Global transcriptome response in Lactobacillus sakei during growth on ribose.

BACKGROUND: Lactobacillus sakei is valuable in the fermentation of meat products and exhibits properties that allow for better preservation of meat and fish. On these substrates, glucose and ribose are the main carbon sources available for growth. We used a whole-genome microarray based on the genome sequence of L. sakei strain 23K to investigate the global transcriptome response of three L. sakei strains when grown on ribose compared with glucose.
RESULTS: The function of the common regulated genes was mostly related to carbohydrate metabolism and transport. Decreased transcription of genes encoding enzymes involved in glucose metabolism and the L-lactate dehydrogenase was observed, but most of the genes showing differential expression were up-regulated. Especially transcription of genes directly involved in ribose catabolism, the phosphoketolase pathway, and in alternative fates of pyruvate increased. Interestingly, the methylglyoxal synthase gene, which encodes an enzyme unique for L. sakei among lactobacilli, was up-regulated. Ribose catabolism seems closely linked with catabolism of nucleosides. The deoxyribonucleoside synthesis operon transcriptional regulator gene was strongly up-regulated, as well as two gene clusters involved in nucleoside catabolism. One of the clusters included a ribokinase gene. Moreover, hprK encoding the HPr kinase/phosphatase, which plays a major role in the regulation of carbon metabolism and sugar transport, was up-regulated, as were genes encoding the general PTS enzyme I and the mannose-specific enzyme II complex (EIIman). Putative catabolite-responsive element (cre) sites were found in proximity to the promoter of several genes and operons affected by the change of carbon source. This could indicate regulation by a catabolite control protein A (CcpA)-mediated carbon catabolite repression (CCR) mechanism, possibly with the EIIman being indirectly involved.
CONCLUSIONS: Our data shows that the ribose uptake and catabolic machinery in L. sakei is highly regulated at the transcription level. A global regulation mechanism seems to permit a fine tuning of the expression of enzymes that control efficient exploitation of available carbon sources.

Background

The Lactobacillus sakei species belongs to the lactic acid bacteria (LAB), a group of Gram-positive organisms with a low G+C content which produce lactic acid as the main end product of carbohydrate fermentation. This trait has, throughout history, made LAB suitable for production of food. Acidification suppresses the growth and survival of undesirable spoilage bacteria and human pathogens. L. sakei is naturally associated with the meat and fish environment, and is important in the meat industry where it is used as starter culture for sausage fermentation [1,2]. The bacterium shows great potential as a protective culture and biopreservative to extend storage life and ensure microbial safety of meat and fish products [3-6]. The genome sequence of L. sakei strain 23K has revealed a metabolic repertoire which reflects the bacterium's adaption to meat products and the ability to flexibly use meat components [7]. Only a few carbohydrates are available in meat and fish, and L. sakei can utilize mainly glucose and ribose for growth, a utilization biased in favour of glucose [7-9]. The species has been observed as a transient member of the human gastrointestinal tract (GIT) [10,11], and ribose may be described as a commonly accessible carbon source in the gut environment [12]. Transit through the GIT of axenic mice gave mutant strains which grow faster on ribose compared with glucose [13].

Glucose is primarily transported and phosphorylated by the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system (PTS). A phosphorylation cascade is driven from PEP through the general components enzyme I (EI) and the histidine protein (HPr), then via the mannose-specific enzyme II complex (EIIman) to the incoming sugar. Moreover, glucose is fermented through glycolysis leading to lactate [7,8,14]. Ribose transport and subsequent phosphorylation are induced by the ribose itself and mediated by a ribose transporter (RbsU), a D-ribose pyranase (RbsD), and a ribokinase (RbsK) encoded by rbsUDK, respectively. These genes form an operon with rbsR which encodes the local repressor RbsR [15,16]. The phosphoketolase pathway (PKP) is used for pentose fermentation ending with lactate and other end products [8,17]. L. sakei also has the ability to catabolize arginine, which is abundant in meat, and to catabolize the nucleosides inosine and adenine, a property which is uncommon among lactobacilli [7,18].

By proteomics, we recently identified proteins involved in ribose catabolism and the PKP to be over-expressed during growth on ribose compared with glucose, while several glycolytic enzymes were less expressed. Moreover, also enzymes involved in pyruvate- and glycerol/glycerolipid metabolism were over-expressed on ribose [19]. Bacteria often use carbon catabolite repression (CCR) in order to control hierarchical utilization of different carbon sources. In low G+C content Gram-positive bacteria, the dominant CCR pathway is mediated by the three main components: (1) catabolite control protein A (CcpA) transcriptional regulator; (2) the histidine protein (HPr); and (3) catabolite-responsive element (cre) DNA sites located in proximity to catabolic genes and operons, which are bound by CcpA [20-23]. The HPr protein has diverse regulatory functions in carbon metabolism depending on its phosphorylation state. In response to high throughput through glycolysis, the enzyme is phosphorylated at Ser46 by HPr kinase/phosphorylase (HPrK/P). This gives P-Ser-HPr which can bind to CcpA and convert it into its DNA-binding-competent conformation. However, when the concentration of glycolytic intermediates drop, the HPrK/P dephosphorylates P-Ser-HPr [20,22-24]. Under low glucose concentrations, HPr is phosphorylated by E1 of the PTS at His15 to give P-His-HPr, which has a catalytic function in the PTS and regulatory functions by phosphorylation of catabolic enzymes and transcriptional regulators with a PTS regulation domain (PRD). Several P-EIIBs also phosphorylate different types of non-PTS proteins and regulate their activities [20-22]. Evidence for regulatory processes resembling glucose repression was shown both during lactose utilization [25] and catabolism of arginine [26,27] in L. sakei. A cre site has been reported upstream of the rbs operon [28], thus CcpA could likely be acting on the rbs operon as well as other catabolic genes and operons in this bacterium.

In the present study, we use a microarray representing the L. sakei 23K genome and an additional set of sequenced L. sakei genes, to investigate the global transcriptome response of three L. sakei strains when grown on ribose compared with glucose. Moreover, we predict the frequency of cre sites presumed to be involved in CCR in the L. sakei 23K genome sequence. Our objective was to identify differentially expressed genes between growth on the two sugars, and to increase the understanding of how the primary metabolism is regulated.

Methods

Bacterial strains, media and growth conditions

L. sakei 23K is a plasmid-cured sausage isolate [29], and its complete genome sequence has been published [7]. L. sakei LS 25 is a commercial starter culture strain for salami sausage [30]. L. sakei MF1053 originates from fermented fish (Norwegian "rakfisk") [9]. The strains were maintained at -80°C in MRS broth (Oxoid) supplemented with 20% glycerol. Growth experiments were performed in a defined medium for lactobacilli [31] supplemented with 0.5% glucose (DMLG) or 0.5% ribose + 0.02% glucose (DMLRg) as described previously [19]. Samples were extracted at three different days from independent DMLG and DMLRg cultures from each strain grown at 30°C to mid-exponential phase (OD600 = 0.5-0.6) for a total of three sample sets (parallels).

Microarrays

The microarrays used have been described by Nyquist et al. [32], and a description is available at http://migale.jouy.inra.fr/sakei/Supplement.html/. 70-mer oligonucleotide probes representing the L. sakei strain 23K genome and an additional set of sequenced L. sakei genes were printed in three copies onto epoxy glass slides (Corning).

RNA extraction

Total RNA extraction was performed using the RNeasy Protect Mini Prep Kit (Qiagen) as described by Rud et al. [33]. The concentration and purity of the total RNA was analysed using NanoDrop ND-1000 (NanoDrop Technologies), and the quality using Agilent 2100 Bioanalyzer (Agilent Technologies). Sample criteria for further use in the transcriptome analysis were A260/A280 ratio superior to 1.9 and 23S/16S RNA ratio superior to 1.6.

cDNA synthesis, labeling, and hybridization

cDNA was synthesized and labeled with the Fairplay III Microarray Labeling Kit (Stratagene, Agilent Technologies) as described previously [34]. After labeling, unincorporated dyes were removed from the samples using the QIAQuick PCR purification kit (Qiagen). The following prehybridization, hybridization, washing, and drying of the arrays were performed in a Tecan HS 400 Pro hybridization station (Tecan) as described by Nyquist et al. [32]. For studying the carbon effects, samples from DMLG and DMLRg were co-hybridized for each of the three strains. Separate hybridizations were performed for each strain on all three biological parallels. In order to remove potential biases associated with labelling and subsequent scanning, a replicate hybridization was performed for each strain for one of the three parallels, where the Cy3 and Cy5 dyes (GE Healthcare) used during cDNA synthesis were swapped. The hybridized arrays were scanned at wavelengths 532 nm (Cy3) and 635 nm (Cy5) with a Tecan scanner LS (Tecan). GenePix Pro 6.0 (Molecular Devices) was used for image analysis, and spots were excluded based on slide or morphology abnormalities.

Microarray data analysis

Downstream analysis was done by the Limma package http://www.bioconductor.org in the R computing environment http://www.r-project.org. Pre-processing and normalization followed a standard procedure using methods described by Smyth & Speed [35], and testing for differential expressed genes were done by using a linear mixed model as described by Smyth [36]. A mixed-model approach was chosen to adequately describe between-array variation and still utilize probe-replicates (three replicates of each probe in each array). An empirical Bayes smoothing of gene-wise variances was conducted according to Smyth et al. [37], and for each gene the p-value was adjusted to control the false discovery rate (FDR), hence all p-values displayed are FDR-adjusted (often referred to as q-values in the literature).

Validation of microarray data by qRT-PCR analysis

The microarray results were validated on selected regulated genes for the LS 25 strain by quantitative real-time reverse transcriptase PCR (qRT-PCR) performed as described previously [38]. Primers and probes (Additional file 1, Table S3) were designed using Primer Express 3.0 (Applied Biosystems). Relative gene expression was calculated by the ΔCmethod, using the DNA gyrase subunit alpha gene (gyrA) as the endogenous reference gene.

Microarray accession numbers

The microarray data have been deposited in the Array Express database http://www.ebi.ac.uk/arrayexpress/ under the accession numbers A-MEXP-1166 (array design) and E-MEXP-2892 (experiment).

Sequence analysis

A prediction of cre sites in the L. sakei 23K genome sequence (GeneBank acc. no. CR936503.1), both strands, was performed based on the consensus sequence TGWNANCGNTNWCA (W = A/T, N = A/T/G/C), confirmed in Gram-positive bacteria [39]. We made a search with the consensus sequence described by the regular expression T-G-[AT]-X-A-X-C-G-X-T-X-[AT]-C-A, allowing up to two mismatches in the conserved positions except for the two center position, highlighted in boldface. All computations were done in R http://www.r-project.org.

Results and Discussion

Selection of L. sakei strains and growth conditions

We have previously investigated L. sakei strain variation [9], and used proteomics to study the bacterium's primary metabolism [19], providing us with a basis for choosing strains with interesting differences for further studies. The starter culture strain LS 25 showed the fastest growth rates in a variety of media, and together with strain MF1053 from fish, it fermented the highest number of carbohydrates [9]. The LS 25 strain belongs to the L. sakei subsp. sakei, whereas the 23K and MF1053 strains belong to L. sakei subsp. carnosus [9,19]. By identification of differentially expressed proteins caused by the change of carbon source from glucose to ribose, LS 25 seemed to down-regulate the glycolytic pathway more efficiently than other strains during growth on ribose [19]. For these reasons, LS 25 and MF1053 were chosen in addition to 23K for which the microarray is based on. Nyquist et al. [32] recently investigated the genomes of various L. sakei strains compared to the sequenced strain 23K by comparative genome hybridization (CGH) using the same microarray as in the present study. A large part of the 23K genes belongs to a common gene pool invariant in the species, and the status for each gene on the array is known for all the three strains [32].

As glucose is the preferred sugar, L. sakei grows faster when glucose is utilized as the sole carbon source compared with ribose [8,9,15]. However, glucose stimulates ribose uptake and a possible co-metabolism of these two sugars present in meat and fish has been suggested, a possibility that give the organism an advantage in competition with other microbiota [15,16,40]. To obtain comparable 2-DE gels between samples issued from bacteria grown on the two carbohydrates in our recent proteomic analysis, growth on ribose was enhanced by adding small amounts of glucose [19]. For the present transcriptome analysis we therefore chose the same growth conditions.

Global gene expression patterns

A microarray representing the L. sakei 23K genome and an additional set of sequenced L. sakei genes was used for studying the effect of carbon source on the transcriptome of L. sakei strains 23K, MF1053 and LS 25. Genes displaying a significant differential expression with a log2 ratio > 0.5 or < -0.5 were classified into functional categories according to the L. sakei 23K genome database http://migale.jouy.inra.fr/sakei/genome-server and are listed in Table 1. The 23K strain showed differential expression for 364 genes within these limits, MF1053 and LS 25 for 223 and 316 genes, respectively. Among these, 88, 47 and 82, respectively, were genes belonging to the category of genes of 'unknown' function. Eighty three genes, the expression of which varied depending on the carbon source, were common to the three strains, among which 52 were up-regulated and 31 down-regulated during growth on ribose (Figure 1). The function of these common regulated genes was mostly related to carbohydrate transport and metabolism (34 genes, Table 1). The reliability of the microarray results was assessed by qRT-PCR analysis using selected regulated genes in the LS 25 strain. As shown in Table S4 in the additional material (Additional file 1), the qRT-PCR results were in agreement with the data obtained by the microarrays.

Table 1

Genes with significant differential expression in three L. sakei strains grown on ribose compared with glucose, FDR adjusted p-value less than 0.01 and log2 of > 0.5 or < -0.5 (log2 values > 1.0 or < -1.0 are shown in bold).

Gene locus	Gene	Description	23K	MF1053	LS 25
Carbohydrate transport and metabolism
Transport/binding of carbohydrates
LSA0185*	galP	Galactose:cation symporter	1.2		1.7
LSA0200*	rbsU	Ribose transport protein	2.8	3.5	4.3
LSA0353*	lsa0353	Putative cellobiose-specific PTS, enzyme IIB	3.6	1.3	2.5
LSA0449*	manL	Mannose-specific PTS, enzyme IIAB	2.1	2.5	1.5
LSA0450*	manN	Mannose-specific PTS, enzyme IIC	1.9	2.0	1.4
LSA0451*	manM	Mannose-specific PTS, enzyme IID	2.4	1.0	2.1
LSA0651*	glpF	Glycerol uptake facilitator protein, MIP family	3.4	4.7	3.4
LSA1050*	fruA	Fructose-specific PTS, enzyme IIABC			0.9
LSA1204*	lsa1204	Putative sugar transporter		1.1
LSA1457*	lsa1457	Putative cellobiose-specific PTS, enzyme IIC		2.3
LSA1462*	ptsI	PTS, enzyme I	0.8	1.7	0.9
LSA1463*	ptsH	Phosphocarrier protein HPr (histidine protein)		1.2	0.9
LSA1533	lsa1533	Putative cellobiose-specific PTS, enzyme IIA		2.5	2.1
LSA1690	lsa1690	Putative cellobiose-specific PTS, enzyme IIC	0.9
LSA1792*	scrA	Sucrose-specific PTS, enzyme IIBCA	0.8		1.1
Metabolism of carbohydrates and related molecules
LSA0123*	lsa0123	Putative sugar kinase, ROK family	1.2
LSA0198	ack1	Acetate kinase (acetokinase)	1.7		1.3
LSA0254*	lsa0254	Putative carbohydrate kinase	2.4	0.8	1.8
LSA0292*	budC	Acetoin reductase (acetoin dehydrogenase) (meso-2,3-butanediol dehydrogenase)	3.4	2.3	3.4
LSA0444	lsa0444	Putative malate dehydrogenase	3.4	D	2.1
LSA0516	hprK	Hpr kinase/phosphorylase	2.0	1.6	1.2
LSA0664*	loxL1N	L-lactate oxidase (N-terminal fragment), degenerate	1.2		0.7
LSA0665*	loxLI	L-lactate oxidase (central fragment), degenerate	1.0
LSA0666*	loxL1C	L-lactate oxidase (C-terminal fragment), degenerate	1.0
LSA0974*	pflB	Formate C-acetyltransferase (pyruvate formate-lyase) (formate acetyltransferase)	4.0
LSA0981	aldB	Acetolactate decarboxylase (alpha-acetolactate decarboxylase)		0.6	1.9
LSA0982	als	Acetolactate synthase (alpha-acetolactate synthase)			1.9
LSA0983	lsa0983	Putative aldose-1 epimerase	0.6
LSA1032	pyk	Pyruvate kinase		-0.7
LSA1080	lsa1080	Myo-inositol monophosphatase	0.6		0.8
LSA1082	pdhD	Pyruvate dehydrogenase complex, E3 component, dihydrolipoamide dehydrogenase	2.8	2.5	2.1
LSA1083	pdhC	Puruvate dehydrogenase complex, E2 component, dihydrolipoamide acetyltransferase	3.4	3.7	2.7
LSA1084	pdhB	Pyruvate dehydrogenase complex, E1 component, beta subunit	3.2	3.3	2.2
LSA1085	pdhA	Pyruvate dehydrogenase complex, E1 component, alpha subunit	2.9	3.5	2.4
LSA1141*	ppdK	Pyruvate phosphate dikinase	1.0		0.9
LSA1188*	pox1	Pyruvate oxidase	2.3	3.1	2.1
LSA1298	ack2	Acetate kinase (acetokinase)	1.1	0.9	0.9
LSA1343*	eutD	Phosphate acetyltransferase (phosphotransacetylase)	2.0	1.0	1.6
LSA1381	lsa1381	Putative acylphosphatase	-0.6	-0.5
LSA1399*	loxL2	L-lactate oxidase	3.4	U
LSA1630	lsa1630	Putative sugar kinase, ROK family	-0.6		-0.6
LSA1640*	nanA	N-acetylneuraminate lyase	2.0		D
LSA1641*	nanE	N-acylglucosamine/mannosamine-6-phosphate 2-epimerase	0.9		D
LSA1643*	lsa1643	Putative sugar kinase, ROK family	1.8
LSA1668	ack3	Acetate kinase (acetokinase)	-0.7		-1.1
LSA1830*	pox2	Pyruvate oxidase	0.7
Intermediary metabolism
LSA0255*	lsa0255	Putative phosphoribosyl isomerase	2.0	1.0	1.6
Specific carbohydrate metabolic pathway
LSA0201*	rbsD	D-ribose pyranase	2.5	2.5	3.4
LSA0202*	rbsK	Ribokinase	3.0	3.9	4.3
LSA0289*	xpk	Xylulose-5-phosphate phosphoketolase	3.2	2.3	2.6
LSA0297	gntZ	6-phosphogluconate dehydrogenase	-1.2	-0.9	-1.7
LSA0298	gntK	Gluconokinase	-0.8
LSA0381	zwf	Glucose-6-phosphate 1-dehydrogenase	-0.6	-0.6	-0.6
LSA0649*	glpK	Glycerol kinase	3.4	4.8	2.1
LSA0650*	glpD	Glycerol-3-phosphate dehydrogenase	2.3	2.2	2.0
LSA0764*	galK	Galactokinase	1.1	0.7	1.8
LSA0765*	galE1	UDP-glucose 4-epimerase			1.2
LSA0766*	galT	Galactose-1-phosphate uridylyltransferase	1.2	0.8	2.0
LSA0767*	galM	Aldose 1-epimerase (mutarotase)	1.3		2.0
LSA1146*	manA	Mannose-6-phosphate isomerase	1.4	1.3	1.5
LSA1531	lsa1531	Putative beta-glucosidase		0.7	0.9
LSA1588	nagA	N-acetylglucosamine-6-phosphate deacetylase	0.6
LSA1685	rpiA	Ribose 5-phosphate epimerase (ribose 5-phosphate isomerase)		1.1	0.8
LSA1710*	lacM	Beta-galactosidase, small subunit (lactase, small subunit)	3.3		1.2
LSA1711*	lacL	Beta-galactosidase, large subunit (lactase, large subunit)	3.0	1.5	1.7
LSA1790*	scrK	Fructokinase		1.0	1.1
LSA1791*	dexB	Glucan 1,6-alpha-glucosidase (dextran glucosidase)			1.1
LSA1795	melA	Alpha-galactosidase (melibiase)			-0.6
Glycolytic pathway
LSA0131	gpm2	Phosphoglycerate mutase		0.7
LSA0206	gpm3	Phosphoglycerate mutase	-0.7	-0.8	-0.9
LSA0609*	gloAC	Lactoylglutathione lyase (C-terminal fragment), authentic frameshift	1.1		0.7
LSA0803	gpm4	Phosphoglycerate mutase	0.5		0.5
LSA1033	pfk	6-phosphofructokinase	-0.6	-1.1	-0.5
LSA1157	mgsA	Methylglyoxal synthase	2.3	1.4	1.7
LSA1179	pgi	Glucose-6-phosphate isomerase	0.5
LSA1527	fba	Fructose-bisphosphate aldolase	-1.0	-0.7	-1.1
LSA1606	ldhL	L-lactate dehydrogenase	-1.0	-0.9	-1.5
Nucleotide transport and metabolism
Transport/binding of nucleosides, nucleotides, purines and pyrimidines
LSA0013	lsa0013	Putative nucleobase:cation symporter	-0.9		-1.5
LSA0055	lsa0055	Putative thiamine/thiamine precursor:cation symporter			1.6
LSA0064	lsa0064	Putative nucleobase:cation symporter		-0.8
LSA0259	lsa0259	Pyrimidine-specific nucleoside symporter	1.5		1.3
LSA0798*	lsa0798	Pyrimidine-specific nucleoside symporter	3.5	2.2	1.7
LSA0799*	lsa0799	Putative purine transport protein	4.4	2.7	2.9
LSA1210	lsa1210	Putative cytosine:cation symporter (C-terminal fragment), authentic frameshift	-0.8		-0.6
LSA1211	lsa1211	Putative cytosine:cation symporter (N-terminal fragment), authentic frameshit	-1.1		-0.9
Metabolism of nucleotides and nucleic acids
LSA0010	lsa0010	Putative nucleotide-binding phosphoesterase			-0.6
LSA0023	lsa0023	Putative ribonucleotide reductase (NrdI-like)	-0.5	D	D
LSA0063	purA	Adenylosuccinate synthetase (IMP-aspartate ligase)		-0.8
LSA0139	guaA	Guanosine monophosphate synthase (glutamine amidotransferase)		-0.5	-0.8
LSA0252	iunH1	Inosine-uridine preferring nucleoside hydrolase	2.6	2.6	1.8
LSA0446	pyrDB	Putative dihydroorotate oxidase, catalytic subunit			0.9
LSA0489	lsa0489	Putative metal-dependent phosphohydrolase precursor	0.5
LSA0533*	iunH2	Inosine-uridine preferring nucleoside hydrolase	1.2
LSA0785	lsa0785	Putative NCAIR mutase, PurE-related protein	-2.3		-1.3
LSA0795*	deoC	2 Deoxyribose-5 phosphate aldolase	4.0	2.1	2.2
LSA0796*	deoB	Phosphopentomutase (phosphodeoxyribomutase)	5.5	4.1	3.2
LSA0797*	deoD	Purine-nucleoside phosphorylase	4.5	2.6	1.9
LSA0801*	pdp	Pyrimidine-nucleoside phosphorylase	1.8
LSA0940	nrdF	Ribonucleoside-diphosphate reductase, beta chain		1.0	0.6
LSA0941	nrdE	Ribonucleoside-diphosphate reductase, alpha chain		1.0	0.6
LSA0942	nrdH	Ribonucleotide reductase, NrdH-redoxin		1.1
LSA0950	pyrR	Bifunctional protein: uracil phosphoribosyltransferase and pyrimidine operon transcriptional regulator	-0.6
LSA0993	rnhB	Ribonuclease HII (RNase HII)			0.6
LSA1018	cmk	Cytidylate kinase			0.6
LSA1097	lsa1097	Putative ADP-ribose phosphorylase, NUDIX family	0.5
LSA1352	lsa1352	Putative phosphomethylpyrimidine kinase	-0.8
LSA1651	lsa1651	Putative purine phosphoribosyltransferase, PRT family		0.8
LSA1661	lsa1661	Putative nucleotide hydrolase, NUDIX family		-0.5
LSA1805	dgk	Deoxyguanosine kinase	-1.0		-0.8
Transcription
Transcription regulation
LSA0130	lsa0130	Putative transcriptional regulator, LacI family	-0.6
LSA0132	lsa0132	Putative transcriptional regulator, MarR family	-0.6
LSA0161	lsa0161	Putative transcriptional regulator, ArsR family	-0.6
LSA0186	lsa0186	Putative transcriptional regulator, LytR family		0.8	0.6
LSA0203	rbsR	Ribose operon transcriptional regulator, LacI family	1.7
LSA0217	lsa0217	Putative thiosulfate sulfurtransferase with a ArsR-HTH domain, rhodanese family		-1.0	-0.7
LSA0229	lsa0229	Putative transcriptional regulator, MerR family (N-terminal fragment), authentic frameshift	-0.5
LSA0269	lsa0269	Putative transcriptional regulator, TetR family			-0.6
LSA0293	lsa0293	Putative DNA-binding protein, XRE family			-0.6
LSA0356	rex1	Redox-sensing transcriptional repressor, Rex	-0.8	-0.5	-0.9
LSA0603	cggR	Glycolytic genes regulator		-0.6	-0.6
LSA0669	lsa0669	Putative transcription regulator, TetR family		-0.6
LSA0783	lsa0783	Putative transcriptional regulator, Fnr/Crp Family	-0.6
LSA0800	deoR	Deoxyribonucleoside synthesis operon transcriptional regulator, GntR family	3.8	2.1	1.9
LSA0835	lsa0835	Putative DNA-binding protein, XRE family	-0.6
LSA0848	rex	Redox-sensing transcriptional repressor, Rex	1.6	0.7
LSA0972	lsa0972	Putative transcriptional regulator, LysR family	0.9
LSA1201	lsa1201	Putative transcriptional regulator, GntR family	1.4	D	D
LSA1322	glnR	Glutamine synthetase transcriptional regulator, MerR family	-1.4	-1.3
LSA1351	lsa1351	Putative transcritional regulator with aminotransferase domain, GntR family		-0.5	-0.6
LSA1434	lsa1434	Putative transcriptional regulator, DUF24 family (related to MarR/PadR families)	-0.8
LSA1449	spxA	Transcriptional regulator Spx	1.0		0.6
LSA1521	lsa1521	Putative transcriptional regulator, TetR family	0.6
LSA1554	lsa1554	Putative transcriptional regulator, LacI family	-0.7	-0.9	-0.5
LSA1587	lsa1587	Putative transcriptional regulator, GntR family	0.6
LSA1611	lsa1611	Putative DNA-binding protein, PemK family		-0.5	-0.7
LSA1653	lsa1653	Putative transcriptional regulator, MarR family			-0.6
LSA1692	lsa1692	Putative transcriptional regulator, GntR family	0.7		0.7
CoEnzyme transport and metabolism
Metabolism of coenzymes and prostethic groups
LSA0041	panE	2-dehydropantoate 2-reductase		0.8
LSA0057	thiE	Thiamine-phosphate pyrophosphorylase (thiamine-phosphate synthase)			1.9
LSA0058	thiD	Phosphomethylpyrimidine kinase (HMP-phosphate kinase)			1.4
LSA0059	thiM	Hydroxyethylthiazole kinase (4-methyl-5-beta-hydroxyethylthiazole kinase)	1.0		1.8
LSA0183	lsa0183	Putative hydrolase, isochorismatase/nicotamidase family	-0.7
LSA0840	lsa0840	Putative glutamate-cysteine ligase	0.6
LSA0947	fhs	Formate-tetrahydrofolate ligase (formyltetrahydrofolate synthetase)	0.6
LSA0980	lsa0980	Putative hydroxymethylpyrimidine/phosphomethylpyrimidine kinase, PfkB family	0.6
LSA1101	folK	2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase	0.6	U
LSA1614	acpS	Holo-[acyl-carrier protein] synthase (holo-ACP synthase) (4'-phosphopantetheine transferase AcpS)	-1.0	-0.9	-0.9
LSA1664	lsa1664	Putative dihydrofolate reductase	1.6	1.1	1.5
Energy production and conversion
Membrane bioenergetics (ATP synthase)
LSA1125	atpC	H(+)-transporting two-sector ATPase (ATP synthase), epsilon subunit	0.6
LSA1126	atpD	H(+)-transporting two-sector ATPase (ATP synthase), beta subunit			0.6
LSA1127	atpG	H(+)-transporting two-sector ATPase (ATP synthase), gamma subunit			0.8
LSA1128	atpA	H(+)-transporting two-sector ATPase (ATP synthase), alpha subunit			0.6
LSA1129	atpH	H(+)-transporting two-sector ATPase (ATP synthase), delta subunit			0.6
LSA1130	atpF	H(+)-transporting two-sector ATPase (ATP synthase), B subunit			0.5
LSA1131	atpE	H(+)-transporting two-sector ATPase (ATP synthase), C subunit			0.7
Inorganic ion transport and metabolism
Transport/binding of inorganic ions
LSA0029	lsa0029	Putative ion Mg(2+)/Co(2+) transport protein, hemolysinC-family			-0.7
LSA0134	lsa0134	Putative Na(+)/H(+) antiporter			-0.6
LSA0180	mtsC	Manganese ABC transporter, ATP-binding subunit	-0.8
LSA0181	mtsB	Manganese ABC transporter, membrane-spanning subunit	-0.8		-1.0
LSA0182	mtsA	Manganese ABC transporter, substrate-binding lipoprotein precursor	-0.7		-0.6
LSA0246	mntH1	Mn(2+)/Fe(2+) transport protein	-0.9		-1.3
LSA0283	lsa0283	Putative zinc/iron ABC transporter, ATP-binding subunit			-0.5
LSA0284	lsa0284	Putative zinc/iron ABC transporter, membrane-spanning subunit			-0.6
LSA0399	lsa0399	Iron(III)-compound ABC transporter, substrate-binding lipoprotein precursor	1.1	0.9
LSA0400	lsa0400	Iron(III)-compound ABC transporter, ATP-binding subunit		0.7
LSA0401	lsa0401	Iron(III)-compound ABC transporter, membrane-spanning subunit			0.5
LSA0402	lsa0402	Iron(III)-compound ABC transporter, membrane-spanning subunit	0.5		0.6
LSA0503	pstC	Phosphate ABC transporter, membrane-spanning subunit	0.5
LSA0504	pstA	Phosphate ABC transporter, membrane-spanning subunit	0.6
LSA0781	lsa0781	Putative cobalt ABC transporter, membrane-spanning/permease subunit	-0.9
LSA0782	lsa0782	Putative cobalt ABC transporter, membrane-spanning/permease subunit	-2.1
LSA1166	lsa1166	Putative potassium transport protein	0.7
LSA1440	cutC	Copper homeostasis protein, CutC family	-0.6
LSA1460	atkB	Copper-transporting P-type ATPase	0.6
LSA1638	lsa1638	Putative large conductance mechanosensitive channel		-1.0	-0.8
LSA1645	lsa1645	Putative Na(+)/(+) antiporter	1.4		D
LSA1699	mntH2	Mn(2+)/Fe(2+) transport protein			-0.6
LSA1703	lsa1703	Putative Na(+)/H(+) antiporter	-1.2
LSA1704	lsa1704	Putative calcium-transporting P-type ATPase			-0.8
LSA1735	lsa1735	Putative cobalt ABC transporter, membrane-spanning subunit			-0.6
LSA1736	lsa1736	Putative cobalt ABC transporter, ATP-binding subunit	-0.6
LSA1737	lsa1737	Putative cobalt ABC transporter, ATP-binding subunit	-0.7
LSA1838	lsa1838	Putative metal ion ABC transporter, membrane-spanning subunit			-0.5
LSA1839	lsa1839	Putative metal ion ABC transporter, substrate-binding lipoprotein precursor			-0.6
Amino acid transport and metabolism
Transport/binding of amino acids
LSA0125	lsa0125	Putative amino acid/polyamine transport protein	0.6
LSA0189	lsa0189	Putative amino acid/polyamine transport protein			-0.7
LSA0311	lsa0311	Putative glutamate/aspartate:cation symporter	-1.1		-1.0
LSA1037	lsa1037	Putative amino acid/polyamine transport protein	1.0	0.8	0.5
LSA1219	lsa1219	Putative cationic amino acid transport protein	0.7
LSA1415	lsa1415	Putative amino acid/polyamine transport protein	1.1		0.7
LSA1424	lsa1424	Putative L-aspartate transport protein	-1.4	-0.9	-1.2
LSA1435	lsa1435	Putative amino acid:H(+) symporter	1.0		0.8
LSA1496	lsa1496	Putative glutamine/glutamate ABC transporter, ATP-binding subunit		1.2
LSA1497	lsa1497	Putative glutamine/glutamate ABC transporter, membrane-spanning/substrate-binding subunit precursor		0.7
Transport/binding of proteins/peptides
LSA0702	oppA	Oligopeptide ABC transporter, substrate-binding lipoprotein precursor		1.3	1.0
LSA0703	oppB	Oligopeptide ABC transporter, membrane-spanning subunit		0.8	0.8
LSA0704	oppC	Oligopeptide ABC transporter, membrane-spanning subunit		1.8	1.0
LSA0705	oppD	Oligopeptide ABC transporter, ATP-binding subunit		1.2	1.1
LSA0706	oppF	Oligopeptide ABC transporter, ATP-binding subunit		1.2	1.2
Protein fate
LSA0053	pepO	Endopeptidase O	0.6
LSA0133	pepR	Prolyl aminopeptidase	1.5
LSA0226	pepN	Aminopeptidase N (lysyl-aminopeptidase-alanyl aminopeptidase)			-0.7
LSA0285	pepF1	Oligoendopeptidase F1			-0.7
LSA0320	pepD3	Dipeptidase D-type (U34 family)		-0.8	-0.5
LSA0424	pepV	Xaa-His dipeptidase V (carnosinase)	1.6
LSA0643	pepX	X-Prolyl dipeptidyl-aminopeptidase	0.6
LSA0888	pepT	Tripeptide aminopeptidase T	0.6
LSA1522	pepS	Aminopeptidase S	0.5
LSA1686	pepC1N	Cysteine aminopeptidase C1 (bleomycin hydrolase) (N-terminal fragment), authentic frameshift		1.6
LSA1688	pepC2	Cysteine aminopeptidase C2 (bleomycin hydrolase)		0.7
LSA1689	lsa1689	Putative peptidase M20 family	1.0		1.1
Metabolism of amino acids and related molecules
LSA0220_c	dapE	Succinyl-diaminopimelate desuccinylase	-1.4		-1.5
LSA0316	sdhB	L-serine dehydratase, beta subunit (L-serine deaminase)	-0.7
LSA0370*	arcA	Arginine deiminase (arginine dihydrolase)	1.9
LSA0372*	arcC	Carbamate kinase	0.5
LSA0463	lsa0463	Putative 2-hydroxyacid dehydrogenase	-0.7
LSA0509	kbl	2-amino-3-ketobutyrate coenzyme A ligase (glycine acetyltransferase)	1.5
LSA0510	lsa0510	L-threonine dehydrogenase (N-terminal fragment), authentic frameshift	2.0	0.5
LSA0572*	tdcB	Threonine deaminase (threonine ammonia-lyase, threonine dehydratase, IlvA homolog)	2.2		1.7
LSA0922	serA	D-3-phosphoglycerate dehydrogenase	0.9
LSA1134	glyA	Glycine/Serine hydroxymethyltransferase		0.7
LSA1321	glnA	Glutamate-ammonia ligase (glutamine synthetase)	-1.3	-1.0
LSA1484	mvaS	Hydroxymethylglutaryl-CoA synthase	-0.7	-0.6	-0.7
LSA1693	asnA2	L-asparaginase	0.8
Lipid transport and metabolism
Metabolism of lipids
LSA0045	cfa	Cyclopropane-fatty-acyl-phospholipid synthase	-1.3	-1.4	-1.4
LSA0644	lsa0644	Putative acyl-CoA thioester hydrolase	0.6
LSA0812	fabZ1	(3R)-hydroxymyristoyl-[acyl-carrier protein] dehydratase		-0.7	0.5
LSA0813	fabH	3-oxoacyl-[acyl carrier protein] synthetase III			0.6
LSA0814	acpP	Acyl carrier protein			0.6
LSA0815	fabD	Malonyl-CoA:ACP transacylase		-0.7	0.7
LSA0816	fabG	3-oxoacyl-acyl carrier protein reductase		-0.7
LSA0817	fabF	3-oxoacyl-[acyl carrier protein] synthetase II		-0.7
LSA0819	fabZ	(3R)-hydroxymyristoyl-[acyl carrier proetin] dehydratase			0.7
LSA0820	accC	Acetyl-CoA carboxylase (biotin carbooxylase subunit)		-0.7
LSA0821	accD	Acetyl-CoA carboxylase (carboxyl transferase beta subunit)			0.8
LSA0822	accA	Acetyl-CoA carboxylase (carboxyl transferase alpha subunit)			0.6
LSA0823	fabI	Enoyl [acyl carrier protein] reductase			0.9
LSA0891	lsa0891	Putative lipase/esterase	1.2
LSA1485	mvaA	Hydroxymethylglutaryl-CoA reductase	-0.5
LSA1493	lsa1493	Putative diacylglycerol kinase	-0.6	-0.9	-0.7
LSA1652	ipk	4-diphosphocytidyl-2-C-methyl-D-erythritol kinase	-0.6		-0.7
Secondary metabolites transport and metabolism
Transport/binding proteins and lipoproteins
LSA0046	lsa0046	Putative transport protein	-1.0	-0.6	-1.3
LSA0089	lsa0089	Putative drug transport protein	-2.1	-0.9	-0.8
LSA0094	lsa0094	Putative transport protein, Major Facilitator Super (MFS) family transporter	-0.7		-0.7
LSA0095	lsa0095	Putative transport protein	1.3	0.5
LSA0128	lsa0128	Putative antimicrobial peptide ABC exporter, membrane-spanning/permease subunit			-0.5
LSA0187	lsa0187	Putative drug-resistance ABC transporter, two ATP-binding subunits		0.7
LSA0219_b	lsa0219_b	Putative cyanate transport protein	-0.6
LSA0232	lmrA	Multidrug ABC exporter, ATP-binding and membrane-spanning/permease subunits	-0.7		-0.7
LSA0270	lsa0270	Putative multidrug ABC exporter, membrane-spanning/permease subunit	-0.7
LSA0271	lsa0271	Putative multidrug ABC exporter, ATP-binding subunit	-0.7		-0.6
LSA0272	lsa0272	Putative multidrug ABC exporter, ATP-binding and membrane-spanning/permease subunits	-0.6		-0.6
LSA0308	lsa0308	Putative drug:H(+) antiporter			-0.7
LSA0376	lsa0376	Putative transport protein	0.7
LSA0420	lsa0420	Putative drug:H(+) antiporter (N-terminal fragment), authentic frameshift	-0.8		-1.1
LSA0469	lsa0469	Putative drug:H(+) antiporter	-0.6		-0.5
LSA0788	lsa0788	Putative facilitator protein, MIP family	-2.6
LSA0936	lsa0936	Putative drug ABC exporter, membrane-spanning/permease subunit	1.1
LSA0937	lsa0937	Putative drug ABC exporter, membrane-spanning/permease subunit	1.3
LSA0938	lsa0938	Putative drug ABC exporter, ATP-binding subunit	1.2
LSA0963	lsa0963	Integral membrane protein, hemolysin III related
LSA1088	lsa1088	Putative multidrug ABC exporter, ATP-binding and membrane-spanning/permease subunits	0.5
LSA1261	lsa1261	Putative autotransport protein	0.5
LSA1340	lsa1340	Putative transport protein		-0.7
LSA1366	lsa1366	Putative ABC exporter, ATP-binding subunit	-0.8		-1.0
LSA1367	lsa1367	Putative ABC exporter, membrane-spanning/permease subunit	-0.8	-0.5	-0.8
LSA1420	lsa1417	Putative lipase/esterase		-1.1
LSA1621	lsa1621	Putative drug:H(+) antiporter		-1.1
LSA1642	lsa1642	Putative Solute:Na(+) symporter	3.4	1.8	D
LSA1872	lsa1872	Putative drug:H(+) antiporter		0.7
LSA1878	lsa1878	Putative drug resistance ABC transporter, two ATP-binding subunits	-0.6
Detoxification
LSA0772	lsa0772	Hypothetical protein (TelA, telluric resistance family)	1.0		0.7
LSA1317	lsa1317	Putative chromate reductase	0.6	-0.7
LSA1450	lsa1450	Putative metal-dependent hydrolase (beta-lactamase family III)			0.6
LSA1776	lsa1776	Putative 4-carboxymuconolactone decarboxylase	0.6		D
Translation, ribosomal structure and biogenesis
Translation initiation
LSA1135	lsa1135	Putative translation factor, Sua5 family		0.7	0.6
Translation elongation
LSA0251	efp1	Elongation factor P (EF-P)	0.5
LSA1063	tuf	Elongation factor Tu (EF-Tu)	0.6
Ribosomal proteins
LSA0011	rplI	50S Ribosomal protein L9			-0.8
LSA0266	rpsN	30S ribosomal protein S14		0.7	-0.5
LSA0494	lsa0494	30S ribosomal interface protein S30EA	1.7
LSA0696	rpmB	50S ribosomal protein L28			0.8
LSA1017	rpsA	30S Ribosomal protein S1	0.9		0.6
LSA1333	rpmG	50S ribosomal protein L33			0.6
LSA1666	rplL	50S ribosomal protein L7/L12	-0.6
LSA1676	rpmG2	50S ribosomal protein L33			-0.6
LSA1750	rplF	50S ribosomal protein L6		0.6
LSA1755	rpsQ	30S ribosomal protein S17		0.5
LSA1761	rplB	50S ribosomal protein L2		0.6
LSA1765	rpsJ	30S ribosomal protein S10	-0.7
Protein synthesis
LSA0377	tgt	Queuine tRNA-ribosyltransferase	-0.6
LSA1546	gatB	Glutamyl-tRNA amidotransferase, subunit B		-0.5
LSA1547	gatA	Glutamyl-tRNA amidotransferase, subunit A	-0.5		-0.5
RNA restriction and modification
LSA0437	lsa0437	Hypothetical protein with an RNA-binding domain	-0.7
LSA0443	lsa0443	Putative single-stranded mRNA endoribonuclease	2.7		1.9
LSA0738	dtd	D-tyrosyl-tRNA(tyr) deacylase	0.5
LSA0794	trmU	tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase		-0.9
LSA1534	lsa1534	Putative ATP-dependent RNA helicase		0.9
LSA1615	lsa1615	Putative ATP-dependent RNA helicase	-0.7	-0.8	-1.0
LSA1723	truA	tRNA pseudouridylate synthase A (pseudouridylate synthase I)	-0.7		-0.6
LSA1880	trmE	tRNA modification GTPase trmE	-0.7
Aminoacyl-tRNA synthetases
LSA0880	glyQ	Glycyl-tRNA synthetase, alpha subunit		0.7
LSA0881	glyS	Glycyl-tRNA synthetase, beta subunit		0.7
LSA1400	thrS	Threonyl-tRNA synthetase	0.6
LSA1681	cysS	Cysteinyl-tRNA synthetase	-0.6
DNA replication, recombination and repair
DNA replication
LSA0221	lsa0221	Putative transcriptional regulator, LysR family (C-terminal fragment), degenerate	-0.8	-0.9	-1.1
LSA0976	parE	Topoisomerase IV, subunit B		0.5
Transposon and IS
LSA1152_a	tnpA3-ISLsa1	Transposase of ISLsa1 (IS30 family)	-0.6
Phage-related function
LSA1292	lsa1292	Putative prophage protein	0.6
LSA1788	lsa1788	Putative phage-related 1,4-beta-N-acetyl muramidase (cell wall hydrolase)	-1.0	D	D
DNA recombination and repair
LSA0076	lsa0076	Putative DNA invertase (plasmidic resolvase)	-1.1	-1.5	-1.4
LSA0366	ruvA	Holliday junction DNA helicase RuvA			-0.5
LSA0382	dinP	DNA-damage-inducible protein P	-0.5
LSA0487	recA	DNA recombinase A	-0.8		-1.1
LSA0523	uvrB	Excinuclease ABC, subunit B	-0.7		-0.5
LSA0524	uvrA1	Excinuclease ABC, subunit A	-1.2		-0.7
LSA0910	rexAN	ATP-dependent exonuclease, subunit A (N-terminal fragment), authentic frameshift	0.6
LSA0911	rexAC	ATP-dependent exonuclease, subunit A (C-terminal fragment), authentic frameshift	0.7
LSA0912	lsa0912	Putative ATP-dependent helicase, DinG family	0.6		0.8
LSA1162	lsa1162	DNA-repair protein (SOS response UmuC-like protein)		0.8	-0.6
LSA1405	fpg	Formamidopyrimidine-DNA glycosylase	-0.5	-0.6	-0.6
LSA1477	recX	Putative regulatory protein, RecX family	-0.6
LSA1843	ogt	Methylated-DNA-protein-cysteine S-methyltransferase	-0.6
DNA restriction and modification
LSA0143	lsa0143	Putative adenine-specific DNA methyltransferase	-0.7	D	D
LSA0921	lsa0921	Putative adenine-specific DNA methyltransferase	0.8
LSA1299	lsa1299	Putative adenine-specific DNA methyltransferase	0.9	0.7	1.2
Information pathways
LSA0326	lsa0326	Putative DNA helicase		-0.6	U
DNA packaging and segregation
LSA0135	lsa0135	Hypothetical integral membrane protein, similar to CcrB			-0.6
LSA1015	hbsU	Histone-like DNA-binding protein HU	1.0		0.9
Cell division and chromosome partitioning
Cell division
LSA0755	divIVA	Cell-division initiation protein (septum placement)			0.5
LSA0845	lsa0845	Putative negative regulator of septum ring formation	0.7		0.6
LSA1118	lsa1118	Rod-shape determining protein		0.6	0.5
LSA1597	ftsH	ATP-dependent zinc metalloendopeptidase FtsH (cell division protein FtsH)			-0.6
LSA1879	gidA	Cell division protein GidA	-0.6
Cell envelope biogenesis, outer membrane
Cell wall
LSA0280	murE	UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase	-0.6	-0.6	-0.7
LSA0621	pbp2A	Bifunctional glycolsyltransferase/transpeptidase penicillin binding protein 2A			0.7
LSA0648	lsa0648	Putative penicillin-binding protein precursor (beta-lactamase class C)			1.0
LSA0862	lsa0862	N-acetylmuramoyl-L-alanine amidase precursor (cell wall hydrolase) (autolysin)	0.6		0.8
LSA0917	pbp1A	Bifunctional glycosyltransferase/transpeptidase penicillin-binding protein 1A			0.5
LSA1123	murA1	UDP-N-acetylglucosamine 1-carboxyvinyltransferase I		-0.5
LSA1334	pbp2B2	Bifuntional dimerisation/transpeptidase penicillin-binding protein 2B		0.7	0.7
LSA1437	lsa1437	N-acetylmuramoyl-L-alanine amidase precursor (cell wall hydrolase) (autolysin)		-0.7
LSA1441	bacA	Putative undecaprenol kinase (bacitracine resistance protein A)		0.6
LSA1613	alr	Alanine racemase	-0.8	-0.9	-0.7
LSA1616	murF	UDP-N-acetylmuramoyl-tripeptide--D-alanyl-D-alanine ligase			-0.5
Cell envelope and cellular processes
LSA0162	lsa0162	Putative Bifunctional glycosyl transferase, family 8		-1.2	-1.5
LSA1246	lsa1246	Putative glycosyl transferase, family 2		-0.9
LSA1558	lsa1558	Putative extracellular N-acetylmuramoyl-L-alanine amidase precursor (cell wall hydrolase/Lysosyme subfamily 2)			-0.6
Cell motility and secretion
Protein secretion
LSA0948	lspA	Signal peptidase II (lipoprotein signal peptidase) (prolipoprotein signal peptidase)			0.5
LSA1884	oxaA2	Membrane protein chaperone oxaA			-0.6
Signal transduction
Signal transduction
LSA0561	sppKN	Two-component system, sensor histidine kinase, (SppK fragment), degenerate		0.5
LSA0692	lsa0692	Putative serine/threonine protein kinase		0.5	0.6
LSA1384	lsa1384	Two-component system, response regulator		0.5
Post translational modifications, protein turnover, chaperones
Protein folding
LSA0050	lsa0050	Putative molecular chaperone, small heat shock protein, Hsp20 family			-0.7
LSA0082	htrA	Serine protease HtrA precursor, trypsin family		-0.6
LSA0207	clpL	ATPase/chaperone ClpL, putative specificity factor for ClpP protease	0.6
LSA0358	groS	Co-chaperonin GroES (10 kD chaperonin) (protein Cpn10)			-0.5
LSA0359	groEL	Chaperonin GroEL (60 kDa chaperonin) (protein Cpn60)			-0.5
LSA0436	lsa0436	Putative peptidylprolyl isomerase (peptidylprolyl cis-trans isomerase) (PPIase)			-0.6
LSA0984	hslU	ATP-dependent Hsl protease, ATP-binding subunit HslU	0.7		0.7
LSA1465	clpE	ATPase/chaperone ClpE, putative specificity factor for ClpP protease	-0.7	-0.6	-0.6
LSA1618	htpX	Membrane metalloprotease, HtpX homolog		0.8
Adaption to atypical conditions
LSA0170	lsa0170	Putative general stress protein	0.5		-1.5
LSA0247	usp2	Similar to universal stress protein, UspA family			-0.5
LSA0264	lsa0264	Putative glycine/betaine/carnitine/choline transport protein	-0.6		-0.6
LSA0513	lsa0513	Putative stress-responsive transcriptional regulator		-0.8
LSA0552	lsa0552	Organic hydroperoxide resistance protein		0.6
LSA0616	lsa0616	Putative glycine/betaine/carnitine/choline ABC transporter, ATP-binding subunit	0.9
LSA0617	lsa0617	Putative glycine/betaine/carnitine/choline ABC transporter, membrane-spanning subunit	1.3
LSA0618	lsa0618	Putative glycine/betaine/carnitine/choline ABC transporter, substrate-binding lipoprotein	0.6
LSA0619	lsa0619	Putative glycine/betaine/carnitine/choline ABC transporter, membrane-spanning subunit	1.5	0.5
LSA0642	usp3	Similar to universal stress protein, UspA	0.9
LSA0768	csp1	Similar to cold shock protein, CspA family	2.1	0.6	1.8
LSA0836	usp6	Similar to universal stress protein, UspA family	0.6
LSA0946	csp4	Similar to cold shock protein, CspA family	0.6
LSA1110	lsa1110	Putative NifU-homolog involved in Fe-S cluster assembly		0.6
LSA1111	lsa1111	Putative cysteine desulfurase (class-V aminotransferase, putative SufS protein homologue)		0.7
LSA1173	usp4	Similar to universal stress protein, UspA family	1.5	-2.1
LSA1694	lsa1694	Putative glycine/betaine/carnitine ABC transporter, substrate binding lipoprotein precursor	-1.7		-1.1
LSA1695	lsa1695	Putative glycine/betaine/carnitine ABC transporter, membrane-spanning subunit	-2.1	-2.0	-1.9
LSA1696	lsa1696	Putative glycine/betaine/carnitine ABC transporter, ATP-binding subunit	-1.6		-0.9
LSA1870	lsa1870	Putative glycine betaine/carnitine/choline ABC transporter, ATP-binding subunit	-0.6		-0.6
Protein modification
LSA0865	lsa0865	Putative protein methionine sulfoxide reductase		-0.6
LSA0866	msrA	Protein methionine sulfoxide reductase		-0.7
LSA0934	lplA	Lipoate-protein ligase	1.6	1.4	1.0
LSA0973	pflA	Pyruvate formate-lyase activating enzyme	1.7
General function prediction only
Miscellaneous
LSA0030	lsa0030	Putative aldo/keto reductase (oxidoreductase)		-0.7	-0.8
LSA0120	lsa0120	Putative GTP-binding protein	-0.5
LSA0164	lsa0164	Putative serine/tyrosine protein phosphatase	0.2	-1.1	-1.2
LSA0165	lsa0165	Putative oxidoreductase, short chain dehydrogenase/reductase family		-0.9	-1.2
LSA0218	trxA1	Thioredoxin		-0.9
LSA0258	lsa0258	Putative iron-containing alcohol dehydrogenase	1.6	0.5	1.6
LSA0260	lsa0260	Putative aldo/keto reductase (oxidoreductase)	1.9	1.2	1.7
LSA0312	lsa0312	Putative NADH oxidase	-0.9		-1.0
LSA0324	lsa0324	Putative hydrolase, haloacid dehalogenase family (N-terminal fragment), authentic frameshift	1.9
LSA0325	lsa0325	Putative hydrolase, haloacid dehalogenase family (C-terminal fragment), authentic frameshift	1.8
LSA0350	lsa0350	Putative N-acetyltransferase, GNAT family	-0.5
LSA0369	lsa0369	Putative N-acetyltransferase, GNAT family	-0.5		-0.5
LSA0384	lsa0384	Putative phosphoesterase, DHH family	-0.5
LSA0403	lsa0403	Putative thioredoxin reductase		0.9
LSA0447	lsa0447	Putative hydrolase, haloacid dehalogenase family			0.6
LSA0475	lsa0475	Putative N-acetyltransferase, GNAT family		-0.6
LSA0520	trxB2	Thioredoxin reductase	-0.8
LSA0575	npr	NADH peroxidase	1.0	U
LSA0802	nox	NADH oxidase	1.5
LSA0806	lsa0806	Putative N-acetyltransferase, GNAT family	0.6
LSA0831	lsa0831	Putative nitroreductase (oxidoreductase)		1.6
LSA0896	sodA	Iron/Manganese superoxide dismutase	3.4	1.7	1.7
LSA0925	adh	Putative zinc-containg alcohol dehydrogenase (oxidoreductase)	0.5
LSA0971	ppa	Inorganic pyrophosphatase (pyrophosphate phosphohydrolase)	0.7
LSA0994	lsa0994	Putative GTP-binding protein			0.6
LSA1016	engA	Putative GTP-binding protein	0.6		0.7
LSA1045	obgE	Putative GTP-binding protein	0.6
LSA1153	lsa1153	Hypothetical protein, CAAX protease family	0.5
LSA1311	lsa1311	Hypothetical protein containing a possible heme/steroid binding domain	0.7	-0.6
LSA1320	lsa1320	Putative NADPH-quinone oxidoreductase		-0.8
LSA1345	lsa1345	Putative hydrolase, haloacid dehalogenase family	0.5
LSA1349	lsa1349	Putative N-acetyltransferase, GNAT family		-0.5
LSA1365	lsa1365	Hypothetical protein		-0.5	-0.7
LSA1368	lsa1368	Hypothetical protein	0.9		0.6
LSA1371	lsa1371	Hypothetical membrane protein	0.6
LSA1395	lsa1395	Putative zinc-containing alcohol dehydrogenase (oxidoreductase)	0.9
LSA1427	lsa1427	Putative hydrolase, haloacid dehalogenase	1.3		0.6
LSA1472	lsa1472	Putative N-acetyl transferase, GNAT family	0.6
LSA1535	lsa1535	Putative oxidoreductase	0.5	1.1	0.7
LSA1553	lsa1553	Putative hydrolase, haloacid dehalogenase family	-0.6
LSA1559	lsa1559	Putative oxidoreductase	0.6	1.1	0.7
LSA1702	lsa1702	Putative zinc-containing alcohol dehydrogenase (oxidoreductase)	1.1
LSA1712	lsa1712	Putative nitroreductase (oxidoreductase)		-0.7	-0.8
LSA1832	lsa1832	Putative zinc-containing alcohol dehydrogenase (oxidoreductase)		1.0
LSA1835	lsa1835	Putative zinc-containing alcohol dehydrogenase (oxidoreductase)	-0.7		-1.0
LSA1867	lsa1867	Putative acetyltransferase, isoleucine patch superfamily	-0.5	-0.6	-0.7
LSA1871	gshR	Glutathione reductase	-0.6
Unknown
Proteins of unknown function that are similar to other proteins
LSA0018	lsa0018	Hypothetical protein		0.5
LSA0027	lsa0027	Hypothetical protein			-1.1
LSA0028	lsa0028	Hypothetical protein, DegV family	-0.5
LSA0044	lsa0044	Hypothetical protein			-0.7
LSA0061	lsa0061	Hypothetical extracellular protein precursor	-0.5
LSA0106	lsa0106	Hypothetical cell surface protein precursor	0.5
LSA0160	lsa0160	Hypothetical protein	-0.7
LSA0166	lsa0166	Hypothetical Integral membrane protein			-1.2
LSA0190	lsa0190	Hypothetical integral membrane protein	-0.7		-0.6
LSA0191	lsa0191	Hypothetical integral membrane protein	-0.6		-0.6
LSA0199	lsa0199	Hypothetical protein	1.1	1.0	1.1
LSA0208	lsa0208	Hypothetical integral membrane protein	0.7
LSA0235	lsa0235	Hypothetical extracellular protein precursor	2.1	1.6	1.7
LSA0236	lsa0236	Hypothetical extracellular peptide precursor	2.0	1.3	1.5
LSA0244	lsa0244	Hypothetical integral membrane protein			-0.5
LSA0245	lsa0245	Hypothetical lipoprotein precursor	-0.9	-1.0	-1.1
LSA0249	lsa0249	Hypothetical protein	1.1	1.0
LSA0263	lsa0263	Hypothetical integral membrane protein	-0.6		-0.9
LSA0300	lsa0300	Hypothetical protein			0.7
LSA0315	lsa0315	Hypothetical protein	-0.7
LSA0319	lsa0319	Hypothetical protein		-0.8	-0.8
LSA0323	lsa0323	Hypothetical protein			-0.5
LSA0337	lsa0337	Hypothetical protein	-0.7
LSA0348	lsa0348	Hypothetical integral membrane protein	-0.9		-0.7
LSA0352	lsa0352	Hypothetical integral membrane protein	-0.6
LSA0354	lsa0354	Hypothetical integral membrane protein			-1.1
LSA0388	lsa0388	Hypothetical protein		-0.6
LSA0389	lsa0389	Hypothetical protein		-0.7	-0.7
LSA0390	lsa0390	Hypothetical protein		-0.5
LSA0409	lsa0409	Hypothetical integral membrane protein			-0.8
LSA0418	lsa0418	Hypothetical protein			-0.8
LSA0464	lsa0464	Hypothetical protein		-0.6
LSA0470	lsa0470	Hypothetical protein	0.9		0.7
LSA0512	lsa0512	Hypothetical protein		-0.6
LSA0515	lsa0515	Hypothetical integral membrane protein		-0.5
LSA0536	lsa0536	Hypothetical protein		0.7
LSA0716	lsa0716	Hypothetical protein			0.6
LSA0752	lsa0752	Hypothetical protein	0.5		0.6
LSA0757	lsa0757	Hypothetical protein		0.8
LSA0773	lsa0773	Hypothetical protein	0.9		0.6
LSA0784	lsa0784	Hypothetical protein	-2.6
LSA0786	lsa0786	Hypothetical protein	-2.0
LSA0787	lsa0787	Hypothetical protein	-1.7
LSA0790	lsa0790	Hypothetical protein, ATP utilizing enzyme PP-loop family	-2.5
LSA0827	lsa0827	Hypothetical lipoprotein precursor	0.8		U
LSA0828	lsa0828	Hypothetical protein	0.7
LSA0829	lsa0829	Hypothetical integral membrane protein			0.5
LSA0874	lsa0874	Hypothetical protein	0.5
LSA0901	lsa0901	Hypothetical protein			0.5
LSA0913	lsa0913	Hypothetical extracellular protein precursor	0.5		0.7
LSA0919	lsa0919	Hypothetical protein			0.7
LSA0933	lsa0933	Hypothetical protein	0.6		0.6
LSA0961	lsa0961	Hypothetical protein, DegV family		-0.5
LSA0968	lsa0968	Hypothetical integral membrane protein	0.7
LSA0977	lsa0977	Hypothetical integral membrane protein	0.7		0.8
LSA0987	lsa0987	Hypotehtical protein, GidA family (C-terminal fragment)	0.5
LSA0996	lsa0996	Hypothetical protein			0.5
LSA1003	lsa1003	Hypothetical protein	2.0		1.2
LSA1005	lsa1005	Hypothetical membrane protein	0.9	0.6	0.7
LSA1008	lsa1008	Putative extracellular chitin-binding protein precursor		0.9	1.2
LSA1027	lsa1027	Hypothetical protein			0.6
LSA1047	lsa1047	Hypothetical protein	3.5	1.2	1.3
LSA1064	lsa1064	Hypothetical protein	0.5		0.7
LSA1075	lsa1075	Hypothetical protein			0.5
LSA1078	lsa1078	Hypothetical protein			0.6
LSA1081	lsa1081	Hypothetical protein	1.0		1.0
LSA1091	lsa1091	Hypothetical protein			0.6
LSA1096	lsa1096	Hypothetical protein	0.6
LSA1124	lsa1124	Hypothetical protein		-0.7
LSA1154	lsa1154	Hypothetical protein	0.6		0.6
LSA1158	lsa1158	Hypothetical protein	1.7	1.4
LSA1189	lsa1189	Hypothetical integral membrane protein	-1.6		-1.1
LSA1282	lsa1282	Hypothetical protein		-0.5
LSA1296	lsa1296	Hypothetical integral membrane protein		-1.2	-0.8
LSA1342	lsa1342	Hypothetical protein		-0.7
LSA1346	lsa1346	Hypothetical protein	0.8
LSA1350	lsa1350	Hypothetical protein		-0.6	-1.0
LSA1353	lsa1353	Hypothetical integral membrane protein	-0.9	-0.5
LSA1446	lsa1446	Hypothetical protein	-0.6	-0.6	-0.7
LSA1466	lsa1466	Hypothetical protein	0.6
LSA1467	lsa1467	Hypothetical protein		-0.6	-1.1
LSA1524	lsa1524	Hypothetical protein	0.7
LSA1540	lsa1540	Hypothetical extracellular protein precursor	0.7
LSA1563	lsa1563	Hypothetical integral membrane protein		-0.6	-0.6
LSA1610	lsa1610	Hypothetical integral membrane protein	-0.7		-0.9
LSA1617	lsa1617	Hypothetical protein			-0.7
LSA1620	lsa1620	Hypothetical protein			-0.6
LSA1623	lsa1623	Hypothetical integral membrane protein	-0.5		-0.6
LSA1637	lsa1637	Hypothetical integral membrane protein, TerC family	-1.7	-1.0	-1.6
LSA1644	lsa1644	Hypothetical protein	1.7		D
LSA1649	lsa1649	Hypothetical extracellular protein precursor			-0.5
LSA1659	lsa1659	Hypothetical protein	-0.5
LSA1662	lsa1662	Hypothetical protein	-1.0	-0.6	-0.7
LSA1663	lsa1663	Hypothetical protein	-0.8
LSA1678	lsa1678	Hypothetical protein	-0.6
LSA1680	lsa1680	Hypothetical protein	-0.6
LSA1716	lsa1716	Hypothetical protein		-0.5
LSA1822	lsa1822	Hypothetical protein			-0.5
LSA1828	lsa1828	Hypothetical integral membrane protein	0.6	0.7
LSA1850	lsa1850	Hypothetical protein		-0.6
LSA1876	lsa1876	Hypothetical integral membrane protein			-0.6
LSA1877	lsa1877	Hypothetical protein			-0.6
Proteins of unknown function only similar to other proteins from the same organism
LSA1159	lsa1159	Hypothetical cell surface protein precursor	2.0		0.5
LSA1165	lsa1165	Hypothetical cell surface protein precursor	1.8
LSA1700	lsa1700	Hypothetical protein	2.1	0.8
LSA1814	lsa1814	Hypothetical protein			-0.5
Proteins of unknown function. without similarity to other proteins
LSA0065	lsa0065	Hypothetical integral membrane protein	-0.5
LSA0093	lsa0093	Hypothetical integral membrane protein	-0.9		-1.2
LSA0121	lsa0121	Hypothetical small peptide	-0.7	-0.6	-0.5
LSA0163	lsa0163	Hypothetical protein		-1.1	-1.3
LSA0167	lsa0167	Hypothetical protein			-1.4
LSA0168	lsa0168	Hypothetical protein			-1.4
LSA0188	lsa0188	Hypothetical small peptide			-0.8
LSA0256_a	lsa0256_a	Hypothetical protein	2.3	1.0	2.2
LSA0257	lsa0257	Hypothetical protein	1.4
LSA0281	lsa0281	Hypothetical lipoprotein precursor		-0.5	-0.6
LSA0301	lsa0301	Hypothetical protein			0.6
LSA0334	lsa0334	Hypothetical extracellular protein precursor	1.1
LSA0339	lsa0339	Hypothetical protein	-0.5
LSA0378	lsa0378	Hypothetical protein	-0.7
LSA0514	lsa0514	Hypothetical small extracellular protein precursor		-0.8
LSA0534	lsa0534	Hypothetical cell surface protein precursor (with LPQTG sorting signal)	1.0		D
LSA0576	lsa0576	Hypothetical protein	0.5	D
LSA0641	lsa0641	Hypothetical extracellular peptide precursor		-0.5
LSA0647	lsa0647	Hypothetical extracellular protein precursor	0.6
LSA0667	lsa0667	Hypothetical protein	1.0		0.9
LSA0753	lsa0753	Hypothetical integral membrane protein			0.5
LSA0789	lsa0789	Hypothetical protein	-1.9
LSA0837	lsa0837	Hypothetical protein	1.2	1.3	1.4
LSA0885	lsa0885	Hypothetical protein	1.8
LSA0902	lsa0902	Hypothetical protein	0.7	D
LSA0945	lsa0945	Hypothetical protein			0.9
LSA1019	lsa1019	Hypothetical cell surface protein precursor			0.8
LSA1035	lsa1035	Hypothetical small integral membrane protein			0.6
LSA1086	lsa1086	hypothetical protein	0.8		0.5
LSA1104	lsa1104	Hypothetical protein	-0.5
LSA1155	lsa1155	Hypothetical integral membrane protein	0.5
LSA1174	lsa1174	Hypothetical protein	1.0
LSA1176	lsa1176	Hypothetical protein		-1.0	U
LSA1319	lsa1319	Hypothetical small protein		-0.8
LSA1408	lsa1408	Hypothetical protein			0.6
LSA1464	lsa1464	Hypothetical protein	-0.6
LSA1478	lsa1478	Hypothetical protein	-0.7	-0.6	-0.6
LSA1480	lsa1480	Hypothetical membrane protein	0.5	D
LSA1524	lsa1524	Hypothetical protein	0.8
LSA1539	lsa1539	Hypothetical protein	0.9
LSA1713	lsa1713	Hypothtical small peptide			-0.6
LSA1787	lsa1787	Hypothetical cell surface protein precursor	-0.5	U
LSA1820	lsa1820	Hypothetical cell surface protein precursor			-0.6
LSA1821	lsa1821	Hypothetical cell surface protein precursor		-0.6
LSA1845	lsa1845	Hypothetical small protein		0.8
LSA1848	lsa1848	Hypothetical protein			-0.5
LSA1851	lsa1851	Hypothetical extracellular small protein	-0.6		-0.7
LSA1883	lsa1883	Hypothetical small protein	1.2		1.5
Bacteriocin associated genes
SKP0001	sppIP	Bacteriocin sakacin P inducing peptide	D	0.5	D
SKP0006	sppT	Sakacin P ABC transporter	D	0.6	D
SKP0007	sppE	Sakacin P accesory transport protein	D	0.6	D

The microarray used has been described previously [32]. Asterix (*) relates the gene to Table 2. D and U refer to genes classified as 'divergent' and 'uncertain', respectively, by CGH analysis [32]. Genes encoding proteins with a change in expression according to McLeod et al. [19], are underlined.

Figure 1

Venn diagram showing the number of unique and common up- and down-regulated genes in .

Several of the up-regulated genes are located in operons, an organisation believed to provide the advantage of coordinated regulation. In addition, in order to discriminate genes induced by growth on ribose from those repressed by glucose (submitted to CCR mediated by CcpA), a search of the complete genome sequence of L. sakei 23K [7] was undertaken, with the aim to identify putative cre sites. The search revealed 1962 hits, most of which did not have any biological significance considering their unsuitable location in relation to promoters. Relief of CcpA-mediated CCR likely occur for many of the up-regulated genes in the category of carbohydrate transport and metabolism. Putative cre sites were identified in their promoter region, as well as for some genes involved in nucleoside and amino acid transport and metabolism (Table 2). In the other gene categories, the presences of putative cre sites were rare. With regard to gene product, the L. sakei genome shares high level of conservation with Lactobacillus plantarum [7], and high similarity of catabolic operon organization. The role of CcpA in CCR in L. plantarum has been established, and was shown to mediate regulation of the pox genes encoding pyruvate oxidases [41,42]. During growth on ribose, L. plantarum induces a similar set of genes as observed in the present study, and putative cre sites were identified in the upstream region of several genes involved [33].

Table 2

Putative cre sites present in the promoter region of some L. sakei genes up-regulated in the present study.

Gene locus	Gene	cre site sequencea	Positionb	Co-transcribed genes/operonc	Gene locus
LSA0123	lsa0123	TGAAAGCGTTACAA	-93
LSA0185	galP	GAACATCGTTATCA	-46
LSA0200	rbsU	GTAAACCGTTTTCA	-113	rbsUDK	LSA0200-0202
LSA0254	lsa0254	TGTAAGCGTTTTAT	-56	lsa0254-lsa0255-lsa0256_a	LSA0254-0256_a
LSA0289	xpk	CTATTACGATGACA	-8
LSA0292	budC	TGTAACCGTTTTAA	-51
LSA0353	lsa0353	AGAAAGCGCTTATA	-102
LSA0370	arcA	TGAAAGCGATTACC	-58	arcA-arcBe-arcC-arcTe-arcDe	LSA0370-0374
LSA0449	manL	TGTTAGCGTTTTTA	-56	manL-manM-manN	LSA0449-0451
LSA0533	iunH2	AAAAAGCGTTCACA	-35
LSA0572	tdcB	TGAAAACGTTCTAA	-134
LSA0608	Glo AN	TGTAACCGTTTTAA	-100	gloAN-gloAC	LSA0608-0609
LSA0649	glpK	AGGAAACGTTTTCC	-42	glpK-glpD-glpF	LSA0649-0651
LSA0664	loxL1	AGAAAGCGAGTACA	-82	loxL1N-loxLI-loxL1C	LSA0664-0666
LSA0764	galK	TGAAAGCGATTAAT	-30	galK-galE1-galT-galM	LSA0764-0767
LSA0795	deoC	TGAAAGCGTTAACA	-33	deoC-deoB-deoD-lsa0798-lsa0799-deoR-pdp	LSA0795-0801
LSA0974	pflB	TACGAACGCTTACA	-147	pflB-pflA	LSA0974-0973
LSA1048	fruRe	TGTAAACGATGACA	-39	fruRe-fruKe-fruA	LSA1048-1050
LSA1141	ppdK	GGTTATCGATAAAA	-29
LSA1146	manA	CGAAATCGCTTTAA	-98
LSA1188	pox1	TGTAATCGATTTCA	-88
LSA1204	lsa1204	TGTAATCGTTTTTT	-127
LSA1343	eutD	GTAAAACGCTCTCA	-94
LSA1399	loxL2	TGTAAACGATTTCA	-42
LSA1457	lsa1457	TGATAACGCTTACA	-85
LSA1463d	ptsH	TGAAAGCGGTATAG	-161	ptsHI	LSA1463-1462
LSA1641	nanE	TGTAAGCGGTTAAT	-85	nanE-nanA	LSA1641-1640
LSA1643	lsa1643	TGATAACGCTTACA	-31
LSA1651	lsa1651	GGTAAGCGGTTAAA	-148
LSA1711	lacL	TGAAACCGTTTTAA	-36	lacL-lacM	LSA1711-1710
LSA1792	scrA	TGTAAACGGTTGTA	-78	scrA-dexB-scrK	LSA1792-1790
LSA1830	pox2	TTGTAACGCTTACA	-70

The identification is based on the genome sequence of L. sakei strain 23K, and the consensus sequence TGWNANCG NTNWCA (W = A/T, N = A/T/G/C), confirmed in Gram-positive bacteria [39] was used in the search, allowing up to two mismatches (underlined) in the conserved positions except for the two center positions, highlighted in boldface.

a mismatch to consensus sequence is underlined

b position of cre in relation to the start codon

c suggested co-transcribed genes or genes organized in an operon

d cre in preceding gene encoding hypothetical protein

e gene not regulated in this study

Ribose catabolism and PKP

Confirming its major role in ribose transport and utilization in L. sakei, and in agreement with previous findings [16], our microarray data revealed a strong up-regulation (Table 1; log2 = 2.8-4.3) of rbsUDK. The genes encoding an additional putative carbohydrate kinase belonging to the ribokinase family and a putative phosphoribosyl isomerase, lsa0254 and lsa0255, respectively, previously suggested to be involved in catabolism of ribose in L. sakei [7], were induced in all the strains (Table 1). Recent CGH studies revealed that some L. sakei strains which were able to grow on ribose did not harbour the rbsK gene, whereas lsa0254 was present in all strains investigated [32]. This second ribokinase could therefore function as the main ribokinase in some L. sakei strains. The rbsK sequence could also differ considerably from that of 23K in these strains. The PKP showed an obvious induction with an up-regulation (2.2-3.2) of the xpk gene encoding the key enzyme xylulose-5-phosphate phosphoketolase (Xpk). This enzyme connects the upper part of the PKP to the lower part of glycolysis by converting xylulose-5-phosphate into glyceraldehyde-3-phosphate and acetyl-phosphate. Acetyl-phosphate is then converted to acetate and ATP by acetate kinase (Ack). Supporting our results, previous proteomic analysis showed an over-expression of RbsK, RbsD and Xpk during growth on ribose [15,16,19]. The induction of ribose transport and phosphorylation, and increased phosphoketolase and acetate kinase activities were previously observed during growth on ribose [15]. Three genes encoding Ack are present in the 23K genome [7], as well as in MF1053 and LS 25 [32]. A preferential expression of different ack genes for the acetate kinase activity seem to exist. The ack2 gene was up-regulated in all the strains, while ack1 was up-regulated and ack3 down-regulated in 23K and LS 25 (Table 1). An illustration of the metabolic pathways with genes affected by the change of carbon source from glucose to ribose in L. sakei is shown in Figure 2.

Figure 2

Overview of the glycolysis, phosphoketolase pathway and nucleoside catabolic pathway affected by the change of carbon source from glucose to ribose in three . Genes which expression is up- or down-regulated are indicated with upward and downward pointing arrows, respectively, and are listed in Table 1. Black arrows indicate regulation in all three strains, and grey arrows indicate regulation in one or two strains. Schematic representation of CcpA-mediated CCR pathway is shown in the upper right corner. EII, enzyme II of the phosphotransferase system (PTS); EI, enzyme I, HPr, Histidine-containing protein; T, transport protein; P, phosphate; HPrK/P, HPr kinase/phosphatase; G6P, glucose-6-phosphate; F6P; fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; G3P, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; Gly3P, glycerol-3-phosphate; X5P, xylulose-5-phosphate; 1,3PG, 1,3-phosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolepyruvate; glk, glucokinase; pgi, phosphoglucoisomerase; fbp, fructose-1,6-bisphosphatase; tpi, triose-phosphate isomerase; gap, glyceraldehyde-3-phosphate dehydrogenase; pgk, phosphoglycerate kinase; eno, enolase; rpi, ribose-5-phosphate isomerase; rpe, ribulose-phosphate 3-epimerase.

As a consequence of the pentose-induced PKP, genes involved in PKP-metabolism of glucose, such as gntZ, gntK and zwf, were down-regulated (Table 1, Figure 2). The glycolytic pathway was clearly repressed, supporting previous findings [15,19]. Among these genes were pfk (0.5-1.1) encoding 6-phosphofructokinase (Pfk), and fba (0.7-1.1) coding for fructose-bisphosphate aldolase, both acting at the initial steps of glycolysis. In addition, gpm3 encoding one of the five phosphoglycerate mutases present in the 23K genome, acting in the lower part of glycolysis, was also down-regulated (0.7-0.9). MF1053 down-regulated pyk (0.7) encoding pyruvate kinase (Pyk) that competes for PEP with the PTS (Figure 2). Its activity results in the production of pyruvate and ATP, and it is of major importance in glycolysis and energy production in the cell. MF1053 also showed a stronger down-regulation of pfk than the other strains (Table 1). Similar to several other lactobacilli, pfk is transcribed together with pyk [43,44], and in many microorganisms the glycolytic flux depends on the activity of the two enzymes encoded from this operon [43,45]. At the protein level, we previously observed both Pfk and Pyk expressed at a lower level for all the three strains [19], however this was not confirmed at the level of gene expression for 23K and LS 25. We could also not confirm the lower protein expression of glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and enolase previously seen in LS 25 [19]. The latter three enzymes are encoded from the central glycolytic operon (cggR-gap-pgk-tpi-eno) together with triose-phosphate isomerase and the putative central glycolytic genes regulator (CggR) [46]. Besides the cggR gene being down-regulated in MF1053 and LS 25, no change in gene expression was seen of these central glycolytic genes. Thus at the transcription level it is not obvious that the LS 25 strain down-regulate the glycolytic pathway more efficiently than the other strains, as previously suggested [19].

Interestingly, all the strains showed an induction (1.4-2.3) of mgsA encoding methylglyoxal synthase, which catalyzes the conversion of dihydroxyacetone-phosphate to methylglyoxal (Figure 2). The presence of this gene is uncommon among LAB and so far a unique feature among the sequenced lactobacilli. The methylglyoxal pathway represents an energetically unfavourable bypass to the glycolysis. In E. coli, this bypass occurs as a response to phosphate starvation or uncontrolled carbohydrate metabolism, and enhanced ribose uptake was shown to lead to the accumulation of methylglyoxal [47,48]. As suggested by Chaillou et al. [7], such flexibility in the glycolytic process in L. sakei may reflect the requirement to deal with glucose starvation or to modulate carbon flux during co-metabolism of alternative carbon sources. Breakdown of methylglyoxal is important as it is toxic to the cells [49]. An induction of the lsa1158 gene contiguous with mgsA was seen for 23K and MF1053. This gene encodes a hypothetical protein, also suggested as a putative oxidoreductase, which may reduce methylglyoxal to lactaldehyde [7]. However, no induction of the adhE (lsa0379) gene encoding an iron-containing aldehyde dehydrogenase suggested to further reduce lactaldehyde to L-lactate [7] was seen. By CGH [32]lsa1158 and adhE were present in all the L. sakei strains investigated, whereas mgsA was lacking in some strains, indicating that the MgsA function is not vital.

Pyruvate metabolism

Pyruvate is important in both glycolysis and PKP. It can be converted into lactate by the NAD-dependent L-lactate dehydrogenase, which regenerates NAD+ and maintains the redox balance. This enzyme is encoded by the ldhL gene which was down-regulated (0.7-1.4) in all three strains, in accordance with previous findings [50], and the down-regulation was strongest for the LS 25 strain. At the protein level, only LS 25 showed a lower expression of this enzyme during growth on ribose [19]. Genes responsible for alternative fates of pyruvate (Figure 2) were highly induced in all the strains, however with some interesting strain variation (Table 1). The shift in pyruvate metabolism can benefit the bacteria by generating ATP, or by gaining NAD+ for maintaining the redox balance and may lead to various end products in addition to lactate [51].

In all the strains, a strongly up-regulated (2.1-3.0) pox1 gene was observed, and in 23K an up-regulated pox2 (0.7), encoding pyruvate oxidases which under aerobic conditions convert pyruvate to acetyl-phosphate with hydrogen peroxide (H2O2) and CO2 as side products. Accumulation of peroxide ultimately leads to aerobic growth arrest [52]. H2O2 belongs to a group of compounds known as reactive oxygen species and reacts readily with metal ions to yield hydroxyl radicals that damage DNA, proteins and membranes [53]. Remarkable differences in redox activities exist among Lactobacillus species and L. sakei is among those extensively well equipped to cope with changing oxygen conditions, as well as dealing effectively with toxic oxygen byproducts [7]. 23K up-regulated npr (1.0) encoding NADH peroxidase which decomposes low concentrations of H2O2 to H2O and O2, and all the strains up-regulated the sodA gene (1.7-3.4) encoding a superoxide dismutase which produces hydrogen peroxide from superoxide (O2-). Various oxidoreductases showed an up-regulation in all the strains (Table 1), indicating the need for the bacterium to maintain its redox balance.

The pdhABCD gene cluster encoding components of the pyruvate dehydrogenase enzyme complex (PDC) which transforms pyruvate into acetyl-CoA and CO2 were among the strongly up-regulated (2.1-3.7) genes. The eutD gene encoding a phosphate acetyltransferase which further forms acetyl-phosphate from acetyl-CoA was also induced (1.0-2.0). Pyruvate can be transformed to acetolactate by acetolactate synthase and further to acetoin by acetolactate decarboxylase, before 2,3-butanediol may be formed by an acetoin recuctase (Figure 2). While the budC gene encoding the acetoin reductase showed a strong up-regulation in all three strains, the als-aldB operon was only strongly up-regulated in LS 25 (1.9). Pyruvate formate lyase produces acetyl-CoA and formate from pyruvate. Only in 23K, the pflAB genes encoding formate C-acetyltransferase and its activating enzyme involved in formate formation were strongly up-regulated (4.0 and 1.7, respectively). This strain was the only one to strongly induce L-lactate oxidase encoding genes which are responsible for conversion of lactate to acetate when oxygen is present (Table 1). In 23K and LS 25, the ppdK gene coding for the pyruvate phosphate dikinase involved in regenerating PEP, was induced, as was also lsa0444 encoding a putative malate dehydrogenase that catalyzes the conversion of malate into oxaloacetate using NAD+ and vice versa (Table 1).

During growth on ribose, L. sakei was shown to require thiamine (vitamine B1) [15]. The E1 component subunit α of the PDC, as well as Pox and Xpk, require thiamine pyrophosphate, the active form of thiamine, as a coenzyme [54]. This could explain the induction of the thiMDE operon and lsa0055 in LS 25, as well as lsa0980 in 23K, encoding enzymes involved in thiamine uptake and biosynthesis (Table 1). The up-regulation of lsa1664 (1.1-1.6) encoding a putative dihydrofolate reductase involved in biosynthesis of riboflavin (vitamin B2) in all the strains could indicate a requirement for flavin nucleotides as enzyme cofactors. Riboflavin is the precursor for flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) redox cofactors in flavoproteins, and the E3 component of PDC as well as glycerol-3-phosphate dehydrogenase encoded from the up-regulated glpD, are among enzymes requiring FAD. Another cofactor which seems to be important during growth on ribose is lipoate, essential of the E2 component of the PDC. An up-regulation of lplA (1.0 - 1.6) encoding lipoate-protein ligase, which facilitates attachment of the lipoyl moiety to metabolic enzyme complexes, was seen in all the strains, allowing the bacterium to scavenge extracellular lipoate [55,56].

Nucleoside catabolism

The L. sakei genome contains a multiplicity of catabolic genes involved in exogenous nucleoside salvage pathways, and the bacterium has been shown to catabolize inosine and adenosine for energy [7]. Three iunH genes are present in the 23K genome, which encode inosine-uridine preferring nucleoside hydrolases responsible for conversion of inosine to ribose and purine base. The iunH1 gene was up-regulated in all the strains when grown on ribose (1.8-2.6), as was also the iunH2 gene in 23K (1.2). The deoC gene encodes a deoxyribose-phosphate aldolase, and is located in an operon structure preceding the genes deoB, deoD, lsa0798, lsa0799, deoR and pdp which encode phosphopentomutase, purine nucleoside phosphorylase, pyrimidine-specific nucleoside symporter, a putative purine transport protein, the deoxyribonucleoside synthesis operon transcriptional regulator (DeoR), and a pyrimidine-nucleoside phosphorylase, respectively. The complete operon was induced in all the strains, except for pdp only induced in 23K (Table 1). The phosphorylases catalyze cleavage of ribonucleosides and deoxyribonucleosides to the free base pluss ribose-1-phosphate or deoxyribose-1-phosphate. The bases are further utilized in nucleotide synthesis or as nitrogen sources. The pentomutase converts ribose-1-phosphate or deoxyribose-1-phosphate to ribose-5-phosphate or deoxyribose-5-phosphate, respectively, which can be cleaved by the aldolase to glyceraldehyde-3-phosphate and acetaldehyde. Glyceraldehyde-3-phosphate enters the glycolysis, while a putative iron containing alcohol dehydrogenase, encoded by lsa0258 up-regulated in all the strains (0.5-1.6), could further reduce acetaldehyde to ethanol (Figure 2). The obvious induced nucleoside catabolism at the level of gene expression was not seen by proteomic analysis [19].

Genes involved in glycerol/glycerolipid/fatty acid metabolism

During growth on ribose, a strong induction of the glpKDF operon encoding glycerol kinase (GlpK), glycerol-3-phosphate dehydrogenase (GlpD), and glycerol uptake facilitator protein was observed (Table 1), which is in correlation with the over-expression of GlpD and GlpK seen by proteomic analysis [19]. GlpD is FADH2 linked and converts glycerol-3-phosphate to dihydroxyacetone-phosphate. An over-expression of GlpD was also reported when L. sakei was exposed to low temperature [57]. A glpD mutant showed enhanced survival at low temperature, and it was suggested that this was a result of the glycerol metabolism being redirected into phosphatidic acid synthesis which leads to membrane phospholipid biosynthesis [57]. Nevertheless, a down-regulation was observed of the lsa1493 gene (0.6-0.9) encoding a putative diacylglycerol kinase involved in the synthesis of phosphatidic acid, and of cfa (1.3-1.4) encoding cyclopropane-fatty-acyl-phospholipid synthase directly linked to modifications in the bacterial membrane fatty acid composition that reduce membrane fluidity and helps cells adapt to their environment [58]. Interestingly, LS 25 up-regulated several genes (LSA0812-0823), including accD and accA encoding the α- and ß-subunits of the multi-subunit acetyl-CoA carboxylase (Table 1). This is a biotin-dependent enzyme that catalyzes the irreversible carboxylation of acetyl-CoA to produce malonyl-CoA, an essential intermediate in fatty acid biosynthesis. In B. subtilis, the malonyl-CoA relieves repression of the fab genes [59]. We observed that also acpP, fabZ1, fabH, fabD and fabI (Table 1) encoding enzymes involved in fatty acid biosynthesis were induced in LS 25. The altered flux to malonyl-CoA may be a result of the decreased glycolytic rate. MF1053, on the other hand, showed a down-regulation of several genes in the same gene cluster. A higher level of acetate is produced when the bacterium utilizes ribose, and acetate lowers the pH and has a higher antimicrobial effect than lactate. Changes in the phospholipid composition could be a response to changes in intracellular pH. Protons need to be expelled at a higher rate when the pH drops. The LS 25 strain which showed faster growth rates than the other strains [9], was the only strain to up-regulate the F0F1 ATP synthase (Table 1), which at the expense of ATP expels protons during low pH.

Regulation mechanisms

Little is known about the regulation of catabolic pathways in L. sakei. Starting from ribose uptake, the rbs operon may be both relieved from repression and ribose induced. Presumably, a dual regulation of this operon by two opposite mechanisms, substrate induction by ribose and CCR by glucose may occur in L. sakei. The ccpA gene was not regulated, consistent with this gene commonly showing constitutive expression in lactobacilli [42,60]. The local repressor RbsR is homologous with CcpA, both belonging to the same LacI/GalR family of transcriptional regulators. RbsR was proposed to bind a cre-like consensus sequence located close to a putative CcpA cre site, both preceding rbsU [28]. RbsR in the Gram-positive soil bacterium Corynebacterium glutamicum was shown to bind a cre-like sequence, and using microarrays, the transcription of no other genes but the rbs operon was affected positively in an rbsR deletion mutant. It was concluded that RbsR influences the expression of only the rbs operon [61]. Similarily, in the L. sakei sequence, no other candidate members of RbsR regulation could be found [28]. However, experiments are needed to confirm RbsR binding in

L. sakei. In Bacillus subtilis, RbsR represent a novel interaction partner of P-Ser-HPr in a similar fashion to CcpA [62]. The P-Ser-HPr interaction is possible also in L. sakei as the bacterium exhibits HPr-kinase/phosphatase activity.

A putative cre site is present in the promoter of lsa0254 encoding the second ribokinase (Table 2), and this gene is preceeded by the opposite oriented gene lsa0253 encoding a transcriptional regulator with a sugar binding domain which belongs to the GntR family. This family of transcriptional regulators, as well as the LacI family which RbsR and CcpA belong to, are among the families to which regulators involved in carbohydrate uptake or metabolism usually belong [63]. The GntR-type regulator could possibly be involved in regulating the expression of the second ribokinase, or of the inosine-uridine preferring nucleoside hydrolase encoding iunH1 gene which is located further upstream of lsa0254. C. glutamicum possesses an operon encoding a ribokinase, a uridine transporter, and a uridine-preferring nucleoside hydrolase which is co-controlled by a local repressor together with the RbsR repressor of the rbs operon [60,61,64]. It is possible that such co-control could exist also in L. sakei. Ribose as well as nucleosides are products of the degradation of organic materials such as DNA, RNA and ATP. The simultaneous expression of the rbs and deo operons as well as the other genes involved in ribose and nucleoside catabolism (Figure 2) allows the bacterium to access the different substrates simultaneously and use both ribose as well as nucleosides as carbon and energy source. DeoR shows 51% identity to the B. subtilis DeoR repressor protein [65,66]. Genes encoding deoxyribose-phosphate aldolase, nucleoside uptake protein and pyrimidine nucleoside phosphorylase in B. subtilis are organized in a dra-nupC-pdp operon followed by deoR, and ribose was shown to release DeoR from DNA binding and thus repression of the operon genes are alleviated [65-67]. The B. subtilis pentomutase and purine-nucleoside phosphorylase are encoded from a drm-pupG operon which is not negatively regulated by DeoR, though both operons are subject to CcpA mediated CCR [65,66,68]. As a cre site is found preceding the L. sakei deoC (Table 2), the operon could be regulated by CcpA as well. It is interesting that deoR is the only strongly induced transcriptional regulator gene in all three strains, and the encoded regulator has sigma (σ) factor activity. We can only speculate whether it could function as activator of transcription on some of the regulated genes in this study.

Expression of the Xpk encoding gene of Lactobacillus pentosus was reported to be induced by sugars fermented through the PKP and repressed by glucose mediated by CcpA [69]. Indeed, the cre site overlapping ATG start codon of L. sakei xpk (Table 2) indicates relief of CcpA-mediated CCR during growth on ribose. Also for several genes involved in alternative fates of pyruvate, putative cre sites were present (Table 2).

Several genes and operons involved in transport and metabolism of various carbohydrates such as mannose, galactose, fructose, lactose, cellobiose, N-acetylglucosamine, including putative sugar kinases and PTSs, were induced during growth on ribose (Table 1), and as shown in Table 2, putative cre sites are located in the promoter region of many of these up-regulated genes and operons. 23K showed an up-regulation of genes involved in the arginine deiminase pathway, and 23K and LS 25 showed an up-regulated threonine deaminase (Table 1). The arcA and tdcB both have putative cre sites in their promoter regions (Table 2). Thus ribose seems to induce a global regulation of carbon metabolism in L. sakei.

A putative cre site precedes the glp operon (Table 2), suggesting regulation mediated by CcpA. However, regulation of the L. sakei GlpK may also occur by an inducer exclusion-based CcpA-independent CCR mechanism as described in enterococci and B. subtilis [70,71], and as previously suggested by Stentz et al. [15]. By this mechanism, glycerol metabolism is regulated by PEP-dependent, EI- and HPr-catalyzed phosphorylation of GlpK in response to the presence or absence of a PTS substrate. In the absence of a PTS sugar, GlpK is phosphorylated by P-His-HPr at a conserved histidyl residue, forming the active P-GlpK form, whereas during growth on a PTS sugar, phosphoryl transfer flux through the PTS is high, concentration of P-His-HPr is low, and GlpK is present in a less active dephospho form [20,70,71]. This conserved histidyl residue (His232) is present in L. sakei GlpK [20], and Stentz et al. [15] reported that whereas L. sakei can grow poorly on glycerol, this growth was abolished in ptsI mutants.

Mannose-PTS

As mentioned in the introduction, the PTS plays a central role, in both the uptake of a number of carbohydrates and regulatory mechanisms [20-22]. Encoding the general components, ptsH showed an up-regulation in MF1053 and LS 25 (1.2 and 0.9, respectively), while all the strains up-regulated ptsI (0.8-1.7). The manLMN operon encoding the EIIman complex was surprisingly strongly up-regulated during growth on ribose in all the strains (Table 1). By proteomic analysis, no regulation of the PTS enzymes was seen [19]. The expression of HPr and EI in L. sakei during growth on glucose or ribose was previously suggested to be constitutive [14], and in other lactobacilli, the EIIman complex was reported to be consistently highly expressed, regardless of carbohydrate source [72-74]. Notably, PEP-dependent phosphorylation of PTS sugars has been detected in ribose-grown cells, indicating that the EIIman complex is active, and since no transport and phosphorylation via EIIman occurs, the complex is phosphorylated, while it is unphosphorylated in the presence of the substrates of the EIIman complex [8,73]. The stimulating effect exerted by small amounts of glucose on ribose uptake in L. sakei, which has also been reported in other lactobacilli [74,75], was suggested to be caused by dephosphorylation of the PTS proteins in the presence of glucose, as a ptsI mutant lacking EI, as well as P-His-HPr, was shown to enhance ribose uptake [15,16,76]. Stentz et al. [15] observed that a L. sakei mutant (strain RV52) resistant to 2 deoxy-D-glucose, a glucose toxic analog transported by EIIman, and thus assumed to be affected in the EIIman, did not show the same enhanced uptake [15]. It was concluded that EIIman is not involved in the PTS-mediated regulation of ribose metabolism in L. sakei. The mutation was though not reported verified by sequencing [15], and other mutations could be responsible for the observed phenotype. The L. sakei EIIABman, EIICman and EIIDman show 72, 81, and 82% identity, respectively, with the same enzymes in L. casei, in which mutations rendering the EIIman complex inactive were shown to derepress rbs genes, resulting in a loss of the preferential use of glucose over ribose [75]. Furthermore, in L. pentosus, EIIman was shown to provide a strong signal to the CcpA-dependent repression pathway [73]. The hprK gene encoding HPrK/P which controls the phosphorylation state of HPr was strongly up-regulated (1.2-2.0) in all three strains. HPrK/P dephosphorylates P-Ser-HPr when the concentration of glycolytic intermediates drop, which is likely the situation during growth on ribose [20,22,24].

Numerous genes encoding hypothetical proteins with unknown function were also found to be differentially expressed (Table 1), as well as several other genes belonging to various functional categories. For most of these, their direct connection with ribose metabolism is unknown, and is likely an indirect effect.

Conclusions

The ability to ferment meat and fish is related to the capacity of the bacterium to rapidly take up the available carbohydrates and other components for growth. The importance of this process, especially to the meat industry, stimulates research aimed at understanding the mechanisms for transport and metabolism of these compounds, with the ultimate goal to be able to select improved strains. Genome-wide transcriptome analyses with DNA microarrays efficiently allowed the identification of genes differentially expressed between growth on the two carbohydrates which L. sakei can utilize from these substrates. Moreover, microarrays were a powerful tool to increase the understanding of the bacterium's primary metabolism and revealed a global regulatory mechanism. In summary, the ribose uptake and catabolic machinery is highly regulated at the transcription level, and it is closely linked with catabolism of nucleosides. A global regulation mechanism seems to permit a fine tuning of the expression of enzymes that control efficient exploitation of available carbon sources.

Abbreviations

PKP: phosphoketolase pathway; PEP: phosphoenolpyruvate; PTS: PEP-dependent carbohydrate phosphotransferase system; CCR: carbon catabolite repression; cre: catabolite responsive element; RbsK: ribokinase; RbsD: D-Ribose pyranase; Xpk: xylulose-5-phosphate phosphoketolase; Ack: Acetate kinase, Pfk: 6-phosphofructokinase; Pyk: pyruvate kinase; PDC: pyruvate dehydrogenase complex; GlpD: glycerol-3-phosphate dehydrogenase; GlpK: glycerol kinase; EII: enzyme II; EI: enzyme I; HPr: histidine protein; HPrK/P: HPr kinase/phosphatase; DeoR: deoxyribonucleoside synthesis operon transcriptional regulator.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

AM participated in the study design, conducted the experimental work, analyzed and interpreted data, and wrote the manuscript. LS conducted the statistical analysis. KN and LA conceived the study, participated in the study design process and reviewed the manuscript. All authors read and approved the final manuscript.

Additional file 1

Table S3. Primer and probe sets used for qRT-PCR. Presents the primer and probe sets used for validation of microarray data by qRT-PCR analysis. Table S4. Comparison of microarray data with qRT-PCR results of . . Presents gene regulation values (log2) from the qRT-PCR analysis in comparison with microarray data.

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