Insertion elements on lactococcal proteinase plasmids

DNA segments of 809 and 808 nucleotides, with 18-base-pair terminal inverted repeats, are present on the proteinase plasmids pWV05 from Lactococcus lactis subsp. cremoris Wg2 and pSK111 from L. lactis subsp. cremoris SK11, respectively. These DNA segments are highly similar: 77% identical nucleotides and both contain an open reading frame that can encode a protein of 226 amino acids. Furthermore, both DNA segments are located downstream of the proteinase maturation gene prtM, but they differ individually in their orientation with respect to the prtM gene. On the basis of the striking similarity between ISS1, an 808-base-pair insertion sequence (IS) from L. lactis subsp. lactis ML3 lactose plasmid pSK08, and the DNA segments of pWV05 and pSK111, we propose that these DNA segments comprise IS elements. The IS elements from strains Wg2 and SK11 were named ISS1W and ISS1N, respectively. On pWV05, ISS1W is flanked on one side by only part of a second IS element, indicating that pWV05 evolved as a deletion derivative of a precursor plasmid that carried at least two IS elements.     

Insertion elements on lactococcal proteinase plasmids.

DNA segments of 809 and 808 nucleotides, with 18-base-pair terminal inverted repeats, are present on the proteinase plasmids pWV05 from Lactococcus lactis subsp. cremoris Wg2 and pSK111 from L. lactis subsp. cremoris SK11, respectively. These DNA segments are highly similar: 77% identical nucleotides and both contain an open reading frame that can encode a protein of 226 amino acids. Furthermore, both DNA segments are located downstream of the proteinase maturation gene prtM, but they differ individually in their orientation with respect to the prtM gene. On the basis of the striking similarity between ISS1, an 808-base-pair insertion sequence (IS) from L. lactis subsp. lactis ML3 lactose plasmid pSK08, and the DNA segments of pWV05 and pSK111, we propose that these DNA segments comprise IS elements. The IS elements from strains Wg2 and SK11 were named ISS1W and ISS1N, respectively. On pWV05, ISS1W is flanked on one side by only part of a second IS element, indicating that pWV05 evolved as a deletion derivative of a precursor plasmid that carried at least two IS elements.     

Cloning and partial sequencing of the proteinase gene complex from Lactococcus lactis subsp. lactis UC317.

The proteinase genes from Lactococcus lactis subsp. lactis UC317 were identified on a plasmid, pCI310, which is a deletion derivative of a cointegrate between pCI301, the 75 kb Lac Prt plasmid from UC317 and the 38.5 kb cryptic plasmid from that strain. The prt genes were cloned using a replacement cloning strategy whereby fragments from pCI310 were exchanged with the equivalent fragments in pNZ521, which contains the cloned proteinase genes from L. lactis subsp. lactis SK112. This generated two plasmids which encoded a cell-envelope-associated and a secreted proteinase, respectively. Specific regions of the UC317 structural prtP gene known to encode seven of the amino acids essential for substrate cleavage specificity were sequenced and compared with the known sequences of prt genes from L. lactis strains SK112, Wg2 and NCDO763. In spite of various differences that were detected in the nucleotide sequence of this region, it appears that these seven amino acids in strains UC317 and NCDO763 are identical, and represent a combination of three of the amino acids from SK112 and four from Wg2. These results indicate that the UC317 proteinase is a natural hybrid of the SK112 and Wg2 proteinases.     

Cloning, sequencing and expression of the gene encoding the cell-envelope-associated proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151.

The gene encoding the cell-envelope-associated proteinase of Lactobacillus paracasei subsp. paracasei NCDO 151 (formerly Lactobacillus casei NCDO 151) was cloned and sequenced. The gene was located on the chromosome and encoded a polypeptide of 1902 amino acids. The proteinase is N-terminally cleaved upon maturation. It shows extensive homology to the Lactococcus lactis subsp. cremoris Wg2 proteinase. Similar to the situation in Lactococcus, a maturation gene was found upstream of the proteinase gene. The cloned proteinase gene was expressed in Lactobacillus plantarum. However, no expression was observed when the gene was cloned in Lactococcus lactis.     

Chromosomal stabilization of the proteinase genes in Lactococcus lactis.

The plasmid-encoded proteinase genes prtP and prtM of Lactococcus lactis subsp. cremoris Wg2 were integrated by a Campbell-like mechanism into the L. lactis subsp. lactis MG1363 chromosome by using the insertion vector pKLG610. Two transformants were obtained that differed in the number of amplified pKLG610 copies in head-to-tail arrangements on their chromosomes; MG610 contained approximately two copies, and MG611 contained about eight copies. The amplifications were stably maintained during growth in milk in the absence of antibiotics. The proteolytic activity of strain MG611 was approximately 11-fold higher than that of strain MG610 and about 1.5 times higher than that of strain MG1363(pGKV552), which carried the proteinase genes on an autonomously replicating plasmid with a copy number of approximately 5. All three strains showed rapid growth in milk with concomitant rapid production of acid. The results suggest that a limited number of copies of the proteinase genes prtP and prtM per genome is sufficient for good growth in milk.     

Chromosomal stabilization of the proteinase genes in Lactococcus lactis.

The plasmid-encoded proteinase genes prtP and prtM of Lactococcus lactis subsp. cremoris Wg2 were integrated by a Campbell-like mechanism into the L. lactis subsp. lactis MG1363 chromosome by using the insertion vector pKLG610. Two transformants were obtained that differed in the number of amplified pKLG610 copies in head-to-tail arrangements on their chromosomes; MG610 contained approximately two copies, and MG611 contained about eight copies. The amplifications were stably maintained during growth in milk in the absence of antibiotics. The proteolytic activity of strain MG611 was approximately 11-fold higher than that of strain MG610 and about 1.5 times higher than that of strain MG1363(pGKV552), which carried the proteinase genes on an autonomously replicating plasmid with a copy number of approximately 5. All three strains showed rapid growth in milk with concomitant rapid production of acid. The results suggest that a limited number of copies of the proteinase genes prtP and prtM per genome is sufficient for good growth in milk.     

The plasmid-encoded lactococcal envelope-associated proteinase is encoded by a chromosomal gene in Lactococcus lactis subsp. cremoris BC101.

The plasmid-free strain Lactococcus lactis subsp. cremoris BC101 produced an extracellular proteinase physicochemically similar to the proteinase encoded by the plasmid-linked prtP gene of other lactococcal strains. The absence of detectable plasmids in strain BC101 indicated that the prtP proteinase gene may be chromosomally located. The chromosomal linkage of the prtP proteinase gene in BC101 was confirmed by pulsed-field electrophoresis of chromosomal DNA and hybridization, using as a probe the plasmid-linked prtP gene from L. lactis subsp. cremoris Wg2. The prtM gene necessary for the maturation of the proteinase was also chromosomally located adjacent to prtP in BC101. By using as a hybridization probe the ISS1-like element ISS1W, which is found adjacent to the proteinase genes in both pWV05 and pSK111, specific homology to the chromosomal fragment containing the proteinase gene was found. DNA sequencing of a polymerase chain reaction product of chromosomal DNA upstream from prtM revealed a 123-nucleotide sequence which was 100% identical to the equivalent sequence in the ISS1W-containing plasmid. The terminal inverted repeat (18 nucleotides) of the ISS1W element was found in this sequenced DNA. These findings suggest that the chromosomal proteinase gene is organized in a fashion similar to that of the plasmid-linked proteinase gene.     

The plasmid-encoded lactococcal envelope-associated proteinase is encoded by a chromosomal gene in Lactococcus lactis subsp. cremoris BC101.

The plasmid-free strain Lactococcus lactis subsp. cremoris BC101 produced an extracellular proteinase physicochemically similar to the proteinase encoded by the plasmid-linked prtP gene of other lactococcal strains. The absence of detectable plasmids in strain BC101 indicated that the prtP proteinase gene may be chromosomally located. The chromosomal linkage of the prtP proteinase gene in BC101 was confirmed by pulsed-field electrophoresis of chromosomal DNA and hybridization, using as a probe the plasmid-linked prtP gene from L. lactis subsp. cremoris Wg2. The prtM gene necessary for the maturation of the proteinase was also chromosomally located adjacent to prtP in BC101. By using as a hybridization probe the ISS1-like element ISS1W, which is found adjacent to the proteinase genes in both pWV05 and pSK111, specific homology to the chromosomal fragment containing the proteinase gene was found. DNA sequencing of a polymerase chain reaction product of chromosomal DNA upstream from prtM revealed a 123-nucleotide sequence which was 100% identical to the equivalent sequence in the ISS1W-containing plasmid. The terminal inverted repeat (18 nucleotides) of the ISS1W element was found in this sequenced DNA. These findings suggest that the chromosomal proteinase gene is organized in a fashion similar to that of the plasmid-linked proteinase gene.     

Monoclonal Antibodies to the Cell-Wall-Associated Proteinase of Lactococcus lactis subsp. cremoris Wg2

Twelve monoclonal antibodies directed to the cell-wall-associated proteinase of Lactococcus lactis subsp. cremoris Wg2 were isolated after immunization of BALB/c mice with a partially purified preparation of the proteinase. The monoclonal antibodies reacted with the 126-kilodalton proteinase band in a Western immunoblot. All but one of the monoclonal antibodies reacted with protein bands with a molecular weight below 126,000, possibly degradation products of the proteinase. The monoclonal antibodies could be divided into six groups according to their different reactions with the proteinase degradation products in the Western blot. Different groups of monoclonal antibodies reacted with different components of the L. lactis subsp. cremoris Wg2 proteinase. Crossed immunoelectrophoresis showed that monoclonal antibody groups I, II, and III react with proteinase component A and that groups IV, V, and VI react with proteinase component B. The isolated monoclonal antibodies cross-reacted with the proteinases of other L. lactis subspecies. Monoclonal antibodies of group IV cross-reacted with proteinase component C of other L. lactis subsp. cremoris strains. The molecular weight of the proteinase attached to the cells of L. lactis subsp. cremoris Wg2 was 200,000, which is different from the previously reported values. This could be analyzed by immunodetection of the proteinase on a Western blot. This value corresponds to the molecular weight calculated from the amino acid sequence of the cloned L. lactis subsp. cremoris Wg2 proteinase gene.     

Identification of Lactococcus lactis genes required for bacteriophage adsorption

The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and phi 645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that phi645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that phi 645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and phi 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and phi 645 that showed no homology in the C-terminal part.     

Primary structure and organization of the gene for a procaryotic, cell envelope-located serine proteinase

We have determined the complete nucleotide sequence of the gene for the cell envelope-located proteinase of Lactococcus lactis SK11. The gene contains a very AT-rich promoter region followed by the coding sequence of a protein of 1962 amino acids. Comparison of the NH2-terminal amino acid sequence of the mature proteinase and the expected primary translation product of the proteinase gene indicates that the enzyme is probably synthesized as a pre-pro-protein. This is confirmed by expression studies of the proteinase gene in Escherichia coli. The amino acid sequence of the proteinase shows significant homology to a number of serine proteinases of the subtilisin family. Compared with the related proteinase of L. lactis Wg2, the proteinase of L. lactis SK11 contains a 60-amino acids duplication and a total of 44-amino acid substitutions, some of which may account for the different cleavage specificity of both enzymes. Furthermore, a region was identified in the Lactococcus proteinase, which shows homology to the membrane-anchoring domains of a number of proteins from other Gram-positive bacteria.     

Cloning and expression of the Lactococcus lactis subsp. cremoris SK11 gene encoding an extracellular serine proteinase

The Lactococcus lactis subsp. cremoris SK11 plasmid-located prtP gene, encoding a cell-envelope-located proteinase (PrtP) that degrades alpha s1-, beta- and kappa-casein, was identified in a lambda EMBL3 gene library in Escherichia coli using immunological methods. The complete prtP gene could not be cloned in E. coli and L. lactis on high-copy-number plasmid vectors. However, using a low-copy-number vector, the complete prtP gene could be cloned in strains MG1363 and SK1128, proteinase-deficient derivatives of L. lactis subsp. lactis 712 and L. lactis subsp. cremoris SK11, respectively. The proteinase deficiency of these hosts was complemented to wild-type (wt) levels by the cloned SK11 prtP gene. The caseinolytic specificity of the proteinase specified by the cloned prtP gene was identical to that encoded by the wt proteinase plasmid, pSK111. The expression of recombinant plasmids containing 3' and 5' deletions of prtP was analyzed with specific attention directed towards the location of the gene products. In this way the expression signals of prtP were localized and overproduction was obtained in L. lactis subsp. lactis. Furthermore, a region at the C terminus of PrtP was identified which is involved in cell-envelope attachment in lactococci. A deletion derivative of prtP was constructed which specifies a C-terminally truncated proteinase that is well expressed and fully secreted into the medium, and still shows the same capacity to degrade alpha s1-, beta- and kappa-casein.     

Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis.

A pair of vectors for expression of heterologous genes in Lactococcus lactis was constructed. In addition to an origin of replication that has a broad host range, these vectors contain a multiple cloning site flanked by gene expression signals originating from L. lactis subsp. cremoris Wg2. The two vectors, about 3.7 kilobase pairs in size, differ only in the type of antibiotic resistance they confer to their hosts. pMG36 carries a kanamycin resistance marker, which was replaced by an erythromycin resistance marker in pMG36e. As an example of the use of these vectors, the hen egg white lysozyme-coding sequence was inserted. A fusion protein of the expected size was detected in a transformed L. lactis subsp. lactis strain by using Western blotting (immunoblotting).     

Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis

A pair of vectors for expression of heterologous genes in Lactococcus lactis was constructed. In addition to an origin of replication that has a broad host range, these vectors contain a multiple cloning site flanked by gene expression signals originating from L. lactis subsp. cremoris Wg2. The two vectors, about 3.7 kilobase pairs in size, differ only in the type of antibiotic resistance they confer to their hosts. pMG36 carries a kanamycin resistance marker, which was replaced by an erythromycin resistance marker in pMG36e. As an example of the use of these vectors, the hen egg white lysozyme-coding sequence was inserted. A fusion protein of the expected size was detected in a transformed L. lactis subsp. lactis strain by using Western blotting (immunoblotting).     

Is protein phosphorylation a control mechanism for the degradation of caseins by lactic acid bacteria? The detection of an extracellular acid phosphatase activity

The existence of an extracellular acid phosphatase activity in Lactobacillus delbrueckii subsp. bulgaricus ACADC235 and in Lactococcus lactis subsp. cremoris Wg2 is reported in the present work. Acid phosphatase ( p -nitrophenyl phosphatase, pNPPase) activity was detected both in whole cells and in crude cell-wall extracts and coelutes from Sepharose 6B together with a fraction of proteinase activity and a high Mr phosphopolyanionic material. It represents a putative protein phosphatase as it efficiently dephosphorylates casein and phosvitin. This extracellular acid phosphatase activity seems to play a role in the control of casein proteolysis by lactic acid bacteria since dephosphorylation of casein enhances its degradation by the cell wall-bound proteinase.     

限制和修饰系统LlaBIII在构建抗噬菌体菌株中的作用

限制和修饰 (ｒｅｓｔｒｉｃｔｉｏｎａｎｄｍｏｄｉｆｉｃａｔｉｏｎ ,Ｒ Ｍ)系统是指由限制性内切酶和甲基化酶组成的单亚基或多亚基复合酶系统 ,两者通常成对出现 ,具有相同的ＤＮＡ识别位点 ,其作用相反。Ｒ Ｍ系统在原核生物中普遍存在 ,在保护细胞免遭外源病毒侵害方面具有重要作用[1] 。作为发酵剂的乳酸乳球菌在乳制品发酵中具有重要作用 ,但这类菌株极易遭受噬菌体感染 ,导致菌株产酸力降低 ,甚至发酵失败 ,造成严重的经济损失。所以在乳制品发酵过程中防止噬菌体感染就成为十分重要的问题。通过自然筛选或诱变处理等手段筛选噬菌…     

Engineering of the Lactococcus lactis serine proteinase by construction of hybrid enzymes

Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of the prtP genes of Lactococcus lactis strains Wg2 and SK11. These plasmids were introduced into the plasmid-free strain L. lactis MG1363. The serine proteinases produced by these L. lactis strains were isolated, and their cleavage specificity and rate towards alpha s1- and beta-casein was investigated. The catalytic properties of both the SK11 and Wg2 proteinases, which differ in 44 out of 1902 amino acid residues, could be changed dramatically by the reciprocal exchange of specific fragments between the two enzymes. As a result, various L. lactis strains were constructed having new proteolytic properties that differ from those of the parental strains. Furthermore, two segments in the proteinase could be identified that contribute significantly to the cleavage specificity towards casein; within these two segments, several amino acid residues were identified that are important for substrate cleavage rate and specificity. The results also indicate that the lactococcal proteinase has an additional domain involved in substrate binding compared with the related subtilisins. This suggests that the 200 kd L. lactis proteinase may be the representative of a new subclass of subtilisin-like enzymes.     

Mechanism of Proteinase Release from Lactococcus lactis subsp. cremoris Wg2

The procedure generally used for the isolation of extracellular, cell-associated proteinases of Lactococcus lactis species is based on the release of the proteinases by repeated incubation and washing of the cells in a Ca²⁺-free buffer. For L. lactis subsp. cremoris Wg2, as many as five incubations for 30 min at 29°C are needed in order to liberate 95% of the proteinase. Proteinase release was not affected by chloramphenicol, which indicates that release is not the result of protein synthesis during the incubations. Ca²⁺ inhibited, while ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) stimulated, proteinase release from the cells. The pH optimum for proteinase release ranged between 6.5 and 7.5, which was higher than the optimum pH of the proteinase measured for casein hydrolysis (i.e., 6.4). Treatment of cells with the serine proteinase inhibitor phenylmethylsulfonyl fluoride prior to the incubations in Ca²⁺-free buffer reduced the release of the proteinase by 70 to 80%. The residual proteinase remained cell associated but could be removed by the addition of active L. lactis subsp. cremoris Wg2 proteinase. This suggests that proteinase release from cells of L. lactis subsp. cremoris Wg2 is the result of autoproteolytic activity. From a comparison of the N-terminal amino acid sequence of the released proteinase with the complete amino acid sequence determined from the nucleotide sequence of the proteinase gene, a protein of 180 kilodaltons would be expected. However, a proteinase with a molecular weight of 165,000 was found, which indicated that further hydrolysis had occurred at the C terminus.