cII Gene Products Research Articles

Toxicity of the bacteriophage λ cII gene product to Escherichia coli arises from inhibition of host cell DNA replication

The bacteriophage λ cII gene codes for a transcriptional activator protein which is a crucial regulator at the stage of the “lysis-versus-lysogeny” decision during phage development. The CII protein is highly toxic to the host, Escherichia coli, when overproduced. However, the molecular mechanism of this toxicity is not known. Here we demonstrate that DNA synthesis, but not total RNA synthesis, is strongly inhibited in cII-overexpressing E. coli cells. The toxicity was also observed when the transcriptional stimulator activity of CII was abolished either by a point mutation in the cII gene or by a point mutation, rpoA341, in the gene coding for the RNA polymerase α subunit. Moreover, inhibition of cell growth, caused by both wild-type and mutant CII proteins in either rpoA+ or rpoA341 hosts, could be relieved by overexpression of the E. coli dnaB and dnaC genes. In vitro replication of an oriC-based plasmid DNA was somewhat impaired by the presence of the CII, and several CII-resistant E. coli strains contain mutations near dnaC. We conclude that the DNA replication machinery may be a target for the toxic activity of CII.

The Escherichia coli RNA polymerase alpha subunit and transcriptional activation by bacteriophage lambda CII protein.

Bacteriophage lambda is not able to lysogenise the Escherichia coli rpoA341 mutant. This mutation causes a single amino acid substitution Lys271Glu in the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). Our previous studies indicated that the impaired lysogenisation of the rpoA341 host is due to a defect in transcriptional activation by the phage CII protein and suggested a role for alphaCTD in this process. Here we used a series of truncation and point mutants in the rpoA gene placed on a plasmid to investigate the process of transcriptional activation by the cII gene product. Our results indicate that amino-acid residues 265, 268 and 271 in the a subunit may play an important role in the CII-mediated activation of the pE promoter (most probably residue 271) or may be involved in putative interactions between alphaCTD and an UP-like element near pE (most probably residues 265 and 268). Measurement of the activity of pE-lacZ, pI-lacZ and p(aQ)-lacZ fusions in the rpoA+ and rpoA341 hosts demonstrated that the mechanism of activation of these CII-dependent promoters may be in each case different.

Impaired lysogenisation of the Escherichia coli rpoA341 mutant by bacteriophage lambda is due to the inability of CII to act as a transcriptional activator.

The C-terminus of the alpha subunit of Escherichia coli RNA polymerase is known to function in transcriptional activation at certain promoters. This region was previously shown to be necessary for full activation of the pE promoter by the phage lambda CII protein in vitro. In this work we investigated the inability of phage lambda to follow the lysogenic pathway in cells carrying the point mutation rpoA341 (a change of lysine 271 to glutamic acid). We found that neither overexpression of the cII gene nor stabilisation of the CII protein by the can1 mutation or by cIII gene overexpression was able to suppress the block in lysogenisation. In contrast, the lambda cin1 phage, which carries a CII-independent promoter for the expression of the cI gene, was able to efficiently lysogenise the rpoA341 mutant strain. Furthermore, the rpoA341 mutation prevented the activation of pE-lacZ and pI-lacZ transcriptional fusions by CII. Therefore we conclude that transcriptional activation by the cII gene product is abolished by the rpoA341 mutation, most probably due to impaired interaction between the CII activator and mutant RNA polymerase. The inability of RNA polymerase to respond to CII results in the impairment of lysogenisation of the rpoA341 mutant by phage lambda.

The CII protein of bacteriophage 186 establishes lysogeny by activating a promoter upstream of the lysogenic promoter.

We have shown previously that the cII gene product of the non-lambdoid temperate bacteriophage 186 is required for the establishment of lysogeny. We show here that CII, a potential helix-turn-helix DNA-binding protein, establishes lysogeny by activating a promoter (PE) which spans the apl/cII intergenic region, upstream of the lysogenic promoter, PL. The start site of the PE transcript (+1) has been mapped by primer extension and we have identified the CII binding determinants at PE by DNase I footprinting. CII binds to inverted repeat sequences separated by two turns of the helix, with binding half-sites centred at the 38 and -58 positions of PE. Oligomerisation studies with purified CII protein indicate that a CII tetramer may be the species that binds to this site. We also show that PE is subject to direct negative feedback by the CI repressor.

Monoclonal antibodies to a proenkephalin A fusion peptide synthesized in Escherichia coli recognize novel proenkephalin A precursor forms.

Monoclonal antibodies have been generated to a chimeric peptide comprised of Escherichia coli beta-galactosidase fused to the amino acid sequence 69-207 of human preproenkephalin A. Two monoclonal antibodies, PE-1 and PE-2, were identified by their ability to recognize the same segment of proenkephalin A fused to the cII gene product of the E. coli bacteriophage lambda. The binding domains of PE-1 and PE-2 have been broadly located, with respect to the primary translation product, within the amino acid sequences 152-207 and 84-131, respectively. Immunoblot analysis of total bovine adrenomedullary chromaffin granule lysate reveals PE-1 and PE-2 immunoreactive forms of observed molecular mass 35, 33, 29, 24, 22, and 15 kDa, and an 18-kDa PE-1 immunoreactive form. Separation of granule membranes from their contents reveals differential membrane association of these high molecular weight polypeptides. There is preliminary evidence that PE-1 may be detecting a subset of polypeptides where shortening from the NH2 terminus has occurred. We postulate that the 35-kDa form represents the intact bovine enkephalin precursor of predicted molecular mass 27.3 kDa. This experimental approach should be generally applicable to the generation of antibodies which will recognize intact peptide precursors together with their post-translational cleavage products.

Kinetic analysis of mutations affecting the cII activation site at the PRE promoter of bacteriophage lambda.

Abortive initiation and run-off transcription assays were used to study the effects of cy mutations on activation of the phage lambda PRE promoter by cII gene product. Six point mutations in the repeated T-T-G-C sequences that flank the -35 consensus region of PRE decreased the apparent affinity of the promoter for cII protein by factors of 4-16 relative to the wild-type affinity. Kinetic analyses of transcription initiation in the presence and absence of cII protein demonstrated that five of the six mutations did not significantly affect the intrinsic interaction of RNA polymerase with PRE. Thus, these mutations differ from other cy mutations, including those in the -35 consensus region, which affect the formation of polymerase-PRE closed complexes or the isomerization of closed complexes to open complexes but do not affect the binding of cII protein. A sixth T-T-G-C mutation, cy3001, may affect intrinsic initiation by RNA polymerase as well as cII binding.

Evidence that the cro repressor inhibits expression of the bacteriophage λP gene at high multiplicities of infection

The activity of the lambda P gene product at various multiplicities of infection (m.o.i.) was examined in CI- conditions using an assay which measures the disappearance of the rapidly-sedimenting closed-circular (c.c.) form of phage DNA. When cells were infected with lambda CI857 at multiplicities of 5 phage/cell or less, between 65%-75% c.c. DNA was lost during incubation. If the multiplicity of infection was increased to 10 phage/cell or greater, a marked inhibition in the cleavage of c.c. DNA was observed. When bacteria were infected with either lambda CI857CII2002 or lambda CI857cro27 at low m.o.i., the usual 65%-75% decrease in the percentage of c.c. phage DNA occurred during incubation. In contrast, no losses in c.c. DNA were noted after infection with lambda CI857cro27susP3. At high m.o.i., the cleavage of c.c. DNA was inhibited after infection with lambda CI857CII2002, but not after infection with lambda CI857cro27. It is concluded that at high m.o.i. in CI- infections, the expression of gene P is unaffected by the CII gene product, but is inhibited by the increased intracellular levels of cro protein.

The cIII gene and protein of bacteriophage λ

The cIII and cII gene products of bacteriophage λ control the lysogenic response through positive regulation of the viral repressor and integration genes and negative regulation of lytic functions. Although many aspects of cII action have been defined biochemically, little is known about cIII. As a first step in defining the molecular role of cIII in the regulation of lysogeny, we have determined the precise location and DNA sequence of the cIII gene. In addition, we have identified the cIII gene product as a polypeptide with a molecular weight of approximately 6000.

Coordinated regulation of the cI and int genes in coliphage λ and specificity of the cII/cIII activators

Expression of the cI (repressor) and int (integrase) genes in the λ lysogenic pathway has been studied by following protein synthesis in uv-irradiated cells. Gene expression following infection by λ +, a phage carrying an intact repressor gene, and by λ vir, a phage carrying operator mutations with reduced affinity for the repressor, substantiated previous findings on the coordinated regulation of these two genes, and showed that their expression can be dissociated from that of other early genes. Experiments with λ hybrid phages showed that the cII gene products of λ imm21 and of λ imm22, which are of origin of phages 21 and P22, can activate Int synthesis from P I of λ origin. Possible homology between the various cII genes is discussed.

The regulation of integrative recombination by the b2 region and the cII gene of bacteriophage λ

We have used a PP′ × BB′ prophage excision system to study the regulation of int expression in bacteriophage λ. The assay is based on heteroimmune infection of a lysogen [carrying a λ int −gal prophage flanked by the phage (PP′) and the bacterial (BB′) attachment sites] with an int + helper. The Int function provided by the helper excises the gal prophage, which subsequently is detected in the lysate. This Int-promoted prophage excision depends on int + and cII + genes, is independent of Rec and Red systems, is thermolabile, and depends on a high multiplicity of infection with the cII + helper, in agreement with known characteristics of integrative recombination. While λ cII − is defective in helping integrative recombination, λ b2 cII − is proficient in promoting the reaction. A cis-trans complementation test shows that the inhibitor in the b2 region acts in cis to the active int gene. These results suggest that the b2 region contains a nondiffusible inhibitor of integration which is overcome by the cII gene product. We discuss the possible role of a b2 inhibitory site on Int expression from p I or p L promoters.

A pleiotropic regulatory mutation in lambda bacteriophage.

Lambda bacteriophage mutants, lambdasar, were isolated. These mutants can form plaques on a non lysogenic lawn and are unable to grow on nonimmune (imm-), cro constitutive hosts. Analysis of the restriction of lambdasar by a set of defective lysogens suggested that both the cro and cII gene products participate in the inhibition. The sar mutations were mapped in the ori region between the genes cII and O. Complementation experiments showed that under the restrictive conditions lamdasar is defective in the expression of both the N and the O genes. Transcription analyses support these findings, as lambdasar is unable to serve as a template for transcription after infecting cro constitutive hosts. In addition lambdasar does not replicate under the restrictive conditions, although its DNA can bind to the host membrane to some extent. The Sar phenotype can be relieved by removing sites of action of cro either by a V2 mutation or by substituting the lambda immunity region by imm434 or imm21. Similarly introducing a cy mutation, which interferes with the action of the cII gene product, also eliminates the Sar effect. The sar mutation can suppress cy mutations as manifested in plaque morphology, lysogenization frequency, cI repressor synthesis and the expression of rex function. Suppression takes place only when the sar mutation is present in cis to cy and it requires the action of the cII and cIII gene products. It is suggested that the sar mutation suppresses cy by activating a new promoter for repressor synthesis, pro. The results also suggest that the cII and cIII gene products may act at a site other than y.