Bacterial Attachment Site attB Research Articles

A novel genetic tool for metabolic optimization of Corynebacterium glutamicum: efficient and repetitive chromosomal integration of synthetic promoter-driven expression libraries.

Fine-tuning the expression level of multiple genes is usually pivotal for metabolic optimization. We have developed a tool for this purpose for the important industrial workhorse Corynebacterium glutamicum that allows for the introduction of synthetic promoter-driven expression libraries of arbitrary genes. We first devised a method for introducing genetic elements into the chromosome repeatedly, relying on site-specific recombinases and the vector pJS31 serving as the carrier. The pJS31 vector contains a synthetic cassette including a phage attachment site attP for integration, a bacterial attachment site attB for subsequent integration, a multiple cloning site, and two modified loxP sites to facilitate easy removal of undesirable vector elements. Meanwhile, we constructed a derivative of the wild-type strain ATCC 13032 carrying an attB site in its chromosome (JS34) and demonstrated that pJS31 readily could integrate into the attB site in this strain providing expression of the corresponding integrase. Subsequent expression of the Cre recombinase promoted recombination between the modified loxP sites, resulting in a strain only retaining the target insertions and an attB site. To simplify the procedure, non-replicating circular expression units for the phage integrase and the Cre recombinase were used. As a showcase, we used the tool to construct a battery of strains simultaneously expressing the two reporter genes, lacZ (encoding β-galactosidase) and gusA (encoding β-glucuronidase), to arbitrary levels. In principle, an unlimited number of genes, whether native, heterologous, or synthetic, can be introduced using the developed approach, and this should greatly facilitate metabolic optimization of this important platform organism.

1097. Complete and Persistent Phenotypic Correction of Phenylketonuria in Mice by Site-Specific Genome Integration of Phenylalanine Hydorxylase cDNA

Top of pageAbstract Phenylketonuria (PKU) is a metabolic disorder that predisposes affected children to severe and irreversible mental retardation, and is secondary to a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH), that catalyses the conversion of phenylalanine to tyrosine. In an attempt to correct this metabolic deficiency, we explored the potential of using a bacteriophage integrase system to achieve site-specific genome integration of murine PAH cDNA in the livers of Phenylketonuric mice. The integrase from Streptomyces phage |[Phi]|BT1 mediates efficient recombination between the phage attachment site attP and the bacterial attachment site attB, results in the integration of phage DNA into the bacterial genome in a site-specific manner. Here we showed that this phage enzyme functioned efficiently both in Escherichia coli and mouse cells in vitro. Several naturally occurring |[ldquo]|pseudo|[rdquo]| attP sites in the mouse genome, which contain partial sequence identity to the wild type attP site and are recognizable by the phage integrase, have been identified and molecularly characterized. In the presence of the phage integrase, naked plasmid DNA containing a wild-type attB site can be integrated specifically into these pseudo attP site permanently both in culture mouse cells and in the livers of mice. We further demonstrated that the addition of nuclear localization signal sequences to the C-terminus of the phage integrase enzyme enhanced the efficiency for transgene integration into the mouse genome. Using this phage integration system we delivered mouse PAH cDNA to the livers of PKU mice by hydrodynamic injection of plasmid DNA and showed that serum phenylalanine levels in the treated mice decreased significantly and persistently. After 3 applications, serum phenylalanine levels in all treated mice were reduced to the normal range and remained stable for more than 6 weeks. Furthermore, their fur color also changed from gray to black, indicating the reconstitution of melatonin biosynthesis as a result of available tyrosine derived from phenylalanine hydroxylation. Thus the bacterio-phage integrase represents an effective site-specific genome integration system in mammalian cells and can be of great value in plasmid DNA-mediated gene therapy of a multitude of genetic disorders.

Genomic analysis of Clostridium perfringens bacteriophage phi3626, which integrates into guaA and possibly affects sporulation.

Two temperate viruses, phi3626 and phi8533, have been isolated from lysogenic Clostridium perfringens strains. Phage phi3626 was chosen for detailed analysis and was inspected by electron microscopy, protein profiling, and host range determination. For the first time, the nucleotide sequence of a bacteriophage infecting Clostridium species was determined. The virus belongs to the Siphoviridae family of the tailed phages, the order Caudovirales. Its genome consists of a linear double-stranded DNA molecule of 33,507 nucleotides, with invariable 3'-protruding cohesive ends of nine residues. Fifty open reading frames were identified, which are organized in three major life cycle-specific gene clusters. The genes required for lytic development show an opposite orientation and arrangement compared to the lysogeny control region. A function could be assigned to 19 gene products, based upon bioinformatic analyses, N-terminal amino acid sequencing, or experimental evidence. These include DNA-packaging proteins, structural components, a dual lysis system, a putative lysogeny switch, and proteins that are involved in replication, recombination, and modification of phage DNA. The presence of genes encoding a putative sigma factor related to sporulation-dependent sigma factors and a putative sporulation-dependent transcription regulator suggests a possible interaction of phi3626 with onset of sporulation in C. perfringens. We found that the phi3626 attachment site attP lies in a noncoding region immediately downstream of int. Integration of the viral genome occurs into the bacterial attachment site attB, which is located within the 3' end of a guaA homologue. This essential housekeeping gene is functionally independent of the integration status, due to reconstitution of its terminal codons by phage sequence.

Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phiC31 site-specific recombination system.

The site-specific recombination system used by the Streptomyces bacteriophage phiC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the phiC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attBxattP recombination was precise. In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the phiC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.

Sequence analysis and characterization of phi O1205, a temperate bacteriophage infecting Streptococcus thermophilus CNRZ1205.

The complete nucleotide sequence of phi O1205, a temperate bacteriophage infecting Streptococcus thermophilus strain CNRZ1205, was determined. The phage genome has a unit length of 43,075 bp and appears to be packaged by the so-called headful mechanism. The genomic organization and structure of phi O1205 resemble those of several temperate lactococcal phages that display a life-cycle-specific organization, where ORFs believed to be involved in the lysogenic life-cycle are clustered and arranged in an orientation opposite to the ORFs supposedly involved in the lytic life-cycle. Database searches revealed putative functions for several identified ORFs and further indicated that phi O1205 is genetically related to a particular group of lactococcal phages. Three genes encoding the major structural proteins were identified on the phi O1205 genome. The phage attachment site attP, the bacterial attachment site attB, and the two phage/chromosome junctions attL and attR were identified and found to contain a 40 bp common core sequence.

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.