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Journal of Bacteriology, November 2009, p. 6779-6781, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.01150-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

COMMENTARY

A New Twist on a Classic Paradigm: Illumination of a Genetic Switch in Vibrio cholerae Phage CTX

Bryce E. Nickels^*

Department of Genetics and Waksman Institute, Rutgers University, Piscataway, New Jersey 08854

Regulatory circuits that control the life cycles of bacteriophage have served as important models for understanding complex regulatory networks in all organisms. Many lysogenic bacteriophage can exist in a dormant state in which the phage DNA is integrated into the bacterial chromosome. Maintenance of this "prophage state" is achieved through finely tuned regulatory circuits built in a manner that enables prophage induction, i.e., the production of phage particles, to occur in a rapid and efficient manner upon exposure to acute cellular stress. The paradigm for such regulatory circuits is the classic "genetic switch" of bacteriophage (8), in which prophage induction is governed by a phage-encoded DNA binding protein, CI, which represses expression of the genes that control phage production and cell lysis. Proper functioning of the switch requires that the intracellular levels of CI be precisely controlled, which is accomplished through the ability of CI to regulate expression of its own gene through an auto-feedback loop. In this issue of the Journal of Bacteriology, Kimsey and Waldor illuminate features of a regulatory circuit that controls production of the filamentous bacteriophage CTX in Vibrio cholerae (5), which bears similarities to the genetic switch of . The CTX switch is governed by two transcription factors, the host-encoded SOS response regulator LexA and the phage-encoded repressor RstR. Prior work had suggested that the intracellular levels of RstR must be precisely controlled to ensure the proper functioning of the CTX switch. Kimsey and Waldor's study now provides evidence that control of the intracellular levels of RstR is mediated, in part, through a unique LexA-dependent auto-feedback loop. Kimsey and Waldor propose that these novel features of the CTX switch allow the circuit to exhibit transient, reversible behavior upon induction.

Vibrio cholerae bacteriophage CTX.

The lysogenic filamentous phage CTX, which carries genes encoding cholera toxin, infects the gram-negative bacterium Vibrio cholerae, the causative agent of epidemic cholera (12). The activity of cholera toxin is primarily responsible for the profuse secretory diarrhea that is the hallmark of cholera and which enables the dissemination of Vibrio cholerae. Thus, the horizontal transfer of CTX is an important contributor to the emergence of new pathogenic strains of Vibrio cholerae.

CTX is an unusual filamentous bacteriophage because its DNA becomes incorporated into the Vibrio cholerae genome, leading to the establishment of a lysogenic program of gene expression (7). In contrast to other lysogenic phage such as bacteriophage , CTX does not enter into a lytic cycle and kill its host. Rather, CTX and Vibrio cholerae seem to have coevolved to the mutual benefit of both organisms (7). In particular, the replication and dissemination of the host Vibrio cholerae are facilitated by the production of cholera toxin from the CTX chromosome. Furthermore, Vibrio cholerae-encoded factors are required throughout the CTX life cycle, enabling the stable integration of CTX DNA into the bacterial chromosome and the production and secretion of phage particles.

The CTX switch: the roles of RstR and LexA.

In the context of the CTX lysogen, the genes required for CTX replication and morphogenesis, under the control of phage promoter P_A, are repressed. During prophage induction, triggered by agents that cause DNA damage, P_A transcription is derepressed, resulting ultimately in the production of phage particles that are secreted from the cell (2, 7, 11). Because prophage induction does not result in cell lysis, CTX presumably is able to reestablish the lysogenic program and once again enter into a quiescent state. Thus, unlike the genetic switch of , which once flipped, irreversibly commits the phage to the lytic program, the CTX switch must have distinct features that enable it to display reversible behavior upon induction.

Prior work has established that control of gene expression from P_A is governed by two DNA-binding proteins, the phage-encoded RstR and the host-encoded SOS response regulator LexA (Fig. 1A). In the context of the lysogen, RstR and LexA function in an independent manner to repress P_A transcription. RstR inhibits transcription from P_A by binding to the following three sites in the P_A promoter region (4): a high-affinity operator, O1, and two weaker operators, O2 and O3. LexA inhibits transcription from P_A by binding a single site (SOS box) that overlaps with the RstR O2 operator (9), and as expected based on this overlap, LexA binding to the SOS box and RstR binding to the O2 operator are mutually exclusive (9).

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FIG. 1. Genetic switches of CTX and phage . (A) CTX switch. Diagrams show occupancy of operator sites by RstR tetramers (green ovals) or LexA dimers (purple ovals) under normal growth conditions and after prophage induction. Kimsey and Waldor's work indicates that under normal growth conditions, RstR (bound to O1) together with LexA activates transcription of PR, which controls the expression of rstR. When RstR levels become too high, RstR displaces LexA from DNA (occupying O2 and O3), and PR is repressed. DNA damage activates the SOS response, leading to the autocleavage of LexA and derepression of PA. When the levels of LexA return to normal, the switch is reestablished. (B) Bacteriophage switch. Diagrams show occupancy of operator sites by CI dimers (red dumbbells) under normal growth conditions and after prophage induction. Under normal growth conditions, CI represses transcription from PR by binding operators OR1 and OR2, and the CI dimer bound to OR2 activates transcription of PRM. When CI levels become too high, CI occupies OR3 and represses PRM. DNA damage activates the SOS response, leading to the autocleavage of CI and derepression of PR. In contrast to the CTX switch, the switch commits the cell to a lysis program of gene expression, and the switch is not reestablished. The phage-encoded protein Cro (blue ovals) helps to prevent reestablishment of lysogeny by binding to OR3 and repressing transcription of PRM (10). (Adapted from reference 11 with permission of the publisher.)

Prophage induction in response to DNA damage occurs via the RecA-stimulated autocleavage of LexA, which leads to partial derepression of P_A (9). This finding suggested that under normal growth conditions, the intracellular levels of LexA and RstR are such that LexA occupies the SOS box DNA. Furthermore, because the accumulation of high levels of RstR would cause RstR to occupy the O2 site and prevent LexA from binding to the SOS box, these findings raised the possibility that the phage may possess a mechanism to tightly regulate the levels of RstR. Kimsey and Waldor therefore sought to determine how RstR levels are regulated in the context of the prophage. To do this, they first identified the promoter that controls the expression of the rstR gene, P_R, which is located immediately upstream of P_A, divergently oriented and embedded among the three RstR operators and the SOS box (Fig. 1). The finding that the RstR operators and the SOS box overlap with P_R immediately suggested that RstR and LexA might be involved in regulating transcription from P_R. To explore this possibility, Kimsey and Waldor performed a series of in vivo and in vitro experiments revealing that RstR regulates the expression of its own gene. In particular, when the concentration of RstR is such that RstR occupies only O1, RstR activates transcription of P_R. Furthermore, when the concentration of RstR is sufficiently high so that O1, O2, and O3 are occupied, RstR represses P_R transcription. Surprisingly, Kimsey and Waldor also found that the ability of RstR to activate transcription from P_R absolutely depends upon LexA being bound to the SOS box. Taken together, Kimsey and Waldor's findings provide evidence that the intracellular levels of RstR are maintained via a LexA-dependent auto-feedback loop. This feature of the CTX switch is designed to ensure that the intracellular levels of RstR are such that P_A is repressed (i.e., O1 is occupied by RstR) but low enough to allow the circuit to respond to changes in the levels of LexA (i.e., O2 is not occupied by RstR).

Comparisons between the CTX switch and the switch.

The classic "genetic switch" of phage is governed by a phage-encoded DNA-binding protein, CI, which represses transcription of genes that commit the phage to the lysis pathway (8). In particular, CI inhibits transcription from lytic promoters P_R and P_L by binding to a series of operator sites embedded within the P_R and P_L promoter regions and forming higher-order oligomers (Fig. 1B). To ensure that the lysogen remains stable and that efficient prophage induction can occur in response to acute cellular stress, the switch contains features that tightly regulate the intracellular levels of CI. Specifically, control of CI levels is mediated by an auto-feedback loop that regulates the phage cI promoter P_RM. P_RM is located upstream of P_R and divergently arranged such that the two promoters share a common set of three CI operators. Thus, when CI binds to operator O_R2, it simultaneously represses P_R transcription while activating transcription of P_RM. When CI levels become high enough so that operator site O_R3 is occupied, CI represses transcription of P_RM (3). In response to agents that cause DNA damage, RecA stimulates the autocleavage of CI (6), which leads to the derepression of the lysis genes.

Even though filamentous phages and lambdoid phages are unrelated, the CTX regulatory switch has a number of features reminiscent of the switch. First, the promoters that control the CTX switch and the switch are divergently oriented and share a common set of operator sites. Second, both the CTX switch and the switch rely upon a phage-encoded DNA-binding protein (CI or RstR) to repress transcription of genes involved in phage particle production. Third, the levels of both CI and RstR are tightly controlled through an autoregulatory feedback loop. Maintenance of the proper levels of CI or RstR ensures that efficient prophage induction can occur in response to DNA damage. In the case of the CTX switch, if the levels of RstR become too high, the switch would no longer be responsive to the intracellular levels of LexA, and prophage induction would not occur in response to DNA damage (Fig. 1A).

While sharing certain similarities with the switch, the CTX switch also bears a number of distinct features. First, whereas during lysogeny the switch is governed by a single phage-encoded factor, the CTX switch is governed by two transcription factors, the host-encoded SOS response regulator LexA and the phage-encoded repressor RstR. Second, unlike CI, which can activate transcription of its own gene in the absence of additional factors, RstR requires the presence of LexA to activate transcription of its own gene. Third, whereas prophage induction in the context of CTX occurs due to the RecA-stimulated autocleavage of the host-encoded LexA, prophage induction in the context of occurs due to the RecA-stimulated autocleavage of CI (whose cleavage site mimics that of LexA). Finally, while induction of the lysis genes in the context of is irreversible, the CTX switch is apparently able to eventually return to the quiescent state. Kimsey and Waldor propose that incorporation of LexA into the CTX circuit may, in large part, account for the differences in behavior of the two switches.

Future questions.

The study by Kimsey and Waldor provides important insight regarding the control of the CTX lysogenic switch and raises a number of further questions (5). First, precisely how do the features of the CTX switch identified in their study enable the switch to exhibit transient, reversible kinetics? Second, what additional mechanisms govern the reestablishment of the CTX switch after prophage induction? Reestablishment of the CTX switch would presumably require a basal amount of RstR to remain present in the cell, thus allowing the switch to be reestablished once LexA levels are restored. Whether the levels of RstR are subject to an additional level of regulation (e.g., through effects on protein stability) remains to be determined. Third, do any other host- or phage-encoded factors impact the kinetics of the switch? For example, a proposed role of a phage-encoded antirepressor (RstC) in delaying the reestablishment of the switch has not been explored (1, 7). Finally, what is the mechanistic basis for the dual requirements for both LexA and RstR in the activation of P_R? Kimsey and Waldor provide experimental data that rule out a model whereby the increase in P_R transcription is an indirect consequence of the RstR- and LexA-mediated repression of P_A transcription (i.e., "transcriptional interference"). Thus, it appears likely that both RstR and LexA directly contact RNA polymerase or that one of these factors alters the geometry of the RNA polymerase-promoter complex to facilitate productive interaction between RNA polymerase and the other factor.

ACKNOWLEDGMENTS

I thank Matt Waldor and Ann Hochschild for helpful discussion.

 
* Mailing address: Department of Genetics and Waksman Institute, Rutgers University, Piscataway, NJ 08854. Phone: (732) 445-6852. Fax: (732) 445-5735. E-mail: bnickels{at}waksman.rutgers.edu

FOOTNOTES

Published ahead of print on 11 September 2009.

The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.

REFERENCES

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Journal of Bacteriology, November 2009, p. 6779-6781, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.01150-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Related articles in JB:

Vibrio cholerae LexA Coordinates CTX Prophage Gene Expression

Harvey H. Kimsey and Matthew K. Waldor
JB 2009 191: 6788-6795. [Abstract] [Full Text]  

This Article FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Related articles in JB Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Google Scholar Articles by Nickels, B. E. PubMed PubMed Citation Articles by Nickels, B. E.