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.     

Isolation of Lactococcus lactis nonsense suppressors and construction of a food-grade cloning vector

Nonsense suppressor strains of Lactococcus lactis were isolated using plasmids containing nonsense mutations or as revertants of a nonsense auxotrophic mutant. The nonsense suppressor gene was cloned from two suppressor strains and the DNA sequence determined. One suppressor is an ochre suppressor with an altered tRNA_gin and the other an amber suppressor with an altered tRNA_ser. The nonsense suppressors allowed isolation of nonsense mutants of a lytic bacteriophage and suppressible auxotrophic mutants of L. lactis MG1363. A food-grade cloning vector based totally on DNA from Lactococcus and a synthetic polylinker with 11 unique restriction sites was constructed using the ochre suppressor as a selectable marker. Selection, following etectroporation of a suppressible purine auxotroph, can be done on purine-free medium. The pepN gene from L. lactis Wg2 was subcloned resulting in a food-grade plasmid giving a four- to fivefold increase in lysine aminopeptidase activity.     

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.     

Construction and Application of a Food-Grade Expression System for Lactococcus lactis

A food-grade host/vector expression system for Lactococcus lactis was constructed using alanine racemase gene (alr) as the complementation marker. We obtained an alanine racemase auxotrophic mutant L. lactis NZ9000Δalr by double-crossover recombination using temperature-sensitive integration plasmid pG⁺host9 and a food-grade vector pALR with entirely lactococcal DNA elements, including lactococcal replicon, nisin-inducible promoter PnisA and the alr gene from Lactobacillus casei BL23 as a complementation marker. By using the new food-grade host/vector system, the green fluorescent protein and capsid protein of porcine circovirus type II were successfully overexpressed under the nisin induction. These results indicate that this food-grade host/vector expression system has application potential as an excellent antigen delivery vehicle, and is also suitable for the use in the manufacture of ingredients for the food industry.     

Molecular Characterization of a Theta Replication Plasmid and Its Use for Development of a Two-Component Food-Grade Cloning System for Lactococcus lactis

pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative cis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-acting ori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is a repB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis if trans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactis and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.     

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.     

Yeast suppressors of UAA and UAG nonsense codons work efficiently in vitro via tRNA

A cell-free protein-synthesizing system, containing an S-100 fraction from yeast, ribosomal subunits from Krebs ascites cells, and ribosome initiation factors from rabbit reticulocytes, translates yeast, adenovirus, and rabbit globin messenger RNAs and the RNA from bacteriophage Qβ. An amber mutation in the Qβ synthetase gene is suppressed in vitro if the S-100 fraction is from yeast strains carrying amber suppressor mutations. Suppressor SUP6-2 gives 16% suppression, and the recessive lethal suppressor RL-1 gives 50% suppression. Extracts from strain FM6, which has the ochre suppressor SUP4-1, give a longer protein product from the normal synthetase gene of Qβ with an efficiency of 63%. This implies that UAA is the terminator for the synthetase gene, and that synthesis of this read through protein can be used as an assay for ochre suppression. Suppression in each of these cases is mediated by tRNA, since purified tRNA is the only fraction from suppressing strains that is required in an otherwise nonsuppressing cell-free system.     

Identification of the Propionicin F Bacteriocin Immunity Gene (pcfI) and Development of a Food-Grade Cloning System for Propionibacterium freudenreichii

This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 10⁷ transformants/μg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive P_pampS promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by ~91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.     

Use of GFP to trace the colonization of Lactococcus lactis WH-C1 in the gastrointestinal tract of mice

A new expression vector for Lactococcus was constructed using nisI as a selection marker and GFP as a reporter protein to explore the colonization characteristics in vivo of Lactococcus lactis WH-C1. By high expression of GFP, it was shown WH-C1 could pass through the stomach and survive in the gastrointestinal tract.     

A functional requirement for modification of the wobble nucleotide in the anticodon of a T4 suppressor tRNA

Temperature-sensitive mutants of E. coli have been isolated which restrict the growth of strains of bacteriophage T4 which are dependent upon the function of a T4-coded amber or ochre suppressor transfer RNA. One such mutant restricts the growth of certain ochre but not amber suppressor-requiring phage. Analysis of the T4 tRNAs synthesized in this host revealed that many nucleotide modifications are significantly reduced. The modifications most strongly affected are located in the anticodon regions of the tRNAs. The T4 ochre suppressor tRNAs normally contain a modified U residue in the wobble position of the anticodon; it has been possible to correlate the absence of this specific modification in the mutant host with the restriction of suppressor activity. Furthermore, the extent of this restriction varies dramatically with the site of the nonsense codon, indicating that the modification requirement is strongly influenced by the local context of the mRNA. An analysis of spontaneous revertants of the E. coli ts mutant indicates that temperature sensitivity, restriction of phage suppressor function, and undermodification of tRNA are the consequences of a single genetic lesion. The isolation of a class of partial revertants to temperature insensitivity which have simultaneously become sensitive to streptomycin suggests that the translational requirement for the anticodon modification can be partially overcome by a change in the structure of the ribosome.     

Adhesive properties of predominant bacteria in raw cow’s milk to bovine mammary gland epithelial cells

Various bacteria have been found in raw cow’s milk, and identifying milk microflora and its functions is critical for maintaining cow health and farm hygiene. Although studies on pathogens and spoilage bacteria in milk have been widely reported, the relationship between milk bacteria, including nonpathogenic bacteria, and the bovine udder is poorly understood. We investigated milk microflora over 1 year using a culture-dependent method and culture-independent analysis by denaturing gradient gel electrophoresis. Among 240 isolates, Lactococcus lactis (81/240) was predominant. The predominant genera were Lactococcus, Stenotrophomonas, Microbacterium, Chryseobacterium, Serratia and Pseudomonas. Among seven strains belonging to these predominant genera, two strains of L. lactis (ssp. lactis and ssp. cremoris) exhibited the highest adherence to bovine mammary gland epithelial cells (BMECs) derived from the bovine udder; 3.4 % of the inoculated bacteria adhered to BMECs. This was followed by Serratia sp. (1.6 %), Microbacterium sp. (0.8 %), Stenotrophomonas maltophilia (0.5 %), Pseudomonas sp. (0.3 %) and Chryseobacterium sp. (0.1 %). The two L. lactis isolates exhibited higher adherence to BMECs than type strains and isolates of various origins.     

Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells

We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair. We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells. Activity of each suppressor tRNA is dependent upon the expression of E.coli glutaminyl-tRNA synthetase, indicating that none of the suppressor tRNAs are aminoacylated by any of the twenty aminoacyl-tRNA synthetases in the mammalian cytoplasm. Amber, ochre and opal suppressor tRNAs with a wide range of activities in suppression (increases of up to 36, 156 and 200-fold, respectively) have been generated by introducing further mutations into the suppressor tRNA genes. The most active suppressor tRNAs have been used in combination to concomitantly suppress two or three termination codons in an mRNA. We discuss the potential use of these 21st synthetase-suppressor tRNA pairs for the site-specific incorporation of two or, possibly, even three different unnatural amino acids into proteins and for the regulated suppression of amber, ochre and opal termination codons in mammalian cells.     

<Emphasis Type="Italic">Lactococcus lactis</Emphasis> M4, a potential host for the expression of heterologous proteins

Background  

Many plasmid-harbouring strains of Lactococcus lactis have been isolated from milk and other sources. Plasmids of Lactococcus have been shown to harbour antibiotic resistance genes and those that express some important proteins. The generally regarded as safe (GRAS) status of L. lactis also makes it an attractive host for the production of proteins that are beneficial in numerous applications such as the production of biopharmaceutical and nutraceutical. In the present work, strains of L. lactis were isolated from cow's milk, plasmids were isolated and characterised and one of the strains was identified as a potential new lactococcal host for the expression of heterologous proteins.     

Evidence that the supE44 Mutation of Escherichia coli Is an Amber Suppressor Allele of glnX and that It Also Suppresses Ochre and Opal Nonsense Mutations

Translational readthrough of nonsense codons is seen not only in organisms possessing one or more tRNA suppressors but also in strains lacking suppressors. Amber suppressor tRNAs have been reported to suppress only amber nonsense mutations, unlike ochre suppressors, which can suppress both amber and ochre mutations, essentially due to wobble base pairing. In an Escherichia coli strain carrying the lacZU118 episome (an ochre mutation in the lacZ gene) and harboring the supE44 allele, suppression of the ochre mutation was observed after 7 days of incubation. The presence of the supE44 lesion in the relevant strains was confirmed by sequencing, and it was found to be in the duplicate copy of the glnV tRNA gene, glnX. To investigate this further, an in vivo luciferase assay developed by D. W. Schultz and M. Yarus (J. Bacteriol. 172:595-602, 1990) was employed to evaluate the efficiency of suppression of amber (UAG), ochre (UAA), and opal (UGA) mutations by supE44. We have shown here that supE44 suppresses ochre as well as opal nonsense mutations, with comparable efficiencies. The readthrough of nonsense mutations in a wild-type E. coli strain was much lower than that in a supE44 strain when measured by the luciferase assay. Increased suppression of nonsense mutations, especially ochre and opal, by supE44 was found to be growth phase dependent, as this phenomenon was only observed in stationary phase and not in logarithmic phase. These results have implications for the decoding accuracy of the translational machinery, particularly in stationary growth phase.Translation termination is mediated by one of the three stop codons (UAA, UAG, or UGA). When such stop codons arise in coding sequences due to mutations, referred to as nonsense mutations, they lead to abrupt arrest of the translation process. However, the termination efficiency of such nonsense codons is not 100%, as certain tRNAs have the ability to read these nonsense codons. Genetic code ambiguity is seen in several organisms. Stop codons have been shown to have alternate roles apart from translation termination. In organisms from all three domains of life, UGA encodes selenocysteine through a specialized mechanism. In Methanosarcinaceae, UAG encodes pyrrolysine (3). UAA and UAG are read as glutamine codons in some green algae and ciliates such as Tetrahymena and Diplomonads (24), and UAG alone encodes glutamine in Moloney murine leukemia virus (32). UGA encodes cysteine in Euplotes; tryptophan in some ciliates, Mycoplasma species, Spiroplasma citri, Bacillus, and tobacco rattle virus; and an unidentified amino acid in Pseudomicrothorax dubius and Nyctotherus ovalis (30). In certain cases the context of the stop codon in translational readthrough has been shown to play a role; for example, it has been reported that in vitro in tobacco mosaic virus, UAG and UAA are misread by tRNA^Tyr in a highly context-dependent manner (34, 9).Termination suppressors are of three types, i.e., amber, ochre, and opal suppressors, which are named based on their ability to suppress the three stop codons. Amber suppressors can suppress only amber codons, whereas ochre suppressors can suppress ochre codons (by normal base pairing) as well as amber codons (by wobbling) and opal suppressors can read opal and UGG tryptophan codon in certain cases. As described by Sambrook et al. (27), a few amber suppressors can also suppress ochre mutations by wobbling. The suppression efficiency varies among these suppressors, with amber suppressors generally showing increased efficiency over ochre and opal suppressors. supE44, an amber suppressor tRNA, is an allele of and is found in many commonly used strains of Escherichia coli K-12. Earlier studies have shown that supE44 is a weak amber suppressor and that its efficiency varies up to 35-fold depending on the reading context of the stop codon (8).Translational accuracy depends on several factors, which include charging of tRNAs with specific amino acids, mRNA decoding, and the presence of antibiotics such as streptomycin and mutations in ribosomal proteins which modulate the fidelity of the translational machinery. Among these, mRNA decoding errors have been reported to occur at a frequency ranging from about 10⁻³ to 10⁻⁴ per codon. Translational misreading errors also largely depend on the competition between cognate and near-cognate tRNA species. Poor availability of cognate tRNAs increases misreading (18).Several studies with E. coli and Saccharomyces cerevisiae have shown the readthrough of nonsense codons in suppressor-free cells. In a suppressor-free E. coli strain, it has been shown in vitro that glutamine is incorporated at the nonsense codons UAG and UAA (26). It has been reported that overexpression of wild-type tRNA^Gln in yeast suppresses amber as well as ochre mutations (25). In this study, we have confirmed the presence of an amber suppressor mutation in the glnX gene in a supE44 strain by sequence analysis. This was done essentially because we observed that supE44 could also suppress lacZ ochre mutations, albeit inefficiently. On further investigation using an in vivo luciferase reporter assay system for tRNA-mediated nonsense suppression (28), we found that the efficiency of suppression of amber lesion by supE44 is significantly higher than that reported previously in the literature. An increased ability to suppress ochre and opal nonsense mutations was observed in cells bearing supE44 compared to in the wild type. Such an effect was observed only in the stationary phase and was abolished in logarithmic phase.     

Identification of transfer RNA suppressors in Escherichia coli. II. Duplicate genes for tRNA2Gln.

The number of gene copies for tRNA₂^Gln in λpsu⁺2 was determined by genetic and biochemical studies. The transducing phage stimulates the production of the su⁺2 (amber suppressor) and su°2 glutamine tRNAs and methionine tRNA_m. When the su⁺2 amber suppressor was converted to an ochre suppressor by single-base mutation, the phage stimulated ochre-suppressing tRNA₂^Gln, instead of the amber-suppressing tRNA₂^Gln. From the transducing phage carrying the ochre-suppressing allele, strains carrying both ochre and amber suppressors were readily obtainable. These phages stimulated both ochre-suppressing and amber-suppressing tRNA₂^Gln, but not the non-suppressing form. We conclude that the original transducing phage carries two tRNA₂^Gln genes, one su⁺2 and one su°2. The transducing phage carrying two suppressors, ochre and amber, segregates one-gene derivatives that encode only one or the other type of suppressor tRNA. These derivatives apparently arise by unequal recombination involving the two glutamine tRNA genes in the parental phage. This segregation is not accompanied by the loss of the tRNA_m^Met gene. Based on these results, it is suggested that Escherichia coli normally carries in tandem two identical genes specifying tRNA₂^Gln at 15 minutes on the bacterial chromosome. su⁺2 mutants may arise by single-base mutations in the anticodon region of either of these two, leaving the other intact. By double mutations, tRNA₂^Gln genes could also become ochre suppressors. A tRNA_m^Met gene is located near, but not between, these two tRNA₂^Gln genes.     

Bacteriophage T4 transfer RNA. I. Isolation and characterization of two-phage-coded nonsense suppressors

Two phage-coded nonsense suppressors, psuf_a⁺ and psu_b⁺, have been isolated and characterized. Both were isolated as pseudo-wild type revertants of phage strains which carry multiple amber mutations. psu_a⁺ is an amber suppressor which occurs at a frequency of 10⁻¹¹ to 10⁻¹² and is indistinguishable from wild type phage in its growth on both B and K strains of Escherichia coli bacteria. psu_b⁺ may be either an amber or an ochre suppressor, which occurs at a frequency of 10⁻⁷ to 10⁻¹⁰ and makes small plaques on B strains, but grows very poorly or not at all on K strains. Phage with the characteristics of psu_a⁺ occur in populations of psu_b⁺ phage at a frequency of 10⁻⁴. Both suppressors insert serine in response to the amber codon at an efficiency of about 45%.     

Somatic homologous recombination in planta: The recombination frequency is dependent on the allelic state of recombining sequences and may be influenced by genomic position effects

The Escherichia coli sodA gene encoding the antioxidant enzyme Mn-containing superoxide dismutase (MnSOD), was cloned in the expression vector pMG36e. This vector has a multiple cloning site down-stream of a promoter and Shine-Dalgarno sequences derived from Lactococcus. The protein-coding region of sodA from E. coli was amplified by the polymerase chain reaction, using a thermocycler and Taq DNA polymerase before cloning into pMG36e. When introduced into E. coli, the recombinant plasmid expressed the predicted fusion protein, both in the presence and absence of oxygen. The expression of the fusion protein in E. coli was verified by SOD assays, activity gels and Western blots. The recombinant plasmid was also introduced into Lactococcus lactis, which contains a resident SOD, and into Lactobacillus gasseri, which is devoid of SOD. Transformed lactococci expressed an active SodA fusion protein plus an active hybrid protein composed of subunits of the Lactococcus and the recombinant E. coli enzymes. Transformants of L. gasseri expressed only the fusion SodA protein, which was enzymatically active.     

Nisin-selectable food-grade secretion vector for Lactococcus lactis

A nisin-resistant Lactococcus lactis strain TML01 was isolated from crude milk. A gene with 99% homology to the nisin-resistance gene, nsr, was identified. The food-grade secretion plasmid, pLEB690 (3746 bp), was constructed based on this novel nsr gene enabling primary selection with up to 5 μg nisin/ml. The functionality of pLEB690 as a secretion vector was shown by expressing and secreting the pediocin AcH gene papA in L. lactis. pLEB690 is therefore, a functional food-grade secretion vector potentially useful for the food industry.     

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.     

The Schizosaccharomyces pombe sup3-i suppressor recognizes ochre, but not amber codons in vitro and in vivo.

The inefficient suppressor sup3-i of the fission yeast Schizosaccharomyces pombe is an ochre suppressor. Sup3-i was derived from the efficient serine inserting UGA suppressor sup3-e. The cloning and sequencing of the sup3-i gene indicate that the suppressor is different from the parent sup3-e by a C----T substitution in the sequence coding for the middle position of the anticodon. In vitro translation assays supplemented with purified sup3-i tRNA and programmed with Xenopus globin mRNAs lead to the accumulation of a readthrough product in response to UAA termination signals, but not in response to UGA termination codons. Transformation of Saccharomyces cerevisiae nonsense mutant strains with plasmid DNA carrying the S. pombe sup3-i gene, led to ochre, but not amber or UGA suppression in vivo.