Stimulation of IS1 excision by bacteriophage P1 ref function.

Lysogenization by a c1ts variant of coliphage P1, P1c1.100, markedly increased the frequency of reversion of a galT::IS1 mutation. The formation of Gal+ colonies presumably occurs by microhomologous recombination between the 9-base-pair repeats in galT (CGCCGCTAC) generated by the transposition of IS1. The responsible P1 gene, ref, has been cloned and sequenced. ref encodes a 22.8-kilodalton protein and is located near the P1 site-specific recombination function, cre. Expression of ref was repressed by P1 c+. The absence of a distinctive ribosome-binding site is consistent with a poor translation of ref from an expression vector in vivo. Placement of a ribosome-binding site before ref resulted in the extensive synthesis of the Ref protein. Ref stimulated precise excision in recB or himA cells, but not in recA mutants. Ref was active in lexA3 mutants, suggesting that the recombination activity of RecA was directly involved in the reaction. We have constructed a P1c1.100 ref::Tn10 mutant. The absence of Ref did not appear to restrict dramatically the ability of P1 to grow lytically or to form lysogens. Thus, the role of ref in the physiology of P1 remains to be determined.     

A cloned DNA fragment from bacteriophage P1 enhances IS2 insertion

Summary A 1.75 kb DNA segment of the bacteriophage P1 genome is known to serve as a preferred target for IS2 insertions. The presence of this fragment in a plasmid expressing the galK gene dramatically increases the proportion of IS2 insertions among spontaneous galK ^- mutants. Subfragments from two different parts of the 1.75 kb segment independently stimulate IS2 insertion, while another subfragment does not. In the plasmids studied IS2 elements not only insert into the cloned P1 fragment but also into parts of the galK gene with similar probability and mostly in one orientation. Many insertion sites are unique but several specific sites within the preferred target are repeatedly used for IS2 integration. The experimental data are compatible with a proposed cooperative mechanism, according to which more than one attracting sequence on the same plasmid might significantly enhance the probability of a particular target region to attract IS2.     

The sequence of the bacteriophage P1 genome region serving as hot target for IS2 insertion.

A restriction fragment of the bacteriophage P1 genome known to serve as a hot target for IS2 insertion in its host, Escherichia coli K12, was entirely sequenced. It is 1756 bp long and it contains four long open reading frames, all in the same orientation. The two middle frames overlap partially. Eight of the nine studied IS2 insertions affecting phage reproduction map within three of these reading frames. No common feature was found between the nine target sites which have served for IS2 integration. However, there are two structural elements which might possibly contribute to rendering the studied DNA segment a hot region for IS2 insertion. The first is formed by two neighbouring, 30 and 40 bp regions of homology with an internal segment of IS2. The second is the pentanucleotide 5' GGTAT3', which is carried nine times in the sequenced fragment and which is found always in at least one copy within a variable distance of less than 100 bp of each inserted IS2 element.     

Transposition of IS1-lambdaBIO-IS1 from a bacteriophage lambda derivative carrying the IS1-cat-IS1 transposon (Tn9)

Summary Tn9 is a transposable element in which a gene (cat) determining chloramphenicol resistance is flanked by directly repeated sequences that are homologous to the insertion sequence IS1. We show here that infection of Escherichia coli K12 (under Rec^- Red^- Int^- conditions) with a bio transducing phage carrying Tn9 results in the formation of bio transductants as frequently as cat transductants (about 1 per 10⁶ to 10⁷ infected cells). Most of the bio transductants do not carry cat, just as most of the cat transductants do not carry bio. In spite of the absence of cat, the bio prophage can transpose a second time, from the E. coli chromosome to different sites on an F gal plasmid. Analysis of the structure of the transposed bio element, by restriction nuclease digestion and by electron microscopy, demonstrates that the integrated bio prophage is flanked by directly repeated IS1 elements. We conclude that there is no genetic information for the ability to transpose encoded in the non-repeated portion of Tn9, i.e. that the directly repeated IS1 elements alone are responsible for Tn9 transposition.     

Use of bacteriophage P1 as a vector for Tn5 insertion mutagenesis.

Infection of a strain lysogenic for bacteriophage P1 CM with P1::Tn5 followed by simultaneous selection for the chloroamphenicol resistance associated with the resident prophage and the kanamycin resistance associated with Tn5 results in a large number of independent Tn5 insertion mutations. This superinfection-selection protocol is a fast, easy, and safe way to isolate null mutations in enteric bacteria without generating unwanted cryptic mutations elsewhere in the genome.     

Plaque forming specialized transducing phage P1: Isolation of P1CmSmSu, a precursor of P1Cm

Summary E. coli strains lysogenic for various types of P1-R hybrids were isolated. These carry all the essential genes for vegetative phage production and lysogenization including P1 immunity and P1 incompatibility, together with drug resistance genes derived from the R plasmid NR1. In particular, P1Cm and P1CmSmSu derivatives were studied. When strains lysogenic for these phages were induced in the absence of helper phage, yields of phage particles as high as with wild type P1 were obtained. All P1Cm phages isolated were of plaque forming type and usually every plaque contained Cm^r lysogens. Lysates of P1CmSmSu lysogens transduced Cm^rSm^rSu^r at high frequency and they formed plaques with an efficiency of 10^-4 to 10^-2 per phage particle. Only a minority of these plaques contained drug resistant bacteria. Cm^rSm^rSu^r transductants isolated from bacteria infected at a high multiplicity with phage P1CmSmSu were lysogens for the original P1CmSmSu. In contrast, Cm^rSm^rSu^r transductants isolated after infection at low multiplicity appeared to carry the Cm^rSm^rSu^r markers integrated into the host chromosome. The results described suggest that P1CmSmSu prophages carry the resistance genes transposed into the P1 genome without in principle causing a loss of essential gene functions. However, since these prophages are longer than the wild type P1 genome, the DNA packaged into phage particles has a reduced redundancy which seriously affects the reproduction and lysogenization abilities.Plaque forming P1Cm can be obtained from P1CmSmSu. Thus, P1CmSmSu is a precursor of P1Cm. P1Cm is also obtainable from P1 and NR1 under the recA ^- condition. The mechanism of formation of plaque forming P1Cm is discussed.     

Expression of a proteolipid gene from a high-copy-number plasmid confers trifluoperazine resistance to Saccharomyces cerevisiae.

A wild-type haploid yeast strain was transformed with a library of wild-type yeast DNA fragments ligated into a high-copy-number plasmid vector (YEp24). The pooled URA+ transformants were plated on rich medium containing a lethal concentration of trifluoperazine (TFP). Plasmids rescued into Escherichia coli from TFP-resistant yeast colonies contained overlapping DNA fragments from a unique region of yeast chromosome XVI. Deletion and disruption experiments, mini-Tn10 LUK hop analysis, and DNA sequencing defined a novel gene with significant amino acid identity to bovine and yeast vacuoletype proteolipid subunits. This is the second locus identified that can be altered to confer TFP resistance to Saccharomyces cerevisiae and that has significant amino acid identity to a vacuolar ATPase subunit. This suggests that a target for TFP in S. cerevisiae is the electrogenic membranes of the vacuolar network and that alteration of expression or activity of vacuolar proton ATPase subunits is a general mechanism for TFP resistance in this yeast.     

Analysis of IS1236-mediated gene amplification events in Acinetobacter baylyi ADP1

Recombination between insertion sequence copies can cause genetic deletion, inversion, or duplication. However, it is difficult to assess the fraction of all genomic rearrangements that involve insertion sequences. In previous gene duplication and amplification studies of Acinetobacter baylyi ADP1, an insertion sequence was evident in approximately 2% of the characterized duplication sites. Gene amplification occurs frequently in all organisms and has a significant impact on evolution, adaptation, drug resistance, cancer, and various disorders. To understand the molecular details of this important process, a previously developed system was used to analyze gene amplification in selected mutants. The current study focused on amplification events in two chromosomal regions that are near one of six copies of the only transposable element in ADP1, IS1236 (an IS3 family member). Twenty-one independent mutants were analyzed, and in contrast to previous studies of a different chromosomal region, IS1236 was involved in 86% of these events. IS1236-mediated amplification could occur through homologous recombination between insertion sequences on both sides of a duplicated region. However, this mechanism presupposes that transposition generates an appropriately positioned additional copy of IS1236. To evaluate this possibility, PCR and Southern hybridization were used to determine the chromosomal configurations of amplification mutants involving IS1236. Surprisingly, the genomic patterns were inconsistent with the hypothesis that intramolecular homologous recombination occurred between insertion sequences following an initial transposition event. These results raise a novel possibility that the gene amplification events near the IS1236 elements arise from illegitimate recombination involving transposase-mediated DNA cleavage.     

Expression of the bacteriophage P1 cin recombinase gene from its own and heterologous promoters

The cin recombinase of bacteriophage P1, a protein that catalyses site-specific DNA inversions, has been identified and its structural gene has been cloned under the control of different promoters. One of the DNA sequences used for the site-specific recombination, cixL, overlaps with the 3' end of the gene, but we show that the presence of this site does not affect cin gene expression from strong promoters. To assay cin activity we have constructed plasmids that carry antibiotic resistance genes within the invertible segment that are transcribed from promoters outside the segment. DNA inversion switches on or off genes for chloramphenicol or kanamycin resistance. These tester plasmids are used to study cin-mediated DNA inversion both in vivo and in vitro.     

The generation of concatemeric plasmid DNA in Bacillus subtilis as a consequence of bacteriophage SPP1 infection.

Bacteriophage SPP1 infection of Bacillus subtilis cells bearing plasmids induces the synthesis of multigenome-length plasmid molecules. Two independent pathways can account for this synthesis. In one of those, homology to the phage genome is required, whereas in the other such homology is not a prerequisite. In wild type cells both modes overlap. In dnaB(Ts), at non permissive temperature, or in recE polA strains the main concatemeric plasmid replication mode is the homology-dependent plasmid (hdp) mode. The rate of recombination-dependent concatemeric plasmid DNA synthesis is a consequence of a phage-plasmid interaction which leads to chimeric phage::plasmid DNA. The second mode, which is an homology-independent plasmid (hip) mode seems to be triggered upon the synthesis of a phage encoded product(s) (e.g. inactivation of the exonuclease V enzyme).     

Characterization of P1argF derivatives from Escherichia coli K12 transduction. I. IS1 elements flank the argF gene segment

Summary Specialized transducing derivatives of the temperate bacteriophage P1 (P1std) are selected by transduction into recipients with deletions in the corresponding genes (Stodolsky 1973). When Escherichia coli K12 strains are used as donors in such transduction experiments, P1argF derivatives can be selected. The argF gene is unique to these strains (Glansdorff et al. 1967). Under these experimental conditions P1argF are formed with frequencies 10,000 times greater than other P1std. The majority of the P1argF derivatives that have been analyzed are indistinguishable by cleavage analyses. One such derivative, P1argF5 has been characterized in detail. Heteroduplex analysis against P1, P7, and P1CmO identified an 11 kb insertion of DNA precisely at the naturally occurring IS1 locus of P1. Cleavage analysis with EcoRI, BamHI and PstI confirmed this finding. To further define the argF insertion, a P1Cm13argF derivative was constructed having the IS1 sequences of Cm13 and argF in opposite orientation. Intrastrand annealing of P1Cm13argF5 DNA established that the argF segment is flanked by directly repeated IS1 sequences. The IS1-argF-IS1 segment is desigmated Tn2901. The assignment of the map position of the argF gene within the 11 kb insert of P1argF5 is discussed. The evolutionary significance of this finding and a model for P1argF formation is also presented.     

Mutator and antimutator effects of the bacteriophage P1 hot gene product

The Hot (homolog of theta) protein of bacteriophage P1 can substitute for the Escherichia coli DNA polymerase III theta subunit, as evidenced by its stabilizing effect on certain dnaQ mutants that carry an unstable polymerase III epsilon proofreading subunit (antimutator effect). Here, we show that Hot can also cause an increase in the mutability of various E. coli strains (mutator effect). The hot mutator effect differs from the one caused by the lack of theta. Experiments using chimeric theta/Hot proteins containing various domains of Hot and theta along with a series of point mutants show that both N- and C-terminal parts of each protein are important for stabilizing the epsilon subunit. In contrast, the N-terminal part of Hot appears uniquely responsible for its mutator activity.     

In pursuit of a molecular mechanism for adaptive gene amplification

"Adaptive" or "stationary-phase" mutation is a collection of apparent stress responses in which cells exposed to a growth-limiting environment generate genetic changes, some of which can allow resumption of rapid growth. In the well-characterized Lac system of Escherichia coli, reversions of a lac frameshift allele give rise to adaptive point mutations. Also in this system, adaptive gene amplification has been documented as a separate and parallel response that allows growth on lactose medium without acquisition of a compensatory frameshift mutation. In amplification, the DNA region containing the weakly functional lac allele becomes amplified to multiple copies, which produce sufficient enzyme activity to allow growth on the otherwise growth-limiting lactose medium. The amplifications are "adaptive" in that they occur after cells encounter the growth-limiting environment. Adaptive amplification is a reversible genetic change that allows adaptation and growth. It may be similar to chromosomal instability observed in the origins and progression of many cancers. We explore possible molecular mechanisms of adaptive amplification in the bacterial system and note parallels to chromosomal instability in other systems.     

In vitro activation of bacteriophage P2 late gene expression by extracts from phage P4-infected cells.

We have used a cell-free, DNA-dependent protein-synthesizing system to study the stimulation of phage P2 late gene expression by satellite phage P4. An activity is present in extracts prepared from P4-infected cells, which, when added to the in vitro system with P2 DNA template, stimulates the synthesis of a number of P2 proteins. These stimulated proteins include the major P2 capsid protein (N gene product) and a major component of the P2 phage tail (FII gene product). Extracts prepared from P4-infected cells are also able to stimulate the synthesis from P4 DNA of two low-molecular-weight proteins (18,500 and 17,000 Mr). The stimulating activity has no effect on the synthesis of proteins from lambda plac5 template. Extracts prepared from cells infected with P4 alpha amber mutants lack this stimulating activity.