Regulation of Repressor Expression in λ

Using a module exchange approach, we have tested a longstanding model for the role of Cro repressor in 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 R3 operator, thereby repressing the cI gene and making the switch irreversible. Here we tested this model by replacing 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.circuit design ͉ epigenetic switch ͉ gene regulation ͉ systems biology ͉ threshold behavior A cell infected with can follow either of two exclusive pathways (1, 2). In the lytic pathway, a temporal pattern of gene expression occurs, the viral DNA replicates, and Ϸ100 new virions are made and released by cell lysis. In the alternative lysogenic pathway, the phage DNA is integrated into the host genome, and its lytic genes are repressed by CI repressor, resulting in a stable association with the host termed the lysogenic state. The initial choice between these two pathways is largely determined by the level of a viral regulatory protein, CII. If CII levels are high, it stimulates three promoters that act in various ways (3) to favor the lysogenic pathway. CII is unstable but is stabilized by another viral protein, CIII. Infection with several phages favors high CII levels and the lysogenic pathway (1, 4); poorly understood physiological factors also likely help to regulate CII levels.The lysogenic state is highly stable; however, it can switch to the lytic state in an epigenetic switch called prophage induction (2, 5). Upon induction of the host SOS system (6) by DNA damage, such as from UV irradiation, RecA protein is activated to a form that mediates specific cleavage of CI (7, 8), inactivating CI and allowing expression of lytic genes and the lytic pathway.The central events that control and stabilize the regulatory states occur in the O R region (Fig. 1A). This region contains two promoters: the lytic promoter P R , from which cro and several early lytic genes are expressed, and P RM , from which cI is expressed in a lysogen. O R also contains three operator sites to which both Cro and CI bind. In addition, both proteins bind to three operators in the O L region; binding ...

Section: Resultsmentioning

confidence: 99%

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

Atsumi

Little²

2006

“…A small number of phage functions are specifically required for carrying out the lysogenic response (9, 10). Studies using have unraveled key processes in its gene regulation and developmental pathways, suggesting the presence of a genetic switch (8,11). The regulatory network is composed of both phage and host functions, which respond to each other, and to external factors such as the physiological conditions of the host.…”

mentioning

confidence: 99%

Quantitative kinetic analysis of the bacteriophage λ genetic network

Kobiler

Rokney

Friedman

et al. 2005

104

170

The lysis-lysogeny decision of bacteriophage has been a paradigm for a developmental genetic network, which is composed of interlocked positive and negative feedback loops. This genetic network is capable of responding to environmental signals and to the number of infecting phages. An interplay between CI and Cro functions suggested a bistable switch model for the lysis-lysogeny decision. Here, we present a real-time picture of the execution of lytic and lysogenic pathways with unprecedented temporal resolution. We monitor, in vivo, both the level and function of the CII and Q gene regulators. These activators are cotranscribed yet control opposite developmental pathways. Conditions that favor the lysogenic response show severe delay and down-regulation of Q activity, in both CII-dependent and CII-independent ways. Whereas CII activity correlates with its protein level, Q shows a pronounced threshold before its function is observed. Our quantitative analyses suggest that by regulating CII and CIII, Cro plays a key role in the ability of the genetic network to sense the difference between one and more than one phage particles infecting a cell. Thus, our results provide an improved framework to explain the longstanding puzzle of the decision process.gene regulation ͉ lysogeny ͉ lysis ͉ green fluorescent protein B acteriophages are the most abundant species in nature and play an immense role in the turnover of bacterial ecosystems (1, 2). Yet, some bacteria and phages exist in symbiotic relationships with phage present in a dormant, lysogenic (prophage) state (3-6). Lambdoid prophages, among other types, are responsible for the expression and release of pathogenic toxins (7)., itself, is a temperate phage, which undergoes either lytic or lysogenic development (5,8). A small number of phage functions are specifically required for carrying out the lysogenic response (9, 10). Studies using have unraveled key processes in its gene regulation and developmental pathways, suggesting the presence of a genetic switch (8,11). The regulatory network is composed of both phage and host functions, which respond to each other, and to external factors such as the physiological conditions of the host. As examples, lysogeny is preferred upon infection of starved cells or when cells are infected at high multiplicity of infection (moi) (12, 13).Although the interactions and structure of the genetic network have been extensively described, many fundamental issues still remain elusive and deserve further attention. For instance, the complex negative control of lytic functions during the lysogenic response has been generally ignored, and the relative importance of different key regulators in determining the decision is poorly understood, in particular for different values of moi. Theoretical studies have provided detailed predictions of the execution of lytic and lysogenic pathways (14-17). These predictions have not been adequately tested in an experiment.We addressed these issues by using GFP reporter fusions that are activated after p...

“…Each promoter is associated with a CI recognition site or operator, O L (composed of three adjacent subsites, O L 1, O L 2, O L 3) and O R (O R 1, O R 2, O R 3). O R is associated with promoter P RM (promoter for maintenance of repressor synthesis), which directs transcription of the cI gene in the prophage state (4)(5)(6). Each subsite binds a CI dimer (7).…”

mentioning

confidence: 99%

Multilevel autoregulation of λ repressor protein CI by DNA looping in vitro

Lewis

Zurla

et al. 2011

The prophage state of bacteriophage λ is extremely stable and is maintained by a highly regulated level of λ repressor protein, CI, which represses lytic functions. CI regulates its own synthesis in a lysogen by activating and repressing its promoter, P RM . CI participates in long-range interactions involving two regions of widely separated operator sites by generating a loop in the intervening DNA. We investigated the roles of each individual site under conditions that permitted DNA loop formation by using in vitro transcription assays for the first time on supercoiled DNA that mimics in vivo situation. We confirmed that DNA loops generated by oligomerization of CI bound to its operators influence the autoactivation and autorepression of P RM regulation. We additionally report that different configurations of DNA loops are central to this regulation-one configuration further enhances autoactivation and another is essential for autorepression of P RM .activation | cooperativity | repression B acteriophage lambda (λ) of Escherichia coli can grow either in a lytic or lysogenic mode. In a lysogenic cell, the phageencoded λ repressor protein (CI) prevents lytic growth by directly repressing two promoters needed to express lytic functions, P L and P R (1-3). Each promoter is associated with a CI recognition site or operator,. O R is associated with promoter P RM (promoter for maintenance of repressor synthesis), which directs transcription of the cI gene in the prophage state (4-6). Each subsite binds a CI dimer (7).The CI protein autoregulates its synthesis. At low cellular CI concentration, CI enhances its own synthesis from P RM ; when high, CI represses P RM (1,8,9). It was originally believed that both positive and negative autoregulations are achieved exclusively by the action of CI dimers at the P RM -O R -P R sequence of the phage genome (1, 10) (Fig. 1A), based on the following observations. (i) There is a hierarchy of intrinsic binding affinities of a CI dimer to individual operators sites: (11)(12)(13)(14)(15)(16)(17); (ii) CI bound to the intrinsically weak O R 2 site is strengthened by cooperative interactions with CI bound to the stronger adjacent O R 1 site, and the ensemble of two CI dimers at the O R 1 ∼ O R 2 sites represses P R and activates P RM (13, 16) (Fig. 1A); (iii) at high CI concentrations, a CI dimer can bind to the weakest operator site, O R 3, repressing P RM (1, 2) (Fig. 1C). Incidentally, a second pair of CI dimers binds cooperatively to O L 1 ∼ O L 2, and represses P L (7).This model was recently modified on the basis of physical and genetic experiments showing that a CI tetramer cooperatively bound to O R 1 ∼ O R 2 interacts with a tetramer cooperatively bound to O L 1 ∼ O L 2, located 2.3 kbp away (18-21), resulting in the looping out of the intervening DNA, as shown by electron and atomic force microscopy (18,22,23). DNA loop was also observed by cooperative interactions of CI at sites separated by only five and six helical turns (23, 24). The kinetic and thermodynamic properties ...