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

<p>An important trait of the lactic acid bacterium <em>Lactococcus lactis</em> , 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 <sup>lac</SUP>), have been well established (Fig. 1). However, except for the molecular cloning and characterization of the plasmid-located phospho-B-galactosidase gene (Boizet <em>et al.</em> , 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 <em>L. lactis.</em> In addition, since high lactose-specific enzyme activities are observed during growth on lactose, which are repressed during growth on glucose, expression of the <em>lac</em> 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 <em>Lactococci</em> . Isolation and elucidation of the mechanism of control of the <em>lac</em> 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 <em>L. lactis</em> subsp. <em>lactis.</em> In addition, several <em>cis</em> - and <em>trans</em> -acting factors that are involved in the regulation of their expression were identified.<p>In <strong>Chapter 1</strong> 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.<p>The characterization of the genetic determinants for lactose metabolism, including the PEP-PTS <sup>lac</SUP>(LacEF), phospho-β-galactosidase (LacG) and tagatose-6-phosphate pathway enzymes (LacABCD), is presented in <strong>Chapters 2</strong> and <strong>3</strong> . The <em>lac</em> genes of the <em>L. lactis</em> subsp. <em>lactis</em> 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 <em>lacABCDFEGX</em> (Fig. 1). The <em>lacE</em> and <em>lacF</em> genes encode the PEP-PTS <sup>lac</SUP>proteins Enzyme II <sup>lac</SUP>(62 kDa) and Enzyme III <sup>lac</SUP>(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 <sup>lac</SUP>is active as a trimer. The identity of the <em>lacF</em> gene was confirmed by complementation of <em>lacF</em> deficiency in <em>L. lactis</em> 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 <em>L.</em> lactis lacE and lacF genes and those of <em>Lactobacillus c</em> a <em>sei</em> and <em>Staphylococcus aureus</em> . In addition, the deduced <em>L. lactis</em> LacE and LacF amino acid sequences were homologous to those of CelA, CelB and CelC that are involved in the cellobiose PTS of <em>Escherichia coli</em> (Reizer <em>et al.,</em> 1990). The <em>lacG</em> 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 <em>L. lactis</em> phospho-β-galactosidase has been purified from an overexpressing <em>E.coli</em> strain (De Vos and Simons, 1988) and belongs to the superfamily of β-glycohydrolases (Hassouni <em>et al.</em> 1992). The tagatose-6-phosphate pathway enzymes were shown to be encoded by the <em>lacABCD</em> 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 <em>lac</em> operon, the <em>lacAB</em> genes. Galactose-6- phosphate activities were only observed in <em>E.coli</em> cells overexpressing both the <em>lacA</em> and <em>lacB</em> genes, whereas no activity was found in cells expressing solely LacA (15 kDa) or LacB (19 kDa). The <em>lacC</em> and <em>lacD</em> 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 <em>E.coli</em> cells. The deduced amino acid sequences of the <em>lacABCD</em> genes appeared to be strongly homologous to those of <em>S. aureus</em> and <em>S</em> . <em>mutans</em> (Jagusztyn- Krynicka <em>et</em><em>al.</em> 1992). In addition, the <em>L. lactis</em> LacC sequence is homologous to the <em>E. coli</em> enzyme phosphofructosekinase B, that catalyzes the phosphorylation of tagatose-6- phosphate in the galactitol catabolic pathway. The function of the distal <em>lacX</em> gene, encoding a 34-kDa protein, is still unclear. No significant homology was found with other sequences in DNA or protein databases. However, the <em>lacX</em> gene seems not to be essential for lactose catabolism, since <em>L. lactis</em> strains in which transcription of <em>lacX</em> was prevented did not show significantly altered growth characteristics or phospho-β-galactosidase activities during growth on lactose (Simons <em>et al.,</em> 1993). Northern-blot analysis showed that the <em>lac</em> genes are transcribed as two 6.0- and 8.0-kb polycistronic transcripts, of the <em>lacABCDFE</em> and <em>lacABCDFEGX</em> genes, respectively. An inverted repeat which is located between the <em>lacE</em> and <em>lacG</em> genes could function as the transcription termination site for the 6.0-kb transcript. In cells shifted from glucose to lactose, <em>lac</em> operon transcription was induced similarly as lactose enzyme activities (approximately 5-10 fold), indicating that the expression of the <em>lac</em> operon is regulated at the transcriptional level. The 3' end of the <em>lacABCDFEGX</em> operon appeared to be followed by an <em>iso</em> -IS <em>S1</em> element ( <strong>Chapter 4</strong> ). This element is flanked by 16-bp inverted repeats and contains a divergently transcribed gene ( <em>orf1</em> ) encoding a putative transposase that is highly homologous to that of other <em>iso</em> -IS <em>S1</em> 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.<p>Transcription of the <em>lacABCDFEGX</em> operon was found to be regulated by the product of the divergently transcribed 0.8-kb <em>lacR</em> gene ( <strong>Chapter 5</strong> ). The <em>lacR</em> gene was characterized by overexpression in <em>E.coli</em> and DNA sequencing and found to encode a 28- kDa protein. Northern-blot analysis showed that, in contrast to the <em>lacABCDFEGX</em> genes, the <em>lacR</em> gene is induced during growth on glucose. The deduced amino acid sequence of LacR appeared to be homologous to those of the <em>E. coli</em> DeoR, GutR, and FucR, <em>S.aureus</em> and <em>S.mutans</em> LacR, and <em>Agrobacterium tumefaciens</em> 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 <em>E. coli</em> 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 <em>L.lactis</em> 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 <em>lacR</em> gene product as a repressor was demonstrated after introduction of multiple copies of the <em>lacR</em> gene in <em>L. lactis</em> strain MG5267, that contains a single chromosomal copy of the pMG820 <em>lac</em> operon. Whereas no effects were observed during growth on glucose, significant decreased growth rates and <em>lac</em> operon activities were observed during growth on lactose, indicating that <em>lacR</em> specifically represses expression of the <em>lac</em> operon.<p>Characterization of the <em>lac</em> promoter and modulation of promoter activity by the <em>lacR</em> gene product is presented in <strong>Chapter 6</strong> . The transcription initiation site of the <em>lac</em> promoter was determined by primer extension mapping. The <em>lac</em> 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 <em>lacR</em> promoter (Fig. 1). The effects on <em>lac</em> promoter activity of flanking sequences and the <em>lacR</em> gene were studied in <em>L. lactis</em> and <em>E. coli</em> by using transcriptional fusions with a promoterless chloramphenicol acetyltransferase ( <em>cat</em> -86) gene. In the presence of the <em>lacR</em> gene both in <em>L. lactis</em> and <em>E. coli,</em> significantly decreased CAT activities were observed, indicating that the <em>lacR</em> gene product represses <em>lac</em> promoter activity. In addition, to obtain inducible CAT-activities a <em>lac</em> 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 <em>lac</em> promoter significantly contribute to the promoter efficiency in <em>L. lactis.</em> Enhancement of promoter activity in <em>L. lactis</em> of up to 38-fold was observed.<p><img src="/wda/abstracts/i1628_1.gif" height="757" width="600"/><p>The interaction between the LacR repressor and the <em>lac</em> promoter region is described in <strong>Chapter 7</strong> . For this purpose, LacR was overexpressed in <em>E. coli</em> 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, <em>lacO1</em> and <em>lacO2,</em> in the intercistronic region between the <em>lacA</em> and <em>lacR</em> genes (Fig. 1). The <em>lacO1</em> operator is located at positions -31 to +6 and -96 to -59 relative to the transcription initiation sites of the <em>lac</em> operon and <em>lacR</em> gene, respectively. The distances between <em>lacO1</em> and transcription initiation sites of the <em>lac</em> operon and <em>lacR</em> gene are comparable to those often observed for repressor and activator binding sites, respectively, as is illustrated in Fig. 2. The <em>lacO2</em> operator is located at positions -313 to -278 and +188 to +223 relative to the transcription initiation sites of the <em>lac</em> operon and the <em>lacR</em> gene, respectively. Since a TGTTT sequence is present as an inverted repeat in <em>lacO1</em> and as a direct repeat in <em>lacO2,</em> we proposed that the TGTTT box comprises the LacR recognition sequence. Titration experiments with purified LacR and DNA-fragments containing <em>lacO1, lac02,</em> or both <em>(lacO1O2)</em> showed that <em>lacO1</em> and <em>(lacO1O2)</em> have a three-fold higher affinity than <em>lacO2,</em> for LacR binding. This indicated that the presence of <em>lac02 in cis</em> does not significantly enhance binding <em>of</em> LacR to <em>lacO1. To</em> identify the metabolite that induces <em>lac</em> operon expression during growth on lactose, gel mobility shift assays were carried out with the LacR repressor and <em>(lacO1O2)</em> in the presence <em>of</em> various phosphorylated monosaccharide intermediates from the tagatose-6-phosphate and glycolytic pathways. Dissociation of the LacR <em>-lacO1O2</em> complex was observed only in the presence of tagatose-6- phosphate, which is an intermediate of <em></em> 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 <em>lac</em> 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.<p>In order to study whether the LacR repressor is the only determinant in the control <em>of lac</em> operon expression and to develop an expression system in <em>L.lactis</em> that allowed screening <em>of</em> mutated <em>lacR</em> genes, the chromosomally located <em>lacR</em> gene <em>of</em> strain MG5267 was deleted by replacement recombination ( <strong>Chapter 8</strong> ). The resulting strain was designated <em>L.lactis</em> NZ3015. As expected, determination <em>of</em> phospho-β-galactosidase (LacG) and lactose phosphotransferase (LacEF) activities, and <em>lac</em> mRNA levels of <em></em> lactose- and glucose-grown NZ3015 cells showed that expression of <em></em> the <em>lac</em> operon was significantly derepressed in the glucose-grown cells. However, approximately one fifth <em>of</em> the wild-type regulation level remained, as was demonstrated by the 1.6-fold (average) higher <em>lac</em> operon activities during growth on lactose than on glucose. This indicates that an additional control circuit is involved in the regulation of the <em>lac</em> operon. Since the RNA- studies demonstrated that this regulatory circuit mediates <em>lac</em> operon expression at the transcriptional level, we searched for DNA sequences in the <em>lac</em> promoter region that were homologous to a putative glucose-responsive-element (GRE) from <em>Bacillus.</em> Five basepairs downstream of the <em>lacO1</em> operator a sequence was detected that showed strong homology to the <em>Bacillus</em> GRE sequence. The <em>L. lactis</em> GRE sequence was also found in the promoter region of the <em>S.aureus lac</em> operon, that is strongly homologous to that <em>of L. lactis.</em><p><img src="/wda/abstracts/i1628_2.gif" height="553" width="600"/><p>The last two <strong>Chapters 9</strong> and <strong>10</strong> present the identification <em>of</em> amino acids in the <em>L. lactis</em> 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 <em>lac</em> operon expression in the LacR-deficient strain NZ3015 and wild-type strain MG5267, after introduction of mutated <em>lacR</em> genes. Since LacR belongs to the <em>E.coli</em> 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 <em>lacO1O2</em> 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 <em>lacO1O2</em> and the mutant proteins was affected by tagatose-6-phosphate, whereas the complex between <em>lacO1O2</em> 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 <em>in vivo</em> tagatose-6-phosphate is the inducer of the L.lactis lac operon.<p>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 <em>in</em> 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 <em>lacO1</em> operator and the <em>E.coli</em> LacI repressor variant 44 and its corresponding operator, a model for the binding of LacR to the <em>lacO1</em> operator was presented.<p>Based on the studies presented in this thesis a model for the action of the LacR repressor in the regulation of the <em>L.lactis lac</em> operon is proposed. Below, three stages of the model will be discussed. <strong></strong><p><strong>1. Binding of LacR repressor to operator <em>lacO1</em> during growth on glucose results in autoactivation of <em>lacR</em> gene expression</strong> . The induction of <em>lacR</em> on glucose and the high affinity of the LacR repressor for <em>lacO1</em> are evident from the Northern-studies (Chapter 5) and gel mobility shift titration experiments (Chapter 7), respectively. The distance between location of <em>lacO1</em> and the <em>lacR</em> transcription initiation site coincides with the distance that is commonly observed for an activator (Fig. 2). The involvement of <em>lacO1</em> in the regulation of <em>lacR</em> is supported by the observation that partial deletion of <em>lacO1</em> resulted in the loss of <em>lacR</em> regulation (Chapter 9, Fig. 3). However, no experimental data have been generated to establish the <u>direct</u> involvement of LacR in activating expression of its own gene. Since the transcriptional fusion studies (Chapter 6) showed that <em>lacO1</em> alone is incapable of regulating CAT-expression, we presume that no repression of <em>lac</em> operon expression occurs at this stage.<p><strong>2. Binding of <em></em> LacR repressor to <em>lacO2</em> at incr