Allele-specific complementation of an Escherichia coli leuB mutation by a Lactobacillus bulgaricus tRNA gene

Hottinger, Herbert; Ohgi, Tadaaki; Zwahlen, Marie-Camille; Dhamija, Sunder S.; Söll, Dieter

doi:10.1016/0378-1119(87)90215-0

Cited by 20 publications

(7 citation statements)

References 25 publications

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“…Our data do not indicate the length of the overlap region but highlight the importance of the CCT sequence in the strand exchange reaction. This result is further supported by the fact that in L. lactis MG1363, pMC1 did not integrate into the 70% identical tRNA Ser (UCG) gene whose sequence of the suppressor (supD) is known (20). This was presumably because this gene carries a CCC trinucleotide instead of CCT at the 5Ј end of the relatively well conserved core (10 of the 17 nt are conserved [ Fig.…”

Section: Discussionmentioning

confidence: 91%

Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4

et al. 1997

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Bacteriophage mv4 is a temperate phage infecting Lactobacillus delbrueckii subsp. bulgaricus. During lysogenization, the phage integrates its genome into the host chromosome at the 3 end of a tRNA Ser gene through a site-specific recombination process (L. Dupont et al., J. Bacteriol., 177:586-595, 1995). A nonreplicative vector (pMC1) based on the mv4 integrative elements (attP site and integrase-coding int gene) is able to integrate into the chromosome of a wide range of bacterial hosts, including Lactobacillus plantarum, Lactobacillus casei (two strains), Lactococcus lactis subsp. cremoris, Enterococcus faecalis, and Streptococcus pneumoniae. Integrative recombination of pMC1 into the chromosomes of all of these species is dependent on the int gene product and occurs specifically at the pMC1 attP site. The isolation and sequencing of pMC1 integration sites from these bacteria showed that in lactobacilli, pMC1 integrated into the conserved tRNA Ser gene. In the other bacterial species where this tRNA gene is less or not conserved, secondary integration sites either in potential protein-coding regions or in intergenic DNA were used. A consensus sequence was deduced from the analysis of the different integration sites. The comparison of these sequences demonstrated the flexibility of the integrase for the bacterial integration site and suggested the importance of the trinucleotide CCT at the 5 end of the core in the strand exchange reaction.The possibilities for genetic manipulation of the lactobacilli, a group of organisms of considerable economic importance, are limited by the lack of genetic tools such as efficient gene transfer systems and vectors for stable maintenance of genes.Integrative vectors are well adapted for chromosomal integration of cloned DNA sequences. Bacteriophage site-specific integration elements have been used to develop integrative vectors for multiple species such as Escherichia coli (3), Mycobacterium spp. (26, 45), Staphylococcus aureus (25), Arthrobacter aureus (28), Lactococcus lactis (12, 46), and Lactobacillus gasseri (37). Wide-spectrum integrative vectors for actinomycetes have also been constructed by using the integrative function of Streptomyces plasmid pSAM2 (1,31,32,43).We previously characterized the genetic elements required for the integration of the temperate phage mv4 into the Lactobacillus delbrueckii subsp. bulgaricus chromosome (15). This integration occurs by specific recombination between the phage attachment site attP and the bacterial attachment site attB. These two sites have a 17-nucleotide (nt)-long identical sequence defined as the core, within which the crossover takes place during the recombination event. This mechanism follows the Campbell model (8) and is catalyzed by the phage-encoded recombinase belonging to the integrase family. Analysis of the attB site revealed that mv4 DNA integrates into a tRNA gene (tRNA Ser ), a phenomenon common to many bacterial species (4,6,7,16,19,21,26,32,33,38,47). A nonreplicative vector, pMC1, bearing the phage attP site ...

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Section: Discussionmentioning

confidence: 91%

Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4

et al. 1997

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“…Genomic DNA from L. bulgaricus was prepared by conventional methods (9). The DNA was digested with Sau3A (0.1 U/,ug of DNA at 37°C for 30 min) to maximize fragments of 6 to 20 kb.…”

Section: Methodsmentioning

confidence: 99%

Lactose metabolism in Lactobacillus bulgaricus: analysis of the primary structure and expression of the genes involved

1991

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The genes coding for the lactose permease and 0-galactosidase, two proteins involved in the metabolism of lactose by LactobaciUlus bulgaricus, have been cloned, expressed, and found functional in Escherichia coli. The nucleotide sequences of these genes and their flanking regions have been determined, showing the presence of two contiguous open reading frames (ORFs). One of these ORFs codes for the lactose permease gene, and the other codes for the P-galactosidase gene. The lactose permease gene is located in front of the I-galactosidase gene, with 3 bp in the intergenic region. The two genes are probably transcribed as one operon. Primer extension studies have mapped a promoter upstream from the lactose permease gene but not the ,-galactosidase gene. This promoter is similar to those found in E. coli with general characteristics of GC-rich organisms. In addition, the sequences around the promoter contain a significantly higher number of AT base pairs (80%) than does the overall L. bulgaricus genome, which is rich in GC (GC content of 54%). The amino acid sequences obtained from translation of the ORFs are found to be highly homologous (similarity of 75%) to those from Streptococcus thermophilus. The first 460 amino acids of the lactose permease shows homology to the melibiose transport protein of E. coli. Little homology was found between the lactose permease of L. bulgaricus and E. coli, but the residues which are involved in the binding and the transport of lactose are conserved. The carboxy terminus is similar to that of the enzyme Ill of several phosphoenolpyruvate-dependent phosphotransferase systems.Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) and Streptococcus thermophilus are dairy lactic acid bacteria that are widely used in the fermentation of milk (25). During their growth in milk, lactose is used as the primary energy source. Two systems for transport and metabolism of lactose are known in lactic acid bacteria: (i) a phosphoenolpyruvate (PEP) lactose phosphotransferase system (PTS) with a phospho-p-galactosidase enzyme (21) and (ii) a lactose permease system with a P-galactosidase. The PEP-PTS system is found in many species, including lactococci (reviewed in references 30 and 31).L. bulgaricus utilizes lactose via the second pathway. Lactose is brought into the cell as the free sugar and cleaved by P-galactosidase. The glucose moiety of the sugar is further metabolized while the galactose moiety is released (8, 23). The lactose operon of Escherichia coli, which also has a lactose permease (lacY) and 3-galactosidase (lacZ), has been extensively studied, and the proteins involved are being characterized (1-4, 11, 13, 24). Not much is known, however, about the system in lactic acid bacteria. Recently, there has been a report on the cloning and sequencing of the ,-galactosidase gene from L. bulgaricus (28). This group found that ,B-galactosidase from L. bulgaricus has an average similarity of 34% to that from E. coli, with stretches of high homology around the regions involved in activit...

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“…Only recently have attempts to introduce DNA into L. bulgaricus by transfection or conjugation been reported (2,24). The only genes isolated and analyzed so far are the P-galactosidase (p-gal) gene (44; P. Leong-Morgenthaler and M.-C. Zwahlen, personal communication) and several tRNA genes (16,36,37).…”

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Spontaneous deletion formation within the beta-galactosidase gene of Lactobacillus bulgaricus

Mollet

Delley

1990

J Bacteriol

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To investigate the genetic stability of the dairy organism Lactobacillus bulgaricus, we have analyzed 107 spontaneous mutations of the 3-galactosidase gene 'of this organism. Ten of these mutations were DNA rearrangements giving rise to different deletions, located predominantiy within a small hot spot area. The DNA sequences of the different deletion junctions' have been determined. The analysis showed that the deletions can be divided into two classes,-depending on the presence of short direct-repeat sequences at the deletion endpoints and on the length of the deleted sequences. Possible mechanisms of these deletion formations and the involvement of inverted-repeat sequences that may enhance slipped DNA mispafring are discussed.72, No. 10

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Allele-specific complementation of an Escherichia coli leuB mutation by a Lactobacillus bulgaricus tRNA gene

Cited by 20 publications

References 25 publications

Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4

Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4

Lactose metabolism in Lactobacillus bulgaricus: analysis of the primary structure and expression of the genes involved

Spontaneous deletion formation within the beta-galactosidase gene of Lactobacillus bulgaricus

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