Effective plasmid pX3 transduction in Lactobacillus delbrueckii by bacteriophage LL-H

High-frequency plasmid transductions in Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus strains mediated by pac-type bacteriophages were observed and further investigated. The frequency of plasmid transduction by phages LL-H and LL-S attained levels of from 0.10 to about 1 with plasmid pX3, but only about 2 × 10⁻² with plasmid pJK650. Infection of L. delbrueckii subsp. lactis strain LKT(pX3) or ATCC 15808(pX3) with phage LL-H resulted in intensive concatemerization of plasmid pX3, and most progeny phage particles contained concatemers of plasmid DNA instead of phage LL-H DNA. The synthesis of phage LL-H DNA was depressed. No evident homology or recombination was observed between phage LL-H DNA and plasmid pX3. The unusually high frequency of plasmid pX3 transduction by phage LL-H could be considered to result from specific interaction(s) between a particular phage and plasmid. These interactions may include pX3-mediated blockage of phage LL-H DNA replication and effective use of a particular pac-like site located about 1 kb from BglII in the smaller NdeI-BglII fragment of plasmid pX3. Phage LL-H together with plasmid vector pX3 could be used as effective plasmid transduction tools for genetic engineering of L. delbrueckii subsp. lactis and subsp. bulgaricus strains.     

Genetic and biochemical characterization of the Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H lysin.

LL-H, a virulent phage of Lactobacillus delbrueckii subsp. lactis, produces a peptidoglycan-degrading enzyme, Mur, that is effective on L. delbrueckii, Lactobacillus acidophilus, Lactobacillus helveticus, and Pediococcus damnosus cell walls. In this study, the LL-H gene mur was cloned into Escherichia coli, its nucleotide sequence was determined, and the enzyme produced in E. coli was purified and biochemically characterized. Mur was purified 112-fold by means of ammonium sulfate precipitation and cation-exchange chromatography. The cell wall-hydrolyzing activity was found to be associated with a 34-kDa protein. The C-terminal domain of Mur is not essential for catalytic activity since it can be removed without destroying the lytic activity. The N-terminal sequence of the purified lysin was identical to that deduced from the nucleotide sequence, but the first methionine is absent from the mature protein. The N-terminal part of this 297-amino-acid protein had homology with several Chalaropsis-type lysozymes. Reduction of purified and Mur-digested L. delbrueckii cell wall material with labeled NaB3H4 indicated that the enzyme is a muramidase. The temperature optimum of purified Mur is between 30 and 40 degrees C, and the pH optimum is around 5.0. The LL-H lysin Mur is stable at temperatures below 60 degrees C.     

Physical and genetic characterization of the genome of Lactobacillus lactis bacteriophage LL-H.

Bacteriophage LL-H is a virulent phage of Lactobacillus lactis LL23. A restriction map of the phage genome was constructed with various restriction endonucleases. This chromosome has a 34-kilobase size and seems to be circularly permuted. We used a bank of LL-H restriction fragments to study the expression of five of the seven main phage particle proteins. Immunoblotting experiments permitted the mapping on the chromosome of several genes coding for phage particle proteins. We also show that the gene of the main capsid protein is expressed from its own promoter in an Escherichia coli strain.     

Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum.

Temperate phage mv4 integrates its DNA into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus strains via site-specific recombination. Nucleotide sequencing of a 2.2-kb attP-containing phage fragment revealed the presence of four open reading frames. The larger open reading frame, close to the attP site, encoded a 427-amino-acid polypeptide with similarity in its C-terminal domain to site-specific recombinases of the integrase family. Comparison of the sequences of attP, bacterial attachment site attB, and host-phage junctions attL and attR identified a 17-bp common core sequence, where strand exchange occurs during recombination. Analysis of the attB sequence indicated that the core region overlaps the 3' end of a tRNA(Ser) gene. Phage mv4 DNA integration into the tRNA(Ser) gene preserved an intact tRNA(Ser) gene at the attL site. An integration vector based on the mv4 attP site and int gene was constructed. This vector transforms a heterologous host, L. plantarum, through site-specific integration into the tRNA(Ser) gene of the genome and will be useful for development of an efficient integration system for a number of additional bacterial species in which an identical tRNA gene is present.     

Unusual structure of the attB site of the site-specific recombination system of Lactobacillus delbrueckii bacteriophage mv4

The temperate phage mv4 integrates its genome into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus by site-specific recombination within the 3' end of a tRNA(Ser) gene. Recombination is catalyzed by the phage-encoded integrase and occurs between the phage attP site and the bacterial attB site. In this study, we show that the mv4 integrase functions in vivo in Escherichia coli and we characterize the bacterial attB site with a site-specific recombination test involving compatible plasmids carrying the recombination sites. The importance of particular nucleotides within the attB sequence was determined by site-directed mutagenesis. The structure of the attB site was found to be simple but rather unusual. A 16-bp DNA fragment was sufficient for function. Unlike most genetic elements that integrate their DNA into tRNA genes, none of the dyad symmetry elements of the tRNA(Ser) gene were present within the minimal attB site. No inverted repeats were detected within this site either, in contrast to the lambda site-specific recombination model.     

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

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.     

Complete genome sequence of Lactobacillus delbrueckii subsp. bulgaricus strain ND02

Lactobacillus delbrueckii subsp. bulgaricus strain ND02 is a Chinese commercial dairy starter used for the manufacture of yoghurt. It was isolated from naturally fermented yak milk in Qinghai, China. Here, we report the main genome features of ND02 and several differences with two other published genomes of Lactobacillus delbrueckii subsp. bulgaricus strains.     

动物基因组定点整合转基因技术研究进展

传统转基因技术,如显微注射、转座子、慢病毒转染等将目的基因插入基因组内的整合方式是随机的,这些随机整合对后期转基因动物品系组建和育种带来诸多不利,因此有研究人员提出了定点整合转基因技术。目前该技术的定点整合效率非常低,主要取决于两个方面：一是靶位点产生DNA双链断裂(double-strand break, DSB)的效率;二是断裂后的靶位点与携带同源臂及外源基因的供体质粒发生同源重组的效率,其中同源重组修复(homologous recombination repair, HDR)是基因组定点整合最为依赖的修复机制。靶位点产生DSB后,机体的DNA修复既可能发生HDR,也可能发生非同源末端连接(nonhomologous end joining, NHEJ),并且两者之间存在竞争关系,因此激活HDR或抑制NEHJ都可提高定点整合转基因的效率。本文结合影响定点整合的因素,对提高定点整合效率最新探索方面进行了综述。     

Bacterial interspersed mosaic elements (BIMEs) are present in the genome of Klebsiella

Bacterial interspersed mosaic elements (BIMEs) constitute a family of highly repetitive sequences containing palindromic units (PUs), also called repetitive extragenic palindromes (REPs). BIMEs were originally described in Escherichia coli and Salmonella typhimurium. We show here, by determining the nucleotide sequence of two intergenic regions of Klebsiella pneumoniae, by computer searches, and by hybridization, that sequences with similar characteristics are found in the genome of several Klebsiella species. This reinforces the idea that BIMEs play general and important roles in enterobacteria such as in the organization of the bacterial chromosome.     

Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis by SDS-PAGE of cell-wall proteins

AIMS: In the present study, a method based on SDS-PAGE fingerprinting of surface layer proteins was developed to identify Lactobacillus delbrueckii subsp. bulgaricus and subsp. lactis dairy isolates. METHODS AND RESULTS: The two subspecies, identified by species-specific PCR, were characterized by different SDS-PAGE cell-wall protein profiles; subspecies bulgaricus showed one band of about 31 kDa which, in some cases, was observed at a doublet, and subspecies lactis showed one band of about 21 kDa or 18 kDa. CONCLUSION: The sensitivity of this procedure for discriminating between the two subspecies was very high. The different types of SDS-PAGE profile for cell-wall proteins of the strains studied in this work did not seem to be correlated to the different dairies of origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The method appears to be an efficient taxonomic tool. It has the advantage of easy gel interpretation over fingerprinting of whole-cell protein extracts, and may be used as an alternative to established PCR-based techniques which, though rapid and safe, require expensive instruments and reagents.     

Scarless engineering of the Drosophila genome near any site-specific integration site

We describe a simple and efficient technique that allows scarless engineering of Drosophila genomic sequences near any landing site containing an inverted attP cassette, such as a MiMIC insertion. This two-step method combines phiC31 integrase-mediated site-specific integration and homing nuclease-mediated resolution of local duplications, efficiently converting the original landing site allele to modified alleles that only have the desired change(s). Dominant markers incorporated into this method allow correct individual flies to be efficiently identified at each step. In principle, single attP sites and FRT sites are also valid landing sites. Given the large and increasing number of landing site lines available in the fly community, this method provides an easy and fast way to efficiently edit the majority of the Drosophila genome in a scarless manner. This technique should also be applicable to other species.     

Characterization of genetic elements required for site-specific integration of the temperate lactococcal bacteriophage phi LC3 and construction of integration-negative phi LC3 mutants.

The genetic elements required for the integration of the temperate lactococcal bacteriophage phi LC3 into the chromosome of its bacterial host, Lactococcus lactis subsp. cremoris, were identified and characterized. The phi LC3 phage attachment site, attP, was mapped and sequenced. DNA sequence analysis of attP and of the bacterial attachment site, attB, as well as the two phage-host junctions, attR and attL, in the chromosome of a phi LC3 lysogen, identified a 9-bp common core region, 5'-TTCTTCATG'-3, within which the strand exchange reaction takes place during integration. The attB core sequence is located within the C-terminal part of an open reading frame of unknown function. The phi LC3 integrase gene (int), encoding the phi LC3 site-specific recombinase, was identified and is located adjacent to attP. The phi LC3 Int protein, as deduced from the nucleotide sequence, is a basic protein of 374 amino acids that shares significant sequence similarity with other site-specific recombinases of the integrase family. Phage phi LC3 int- and int-attP-defective mutants, conferring an abortive lysogenic phenotype, were constructed.     

Diversity in specificity of the extracellular proteinases in Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus

AIMS: To investigate the diversity in specificity of cell-bound extracellular proteinases in Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus. METHODS AND RESULTS: HPLC analysis of whole-cell preparations of 14 Lact. delbrueckii subsp. bulgaricus and eight Lact. helveticus strains incubated with alpha (s1)-casein (f 1-23) detected at least six distinct proteolytic patterns. Differences between groups were found in both the primary and secondary specificity toward alpha(s1)-casein (f 1-23) and its breakdown products. No correlation was found between the o-phthaldialdehyde (OPA) general proteolysis analysis and alpha(s1)-casein (f 1-23) cleavage profiles. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: Using the alpha(s1)-CN (f 1-23) method, six patterns of proteolysis were found in the dairy lactobacilli tested. Understanding the influence of Lactobacillus proteinase specificity on casein degradation should facilitate efforts to develop starter cultures that predictably improve the functional properties of Mozzarella cheese.     

Characterization of the genetic elements required for site-specific integration of plasmid pSE211 in Saccharopolyspora erythraea.

The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site. Restriction analysis of the integrated plasmid, pSE211int, and adjacent chromosomal sequences allowed identification of attP, the plasmid attachment site. Nucleotide sequencing of attP, attB, attL, and attR revealed a 57-base-pair sequence common to all sites with no duplications of adjacent plasmid or chromosomal sequences in the integrated state, indicating that integration takes place through conservative, reciprocal strand exchange. An analysis of the sequences indicated the presence of a putative gene for Phe-tRNA at attB which is preserved at attL after integration has occurred. A comparison of the attB site for a number of actinomycete plasmids is presented. Integration at attB was also observed when a 2.4-kilobase segment of pSE211 containing attP and the adjacent plasmid sequence was used to transform a pSE211- host. Nucleotide sequencing of this segment revealed the presence of two complete open reading frames (ORFs) and a segment of a third ORF. The ORF adjacent to attP encodes a putative polypeptide 437 amino acids in length that shows similarity, at its C-terminal domain, to sequences of site-specific recombinases of the integrase family. The adjacent ORF encodes a putative 98-amino-acid basic polypeptide that contains a helix-turn-helix motif at its N terminus which corresponds to domains in the Xis proteins of a number of bacteriophages. A proposal for the function of this polypeptide is presented. The deduced amino acid sequence of the third ORF did not reveal similarities to polypeptide sequences in the current data banks.     

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 10⁴ transformants per μg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii.     

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids.

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 10(4) transformants per microg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii.     

Optimization of the freeze-drying media and survival throughout storage of freeze-dried Lactobacillus gasseri and Lactobacillus delbrueckii subsp. delbrueckii for veterinarian probiotic applications

The use of lactic acid bacteria (LAB) as vaginal probiotic cultures depends on the preservation technologies employed by the related industries.A full two-factor analysis of variance (ANOVA), considering medium and strain, of the decrease in bacterial viability during freeze-drying was applied. Lactobacillus gasseri CRL1421 was significantly more resistant than L. gasseri CRL1412 to the process. L. gasseri CRL1412 suspended in skim milk showed a significantly higher resistance than when it was suspended in water, but lactose or sucrose did not significantly increase its viability after lyophilization. Lactobacillus delbrueckii subsp. delbrueckii CRL1461, an autoaggregative strain, was significantly more sensitive to freeze-drying under the assessed conditions.The dried cultures were included in two pharmaceutical forms and viability was monitored during 270 days of storage. Although the microorganisms studied belonged to the same species, the optimal storage conditions were different for each of them.Our results can be applied to the design of a veterinarian probiotic to prevent metritis in diary postpartum cows.     

Primary structure and features of the genome of the Lactobacillus gasseri temperate bacteriophage (phi)adh.

The complete DNA sequence of the Lactobacillus (Lb.) gasseri temperate phage (phi)adh was determined. The linear and double-stranded genome consists of 43.785bp with a G+C content of 35. 3% and 3' protruding cohesive ends of 12nt. Sixty-two possible ORFs were identified. On the basis of homology comparisons, some of them could be assigned to possible functions, such as a helicase, a nucleic acid polymerase and a protease. In a non-coding area of the (phi)adh genome, structural features of a potential replication origin were detected. After subcloning, this region was functional as a replicon in Lb. gasseri and Lactococcus lactis. N-terminal aa sequencing and electron microscopic analysis of intact and defective phage particles enabled the identification of two capsid protein genes. One of their products, the major head protein, seems to be processed on the posttranslational level.     

Engineering the mouse genome by site-specific recombination.

Site-specific recombination systems are powerful tools for introducing predetermined modifications into eukaryotic genomes. Recent advances allow the manipulation of chromosomal DNA in a spatially and temporally controlled manner in mice, offering unprecedented possibilities for studying mammalian genome function and for generating animal models for human diseases.