Applied and Environmental Microbiology, July 2003, p . 4049-4056, Vol . 69, No . 7

Characterization of Genes Involved in the Metabolism of -Galactosides by Lactococcus raffinolactis

Isabelle Boucher, Christian Vadeboncoeur, and Sylvain Moineau^*

Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Groupe de Recherche en Écologie Buccale (GREB), Faculté de Médecine Dentaire, Université Laval, Québec, Canada G1K 7P4

Received 9 December 2002/ Accepted 31 March 2003

L . raffinolactis has also the peculiar property of being able to ferment -galactosides, such as melibiose and raffinose . This characteristic is attributed mainly to the activity of an -galactosidase (Aga) . Following the hydrolysis of -galactosides by Aga, the -galactose subunit is released and can be degraded through two distinct metabolic pathways: the tagatose 6-phosphate pathway and the Leloir pathway . The substrate for the tagatose 6-phosphate pathway is the galactose 6-phosphate, resulting from the transport and phosphorylation of melibiose by the sugar phosphotransferase transport system (PTS) . Galactose 6-phosphate is metabolized to triose-phosphates by three enzymes: galactose 6-phosphate isomerase (LacA and LacB), tagatose 6-phosphate kinase (LacC), and tagatose 1,6-bisphosphate aldolase (LacD) . On the other hand, intracellular nonphosphorylated galactose molecules are degraded by the Leloir pathway . -Galactose is first transformed into galactose 1-phosphate by a galactokinase (GalK) . Then, two additional enzymes, namely, galactose 1-phosphate uridylyltransferase (GalT) and UDP-galactose 4-epimerase (GalE), are responsible for converting the galactose 1-phosphate to glucose 1-phosphate, a glycolysis precursor . Both pathways are found in L . lactis (2, 29, 30), and their genetic determinants usually form an operon (9, 13, 14, 32) .

A few -galactosidase genes have also been cloned and sequenced in LAB . In Streptococcus mutans, the -galactosidase gene is associated with the multiple-sugar metabolism operon (24) . This Aga is essential for growth in the presence of melibiose and raffinose . The expression of S . mutans -galactosidase is activated by a transcriptional regulator of the AraC/XylS family . In Streptococcus pneumoniae, Aga is essential for raffinose utilization and its activity is stimulated by raffinose and catabolite repressed by sucrose but not glucose (23) . The expression of Aga is also stimulated by two gene products, one of which belongs to the AraC/XylS family . In Carnobacterium piscicola, the -galactosidase determinant is grouped with two ß-galactosidase genes and both enzymatic activities are repressed in the presence of glucose or lactose during growth (6) . Similarly, the -galactosidase gene from Lactobacillus plantarum (melA) is clustered with lacA and lacM, encoding a heterodimeric ß-galactosidase (21, 27) . Transcription of melA is independent of surrounding genes and is induced by melibiose and partially repressed by glucose (27) .

In this work, we present the characterization and transcriptional analysis of a 12-kb chromosomal segment of L . raffinolactis ATCC 43920 (also known as NCDO617) containing the -galactosidase gene and its transcriptional regulator as well as genes involved in the Leloir pathway of galactose degradation . This study provides the first genetic analysis of L . raffinolactis through its -galactosides metabolism .

 
DNA techniques.
Routine DNA manipulations were carried out according to standard procedures (25) . Restriction enzymes, alkaline phosphatase, RNase-free DNase, RNase inhibitor (Roche Diagnostics), and T4 DNA ligase (Invitrogen Life Technologies) were used according to the suppliers' instructions . All primers used in this study were obtained from Invitrogen Life Technologies and are listed in Table 2 . Transformations of E . coli (25) and L . lactis (16) were performed as described elsewhere . Plasmid DNA from E . coli and L . lactis was isolated as previously described (5, 11) . Total lactococcal DNA was obtained as described by Boucher et al . (5) . When needed, large amounts of E . coli plasmid DNA were isolated with a Qiagen Plasmid Maxi kit .

 
Sequencing of the aga locus from L . raffinolactis ATCC 43920.
The 4-kb EcoRI/HindIII DNA fragment from L . raffinolactis containing the aga gene (5) was used as a probe in Southern hybridizations to target a larger chromosomal region . The fragment was labeled using a DIG High-Prime DNA labeling kit (Roche Diagnostics) . Prehybridization, hybridization, and posthybridization washes and detection by chemiluminescence were performed as suggested by the manufacturer (Roche Diagnostics) . An 8-kb KpnI/NheI fragment was isolated as previously described (5) and cloned into pBS to construct pRAF110, and the DNA sequences on both strands were determined by primer walking . Primer raf34, complementary to the galT gene, was then used to target additional chromosomal restriction fragments covering the area downstream of this gene on Southern hybridizations . A 5-kb ClaI/SpeI fragment was cloned into pBS to construct pRAF102, and the cloned DNA was sequenced on both strands by primer walking . Finally, a 3-kb KpnI fragment was used as the substrate for inverse PCR (4), and the resulting amplicon was also sequenced . DNA sequencing was carried out by the DNA sequencing service at the Université Laval by means of an ABI Prism 3100 apparatus . Sequence analyses were performed using the Wisconsin Package software (version 10.3) of the Genetics Computer Group (7) .

Transcriptional analysis of the aga locus.
Total RNA was isolated from L . raffinolactis by means of the same procedure used with L . lactis (5) . Approximately 5 µg of material was separated on agarose-formaldehyde electrophoresis gel as described by Sambrook and Russell (25) . Nucleic acids were transferred to a positively charged nylon membrane (Roche Diagnostics) and fixed by UV exposure . PCR amplicons were labeled with ³²P by using High Prime (Roche Diagnostics) and used as probes . Prehybridization (3 h) and hybridization (overnight) were performed in DIG Easy Hyb (Roche Diagnostics) at 50°C, and two posthybridization washes were made at the same temperature in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 sodium citrate)-0.1% sodium dodecyl sulfate prior to film exposure (Kodak X-Omat AR) . Stripping of the membrane was performed by immersion in boiling 0.1% sodium dodecyl sulfate (25) . The membrane was successively hybridized with the different probes in the following order: B, D, A, and C (Fig . 1) . Probes A, B, C, and D were obtained by PCR amplification of L . raffinolactis DNA with primers raf32 and raf33, primers raf5 and raf13, primers raf9 and raf11, and primers raf48 and raf57, respectively .

 
Localization of the transcriptional initiation site of aga.
The transcriptional initiation site of aga was established through 5' rapid amplification of cDNA ends (RACE) essentially as described by Sambrook and Russell (25) . RNA was isolated from L . raffinolactis cells grown in raffinose . DNase treatments and cDNA synthesis were performed using 20 µg of total RNA and the aga-specific primer raf67 (5) . Free nucleotides and primers were removed by precipitating the cDNA twice in 2.5 M ammonium acetate with 3 volumes of 95% ethyl alcohol . cDNA was then dissolved in double-distilled water to a final volume of 20 µl . cDNA (13 µl) was used for poly(dG) tailing with terminal deoxyribonucleotidyl transferase, as recommended by the manufacturer (Amersham Pharmacia Biotech) . Tailed DNA was diluted to 1 ml with 10 mM Tris-HCl, pH 8.5, and 5 µl was used for PCR amplification with a poly(dC) primer (CB3) and the aga-specific primer raf73 . The PCR product was purified with silica as previously described (10) and sequenced using primer raf73 . The transcriptional initiation site of aga was also determined by primer extension analysis essentially as described by Sambrook and Russell (25) .

-Galactosidase assay.
-Galactosidase activity was evaluated as described by Boucher et al . (5) . Briefly, 10-ml cell cultures (optical density at 600 nm, 0.5) were washed and lysed with glass beads . The cell lysate was cleared by centrifugation, and the protein content was determined with the Bio-Rad DC protein assay reagent . The -galactosidase activity was assayed at 30°C and at pH 7.0 with p-nitrophenyl--D-galactopyranoside (Sigma) as the substrate (22) .

ß-Galactosidase and phospho-ß-galactosidase assays.
Both the ß-galactosidase and phospho-ß-galactosidase enzymatic activities were detected as described for the -galactosidase assay but with cells grown in LM17 medium (optical density at 600 nm, 0.5) . The enzyme assays were performed at 30°C and at pH 7.0 with o-nitrophenyl-ß-D-galactopyranoside (Sigma-Aldrich) as the substrate for ß-galactosidase and o-nitrophenyl-ß-D-galactopyranoside 6-phosphate (Sigma-Aldrich) as the substrate for phospho-ß-galactosidase . The presence of the enzyme activity was confirmed by the appearance of a yellow color after the cells were incubated overnight . The industrial strain Streptococcus thermophilus SMQ-301 (31) was used as a positive control for ß-galactosidase, and L . lactis subsp . cremoris SMQ-754 was used as a positive control for phospho-ß-galactosidase .

Nucleotide sequence accession number.
The GenBank accession number assigned to the nucleotide sequence of the aga locus of L . raffinolactis ATCC 43920 is AY164273 .

Sequence analysis of the aga locus from L . raffinolactis ATCC 43920.
A 4-kb DNA region involved in -galactoside fermentation was previously cloned and sequenced from L . raffinolactis ATCC 43920 and used as a dominant selectable marker in a food-grade cloning vector for the genetic modification of L . lactis (5) . In our study, this region was further characterized, and an additional 8 kb of DNA was sequenced (Fig . 1A) This DNA segment possessed a G+C content of 39.7%, which is in agreement with the previous estimated values of 40.3 to 41.5% (17) . The analysis of the 12-kb DNA fragment revealed nine open reading frames (ORFs) (Fig . 1A and Table 3) . Putative functions were attributed to products of eight ORFs based on motifs in the peptide chains, and/or extensive amino acid similarity with proteins of known function (see Table 3 for details) .

 
Sequence analysis also revealed the presence of three putative promoters upstream from the genes aga (TTGACA-N₁₇-TATAAT) (5), orf2 (TTGAAA-N₁₇-TAAAGT), and galR (TTGTTT-N₂₀-TAAAAT) . Two regions containing inverted repeats, able to form a stem-loop structure and to act as intrinsic transcriptional terminators, were found downstream from galR (G^25°C = -17.6 kcal/mol) and galT (G^25°C = -13.2 kcal/mol) . Two catabolite-responsive element sequences were also identified in the 12-kb fragment of L . raffinolactis . Catabolite-responsive element sequences were found in the -35 region of the promoters upstream from aga and orf2, indicating that these genes might be subjected to catabolite repression (18) . Finally, a potential binding site for GalR was identified in the -10 box region of the aga promoter (Fig . 2) . The N-terminal helix-turn-helix motif of GalR and its putative binding site are similar to cognate regions characterized for S . mutans (1) and S . thermophilus (34) .

 
These results suggested that (i) aga, galK, and galT formed a transcriptional unit expressed from a promoter located upstream from aga and ending at the terminator identified downstream from galT; (ii) galR was transcribed independently from the aga-galKT operon and its transcription ended in the galR-aga intergenic region; and (iii) orf2, orf3, and orf4 may also be expressed independently from at least one promoter located upstream from orf2 .

mRNA analysis of the 12-kb DNA fragment from L . raffinolactis ATCC 43920.
To determine the number and size of transcripts in this chromosomal region, Northern blot analyses were performed on L . raffinolactis cells grown on various sugars (Fig . 1B) . The gene galR was expressed, under every condition tested, as a monocistronic mRNA 1 kb in size . The transcript size was in agreement with the gene size and suggested transcription from a promoter located upstream from galR and termination at the stem-loop structure identified in the galR-aga intergenic region .

The aga-galKT mRNA was 5 kb in size, which corresponded to a transcriptional unit that started at the proposed promoter upstream from aga and ended at the terminator downstream from galT . The transcription of this operon was stimulated by galactosides such as lactose, melibiose, and raffinose (Fig . 1B) . Overexposure of the Northern membrane revealed a weak aga-galKT transcript of the same size in cells grown on galactose, while no signal was detected in glucose-grown cells (data not shown) .

Finally, the three genes downstream from galT were expressed independently from the aga operon in cells grown on all the sugars tested (Fig . 1B) .

Identification of the transcription start site of the aga operon.
The RACE-PCR technique was used to identify the transcription initiation site of the aga-galKT operon (Fig . 3) . The transcription was initiated at the A position located 7 bases downstream from the last T position of the -10 box identified on the DNA coding strand . An identical transcription initiation site was confirmed by primer extension analysis (data not shown) .

 
-Galactosidase activity in L . raffinolactis ATCC 43920.
The -galactosidase activity was measured in cell lysates of L . raffinolactis grown in the presence of various sugars (Table 4) . Aga expression was strongly stimulated by the galactosides lactose, melibiose, and raffinose but poorly stimulated by galactose (Table 4), which induced fivefold less Aga activity than did melibiose . Virtually no Aga activity was detected in L . raffinolactis cells cultivated in the presence of glucose (Table 4) . The finding that lactose induced aga in L . raffinolactis contrasted with the observation that lactose did not induce aga in the industrial lactose-positive strain L . lactis SMQ-741 (5) . This difference most likely resulted from the fact that these two lactococcal species do not metabolize lactose the same way . L . lactis transports lactose by the PTS, and the galactose 6-phosphate produced following hydrolysis by phospho-ß-galactosidase is metabolized via the tagatose 6-phosphate pathway (2, 29) . Both ß-galactosidase and phospho-ß-galactosidase activities were detected in lactose-grown L . raffinolactis cells (data not shown), suggesting that the galactose moiety of lactose could be metabolized by the Leloir and the tagatose 6-phosphate pathways . Thus, the metabolism of lactose by L . raffinolactis generated intracellular galactose 6-phosphate as well as free galactose and galactose derivatives of the Leloir pathway . These results suggested that galactose or an intermediate of the Leloir pathway, but not galactose-6 phosphate, is involved in aga induction in lactococci . However, since extracellular galactose barely induced the aga operon in L . raffinolactis (Table 4), we suggest that aga induction in lactococci is optimally driven by a metabolite derived from galactoside metabolism .

 
-Galactosidase activity in L . lactis MG1363 in the presence or absence of GalR.
A potential GalR binding site was found in the promoter region of the aga operon . To determine whether GalR acted as a transcriptional regulator of the aga operon, gene inactivation and allelic replacement were attempted in L . raffinolactis . Despite several attempts using various strategies, we failed to obtain a galR mutant derivative in this species . Thus, the function of L . raffinolactis GalR was assessed in L . lactis subsp . cremoris MG1363, which does not display any -galactosidase activity .

The aga gene was cloned into the high-copy-number plasmid vector pNZ123, alone (pRAF301) or in combination with galR (pRAF300) . aga was cloned on a high-copy-number vector to minimize the potential effects of endogenous L . lactis regulators . The recombinant plasmids were separately introduced into L . lactis MG1363, and the -galactosidase activity was evaluated after growth in the presence of glucose, galactose, and melibiose (Table 5) . It is noteworthy that this L . lactis strain cannot utilize lactose . Interestingly, the -galactosidase activity was at least 20-fold higher in cells containing pRAF301 (aga) than in those with pRAF300 (aga and galR) . Therefore, aga was expressed from its own promoter without the need for GalR-mediated activation, and GalR clearly repressed Aga activity in L . lactis . These results suggest that GalR acted as a repressor in L . raffinolactis .

 
Organization of the -galactosidase genes in LAB.
The genes coding for the Leloir pathway enzymes generally form operons (14, 32) . In L . lactis subsp . cremoris MG1363, the order of the five genes required for galactose utilization is galPMKTE, where galP (formerly galA) encodes a galactose transporter (5, 13, 14) . The gal genes of L . lactis subsp . lactis NCDO2054 and IL1403 are similarly organized but include the insertion of two genes (lacA and lacZ) between galT and galE (3, 33) . In L . raffinolactis ATCC 43920, the gal genes are not grouped around galM, and galE genes could not be found on the 12-kb segment of the DNA sequenced .

Conversely, -galactosidase determinants are heterogeneous in terms of their genetic positioning . In L . raffinolactis, the -galactosidase gene is clustered with galK and galT to form an operon . The -galactosidase gene organization is known for six other LAB genera (Fig . 4) . In a general manner, -galactosidase genes were found with genes coding for a diversity of enzymes and transporters involved in the metabolism of various sugars . In Lactobacillus plantarum, the melA gene encoding -galactosidase was also located near genes involved in the Leloir pathway of galactose metabolism, namely, galM3, galK, galE4, and galT (21, 27) . Surprisingly, the organization found in Leuconostoc lactis appeared to be similar to that found in L . raffinolactis (13) . Unfortunately, the DNA sequence required to support investigations concerning possible horizontal gene transfer between the two bacteria was not available .

 
-Galactoside metabolism is also found in some L . lactis subsp . lactis strains isolated from fruits and vegetables where raffinose is readily available (19, 20) . In the -galactosides-fermenting strain KF292 of L . lactis, we have found a DNA region encompassing the galR and aga genes that is over 90% identical to a DNA segment of L . raffinolactis ATCC 43920 (data not shown) . Most L . lactis strains used by the dairy industry are melibiose and raffinose negative, and they were isolated from raw milk or fermented milk products . On the other hand, L . lactis strains found on plant material are able to utilize these sugars . Therefore, the presence or absence of an -galactosides metabolism in lactococcal strains is likely related to their distinct ecological adaptation .

Conclusion.
Over the years, research on LAB genetics has focused mainly on the industrial applications of these organisms . Consequently, certain lactococcal species, such as L . raffinolactis, remain uncharacterized at the genetic level . This study provides the first genetic analysis of L . raffinolactis. In this species, -galactoside metabolism was driven by an operon that contains the three genes aga, galK, and galT . The gene aga encoded an -galactosidase, while galK and galT most likely encoded, respectively, a galactokinase and a galactose 1-phosphate-uridylyltransferase, two enzymes of the Leloir pathway . The expression of this operon is controlled by a gene located upstream, galR . The product of galR belonged to the LacI/GalR family of transcriptional regulators and acted as a repressor of the gene cluster .

We are grateful to William Kelly for providing the -galactoside-fermenting strain L . lactis KF292 .

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