Plasmid-associated Bacteriocin Production by and Immunity of Lactobacillus acidophilus TK8912

Lactobacillus acidophilus TK8912 produces an antibacterial substance, designated acidocin 8912, which is active against strains of Lactobacillus and Lactococcus. Of all conditions tested, the production of acidocin 8912 was maximum at 30°C in MRS broth. Acidocin 8912 was stable to heat treatment (120°C for 20min), but completely inactivated by protease treatment. Curing a plasmid pLA103 resulted in the loss of both acidocin 8912 production (Acd⁺) and host immunity (Acd^r). A plasmid-cured strain, TK1–4 (Acd^- Acd⁸), was transformed to Acd⁺Acd^r with the pLA103 plasmid. These results provided the first direct evidence in lactobacilli for involvement of this plasmid in bacteriocin production and immunity.     

Sequencing and analysis of three plasmids from <Emphasis Type="Italic">Lactobacillus casei</Emphasis> TISTR1341 and development of plasmid-derived <Emphasis Type="Italic">Escherichia coli–L. casei</Emphasis> shuttle vectors

Pyrosequencing followed by conventional PCR and sequencing was used to determine the complete nucleotide sequence of three plasmids (pRCEID2.9, pRCEID3.2, and pRCEID13.9) from the Lactobacillus casei strain TISTR1341. The plasmid sequences were found to be almost identical, respectively, to those of pLA106, pLA105, and pLA103 from Lactobacillus acidophilus strain TK8912, suggesting that these strains may be related. Sequence analysis and comparison indicated that pRCEID2.9 replicates by a rolling circle (RC) mechanism, while pRCEID3.2 and pRCEID13.9 probably follow a theta-type mode of replication. Replicons of pRCEID2.9 and pRCEID13.9 were used to develop Escherichia coli/L. casei compatible shuttle vectors, which were stably maintained in different genetic backgrounds. Real-time quantitative PCR analysis showed copy numbers of around 4 and 15, respectively, for the pRCEID13.9- and pRCEID2.9-derived shuttle vectors per chromosome equivalent. The functionality of vector pRCEID-LC13.9 was proved by cloning and expressing in L. casei of a green fluorescent protein gene variant from Aequorea victoria under the control of the promoter from a homologous lactate dehydrogenase gene. The new vectors might complement those currently in use for the exploitation of L. casei as a cellular factory and in other biotechnological applications.     

Isolation and Characterization of Plasmid DNA in Streptococcus cremoris

Nine industrially important strains of Streptococcus cremoris (HP, AM₂, ML₁, WC, C₃, R₁, E₈, KH, and Wg₂) were shown to possess a diversity of plasmid molecules. Molecular weights of plasmids were determined from their relative mobilities after agarose gel electrophoresis and via electron microscopy. To illustrate the varied plasmid sizes, strain HP contained plasmids of 26, 18, 8.5, 3.3, and 2 megadaltons (Mdal); strain ML₁ contained plasmids of 29, 18, 9, 4, 2.2, and 1.8 Mdal; and strain AM₂ had plasmids of 42, 27, 16, and 8.4 Mdal. The numbers of plasmids observed in the other strains were 6, 5, 5, 7, 5, and 4 for C₃, E₈, KH, R₁, WC, and Wg₂, respectively. A spontaneous proteinase-negative (Prt⁻) mutant of HP was missing the 8.5-Mdal plasmid, which suggests that in this strain proteinase activity could be linked to this particular plasmid. A lactose-negative (Lac⁻) Prt⁻ mutant of ML₁ lacked the 2.2-Mdal plasmid. Under the conditions employed, antibiotic sensitivity and heavy-metal susceptibility did not correlate with the missing plasmid in Prt⁻ HP or in the Lac⁻ Prt⁻ ML₁. Curing experiments with AM₂, using acridine dyes and elevated temperatures, did not yield Lac⁻ variants. AM₂ was also cultured at high dilution rates in a chemostat for 168 h by using a buffered milk or lactic broth at 18 or 32°C with no selection of Lac⁻ derivatives. The inability to obtain Lac⁻ variants under conditions known to facilitate plasmid elimination suggests that lactose metabolism is not plasmid-mediated in AM₂.     

Cloning and nucleotide sequence of the gene for acidocin 8912, a bacteriocin from Lactobacillus acidophilus TK8912

K. KANATANI, T. TAHARA, M. OSHIMURA, K. SANO AND C. UMEZAWA. 1995. Acidocin 8912 is a bacteriocin produced by Lactobacillus acidophilus TK8912. The acidocin 8912 structural gene, acdT , was cloned and determined. It was located on the 14–kb plasmid pLA103 and encoded a 46 amino acid precursor including a 20 amino acid N-terminal extension. The precursor sequence of the acdT gene shows a conservation of the general structural characteristics of the bacteriocin precursors from some lactic acid bacteria.     

Transduction of Lactose Metabolism by Streptococcus cremoris C3 Temperate Phage

Temperate phage was induced from Streptococcus cremoris C3 and morphologically characterized by high-resolution electron micrographic techniques. Interspecies genetic transfer of lactose-fermenting ability by the temperate phage was demonstrated, using two lactose-negative (Lac⁻) S. lactis strains as recipients. Plasmid transfer was confirmed by agarose gel electrophoresis. Transductant plasmid profiles were of three types—those containing no visible plasmid deoxyribonucleic acid, those possessing a 23-megadalton (Mdal) plasmid, and those containing a 23-Mdal plasmid and a 30-Mdal plasmid. A Lac⁺ transductant could serve as a donor of the lac determinants during solid-surface matings. These results add to previously published reports of inter- and intraspecies genetic transfer in dairy starter cultures.     

Overproduction of Gene Products by the Super-High-Copy-Number Plasmid pNR333

The plasmid pNR333 is a kanamycin-resistant, deletion derivative of pNR113 with an extremely high copy number in Escherichia coli and in Proteus mirabilis. In order to determine the usefulness of pNR333 as a replication gene of vector, the genes encoding chloramphenicol acetyltransferase (CAT) and β-galactosidase (β-gal) were cloned individually into both pNR333 and other low-copy-number plasmids. The expression of the cloned genes was compared by measuring the specific activity of each enzyme and the amounts of the proteins produced. A hybrid plasmid pNR333-cat expressed 53 times as much activity of CAT as the low-copy plasmid S-a which had a copy number of four. The lacZ gene cloned in pNR333 produced 17 times as much β-gal as in the low-copy-number plasmid pNR1150. These results suggest that pNR333 is a useful vector plasmid for producing a large amount of polypeptides in E. coli hosts.     

Identification of the replication region of Lactobacillus acidophilus plasmid pLA103

Abstract The structure of the region necessary for replication of the plasmid pLA103 from Lactobacillus acidophilus TK8912 has been characterized. Sequence analysis revealed that the replication region contained an open reading frame (OrfA) encoding a 282-amino acid peptide preceded by a 22-bp tandem repeat sequence region. The predicted OrfA protein showed homology to the replication protein of a plasmid from Pediococcus halophilus . The plasmid containing the repeat sequence region preceding OrfA was able to replicate in the Lactobacillus host when provided with OrfA in trans , suggesting that the repeat sequence region contains the origin sequence essential for the pLA103 replication.     

Plasmid Profiles of Lactose-Negative and Proteinase-Deficient Mutants of Streptococcus lactis C10, ML3, and M18

Lactose- and proteinase-negative (Lac⁻ Prt⁻) mutants of Streptococcus lactis C10, ML3, and M18 were isolated after treatment with ethidium bromide. The Lac⁻ Prt⁻ mutants of C10 were missing a 40-megadalton plasmid. A 33-megadalton plasmid was absent in the ML3 mutants, and the M18 variants lacked a 45-megadalton plasmid. The results suggest a linkage of these metabolic traits to the respective plasmids. The possible complexity of the interrelationship between lactose metabolism and proteinase activity is presented.     

Cotransformation of lactococcin-producing, 2.0-mega dalton and erythromycin-resistant pGB 301 plasmids toLactococcus lactis subsp.lactis protoplast

Protoplasts of plasmid-freeLactococcus lactis subsp.lactis LM 0230 and PC₄ strains were cotransformed successfully with the plasmid pools ofL. lactis subsp.lactis 484, a lactosefermenting (Lac⁺), lactococcin-producing (Lap⁺), lactococcin-resistant (Lap^r), sucrosefermenting (Suc⁺) wild strain, its derivatives, and pGB 301 erythromycin resistance plasmid (Ery^r) at the frequencies of 10⁴ transformants/g of DNA. PC₄ protoplasts were transformed at slightly lower frequencies that LM 0230 protoplasts when the same plasmid combinations were used for transformation. Agarose gel electrophoresis of plasmids from three groups of transformants, namely, Lac^–Lap^–Ery^r, Lac⁺Suc⁺Lap⁺Lap^rEry^r, and Lac^–Suc⁺Lap⁺ Lap^rLap^r, confirmed that 2.0 and 65.0 megadalton (MDa) plasmids carried genes for Suc⁺Lap⁺Lap^r and Lac⁺ phenotypes respectively. The protoplasts could be transformed with low-molecular-weight 2.0 MDa Lap plasmid at a relatively higher frequency than those with high-molecular-weight 65.0 MDa Lac plasmid. All the transformants resembled parent culture 484 in terms of lactic acid production (0.810–0.840%), milk curdling time (6 h), and lactococcin activity (7–12 mm, zone of inhibition) againstListeria monocytogenes, Salmonella typhi, andStaphylococcus aureus. The plasmids and their respective phenotypes in PC₄ transformants were genetically more stable than those of LM 0230 protoplasts. The marker plasmid pGB 301 disappeared more frequently from the transformants when present in association with the lowmolecular-weight, high-copy-number 2.0 MDa plasmid, thereby suggesting the incompatibility of these two plasmids.     

Growth Kinetics of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> ssp. <Emphasis Type="Italic">diacetylactis</Emphasis> Harboring Different Plasmid Content

The effect of plasmid content on growth of Lactococcus lactis ssp. diacetylactis harboring different plasmids and on plasmid stability was studied. Strain DRC-2C is a plasmid Lac⁺- and Prt⁺-free strain. Strain DRC-2 utilizes lactose as carbohydrate and has proteinase activity. The plasmid-free strain DRC-2C exhibited none of these features. Plasmid-encoded properties were clearly identified. Results showed that plasmid content decreased bacterial growth in terms of the specific growth rate determined. Slightly lower specific growth rate and lactic acid production were observed in the strain of higher plasmid content owing to the plasmid presence, causing metabolic burden to the host cell. The plasmid profile results showed that the number of bands in the two strains before and after fermentation were the same. This indicated that the plasmids were stably maintained and unchanged during the fermentation. Received: 27 July 2002 / Accepted: 27 August 2002     

Stabilization of Lactose Metabolism in Streptococcus lactis C2

The integration of the lactose plasmid from lactic streptococci into the host chromosome could stabilize this trait for dairy fermentations. Sixty lactose-positive (Lac⁺) transductants of lactose- and proteinase-negative (Lac⁻ Prt⁻) LM0220 were induced for temperature phage by UV irradiation or mitomycin C. Four of the transductants, designated KB18, KB21, KB54, and KB58, yielded lysates demonstrating less than one Lac⁺ transductant per 0.2 ml of phage lysate. Successive transferring in the presence of acriflavine did not yield Lac⁻ segregants from KB18, KB21, KB54, or KB58, whereas Streptococcus lactis C2 (parent culture) and three other Lac⁺ transductants showed 12 to 88% conversion from Lac⁺ to Lac⁻ within 6 to 10 repetitive transfers. When grown in continuous culture, KB21 did not show any Lac⁻ variants in 168 h, while S. lactis C2 had 96% conversion from Lac⁺ to Lac⁻ in 144 h. Agarose gel electrophoresis of plasmid DNA isolated from KB18, KB21, KB54, and KB58 revealed that the lactose plasmid, pLM2103, normally present in Lac⁺ transductants, was missing. This suggested integration of the transferred lactose plasmid into the chromosome. In contrast to phage lysates induced from S. lactis C2, which exhibited an exponential decrease in the number of Lac⁺ transductants after exposure to small doses of UV irradiation, the transduction frequency for lactose metabolism was stimulated by UV irradiation of lysates from KB58. The latter indicated chromosomal linkage for lac and that integration of the lactose genes plasmid into the chromosome had occurred.     

Involvement of a transmissible plasmid in heat-stable exotoxin and delta-endotoxin production inBacillus thuringiensis subspeciesdarmstadiensis

A strain ofBacillus thuringiensis subsp.darmstadiensis (serotype 10), which produces heat-stable exotoxin and delta-endotoxin (Exo⁺Cry⁺), was used for curing and conjugation-like transformation experiments. After treatment with sodium dodecyl sulfate, nine independent mutants that lacked exotoxin productivity (Exo^–) were obtained. Agarose gel electrophoresis showed that all Exo^– strains had lost a plasmid, whose size was 62 megadaltons (Mdal). WhenB. thuringiensis was mated with a streptomycin-resistant (Str^r)B. cereus strain, five Exo⁺Str^r transformants that had acquired the 62-Mdal plasmid were isolated. Furthermore, the Cry⁺ phenotype was consistently associated with the Exo⁺ phenotype. These results indicate that a transmissible plasmid is involved in production of both heat-stable exotoxin and delta-endotoxin.     

Recombination between an Flac and a mini-FKm plasmid deleted for an origin of replication

The mini-F plasmid specifying resistance to kanamycin (Km), pML31, contains an origin of replication at kilobase coordinate 42.6 in the F DNA sequences. In previous research we found that this origin could be deleted by recombinant DNA techniques without the loss of plasmid maintenance functions. In this report we show that the deleted plasmid, designated pMF21, has normal incompatibility properties and a recA⁺-dependent ability to form cointegrates with an Flac plasmid. By comparison, pML31 does not form cointegrates with the Flac plasmid at a detectable frequency. The frequency for spontaneous loss of the Lac⁺ phenotype in strains containing pMF21:Flac cointegrates resembles that of the Flac plasmid; however, in some Lac⁻ variants the Km^r phenotype is retained. Examination of the plasmid DNA in four of these Lac⁻Km^r clones revealed two with normal pMF21 plasmids and two with plasmids intermediate in size between pMF21 and the Flac.     

Conjugal transfer of lactose-fermenting ability fromStreptococcus lactis C2 toLeuconostoc cremoris CAF7 yieldsLeuconostoc that ferment lactose and produce diacetyl

Summary Conjugation between lactose-fermenting (Lac⁺)Streptococcus lactis C2 and Lac^– Leuconostoc cremoris CAF7 was performed. The frequency of Lac⁺ transfer was 1.5 · 10^–2 per donor cell. Lac⁺ Leuconostoc transconjugants could ferment lactose significantly faster than wild-type cells. When grown in litmus milk fortified with 0.2% yeast extract, Lac⁺ transconjugants reached pH 4.68 within 24 h at 30°C and produced diacetyl. The identity of the transconjugants asLeuconostoc derivatives was confirmed by their resistance to phage c2 and to vancomycin (>500 g/ml), and by growth on selective medium containing azide. Plasmid profiles of 10 transconjugants showed two unique patterns. A novel enlarged plasmid was found. Southern blot hybridization revealed some homology with the 30 Md Lac⁺ plasmid of donor, recipient and the transconjugants, as well as with some of the remaining plasmids of the donor.Technical Paper No. 7953, Oregon Agricultural Experiment Station.     

Properties of Lactose Plasmid pLY101 in Lactobacillus casei

A starter strain, Lactobacillus casei C257, was found to carry a lactose plasmid, pLY101. Restriction mapping showed that pLY101 DNA was 68.2 kilobases long. Since a non-lactose-utilizing variant of C257, MSK248, lost phospho-β-galactosidase (P-β-gal) activity and pLY101 DNA had a sequence(s) homologous to the streptococcal fragment including a P-β-gal gene, pLY101 is likely to encode a P-β-gal gene required for lactose metabolism in C257. MSK248 grew in galactose medium at a rate identical to that of C257 and retained phosphoenolpyruvate-dependent phosphotransferase system activity for lactose similar to that of C257. Therefore, the C257 chromosome appears to encode a complete set of genes for the lactose-phosphotransferase system and the predominant galactose metabolic pathway in C257. pLY101 DNA had a sequence homologous to a lactobacillus insertion sequence, ISL1, which mapped more than 12 kilobases from the sequence homologous to the streptococcal P-β-gal fragment.     

Cloning and sequencing of plasmid pLC494 isolated from human intestinal Lactobacillus casei: construction of an Escherichia coli-Lactobacillus shuttle vector

The complete nucleotide sequence of plasmid pLC494 isolated from Lactobacillus casei L-49 was determined. Plasmid pLC494 is an 8846-bp long circular molecule with a G+C content of 41.5%. Two putative open reading frames, ORF4 (282 amino acids) and ORF5 (169 amino acids), were identified as replication proteins A and B that revealed 100 and 99% similarity, respectively, with the replication proteins of plasmid pLA103 from Lactobacillus acidophilus TK8912. Upstream of ORF4 were the four repeat regions (three perfect 22-bp repeats and one imperfect motif), a putative ribosome binding site, a -10 region, and a -35 region. The shuttle vector pJLE4942 (5318 bp) was constructed using repA from pLC494, a multiple cloning site, ColE1 ori, the ori of gram-negative bacteria from vector pUC19, and the chloramphenicol resistance gene from pJIR418 as a selection marker. Transformation of several lactic acid bacteria with the vector pJLE4942 indicated that this vector might be useful as a genetic tool for the intestinal lactobacilli.     

Cloning of the β-Isopropylmalate Dehydrogenase Gene from Acetobacter aceti and Its Use for Construction of a New Host-vector System for Acetobacter

The β-isopropylmalate dehydrogenase (β-IPMDH) gene of Acetobacter aceti No. 1023, which complemented the leuB mutation of Escherichia coli, was cloned and expressed in E. coli. Plasmids pCAL1 and pCAL4, carrying a 5.44 kilobase pairs (kb) HindIII-fragment in the opposite orientation, conferred the same β-IPMDH activity as that of the wild type strain of E. coli. Restriction mapping and deletion analysis indicated that the β-IPMDH gene was located on a 1.65 kb AatII-HindIII fragment. Plasmids pMVL1 and pMVL2, composing the cloned β-IPMDH gene and plasmid pMV102, a plasmid indigenous to Acetobacter, were constructed as plasmid cloning vectors which allow selection of leu⁺ transformants in an A. aceti leu^- host. The A. aceti leu^- host was obtained through the insertional inactivation occurring as a result of homologous recombination between the chromosome of A. aceti and the cloned β-IPMDH gene, which was disrupted by insertion of the kanamycin resistance gene from pACYC177 into the BamHI site in the AatII-HindIII fragment. This system constitutes a relatively simple technique for gene disruption or replacement in Acetobacter that requires a single transformation.     

Characterization of Plasmid Deoxyribonucleic Acid in Streptococcus lactis subsp. diacetylactis: Evidence for Plasmid-Linked Citrate Utilization

The use of Streptococcus diacetylactis as a flavor producer in dairy fermentations is dependent upon its ability to produce diacetyl from citrate. Treatment of S. diacetylactis strains 18-16 and DRC1 with acridine orange resulted in the conversion of approximately 2% of the DRC1 population and 20% of the 18-16 population to citrate negative, which is indicative of the involvement of plasmid deoxyribonucleic acid (DNA). Growth in the presence of acridine orange also resulted in the appearance of 2% lactose-negative derivatives in S. diacetylactis 18-16 and 99% lactose-defective, proteinase-negative derivatives in S. diacetylactis DRC1. Cesium chloride-ethidium bromide equilibrium density gradients of cleared lysate material from each strain revealed the presence of covalently closed circular DNA. Samples of this covalently closed circular DNA were subjected to agarose gel electrophoresis to determine the plasmid composition of each strain. S. diacetylactis 18-16 was found to possess six plasmids, of approximately 41, 28, 6.4, 5.5, 3.4, and 3.0 megadaltons (Mdal). S. diacetylactis DRC1 contained six plasmids, of approximately 41, 31, 18, 5.5, 4.5, and 3.7 Mdal. Variants of S. diacetylactis 18-16 which failed to produce acetoin plus diacetyl from citrate (citrate negative) were missing a 5.5-Mdal plasmid. Lactose-negative mutants of the same strain were devoid of a 41-Mdal plasmid. Lactose-defective, proteinase-negative mutants of S. diacetylactis DRC1 were missing a 31-Mdal plasmid. The citrate-negative mutants of S. diacetylactis DRC1 isolated in this study did not possess a 5.5-Mdal plasmid. Thus, we have evidence that there is a correlation between the ability to utilize citrate and the presence of a 5.5-Mdal plasmid. A relationship was also noted between lactose fermentation and proteinase activity and plasmid DNA in S. diacetylactis.     

Growth ofZymomonas on lactose: Gene cloning in combination with mutagenesis

Summary Wild-type strains ofZymomonas mobilis have a limited substrate range of glucose, fructose and sucrose. In order to expand this substrate range, transconjugants ofZ. mobilis containing Lac⁺ plasmids have been constructed. Although -galactosidase is expressed in such strains, they lack the ability to grow on lactose. We now report the development ofZ. mobilis strains capable of growth on lactose. This was achieved in two stages. First, a broad host range plasmid was constructed (pRUT102) which contained the lactose operon under the control of aZ. mobilis promoter plus genes for galactose utilization.Z. mobilis CP4.45 containing pRUT102 was then subjected to mutagenesis combined with continued selection pressure for growth on lactose. One strain,Z. mobilis SB6, produced a turbid culture that yielded 0.25% ethanol from 5% lactose (plus 2% yeast extract) in 15 days.