Isolation and preliminary characterization of Escherichia coli mutants resistant to lethal action of the bacteriophage λ P gene

Abstract: Transforming Growth Factor (TGF)-β family, including TGF-β, bone morphorgenic protein (BMP), and activn, plays an important role in essential cellular functions such as proliferation, differentiation, apoptosis, tissue remodeling, angiognesis, immune responses, and cell adhesions. TGF-β predominantly transmits the signals through serine/ threonine receptor kinases and cytoplasmic proteins called Smads. Since the discovery of TGF-β in the early 1980s, the dysregulation of TGF-β/Smad signaling has been implicated in the pathogenesis of human diseases. Among signal transducers in TGF-β/Smad signaling, inhibitory Smads (I-Smads), Smad6 and Smad7, act as major negative regulators forming autoinhibitory feedback loops and mediate the cross-talking with other signaling pathways. Expressions of I-Smads are mainly regulated on the transcriptional levels and post-translational protein degradations and their intracellular levels are tightly controlled to maintain the homeostatic balances. However, abnormal levels of I-Smads in the pathological conditions elicit the altered TGF-β signaling in cells, eventually causing TGF-β-related human diseases. Thus, exploring the molecular mechanisms about the regulations of ISmads may provide the therapeutic clues for human diseases induced by the abnormal TGF-β signaling.

Abstract: 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.

Abstract: A cloned rpoB gene, specifying an apparently mutant RNA polymerase beta subunit, protected Escherichia coli against the cytocidal effects of the E3 protein of bacteriophage SPO1, suggesting that RNA polymerase is the primary cellular target of the E3 protein. Two segments of the wild-type E. coli genome, one of which specifies a suppressor of dnaK mutations, and thus, possibly, a molecular chaperone, also provided protection when overexpressed, but wild-type rpoB did not.

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.

Abstract: Bacteriophage lambda, having a mutation replacing glycine by glutamic acid at the 48th codon of cro, kills the host under N- conditions; we call this the hk mutation. In lambda N-N-cl-hk phage-infected bacteria, the late gene R is expressed to a significant level, phage DNA synthesis occurs with better efficiency, and the Cro activity is around 20% less, all compared to those in lambda N-N-cl-hk(+)-infected bacteria. Segments of lambda DNA from the left of pR to the right of tR2, carrying cro, cII, O, P, and the genes of the nin5 region from the above hk and hk+ phages, were cloned in pBR322. Studies with these plasmids and their derivatives having one or more of the lambda genes deleted indicate that the hk mutation is lethal only when a functional P gene is also present. When expression of P from pR is elevated, due to the deletion of tR1, host killing also occurs without the hk mutation. We conclude that the higher levels of P protein, produced either (1) when cro has the hk mutation or (2) when tR1 is deleted, are lethal to the host. We also show that due to the hk mutation, the Cro protein becomes partially defective in its negative regulation at pR, resulting in the expression of P to a lethal level even in the absence of N protein-mediated antitermination. This P protein-induced host killing depends neither on lambda DNA replication nor on any other gene functions of the phage.

Abstract: A new protein has been isolated from E. coli which causes specific termination and release of RNA during synthesis in vitro. It has been given the name ρ-factor.

Abstract: A bacterial mutation affecting λ DNA replication, called groPC756, has been mapped between the thr and leu bacterial loci. Most of the parental λ DNA does not undergo even one round of replication in this host. Lambda mutants, called π, which map in the λ P gene are able to overcome the inhibitory effect of the groPC756 mutation. It is shown that the mutation at the groPC locus also interferes with bacterial growth at 42°C. A λ-transducing phage, carrying the groPC+ allele, was isolated as a plaqueformer on groPC756 bacteria. Upon lysogenization, it restores both the gro + and temperature resistant phenotypes.

Abstract: Binding of the O protein of phage lambda to the replication origin (ori lambda) results in the formation of an organized nucleoprotein structure termed the O-some The O-some serves to localize and initiate a six-protein sequential reaction that provides for localized unwinding of the origin region, the critical prepriming step for precise initiation of DNA replication By the use of electron microscopy of gold-tagged antibody complexes, we have defined four stages of protein association and dissociation reactions that are involved in the prepriming pathway First, as defined previously, O protein binds to multiple DNA sites and self-associates to form the O-some Second, lambda P and host DnaB proteins add to the O-some to generate an OPDnaBori lambda complex Addition of the DnaK and DnaJ proteins yields a third stage complex containing DnaK, DnaJ, O, P, and DnaB With the addition of ATP and single-strand binding protein (SSB), the P protein is largely removed, and the DnaB acts as a helicase to generate locally unwound, SSB-coated single strand DNA Thus, the initiation of lambda DNA replication requires ordered assembly and partial disassembly of specialized nucleoprotein structures The disassembly activity of DnaK and DnaJ may be their general role in the heat shock response

Abstract: The isolation of a bacterial mutation in a gene, designated groPC, which affects the growth of phages lambda and P2 is described. Lambda replication is severely limited in the strain, and some lambda π mutations, which map in (or near) the P gene, allow growth. The gro mutation, groPC259, is recessive to wild type and maps between threonine (thr) and diaminopimelate (dapB) on the E. coli chromosome. The possibility that the groPC gene is concerned with host DNA replication is discussed.

Abstract: λc17 is a mutant of λ originally recognized by Pereira da Silva because of its nearly clear plaque morphology on a sensitive host and its ability to give virulence when combined in cis with certain cI mutations. Experiments are presented which show that c17 confers on the chromosome which carries it two unique and distinct properties: constitutive function of genes O and P and loss of normal sensitivity to replication inhibition. These properties lead to constitutive replication of λc17 DNA in the immune host and to host killing, even though λc17 does not lyse an immune host and release progeny phage unless it also carries a cI mutation. A model is presented to explain λc17cI virulence that proposes that those λc17 genes which are subject to cI repression in the immune host can be derepressed if phage replication produces too many phage chromosomes for the cI repressor to control. It is proposed that this derepression can occur only if the replicating element is unable to augment the level of free repressor produced by the prophage, i.e., if it contains a cI mutation.