A Potential Food-Grade Cloning Vector for Streptococcus thermophilus That Uses Cadmium Resistance as the Selectable Marker

A potential food-grade cloning vector, pND919, was constructed and transformed into S. thermophilus ST3-1, a plasmid-free strain. The vector contains DNAs from two different food-approved organisms, Streptococcus thermophilus and Lactococcus lactis. The 5.0-kb pND919 is a derivative of the cloning vector pND918 (9.3 kb) and was constructed by deletion of the 4.3-kb region of pND918 which contained DNA from non-food-approved organisms. pND919 carries a heterologous native cadmium resistance selectable marker from L. lactis M71 and expresses the Cd^r phenotype in S. thermophilus transformants. With the S. thermophilus replicon derived from the shuttle vector pND913, pND919 is able to replicate in the two S. thermophilus industrial strains tested, ST3-1 and ST4-1. Its relatively high retention rate in S. thermophilus further indicates its usefulness as a potential food-grade cloning vector. To our knowledge, this is the first report of a replicative potential food-grade vector for the industrially important organism S. thermophilus.     

Development of food-grade cloning and expression vectors for Lactococcus lactis

AIMS: To develop food-grade cloning and expression vectors for use in genetic modification of Lactococcus lactis. METHODS AND RESULTS: Two plasmid replicons and three dominant selection markers were isolated from L. lactis and used to construct five food-grade cloning vectors. These vectors were composed of DNA only from L. lactis and contained no antibiotic resistance markers. Three of the vectors (pND632, pND648 and pND969) were based on the same plasmid replicon and carried, either alone or in combination, the three different selectable markers encoding resistance to nisin, cadmium and/or copper. The other two (pND965DJ and pND965RS) were derived from a cadmium resistance plasmid, and carried a constitutive promoter and a copper-inducible promoter, respectively, immediately upstream of a multicloning site. All vectors were stable in L. lactis LM0230 for at least 40 generations without selection pressure. The two groups of vectors were compatible in L. lactis LM0230. The vectors pND648 and pND965RS, as representatives of the two groups, were transferred successfully by electroporation into and maintained in an industrial strain of L. lactis. The usefulness of the vectors was further demonstrated by expressing a phage resistance gene (abiI) in another industrial strain of L. lactis. CONCLUSIONS: The five food-grade vectors constructed are potentially useful for industrial strains of L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: These vectors represent a new set of molecular tools useful for food-grade modifications of L. lactis.     

Molecular Properties of Streptococcus thermophilus Plasmid pER35 Encoding a Restriction Modification System

Bacteriophage attack on lactic fermentation bacteria (LFB) is costly to the dairy industry because it results in product loss. One mechanism used by LFB to protect themselves from bacteriophage attack is restriction of foreign DNA. Three plasmids, pER16, pER35, and pER36, from three different strains of the thermotolerant dairy fermentation bacterium Streptococcus thermophilus were sequenced. One of these plasmids, pER35, isolated from S. thermophilus ST135, encoded a type IC restriction-modification (R-M) system very similar to those encoded on plasmids pIL2614 in Lactococcus lactis subsp. lactis and pND861 in Lactococcus lactis biovar diacetylactis. The high degree of identity between the R-M systems encoded on pER35, pIL2614, and pND861 indicated the potential for horizontal transfer of these genes between different species of lactic fermentation bacteria. Similar to the functional R-M system encoded on pIL2614 that protects the mesophilic L. lactis subsp. lactis against phage attack, the R-M system on pER35 most likely functions in the same role in S. thermophilus ST135. The plasmid pER16 was found to encode the specificity subunit of the R-M system, but not the R or M subunits. In addition, all three plasmids encoded proteins that are present on other S. thermophilus plasmids, including a protein for rolling-circle replication (RepA) and a low-molecular-weight stress protein (Hsp). The presence of a complete R-M system encoded on a plasmid in S. thermophilus, a species that often lacks plasmids, is novel and may be beneficial for protecting S. thermophilus from bacteriophage attack under dairy fermentation conditions.     

Cloning and expression of the beta-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli

The beta-D-galactosidase (beta-gal) gene from Streptococcus thermophilus was cloned to isolate and characterize it for potential use as a selection marker in a food-grade cloning vector. Chromosomal DNA from S. thermophilus 19258 was cleaved with the restriction enzyme PstI and ligated to pBR322 for transformation into Escherichia coli JM108. A beta-galactosidase-positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. This transformant possessed a single plasmid, designated pRH116, which contained, in addition to the vector DNA, a 7.0-kilobase (kb) PstI insertion fragment coding for beta-gal activity. An extract from JM108(pRH116) contained a beta-gal protein with the same electrophoretic mobility as the beta-gal from S. thermophilus 19258. Compared with the beta-gal from E. coli HB101, the S. thermophilus beta-gal was of lower molecular weight. A restriction map of pRH116 was constructed from cleavage of both the plasmid and the purified insert. The construction of deletion derivatives of pRH116 with BglII, BstEII, and HindIII revealed the approximate location of the gene on the 7.0-kb fragment. The beta-gal gene was further localized to a 3.85-kb region.     

Cloning and expression of the beta-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli.

The beta-D-galactosidase (beta-gal) gene from Streptococcus thermophilus was cloned to isolate and characterize it for potential use as a selection marker in a food-grade cloning vector. Chromosomal DNA from S. thermophilus 19258 was cleaved with the restriction enzyme PstI and ligated to pBR322 for transformation into Escherichia coli JM108. A beta-galactosidase-positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. This transformant possessed a single plasmid, designated pRH116, which contained, in addition to the vector DNA, a 7.0-kilobase (kb) PstI insertion fragment coding for beta-gal activity. An extract from JM108(pRH116) contained a beta-gal protein with the same electrophoretic mobility as the beta-gal from S. thermophilus 19258. Compared with the beta-gal from E. coli HB101, the S. thermophilus beta-gal was of lower molecular weight. A restriction map of pRH116 was constructed from cleavage of both the plasmid and the purified insert. The construction of deletion derivatives of pRH116 with BglII, BstEII, and HindIII revealed the approximate location of the gene on the 7.0-kb fragment. The beta-gal gene was further localized to a 3.85-kb region.     

Cloning Vectors for Lactococci Based on a Plasmid Encoding Resistance to Cadmium

An 8.8-kb plasmid (pND302) was identified in Lactococcus lactis spp lactis M71 which encodes cadmium resistance (Cd^R). Most of the commercial lactococcal strains tested were sensitive to cadmium. Therefore, Cd^R should provide a useful selectable marker for constructing cloning vectors in lactococci. pND302 was mapped with a number of restriction enzymes and found to contain a unique EcoRI site suitable for cloning. Two E. coli/L. lactis shuttle cloning vectors, pND304 and pND624, were constructed by subcloning of the E. coli plasmids pBR322 and pGEM-7Zf(+) containing a 1.6-kb gene encoding nisin resistance (Nis^R) of lactococcal origin into the EcoRI site of pND302, separately. The E. coli DNA component of pND624 was removed and the resulting plasmid, pND625, consisted of only lactococcal DNA, expressing Nis^R and Cd^R, with two synthetic polylinkers that contain multiple restriction sites for versatile cloning. Both pND302 and pND625 can be transformed by electroporation into L. lactis LMO230 at 10³/μg DNA and maintained stably in LMO230. The results indicated that pND302 and pND625 are potential food-grade cloning vectors for lactococci. Received: 27 November 1995 / Accepted: 29 December 1995     

Additive Effects of Two Different Plasmid-Linked Restriction and Modification Systems in Lactococcus

Two plasmids, pND801 and pND802, encoding different restriction and modification systems were isolated from Lactococcus lactis ssp. lactis LL42-1 and Lactococcus lactis ssp. cremoris LC14-1, respectively. pND802 contained one Sphl restriction enzyme site and the whole plasmid was cloned into the Sphl site of the streptococcal/ E. coli shuttle vector pSA3 generating the plasmid pND803. pND803 was stably maintained in L.lactis MG1363 harbouring pND801. The combination of the two R/M systems within L.lactis MG1363 resulted in an additive resistance towards both isometric phage and prolate phage.     

A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI

A new food-grade cloning vector for lactic acid bacteria was constructed using the nisin immunity gene nisI as a selection marker. The food-grade plasmid, pLEB590, was constructed entirely of lactococcal DNA: the pSH71 replicon, the nisI gene, and the constitutive promoter P45 for nisI expression. Electroporation into Lactococcus lactis MG1614 with 60 international units (IU) nisin/ml selection yielded approximately 10⁵ transformants/μg DNA. MG1614 carrying pLEB590 was shown to be able to grow in medium containing a maximum of 250 IU nisin/ml. Plasmid pLEB590 was succesfully transformed into an industrial L. lactis cheese starter carrying multiple cryptic plasmids. Suitability for molecular cloning was confirmed by cloning and expressing the proline iminopeptidase gene pepI from Lactobacillus helveticus in L. lactis and Lb. plantarum. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in lactic acid bacteria in order to construct improved starter bacteria for food applications. Electronic Publication     

A food-grade approach for functional analysis and modification of native plasmids in Lactococcus lactis

While plasmids from lactic acid bacteria possess many traits that are of industrial value, their exploitation is often frustrated by an inability to conduct food-grade engineering of native plasmids or to readily screen for their transfer. Here we describe a system that uses a RepA(+) temperature-sensitive helper plasmid and a RepA(-) cloning vector to overcome these problems while maintaining the food-grade status of the native plasmid. This strategy was used to precisely delete ltnA1 alone, or in conjunction with ltnA2 (encoding the structural proteins of the lantibiotic lacticin 3147), from the native 60.2-kb plasmid pMRC01 and to select for the transfer of pMRC01 between Lactococcus lactis strains.     

Development of an antibiotic-free plasmid selection system based on thymine auxotrophy in Lactococcus lactis

Lactococcus lactis has become the best studied species of the lactic acid bacteria (LAB) clade and an ideal cell factory for heterogenous proteins. We have constructed an antibiotic-free expression vector, pMG-thyA, using thymidine synthase gene thyA as the selection marker. The thyA gene was cloned from the food industry strain Streptococcus thermophilus St-JY and was used to replace the erythromycin resistance genes on L. lactis expression vector pMG36e in order to construct pMG-thyA. The construction of the new vector and thyA-null host L. lactis MG1363-TT yielded an antibiotic-free expression system. The α-amylase gene (amy) was cloned onto the multiple cloning site of the vector pMG-thyA as a reporter gene, yielding the recombinant plasmid pMGthyA-amy. This plasmid was electroporated into L. lactis MG1363-TT, and the recombinant strain grown on SA plates containing 0.5 % (w/v) soluble starch formed distinct bacterial colonies and clear zones (halo) around the colonies following the addition of iodine solution. These research findings lay the foundation for food-grade expression in L. lactis.     

Comparative sequence analysis of plasmids from Lactobacillus delbrueckii and construction of a shuttle cloning vector

While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with Lactobacillus delbrueckii, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of Lactobacillus delbrueckii subsp. bulgaricus was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic ori and encoded six open reading frames (ORFs), of which functions could be predicted for three-a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available L. delbrueckii plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for L. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from Streptococcus thermophilus. This observation and the ability of the replicon to function in S. thermophilus support the idea that the origin of plasmids in L. delbrueckii was likely from S. thermophilus. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from L. delbrueckii has previously been constructed, an Escherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A ori, the chloramphenicol resistance gene of pCI372, and the lacZ polylinker from pUC18. This cloning vector was successfully introduced into E. coli, L. delbrueckii subsp. bulgaricus, S. thermophilus, and Lactococcus lactis. This shuttle cloning vector provides a new tool for molecular analysis of Lactobacillus delbrueckii and other lactic acid bacteria.     

Plasmid-encoded copper resistance in Lactococcus lactis

A 54-kb plasmid (pND306) from Lactococcus lactis subsp. lactis 1252D encoded resistance to both Cu and Sn . The copper resistance determinant was subcloned on a 12.8-kb PvuII DNA fragment and mapped using a number of restriction endonucleases. Six other copper resistant lactococcal strains were also identified and all contained multiple plasmids. Plasmids in five of these strains showed strong hybridization with a probe made using the 12.8-kb DNA fragment, however no chromosomal homologs were detected. The copper resistance determinant was further isolated as a 10.6-kb SphI fragment and used to construct pND968 that expresses resistance to both copper and nisin.     

ThyA as a selection marker in construction of food-grade host-vector and integration systems for Streptococcus thermophilus

We constructed food-grade host-vector and integration systems for Streptococcus thermophilus by using a thymidylate synthase gene (thyA) as the selection marker. Two thyA genes, thyA(St) and thyA(Lb), were cloned from S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, respectively. Thymidine-requiring mutants of S. thermophilus were obtained after successive cultures in the presence of trimethoprim, and one of them, TM1-1, was used as the host. Food-grade vectors were constructed by using either thyA(St) or thyA(Lb) as the selection marker. Transformants of TM1-1 created by using these vectors were selected for thymidine autotrophy as efficiently as for erythromycin resistance. By using the host-vector system developed in this way, a foreign amylase gene (amyA) was expressed in TM1-1 and was also integrated into the chromosome by use of a temperature-sensitive integration vector constructed with thyA(Lb) as the selection marker via a double-crossover event. The results obtained show that thyA is an efficient and safe selection marker for S. thermophilus that is suitable for food applications.     

Expression of a Lactococcus lactis Phage Resistance Mechanism by Streptococcus thermophilus

The 7.8-kb lactococcal plasmid pSRQ700 encodes the LlaII restriction/modification system which recognizes and cleaves the sequence 3(prm1)-GATC-5(prm1). When the plasmid pSRQ700 is introduced into a phage-sensitive Lactococcus lactis strain, strong phage resistance is conferred by the LlaII system. In this report, we show that pSRQ700 cannot replicate in Streptococcus thermophilus. However, if cloned into the vector pNZ123, the native LlaII system is expressed and strong phage resistance is conferred to various industrial S. thermophilus strains. Resistance against phages isolated from yogurt and mozzarella wheys was observed. To our knowledge, this is the first report of increased phage resistance in S. thermophilus.     

A novel vector for lactic acid bacteria that uses a bile salt hydrolase gene as a potential food-grade selection marker

A novel vector pM4aB for lactic acid bacterial was developed using a bile salt hydrolase gene from Lactobacillus plantarum as a potential food-grade selection marker. The 3.0-kb pM4aB consisted of the replicon of Lactobacillus plasmid pM4, a multiple cloning site and the bsh gene, which was constructed by elimination of a 5.5-kb non-food-grade DNA fragment from an 8.5-kb intermediate vector pBEmpM4aB. For electroporation into Lactobacillus paracasei X9, a high transformation efficiency of 4.0±1.0×10(4) CFU/μg plasmid DNA was yielded with 0.1% (wt/vol) glycodeoxycholic acid sodium selection. A high segregation stability of the vector was also observed as only 0.1% plasmid was lost after 50 generations of growth without selection pressure. The application potential of pM4aB was further confirmed by expression of a catalase gene from Lactobacillus sakei in L. paracasei. These results revealed that the novel vector pM4aB constructed in this study would be a useful tool for genetic modification of the industrially important LAB.     

Molecular cloning of lactose genes in dairy lactic streptococci: the phospho-beta-galactosidase and beta-galactosidase genes and their expression products

The mesophilic (S. lactis and S. cremoris) and thermophilic (S. thermophilus) dairy lactic streptococci, which are used in industrial dairy fermentations, contain two different lactose hydrolysing enzymes, a phospho-beta-galactosidase and a beta-galactosidase. The central role of these enzymes in the pathways used for lactose transport and degradation is discussed along with their properties and distributions in lactic streptococci. In addition, recent results on the cloning, expression and sequence organization of the genes for the mesophilic phospho-beta-galactosidase and thermophilic beta-galactosidase are reviewed. Original data are presented concerning heterologous gene expression in the study of lactose hydrolysis in lactic streptococci. These include 1) the purification of the S. lactis phospho-beta-galactosidase from an overproducing Escherichia coli, and 2) the expression of the E. coli beta-galactosidase (lacZ) gene in S. lactis employing a lactic streptococcal expression vector.     

Food-grade host/vector expression system for <Emphasis Type="Italic">Lactobacillus casei</Emphasis> based on complementation of plasmid-associated phospho-β-galactosidase gene <Emphasis Type="Italic">lacG</Emphasis>

A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-beta-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-beta-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.     

A food-grade cloning system for industrial strains of Lactococcus lactis

We have previously reported the construction of a food-grade cloning vector for Lactococcus using the ochre suppressor, supB, as the selective marker. This vector, pFG1, causes only a slight growth inhibition in the laboratory strain MG1363 but is unstable in the industrial strains tested. As supB suppresses both amber and ochre stop codons, which are present in 82% of all known lactococcal genes, this undesirable finding may result from the accumulation of elongated mistranslated polypeptides. Here, we report the development of a new food-grade cloning vector, pFG200, which is suitable for overexpressing a variety of genes in industrial strains of Lactococcus lactis. The vector uses an amber suppressor, supD, as selectable marker and consists entirely of Lactococcus DNA, with the exception of a small polylinker region. Using suppressible pyrimidine auxotrophs, selection and maintenance are efficient in any pyrimidine-free medium including milk. Importantly, the presence of this vector in a variety of industrial strains has no significant effect on the growth rate or the rate of acidification in milk, making this an ideal system for food-grade modification of industrially relevant L. lactis strains. The usefulness of this system is demonstrated by overexpressing the pepN gene in a number of industrial backgrounds.     

Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis

Cloning vector plasmids have been constructed on the basis of the broad host range plasmid pAM beta 1 and used for the cloning of a nisin resistance determinant in Streptococcus lactis. They incorporate several desirable features for gene cloning in S. lactis and other transformable Gram-positive bacteria. They carry an easily selectable erythromycin resistance marker, are present at low (6-9) or high (45-85) copy number in S. lactis and possess a convenient polyrestriction site sequence. A significant advantage of these plasmids is their capability to carry and stably maintain very large cloned DNA fragments (up to 30 kilobases).