Nucleotide sequence of a secondary attachment site for bacteriophage lambda on the Escherichia coli chromosome.

The nucleotide sequence of a secondary attachment site for bacteriophage lambda was determined in a region near the rrnB gene at 88 min on the E. coli chromosome. The sequence has a 8 base pair interrupted homology GCT TTTTA to the common core of the primary attachment site (attB) and the corresponding phage sequence (attP). The site of crossover during integration lies probably between nucleotides -3 and +1. The flanking regions have no obvious homology to the arms of either attP or attB.     

Bending of the bacteriophage lambda attachment site by Escherichia coli integration host factor

Escherichia coli integration host factor (IHF) is a small basic protein that is required for efficient integrative recombination of bacteriophage lambda. IHF binds specifically to sequences within attP, the site in bacteriophage lambda that undergoes recombination. It has been suggested that the binding of IHF creates bends in DNA so as to help attP condense into a compact structure that is activated for recombination. In this work we show that IHF binding to either of two sites found within attP does indeed produce bending of DNA. In contrast, the other recombination protein needed for integrative recombination, Int, does not appreciably bend the DNA to which it is bound. In agreement with the proposal that IHF bending is important for creating a condensed attP, bending by IHF persists in the presence of bound Int. Our conclusions about protein-directed bends in DNA are based on the study of the electrophoretic mobility of a set of permuted DNA fragments in the presence or absence of IHF and/or Int. To facilitate this study, we have constructed a novel vector that simplifies the generation of permuted fragments. This vector should be useful in studying the bending of other DNA sequences by specific binding proteins.     

Secondary attachment site for bacteriophage lambda in the guaB gene of Escherichia coli.

lambda gua transducing bacteriophages were used to identify and sequence the secondary attachment site for lambda in the guaB gene of Escherichia coli. The sequence matched the primary core sequence at nine positions, and a putative integrase binding-site overlapped the left core-arm junction. Recombinational crossover occurred between nucleotides -3 and +2 of the core region.     

New shuttle-type vectors for cloning in Escherichia coli and Agrobacterium tumefaciens

The vectors capable of replication in Escherichia coli and Agrobacterium tumefaciens have been constructed on the basis of the plasmid pUB5502. The constructed vectors pVA12, pVA12-2, pVA12-4 contain the mini-replicon and trimethoprim resistance gene (Tp) of a broad host-range plasmid R388 (IncW). The pVA12 vector (8.8 kb) has been constructed by insertion of a kanamycin resistance gene (Km) from the plasmid pUC-4K into a Psti site. It possesses 7 unique restriction sites for XhoI, SmaI, PvuI, PvuII, HindIII, EcoRI, BamHI and the markers for kanamycin and trimethoprim resistance (Km and Tp). The pVA12-2 and pVA12-4 vectors were obtained as a result of changing of the PvuII-EcoI fragment of pVA12 carrying the Tp gene for the PvuII-EcoRI fragment of pBR322 carrying the Tc gene. These plasmids have the same size of 9.7 kb and 8 unique sites for restriction endonucleases XhoI, SmaI, PvuI, PvuII, EcoRI, EcoRV, SalI, BalI and Km and Tc genes. No difference has been registered between the two plasmids by restriction analysis, but pVA12-4 has the dramatically increased copy number in Escherichia coli cells. All three vectors are transferable to Agrobacterium tumefaciens with the same frequencies by transformation or conjugation and do not affect the oncogenicity of pTi.     

Secretion cloning vectors in Escherichia coli

The DNA fragment coding for the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, has been inserted into the high-level expression vectors, pIN-III. A foreign DNA fragment can be cloned in any one of the three reading frames at the unique EcoRI, HindIII or BamHI sites immediately after the ompA signal peptide coding sequence. The cloned foreign gene is under the control of both the lpp promoter and the lac promoter-operator. The expression of the gene is regulated by the lac repressor produced by the same vectors. Using the pIN-III-ompA vector, the DNA fragment coding for only the mature portion of beta-lactamase was inserted into the EcoRI site. Upon induction of gene expression, beta-lactamase was secreted into the periplasmic space. The ompA signal peptide was correctly removed resulting in the production of beta-lactamase with four extra amino acid residues (Gly-Ile-Pro-Gly) at its amino terminus due to the linker sequence in the vector. After a 3-h induction, beta-lactamase was accumulated to 20% of total cellular protein without any detectable accumulation of pro-beta-lactamase. Using oligonucleotide-directed site-specific mutagenesis, we have also removed the linker sequence and upon induction of gene expression, beta-lactamase with the authentic NH2-terminal sequence was produced, in even larger amounts than the beta-lactamase with the linker sequence.     

The secondary attachment site for bacteriophage lambda in the proA/B gene of Escherichia coli

We have determined the nucleotide sequence of a secondary λ attachment site in $\text{pro}\text{A}\text{B}$, a site that accounts for 3% of lysogens isolated from Escherichia coli strains deleted for the primary site. Direct sequence analysis of the transducing bacteriophages carrying the left and right att junctions, as well as the recombinant pro⁺ phage reveals that the $\text{pro}\text{A}\text{B}$ site shares an 11-nucleotide interrupted homology with the core sequence of the primary site. We have compared the $\text{pro}\text{A}\text{B}$att site with other secondary attachment sites to gain insights into the structural features important for λ integration.     

A secondary attachment site for bacteriophage lambda in trpC of E. coli.

We have determined the nucleotide sequence of a secondary lambda attachment site in trpC. Direct sequence analysis of lambdatrp transducing phage DNA fragments carrying the two prophage attachment sites reveals a 6 nucleotide homology in the crossover region which is a subset of the 15 nucleotide core sequence in the primary lambda attachment site: GCTTTTTTATACTAA. This 6 nucleotide sequence is also present in the intact trpC genome at the attachment site, as shown by analysis of trpC mRNA spanning this region.     

Versatile low-copy-number plasmid vectors for cloning in Escherichia coli

Small low-copy-number plasmid vectors were constructed by in vitro and in vivo recombinant DNA techniques. pLG338 and pLG339 are derived from pSC105, have a copy number of six to eight per chromosome, and carry genes conferring resistance to tetracycline and kanamycin. pLG338 (7.3 kb) has unique restriction endonuclease sites for BamHI, SalI, HincII, SmaI, XhoI, EcoRI and KpnI, the first five lying within a drug resistance gene. pLG339 (6.2 kb) lacks the KpnI site, but has unique SphI and PvuII sites. These versatile vectors should be useful for cloning many genes coding for membrane and regulatory proteins which cannot be cloned into high-copy-number plasmids.     

Membrane attachment of folded chromosome of Escherichia coli.

By fractionation of the envelope part of membrane-associated folded chromosomes it is shown that only the outer membrane is bound to the DNA. Experiments with the M-band technique suggest the presence of attachment points for the membrane also in membrane-released folded chromosomes.     

Genetic analysis of Escherichia coli integration host factor interactions with its bacteriophage lambda H'' recognition site.

The bacteriophage P22-based challenge phage system was used to study the binding of integration host factor (IHF) to its H' recognition site in the attP region of bacteriophage lambda. We constructed challenge phages that carried H' inserts in both orientations within the P22 Pant promoter, which is required for antirepressor synthesis. We found that IHF repressed expression of Pant from either challenge phage when expressed from an inducible Ptac promoter on a plasmid vector. Mutants containing changes in the H' inserts that decrease or eliminate IHF binding were isolated by selecting challenge phages that could synthesize antirepressor in the presence of IHF. Sequence analysis of 31 mutants showed that most changes were base pair substitutions within the H' insert. Approximately one-half of the mutants contained substitutions that changed base pairs that are part of the IHF consensus binding site; mutants were isolated that contained substitutions at six of the nine base pairs of the consensus site. Other mutants contained changes at base pairs between the two subdeterminants of the H' site, at positions that are not specified in the consensus sequence, and in the dA + dT-rich region that flanks the consensus region of the site. Taken together, these results show that single-base-pair changes at positions outside of the proposed consensus bases can weaken or drastically disrupt IHF binding to the mutated site.     

A versatile phage lambda expression vector system for cloning in Escherichia coli

By integrating fragments from the expression plasmids pJK2 and pJK4 into a derivative of the bacteriophage lambda, we constructed the phage expression vectors lambda JK2 and lambda JK4, which allow efficient cloning of genomic or cDNA either into the 5' end or the 3' end of the lacZ gene of Escherichia coli. Expression of barrier-free DNA in phase may lead to fusion proteins consisting of active beta-galactosidase (beta Gal) plus an additional polypeptide encoded by the inserted DNA. Analysis of distinct recombinant clones is quick and easy, due to the reversible integration of the plasmid into the genome. As an example, we constructed an expression library of genomic Plasmodium falciparum DNA in lambda JK2. We polymerised (amplified) and expressed a synthetic DNA fragment, which codes for a potential antigenic determinant of the 11-1 gene of Plasmodium falciparum as a fusion to the N terminus of active beta Gal. We demonstrate that such chimeric molecules can be affinity-purified and that polypeptides can be separated from the beta Gal part by cleavage with the protease factor Xa.     

Construction of linear plasmid vectors for cloning in Escherichia coli cells]

Prophage of the temperate coliphage N15 is a linear plasmid with covalently closed ends. The central part of plasmid N15 genome responsible for vegetative phage growth was replaced by DNA fragments containing genes for selective markers which have unique restriction sites. As a result a family of linear plasmid vectors was constructed. Their size is about 20 kb and their capacity is comparable with that of cosmid vectors.     

Preferred secondary attachment site of prophage phi80 on Escherichia coli K-12 chromosome]

The integration frequency of phage att80 immlambdac1857 into the chromosome of a mutant strain H47 Escherichia coli K-12 deleted for the normal prophage insertion site is found to be about 20-fold decreased as compared with its integration into the wild type strain. The most of the resulting lysogens contain the prophage at the secondary attachment site of the mutant bacterial chromosome which is preferentially utilized for prophage insertion. This attachment site (att80-II) is located close to his-genes on the chromosome of H47 strain. Prophage curing procedure of such abnormal lysogens results in the appearance of rare auxotrophic heat-resistant survivors with the His- phenotype. In some cases the prophage insertion can induce an inversion of a neighbouring genetic region. Such lysogens contain the purC gene near prophage located at the att80-II site, and after curing they segregate the heat-resistant His- and Pur- colonies.     

Shuttle vectors for cloning recombinant DNA in Escherichia coli and Streptomyces griseofuscus C581.

The replicon of the Streptomyces plasmid SCP2 was located on a 5.9-kilobase EcoRI-SalI restriction fragment. The SCP2 replicon was combined with Escherichia coli plasmid pBR322 and genes specifying neomycin resistance and thiostrepton resistance in streptomycetes to construct shuttle vectors that are useful for cloning in E. coli and streptomycetes.     

Bacteriophage lambda receptor site on the Escherichia coli K-12 LamB protein.

We have analyzed eight new phage-resistant missense mutations in lamB. These mutations identify five new amino acid residues essential for phage lambda adsorption. Two mutations at positions 245 and 382 affect residues which were previously identified, but lead to different amino acid changes. Three mutations at residues 163, 164, and 250 enlarge and confirm previously proposed phage receptor sites. Two different mutations at residue 259 and one at 18 alter residues previously suggested as facing the periplasmic face. The mutation at residue 18 implicates for the first time the amino-terminal region of the LamB protein in phage adsorption. The results are discussed in terms of the topology of the LamB protein.     

Construction of plasmid vectors for gene cloning in Escherichia coli and Bacillus subtilis

The construction and some properties of new hybrid plasmids which are able to replicate in both Escherichia coli and Bacillus subtilis are presented. A 5.5 Md hybrid plasmid pJP9 was constructed from pBR322 (Tc, Ap) and pUB110 (Nm) plasmids. pIM1 (7.0 Md) and pIM3 (7.7 Md) plasmids are its different erythromycin resistant derivatives. Tetracycline, ampicillin, neomycin and possibly erythromycin resistance genes are expressed in E. coli while neomycin and erythromycin resistance genes are expressed in B. subtilis. Insertional inactivation of only one gene is possible using the pJP9 plasmid as a vector in B. subtilis. However, insertional inactivation of at least two different genes can be achieved and monitored in E. coli and B. subtilis transformants in cloning experiments with PIM1 and pIM3 plasmids. Insertional inactivation of antibiotic resistance genes present in pJP9 plasmid was achieved by cloning of Streptococcus sanguis DNA fragments generated by appropriate restriction endonucleases. The pJP9 plasmid and its derivatives were found to be stable in both hosts cells.     

Escherichia coli plasmid vectors for high-level regulated expression of the bacteriophage lambda xis gene product

The bacteriophage lambda Xis protein is one of the proteins required for site-specific excisive recombination by which the lambda prophage is excised from the Escherichia coli bacterial chromosome. We cloned the lambda xis gene under the control of several prokaryotic promoters to obtain a sufficient source of the protein for biochemical studies. Our results demonstrate that E. coli lac promoter and lambda pL promoter fusions to the xis gene produce high levels of Xis protein. Induction of the expression vectors results in a 10- to 50-fold increase in Xis activity. In addition, one of these plasmids allows the control of xis expression in vivo.