Role of cis-acting sites in stimulation of the phage λ P(RM) promoter by CI-mediated looping.

The lysogenic state of phage λ is maintained by the CI repressor. CI binds to three operators each in the right operator (O(R)) and left operator (O(L)) regions, which lie 2.4 kb apart. At moderate CI levels, the predominant binding pattern is two dimers of CI bound cooperatively at each regulatory region. The resulting tetramers can then interact, forming an octamer and a loop of the intervening DNA. CI is expressed from the P(RM) promoter, which lies in the O(R) region and is subjected to multiple regulatory controls. Of these, the most recently discovered is stimulation by loop formation. In this work, we have investigated the mechanism by which looping stimulates P(RM). We find that two cis-acting sites lying in the O(L) region are involved. One site, an UP element, is required for stimulation. Based on the behavior of other promoters with UP elements located upstream of the -35 region, we suggest that a subunit of RNA polymerase (RNAP) bound at P(RM) binds to the UP element located in the O(L) region. In addition, adjacent to the UP element lies a binding site for integration host factor (IHF); this site plays a less critical role but is required for stimulation of the weak prm240 allele. A loop with CI at the O(L)2 and O(L)3 operators does not stimulate P(RM), while one with CI only at O(L)2 provides some stimulation. We discuss possible mechanisms for stimulation.

Strong enhancement of solar cell efficiency due to quantum dots with built-in charge.

We report a 50% increase in the power conversion efficiency of InAs/GaAs quantum dot solar cells due to n-doping of the interdot space. The n-doped device was compared with GaAs reference cell, undoped, and p-doped devices. We found that the quantum dots with built-in charge (Q-BIC) enhance electron intersubband quantum dot transitions, suppress fast electron capture processes, and preclude deterioration of the open circuit voltage in the n-doped structures. These factors lead to enhanced harvesting and efficient conversion of IR energy in the Q-BIC solar cells.

Evolution of complex gene regulatory circuits by addition of refinements.

How do complex gene regulatory circuits evolve? These circuits involve many interacting components, which work together to specify patterns of gene expression. They typically include many subtle mechanistic features, but in most cases it is unclear whether these features are essential for the circuit to work at all, or if instead they make a functional circuit work better. In the latter case, such a feature is here termed 'dispensable', and it is plausible that the feature has been added at a late stage in the evolution of the circuit. This review describes experimental tests of this question, using the phage λ gene regulatory circuit. Several features of this circuit are found to be dispensable, in the sense that the circuitry works without these features, though not as well as the wild type. In some cases, second-site suppressor mutations are needed to confer near-normal behavior in the absence of such a feature. These findings are discussed here in the context of a two-stage model for evolution of gene regulatory circuits. In this model, a circuit evolves by assembly of a primitive or basic form, followed by adjustment of parameters and addition of qualitatively new features. Pathways are suggested for the addition of such features to a more basic form. Selected examples in other systems are described. Some of the dispensable features of phage λ may be evolutionary refinements. Finding that a feature is dispensable, however, does not prove that it is a late addition - it is possible that it was essential early in evolution, and became dispensable as the circuit evolved. Conversely, a late addition might have become essential. As ongoing work provides additional examples of dispensable features, it may become clearer how often they represent refinements.

Stability and instability in the lysogenic state of phage lambda.

Complex gene regulatory circuits exhibit emergent properties that are difficult to predict from the behavior of the components. One such property is the stability of regulatory states. Here we analyze the stability of the lysogenic state of phage λ. In this state, the virus maintains a stable association with the host, and the lytic functions of the virus are repressed by the viral CI repressor. This state readily switches to the lytic pathway when the host SOS system is induced. A low level of SOS-dependent switching occurs without an overt stimulus. We found that the intrinsic rate of switching to the lytic pathway, measured in a host lacking the SOS response, was almost undetectably low, probably less than 10(-8)/generation. We surmise that this low rate has not been selected directly during evolution but results from optimizing the rate of switching in a wild-type host over the natural range of SOS-inducing conditions. We also analyzed a mutant, λprm240, in which the promoter controlling CI expression was weakened, rendering lysogens unstable. Strikingly, the intrinsic stability of λprm240 lysogens depended markedly on the growth conditions; lysogens grown in minimal medium were nearly stable but switched at high rates when grown in rich medium. These effects on stability likely reflect corresponding effects on the strength of the prm240 promoter, measured in an uncoupled assay system. Several derivatives of λprm240 with altered stabilities were characterized. This mutant and its derivatives afford a model system for further analysis of stability.

RecA-dependent cleavage of LexA dimers.

A critical step in the SOS response of Escherichia coli is the specific proteolytic cleavage of the LexA repressor. This reaction is catalyzed by an activated form of RecA, acting as a co-protease to stimulate the self-cleavage activity of LexA. This process has been reexamined in light of evidence that LexA is dimeric at physiological concentrations. We found that RecA-dependent cleavage was robust under conditions in which LexA is largely dimeric and conclude that LexA dimers are cleavable. We also found that LexA dimers dissociate slowly. Furthermore, our evidence suggests that interactions between the two subunits of a LexA dimer can influence the rate of cleavage. Finally, our evidence suggests that RecA stimulates the transition of LexA from its noncleavable to its cleavable conformation and therefore operates, at least in part, by an allosteric mechanism.

Cooperative DNA binding by CI repressor is dispensable in a phage lambda variant.

Complex gene regulatory circuits contain many interacting components. In principle, all of these components and interactions may be essential to the function of the circuit. Alternatively, some of them may be refinements to a simpler version of the circuit that improve its fitness. In this work, we have tested whether a particular property of a critical regulatory protein, CI, is essential to the behavior of the phage lambda regulatory circuit. In the lysogenic state, CI represses the expression of the lytic genes, allowing a stable lysogenic state, by binding cooperatively to six operators. A mutant phage lacking cooperativity because of a change in cI could not form stable lysogens; however, this defect could be suppressed by the addition of mutations that altered two cis-acting sites but did not restore cooperativity. The resulting triple mutant was able to grow lytically, form stable single lysogens, and switch to lytic growth upon prophage induction, showing a threshold response in switching similar to that of wild-type lambda. We conclude that cooperative DNA binding by CI is not essential for these properties of the lambda circuitry, provided that suppressors increase the level of CI. Unlike wild-type lysogens, mutant lysogens were somewhat unstable under certain growth conditions. We surmise that cooperativity is a refinement to a more basic circuit, and that it affords increased stability to the lysogenic state in response to environmental variations.

Conservation and diversity in the immunity regions of wild phages with the immunity specificity of phage lambda.

The gene regulatory circuitry of phage lambda is among the best-understood circuits. Much of the circuitry centres around the immunity region, which includes genes for two repressors, CI and Cro, and their cis-acting sites. Related phages, termed lambdoid phages, have different immunity regions, but similar regulatory circuitry and genome organization to that of lambda, and show a mosaic organization, arising by recombination between lambdoid phages. We sequenced the immunity regions of several wild phages with the immunity specificity of lambda, both to determine whether natural variation exists in regulation, and to analyse conservation and variability in a region rich in well-studied regulatory elements. CI, Cro and their cis-acting sites are almost identical to those in lambda, implying that regulatory mechanisms controlled by the immunity region are conserved. A segment adjacent to one of the operator regions is also conserved, and may be a novel regulatory element. In most isolates, different alleles of two regulatory proteins (N and CII) flank the immunity region; possibly the lysis-lysogeny decision is more variable among isolates. Extensive mosaicism was observed for several elements flanking the immunity region. Very short sequence elements or microhomologies were also identified. Our findings suggest mechanisms by which fine-scale mosaicism arises.

A synthetic phage lambda regulatory circuit.

Analysis of synthetic gene regulatory circuits can provide insight into circuit behavior and evolution. An alternative approach is to modify a naturally occurring circuit, by using genetic methods to select functional circuits and evolve their properties. We have applied this approach to the circuitry of phage lambda. This phage grows lytically, forms stable lysogens, and can switch from this regulatory state to lytic growth. Genetic selections are available for each behavior. We previously replaced lambda Cro in the intact phage with a module including Lac repressor, whose function is tunable with small molecules, and several cis-acting sites. Here, we have in addition replaced lambda CI repressor with another tunable module, Tet repressor and several cis-acting sites. Tet repressor lacks several important properties of CI, including positive autoregulation and cooperative DNA binding. Using a combinatorial approach, we isolated phage variants with behavior similar to that of WT lambda. These variants grew lytically and formed stable lysogens. Lysogens underwent prophage induction upon addition of a ligand that weakens binding by the Tet repressor. Strikingly, however, addition of a ligand that weakens binding by Lac repressor also induced lysogens. This finding indicates that Lac repressor was present in the lysogens and was necessary for stable lysogeny. Therefore, these isolates had an altered wiring diagram from that of lambda. We speculate that this complexity is needed to compensate for the missing features. Our method is generally useful for making customized gene regulatory circuits whose activity is regulated by small molecules or protein cofactors.

Role of the lytic repressor in prophage induction of phage lambda as analyzed by a module-replacement approach.

Using a module exchange approach, we have tested a long-standing model for the role of Cro repressor in lambda prophage induction. This epigenetic switch from lysogeny to the lytic state occurs on activation of the host SOS system, which leads to specific cleavage of CI repressor. It has been proposed that Cro repressor, which operates during lytic growth and which we shall term the lytic repressor, is crucial to prophage induction. In this view, Cro binds to the O(R)3 operator, thereby repressing the cI gene and making the switch irreversible. Here we tested this model by replacing lambda Cro with a dimeric form of Lac repressor and adding several lac operators. This approach allowed us to regulate the function of the lytic repressor at will and to prevent it from repressing cI, because lac repressor could not repress P(RM) in our constructs. Repression of cI by the lytic repressor was not required for prophage induction to occur. However, our evidence suggests that this binding can make induction more efficient, particularly at intermediate levels of DNA damage that otherwise cause induction of only a fraction of the population. These results indicate that this strategy of module exchange will have broad applications for analysis of gene regulatory circuits.

Positive autoregulation of cI is a dispensable feature of the phage lambda gene regulatory circuitry.

Complex gene regulatory circuits contain many features that are likely to contribute to their operation. It is unclear, however, whether all these features are necessary for proper circuit behavior or whether certain ones are refinements that make the circuit work better but are dispensable for qualitatively normal behavior. We have addressed this question using the phage lambda regulatory circuit, which can persist in two stable states, the lytic state and the lysogenic state. In the lysogenic state, the CI repressor positively regulates its own expression by stimulating transcription from the P(RM) promoter. We tested whether this feature is an essential part of the regulatory circuitry. Several phages with a cI mutation preventing positive autoregulation and an up mutation in the P(RM) promoter showed near-normal behavior. We conclude that positive autoregulation is not necessary for proper operation of the lambda circuitry and speculate that it serves a partially redundant function of stabilizing a bistable circuit, a form of redundancy we term "circuit-level redundancy." We discuss our findings in the context of a two-stage model for evolution and elaboration of regulatory circuits from simpler to more complex forms.

Sequence tolerance of the phage lambda PRM promoter: implications for evolution of gene regulatory circuitry.

Much of the gene regulatory circuitry of phage lambda centers on a complex region called the O(R) region. This approximately 100-bp region is densely packed with regulatory sites, including two promoters and three repressor-binding sites. The dense packing of this region is likely to impose severe constraints on its ability to change during evolution, raising the question of how the specific arrangement of sites and their exact sequences could evolve to their present form. Here we ask whether the sequence of a cis-acting site can be widely varied while retaining its function; if it can, evolution could proceed by a larger number of paths. To help address this question, we developed a lambda cloning vector that allowed us to clone fragments spanning the O(R) region. By using this vector, we carried out intensive mutagenesis of the P(RM) promoter, which drives expression of CI repressor and is activated by CI itself. We made a pool of fragments in which 8 of the 12 positions in the -35 and -10 regions were randomized and cloned this pool into the vector, making a pool of P(RM) variant phage. About 10% of the P(RM) variants were able to lysogenize, suggesting that the lambda regulatory circuitry is compatible with a wide range of P(RM) sequences. Analysis of several of these phages indicated a range of behaviors in prophage induction. Several isolates had induction properties similar to those of the wild type, and their promoters resembled the wild type in their responses to CI. We term this property of different sequences allowing roughly equivalent function "sequence tolerance " and discuss its role in the evolution of gene regulatory circuitry.

Regulatory circuit design and evolution using phage lambda.

Bistable gene regulatory circuits can adopt more than one stable epigenetic state. To understand how natural circuits have this and other systems properties, several groups have designed regulatory circuits de novo. Here we describe an alternative approach. We have modified an existing bistable circuit, that of phage lambda. With this approach, we used powerful genetic selections to identify functional circuits and selected for variants with altered behavior. The lambda circuit involves two antagonistic repressors, CI and Cro. We replaced lambda Cro with a module that included Lac repressor and several lac operators. Using a combinatorial approach, we isolated variants with different types of regulatory behavior. Several resembled wild-type lambda--they could grow lytically, could form highly stable lysogens, and carried out prophage induction. Another variant could form stable lysogens in the presence of a ligand for Lac repressor but switched to the lytic state when the ligand was removed. Several isolates evolved toward a desired behavior under selective pressure. These results strongly support the idea that complex circuits can arise during the course of evolution by a combination of simpler regulatory modules. They also underscore the advantages of modifying a natural circuit as an approach to understanding circuit design, systems behavior, and circuit evolution.