cIII Gene Products Research Articles

Bacteriophage lambda cIII gene product has an additional function apart from inhibition of cII degradation.

For lysogenization of Escherichia coli cells by bacteriophage lambda, functions of three lambda genes called c are necessary. The cI gene codes for a repressor that blocks activities of lytic promoters. However, early after infection, expression of cI is dependent on the function of the cII gene, coding for a specific transcriptional activator. The cII protein is unstable in E. coli cells due to FtsH-mediated proteolysis. The cIII gene product is an inhibitor of the FtsH protease. Here we demonstrate that cIII may have another function apart from inhibition of cII degradation. We found that overexpression of the cII gene results in impaired lysogenization by phage lambda, however simultaneous overexpression of the cIII gene abolished this negative effect on lysogenization. Analysis of cII-mediated transcriptional activation of certain promoters at different levels of cII and cIII proteins in cells confirmed that observed effects cannot be explained assuming that the only role of cIII is inhibition of FtsH-mediated degradation of cII. We propose that cIII has an additional role apart from its well-known function in indirect stabilization of cII. Apparently, cIII influences not only cII level but also activity of this transcriptional stimulator, especially at its high concentrations.

Proteolysis of bacteriophage lambda CII by Escherichia coli FtsH (HflB).

FtsH (HflB) is a conserved, highly specific, ATP-dependent protease for which a number of substrates are known. The enzyme participates in the phage lambda lysis-lysogeny decision by degrading the lambda CII transcriptional activator and by its response to inhibition by the lambda CIII gene product. In order to gain further insight into the mechanism of the enzymatic activity of FtsH (HflB), we identified the peptides generated following proteolysis of the phage lambda CII protein. It was found that FtsH (HflB) acts as an endopeptidase degrading CII into small peptides with limited amino acid specificity at the cleavage site. beta-Casein, an unstructured substrate, is also degraded by FtsH (HflB), suggesting that protein structure may play a minor role in determining the products of proteolysis. The majority of the peptides produced were 13 to 20 residues long.

Translation Control of Gene Expression

The bacteriophage lambda cIII gene product is an early regulator of the lysogenic pathway. The availability of a set of cIII expression mutants allowed us to establish the structure-function relationship of the cIII mRNA. We demonstrated, using defined in vitro systems, that the cIII mRNA is present in two conformations at equilibrium. Mutations that have been shown to lead to cIII overexpression were found to freeze the RNA in one conformation (structure B), and permit efficient binding to the 30S ribosomal subunit. Mutations that have been shown to prevent cIII translation cause the mRNA to assume the alternative conformation (structure A). In this structure, the translation initiation region is occluded, thereby preventing 30S ribosomal subunit binding. Translation of the cIII gene is regulated by RNaseIII. We have localized the RNaseIII responsive element (RRE) to the cIII coding region. We suggest that the regulation of the equilibrium between the two mRNA conformations provides a mechanism for the control of cIII gene expression. The way in which RNaseIII participates in this regulation is as yet unknown.

Functional and structural elements of the mRNA of the cIII gene of bacteriophage lambda

The bacteriophage λ cIII gene product is an early regulatory protein that participates in the lysis-lysogeny decision of the phage following infection. We have previously shown that the translation of the cIII gene is determined by two unique factors: (1) efficient expression is dependent upon the presence of RNaseIII in the cell; (2) alternative mRNA structures of the cIII coding region determine the rate of its translation initiation. In this study we demonstrate the presence of the alternative mRNA structures in vivo. The presence of minor RNaseIII cleavage sites within this region indicate that RNaseIII can differentiate between the two alternative structures. We localize by a deletion analysis the RNaseIII responsive element to the cIII coding region, and suggest that regulation of cIII translation by RNaseIII is achieved through binding to the alternative structures region of the mRNA.

Genetic analysis of the cIII gene of bacteriophage HK022

The cIII gene product of lambdoid bacteriophages promotes lysogeny by stabilizing the phage-encoded CII protein, a transcriptional activator of the repressor and integrase genes. Previous works showed that the synthesis of the bacteriophage lambda CIII protein has specific translational requirements imposed by the structure of the mRNA. To gain insight into the mRNA structure and its role in regulating cIII translation, we undertook a mutational analysis of the cIII gene of the related bacteriophage HK022. Our data support the hypothesis that in HK022, as in lambda, translation initiation requires a specific mRNA structure. In addition, we found that translation of HK022 cIII, like that of lambda, is strongly reduced in a host deficient in the endonuclease RNase III.

Alternative mRNA structures of the cIII gene of bacteriophage λ determine the rate of its translation initiation

The bacteriophage λ cIII gene product has a regulatory function in the lysis-lysogeny decision following infection. The availability of a set of cIII expression mutants allowed us to establish the structure-function relationship of the cIII mRNA. We demonstrate, using defined in vitro systems, that the cIII mRNA is present in two conformations at equilibrium. Mutations that have been shown to lead to cIII overexpression were found to freeze the RNA in one conformation (structure B), and permit efficient binding to the 30 S ribosomal subunit. Mutations that have been shown to prevent cIII translation cause the mRNA to assume the alternative conformation (structure A). In this structure, the translation initiation region is occluded, thereby preventing 30 S ribosomal subunit binding. By varying the temperature or Mg 2+ concentration it was possible to alter the relative proportion of the alternative structures in wild-type mRNA. We suggest that the regulation of the equilibrium between the two mRNA conformations provides a mechanism for the control of cIII gene expression.

Genetic analysis of bacteriophage lambda cIII gene: mRNA structural requirements for translation initiation

The bacteriophage lambda cIII gene product regulates the lysogenic pathway. The cIII gene is located in the leftward operon, which is transcribed from the pL promoter. We have previously shown (S. Altuvia and A. B. Oppenheim, J. Bacteriol. 167:415-419, 1986) that mutations that show elevated expression lie within the cIII coding sequence. We isolated mutants that show decreased CIII activity. All the mutations were found to cause a drastic reduction in the rate of initiation of cIII translation. Several mutations were found to be scattered within the first 40 nucleotides of the cIII coding region. Additional mutations affected the AUG initiation codon, the Shine-Dalgarno sequence, and the upstream RNaseIII processing site. Computer folding of the cIII mRNA suggested the presence of two alternative RNA structures. All the mutations within the coding region that reduce expression reduce the stability of one specific mRNA structure (structure B). Mutations that increase expression lie in the loops of this structure and may in fact stabilize it by interfering with the formation of the alternative structure (structure A). Thus, it appears that a specific mRNA secondary structure at the beginning of the cIII coding region is essential for efficient translation, suggesting that changes in mRNA structure regulate cIII expression.

RNase III stimulates the translation of the cIII gene of bacteriophage lambda.

The bacteriophage lambda cIII gene product regulates the lysogenic pathway by stabilizing the lambda cII regulatory protein. Our results show that the expression of the lambda cIII gene is subject to specific requirements. Tests of a set of cIII-lacZ gene and operon fusions reveal that a sequence upstream of the cIII ribosome binding site is needed for cIII translation. The sequence contains an inefficient RNase III processing site. Furthermore, expression of cIII is drastically reduced in cells lacking RNase III. We have isolated a phage carrying a mutation (r1), which lies in the upstream sequence, that leads to a reduction in cIII translation and inactivates the RNase III processing site. The r1 mutant is nevertheless still dependent on RNase III for cIII translation; r1 reduces cIII translation by a factor of 3 in wild-type cells and by a factor of approximately equal to 30 in an RNase III mutant host. We propose that RNase III stimulates cIII translation by binding to the upstream sequence and thereby exposing the cIII ribosome binding site. This stimulation does not involve RNA cleavage. Consistent with this hypothesis is our finding that, in vitro, unprocessed cIII mRNA is translated, whereas RNase III-cleaved cIII mRNA is not.

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.

Regulation of the establishment of repressor synthesis in bacteriophage λ

The bacteriophage λ cII and cIII gene products positively control the cI gene. In an attempt to define the site of action of cII and cIII, we constructed λ phages in which the lacZ gene is fused to the repressor establishment promoter P E. The cII and cIII genes were supplied by specially constructed plasmids. A DNA sequence extending from the middle of the cro gene to 58 base-pairs upstream of the P E transcription start site responds to cII and cIII. Since this fragment does not contain the oop region, we conclude that this region is dispensable for the positive regulation of cI. Neither P M nor P O are stimulated by cII and cIII, nor do they mediate the response of P E to these regulators. The experiments describe a simple in vivo assay system for the analysis of cII cIII control.

Regulation of the int gene of bacteriophage λ: Activation by the cII and cIII gene products and the role of the P I and P L promoters

The activation of the int gene by the cII and cIII gene products was studied by analysing int expression following infection of UV-irradiated cells by various phage mutants. Residual expression of int, probably from Pl, takes place in the absence of cII/cIII activation. Activation of the int gene, like that of the cI repressor gene, is poor at low multiplicities of infection. The mutation intC, which allows constitutive int expression in the lysogenic state, partially relieves the requirement for cII and cIII activation. The kinetics of Int synthesis after addition of the inhibitor rifampicin suggest that the activation occurs at the transcriptional level.

Interaction of cII, cIII, and cro gene products in the regulation of early and late functions of phage λ

Strains of λ phage containing a mutated cII, cIII, or cy gene in addition to a cro − mutation were constructed. Each single mutant and the double mutants were analyzed for growth behavior and for expression of specific early and late λ proteins. λ c1857 cro 16 is unable to grow well at 30° and at 43°. This strain is incapable of turning off early genes (of the b region, of the P L and P R operons, and the cU and int genes) and is defective in the expression of late functions. The clear mutations permit phage growth at 30°. They relieve the inhibitory effect conferred by the cro − mutation on phage growth at 43° to different extents. Our results also indicate that the cII and cIII proteins do not necessarily act at y in the regulation of late functions.

Lysogenization by bacteriophage lambda IV inhibition of phage DNA synthesis by the products of genes cII and cIII

In direct measurements of phage lambda DNA synthesis, we have detected an inhibition caused by the cII and cIII gene products. This inhibition was more clearly observed when P amber phages were grown in a permissive host, presumably because of the limitation in DNA synthesis due to uncomplete suppression. The inhibition takes place in cells infected at high multiplicity, but not in cells infected at low multiplicity. To explain these findings, we propose a model in which the bacterial population is heterogeneous with respect to its ability to support phage DNA synthesis. An initial limitation caused by host factors would be amplified by the action of the cII and cIII products, at high multiplicity only, and the resulting inhibition would be essential in the "choice" towards lysogeny.

Activation of the lambda int gene by the cII and cIII gene products

The sequential synthesis of λ directed proteins, following infection of uv-irradiated Escherichia coli, has been studied using different λ mutants. The synthesis of phage-specified proteins involved in integration (Int) and repression (the cI repressor) was found to be activated by two other phage-specified proteins, the products of genes cII and cIII. The latter proteins had been thought to be involved only in regulation of cI synthesis, an effect abolished by a cis acting mutation, cy. Although the cII and cIII products regulate int gene expression, the cy mutation has no effect on this process. Moreover, intC, a mutation which maps immediately adjacent to the int gene and results in constitutive Int expression, has no effect on cI synthesis. It appears, therefore, that cII and cIII act at two different promoters to achieve the coordinated expression of the int and cI genes, and thereby ensure the successful establishment of lysogeny.

Internal inversion used to study regulation of the int gene in bacteriophage lambda.

Two events are essential for successful lysogenisation by bacteriophage λ : establishment of repression and integration of the viral genome into the bacterial chromosome. Integration is catalysed by the phage Int function1. The int gene is situated at the distal end of the early leftward N operon, whose transcription is initiated at the PL promoter (Fig. 1). But in addition to PL-promoted transcription, the int gene is specifically activated by the action of the cII and cIII gene products, and we have proposed that the cII and cIII proteins stimulate fast synthesis of Int by acting at the PL promoter (my unpublished work with M. Katzir, M. Belfort and A. Oppenheim). In this respect the int gene behaves similarly to the cI repressor gene, whose fast expression is activated by the gene products of cII and cIII2,3.

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.

Positive and negative regulation by the cII and cIII gene products of bacteriophage λ

Previous experiments have indicated that the cII and cIII gene products of bacteriophage λ serve to establish repression in an infected cell through a bifunctional regulatory activity: an activation of synthesis of cI protein (the maintenance repressor) and an inhibition of production of late lytic proteins. We have investigated further the relationship between the two regulatory activities of c II c III by measurements of genetic and environmental influences on synthesis of endolysin and cI protein. A cy − mutation which exhibits a cis dominant defect in positive regulation of the cI gene also is defective in negative regulation of endolysin. These data are consistent with a mechanism in which the cII and cIII proteins act at a single site in the y-region of λ DNA to provide for both positive and negative regulation. Multiplicity of infection exerts a strong influence on endolysin production, dependent on active cII and cIII genes; higher multiplicity favors repression. However, positive regulation of the cI gene is not substantially more effective at higher multiplicity. Thus there exists a capacity for discoordinate expression of the two regulatory activities of c II c III .

A colicin-tolerant mutant of Escherichia coli with reduced levels of cyclic AMP and a strong bias towards lambda lysogeny.

A colicin-tolerant mutant of Escherichia coli which is temperature-dependent for tolerance and unable to grow at 40° without the addition of salt, plates λ+ wild-type phage with a reduced frequency and very turbid plaques at 30°, failing to form any plaques at 40° even though the cells become phage infected. Phage mutants λcI, λcII, λcY42 and λvir form clear plaques and plate with equal efficiency on the mutant and its parent strain at both temperatures. The results show that over 95% of the phage infected cells become lysogenized after λ+ phage infection. In contrast, the temperate phage P1 formed normal turbid plaques with equal frequency on both bacterial strains, but phage ϕ80 forms normal turbid plaques at 30° and clear (non-lysogenic) plaques at 40°. An investigation of the mutant strain showed that it not only lacked a large molecular weight component from its cell envelope but also had reduced capacity to synthesize cyclic-3′,5′-AMP. It is proposed that the mutation(s) in the colicin-tolerant mutant gives rise to a number of alterations, one of which changes the cell envelope and leads to the reduction of intracellular cyclic AMP and the other giving rise to an altered RNA polymerase specificity. These alterations lead to a changed ratio of the products of the λ-genes involved in the control of lysogeny and give rise to a bias towards the lysogenic response. The cIII gene product of λ is not as essential to lysogeny as cII or cI and therefore possibly acts as an internal control of other λ functions (e.g. cro).

Effect of mutations in the cII and cIII genes of bacteriophage λ on macromolecular synthesis in infected cells

We have investigated the time-course of macromolecular synthesis following infection by cI −, cII − and cIII − mutants of λ in an effort to understand the role of the cII and cIII genes in the establishment of repression and lysogeny. cII − and cIII − mutants of λ begin to synthesize the “late” proteins tail antigen and lysozyme substantially before a cI − mutant; the shift to late messenger RNA production from the head and tail genes also occurs earlier after cII − or cIII − infection. In contrast, no appreciable difference among cI −, cII −, or cIII − mutants was found in synthesis of the “early” protein λ-exonuclease or in the rate of total or λ-specific DNA synthesis. These results suggest that the λ cII and cIII gene products function to delay the lytic response by inhibiting, directly or indirectly, the production of messenger RNA from the late λ genes. When lysozyme synthesis was studied after infection by cI − cII − and cI − cIII − double mutants, results were obtained which were similar to those found in the case of single cII − and cIII − mutants. The results with the double mutants suggest that the cII and cIII gene products may also participate in the regulation of cI represser synthesis or activity.