF_0983	dnaK	chaperone protein DnaK	negative	12.8
	LLKF_1695	thiL	acetyl-CoA acetyltransferase	positive	4.6
	LLKF_0551	dfpA	phosphopantothenoylcysteine decarboxylase	positive	1.5
	LLKF_0663	scrA	PTS system sucrose-specific transporter subunit IIABC	positive	0.5
	LLKF_2232		hypothetical protein	negative	0.9
	LLKF_1965	sufD	SUF system FeS cluster assembly protein SufD	positive	11.3
	LLKF_0510	adaA	methylphosphotriester-DNA alkyltransferase	positive	0.1
	LLKF_1352	gltB	glutamate synthase large subunit	negative	5.1
	LLKF_1018	ribH	riboflavin synthase subunit beta	positive	0.2
	LLKF_0570	yfiE	organic hydroperoxide resistance family protein	positive	24.3
	LLKF_0647	citB	aconitate hydratase	negative	0.3
	LLKF_0471	ligA	NAD-dependent DNA ligase	positive	2.9
	LLKF_0215	yqeL	GTP-binding protein	positive	2.0
	LLKF_0151	ybgA	hypothetical protein	negative	0.3
	LLKF_2444	pp401	phage integrase	positive	2.4
	LLKF_1853		hypothetical protein	positive	7.3
	LLKF_1066	pp137	phage HNH endonuclease	positive	0.7
	LLKF_2398	adhE	alcohol dehydrogenase/ acetaldehyde dehydrogenase	negative	6.5
	LLKF_1858		ABC transporter permease	negative	0.4
	LLKF_1656	yphI	hypothetical protein	positive	0.3
	LLKF_1324	dltC	D-alanine—poly(phosphoribitol) ligase subunit 2	negative	3.2
	LLKF_1284	recA	recombinase recA, C-terminal fragement	negative	0.2
	LLKF_1644	clpB	ATP-dependent Clp protease chaperonin ATPase ClpB	negative	3.1
	LLKF_0873	xseA	exodeoxyribonuclease VII large subunit	positive	2.5
	LLKF_0520	yfcI	metallo-beta-lactamase family protein	positive	0.9
	LLKF_1071	pp142	phage major head protein	positive	1.6
	LLKF_1566	trpA	tryptophan synthase subunit alpha	positive	0.8
	LLKF_1269	ilvC	ketol-acid reductoisomerase	negative	1.8
	LLKF_0822	rnc	ribonuclease III	positive	1.5
	LLKF_1132	cobQ	cobB/cobQ-like glutamine amidotransferase	positive	3.0
	LLKF_1501	pp235	phage terminase large subunit	negative	0.1
	LLKF_1887	pstA	phosphate ABC transporter ATP-binding protein	positive	4.0
	LLKF_1424	pfkA	6-phosphofructokinase	negative	14.2
	LLKF_0854	mutY	A/G-specific adenine DNA glycosylase	positive	1.5
	LLKF_0889	yijB	hypothetical protein	negative	0.2
	LLKF_0505	yfaA	hypothetical protein	positive	0.8
	LLKF_0918	tcsR	Two-component response regulator	positive	1.7
	LLKF_0390	yddD	glyoxalase family protein	positive	0.2
	LLKF_1805	ccpA	catabolite control protein A	positive	4.6
	LLKF_2245		hypothetical protein	negative	2.2
	LLKF_1546	deoC	deoxyribose-phosphate aldolase	positive	3.2
	LLKF_1589		putrescine/ornithine aminotransferase	negative	0.1
	LLKF_0270	nrdD	anaerobic ribonucleoside-triphosphate reductase	negative	11.7
	LLKF_0313		hypothetical protein	negative	0.1
	LLKF_1486	pp220	phage protein	positive	0.5
	LLKF_0104		hypothetical protein	negative	0.2
	LLKF_2239		hypothetical protein	negative	3.0
	LLKF_1351	gltD	glutamate synthase small subunit	negative	3.3
	LLKF_1728	csc2A	c-terminal membrane anchored cell surface protein	negative	0.1
	LLKF_2066	yteB	glycine/D-amino acid oxidase family protein	positive	0.2
	LLKF_0915	rpsN	50S ribosomal protein S14P	negative	0.1
	LLKF_0385	fhuD	ferrichrome ABC transporter substrate-binding protein FhuD	positive	8.9
	LLKF_0640	pfl	formate acetyltransferase	negative	11.3
	LLKF_1348	murI	glutamate racemase	positive	2.2
	LLKF_2368	comGE	competence protein ComGE	negative	0.1
	LLKF_0222	yccJ	hypothetical protein	positive	4.8
SK11	LACR_2496		gluconate kinase	positive
	LACR_2183		manganese transporter NRAMP	positive	1.5
	LACR_2273		hypothetical protein	positive	25.8
	LACR_2219		hypothetical protein	positive	1.1
	LACR_1490		hypothetical protein	positive	0.2
	LACR_C29		hypothetical protein	positive	15.9
	LACR_1011		ABC-type polar amino acid transport system, ATPase component	positive	12.9
	LACR_1370		cation-transporting P-ATPase	positive	20.0
	LACR_1188		hypothetical protein	positive	3.8
	LACR_2217		hypothetical protein	positive	1.0
	LACR_1428		hypothetical protein	positive	14.1
	LACR_1467		hypothetical protein	positive	8.6
	LACR_0359		hypothetical protein	positive	5.3
	LACR_2213		hypothetical protein	positive	3.3
	LACR_2358		integral membrane protein	negative	6.2
	LACR_1427		DeoR family transcriptional regulator	positive	9.4
	LACR_1389		hypothetical protein	positive	7.1
	LACR_0544		hypothetical protein	positive	0.7
	LACR_1168		hypothetical protein	positive	0.5
	LACR_1369		Mn-dependent transcriptional regulator	positive	6.5
	LACR_0743		flavodoxin	positive	2.0
	LACR_1502		hypothetical protein	positive	1.9
	LACR_A11		relaxase/mobilization nuclease domain-containing protein	positive	39.9
	LACR_0543	recU	Holliday junction-specific endonuclease	positive	3.2
	LACR_0274		hypothetical protein	positive	2.4
	LACR_2272		hypothetical protein	positive	5.3
	LACR_1231		hypothetical protein	negative	1.2
	LACR_0805		hypothetical protein	positive	1.3
	LACR_2216		hypothetical protein	positive	2.0
	LACR_1390		transcriptional regulator	positive	19.2
	LACR_2499		hypothetical protein	positive	2.3
	LACR_0927		acetyltransferase	positive	5.2
	LACR_1715		cation transport protein	positive	3.4
	LACR_0774		menaquinone-specific isochorismate synthase	positive	4.0
	LACR_1524		Signal transduction histidine kinase	positive	7.3
	LACR_2012		gamma-aminobutyrate permease related permease	negative	4.4
	LACR_1302	xerS	site-specific tyrosine recombinase XerS	positive	16.5
	LACR_C54		hypothetical protein	positive	4.8
	LACR_0329		acetyltransferase	positive	3.0
	LACR_0302		transcriptional regulator	positive	2.0
	LACR_0398	asnB	asparagine synthetase B	negative	17.5
	LACR_A05		hypothetical protein	positive	3.0
	LACR_2026		ABC-type oligopeptide transport system, periplasmic component	negative	4.4
	LACR_2220		hypothetical protein	positive	1.4
	LACR_2522		hypothetical protein	positive	4.4
	LACR_1437		transposase	positive	9.2
	LACR_1714		ArsR family transcriptional regulator	positive	3.1
	LACR_0904		transcriptional regulator	positive	0.6
	LACR_2151		hypothetical protein	positive	3.1
	LACR_1052		putative exporter of polyketide antibiotics	positive	3.3
	LACR_2126		hypothetical protein	negative	6.1
	LACR_1379		hypothetical protein	positive	1.2
	LACR_1525		hypothetical protein	positive	2.3
	LACR_0781		hypothetical protein	positive	2.5
	LACR_1237	truB	tRNA pseudouridine synthase B	positive	2.0
	LACR_1261		hypothetical protein	positive	1.5
	LACR_C27		pyrrolidone-carboxylate peptidase	positive	6.5
	LACR_1505		transposase	positive	9.0
	LACR_0803		hypothetical protein	positive	1.3
	LACR_2218		hypothetical protein	positive	2.1
	LACR_2270		hypothetical protein	positive	18.7
	LACR_1987	murE	UDP-N-acetylmuramoylalanyl-D-glutamate—2,6-diaminopimelate ligase	positive	3.6
	LACR_1104		hypothetical protein	negative	5.0
	LACR_0812		putative effector of murein hydrolase LrgA	positive	4.1
	LACR_1019		hypothetical protein	negative	4.4
	LACR_1523		DNA-binding response regulator	positive	5.0
	LACR_0804		hypothetical protein	positive	2.2
	LACR_0140		hypothetical protein	positive	0.1
	LACR_0505		hypothetical protein	negative	0.4
	LACR_1362		transcriptional regulator	positive	1.8
	LACR_C28		dienelactone hydrolase family protein	positive	14.6
	LACR_2274		hypothetical protein	positive	12.9
	LACR_1031		lactose transport regulator	positive	2.6
	LACR_1067		amidase	positive	0.5
	LACR_2592		hypothetical protein	positive	0.2
	LACR_1032		tagatose-6-phosphate kinase	positive	4.9
	LACR_0422		transcriptional regulator	positive	0.4
	LACR_0450		hypothetical protein	positive	0.4
	LACR_1982		pleiotropic transcriptional repressor	positive	0.1
	LACR_0809		hypothetical protein	positive	2.3
	LACR_2381	secY	preprotein translocase subunit SecY	negative	21.0
	LACR_2340		hypothetical protein	positive	1.7
	LACR_D08		site-specific recombinase, DNA invertase Pin related protein	negative	11.5
	LACR_1260		hypothetical protein	positive	1.4
	LACR_1122		deoxyuridine 5'-triphosphate nucleotidohydrolase	negative	6.4
	LACR_1079		hypothetical protein	positive	1.5
	LACR_2118		deoxyuridine 5'-triphosphate nucleotidohydrolase	negative	4.6
	LACR_0432		membrane carboxypeptidase (penicillin-binding protein)	positive	2.6
	LACR_0807		sortase (surface protein transpeptidase)	positive	1.2
	LACR_1020		hypothetical protein	negative	4.4
	LACR_1164		hypothetical protein	positive	0.3
	LACR_0301		integrase	positive	2.2
	LACR_2515	ruvB	Holliday junction DNA helicase RuvB	positive	2.8
	LACR_2119		hypothetical protein	negative	1.8
	LACR_0582		dinucleoside polyphosphate hydrolase	positive	1.9
	LACR_0511		hypothetical protein	positive	4.8
	LACR_0775		SSU ribosomal protein S5P alanine acetyltransferase	positive	1.0
	LACR_2134		hypothetical protein	negative	1.9
	LACR_2116		hypothetical protein	negative	1.5
	LACR_2357		hypothetical protein	negative	1.3
	LACR_2558		transcriptional regulator	positive	0.5
	LACR_0956		transcriptional regulator	positive	1.7
	LACR_1891		competence protein	negative	0.2
	LACR_0094		D-tyrosyl-tRNA(Tyr) deacylase	positive	0.7
	LACR_0201		hypothetical protein	negative	5.9
	LACR_2462		transposase	positive	12.1
	LACR_1458		N-acetylglucosamine 6-phosphate deacetylase	positive	3.5
	LACR_C08		acetyltransferase	negative	0.6
	LACR_1266		xanthine/uracil permease	negative	0.9
	LACR_0870		HAD superfamily hydrolase	positive	2.3
	LACR_D23		replication initiator protein	positive	2.5
	LACR_1635		transposase	positive	9.3
	LACR_0715		Mg-dependent DNase	positive	1.4
	LACR_1856		hypothetical protein	positive	1.4
	LACR_0652		XRE family transcriptional regulator	positive	1.5
	LACR_1631	thyA	thymidylate synthase	positive	2.0
	LACR_0249		HAD superfamily hydrolase	negative	1.1
	LACR_0680		transposase	positive	12.4
	LACR_1099		XRE family transcriptional regulator	positive	7.6
	LACR_2061		TIM-barrel fold family protein	negative	11.5
	LACR_1423		hypothetical protein	positive	4.0
	LACR_1063		ribonucleoside-diphosphate reductase class Ib glutaredoxin subunit	positive	10.8
	LACR_0066		transcriptional regulator	positive	2.2
	LACR_C32		transposase	negative	20.6
B
Strain	Locus tag	Gene	Function	Correlation	Slope
IL1403	L162840	yhgC	transcription regulator	negative	0.1
	L79507	yahD	hypothetical protein	positive	2.8
	L0275	dnaN	DNA polymerase III subunit beta	positive	7.1
	L104969	napC	multidrug-efflux transporter	positive	0.2
	L189822	ybiK	hypothetical protein	positive	8.4
	L109527	rsuA	ribosomal small subunit pseudouridine synthase A	negative	0.8
	L84992	ytaB	YtaB	positive	2.9
	L0165	ribA	3,4-dihydroxy-2-butanone 4-phosphate synthase	positive	0.2
	L4822	ptsK	HPr kinase/phosphorylase	positive	5.9
	L196779	yfjD	tRNA/rRNA methyltransferase	negative	1.7
	L180241	mycA	myosin-cross-reactive antigen	positive	5.4
	L7798	ps316	integrase	negative	1.6
	L30663	ycdA	hypothetical protein	negative	0.9
	L20937	ywdF	hypothetical protein	negative	3.3
	L190009	feoB	ferrous ion transport protein B	positive	8.1
	L0016	gpsA	NAD(P)H-dependent glycerol-3-phosphate dehydrogenase	positive	4.8
	L193030	yjjD	ABC transporter permease protein	positive	0.9
	L136552	ybdJ	hypothetical protein	positive	0.2
	L179531	ispB	heptaprenyl diphosphate synthase component II	positive	4.3
	L0241	uxuB	fructuronate reductase	positive	0.1
	L177590	hasC	UTP-glucose-1-phosphate uridylyltransferase	positive	5.7
	L0274	dnaA	chromosomal replication initiation protein	positive	7.2
	L114325	ybbE	hypothetical protein	negative	0.8
	L0298	topA	DNA topoisomerase I	negative	4.3
	L32731	ykdB	hypothetical protein	positive	1.0
	L17893	yebF	transcription regulator	positive	1.2
	L180104	umuC	UmuC	positive	0.3
	L0101	metA	homoserine O-succinyltransferase	positive	1.7
	L197697	yfjE	flavodoxin	negative	1.5
	L200024		hypothetical protein	positive	0.4
	L5776	lgt	prolipoprotein diacylglyceryl transferase	positive	2.0
	L135900	ybdI	hypothetical protein	positive	0.2
KF147	LLKF_2311		family 2 glycosyltransferase	negative	0.3
	LLKF_0448	tcsK	Two-component sensor histidine kinase	negative	5.5
SK11	LACR_0741		hypothetical protein	positive	0.8
	LACR_0891		copper/potassium-transporting ATPase	positive	4.4
	LACR_E7		hypothetical protein	positive	4.1
	LACR_1450		fibronectin-binding protein	positive	1.1
	LACR_0073		esterase	positive	10.0
	LACR_0714		hypothetical protein	positive	3.7
	LACR_C16		replication initiator protein	positive	3.3
	LACR_0074		lactoylglutathione lyase related lyase	positive	7.0
	LACR_1221		hypothetical protein	positive	2.1
	LACR_0072		hypothetical protein	positive	8.5
	LACR_0920		copper-potassium transporting ATPase B	positive	5.0
	LACR_0959		hypothetical protein	positive	1.3
	LACR_0242		saccharopine dehydrogenase related protein	positive	10.1
	LACR_0451		ABC-type multidrug transport system, permease component	positive	3.0
	LACR_0713		acetyltransferase	positive	2.9
	LACR_0452		ABC-type multidrug transport system, ATPase component	positive	5.2
	LACR_0381		hypothetical protein	positive	0.5
	LACR_1506		hypothetical protein	positive	0.3
	LACR_0744		lysophospholipase L1 related esterase	positive	1.5
	LACR_2167		N-acetylmuramoyl-L-alanine amidase	positive	4.5
	LACR_0347		ABC-type multidrug transport system, ATPase and permease component	positive	4.4
	LACR_1291		Beta-xylosidase	positive	0.5
	LACR_1468		orotidine 5'-phosphate decarboxylase	positive	5.4
	LACR_0240		NADPH:quinone reductase related Zn-dependent oxidoreductase	positive	11.5
	LACR_1051		ABC-type multidrug transport system, ATPase component	positive	3.0
	LACR_0075		hypothetical protein	positive	6.7
	LACR_0241		nucleoside-diphosphate sugar epimerase	positive	11.2
	LACR_0105		hypothetical protein	positive	3.3
	LACR_0629		major facilitator superfamily permease	positive	0.3
	LACR_0164		hypothetical protein	positive	3.8
	LACR_2411		hypothetical protein	negative	0.9
	LACR_1362		transcriptional regulator	positive	1.1
	LACR_0982		ring-cleavage extradiol dioxygenase	positive	3.6
	LACR_0742		transcriptional regulator	positive	2.0
	LACR_0537		cysteine synthase	positive	0.4
	LACR_0743		flavodoxin	positive	1.2
	LACR_D16		oligopeptidase O1	negative	11.3
	LACR_2476		transcriptional regulator	positive	5.7
	LACR_0839		sugar metabolism transcriptional regulator	positive	1.7
	LACR_1302	xerS	site-specific tyrosine recombinase XerS	positive	9.7
	LACR_1290		endoglucanase	positive	0.2
	LACR_2355		hypothetical protein	positive	0.8
	LACR_1976		negative regulator of genetic competence, sporulation and motility	positive	2.4
	LACR_1629		transcriptional regulator	positive	2.1
	LACR_1395		hypothetical protein	positive	3.7
	LACR_1922		hypothetical protein	negative	1.1
	LACR_1267		hypothetical protein	positive	0.4
	LACR_2497		6-phosphogluconate dehydrogenase-like protein	positive	0.9
	LACR_0431		tyrosyl-tRNA synthetase	negative	10.0
	LACR_0570	dnaG	DNA primase	positive	2.4
	LACR_0657		adenine phosphoribosyltransferase	negative	5.6
	LACR_2490	recX	recombination regulator RecX	positive	14.2
	LACR_1728		Mg2+ transporter	positive	1.6
	LACR_1751		transposase	positive	3.3
	LACR_0206		glycosyltransferase	negative	1.0
	LACR_1052		putative exporter of polyketide antibiotics	positive	2.0
	LACR_0642		6-phosphogluconate dehydrogenase	negative	5.1
	LACR_0800		XRE family transcriptional regulator	positive	1.2
	LACR_1078		transcriptional regulator	negative	0.1
	LACR_2545		ribosomal small subunit pseudouridine synthase A	negative	1.6
	LACR_2184		oxidoreductase	positive	9.7
	LACR_0212		lipopolysaccharide biosynthesis protein	negative	1.8
	LACR_1105		hypothetical protein	positive	4.3

Correlating gene expressions with robustness towards heat stress (A) or oxidative stress (B) as assessed by a linear model of the strains IL1403, KF147 and SK11. Genes of which expression correlated with survival in more than one strain (including MG1363 [11]) are indicated in bold. Genes are ranked based on the significance of correlation (lowest P-value on top). Slope represents the average slope of the linear models fitting the data of both time points of the stress assay.

In KF147, the operon encoding the pyruvate dehydrogenase complex (pdhABCD) and a lipoate-protein ligase (lplL) as well as an operon encoding a ferrichrome ABC transporter (fhuCDG) and an operon encoding hypothetical proteins and a Gls24 family general stress protein (ytgH) displayed a positive correlation with heat stress survival. Surprisingly, the heat shock genes grpE and dnaK anti-correlated with robustness towards heat stress of KF147 and also their repressor hrcA displayed anti-correlation [24]. The gene fhuC was previously associated with heat stress survival in MG1363 [11], as well as four other genes: uvrC, cysD, cysK and trpA. In contrast to KF147 and MG1363, these transcripts did not show a significant correlation with heat stress survival in IL1403 nor in SK11, although a previous study by Xie et al. did suggest a role of cysK in heat stress survival of IL1403 [13]. Two other genes were found to associate with heat stress survival in both KF147 and SK11 (rarA and yjgE/ LACR_1011) and one in both IL1403 and SK11 (gntK). However, the majority of the correlating genes were shown to associate with stress survival in only one of the strains. In IL1403 the genes aroF and aroH encoding a phospho-2-dehydro-3-deoxyheptonate aldolase anti-correlated with heat stress survival. The gene aroF was previously shown to be upregulated in this strain during osmotic stress [13], suggesting this gene could be involved in a general stress mechanism. In SK11, multiple genes encoding hypothetical proteins were found to correlate with heat stress survival and also a gene encoding a manganese transporter (LACR_2183). Manganese transport was also associated with heat stress survival in an earlier study, where mtsC, encoding part of a manganese ABC transporter was shown to be present in robust strains and absent in sensitive strains within an L. lactis strain collection [5]. Metal ions have several functions in the cell and can be involved in stabilizing proteins, ribosomes and the cell membrane [25, 26]. Because these cellular components are affected during heat stress [8], manganese might have a role in the prevention of damage caused by heat stress.

Similar as for heat stress, the transcriptome signature associated with oxidative stress survival was highly strain-specific, which is exemplified by the fact that only three genes associated with oxidative stress survival in more than one strain. In both IL1403 and SK11 the gene expressions yahD/ LACR_0073, yjjD/ LACR_1052 and rsuA/ LACR_2545 were found to correlate with oxidative stress survival. In IL1403, 32 genes displayed correlation of expression with survival, among which was the gene feoB, which is involved in iron transport and was previously associated with heat stress survival in MG1363 [11]. In SK11, a gene encoding cysteine synthase positively correlated with oxidative stress survival. In MG1363 we previously demonstrated a link between cysteine metabolism and oxidative stress survival [11]. Sulfur-containing amino acids are readily oxidized and, therefore, cysteine metabolism could be involved in oxidative stress survival by affecting the redox balance in the cell. Furthermore, genes associated with oxidative stress survival in SK11 included genes encoding membrane proteins and regulators. For application as indicators for robustness, the genes with a high variation in gene expression (indicated by the slope in Table 2) appear to be most suitable, because they can be detected with methods such as quantitative PCR. For both heat and oxidative stress, none of the genes were associated with survival in more than two strains, although the majority of the genes that displayed correlation with survival are present in all four strains. This lack in overlap demonstrates that the transcriptome signature associated with stress survival is largely strain-dependent, and the complete transcriptome signature associated with robustness in one strain cannot be extrapolated fully to other strains. This indicates that the mechanisms aiming to improve robustness vary among the strains and, therefore, strategies resulting in improved robustness of one strain do not necessarily increase robustness of other strains. To acquire optimal robustness, the fermentation conditions of each strain require individual optimization.

Generic L. lactis genes associated with robustness towards heat or oxidative stress

To establish whether a generic transcriptome signature for L. lactis exists, we searched for single genes with the most significant correlation with robustness towards heat and oxidative stress in all strains. For this, we chose one time point of the stress assay, in which the range between the extreme values of survival in all fermentations was the largest (see Materials and methods). We selected the orthologous groups (OGs) in which the genes of all four strains displayed either a positive or a negative correlation (P < 0.2, assessed with a linear model) of expression level with robustness phenotype and ranked these on average P-value per OG (Table 3). Notably, the top 10 genes included ctsR, encoding a class three stress genes transcriptional repressor, which displayed negative correlation of expression with oxidative stress survival in all four strains. This gene was previously demonstrated to be a key regulator of heat-shock induced gene expression in MG1363 [27]. The observation that the transcript level of this gene appeared in the top 10 list of most significant correlating genes with oxidative stress survival suggests that CtsR is also involved in oxidative stress regulation in L. lactis. Involvement of CtsR in other stress responses besides heat stress response was already suggested by Frees et al., who demonstrated that the CtsR regulon was induced at low pH [28]. Furthermore, in Bacillus subtilis involvement of CtsR in oxidative stress survival has been previously suggested as transcription of the CtsR regulon was increased during oxidative stress [29]. Besides the significant correlation with heat stress survival of KF147, as mentioned in the previous paragraph, the gene lplL also displayed a positive correlation of expression and heat stress survival in the other three strains. This gene was previously demonstrated to be involved in heat shock response in strain IL1403 [13], which further supports the role of this gene in heat stress survival in L. lactis strains in general. Furthermore, the list contained multiple genes encoding for proteins involved in iron(complex) transport (feoA, fhuD, fhuG and fhuB). The fhu operon may be involved in haem uptake, enabling respiration metabolism in L. lactis [30, 31] and was recently demonstrated to be induced in strain MG1363 during the early phase of growth at high oxygen levels [32]. Furthermore, it has been demonstrated that free intracellular iron increases oxidative stress through generation of ROS from hydrogen peroxide by the Fenton reaction, which causes cellular damage and mortality in stationary phase cells of L. lactis [33]. A link between iron metabolism and heat stress survival has been demonstrated in Bacillus licheniformis, where an overlap in response to heat shock and iron limitation was revealed [34]. Taken together, a link between iron metabolism and stress survival in L. lactis appears likely.

Table 3. Generic correlating gene expressions with robustness towards heat stress (A) or oxidative stress (B).

A
locustag IL1403	locustag SK11	locustag KF147	locustag MG1363	gene	function	correlation	average P-value	maximum P-value
L191486	LACR_1356	LLKF_1201	llmg_1317	yljB/nanE	N-acetylmannosamine-6-phosphate 2-epimerase	positive	0.025	0.039
L101688	LACR_1561	LLKF_1575	llmg_1029	ypaA	hypothetical protein	negative	0.026	0.062
L195318	LACR_1054	LLKF_0997	llmg_1551	yjjF/fdhC	formate/nitrite transporter	negative	0.031	0.059
L89001	LACR_1179	LLKF_1106	llmg_1494	ykiI	ABC transporter permease	positive	0.035	0.095
L64373	LACR_0052	LLKF_0039	llmg_0075	lplL	lipoate-protein ligase	positive	0.044	0.116
L143312	LACR_0389	LLKF_0398	llmg_0362	dppA/optS	oligopeptide ABC transporter substrate binding protein	negative	0.046	0.138
L72684	LACR_1157	LLKF_1090	llmg_1513	ykhE	arsenate reductase	negative	0.048	0.103
L18206	LACR_1946	LLKF_1947	llmg_1957	ysdB	sodium ABC transporter ATP-binding protein	negative	0.053	0.106
L192240	LACR_0194	LLKF_0183	llmg_0200	feoA	ferrous iron transport protein A	positive	0.054	0.113
L148945	LACR_1868	LLKF_1872	llmg_0725	yrgF	hypothetical protein	negative	0.057	0.184
B
locustag IL1403	locustag SK11	locustag KF147	locustag MG1363	gene	function	correlation	average P-value	maximum P-value
L0223	LACR_0665	LLKF_0631	llmg_0614	ctsR	class III stress genes transcriptional repressor	negative	0.025	0.055
L128386	LACR_0373	LLKF_0384	llmg_0348	fhuG	ferrichrome ABC transporter permease FhuG	positive	0.030	0.051
L100027	LACR_2040	LLKF_2041	llmg_2036	ytbC	hypothetical protein	negative	0.039	0.127
L0046	LACR_0642	LLKF_0600	llmg_0586	gnd	6-phosphogluconate dehydrogenase	negative	0.044	0.115
L127476	LACR_0372	LLKF_0383	llmg_0347	fhuB	ferrichrome ABC transporter permease protein	positive	0.059	0.109
L117074	LACR_2341	LLKF_2294	llmg_2327	yvdD	glycerol uptake facilitator protein	negative	0.066	0.134
L103246	LACR_1565	LLKF_1577	llmg_1026	ypaC	methylase for ubiquinone/menaquinone biosynthesis	negative	0.077	0.102
L104745	LACR_1567	LLKF_1579	llmg_1024	ypaE	hypothetical protein	negative	0.081	0.118
L162870	LACR_1609	LLKF_1641	llmg_0989	ypgD	ABC transporter ATP binding and permease protein	positive	0.098	0.183
L129403	LACR_0374	LLKF_0385	llmg_0349	fhuD	ferrichrome ABC transporter substrate binding protein	positive	0.099	0.157

Top 10 highest correlating transcript levels with robustness towards heat stress (A) or oxidative stress (B). Average P-value is the average of the P-values of the correlation as assessed by a linear model of the strains MG1363, IL1403, KF147 and SK11 and was used to rank the genes. Maximum P-value indicates the largest P-value of the correlation among the four strains.

Besides the genes that have previously been demonstrated to be involved in stress, the top 10 lists also included genes which to the best of our knowledge have not been associated with stress before. The transcript levels of yljB/nanE, encoding an N-acetylmannosamine-6-phosphate 2-epimerase involved in amino sugar metabolism, displayed the highest correlation in all four strains with robustness towards heat stress. Furthermore, genes encoding transport proteins or hypothetical proteins were among the genes with the most significant correlation of expression and heat or oxidative stress survival in all strains. Revealing the exact mechanism via which the functions encoded by these genes impact on robustness requires additional work.

The strains included in this study varied in type of subspecies, isolation source and general robustness [5] and therefore appear to represent a major part of the L. lactis species. Therefore, it is tempting to suggest that the generic gene expressions associated with robustness in this study can be applied as indicators of robustness for L. lactis strains in general, although individual transcriptome signatures are expected to predict robustness of specific strains more accurately.

Conclusions

In this study we demonstrated that fermentation conditions (e.g. temperature and level of oxygen) have a large impact on heat and oxidative stress survival of L. lactis strains. Therefore, fermentation conditions prior to industrial processing of starter cultures should be carefully selected, and this is true for both intrinsically robust and sensitive strains [5]. The development of a general fermentation strategy for improved robustness of L. lactis starter cultures appears complicated as the effect of fermentation conditions on robustness towards heat and oxidative stress is strain-dependent, even though fermentation conditions have largely similar effects on growth characteristics. The larger part of the transcriptome signatures associated with robustness also appeared strain-specific, indicating that different mechanisms exist to improve robustness. Hence, to obtain optimal robustness in each individual strain tailor-made optimization of fermentation parameters is required. Furthermore, we explored the most significant associations of transcript levels and robustness that overlapped in all four strains, resulting in a generic transcriptome signature associated with robustness in these L. lactis strains, which included both known genes encoding stress related functions and novel genes. This generic transcriptome signature could function as an indicator for robustness and aid the selection of optimal fermentation conditions for optimal robustness during spray drying.

Supporting Information

S1 Fig. DNA microarray hybridization scheme.

Numbers indicate fermentations as presented in Table 1. Samples connected with arrows were hybridized together, the arrow head represents Cy5-labeling, the back end Cy3-labeling.

(TIFF)

S2 Fig. Growth curves during various fermentations.

Growth curves of strains IL1403, KF147 and SK11 in fermentations as presented in Table 1. The data points between the dotted lines indicate the moment of harvesting cells for RNA isolation and stress survival assays.

(TIF)

S3 Fig. Genes expressed by individual fermentation parameters.

Numbers indicate the amount of genes that are differently expressed (P < 0.05) by both the individual fermentation parameter (salt, oxygen, pH and temperature) specified in the top row and in the left column. Bars indicate percentages of overlap of differently expressed genes by both fermentation parameters (full bar = 100%).

(TIF)

S1 Table. Heat and oxidative stress survival at the additional time point.

Survival after 30 minutes of heat stress and after 60 minutes of oxidative stress in the various fermentations of strains IL1403, KF147 and SK11. Survival data represent averages of technical duplicates.

(DOCX)

S2 Table. Correlation fermentation parameters and robustness.

T-test-based correlation of individual fermentation parameters and robustness. Significant differences (P < 0.05) are underlined.

(DOCX)

S1 File. Plots of gene expression and robustness levels in IL1403 (part 1).

Expression levels of genes L0001 –L75633 plotted against survival after 60 minutes heat and 30 min oxidative stress. Survival is expressed as the difference of log CFU/ml after stress and before stress. Numbers indicate fermentations as presented in Table 1. P-values above the plots indicate significance of correlation (assessed by a linear model).

(ZIP)

S2 File. Plots of gene expression and robustness levels in IL1403 (part 2).

Expression levels of genes L75676 –L1889726 plotted against survival after 60 minutes heat and 30 min oxidative stress. Survival is expressed as the difference of log CFU/ml after stress and before stress. Numbers indicate fermentations as presented in Table 1. P-values above the plots indicate significance of correlation (assessed by a linear model).

(ZIP)

S3 File. Plots of gene expression and robustness levels in KF147 (part 1).

Expression levels of genes LLKF_0001 –LLKF_1273 plotted against survival after 10 minutes heat and 30 minutes oxidative stress. Survival is expressed as the difference of log CFU/ml after stress and before stress. Numbers indicate fermentations as presented in Table 1. P-values above the plots indicate significance of correlation (assessed by a linear model).

(ZIP)

S4 File. Plots of gene expression and robustness levels in KF147 (part 2).

Expression levels of genes LLKF_1274 –LLKF_2533 and LLKF_p0001 –LLKF_p0036 plotted against survival after 10 minutes heat and 30 minutes oxidative stress. Survival is expressed as the difference of log CFU/ml after stress and before stress. Numbers indicate fermentations as presented in Table 1. P-values above the plots indicate significance of correlation (assessed by a linear model).

(ZIP)

S5 File. Plots of gene expression and robustness levels in SK11 (part 1).

Expression levels of genes LACR_0001 –LACR_1382 plotted against survival after 10 minutes heat and 30 minutes oxidative stress. Survival is expressed as the difference of log CFU/ml after stress and before stress. Numbers indicate fermentations as presented in Table 1. P-values above the plots indicate significance of correlation (assessed by a linear model).

(ZIP)

S6 File. Plots of gene expression and robustness levels in SK11 (part 2).

Expression levels of genes LACR_1383 –LACR_2610 and LACR_A01 –LACR_E8 plotted against survival after 10 minutes heat and 30 minutes oxidative stress. Survival is expressed as the difference of log CFU/ml after stress and before stress. Numbers indicate fermentations as presented in Table 1. P-values above the plots indicate significance of correlation (assessed by a linear model).

(ZIP)

Data Availability

All relevant data are available in the paper and its Supporting Information files. Microarray data are available from GEO (GSE72045).

Funding Statement

AD was funded by the Kluyver centre for Genomics of Industrial Fermentation which is part of the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research (NWO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.