The Rsb Phosphoregulatory Network Controls Availability of the Primary Sigma Factor in Chlamydia trachomatis and Influences the Kinetics of Growth and Development

Abstract

Chlamydia trachomatis is an obligate intracellular human pathogen that exhibits stage-specific gene transcription throughout a biphasic developmental cycle. The mechanisms that control modulation in transcription and associated phenotypic changes are poorly understood. This study provides evidence that a switch-protein kinase regulatory network controls availability of σ66 , the main sigma subunit for transcription in Chlamydia. In vitro analysis revealed that a putative switch-protein kinase regulator, RsbW, is capable of interacting directly with σ66, as well as phosphorylating its own antagonist, RsbV1, rendering it inactive. Conversely, the putative PP2C-like phosphatase domain of chlamydial RsbU was capable of reverting RsbV1 into its active state. Recent advances in genetic manipulation of Chlamydia were employed to inactivate rsbV1, as well as to increase the expression levels of rsbW or rsbV1, in vivo. Representative σ66-dependent gene transcription was repressed in the absence of rsbV1 or upon increased expression of RsbW, and increased upon elevated expression of RsbV1. These effects on housekeeping transcription were also correlated to several measures of growth and development. A model is proposed where the relative levels of active antagonist (RsbV1) and switch-protein anti-sigma factor (RsbW) control the availability of σ66 and subsequently act as a molecular 'throttle' for Chlamydia growth and development.

The General Stress Response σS Is Regulated by a Partner Switch in the Gram-negative Bacterium Shewanella oneidensis

“…66 is controlled by a canonical RsbVWU-like system, whereas in the case of 70 , the partner-switching mechanism is conserved, but Rsd and HPr, which act as an anti-sigma and an anti-sigma antagonist, respectively, are not RsbVW homologs (50,51). It is thus clear that the partner-switching mechanism is a general pathway to efficiently regulate sigma factors.…”

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

“…Furthermore, the higher MICs required for the delivery of these antibiotics into the inclusion can lead to toxicity in the host cell, which imposes further limits on the use of antibiotics for selection. Several antibiotics have been used successfully in genetic selections, including chloramphenicol, kasugamycin, nalidixic acid, rifampin, spectinomycin, trimethoprim, tetracycline (only for naturally resistant veterinary strains), ␤-lactams (only for LGV serovars), and blasticidin S (12,14,18,19,27,31,(50)(51)(52)(53). However, the use of mutant versions of chlamydial factors, such as 16S rRNA, RpoB, and GyrA, that render them resistant to antibiotics as selectable markers is limited because the gene mutations that confer resistance to these antibiotics are often recessive.…”

“…A second TargeTron vector (pDFTT3-aadA) (Fig. 3), carrying a spectinomycin resistance cassette (aadA), was also generated (53) and successfully adapted to generate a C. trachomatis strain bearing an inactivated rsbV1 gene (rsbV1::GII[aadA]), encoding the anti-anti-sigma factor RsbV1 (52). A similar approach also enabled the generation of a double mutant strain bearing loss-of-function alleles of incA and rsbV1 (incA::GII[aadA] and rsbV1::GII[bla]) (53).…”

CTL0511 from Chlamydia trachomatis Is a Type 2C Protein Phosphatase with Broad Substrate Specificity

“…This is the first study of a "generalist" PP2C from C. trachomatis. RsbU, a sensor phosphatase that functions within the context of a partner-switching mechanism, contains a PP2C domain that has been shown to dephosphorylate a P-Ser within the anti-antisigma factor RsbV1 (70). The defined role of RsbU makes it unlikely to serve as a cognate phosphatase for Pkn1 or PknD.…”