Bacteriophages T7, lambda, P22, and P2/P4 (from Escherichia coli), as well as phi 29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protei ...

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Bacteriophage Protein–Protein Interactions

Bacteriophage λ, rediscovered in the early 1950s, has served as a model in molecular biology studies for decades. Although currently more complex organisms and more complicated biological systems can be studied, this phage is still an excellent model to investigate principles of biological processes occurring at the molecular level. In fact, very few other biological models provide possibilities to examine regulations of biological mechanisms as detailed as performed with λ. In this chapter, recent advances in our understanding of mechanisms of bacteriophage λ development are summarized and discussed. Particularly, studies on (i) phage DNA injection, (ii) molecular bases of the lysis-versus-lysogenization decision and the lysogenization process itself, (iii) prophage maintenance and induction, (iv), λ DNA replication, (v) phage-encoded recombination systems, (vi) transcription antitermination, (vii) formation of the virion structure, and (viii) lysis of the host cell, as published during several past years, will be presented.

Phage λ--new insights into regulatory circuits.

Although most of the 73 open reading frames (ORFs) in bacteriophage λ have been investigated intensively, the function of many genes in host-phage interactions remains poorly understood. Using yeast two-hybrid screens of all lambda ORFs for interactions with its host Escherichia coli, we determined a raw data set of 631 host-phage interactions resulting in a set of 62 high-confidence interactions after multiple rounds of retesting. These links suggest novel regulatory interactions between the E. coli transcriptional network and lambda proteins. Targeted host proteins and genes required for lambda infection are enriched among highly connected proteins, suggesting that bacteriophages resemble interaction patterns of human viruses. Lambda tail proteins interact with both bacterial fimbrial proteins and E. coli proteins homologous to other phage proteins. Lambda appears to dramatically differ from other phages, such as T7, because of its unusually large number of modified and processed proteins, which reduces the number of host-virus interactions detectable by yeast two-hybrid screens.

The Protein Interaction Network of Bacteriophage Lambda with Its Host, Escherichia coli

Bacteriophage λ genome is one of the classical model replicons in studies on the regulation of DNA replication. Moreover, since genes coding for Shiga toxins are located in genomes of lambdoid phages, understanding of mechanisms controlling λ DNA replication may be of bio-medical importance. During lytic development of bacteriophage λ, its genome is replicated according to the θ (circle-to-circle) mode early after infection, and then it is switched to the σ (rolling circle) mode. Two mechanisms of regulation of this switch were proposed recently and both suggested a crucial role for directionality of λ DNA replication. Whereas one hypothesis assumed transient impairment of ClpP/ClpX-mediated proteolysis of the λO initiator protein, another suggested a crucial role for transcriptional activation of the oriλ region and factors involved in the control of the p
 R promoter activity. Here we demonstrate that mutations in clpP and clpX genes had little influence on both directionality of λ DNA replication and appearance of σ replication intermediates. On the other hand, regulators affecting activity of the p
 R promoter (responsible for initiation of transcription, which activates oriλ) directly or indirectly influenced directionality of λ DNA replication to various extents. Therefore, we conclude that regulation of the efficiency of transcriptional activation of oriλ, rather than transient impairment of the λO proteolysis, is responsible for the control of the switch from θ to σ replication, and propose a model for this control.

Switch from theta to sigma replication of bacteriophage lambda DNA: factors involved in the process and a model for its regulation.

Under the condition of expression of lambda P protein at lethal level, the oriC DNA-binding activity is significantly affected in wild-type E. coli but not in the rpl mutant. In purified system, the lambda P protein inhibits the binding of both oriC DNA and ATP to the wild-type DnaA protein but not to the rpl DnaA protein. We conclude that the lambda P protein inhibits the binding of oriC DNA and ATP to the wild-type DnaA protein, which causes the inhibition of host DNA synthesis initiation that ultimately leads to bacterial death. A possible beneficial effect of this interaction of lambda P protein with E. coli DNA initiator protein DnaA for phage DNA replication has been proposed.

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The Bacteriophage λ DNA Replication Protein P Inhibits the oriC DNA- and ATP-binding Functions of the DNA Replication Initiator Protein DnaA of Escherichia coli

Earlier, we reported that the bacteriophage lambda P gene product is lethal to Escherichia coli, and the E. coli rpl mutants are resistant to this lambda P gene-mediated lethality. In this paper, we show that under the lambda P gene-mediated lethal condition, the host DNA synthesis is inhibited at the initiation step. The rpl8 mutation maps around the 83 min position in the E. coli chromosome and is 94 % linked with the dnaA gene. The rpl8 mutant gene has been cloned in a plasmid. This plasmid clone can protect the wild-type E. coli from lambda P gene-mediated killing and complements E. coli dnaAts46 at 42 degrees C. Also, starting with the wild-type dnaA gene in a plasmid, the rpl-like mutations have been isolated by in vitro mutagenesis. DNA sequencing data show that each of the rpl8, rpl12 and rpl14 mutations has changed a single base in the dnaA gene, which translates into the amino acid changes N313T, Y200N, and S246T respectively within the DnaA protein. These results have led us to conclude that the rpl mutations, which make E. coli resistant to lambda P gene-mediated host lethality, are located within the DNA initiator gene dnaA of the host.

Lopa Adhikari

Papers

The Mutation that Makes Escherichia coli Resistant to λ P Gene-mediated Host Lethality Is Located within the DNA Initiator Gene dnaA of the Bacterium