1. Evaluation Summary:

    CarD is an RNA polymerase interacting protein that is essential for mycobacterial viability, the levels of which are important for controlling gene expression in mycobacteria during various stress conditions. This study reports two mechanisms that regulate levels of CarD under stress conditions, including starvation. The authors report that CarD levels are tightly regulated and that there was a dramatic decrease in the levels of CarD when cells switched from the nutrient-rich to the starvation condition. They discovered two synergistic mechanisms that led to this dramatic decrease in CarD. The first is SigF-dependent induction of antisense RNA of CarD (AscarD), which inhibits CarD translation and a second mechanism involving Clp protease-mediated degradation of intracellular CarD. The work will be of interest to researchers studying non-coding RNAs, microbial gene expression, physiology and stress response.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    Manuscript by Li et al. entitled "Clp protease and antisense RNA jointly regulate the global regulator CarD to mediate mycobacterial starvation response" reports two mechanisms that regulate levels of CarD under stress conditions, including starvation. They report that level of CarD was tightly regulated, and there was a dramatic decrease in the levels of CarD when cells switched from the nutrient-rich to the starvation condition. They discovered two synergistic mechanisms that led to this dramatic decrease in the levels of CarD: 1) SigF-dependent induction of antisense RNA of CarD (AscarD), which inhibits CarD translation, and 2) Clp protease-mediated degradation of intracellular CarD.

    CarD is an essential global transcription regulator which activates transcription and modulates the expression of about two-third of genes in M. tuberculosis. It binds RNA polymerase and helps in stabilizing the RNAP open complex. Based on the previous reports, the levels of CarD transcripts increase under stress conditions, including the starvation/stationary phase. Since CarD upregulates the expression of rRNA genes, so this was not clear how levels of genes coding for rRNA goes down while CarD transcripts go up under various stress conditions. This is one of the key unaddressed research gaps in understanding how CarD helps cells adapt under stress conditions. The data presented in the manuscript is an attempt to address this question.

    As stated above, the authors succeeded in discovering two mechanisms that M. tuberculosis employs to tightly regulate the levels of a global transcription regulator. The experiments were well designed, and the data presented in the manuscript support the major conclusion that levels of CarD decrease under various stress conditions. This discovery now helps explain why levels of genes under the control of CarD goes down under stress conditions. This manuscript further reemphasizes to compare both transcriptomics and proteomics data to understand the role of target genes in biological processes. The study also highlights the importance of non-coding RNAs in regulating gene expression.

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  3. Reviewer #2 (Public Review):

    Our understanding of bacterial mechanisms of transcriptional regulation has been pushed forward by studies in a few model organisms that were initially amenable to biochemical experiments. More recently, however, it has become clear that many regulatory pathways are distinct from these model organisms. This fact, coupled with continued appearance of drug-resistant strains of pathogenic bacteria motivates the serious study of important pathways directly in the pathogenic bacteria themselves.

    The mycobacterial transcription factor CarD has been studied via many approaches and the biophysical mechanisms underlying its effect on transcription initiation are well-described. Curiously though, the biological role of CarD is far less well-understood. The authors have approached this question by uncovering two modes of the regulation of CarD concentration during nutrient starvation which provides new insight into how the molecular activity of CarD is beneficial to the bacteria.

    First, they uncover the proteolytic-dependent degradation of CarD in conditions where growth needs to be slowed. Secondly, they describe an anti-sense RNA to the CarD transcript which likely inhibits CarD translation. Combined, these mechanisms reduce the amount of CarD in the cell, which then results in global changes in the gene expression. in particular, a reduction in ribosomal RNA synthesis fits well with the need to slow growth.

    The data are very clear and this work will likely have a large impact on the field. It both sets the stage for future work on the response of Mtb to stress and provides an stress response pathway distinct to that of E. coli and other model systems that may help inform on the regulation in other pathogenic species.

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  4. Reviewer #3 (Public Review):

    This is a study of the regulation of CarD expression by proteolysis and anti sense RNA. CarD is a well-studied RNAP interacting protein with complex roles on RNAP promoter dynamics, as the authors note, but the full mechanisms controlling its cellular levels under various stress conditions are not well defined. One prior report indicated that CarD is subject to proteolysis by the ClpP system. This study demonstrates that CarD protein levels are subject to ClpP proteolysis during stationary phase and that conditional expression of ClpP2 reverses this effect. The authors also identify an antisense RNA at the CarD locus that also negatively regulates CarD protein levels and overexpression of this antisense RNA confers sensitivity to various stresses, phenocopying prior data with CarD depletion.

    Overall, the study is carefully done. The genetics are carefully done and in general the conclusions are well supported. The strongest aspects of the paper are the demonstration of the effect of Clp and asCarD on CarD protein levels. Given the substantial prior literature on CarD and its roles in mycobacterial transcription, this study does add substantial complexity to CarD biology.

    I think the paper could be stronger in two areas:

    1. Although the paper presents a clear picture of these two mechanisms individually on CarD levels, the effects of these mechanisms on cellular phenotypes is less completely explored. For example, for the stationary phase dependent regulation of CarD, does this mechanism effect stationary phase survival? Most of the data shows cell density, but no survival assays are done (as one example). Similarly, what about the the stress conditions examined? Some data is given, but I think more exploration of the cellular effects of these mechanisms would make the paper more appealing to a broad audience. With their ability of tune CarD levels via graded manipulation of asCarD or Clp, the authors could learn much more about the physiologic and global transcriptional effects of these regulatory mechanisms and this would be a major advance for the field.

    2. Although the individual effects of Clp and asCarD are documented, the paper would be stronger if it explored the relative importance and interaction of these two mechanisms in different conditions. Are they simply additive as the model figure suggests? Does the relative importance of each mechanism differ depending on the condition studied?

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