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

Abstract

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.

Phage Lambda P Protein: Trans-Activation, Inhibition Phenotypes and their Suppression

“…They concluded that P-lethality does not involve interaction of P with the host DnaB, DnaJ, or DnaK proteins, which are all essential for λ DNA replication [25,26,27] but is targeted to the E. coli DNA replication initiation protein DnaA, inhibiting its oriC DNA binding [28]. Datta et al [29] identified mutations within the gene dnaA that confer cellular resistance to P-lethality. An alternative hypothesis for the absence of transformants formed by plasmids expressing P was not considered in prior studies, i.e., that P expression from the transformed pBR322-derived plasmids employed interfered with / blocked the initiation of plasmid replication and copy increase within the newly transformed cell.…”

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

“…Transformation and λ P sensitivity of E. coli These were done by the procedures exactly as described in the preceding paper (Datta et al, 2005).…”

“…The Kil protein possibly interacts with certain component (s) of the cell envelope (Greer, 1975b) and inhibits division (Sergueev et al, 2001) (3) The CII protein of this phage has been reported to inhibit host DNA replication (Kedzierska et al, 2003) We showed for the first time that the DNA replication gene P of λ when expressed at elevated level caused bacterial killing even in the absence of phage DNA replication (Maiti et al, 1991a) and that the E. coli rpl mutants which were resistant to λ P gene lethality could be isolated by the mutagenesis of this bacterium (Maiti et al, 1991b), and also the rpl-like mutations have been isolated by in vitro mutagenesis of the dnaA gene is a plasmid (Datta et al, 2005). All those rpl mutations have been located within the bacterial DNA initiator gene dnaA (Datta et al, 2005). All these suggest that the λ P gene-mediated host lethality possibly involves its certain type of interaction with the DnaA protein (the DnaA protein will be called as DnaA in the text) of E. coli.…”

“…The non-lethal interactions of bacteriophage λ genes with those of Escherichia coli have been studied in depth (Friedman et al, 1984). These have been summarized in Datta et al (2005) There are three reports of host toxicity by lambda. (1) Induction of λN − cIts lysogen at 42 o C causes bacterial killing that requires the phage replication genes O and P as well as the right promoter pR (Sly et al, 1968).…”

Inactivation of Indispensable Bacterial Proteins by Early Proteins of Bacteriophages:Implication in Antibacterial Drug Discovery

“…It was indeed shown that expression of P protein from a resident plasmid could restrict the growth of E. coli significantly [20][21]. Interestingly, some dominant point mutations within 200 th , 246 th , and 313 th codons of E. coli dnaA gene nullified P protein-mediated lethality [22]. Besides, P protein also inhibited the ATP and oriC binding activities of DnaA [23].…”