Control of bacteriophage lambda CII activity by bacteriophage and host functions

We have studied the regulation of the lambda cII gene in vivo using cloned lambda fragments. Lambda N protein stimulated cII expression. Surprisingly, although very high cII protein levels were detected by gel electrophoresis, little cII protein activity, measured as stimulation of the lambda pI and pE promoters, was observed. The half-life of cII protein depended critically on its initial level. At low concentrations its half-life was as short as 1.5 min, whereas at high cII protein levels, it could be as long as 22 min. The Escherichia coli mutant ER437 directs lambda towards lysogeny; cII protein was more stable in this strain than in the wild type. On the other hand, although cyclic AMP is required for efficient lysogeny, it did not appear to influence the synthesis, stability, or activity of cII protein.     

hflB, a new Escherichia coli locus regulating lysogeny and the level of bacteriophage lambda cII protein

The level of the viral cII protein has been proposed to be the crucial determinant in the lysis-lysogeny decision of bacteriophage lambda. A new Escherichia coli locus (hflB) has been identified in which a mutation (hflB29) leads to high frequency of lysogeny by lambda. A double mutant defective in both hflB and the previously identified hflA gene displays a more severe Hfl- phenotype than either single mutant. The hflB locus is at 69 minutes on the E. coli map, 85% co-transducible with argG. The hflB29 mutation results in increased stability of the phage cII protein (increasing its half-life twofold) and is recessive to hflB+. We conclude that the hflB+ locus is a negative regulator of cII, perhaps coding for or regulating a protease that acts on cII. In addition, we observe that the can1 mutation, an alteration of the cII gene that results in enhanced lysogenization, leads to increased stability of cII protein. These observations reinforce the view that the level of cII is a key factor in the lysis-lysogeny decision of lambda.     

IHF stimulation of lambda cII gene expression is inhibited by the E. coli NusA protein

The effects of E. coli proteins Integrative Host Factor (IHF) and NusA on the regulation of lambda cII gene expression are presented. As reported previously (Peacock et al. [1984] Proc. Natl. Acad. Sci. USA 81, 6009-6013), IHF stimulates the DNA-directed in vitro synthesis of cII protein or its first dipeptide, fMet-Val. Whereas NusA, by itself, has no effect on cII expression, the presence of NusA inhibits the IHF-mediated stimulation of cII synthesis.     

Hardware (DNA) circuits

A scheme is presented whereby a new genetic control circuit can be introduced into an organism, permitting the experimenter to turn the expression of a given gene (or set of genes) on or off at will. The proposed scheme involves a positive feedback loop--here, a positive regulator, the CII protein of phage lambda, with its structural gene engineered so as to require CII for its expression. This feedback loop creates the possibility of two stable steady states, with gene cII ON or OFF. Genes added downstream of cII and lacking a promoter will follow the same expression as cII. Two additional circuits allow the experimenter to switch at will between the ON and OFF states.     

Nonessential region of bacteriophage P4: DNA sequence, transcription, gene products, and functions

We sequenced the leftmost 2,640 base pairs of bacteriophage P4 DNA, thus completing the sequence of the 11,627-base-pair P4 genome. The newly sequenced region encodes three nonessential genes, which are called gop, beta, and cII (in order, from left to right). The gop gene product kills Escherichia coli when the beta protein is absent; the gop and beta genes are transcribed rightward from the same promoter. The cII gene is transcribed leftward to a rho-independent terminator. Mutation of this terminator creates a temperature-sensitive phenotype, presumably owing to a defect in expression of the beta gene.     

Protein degradation in E. coli: the lon mutation and bacteriophage lambda N and cII protein stability

The Ion gene of E. coli controls the stability of two bacteriophage lambda proteins. The functional half-life of the phage N gene product, measured by complementation, is increased about 5-fold in Ion mutant strains, from 2 min to 10 min. The chemical half-life of N protein, determined by its disappearance on polyacrylamide gels following pulse-chase labeling, increases about three-fold in Ion cells. In contrast to its effect on the N protein, the Ion mutation produces a 50% decrease in the chemical half-life of cII protein. The decay rate of many other phage proteins, including the unstable gene O product, remains unaffected by a host Ion defect. A Ion mutation alters lambda physiology in two ways. First, upon infection, the phage enters the lytic pathway predominantly. This may result from the deficiency of cII protein caused by its decreased stability, since cII product is required for establishment of lysogeny. Second, brief thermal induction of a Ion (lambda c1857) lysogen leads irreversibly to lysis; repression cannot be restablished and the treated cells are committed to forming infective centers. Although N product is normally required for rapid commitment, Ion lysogens become committed more rapidly than Ion+ lysogens, even in the absence of N function. These results identify for the first time native proteins whose stability is affected by the Lon proteolytic pathway. They also indicate that the Lon system may be important in regulating gene expression in E. coli.     

Hepatocarcinogen quinoline induces G:C to C:G transversions in the cII gene in the liver of lambda/lacZ transgenic mice (MutaMouse)

Quinoline is carcinogenic to the liver in rodents, but it is not clear whether it acts by a genotoxic mechanism. We previously demonstrated that quinoline does induce gene mutation in the liver of lambda/lacZ transgenic mice. In the present report, we reveal the molecular nature of the mutations induced by quinoline in the lambda cII gene, which is also a phenotypically selectable marker in the lambda transgene. (The cII gene has 294bp, which enables much easier sequence analysis than the original lacZ gene (3kb)). The liver cII mutant frequency was nine times higher in quinoline-treated mice than in control mice. Sequence analysis revealed that quinoline induced primarily G:C to C:G transversions (25 of 34). Thus, we have confirmed that quinoline is genotoxic in its target organ, and the G:C to C:G transversion is the molecular signature of quinoline-induced mutations.     

The HflB protease of Escherichia coli degrades its inhibitor lambda cIII.

The cIII protein of bacteriophage lambda is known to protect two regulatory proteins from degradation by the essential Escherichia coli protease HflB (also known as FtsH), viz., the lambda cII protein and the host heat shock sigma factor sigma32. lambda cIII, itself an unstable protein, is partially stabilized when the HflB concentration is decreased, and its half-life is decreased when HflB is overproduced, strongly suggesting that it is degraded by HflB in vivo. The in vivo degradation of lambda cIII (unlike that of sigma32) does not require the molecular chaperone DnaK. Furthermore, the half-life of lambda cIII is not affected by depletion of the endogenous ATP pool, suggesting that lambda cIII degradation is ATP independent (unlike that of lambda cII and sigma32). The lambda cIII protein, which is predicted to contain a 22-amino-acid amphipathic helix, is associated with the membrane, and nonlethal overproduction of lambda cIII makes cells hypersensitive to the detergent sodium dodecyl sulfate. This could reflect a direct lambda cIII-membrane interaction or an indirect association via the membrane-bound HflB protein, which is known to be involved in the assembly of certain periplasmic and outer membrane proteins.