WO2003076463A2 - Listeria monocytogenes ctsr mutants presenting low virulence and stress-resistance - Google Patents

Abstract

The present invention relates to Gram positive bacteria with increased stress tolerance, reduced virulence, and/or reduced mobility, as a result of an altered CtsR protein. The alteration of the CtsR protein in particular concerns an alteration of the conserved glycine-rich region in the CtsR protein. The invention further relates to nucleic acid encoding the altered CtsR proteins. The altered Gram positive bacteria may be used in fermentation processes and may be used as delivery vehicles for site specific delivery of therapeutic proteins such as vaccines.

Description

Stress-resistant, low virulence Gram positive bacteria having an altered ctsR gene

Field of the invention The present invention relates to Gram positive bacteria having an alteration in the gene coding for CtsR that results in increased stress-resistance and, in case of pathogenic Gram positive bacteria, reduced virulence. The invention relates to nucleotide sequences encoding the altered CtsR protein and to nucleic acid constructs comprising such nucleotide sequences. The invention further relates to the use of the altered bacteria of the invention in fermentation processes, their use as probiotics and their use as delivery vehicle for therapeutic proteins, including their use as live oral vaccines.

Background of the invention Listeria monocytogenes is a Gram positive facultative anaerobic pathogenic bacterium that can be present in a variety of foods from animal or plant origin. It can cause a severe food-borne illness called listeriosis, due to the ability of this organism to invade and multiply within the host cells. Since this disease is predominantly acquired after consumption of contaminated foods, effective elimination of L. monocytogenes from food products is essential.

Inactivation of bacteria by High Hydrostatic Pressure (HHP) treatment is a relatively novel preservation technique (Rnorr et al, 1998; Smelt, 1998). HHP has detrimental effects on cellular processes, resulting from inhibition of protein and DNA synthesis, perturbation of membranes and membrane-associated processes, and disruption of macromolecular quaternary structures (e.g. protein denaturation)

(Yayanos and Pollard, 1969; Cheftel, 1995; Palou et al., 1999). In general, bacterial growth is inhibited at pressures above 20 MPa, while growth halts at higher pressures. This is a consequence of cessation of DNA-, protein- and RNA-synthesis, which was shown to occur at 50, 58, and 77 MPa, respectively, in Escherichia coli. Cell death of non-piezophilic vegetative bacteria normally occurs at pressures above 200 MPa (Yayanos and Pollard, 1969).

Typical pressures used to inactivate vegetative bacterial cells range from 300 to 700 MPa (Metrick et al., 1989; Patterson et al., 1995; Smelt 1998; Palou et al., 1999). 0178

2 These pressures are applied using an abrupt pressure upshift, and do not allow for bacterial growth and adaptation. Several authors have reported survival of a fraction of a bacterial population upon exposure to hydrostatic pressures that are normally lethal, but it is not known what mechanisms underlie this increased tolerance (Metrick et al., 1989; Hauben et al., 1997).

Some of the factors involved in increased stress tolerance in bacteria have however been identified. E.g. the Class III stress gene Regulator (CtsR) gene encodes a protein that is a negative regulator of the Class III heat shock genes. The CtsR protein acts as a dimer, while the McsA or McsB proteins are believed to play a role in its activity (Derre et al., 2000; Kriiger et al, 2001). CtsR contains domains that are highly conserved amongst low G + C containing Gram positive bacteria, namely, a presumed dimerisation domain and a highly conserved Helix Turn Helix (HTH) domain in the N- terminal region. The central region contains a conserved glycine-rich domain, while the C-terminus is less well conserved (Derre et al., 2000). CtsR is generally the product of the first gene of the clpC operon, that furthermore comprises genes mcsA and mcsB, and clpC (Nair et al, 2000; Kriiger et al, 2001). CtsR negatively controls expression of the Class III heat shock genes clpP, clpE, and of the clpC operon by binding specifically to a direct heptanucleotide repeat in their promoter regions (Derre et al., 1999; Nair et al., 2000). Thereby, it has an autoregulatory function. ClpC and ClpE have ATPase activity and belong to the 100 kDa heat shock protein (HSP100) Clp family of highly conserved molecular chaperones (Schirmer et al., 1996), and the serine protease ClpP is a proteolytic subunit. Clp ATPases regulate ATP-dependent proteolysis and also play a role as molecular chaperones in protein folding and assembly (Wawrzynow et al, 1996). CtsR and Class III heat shock genes are involved in one of the three classes of known heat shock regulatory mechanisms (Class IN is less well defined), and are highly conserved among Gram positive bacteria (Derre et al., 1999, 2000).

In L. monocytogenes, the ClpC and ClpP proteins have been shown to play an important role in adaptive responses, including motility, growth at high temperature and stress tolerance (Kriiger et al., 1994; Msadek et al., 1994, 1998). The majority of the virulence genes in L. monocytogenes are regulated by the pleiotropic regulator PrfA (Cossart and Lecuit, 1998; Kreft and Nazquez-Boland, 2001). Furthermore, the Clp ATPases are required for stress survival and intracellular growth (Rouquette et al., 1996; Gaillot et al., 2000; Nair et al., 2000). In addition, a PrfA-independent virulence protein, SvpA (surface virulence-associated protein), that is controlled by MecA, ClpP, and ClpC, was recently identified (Borezee et al., 2001). There is evidence that there is crosstalk between PrfA and Class III heat shock genes (Ripio et al., 1998), but so far, it is not clear how these interactions take place.

Further insight into these interactions would be required for the isolation and/or construction of defined genetically altered Gram positive bacteria with increased stress tolerance and/or reduced virulence. It is an object of the present invention to provide for such defined genetically altered Gram positive bacteria, as well as for methods for their isolation and/or construction. The thus obtained Gram positive bacteria may be used in fermentation processes, as probiotics, and as delivery vehicle for therapeutic proteins, including their use as live oral vaccines.

Description of the invention The present invention is based on the surprising discovery that in a spontaneous

High Hydrostatic Pressure (HPP) tolerant, i.e. piezotolerant, mutant oϊListeria monocytogenes strain Scott A, named AKOl, several of the phenotypic characteristics of this mutant, such as high piezotolerance, immobility, and reduced virulence, are caused by a mutation in the highly conserved glycine-rich region of CtsR. L. monocytogenes strain AKOl was isolated from a wild type L. monocytogenes Scott A population upon HHP treatment. The AKOl strain showed a -1000-fold higher viability than the wild type upon exposure to 350 MPa. Furthermore, it showed altered morphological characteristics, such as elongation of cells and lack of flagella, and increased resistance to heat, acid, and hydrogen peroxide. Genetic analysis demonstrated that the altered phenotype of the AKOl mutant was the result of a single amino acid deletion in the highly conserved glycine-rich region of CtsR. Definitions A CtsR protein

A CtsR protein is herein defined as a protein having an amino acid sequence identity of at least 30, 33 or 36 % with SEQ ID NO.1 or SEQ ID NO.2. A wild type CtsR protein is (obtainable) from a Gram positive bacterium, in particularly from a Gram positive bacterium having a low genomic G + C content. The CtsR protein is a negative regulator of the Class III heat shock genes, and acts as a di er. The CtsR protein contains highly conserved domains: a presumed dimerisation domain and a highly conserved Helix Turn Helix (HTH) domain in the N-terminal region. The central region contains a conserved glycine-rich domain, while the C-terminus is less well conserved. The CtsR protein is usually the product of the first gene of the clpC operon, that furthermore comprises the genes mcsA and mcsB, and clpC. CtsR negatively controls expression of the Class III heat shock genes clpP, clpE, and of the clpC operon by binding specifically to a direct heptanucleotide repeat (A/GGTCAAA NAN A/GGTCAAA) in their promoter regions.

CtsR proteins from species other than L. monocytogenes may thus be defined by having an amino acid sequence identity of at least 30, 33 or 36 % with SEQ ID NO.1 , more preferably, the CtsR proteins has an amino acid sequence identity with SEQ ID NO. 1 of at least 40, 50, 60, 70 or 80%. Preferred CtsR proteins have an amino acid identity of at least 40, 50, 60, 70 or 80% with SEQ ID NO.2. The amino acid identity between a polypeptide or protein comprised in the term "CtsR protein" and SEQ ID NO. 1 or SEQ ID NO.2 may be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Infomatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SLAM J. Applied Math., 48:1073, 1988. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1):387 (1984), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al, J. Mol. Biol. 215:403-410 (1990)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NTH Bethesda, MD 20894; Altschul, S, et al, J. Mol. Biol. 215:403-410 (1990)). The well-known Smith Waterman algorithm may also be used to determine identity. Preferred parameters for polypeptide sequence comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, WI. The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps). Homologous

The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide may be operably linked to another promoter sequence or, if applicable, another secretory signal sequence and or terminator sequence than in its natural environment.

When used to indicate the relatedness of to nucleic acid sequences the term "homologous" means that one single-stranded nucleic acid sequence may hybridise to a complementary single-stranded nucleic acid sequence. The degree of hybridisation may depend on a number of factors including the amount of identity between the sequences and the hybridisation conditions such as temperature and salt concentration as generally known to the skilled person. Preferably the region of identity is greater than about 5 bp, more preferably the region of identity is greater than 10 bp. Promoter

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most environmental and physiological conditions. An "inducible" promoter is a promoter that is active only under specific environmental or physiological conditions. Operably linked

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.

Detailed description of the invention In a first aspect, the invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding an altered CtsR protein of a Gram positive bacterium. The altered CtsR protein is altered in that it has an amino acid sequence with an alteration in the conserved glycine-rich region that corresponds to amino acid positions 61 to 64 of SEQ ID NO.l with respect to the glycine-rich region of a corresponding wild type CtsR protein. Furthermore, the alteration confers to the altered CtsR protein the ability to increase stress tolerance, to reduce virulence and/or to reduce mobility of a Gram positive bacterium in which the altered CtsR protein is expressed, preferably as sole CtsR protein. A wild type CtsR protein is herein preferably defined as described above.

The conserved glycine-rich region in the CtsR protein is herein understood to mean the region that corresponds to amino acid positions 61 to 64 of SEQ LD NO.l (i.e. the amino acid sequence of the L. monocytogenes strain EGD CtsR protein, Genbank Ace. No. NC_03210, NP_463760.1). The corresponding conserved glycine-rich regions in CtsR proteins of Gram-positive bacteria other than L. monocytogenes may easily be identified by alignment of the particular CtsR amino acid sequence with that of SEQ ID NO. 1 (L. monocytogenes) as described above. Generally, the glycine-rich region in CtsR proteins of Gram positive bacteria consists of four consecutive glycine residues that are usually present between amino acid positions 60 and 70 of the CtsR protein and that are usually directly preceded by two basic amino acids. The glycine-rich region of the CtsR protein of Lactobacillus plantarum strain WCFS1 (SEQ ID NO.2) e.g. is present at amino acid positions 69 to 72 and is directly preceded by a lysine and an arginine residu at positions 67 and 68, respectively.

The ability to increase stress tolerance preferably includes the ability to increase tolerance of at least one of the following stresses: high hydrostatic pressure, heat, acidic conditions (low pH), hydrogen peroxide and other oxygen radical generating agents, high osmolarity, such as e.g. high ionic strength, reduced water activity or drying such as spray drying of e.g. starter cultures. Preferably, the increased tolerance of high hydrostatic pressure means that a treatment of the bacterium at 250 MPa for 20 minutes at 20°C produces at least a 1 log lower reduction of viable counts as compared to the corresponding wild type bacterium, more preferably, this treatment results in a log reduction of viable counts of less than 3.4, 3.0, 2.5, 2.0, or 1.5, or a treatment of the bacterium at 300 MPa for 20 minutes at 20°C results in a log reduction of viable counts of less than 4.4, 4.0, 3.5, 2.5, or 2.0. Preferably, the increased tolerance of heat means that a treatment of the bacterium at 55°C for 20 minutes produces at least a 1 log lower reduction of viable counts as compared to the corresponding wild type bacterium, more preferably this treatment results in a log reduction of viable counts of less than 3.7, 3.5, 3.0, 2.5, 2.0 or 1.5. Preferably, the increased tolerance of acidic conditions means that a treatment of the bacterium at pH 2.5 for 45 minutes produces at least a 1 log lower reduction of viable counts as compared to the corresponding wild type bacterium. Preferably, the increased tolerance of oxygen radical generating agents means that exposure of the bacterium to 0.2%o (w/v) hydrogen peroxide for 40 minutes produces at least a 1 log lower reduction of viable counts as compared to the corresponding wild type bacterium, more preferably, this exposure results in a log reduction of viable counts of less than 4.5, 4.0, 3.5, 2.5, or 2.0. Preferably, the increased tolerance of high osmolarity means that the bacterium growth in medium containing 12.5% (w/v) NaCl at a rate that is at least 1.1 times the growth rate of the corresponding wild type bacterium. Similarly, preferably, the increased tolerance of reduced water activity means that the bacterium growth in medium having a water activity lower than 0.97 at a rate that is at least 1.1 times the growth rate of the corresponding wild type bacterium. Preferably, the increased tolerance of drying means that (spray) drying of the bacterium produces at least a 1 log lower reduction of viable counts as compared to the corresponding wild type bacterium, preferably there is at least a 2, 3, 4, or 5 log lower viable count as compared to the corresponding wild type bacterium. The ability to reduce virulence means that there is at least a 1 log lower viable count in organs or tissues of an infected test animal as compared to the corresponding wild type bacterium, preferably there is at least a 2, 3, 4, or 5 log lower viable count as compared to the corresponding wild type bacterium and most preferably there is no detectable viable count in organs or tissues of an infected test animal. The ability to reduce mobility means that the bacterium is incapable of producing a diffuse red cloudy pattern in Motility Test medium as described in the Examples herein.

In the nucleic acid molecule of the invention, the alteration in the conserved glycine-rich region of the encoded protein preferably consists of a deletion or an insertion of one or more glycines. Thus, one, two, three or four glycines may be deleted or one, two, three or four glycines may be inserted in (or added to) the conserved glycine-rich region. Most preferably, however, a single glycine residue is deleted from or inserted into the altered conserved glycine-rich region of the CtsR protein, such that the total number of glycine residues of the altered glycine-rich region is either 3 or 5. Alternatively, or in addition to the above insertions and deletions of glycine residues, the alteration of the conserved glycine-rich region may comprise one or more replacements of the (remaining) glycine codons in the altered glycine-rich region. Preferably, the alteration in the conserved glycine-rich region comprises a replacement of one or more glycines by an amino acid other than serine or glycine. Preferably, one or more glycines of the conserved glycine-rich region are replaced by amino acids having aliphatic side chains such as alanine, valine, leucine, and isoleucine. Preferably, one or more glycines of the conserved glycine-rich region are replaced by amino acids having amide-containing side chains such as asparagine and glutamine. Preferably, one or more glycines of the conserved glycine-rich region are replaced by amino acids having aromatic side chains such as phenylalanine, tyrosine, and tryptophan.

Preferably, one or more glycines of the conserved glycine-rich region are replaced by amino acids having basic side chains such as lysine, arginine, and histidine. Preferably, one or more glycines of the conserved glycine-rich region are replaced by amino acids having acidic side chains such as glutamic acid and aspartic acid. Preferably, one or more glycines of the conserved glycine-rich region are replaced by amino acids having sulphur-containing side chains such as cysteine and methionine. Preferably, one or more glycines of the conserved glycine-rich region are replaced by threonine or proline. Li the nucleic acid molecule of the invention the amino acid sequence of the altered CtsR protein preferably has an amino acid identity of at least 30, 33, 36, 40, 50, 60, 70 or 80, 90, 95 or 98 % with SEQ ID NO.l or SEQ ID NO.2, over the amino acid sequences outside the altered glycine-rich region. The altered CtsR protein is preferably altered with respect to a wild type CtsR protein from a Gram positive bacterium that belongs to a genus selected from the group consisting of the genera Actinomyces, Bacillus, Bifidobacterium, Carnobacterium, Clostridium, Corynebacterium, Eubacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Mycobacterium, Nocardia, Peptostreptococcus, Pediococcus, Propionibacterium, Staphylococcus and Streptococcus. A particularly preferred altered CtsR protein is altered with respect to a wild type CtsR protein from Lactobacillus plantarum, including a wild type CtsR protein having the amino acid sequence of SEQ ID NO.2, whereby a preferred alteration is the deletion of one glycine residue.

In a further aspect, the invention relates to a nucleic acid construct comprising a nucleotide sequence encoding an altered CtsR protein of a Gram positive bacterium as defined above. Nucleic acid constructs of the invention may be provided using techniques known per se, for which reference is made to standard handbooks such as e.g. Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3^rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York. Usually the nucleic acid constructs of the invention will consist of DNA. Nucleic acid constructs of the invention include e.g. constructs for the multiplication/amplification of the nucleotide sequences encoding the altered CtsR proteins, constructs that are intermediates in a construction route and constructs for expression of the altered CtsR proteins, for inactivation of endogenous wild type copies of the ctsR gene or for replacement of an endogenous wild type copy of the ctsR gene with a nucleotide sequence encoding an altered CtsR protein. Preferred nucleic acid constructs of the invention are expression constructs in which the nucleotide sequence encoding an altered CtsR protein is operably linked to a promoter. The promoter preferably is a promoter that is capable of driving expression in a Gram positive bacterium. The promoter may be a constitutive or an inducible promoter. However, preferably a promoter of a clpC operon of a Gram positive bacterium is used. More preferably the promoter is native to the ctsR gene is used, i.e. the promoter is derived from the wild type ctsR gene that was altered to produce the nucleotide sequence encoding an altered CtsR protein. In the expression construct the promoter may be operably linked to more than one coding sequence, i.e. several coding sequences may be present in a single multicistronic transcription unit regulated by the transcription regulatory sequence. The one or more coding sequences operably linked to the regulatory sequence are preferably each provided with the appropriate signals for initiation of translation such as e.g. a Shine-Dalgarno sequence and a translation initiation codon. In the expression construct, the transcription unit comprising the one or more coding sequences linked to the transcription regulatory sequence is preferably terminated by a suitable transcription terminator, such e.g. tldH or tslP. Suitable bacterial transcription terminator sequences may be identified as described by Ermolaeva et al. (2000).

A further preferred nucleic acid construct of the invention is a vector for allelic replacement of a wild type ctsR sequence. Nucleic acid constructs for allelic replacement in Gram positive bacteria are well known in the art and are usually based on vectors such as plasmids containing an origin of replication that can be conditionally inactivated, such as e.g. a thermosensitive origin of replication, and a selection marker such as an antibiotic resistance marker that allows to select for cells maintaining the vector. When cells containing such replacement constructs are grown under non- permissive conditions for the origin of replication and under selective pressure for the marker, only those cells are selected that maintain the construct without episomal replication, i.e. cells in which the construct has integrated into the bacterial genome, usually by homologous recombination at the locus to be replaced. The cells may subsequently be grown in the absence of selective pressure to cure the cells from the selective marker (and optionally other unnecessary vector sequences). An example of a nucleic acid construct for allelic replacement in Gram positive bacteria is provided in the Examples herein below. In a preferred embodiment, the nucleic acid constructs of the invention, in particular those for allelic replacement contain a bi-directional selection marker that may be selected for both its presence and its absence (see e.g. EP- A-0 635 574). The nucleic acid constructs of the invention may further comprise additional sequence elements for various functions known in the art per se. Such sequence element include e.g.: autonomously replicating sequences or origins of replication for episomal multiplication and maintenance of the construct; sequences for promoting integration of the construct into the host's genome; sequences for homologous recombination; sequences for site-specific integration and/or recombination; selection marker genes; reporter or indicator genes; and sequences for promoting conjugation or other means of transfer of genetic material. Yet another aspect of the invention relates to a Gram positive bacterial host cell comprising a nucleotide sequence encoding an altered CtsR protein of a Gram positive bacterium as defined above. Preferably, the nucleotide sequence encoding the altered CtsR protein of a Gram positive bacterium is the sole expressed ctsR sequence in the bacterial host cell, more preferably, the nucleotide sequence encoding the altered CtsR protein is the sole ctsR sequence present in the bacterial host cell, as may be achieved by allelic replacement of the wild type ctsR sequence as described above. Preferably, in the Gram positive bacterial host cells of the invention, the nucleotide sequence encoding the altered CtsR protein encodes a CtsR protein that is homologous to the Gram positive bacterial host cell except for the alteration in the glycine-rich region. The Gram positive bacterial host cell of the invention, preferably is a host cell that belongs to a genus selected from the group consisting of the genera Actinomyces, Bacillus, Bifidobacterium, Carnobacterium, Clostridium, Corynebacterium, Eubacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Mycobacterium, Nocardia, Peptostreptococcus, Pediococcus, Propionibacterium, Staphylococcus and Streptococcus. As a result of the presence of the nucleotide sequence encoding the altered CtsR protein, the host cells of the invention preferably have one or more of the characteristics of increased stress tolerance, reduced virulence and/or reduced mobility as defined above.

The Gram positive bacterial host cells of the invention may further comprise one or more second or further recombinant nucleotide sequences coding for proteins of interest. The proteins of interest may be therapeutic or industrial proteins or may be proteins that are involved in the biosynthesis of a useful (secondary) metabolite. The protein or polypeptide of interest may have industrial or medicinal (pharmaceutical) applications. Examples of proteins or polypeptides with industrial applications include enzymes such as e.g. lipases (e.g. used in the detergent industry), proteases (used inter alia in the detergent industry, in brewing and the like), cell wall degrading enzymes (such as, cellulases, pectinases, β- 1,3/4- and β-l,6-glucanases, glycohydrolases, rhamnogalacturonases, mannanases, xylanases, pullulanases, galactanases, esterases and the like, used in fruit processing wine making and the like or in feed), phytases, phospholipases, glycosidases (such as amylases, β-glucosidases, arabinofuranosidases, rhamnosidases, apiosidases and the like), dairy enzymes (e.g. chymosin). Mammalian, and preferably human, proteins or polypeptides and/or enzymes with therapeutic, cosmetic or diagnostic applications include but are not limited to insulin, serum albumin (HSA), lactoferrin, hemoglobin α and β, tissue plasminogen activator (tPA), erythropoietin (EPO), tumor necrosis factors (TNF), BMP (Bone Morphogenic Protein), growth factors (G-CSF, GM-CSF, M-CSF, PDGF, EGF, and the like), peptide hormones (e.g. calcitonin, somatomedin, somatotropin, growth hormones, follicle stimulating hormone (FSH) interleukins (IL-x), interferons (IFN-γ), chemokines).

Other examples include antimicriobial peptides such as nisin, pediocin. Also included are bacterial, viral, fungal, protozoan, and parasitic antigens, e.g. for use as vaccines, including e.g. cholera (heat-labile) toxin B-subunit, Hepatitis B virus envelope surface protein, Norwalk virus capsid protein, Human cytomegalovirus glycoprotein B, glycoprotein S, interferon, and transmissible gastroenteritis corona virusreceptors and the like. Further included are genes coding for mutants or analogues of the said proteins.

In a further aspect, the invention relates to methods for producing a fermentation product, whereby these methods comprise culturing a Gram positive bacterial host cell as defined above. The fermentation process may be a process for the production of a fermentation product such as ethanol, lactic acid, acetic acid, succinic acid, amino acids, 1,3-propane-diol, ethylene, glyceroi, or products known as nutraceuticals, such as e.g. vitamins, antioxidants or poly- or oligo-saccharides. It may also be a process for the production of a fermented food product such as a fermented dairy, vegetable or meat product. The fermentation process may also be a process for producing a protein of interest, wherein the method comprises culturing a Gram positive bacterial host cell as defined above, under conditions conducive to the expression of the protein.

In yet a further aspect, the invention relates to a method for site-specific delivery of a therapeutic protein to the gastrointestinal tract of a subject. The method comprises administering a Gram positive bacterial host cell as defined above to the gastrointestinal tract of the subject. The bacterium is preferably orally administered in a composition that is suitable for consumption. A preferred embodiment of such method is a method for vaccinating a subject, wherein the therapeutic protein comprises antigens of a pathogen that provoke a protective immune response against the pathogen.

In another aspect the invention relates to a use of the bacteria as defined above in the manufacture of a medicament for the site-specific delivery of a therapeutic protein to the gastrointestinal tract of a subject. Preferably, the bacteria are used in the manufacture of a medicament for vaccinating a subject.

In yet another aspect, the invention relates to a use of the above defined host cells in a fermentation process, to the use of the above defined host cells as a probiotic or to the use of the above defined host cells as a live oral vaccine.

Description of the figures Figure 1. The CtsR protein of L. monocytogenes AKOl, designated CtsRΔGly, contains 3 glycines versus 4 glycines in the wt CtsR protein of L. monocytogenes ScottA, as a result of (I) a GGT in frame deletion in codon 61, 62, or 63; (II) a GTG deletion in the -2 frame of codon 61, 62, 63, or 64 (a), or the +1 frame of codon 60, 61, 62, or 63 (b); (III) a TGG deletion in the -1 frame of codon 61,62,63, or 64 (a), or the +2 frame of codon 60,61, 62, or 63 (b).

Figure 2. Schematic representation of plasmid pJL3.

Figure 3. Schematic representation of plasmid pJL5.

Figure 4. Log reduction in the viable counts of wild type Listeria monocytogenes ScottA (A), of AKOl (B), strain MBOl containing the wt ctsR gene after double crossover in wt background (C), or MB06, MBl 8 containing the mutant ctsR AGly gene after double crossover (D, E) after HHP treatment at 350 MPa, at 20°C for 20 min. Cells were grown in BHI broth at 30°C under shaking, and values are means of triplicate measurements. Bars represent the S.D. (n=3).

Figure 5. Colony forming units (cfu) of Listeria monocytogenes ScottA (Δ), AKOl (□) and MB 18 (O) in the spleens of infected Balb/c mice through time (days). Arrow indicates infectious dose used through the experiment (log cfu = 5.65) by intraperitoneal route. Figure 6. Specific growth rate of WCFS1 and WCFS1 CtsR dgly at various temperatures.

Figure 7. Viability after hydrogen peroxide treatment of WCFS 1 and WCFS 1 CtsR dgly.

Figure 8. Viability after peroxide treatment of NZ7100 and NZ7100 containing pJL5.

Examples

1. Materials and methods

1.1. Bacterial strains and growth conditions

Wt J. monocytogenes ScottA (Department of Food Science, Wageningen Agricultural University, The Netherlands), L. monocytogenes ScottA AKOl (see below), L. monocytogenes ScottA MB01, MB06 and MB 18 (see below) and Escherichia coli DH5α were used in this study. Strains were routinely grown in Brain Heart Infusion (BHI) broth (Oxoid, Hampshire, England). Erythromycin (Em) (Sigma- Aldrich Chemie, Steinheim, Germany) was used at 5 μg ml^" for L. monocytogenes and 300 μg ml^"1 for E. coli.

L. monocytogenes cultures were incubated overnight at 30°C, and a 0.3 % (v/v) inoculum was then added to 100 ml of BHI broth. Cultures were incubated under shaking (160 rpm), and cells were harvested by centrifugation (10,000x g, 10 min) at mid exponential phase (OD₆₆₀ -0.3 for the wt strain and MB01 or -0.2 for strain AKOl, MB06 and MB18).

The piezotolerant L. monocytogenes AKOl was isolated from a population of J. monocytogenes Scott A that survived a High Hydrostatic Pressure treatment of 400 MPa for 20 min. Cells from a single colony were cultured in BHI broth overnight using a 0.3 (v/v) inoculum and then kept as a stock at -80°C in 15% (v/v) glyceroi. Several test were carried out to confirm the identity of AK 01 as L. monocytogenes, namely, growth on Palcam Listeria selective medium ( Merck, Darmstadt, Germany), carbohydrate utilisation determined by the Apizym identification system (Biomerieux, Marcy-iΕtoile, France) and Ribotyping (TNO Food, Zeist, The Netherlands).

The Lactobacillus plantarum strains used were WCFS1( NCTMB8826 ) and its derivatives: L. plantarum NZ7100: NCIMB8826 containing the nisRK genes integrated into the chromosome (Pavan et al, 2000). L. plantarum WCFS1 CtsR dgly: NCIMB8826 containing mutant ctsR encoding CtsR lacking a glycine-residue in the glycine-rich region as described here. L. plantarum was cultured at 37°C in MRS medium without shaking.

E. coli Ε10 was cultured with shaking at 37°C in TY medium, supplemented with the appropriate antibiotics.

1.2. Materials and Recombinant DNA Techniques Chromosomal DNA isolation, electrophoresis, hybridisation and amplification by

PCR was performed according to standard protocols (Sambrook et al, 1989). Restriction endonucleases and T4 DNA ligase were obtained from Roche (Mannheim, Germany). [³²P]dCTP was obtained from Amersham Biosciences (Buckinghamshire, UK). Plasmid DNA preparation, gel extraction of DNA fragments, and purification of DNA amplified by polymerase chain reaction (PCR) were performed using a QIAquick kit (Qiagen, Hilden, Germany). Oligonucleotides were obtained from Εurogentec (Seraig, Belgium). PCR reactions were performed using PCR High Fidelity (Roche), containing proofreading enzyme activities, according to instructions of the manufacturer. Sequencing of PCR fragments or cloned PCR fragments was performed by Εurogentec or Baseclear (Leiden, The Netherlands).

1.3. Sequencing of the flaA, flaR, cheY, clpC, clpP and ctsR genes in wt . monocytogenes and strain AKOl

The nucleotide sequences of selected genes and their promoter regions were determined in the wt L. monocytogenes and the strain mutant AKOl . Selected DNA fragments were PCR amplified using chromosomal DNA of the two strains as template, and the following primers were used for amplification of the genes: flaAdir and flaArev fox flaA; flaRdir and flaRrev for flaR; CheYdir and CheYrev for cheY; ClpCdir and ClpCrev for clpC; ClpPdir and ClpPrev for clpP; CtsRsall and CtsRecol for ctsR. The nucleotide sequences of these primers are given in Table 1.

Table 1. Nucleotide sequences of primers used in this study Primer name Nucleotide sequence

CheY dir 5^,-ATTACAAATAGGGCGCAGAG-3^Λ

CheY rev 5 -GAAGCTTCTTCTATAAACAG-3^, ClpC dir 5^Λ-CGATTTGGTGTAGAATTAGG-3^λ

ClpC rev 5^,-GACGCGAATCATGATTTTTC-3^,

ClpP dir 5^Λ-CGCTTCAGACTTTATCGTTTGACC-3

ClpP rev 5^,-AATACTAGTGTATACATTCTATGG-3^λ

CtsRsall 5 -GAGAGCGTCGACCGTAGCACAATTCTCGCAT-3^λ CtsRecol 5^,-AAGCTTGAATTCGCCAATGGTAGTTGGGGGC-3^,

CtsRecofw 5 -GATAAAGAATTCCCCGGGGATAACAGGCTTATC-3

CtsRbamrv 5^,-TCCTCAGGATCCAGAACCGCACCATATTCACTC-3^Λ

Detmutld S^{^}-AGATAAGTTTGAATGTGTACCTTC^

Detmutlr 5 -AATAATCCTAATATAGCCACCACCGC-3 FlaA dir 5^,-CGTAAAAACGTTGATAATAAGCCG-3^Λ

FlaA rev 5 -GGGGCTAAGGGTAAACAATGTTCG-3^,

FlaR dir 5^,-TCGCGGTAAGCTAAAAGATG-3^,

FlaR rev S^{^}-GAAGTAATACGTATTATCGC^

1.4. Construction of L. monocytogenes MB06 and MB18 by allelic replacement of wt ctsR with mutant ctsR in L. monocytogenes Scott A

Strain L. monocytogenes AKOl carries a ctsR gene with a 3 basepair (bp) deletion in a triplet GTG repeat region that encodes four glycines between codon 60 (Arg) and codon 65 (Tyr) (Fig. 1). The mutant CtsR protein is designated CtsRΔGly. The gene encoding CtsRΔGly was transferred to a L. monocytogenes ScottA background by allelic exchange of the wt ctsR gene with the mutant ctsR gene of L. monocytogenes AKOl. This procedure was performed using plasmid pAUL-A, containing a thermosensitive replication origin (Chakraborty et al., 1992), as descibed in detail by Schaferkordt et al. (1998). In short, a DNA fragment encompassing the mutant ctsR gene of strain AKOl and -800 bp of its 5' and 3' flanking regions was PCR amplified using genomic DNA of L. monocytogenes AKOl as template, and primers CtsRecofw and CtsRbamrv (Table 1). This fragment was digested with EcoRI and BamΗl, gel purified, and ligated to pAUL-A that had been digested with EcoRI and BamHI.

Transformations were performed using E. coli strain DH5α and cells were plated onto BHI plates containing 300 μg/ml Εm, followed by incubation at the permissive temperature (28-30°C). A plasmid with the correct insert (determined by DNA sequencing) was introduced into L. monocytogenes ScottA by electroporation, and transformants were selected for Εm resistance (5 μg ml-1) at 28-30°C. The plasmid was integrated into the chromosomal target by culturing at the non-permissive temperature of 42°C. Spontaneous excision of the plasmid was achieved by culturing at the permissive temperature in the absence of Εm. Subsequent loss of the plasmid was accomplished by incubation at the non-permissive temperature in the absence of Εm. A PCR based strategy was employed to distinguish L. monocytogenes strains containing the wt ctsR gene and the mutant ctsR gene. The forward primer Detmutld (Table 1) was designed 91 bp upstream from codon 60. The reverse primer Detmutlr (Table 1) encompassed codon 61 (2 basepairs) to 70, and had a single base pair mismatch in the second last nucleotide at the 3' end (... ACCGC-3' instead of ... ACCAC-3 '), adj acent to the MaeT site (ACGT) present in codon 59 and 60 (Fig. 1). PCR amplification rendered a 118 bp DNA fragment containing an intact ell restriction site for the wt ctsR gene, while a 115 bp DNA fragment lacking the Mαell site was obtained for the mutant ctsR gene. PCR reactions were performed on genomic DNA of 19 isolates. Purified PCR products were incubated with Maeϊϊ (Roche), and visualised with ΕtBr on a 5% agarose gel (Nusieve, FMC, Rockland, USA). Two mutant clones, designated MB 06 and MB 18, were identified using this screening method, and sequencing of their complete ctsR genes confirmed the 3 bp deletion between codon 60 and 65 in absence of other mutations in the ctsR gene compared with wt ctsR. Strain MBOl contained the wt ctsR gene after the double crossover procedure, and was used as a control.

1.5. Northern blot analysis Cultures were grown to mid exponential phase of growth at 30°C (see above) and total RNA was extracted using an RNAeasy kit (Qiagen, Hilden, Germany). The RNA was resuspended in DEPC-treated water, and quantified by measuring the A₂₆₀. A total of 5 μg of RNA was denatured by incubation for 30 min at 65 °C in the presence of glyoxal (1M) and DMSO (50%) and run in a 1% agarose gel. The RNA was subsequently transferred to Nytran membranes using a Turboblotter set-up, and crosslinked to the membrane by UN irradiation. Hybridisation with 32P-labelled DΝA fragments was performed at 42°C using Ultrahyb solution (Ambion, Austin, USA), followed by visualisation using autoradiography. Probes were obtained by labelling PCR-amplified genes using Klenow-fragment by standard techniques (Sambrook et al., 1989).

1.6. Protein analysis using two-dimensional gel electrophoresis (2D-E)

Stock cultures of the wt strain and strain AKOl were grown and harvested as described above. The pellet was suspended in water to an OD₆₆₀ of 10. Proteins were extracted and 2D electrophoresis was performed as described by Wouters et al. (1999) and O'Farrell (1975). Proteins that were clearly differentially expressed in mutant AKOl and the wt were isolated by blotting of the proteins in the gel onto membranes, stained with Coomassie blue, and cutting out of the spots. The Ν-terminal sequence of these proteins was determined (Utrecht University, The Netherlands)

1.7. High Hydrostatic Pressure treatment

Mid exponential phase cultures of L. monocytogenes (see above) were subjected to High Hydrostatic Pressure (HHP) treatment as follows. Cells were cultured at 8 or 30°C, and harvested by centrifugation (10,000 x g, 10 min.) at mid exponential phase (OD₆₆₀ 0.3 and 0.2 for the wt and AKOl, respectively). The cells were washed twice in 50 mM N-[2-acetamido]-2-aminoethanesulfonic acid (ACES buffer, Sigma- Aldrich, Steinheim, Germany), pH 7.0. The pellet was resuspended in semi-skimmed milk (Friesche Vlag, Ede, The Netherlands), or in ACES buffer to an OD₆₆₀ of 0.1. 10 ml aliquots were transferred into sterile plastic tubes. ACES buffer was selected since this buffer maintains pH 7.0 during High Pressure treatments. Suspensions were placed in sterile plastic stomacher bags (Seward, London, UK) that were sealed while avoiding excess of air bubbles. These pouches were transferred in glycol, which was the medium through which the pressure was transferred (Resato, Roden, Holland). Subsequently cell suspensions were exposed to pressures of 350 MPa (or 400 MPa in case of the isolation of strain AKOl) in a High Pressure unit (Resato, Roden, Holland) at 20°C for 20 min. The viable numbers of L. monocytogenes were determined in triplicate before and after pressure treatment by plating decimal dilutions of samples in onto BHI agar (1,2 % w/v agar). Plates were incubated at 30°C for 5 days.

1.8. Motility tests

The motility of L. monocytogenes strains was tested on semi-solid Motility Test medium containing 0.4% w/v agar, 10 g l^"1 peptone (Oxoid, Hampshire, England), 5 g 1^{" 1} NaCl (Merck, Darmstadt, Germany), 3 g l^"1 Beef Extract (Becton Dickinson, Sparks, USA), 0.05 g l^"1 2.3.5-Triphenyltetrazolium chloride (Sigma-Aldrich Chemie, Steinheim, Germany). This medium was inoculated withE. monocytogenes by stabbing. After 5 days of incubation at 30°C, isolates that were motile, and therefore able to swarm, showed a red cloudy pattern as a result of reduction of 2,3,5- Triphenyltetrazolium chloride to formazan caused by bacterial metabolism.

1.9. Mouse virulence assays

L. monocytogenes ScottA, the piezotolerant isolate L. monocytogenes AKOl, and L. monocytogenes MB 18 (CtsRΔGly) were analysed for virulence in a murine model of infection. Female Balb/c mice were inoculated intraperitoneously with a 4.5 * 10⁵ cfu inoculum in 200 μl PBS. Numbers of bacteria surviving in mouse spleens were determined for the first three days post-infection.

1.10. Deletion of a glycine residue of CtsR and subsequent allelic replacement inE. plantarum

Deletion of a glycine residue in the glycine-rich region of the Lactobacillus plantarum CtsR was attained by replacement of wild type ctsR by mutant ctsR coding for CtsR lacking one glycine residue in the glycine-rich region. The sequence of the complete genome of L. plantarum. was recently published (Kleerebezem et al, 2003). For this gene replacement, a DCO (double cross over) strategy was used involving pJL3 (Figure 2). E. coli was used as an intermediate host. A PCR based strategy similar as described above for L. monocytogenes was designed to distinguish between wild type and mutant ctsR. Using this strategy, PCR amplification rendered a DNA fragment containing an intact Maell restriction site for wild type ctsR. However, for mutant ctsR, a DNA fragment was obtained in which the restriction site for Maell was replaced by a restriction site for BstUI. A DCO clone, designated WCFS1 CtsR dgly, was isolated and screened for chromosomal replacement of the wild type ctsR gene by the mutant gene using the method described above. Sequencing of part of the ctsR gene of this clone confirmed the expected sequence at the locus of the deletion of the glycine codon.

1.11. Stress treatment in L. plantarum Overnight cultures of the strains WCFS1 and WCFS1 CtsR dgly were diluted

1 : 100 in fresh MRS medium. Cultures were propagated until an OD₆₀o of 1.0 was reached and stress treatments were performed immediately. Hydrogen peroxide (H₂O₂) treatment was performed by addition of 1.0 % of peroxide (w/v) to the cultures. Viable counts were determined just before and at regular time intervals after the addition of the peroxide.

1.12. Inducible overexpression of ctsR in . plantarum and stress treatment

For overexpression of ctsR, the NICE system in L. plantarum WCFS1 containing the lactococcal nisRK genes integrated in the chromosome (NZ7100) was used (Pavan et al, 2000). As shown in Figure 3, a 0.5 kb region containing the L. plantarum ctsR gene was translationally fused to the nisA promoter in the vector pNZ8048, now designated pJL5, using E. coli as an intermediate host.

The strains NZ7100 and NZ7100 containing ρJL5 were grown at 37°C by 1:50 inoculation of fresh MRS and MRS containing 10 μg/ml chloramphenicol with overnight cultures. When the cultures reached an OD₆₀₀ of 0.2, nisin was added at a concentration of 25 ng/ml. The cultures were grown until an OD₆₀₀ of 1.0 and stress treatments were performed immediately.

Hydrogen peroxide (H₂O ) treatment was performed by addition of 0.2 % of peroxide (w/v) to the cultures. Viable counts were determined just before and at regular time intervals after the addition of the peroxide.

Results The piezotolerant strain AKOl shows reduced levels of FlaA protein and flaA mRNA, and increased levels of ClpP protein and clpP mRNA

To identify putative differences in protein expression between the High Hydrostatic Pressure tolerant isolate L. monocytogenes AKOl and wt L. monocytogenes ScottA, we performed 2D gel electrophoresis of the proteins extracted from mid- exponential phase cultures. Compared with the wt, cells of strain AKOl showed increased amounts of ClpP, which was identified by N-terminal amino acid sequencing. The heat shock protein ClpP is a serine protease involved in stress response, intracellular parasitism and virulence (Gaillot et al., 2000). Cells of AKOl showed decreased amounts of two proteins, which were both identified as flagellin, which is the structural protein of the flagellum and it is encoded by the gene flaA (Dons et al, 1992). The presence of two spots identified as flagellin (FlaA) on the 2D gel can be explained by post-translational modification, e.g. glycosylation, as described for flagellar proteins from several bacteria (Dons et al, 1992, Peel et al, 1988). Northern analysis furthermore demonstrated 13 -fold higher clpP mRNA levels and 10-fold lower flaA mRNA levels in strain AKOl than in the wt. The nucleotide sequences of the promoter regions and coding regions of flaA and clpP were analysed to establish whether putative mutations were present that could account for the different expression levels in AKOl and the wt. This analysis demonstrated that the nucleotide sequences of these genes and their promoter regions were identical in both strains. Interestingly, Northern analysis showed a significant 30-fold increase in the levels of ctsR mRNA in strain AKOl compared with the wt.

Sequencing of clpP, flaA, flaR, cheY, clpC and ctsR genes reveals a 3bp deletion in ctsR

The reduced levels of FlaA protein and flaA mRNA, and the increased levels of ClpP protein and clpP mRNA in strain AKOl compared with the wt suggested different transcription regulation of the clpP and flaA genes in the two strains. A number of genes have previously been shown to be involved in stress resistance, motility, and regulation of FlaA or ClpP proteins in L. monocytogenes, namely, cheY (Michel et al, 199%), flaR (Sanchez-Campillio et al, 1995), and clpC ( Nair et al, 2000, Rouquette et al, 1998), clpE (Nair et al, 1999), prfA (Michel et al, 1998), cAeF (Flanary et al., 1999) and ctsR (Nair et al, 2000). The nucleotide sequences of these genes and their -200 bp upstream regions were identical for strain AKOl and the wt strain, except for ctsR. The ctsR gene of AKOl showed a 3 basepair (bp) deletion between codon 60 and 65. This region encodes 4 glycines in wt CtsR protein, and only 3 glycines in the mutated CtsR protein of AKOl, designated CtsRΔGly (Figure 1).

Allelic replacement of ctsR with mutant CtsRΔGly renders the HHP tolerant phenotype

To investigate whether the 3 base-pair deletion in the ctsR gene of strain L. monocytogenes AKOl was exclusively responsible for the observed High Hydrostatic Pressure tolerant phenotype, we constructed strains L. monocytogenes MB06 and MB 18. Both strains carried the mutant ctsR gene, encoding CtsRΔGly, in a wt background after allelic replacement of the native ctsR gene with the mutant ctsR gene from strain AKOl. Upon exposure to HHP treatment, strains MB06 and MB18 showed 2 to 3 log lower reductions in viable numbers than wt L. monocytogenes ScottA (Figure 4). These reductions were similar to those observed for the piezotolerant strain AKOl. HHP treatment of strain MBOl, in which allelic replacement did not take place and served as a control, resulted in reductions in viable numbers that were similar to the wt.

L. monocytogenes expressing the CtsRΔGly protein is non motile

Strains L. monocytogenes MB06 and MB 18, expressing the CtsRΔGly protein, did not swarm in semi-solid motility test medium, whereas the wt and the control strain MBOl clearly showed diffuse growth in the test tubes. The non-motile character of strains MB06 and MB 18 was indistinguishable from that of the piezotolerant strain AKOl. The non-motile phenotype of AKOl is in agreement with the reduced FlaA expression observed in the 2D gel electrophoresis analysis.

L. monocytogenes expressing the CtsRΔGly protein is less virulent than the wt

We compared the virulence properties of wt L. monocytogenes ScottA with those of the piezotolerant strains AKOl and MB 18, carrying the gene encoding CtsRΔGly, by analysing the kinetics of bacterial growth in the spleens of mice infected by the intraperitoneal route. Wt L. monocytogenes ScottA was detected at relatively high levels in the spleens of infected mice at 24 hours post inoculation. The wt strain was subsequently capable of significant growth in infected mice over the following 48 hours, reaching levels of 3.4 * 10⁶ bacteria per organ by day 3 post infection. In contrast, the L. monocytogenes piezotolerant mutants AKOl and MB 18 were detected at low levels in the spleens of infected mice 24 hours following inoculation, suggesting an inability to survive the initial stages of murine infection. The mutant was incapable of significant growth in the spleens of infected mice but did persist in infected spleens over the three days of the study (Figure 5).

Deletion of glycine residue via allelic replacement confers stress resistance to L. plantarum.

To determine the effect of ctsR dgly on the growth rate of L. plantarum, the OD₆₀o of wild type WCFSl and mutant WCFSl CtsR dgly was determined at regular time intervals and at different temperatures. At all temperatures, the specific growth rate of the wild type was higher than the specific growth rate of the mutant. The results are shown in Figure 6. The morphological characteristics of the WCFSl CtsR dgly did not appear to be different from those of the wild type.

Hydrogen peroxide treatment

Hydrogen peroxide treatment of WCFSl and WCFSl CtsR dgly clearly showed that the mutant had a higher resistance to peroxide than the wild type. In Figure 7, a representable graph of the results can be found. Compared to the wild type, deletion of a glycine residue of the glycine-rich region of the CtsR protein resulted in derepression of the Clp proteins, leading to increased resistance to hydrogen peroxide.

Overexpression of wildtype ctsR L. plantarum confers stress sensitivity

As is shown in Figure 8, cultures of the ctsR overexpressing strain NZ7100 containing pJL5 (figure 3) are less resistant to peroxide than the wild type strain

NZ7100. The graph shown in Figure 8 is representable for the results. Overexpression of ctsR using pJL5 inNZ7100 results in overrepression of class III heat shock Clp proteins, leading to increased sensitivity to both hydrogen peroxide and low pH (adjusted with lactic acid) compared to the wild type strain. References

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1482.

SEQUENCE LISTING

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<120> Stress resistant, low virulence Gram positive bacteria

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Lys Glu Ala Arg Leu Met Val Ala Ala Leu Asp Arg Glu Val Leu lie 115 120 125

Leu Pro Leu Pro Asp Arg Asp lie Leu Arg Ser Arg lie Leu Glu Ala 130 135 140

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<400> 2 Met Lys Arg Val Lys His Met Gin Ser Gin Asn lie Ser Asp lie lie 1 5 10 15

Glu Lys Tyr Leu Lys Ser lie Leu Ala Asp Ser Glu His Val Glu lie 20 25 30

Arg Arg Ser Glu lie Ala Asp Leu Phe Asn Val Val Pro Ser Gin lie 35 40 45

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Glu Ser Lys Arg Gly Gly Gly Gly Tyr lie Arg He Glu Lys Val Asn 65 70 75 80

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85 90 95 Asp Ser He Thr Gin Arg Asp Ala Tyr Ala Val Val Gin Ser Leu Tyr 100 105 110

Glu Asp Asp Val Leu Asn Arg Arg Glu Ala Gin Leu He Leu Val Ala 115 120 125

He Asp His Glu Thr Leu Gly Leu Thr Asp Arg Asp Leu Glu Asn Ser 130 135 140

Leu Arg Ala Arg He He He Gly He Leu Asn His Leu Arg Tyr Glu 145 150 155 160

Ser

<210> 3

<211> 20

<212> DNA

<213> Artificial

<400> 3 attacaaata gggcgcagag 20

<210> 4 <211> 20

<212> DNA

<213> Artificial

<400> 4 gaagcttctt ctataaacag 20

<210> 5 <211> 20

<212> DNA

<213> Artificial <400> 5 cgatttggtg tagaattagg

20

<210> 6 <211> 20

<212> DNA

<213> Artificial

<400> 6 gacgcgaatc atgatttttc 20

<210> 7 <211> 24

<212> DNA

<213> Artificial

<400> 7 cgcttcagac tttatcgttt gacc 24

<210> 8

<211> 24

<212> DNA

<213> Artificial

<400> 8 aatactagtg tatacattct atgg 24

<210> 9 <211> 31

<212> DNA

<213> Artificial <400> 9 gagagcgtcg accgtagcac aattctcgca t

31

<210> 10 <211> 31

<212> DNA

<213> Artificial

<400> 10 aagcttgaat tcgccaatgg tagttggggg c 31

<210> 11

<211> 33

<212> DNA

<213> Artificial

<400> 11 gataaagaat tccccgggga taacaggctt ate 33

<210> 12

<211> 33

<212> DNA

<213> Artificial

<400> 12 tcctcaggat ccagaaccgc accatattca etc 33

<210> 13 <211> 24

<212> DNA

<213> Artificial <400> 13 agataagttt gaatgtgtac cttc

24

<210> 14 <211> 26

<212> DNA

<213> Artificial

<400> 14 aataatccta atatagccac caccgc 26

<210> 15

<211> 24

<212> DNA

<213> Artificial

<400> 15 cgtaaaaacg ttgataataa gccg 24

<210> 16

<211> 24

<212> DNA

<213> Artificial

<400> 16 ggggctaagg gtaaacaatg ttcg 24

<210> 17 <211> 20

<212> DNA

<213> Artificial <400> 17 tcgcggtaag ctaaaagatg

20

<210> 18 <211> 20

<212> DNA

<213> Artificial

<400> 18 gaagtaatac gtattatcgc 20

Claims

Claims

1. A nucleic acid molecule comprising a nucleotide sequence encoding an altered CtsR protein of a Gram positive bacterium, whereby the altered CtsR protein is altered in that it has an amino acid sequence with an alteration in the conserved glycine-rich region that corresponds to amino acid positions 61 to 64 of SEQ ID NO.1 with respect to the glycine-rich region of a corresponding wild type CtsR protein, and whereby the alteration confers to the altered CtsR protein at least one ability selected from the abilities to increase stress tolerance, to reduce virulence, and to reduce mobility of a Gram positive bacterium in which the altered CtsR protein is expressed as sole CtsR protein.

2. A nucleic acid molecule according to claim 1, wherein the alteration in the conserved glycine-rich region consists of a deletion or an insertion of one or more glycines.

3. A nucleic acid molecule according to claim 2, wherein the alteration in the conserved glycine-rich region consists of a deletion or insertion of a single glycine.

4. A nucleic acid molecule according to claim 1, wherein the alteration in the conserved glycine-rich region comprises a replacement of one or more glycines by an amino acid other than serine or glycine.

5. A nucleic acid molecule according to any one of claims 1 - 4, wherein the amino acid sequence of the CtsR protein has an amino acid identity of at least 30, 33 or 36 %> with SEQ ID NO.1 over the amino acid sequences outside the altered glycine-rich region.

6. A nucleic acid molecule according to any one of claims 1 - 4, wherein the altered CtsR protein is altered with respect to a wild type CtsR protein from a Gram positive bacterium that belongs to a genus selected from the group consisting of the genera Actinomyces, Bacillus, Bifidobacterium, Carnobacterium, Clostridium, Corynebacterium, Eubacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Mycobacterium, Nocardia, Peptostreptococcus, Pediococcus, Propionibacterium, Staphylococcus and Streptococcus.

7. A nucleic acid construct comprising a nucleotide sequence encoding an altered CtsR protein of a Gram positive bacterium as defined in any one of claims 1 - 6.

8. A nucleic acid construct according to claim 7, wherein the nucleotide sequence is operably linked to a promoter.

9. A nucleic acid construct according to claims 6 or 7, wherein the nucleic acid construct is a vector for allelic replacement of a wild type ctsR sequence.

10. A Gram positive bacterial host cell comprising a nucleotide sequence encoding an altered CtsR protein of a Gram positive bacterium as defined in any one of claims 1 - 6.

11. A Gram positive bacterial host cell according to claim 10, wherein the nucleotide sequence encoding the altered CtsR protein of a Gram positive bacterium is the sole expressed ctsR sequence.

12. A Gram positive bacterial host cell according to claims 10 or 11, wherein the nucleotide sequence encoding the altered CtsR protein of a Gram positive bacterium is the sole ctsR sequence.

13. A Gram positive bacterial host cell according to any one of claims 10 - 12, wherein the nucleotide sequence encoding the altered CtsR protein encodes a CtsR protein that is homologous to the Gram positive bacterial host cell except for the alteration in the glycine-rich region.

14. A Gram positive bacterial host cell according to any one of claims 10 - 13, wherein the host cell belongs to a genus selected from the group consisting of the genera Actinomyces, Bacillus, Bifidobacterium, Carnobacterium, Clostridium, Corynebacterium, Eubacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Mycobacterium, Nocai'dia, Peptostreptococcus, Pediococcus, Propionibacterium, Staphylococcus and Streptococcus.

15. A Gram positive bacterial host cell according to any one of claims 10 - 14, wherein the host cell comprises a second nucleotide sequence coding for a protein of interest.

16. A Gram positive bacterial host cell according to claim 15, wherein the protein of interest is a therapeutic or industrial protein.

17. A method for producing a fermentation product, wherein the method comprises culturing a Gram positive bacterial host cell as defined in any one of claims 10 - 16.

18. A method for producing a protein of interest, wherein the method comprises culturing a Gram positive bacterial host cell as defined in claims 15 or 16, under conditions conducive to the expression of the protein.

19. A method for site-specific delivery of a therapeutic protein to the gastrointestinal tract of a subject, wherein the method comprises administering a Gram positive bacterial host cell as defined in claim 16 to the gastrointestinal tract of the subject.

20. A method according to claim 19 for vaccinating a subject, wherein the therapeutic protein comprises antigens of a pathogen that provoke a protective immune response against the pathogen.

21. Use of a host cell as defined in any one of claims 10 - 16 in a fermentation process.

22. Use of a host cell as defined in any one of claims 10 - 16 as a probiotic.

23. Use of a host cell as defined in any one of claims 10 - 16 as a live oral vaccine.