Characterization of the Lactococcus lactis lactose genes and regulation of their expression

An important trait of the lactic acid bacterium Lactococcus lactis , that is used in industrial dairy fermentations, is the conversion of lactose into lactic acid. The enzymatic steps involved in the breakdown of lactose, that is transported into the cell via a phosphoenolpyruvate-dependent lactose phosphotransferase system (PEP-PTS ^lac), have been well established (Fig. 1). However, except for the molecular cloning and characterization of the plasmid-located phospho-B-galactosidase gene (Boizet et al. , 1988; De Vos and Gasson, 1989), relatively little data have emerged concerning the genetic information for the lactose catabolic enzymes. A solid genetic basis of this key metabolic route is essential for the development of food-grade selection markers and pathway engineering strategies for L. lactis. In addition, since high lactose-specific enzyme activities are observed during growth on lactose, which are repressed during growth on glucose, expression of the lac genes is probably under control of a strong and inducible promoter. Such a promoter would be applicable as a 'genetic switch' in the controlled overexpression of homologous and heterologous genes in Lactococci . Isolation and elucidation of the mechanism of control of the lac promoter would be beneficial for the development of such strains. This thesis describes the characterization and organization of the genes involved in the lactose metabolism of L. lactis subsp. lactis. In addition, several cis - and trans -acting factors that are involved in the regulation of their expression were identified.

In Chapter 1 some background information is given about the enzymology and genetics of lactose metabolism in lactic acid bacteria. In addition, this Chapter provides a brief overview of the various mechanisms that may be involved in the regulation of gene expression in bacteria, and presents the state-of-the-art concerning gene regulation in lactic acid bacteria.

The characterization of the genetic determinants for lactose metabolism, including the PEP-PTS ^lac(LacEF), phospho-β-galactosidase (LacG) and tagatose-6-phosphate pathway enzymes (LacABCD), is presented in Chapters 2 and 3 . The lac genes of the L. lactis subsp. lactis strain MG1820, that are located on the 23.7-kb plasmid pMG820, appeared to be organized in a 7.8-kb operon-structure with the gene order lacABCDFEGX (Fig. 1). The lacE and lacF genes encode the PEP-PTS ^lacproteins Enzyme II ^lac(62 kDa) and Enzyme III ^lac(11 kDa), that are involved in the translocation across the cell membrane and subsequent phosphorylation of lactose (Chapter 2). Crosslinking studies with purified enzyme showed that Enzyme III ^lacis active as a trimer. The identity of the lacF gene was confirmed by complementation of lacF deficiency in L. lactis strain YP2-5, that appeared to contain a G 18E mutation in the deduced LacF protein. Homology was observed between the deduced amino acid sequences of the L. lactis lacE and lacF genes and those of Lactobacillus c a sei and Staphylococcus aureus . In addition, the deduced L. lactis LacE and LacF amino acid sequences were homologous to those of CelA, CelB and CelC that are involved in the cellobiose PTS of Escherichia coli (Reizer et al., 1990). The lacG gene codes for the phospho-β-galactosidase enzyme (54 kDa) that catalyzes the hydrolysis of lactose-6-phosphate into galactose-6-phosphate and glucose (De Vos and Gasson, 1989). The L. lactis phospho-β-galactosidase has been purified from an overexpressing E.coli strain (De Vos and Simons, 1988) and belongs to the superfamily of β-glycohydrolases (Hassouni et al. 1992). The tagatose-6-phosphate pathway enzymes were shown to be encoded by the lacABCD genes (Chapter 3). The first enzyme of the tagatose-6-phosphate pathway, the galactose-6-phosphate isomerase (LacAB), is encoded by the first two genes of the lac operon, the lacAB genes. Galactose-6- phosphate activities were only observed in E.coli cells overexpressing both the lacA and lacB genes, whereas no activity was found in cells expressing solely LacA (15 kDa) or LacB (19 kDa). The lacC and lacD genes encode the tagatose-6-phosphate kinase (33 kDa) and tagatose-1,6-diphosphate aldolase (36 kDa), respectively, as was evident form their enzyme activities in overexpressing E.coli cells. The deduced amino acid sequences of the lacABCD genes appeared to be strongly homologous to those of S. aureus and S . mutans (Jagusztyn- Krynicka etal. 1992). In addition, the L. lactis LacC sequence is homologous to the E. coli enzyme phosphofructosekinase B, that catalyzes the phosphorylation of tagatose-6- phosphate in the galactitol catabolic pathway. The function of the distal lacX gene, encoding a 34-kDa protein, is still unclear. No significant homology was found with other sequences in DNA or protein databases. However, the lacX gene seems not to be essential for lactose catabolism, since L. lactis strains in which transcription of lacX was prevented did not show significantly altered growth characteristics or phospho-β-galactosidase activities during growth on lactose (Simons et al., 1993). Northern-blot analysis showed that the lac genes are transcribed as two 6.0- and 8.0-kb polycistronic transcripts, of the lacABCDFE and lacABCDFEGX genes, respectively. An inverted repeat which is located between the lacE and lacG genes could function as the transcription termination site for the 6.0-kb transcript. In cells shifted from glucose to lactose, lac operon transcription was induced similarly as lactose enzyme activities (approximately 5-10 fold), indicating that the expression of the lac operon is regulated at the transcriptional level. The 3' end of the lacABCDFEGX operon appeared to be followed by an iso -IS S1 element ( Chapter 4 ). This element is flanked by 16-bp inverted repeats and contains a divergently transcribed gene ( orf1 ) encoding a putative transposase that is highly homologous to that of other iso -IS S1 elements. It remains to be determined whether this IS-element, or one of the other IS-elements that have been located on pMG820 (Fig. 1; Van Rooijen, unpublished results), are involved in the conjugal transfer of this or related lactose plasmids.

Transcription of the lacABCDFEGX operon was found to be regulated by the product of the divergently transcribed 0.8-kb lacR gene ( Chapter 5 ). The lacR gene was characterized by overexpression in E.coli and DNA sequencing and found to encode a 28- kDa protein. Northern-blot analysis showed that, in contrast to the lacABCDFEGX genes, the lacR gene is induced during growth on glucose. The deduced amino acid sequence of LacR appeared to be homologous to those of the E. coli DeoR, GutR, and FucR, S.aureus and S.mutans LacR, and Agrobacterium tumefaciens AccR repressors. None of these repressors belongs to one of the known LacI/GaIR or LysR repressor families. Since the DeoR repressor was the first repressor to be identified, this group of repressors was designated the E. coli DeoR family of repressors. Common characteristics of the members of the DeoR family are the presence of a helix-turn-helix motif near their N-termini and a conserved region near their C-termini, that for the L.lactis LacR repressor appeared to be involved in DNA and inducer binding, respectively (see below). In addition, all members have in common that expression of the catabolic operon they control is induced by a phosphorylated sugar, or a derivative thereof. The functionality of the lacR gene product as a repressor was demonstrated after introduction of multiple copies of the lacR gene in L. lactis strain MG5267, that contains a single chromosomal copy of the pMG820 lac operon. Whereas no effects were observed during growth on glucose, significant decreased growth rates and lac operon activities were observed during growth on lactose, indicating that lacR specifically represses expression of the lac operon.

Characterization of the lac promoter and modulation of promoter activity by the lacR gene product is presented in Chapter 6 . The transcription initiation site of the lac promoter was determined by primer extension mapping. The lac promoter canonical -35 and -10 sequences correspond closely to those described for gram-positive bacteria and are located in a back-to-back configuration with those of the divergently orientated lacR promoter (Fig. 1). The effects on lac promoter activity of flanking sequences and the lacR gene were studied in L. lactis and E. coli by using transcriptional fusions with a promoterless chloramphenicol acetyltransferase ( cat -86) gene. In the presence of the lacR gene both in L. lactis and E. coli, significantly decreased CAT activities were observed, indicating that the lacR gene product represses lac promoter activity. In addition, to obtain inducible CAT-activities a lac promoter fragment of at least 0.5 kb was required, suggesting that regions flanking the promoter are involved in regulation. These studies also showed that sequences flanking the lac promoter significantly contribute to the promoter efficiency in L. lactis. Enhancement of promoter activity in L. lactis of up to 38-fold was observed.

The interaction between the LacR repressor and the lac promoter region is described in Chapter 7 . For this purpose, LacR was overexpressed in E. coli and purified in a three-step procedure. Cross-linking studies with glutaraldehyde showed the ability of LacR to generate dimers. Gel-mobility shift assays and DNase I footprinting studies demonstrated the presence of two LacR-binding sites, lacO1 and lacO2, in the intercistronic region between the lacA and lacR genes (Fig. 1). The lacO1 operator is located at positions -31 to +6 and -96 to -59 relative to the transcription initiation sites of the lac operon and lacR gene, respectively. The distances between lacO1 and transcription initiation sites of the lac operon and lacR gene are comparable to those often observed for repressor and activator binding sites, respectively, as is illustrated in Fig. 2. The lacO2 operator is located at positions -313 to -278 and +188 to +223 relative to the transcription initiation sites of the lac operon and the lacR gene, respectively. Since a TGTTT sequence is present as an inverted repeat in lacO1 and as a direct repeat in lacO2, we proposed that the TGTTT box comprises the LacR recognition sequence. Titration experiments with purified LacR and DNA-fragments containing lacO1, lac02, or both (lacO1O2) showed that lacO1 and (lacO1O2) have a three-fold higher affinity than lacO2, for LacR binding. This indicated that the presence of lac02 in cis does not significantly enhance binding of LacR to lacO1. To identify the metabolite that induces lac operon expression during growth on lactose, gel mobility shift assays were carried out with the LacR repressor and (lacO1O2) in the presence of various phosphorylated monosaccharide intermediates from the tagatose-6-phosphate and glycolytic pathways. Dissociation of the LacR -lacO1O2 complex was observed only in the presence of tagatose-6- phosphate, which is an intermediate of the tagatose-6-phosphate pathway. No dissociation was observed with galactose-6-phosphate, tagatose-1,6-diphosphate, glucose-6- phosphate, fructose-6-phosphate and fructose- 1,6-diphosphate. Therefore, it was concluded that tagatose-6-phosphate is the physiological inducer of lac operon expression. This is supported by the observation that lac operon expression is also induced during growth on galactose, that is transported via a galactose-PTS and is metabolized through the tagatose-6-phosphate pathway.

In order to study whether the LacR repressor is the only determinant in the control of lac operon expression and to develop an expression system in L.lactis that allowed screening of mutated lacR genes, the chromosomally located lacR gene of strain MG5267 was deleted by replacement recombination ( Chapter 8 ). The resulting strain was designated L.lactis NZ3015. As expected, determination of phospho-β-galactosidase (LacG) and lactose phosphotransferase (LacEF) activities, and lac mRNA levels of lactose- and glucose-grown NZ3015 cells showed that expression of the lac operon was significantly derepressed in the glucose-grown cells. However, approximately one fifth of the wild-type regulation level remained, as was demonstrated by the 1.6-fold (average) higher lac operon activities during growth on lactose than on glucose. This indicates that an additional control circuit is involved in the regulation of the lac operon. Since the RNA- studies demonstrated that this regulatory circuit mediates lac operon expression at the transcriptional level, we searched for DNA sequences in the lac promoter region that were homologous to a putative glucose-responsive-element (GRE) from Bacillus. Five basepairs downstream of the lacO1 operator a sequence was detected that showed strong homology to the Bacillus GRE sequence. The L. lactis GRE sequence was also found in the promoter region of the S.aureus lac operon, that is strongly homologous to that of L. lactis.

The last two Chapters 9 and 10 present the identification of amino acids in the L. lactis LacR repressor that are involved in the inducer response and binding to DNA, respectively. This was realized by studying the effects on the regulation of lac operon expression in the LacR-deficient strain NZ3015 and wild-type strain MG5267, after introduction of mutated lacR genes. Since LacR belongs to the E.coli DeoR family of repressors, in which all members have in common that their inducer is a phosphorylated sugar, it was anticipated that within this family there will be conserved amino acid residues that are involved the response to the inducer tagatose-6-phosphate. Various amino acid residues in LacR that are conserved in other DeoR family members and located outside the DNA-binding motif, were replaced by alanine or arginine. Cells of strain NZ3015 containing K72A-, K80A-, D210A-, or K213A-LacR, were unable to derepress phospho-β-galactosidase activities during growth on lactose. These low phospho-β-galactosidase activities resulted in significantly decreased growth rates on lactose, and strongly suggested that these LacR mutant proteins had lost their ability to respond to inducer. This hypothesis was verified by carrying out gel mobility shift assays with lacO1O2 operator and purified K72A-, K80A-, D210A-, and K213A-LacR proteins in the presence or absence of the inducer tagatose-6-phosphate. None of the complexes between the lacO1O2 and the mutant proteins was affected by tagatose-6-phosphate, whereas the complex between lacO1O2 and wild-type LacR dissociated in the presence of tagatose-6-phosphate. From these experiments it was concluded that Lys-72, Lys-80, Asp- 210, and Lys-213 are involved in the inducer response of the LacR repressor. It is not yet clear whether these residues are involved in the actual binding of tagatose-6-phosphate or, upon binding, the allosteric transition of LacR into a molecule with a decreased affinity for lacO1O2. In addition, these results confirm that in vivo tagatose-6-phosphate is the inducer of the L.lactis lac operon.

To identify the residues in LacR involved in DNA-binding, amino acid residues in the putative N-terminal DNA-binding domain, that contains a helix-turn-helix motif, were replaced by alanine. The LacR mutants M34A and R38A showed a 10- and 25-fold decrease of the in vivo DNA-binding constant, indicating that Met-34 and Arg-38 are involved in DNA-binding. Two LacR mutants, D30A and D33A, were constructed with a 4-fold increased DNA-binding constant, indicating that it is possible to improve the relatively weak binding of LacR to its operator. Based on the similarities between the LacR repressor and the lacO1 operator and the E.coli LacI repressor variant 44 and its corresponding operator, a model for the binding of LacR to the lacO1 operator was presented.

Based on the studies presented in this thesis a model for the action of the LacR repressor in the regulation of the L.lactis lac operon is proposed. Below, three stages of the model will be discussed.

1. Binding of LacR repressor to operator lacO1 during growth on glucose results in autoactivation of lacR gene expression . The induction of lacR on glucose and the high affinity of the LacR repressor for lacO1 are evident from the Northern-studies (Chapter 5) and gel mobility shift titration experiments (Chapter 7), respectively. The distance between location of lacO1 and the lacR transcription initiation site coincides with the distance that is commonly observed for an activator (Fig. 2). The involvement of lacO1 in the regulation of lacR is supported by the observation that partial deletion of lacO1 resulted in the loss of lacR regulation (Chapter 9, Fig. 3). However, no experimental data have been generated to establish the direct involvement of LacR in activating expression of its own gene. Since the transcriptional fusion studies (Chapter 6) showed that lacO1 alone is incapable of regulating CAT-expression, we presume that no repression of lac operon expression occurs at this stage.

2. Binding of LacR repressor to lacO2 at increasing LacR concentrations during growth on glucose results in repression of lacR gene and lac operon expression . Since it has been shown that lacO2 has a lower affinity for LacR than lacO1 (Chapter 7), lacO2 will only be bound at increasing LacR concentrations. The CAT-reporter studies showed that both lacO1 and lacO2 are required for repression of CAT-activity during growth on glucose (Chapter 6). Therefore, repression of transcription initiation of lac operon occurs when LacR is bound to both lacO1 and lacO2 . The exact repression mechanism has not been elucidated, but might include the formation of a DNA loop between lacO1 and lacO2 , as has been described for other regulatory systems (Matthews, 1992). The postulated repression of lacR expression upon binding of LacR to lacO2 , would prevent the cell from overproduction of LacR due to continuous activation by lacO1 and results in a certain steady state concentration of LacR. However, no experiments have been carried out to establish the role of lacO2 , in the putative autoregulation of lacR .

3. Binding of LacR repressor to tagatose-6-phosphate during growth on lactose results in dissociation of the LacR-operator complex concomitant with the induction of lac operon expression . From the gel mobility shift studies in Chapter 7 it is evident that the LacR- lacO1O2 complex dissociates in the presence of tagatose-6-phosphate, that is an intermediate of the tagatose-6-phosphate pathway. In addition, LacR mutants were constructed, the presence of which in L. lactis resulted in an inability to induce lac operon activity on lactose, that had lost their sensitivity to tagatose-6-phosphate. Therefore, the complex between LacR and tagatose-6-phosphate that is formed during growth on lactose does not bind to the lac operators, resulting in the restoration of transcription initiation from the lac promoter. As a result of the absence of LacR bound to lacO1 the lacR gene is probably no longer (auto)activated resulting in a decreased level of lacR expression. The presence of multiple copies of constitutively expressed lacR results in an additional repression of lac promoter activity during growth on both glucose and lactose (Chapters 5 and 6), suggesting that due to the overproduction of LacR relatively more lacO2 , is bound by LacR. Due to the limited amount of inducer (Chapter 8), it would under these conditions then be theoretically possible that, in contrast to the situation in wild-type cells, lacR expression is induced during growth on lactose. This might be a consequence of the dissociation of only the lacO2 -LacR complex in these cells during growth on lactose. In contrast, in wild-type cells, where the LacR concentration is lower, LacR dissociates from both operators during growth on lactose.

The studies described in this thesis have provided more insight in the genetic basis and regulation of lactose catabolism in L. lactis . Parts of this knowledge have already been used for the development of a food-grade selection system for L. lactis based on the lacF gene (De Vos, 1988). In addition, the lac promoter has already been successfully used for the expression of mutated nisZ genes in L. lactis (Kuipers et al. , 1992). Since L. lactis preferentially metabolizes glucose it should be possible, by the starting the fermentation with a certain amount of glucose, to overexpress genes of interest under control of the lac promoter at a defined stage in a dairy fermentation.