Intermediates in bacteriophage Mu lysogenization of Escherichia coli him hosts

DNA transposition is central to the propagation of temperate phage Mu. A long-standing problem in Mu biology has been the mechanism by which the linear genome of an infecting phage, which is linked at both ends to DNA acquired from a previous host, integrates into the new host chromosome. If Mu were to use its well-established cointegrate mechanism for integration (single-strand nicks at Mu ends, joined to a staggered double-strand break in the target), the flanking host sequences would remain linked to Mu; target-primed replication of the linear integrant would subsequently break the chromosome. The absence of evidence for chromosome breaks has led to speculation that infecting Mu might use a cut-and-paste mechanism, whereby Mu DNA is cut away from the flanking sequences prior to integration. In this study we have followed the fate of the flanking DNA during the time course of Mu infection. We have found that these sequences are still attached to Mu upon integration and that they disappear soon after. The data rule out a cut-and-paste mechanism and suggest that infecting Mu integrates to generate simple insertions by a variation of its established cointegrate mechanism in which, instead of a "nick, join, and replicate" pathway, it follows a "nick, join, and process" pathway. The results show similarities with human immunodeficiency virus integration and provide a unifying mechanism for development of Mu along either the lysogenic or lytic pathway.Mu DNA in a phage head is linear and covalently attached to host chromosomal DNA packaged during the previous round of infection (29). Fifty to 150 bp of host sequences flank the left end of Mu, and 0.5 to 3 kb flank the right end. Upon infection of an Escherichia coli host, noncovalently closed circular forms of Mu have been observed and have been presumed to be integrative precursors (16,26). Infecting DNA integrates into the host genome without prior replication, a process that has been referred to as "conservative" or "nonreplicative" integration (1,15,19). Whether this integration follows the well-established "cointegrate" mechanism described below or occurs by some alternate mechanism is not known. In the ensuing lytic cycle, where Mu DNA is amplified over 100-fold, transposition is known to occur by the cointegrate mechanism (9).Study of Mu transposition using plasmid substrates in vitro established that single-strand cleavages at Mu ends initially liberate free 3Ј OHs which subsequently attack target DNA to generate a strand transfer intermediate (Fig. 1A, panel i) (22). The Mu-target fusion joint leaves 3Ј OHs on target ends, which can be used as primers for replication through Mu, resolving the intermediate to a cointegrate end product, where directly repeated copies of Mu border the target and non-Mu donor sequences (21, 23). The predominant end products of Mu transposition/replication during the lytic cycle are cointegrates (9). If infecting Mu were also to integrate by this mechanism, target-primed replication into the flanking sequences would break the ch...

Section: Resultsmentioning

confidence: 99%

Chromosomal Integration Mechanism of Infecting Mu Virion DNA

Agrawal

Harshey

2006

“…The decision that the virus must make to develop along one of these two pathways is complex (5,28) and can be altered by conditions such as the multiplicity of infection and the genetic composition of the host cell (3,4,8,27). Two viral elements that are critically involved in the lysis-lysogeny decision are the Mu c repressor and its operator site located about 1 kb from the left end of viral DNA.…”

mentioning

confidence: 99%

Temperature-sensitive mutations in the bacteriophage Mu c repressor locate a 63-amino-acid DNA-binding domain

Vogel

Howe

et al. 1991

Phage Mu's c gene product is a cooperative regulatory protein that binds to a large, complex, tripartite 184-bp operator. To probe the mechanism of repressor action, we isolated and characterized 13 phage mutants that cause Mu to undergo lytic development when cells are shifted from 30 to 42°C. This collection contained only four mutations in the repressor gene, and all were clustered near the N terminus. The cts62 substitution of R47-+Q caused weakened specific DNA recognition and altered cooperativity in vitro. A functional repressor with only 63 amino acids of Mu repressor fused to a C-terminal fragment of I-galactosidase was constructed. This chimeric protein was an efficient repressor, as it bound specifically to Mu operator DNA in vitro and its expression conferred Mu immunity in vivo. A DNA looping model is proposed to explain regulation of the tripartite operator site and the highly cooperative nature of repressor binding.The outcome of an infection with bacteriophage Mu is either killing of the host cell or stable integration of phage DNA into the host chromosome and lysogenic growth of the bacterium. The decision that the virus must make to develop along one of these two pathways is complex (5, 28) and can be altered by conditions such as the multiplicity of infection and the genetic composition of the host cell (3,4,8,27). Two viral elements that are critically involved in the lysis-lysogeny decision are the Mu c repressor and its operator site located about 1 kb from the left end of viral DNA.The Mu operator is a large regulatory DNA sequence containing convergent promoters and sites for binding both phage-encoded and host-derived proteins. The two principal promoters that regulate phage development in the lytic and lysogenic pathways are PE and PCM. PE is an early promoter and the control point of a polycistronic operon encoding transposition and regulatory functions (20). The PcM promoter is the start point for a monocistronic transcript that proceeds toward the left end of the phage genome and encodes the repressor (11,18,19). The PE and PcM promoters are regulated by the Mu repressor, which binds in a cooperative manner to nearly 200 bp of operator DNA. The Mu regulatory region is organized into three blocks of DNA sequence designated operator sites 01, 02, and 03 (18, 19). Each operator site contains two or more 14-bp repressor consensus sites; there are 3, 4, and 2 consensus sequences localized in operator sites 01, 02, and 03, respectively (Fig. 1). An integration host factor binding site separates 01 and 02.The Mu repressor is composed of 22,000-Da protomers that oligomerize. At its N terminus, it contains significant structural homology with the N-terminal domain of Mu * Corresponding author. transposase (13) (Fig. 1). Interactions between repressor and supercoiled operator DNA have been modeled in vitro, and the following regulatory pattern was observed. As repressor concentrations were gradually increased, protection from DNase I cleavage was found in 01 and 02 and PE transcription gradu...

“…We suggest that the helping effect could be the result of'provision of Mu B function by auxiliary phage. Supporting evidence is reported in the accompanying paper (5).…”

Section: Resultsmentioning

confidence: 49%

Lysogenization of Escherichia coli him+, himA, and himD hosts by bacteriophage Mu

Bourret

Fox

1988

Self Cite

The possible outcomes of infection of Escherichia coli by bacteriophage Mu include lytic growth, lysogen formation, nonlysogenic surviving cells, and perhaps simple killing of the host. The influence of various parameters, including host himA and himD mutations, on lysogeny and cell survival is described. Mu does not grow lytically in or kill him bacteria but can lysogenize such hosts. Mu ct lysogenizes about 8% of him' bacteria infected at low multiplicity at 37°C. The frequency of lysogens per infected him' cell diminishes with increasing multiplicity of infection or with increasing temperature over the range from 30 to 420C. In him bacteria, the Mu lysogenization frequency increases from about 7% at low multiplicity of infection to approach a maximum where most but not all cells are lysogens at high multiplicity of infection. Lysogenization of him hosts by an assay phage marked with antibiotic resistance is enhanced by infection with unmarked auxiliary phage. This helping effect is possible for at least 1 h, suggesting that Mu infection results in formation of a stable intermediate. Mu immunity is not required for lysogenization of him hosts. We argue that in him bacteria, all Mu genomes which integrate into the host chromosome form lysogens.