Purification and DNA-binding properties of the integrase protein Int encoded by Lactobacillus plantarum phage

The Lactobacillus plantarum temperate phage phi g1e (42,259 bp) encodes an integrase gene int linked to a phage attachment site attP (Kakikawa et al., 1997). To investigate phi g1e recombination, the integrase protein Int was overproduced in Escherichia coli under the T7 promoter, and purified. The Int protein had an apparent molecular mass of 42.0 kDa, corresponding well with that (45.5 kDa) predicted from the DNA sequence. Amino-acid sequencing revealed that the N-terminal 20 amino-acids of the purified Int protein completely coincided with those deduced from the DNA sequence, although deficient in the first methionine. Gel mobility-shift assays demonstrated that Int bound specifically to the attP region. In addition, footprinting analysis showed that Int protected about 35 bases, containing the 24-bp core domain at attP, from DNase I attack. These results are indicative of site-specific interaction of Int with the attP site, the reaction prerequisite for integration and excision of the phi g1e genome into and/or out of the host chromosome.     

Methodological bases for monitoring the health of the population

Bireplicon plasmids were constructed. The plasmids consist of DNAs of the streptomycete plasmid pIJ702, the Escherichia coli plasmid pUC19 and the phi C31 actinophage genome fragment encoding the function of the site-specific integration into the chromosome. Part of the plasmids transformed Streptomyces lividans TK64 to thiostreptone resistance. The DNA transforming activity depended on the mutual orientations of the blocks used for the construction and probably depended on the structural stability of the plasmids in S. lividans. The integrative vectors consisting of the pUC19 plasmid DNA and the phage genome fragment with the integrative function efficiently transformed S. lividans. No phage plagues were detected with the standard procedure of integrants' infection by phi C31 phage, despite the absence of the phi C31 phage repressor gene in the integrated DNA. The phi C31 phage mutants including clear and virulent ones were not capable of lytic growth on the integrants. The region determining the limitation of the phi C31 phage lytic development was localized by the deletion analysis of the bireplicon plasmids. As a result actinomycete monoreplicon plasmids were formed. The region is the 1.3 kb phage fragment whose right end maps at 0.2 kb preceding the right end of the phi C31 phage genome linear map.     

Common elements regulating gene expression in temperate and lytic bacteriophages of Lactococcus species

A phage-inducible middle promoter (P15A10) from the lytic, lactococcal bacteriophage phi 31, a member of the P335 species, is located in an 888-base pair fragment near the right cohesive end. Sequence analysis revealed extensive homology (> 95%) to the right cohesive ends of two temperate phages of the P335 species, phi r1t and phi LC3. Sequencing upstream and downstream of P15A10 showed that the high degree of homology between phi 31 and phi r1t continued beyond the phage promoter. With the exception of one extra open reading frame in phi 31, the sequences were highly homologous (95 to 98%) between nucleotides 13,448 and 16,320 of the published phi r1t sequence. By use of a beta-galactosidase (beta-Gal) gene under the control of a smaller, more tightly regulated region within the P15A10 promoter, P566-888, it was established that mitomycin C induction of a lactococcal strain harboring the prophage phi r1t induced the P566-888 promoter, as determined from an increase in beta-Gal activity. Hybridization of nine other lactococcal strains with 32P-labeled P566-888 showed that the Lactococcus lactis strains C10, ML8, and NCK203 harbored sequences homologous to that of the phage-inducible promoter. Mitomycin C induced the resident prophages in all these strains and concurrently induced the P566-888 promoter, as determined from an increase in beta-Gal activity. DNA restriction analysis revealed that the prophages in C10, ML8, and NCK203 had identical restriction patterns which were different from that of phi r1t. In addition, DNA sequencing showed that the promoter elements in the three phages were identical to each other and to P566-888 from the lytic phage phi 31. These results point to a conserved mechanism in the regulation of gene expression between the lytic phage phi 31 and at least two temperate bacteriophages and provide further evidence for a link in the evolution of certain temperate phages and lytic phages.     

Characterization of the lys2 gene of Penicillium chrysogenum encoding alpha-aminoadipic acid reductase

A DNA fragment containing a gene homologous to LYS2 gene of Saccharomyces cerevisiae was cloned from a genomic DNA library of Penicillium chrysogenum AS-P-78. It encodes a protein of 1409 amino acids (Mr 154859) with strong similarity to the S. cerevisiae (49.9% identity) Schizosaccharomyces pombe (51.3% identity) and Candida albicans (48.12% identity) alpha-aminoadipate reductases and a lesser degree of identity to the amino acid-activating domains of the non-ribosomal peptide synthetases, including the alpha-aminoadipate-activating domain of the alpha-aminoadipyl-cysteinyl-valine synthetase of P. chrysogenum (12.4% identical amino acids). The lys2 gene contained one intron in the 5'-region and other in the 3'-region, as shown by comparing the nucleotide sequences of the cDNA and genomic DNA, and was transcribed as a 4.7-kb monocistronic mRNA. The lys2 gene was localized on chromosome III (7.5 Mb) in P. chrysogenum AS-P-78 and on chromosome IV (5.6 Mb) in strain P2, whereas the penicillin gene cluster is known to be located in chromosome I in both strains. The lys2-encoded protein is a member of the aminoacyladenylate-forming enzyme family with a reductase domain in its C-terminal region.     

The role of supercoiling in mycobacteriophage L5 integrative recombination

The genome of temperate mycobacteriophage L5 integrates into the chromosomes of its hosts, including Mycobacterium smegmatis , Mycobacterium tuberculosis and bacille Calmette-Guérin. This integrase-mediated site-specific recombination reaction occurs between the phage attP site and the mycobacterial attB site and requires the mycobacterial integration host factor. Here we examine the role of supercoiling in this reaction and show that integration is stimulated by DNA supercoiling but that supercoiling of either the attP or the attB substrate enhances recombination. Supercoiling thus facilitates a post-synaptic recombination event. We also show that, while supercoiling is not required for the production of a recombinagenic intasome, a mutant attP DNA deficient in binding of the host factor acquires a dependence on supercoiling for intasome formation and recombination.     

Identification of Delta5-desaturase from Mortierella alpina by heterologous expression in Bakers' yeast and canola

A DNA fragment with homology to Delta6-desaturases from borage and cyanobacteria was isolated after polymerase chain reaction amplification of Mortierella alpina cDNA with oligonucleotide primers corresponding to the conserved regions of known Delta6-desaturase genes. This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 446 amino acids from a M. alpina library. Expression of this open reading frame from an inducible promoter in Saccharomyces cerevisiae in the presence of various substrates revealed that the recombinant product had Delta5-desaturase activity. The effects of growth and induction conditions as well as host strain on activity of the recombinant Delta5-desaturase in S. cerevisiae were evaluated. Expression of the M. alpina Delta5-desaturase cDNA in transgenic canola seeds resulted in the production of taxoleic acid (Delta5,9-18:2) and pinolenic acid (Delta5,9,12-18:3), which are the Delta5-desaturation products of oleic and linoleic acids, respectively.     

In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family

The genome of the broad host range Streptomyces temperate phage, phiC31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase/invertase family of site-specific recombinases. The recombination properties of this novel integrase on the phage and Streptomyces ambofaciens attachment sites, attP and attB, respectively, were investigated in the heterologous host, Escherichia coli, and in an in vitro assay by using purified integrase. The products of attP/B recombination, i.e., attL and attR, were identical to those obtained after integration of the prophage in S. ambofaciens. In the in vitro assay only buffer, purified integrase, and DNAs encoding attP and attB were required. Recombination occurred irrespective of whether the substrates were supercoiled or linear. A mutant integrase containing an S12F mutation was completely defective in recombination both in E. coli and in vitro. No recombination was observed between attB/attB, attP/attP, attL/R, or any combination of attB or attP with attL or attR, suggesting that excision of the prophage (attL/R recombination) requires an additional phage- or Streptomyces-encoded factor. Recombination could occur intramolecularly to cause deletion between appropriately orientated attP and attB sites. The results show that directionality in phiC31 integrase is strictly controlled by nonidentical recombination sites with no requirement to form the topologically defined structures that are more typical of the resolvases/invertases.     

Characterization of the lysogenic bacteriophage MAV1 from Mycoplasma arthritidis

The lysogenic bacteriophage MAV1, which is associated with the arthritogenicity of Mycoplasma arthritidis, was characterized. Several strains of M. arthritidis were examined for their ability to support growth of MAV1. A PFU assay was developed, and the sensitivity of phage to various chemical treatments was assayed. The most notable result was the resistance of MAV1 to proteinase K. The MAV1 genome is a double-stranded, linear DNA molecule of about 16 kb. The site of MAV1 DNA integration in the host chromosome was investigated. The ends of MAV1 DNA were cloned from three independent lysogens shown to have MAV1 DNA inserted at different sites in the host. The nucleotide sequences of the ends of the MAV1 genome and of the MAV1 DNA-chromosomal DNA junctions from each of three lysogens were determined. Sequences flanking the integrated prophage and the ends of native MAV1 DNA were determined, allowing the identification of the phage DNA (attP) and bacterial DNA (attB) recombination sites. Analysis of the left MAV1 DNA-chromosomal DNA junction sites showed a single-base heterogeneity located within MAV1 DNA sequences immediately adjacent to the attB sequence. A model for MAV1 integration-excision is proposed.     

Gene targeting in Penicillium chrysogenum: disruption of the lys2 gene leads to penicillin overproduction

Two strategies have been used for targeted integration at the lys2 locus of Penicillium chrysogenum. In the first strategy the disruption of lys2 was obtained by a single crossing over between the endogenous lys2 and a fragment of the same gene located in an integrative plasmid. lys2-disrupted mutants were obtained with 1.6% efficiency when the lys2 homologous region was 4.9 kb, but no homologous integration was observed with constructions containing a shorter homologous region. Similarly, lys2-disrupted mutants were obtained by a double crossing over (gene replacement) with an efficiency of 0.14% by using two lys2 homologous regions of 4.3 and 3.0 kb flanking the pyrG marker. No homologous recombination was observed when the selectable marker was flanked by short lys2 homologous DNA fragments. The disruption of lys2 was confirmed by Southern blot analysis of three different lysine auxotrophs obtained by a single crossing over or gene replacement. The lys2-disrupted mutants lacked alpha-aminoadipate reductase activity (encoded by lys2) and showed specific penicillin yields double those of the parental nondisrupted strain, Wis 54-1255. The alpha-aminoadipic acid precursor is channelled to penicillin biosynthesis by blocking the lysine biosynthesis branch at the alpha-aminoadipate reductase level.     

Transforming potential of the adenovirus type 5 E4orf3 protein

Previous observations that the adenovirus type 5 (Ad5) E4orf6 and E4orf3 gene products have redundant effects in viral lytic infection together with the recent findings that E4orf6 possesses transforming potential prompted us to investigate the effect of E4orf3 expression on the transformation of primary rat cells in combination with adenovirus E1 oncogene products. Our results demonstrate for the first time that E4orf3 can cooperate with adenovirus E1A and E1A plus E1B proteins to transform primary baby rat kidney cells, acting synergistically with E4orf6 in the presence of E1B gene products. Transformed rat cells expressing E4orf3 exhibit morphological alterations, higher growth rates and saturation densities, and increased tumorigenicity compared with transformants expressing E1 proteins only. Consistent with previous results for adenovirus-infected cells, the E4orf3 protein is immunologically restricted to discrete nuclear structures known as PML oncogenic domains (PODs) in transformed rat cells. As opposed to E4orf6, the ability of E4orf3 to promote oncogenic cell growth is probably not linked to a modulation of p53 functions and stability. Instead, our results indicate that the transforming activities of E4orf3 are due to combinatorial effects that involve the binding to the adenovirus 55-kDa E1B protein and the colocalization with PODs independent from interactions with the PML gene product. These data fit well with a model in which the reorganization of PODs may trigger a cascade of processes that cause uncontrolled cell proliferation and neoplastic growth. In sum, our results provide strong evidence for the idea that interactions with PODs by viral proteins are linked to oncogenic transformation.     

Sp1 phosphorylation by Erk 2 stimulates DNA binding

The temperate mycobacteriophage L5 integrates site specifically into the genomes of Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium bovis bacillus Calmette-Guérin. This integrative recombination event occurs between the phage L5 attP site and the mycobacterial attB site and requires the phage-encoded integrase and mycobacterial-encoded integration host factor mIHF. Here we show that attP, Int-L5, and mIHF assemble into a recombinationally active complex, the intasome, which is capable of attB capture and formation of products. The arm-type integrase binding sites within attP play specialized roles in the formation of specific protein-DNA architectures; the intasome is constructed by the formation of intramolecular integrase bridges between one pair of sites, P4-P5, and the attP core, while an additional pair of sites, P1-P2, is required for interaction with attB.     

AbiQ, an abortive infection mechanism from Lactococcus lactis

Lactococcus lactis W-37 is highly resistant to phage infection. The cryptic plasmids from this strain were coelectroporated, along with the shuttle vector pSA3, into the plasmid-free host L. lactis LM0230. In addition to pSA3, erythromycin- and phage-resistant isolates carried pSRQ900, an 11-kb plasmid from L. lactis W-37. This plasmid made the host bacteria highly resistant (efficiency of plaquing <10(-8)) to c2- and 936-like phages. pSRQ900 did not confer any resistance to phages of the P335 species. Adsorption, cell survival, and endonucleolytic activity assays showed that pSRQ900 encodes an abortive infection mechanism. The phage resistance mechanism is limited to a 2.2-kb EcoRV/BclI fragment. Sequence analysis of this fragment revealed a complete open reading frame (abiQ), which encodes a putative protein of 183 amino acids. A frameshift mutation within abiQ completely abolished the resistant phenotype. The predicted peptide has a high content of positively charged residues (pI = 10.5) and is, in all likelihood, a cytosolic protein. AbiQ has no homology to known or deduced proteins in the databases. DNA replication assays showed that phage c21 (c2-like) and phage p2 (936-like) can still replicate in cells harboring AbiQ. However, phage DNA accumulated in its concatenated form in the infected AbiQ+ cells, whereas the AbiQ- cells contained processed (mature) phage DNA in addition to the concatenated form. The production of the major capsid protein of phage c21 was not hindered in the cells harboring AbiQ.     

Cognition in children does not suffer from very low lead exposure

It was previously demonstrated that while lysogenic development of bacteriophage lambda in Escherichia coli proceeds normally at low temperature (20-25 degrees C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the pE promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage lambda cIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37 degrees C, while no phage progeny are observed at 20 degrees C. Contrary to previous reports, it is possible to demonstrate that pE promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20 degrees C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage lambda is neither inhibited at 20 degrees C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between lambda phage and lambda plasmid DNA at low temperature.     

Recurrence of recipient Langerhans'' cell histiocytosis following bilateral lung transplantation

The DNA of the temperate bacteriophage Aaphi23 isolated from the oral bacterium Actinobacillus actinomycetemcomitans was examined structurally both in the phage head and in the prophage. The DNA in phage particles comprises 44 kb linear molecules with a terminal redundancy of 1.6 kb, which represent circular permutations. Thus, DNA is packaged into phage heads by the headful mechanism. The Aaphi23 prophage is integrated into the host chromosome.