Interdomain Communication in the Mycobacterium tuberculosis Environmental Phosphatase Rv1364c

Abstract

An "environmental phosphatase" controls bacterial transcriptional responses through alternative sigma factor subunits of RNA polymerase and a partner switching mechanism has been proposed to mediate phosphatase regulation. In many bacteria, the environmental phosphatase and multiple regulators are encoded in separate genes whose products form transient complexes. In contrast, in the Mycobacterium tuberculosis homolog, Rv1364c, the phosphatase is fused to two characteristic regulatory modules with sequence similarities to anti-sigma factor kinases and anti-anti-sigma factor proteins. Here we exploit this fusion to explore interactions between the phosphatase and the regulatory domains. We show quantitatively that the anti-sigma factor kinase domain activates the phosphatase domain, the kinase-phosphatase fusion protein autophosphorylates in Escherichia coli, and phosphorylation is antagonized by the phosphatase activity. Small angle x-ray scattering defines solution structures consistent with the interdomain communication observed biochemically. Taken together, these data indicate that Rv1364c provides a single chain framework to understand the structure, function, and regulation of environmental phosphatases throughout the bacterial kingdom.One third of the world population is seropositive for Mycobacterium tuberculosis (Mtb).3 Whereas most anti-microbial therapeutics target actively growing bacteria, Mtb can evade current drugs by converting to a persistent, metabolically suppressed state (1). Moreover, Mtb survives in distinct environments in vivo by adjusting transcriptional programs. Little is known about the transcriptional cues that mediate such developmental transitions, and defining their underlying mechanisms remains the focus of much mycobacterial research (2-6). The Mtb genome encodes 13 sigma factor homologs, several of which play essential roles in disease (7).Diverse bacteria employ sigma factors to mediate the transcriptional programs that drive life cycle transitions (8, 9). Alternative sigma factors, unique RNA-polymerase subunits that mediate promoter choice, are known to respond to environmental cues such as heat and limited energy (10) to drive the transition into spore formation (11) and stationary phase (12). As reviewed in Ref. 9, anti-sigma factors exemplified by the Bacillus subtilis regulator of sigma B W (RsbW) are capable of binding sigma factors and sequestering them away from RNA polymerase (Fig. 1). Through a partner switching mechanism, anti-anti-sigma factor proteins such as RsbV bind the antisigma factors, freeing the cognate sigma factors to activate transcription. Anti-sigma factors, which display homology to histidine kinases, phosphorylate their anti-anti-sigma factor antagonists on a serine or threonine residue (9). A master "environmental phosphatase," such as RsbU in B. subtilis, reverses this phosphorylation and restores the complexes of anti-and anti-anti-sigma factors (13).These RsbU-like Ser/Thr phosphatases are regulated by proteins with remarkable homologies to the ...

Spectroscopic investigations on dermorphin and its [L‐Ala2] analog

“…The pK, values of the ionizable groups of DM and LDM can be considered as typical of short, conformationally disordered peptides. For the dipeptide H-Tyr-Tyr-OH a pK, of 7.68 has been reported for the a-amino group and 9.80, 10.26 for the two tyrosine side chains (41). The absence of a terminal carboxyl group (negatively charged at the pH values necessary to deprotonate the a-amino and the phenol groups) is expected to lower both pK,'s, as indeed observed in our case.…”

The sigma factors of Mycobacterium tuberculosis: regulation of the regulators

“…The anti-r factor domain activates the phosphatase domain through direct interaction, while its kinase activity in turn is antagonized by the phosphatase domain. This entire module has been proposed to regulate the environmental phosphatase, RsbU, of Rv1364c [122] akin to the RsbU-RsbT-RsbS system of B. subtilis [117]. However, the signal governing this cascade, and other possible regulators playing a role in this pathway, still needs to be determined.…”

Regulation of σ factors by conserved partner switches controlled by divergent signalling systems

“…Notably, the protein Rv1364c is a PSS module organized in four domains corresponding to a PAS, a phosphatase, an anti-r factor and an anti-r factor antagonist domain ( Fig. 2D) (Parida et al, 2005;Sachdeva et al, 2008;Greenstein et al, 2009;Malik et al, 2009;Jaiswal et al, 2010;King-Scott et al, 2011). Interestingly, Rv1364c seems to detect signals itself without upstream sensing module.…”