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A bacterial control circuit integrates polar localization and proteolysis of key regulatory proteins with a phospho-signaling cascade.

ABSTRACT: Dynamic protein localization is an integral component of the regulatory circuit that drives the Caulobacter cell cycle. The ClpXP protease is localized to the Caulobacter cell pole, where it catalyzes the degradation of the CtrA master regulator at specific times in the cell cycle. Clearance of active CtrA at the G1/S transition allows the initiation of DNA replication and cell-cycle progression. The polar localization of ClpXP is dependent on the polar positioning of the CpdR single-domain response regulator. Only the unphosphorylated form of CpdR localizes and activates ClpXP. We demonstrate that another single domain response regulator, DivK, promotes the polar accumulation of unphosphorylated CpdR and that CpdR is subsequently degraded at the cell pole by the localized ClpXP protease. Thus, CpdR function is regulated by a feedback loop that incorporates its differential phosphorylation, the transient polar localization and activity of the ClpXP protease, and the clearance of the CpdR by polar ClpXP that, in turn, releases ClpXP from the pole relieving the degradation of CtrA. CtrA approximately P then accumulates and activates the transcription of cpdR, completing the regulatory loop, establishing an integrated network that controls a robust cell-cycle transition.

SUBMITTER: Iniesta AA

PROVIDER: S-EPMC2575466 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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A bacterial control circuit integrates polar localization and proteolysis of key regulatory proteins with a phospho-signaling cascade.

Iniesta Antonio A AA Shapiro Lucy L

Proceedings of the National Academy of Sciences of the United States of America 20081022 43

Dynamic protein localization is an integral component of the regulatory circuit that drives the Caulobacter cell cycle. The ClpXP protease is localized to the Caulobacter cell pole, where it catalyzes the degradation of the CtrA master regulator at specific times in the cell cycle. Clearance of active CtrA at the G1/S transition allows the initiation of DNA replication and cell-cycle progression. The polar localization of ClpXP is dependent on the polar positioning of the CpdR single-domain resp ...[more]

PMID: 18946044

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Project description:In Caulobacter crescentus, timely degradation of several proteins by the ClpXP protease is critical for proper cell cycle progression. During the cell cycle, the ClpXP protease, the substrate CtrA, and many other proteins are localized to the stalked pole dependent on a polar interaction hub composed of PopZ protein oligomers. Prior work suggests that the localization of ClpXP, protease substrates, and cofactors is needed for recognition of substrates, such as CtrA, by ClpXP. Here, we formally test this hypothesis by examining the role of PopZ in ClpXP activity and find, surprisingly, that CtrA degradation is enhanced in cells lacking polar localization due to loss of PopZ. The ClpXP adaptor CpdR is required for this enhanced degradation of CtrA and other adaptor-dependent substrates, but adaptor-independent substrate degradation is not affected upon loss of PopZ. We find that overexpression of PopZ also leads to faster degradation of CtrA but is likely due to nonphysiologically relevant recognition of CtrA by ClpXP alone, as loss of CpdR does not affect this enhancement. Our main conclusion is that loss of PopZ, and therefore loss of polar localization, does not result in the loss of ClpXP-regulated proteolysis, as would be predicted from a model which requires polar localization of ClpXP for its activation. Rather, our data point to a model where PopZ normally restrains ClpXP proteolysis by promoting the inactivation of the CpdR adaptor, perhaps through the activity and localization of the CckA kinase.IMPORTANCE Regulated proteolysis is critical for the cell cycle progression of bacteria, such as Caulobacter crescentus According to one model, this regulated proteolysis requires localization of the ClpXP protease at the stalked pole for its subsequent degradation of substrates, such as CtrA. This study offers evidence that supports an alternative model to explain how localization might influence protein degradation. Using a delocalized in vivo system created by the deletion of a polar organizing protein, PopZ, we show that activation of the ClpXP protease is independent of its polar localization. The data point to a role for PopZ in restraining ClpXP activity, likely by controlling the activity of upstream regulators of protease activity, such as CckA, though changes in its localization.

Project description:Global atmospheric loading of the climate-active gas nitrous oxide (N2O) continues to increase. A significant proportion of anthropogenic N2O emissions arises from microbial transformation of nitrogen-based fertilizers during denitrification, making microbial N2O emissions a key target for greenhouse gas reduction strategies. The genetic, physiological, and environmental regulation of microbially mediated N2O flux is poorly understood and therefore represents a critical knowledge gap in the development of successful mitigation approaches. We have previously mapped the transcriptional landscape of the model soil-denitrifying bacterium Paracoccus denitrificans Here, we show that a single bacterial small RNA (sRNA) can control the denitrification rate of P. denitrificans by stalling denitrification at nitrite reduction to limit production of downstream pathway intermediates and N2O emissions. Overexpression of sRNA-29 downregulates nitrite reductase and limits NO and N2O production by cells. RNA sequencing (RNA-seq) analysis revealed 53 genes that are controlled by sRNA-29, one of which is a previously uncharacterized GntR-type transcriptional regulator. Overexpression of this regulator phenocopies sRNA-29 overexpression and allows us to propose a model whereby sRNA-29 enhances levels of the regulator to repress denitrification under appropriate conditions. Our identification of a new regulatory pathway controlling the core denitrification pathway in bacteria highlights the current chasm in knowledge regarding genetic regulation of this pivotal biogeochemical process, which needs to be closed to support future biological and chemical N2O mitigation strategies.IMPORTANCE N2O is an important greenhouse gas and a major cause of ozone depletion. Denitrifying bacteria play vital roles in the production and consumption of N2O in many environments. Complete denitrification consists of the conversion of a soluble N-oxyanion, nitrate (NO3 -), to an inert gaseous N-oxide, dinitrogen (N2). Incomplete denitrification can occur if conditions are prohibitive, for example, under conditions of low soil copper concentrations, leading to emission of N2O rather than N2 Although enzymatically well characterized, the genetic drivers that regulate denitrification in response to environmental and physiological cues are not fully understood. This study identified a new regulatory sRNA-based control mechanism for denitrification in the model denitrifying bacterium P. denitrificans Overexpression of this sRNA slows the rate of denitrification. This report highlights that there are gaps in understanding the regulation of this important pathway which need to be filled if strategies for N2O mitigation can be rationally and carefully developed.

Dataset Information

A bacterial control circuit integrates polar localization and proteolysis of key regulatory proteins with a phospho-signaling cascade.

Publications

A bacterial control circuit integrates polar localization and proteolysis of key regulatory proteins with a phospho-signaling cascade.

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