In this work, we identified a regulon controlled by the copper-sensing repressor RicR. We propose that under low copper conditions RicR represses gene expression, whereas exposure to excess copper results in RicR dissociation from DNA and the induction of lpqS, Rv2963, mymT, socAB and ricR. Consistent with this hypothesis, disruption of ricR resulted in the constitutive expression of all five loci in the absence of excess copper. Based on our copper sensitivity assays, we propose that the RicR regulon plays a protective role against copper toxicity. It is also possible that one or more of the regulated genes is not involved in counteracting the effects of copper toxicity, but uses copper as an in vivo cue to trigger gene expression. It is intriguing that RicR regulates several genes that are only found in pathogenic Mycobacteria, suggesting that these genes may be required for growth or survival in a specific niche in an animal host.
Of the genes that are controlled by RicR, mymT
is the only one that has been previously characterized (Gold et al., 2008
). MymT is a copper metallothionein that binds up to six Cu(I) ions. As would be predicted for a metallothionein gene, mymT
is highly induced in the presence of copper (, ) and appears to protect Mtb
from the toxic effects of copper. However, a mymT
null mutant is no less virulent than WT Mtb
in a mouse model of infection (Gold et al., 2008
). It is possible that all RicR-regulated genes need to be deleted or constitutively repressed in order to observe a measurable virulence phenotype, a hypothesis we are currently testing. Alternatively, a mouse model of infection may not be appropriate for examining the effects of copper on Mtb
survival in vivo.
Because the RicR regulon is induced in copper, we propose that gene products in addition to MymT may also play a role in combating copper toxicity. Rv2963 is a putative permease that contains a carboxy(C)-terminus rich in histidines, which can potentially bind copper. LpqS is a probable lipoprotein that also has a histidine-rich region beginning 30 amino acids from its amino(N)-terminus. These histidines are predicted to remain after possible lipoprotein processing and lipidation (Sander et al., 2004
). As a putative lipoprotein, LpqS may work with Rv2963 to export copper. That lipoproteins associate with permeases to export ligands has precedence in Mtb
. LprG (Rv1411c) and Rv1410c transport ethidium bromide (Farrow & Rubin, 2008
), while LppX (Rv2945c) is proposed to work with MmpL7 (Rv2942) to export phthiocerol dimycocerosate across the cell membrane (Camacho et al., 1999
, Cox et al., 1999
Divergently expressed from the lpqS
promoter is Rv0846c, a predicted multi-copper oxidase whose expression is induced by copper (Ward et al., 2008
). We also found that it is over-expressed in the ricR
mutant (). It is possible that RicR bound to the lpqSp
palindrome represses the divergently expressed Rv0846c, especially if RicR, like other metalloregulators, induces long-range changes in DNA structure (Iwig & Chivers, 2009
). It was proposed that Rv0846c is chaperoned to the twin-arginine transport system to export copper (Ward et al., 2008
); Rv0846c may function like E. coli
CueO, a "cuproxidase" that oxidizes Cu(I) to the less toxic Cu(II) outside of the cell (Kosman, 2010
In addition to LpqS and Rv2963, no function has been assigned to the previously unidentified socAB
operon. Although socAB
is transcribed (Fig. S2
), it is unknown if the message is translated or if it is actually a small non-coding RNA. The role of socAB
and the other RicR-regulated genes in protecting Mtb
against copper toxicity is currently under investigation.
Despite the successful identification of the regulator of a new copper responsive regulon, we still do not understand why the RicR regulon is repressed in proteasome degradation-defective Mtb. The simplest model is that RicR is itself a proteasome substrate. The accumulation of RicR in the pafA and mpa mutants might result in the repression of the regulon under normal culture conditions. However, neither endogenous nor ectopically produced RicR levels were altered in proteasome degradation-defective strains under all conditions tested so far (data not shown). Another possibility is that the down-regulation of the RicR regulon in the mpa and pafA mutants is due to the accumulation of one or more copper-binding proteins that are normally proteasome substrates. This may result in the sequestration of available copper in the cell, mimicking copper-depleted conditions that result in RicR regulon repression. A similar explanation may account for the induction of the Zur regulon, where sequestered zinc would lead to higher gene expression. Ongoing studies are attempting to determine how proteasome activity affects these two metal-dependent regulons.
Prior to our work, CsoR was the only known copper responsive regulator in Mtb
, controlling the expression of ctpV
, which encodes a predicted membrane P-type ATPase. CtpV is proposed to be a copper efflux pump based on its regulation by CsoR and its similarity to other copper transporters (Liu et al., 2007
). Supporting this hypothesis, a ctpV
mutant is more susceptible to killing by high levels of copper than WT Mtb
, presumably because the mutant cannot effectively export copper (Ward et al.
). Importantly, the ctpV
mutant is attenuated in an animal infection model, supporting the notion that the maintenance of copper homeostasis is important in vivo. Here, we present data that suggest the RicR regulon, which does not include ctpV
, is also protective against high levels of copper in vitro. Taken together, we propose that Mtb
has at least two mutually exclusive but parallel copper inducible pathways controlled by RicR and CsoR (). It remains to be determined if these two pathways represent a redundant response to copper, or if Mtb
has evolved a graded response to copper toxicity; if so, this would predict that the Cu(I) binding affinities are different for RicR v. CsoR, a hypothesis currently under investigation (B. Keste, D. Giedroc, personal communication).
Model for a dual copper response in Mtb
Little is understood about copper homeostasis during a bacterial infection. The exposure of microbes to excess copper could perhaps act as an innate immune defense, a hypothesis supported by a report suggesting that the mammalian ATP7A, a copper-transporting ATPase, provides some bactericidal activity in macrophages (White et al., 2009
). Furthermore, copper levels are elevated in cultured macrophages infected with Mtb
(Wagner et al., 2005
). Copper has long been used as an anti-bacterial agent in laboratory and medical settings (Mikolay et al., 2010
), thus perhaps it would not be surprising if animals have developed ways to mobilize cellular copper to battle invading microbes.