Analysis of MreB interactors in Chlamydia reveals a RodZ homolog but fails to detect an interaction with MraY

Abstract

Chlamydia is an obligate intracellular bacterial pathogen that has significantly reduced its genome in adapting to the intracellular environment. One class of genes for which the bacterium has few annotated examples is cell division, and Chlamydia lacks FtsZ, a central coordinator of the division apparatus. We have previously implicated MreB as a potential substitute for FtsZ in Chlamydia (Ouellette et al., 2012). Thus, to identify new chlamydial cell division components, we searched for proteins that interacted with MreB. We performed a small-scale screen using a Gateway® compatible version of the Bacterial Adenylate Cyclase Two Hybrid (BACTH) system, BACTHGW, to detect proteins interacting with chlamydial MreB and identified a RodZ (YfgA) homolog. The chlamydial RodZ aligns well with the cytoplasmic domain of E. coli RodZ but lacks the periplasmic domain that is dispensable for rod cell shape maintenance in E. coli. The expression pattern of yfgA/rodZ was similar to that of mreB and ftsI, suggesting that these genes may operate in a common functional pathway. The chlamydial RodZ correctly localized to the membrane of E. coli but was unable to complement an E. coli rodZ mutant strain, likely because of the inability of chlamydial RodZ to interact with the native E. coli MreB. Finally, we also tested whether chlamydial MreB could interact with MraY, as suggested by Gaballah et al. (2011). However, we did not detect an interaction between these proteins even when using an implementation of the BACTH system to allow native orientation of the N- and C-termini of MraY in the periplasm. Thus, further work will be needed to establish this proposed interaction. In sum, we have added to the repertoire of potential cell division proteins of Chlamydia.

Evolutionary Cell Biology of Division Mode in the Bacterial Planctomycetes-Verrucomicrobia- Chlamydiae Superphylum

“…Thus, important modifications of division mode due to the lack of RodZ are expected. This is in agreement with the fact that the chlamydial RodZ is truncated, lacking its C-terminal periplasmic domain, and cannot complement an E. coli RodZ mutant (Ouellette et al, 2014). …”

Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB

“…Furthermore, rod‐shaped morphology of rodZ deficient E. coli was partially restored with complementation of CT009 supporting structural observations and providing evidence that CT009 functions as a homolog to RodZ. Bacterial two‐hybrid analyses demonstrated interaction with the key morphogenic protein MreB, similar to previous report (Ouellette et al ., ). Mutagenesis of two amino acids in CT009 that are predicted to be central to interactions with MreB provided additional level of specificity for these interactions.…”

“…Interestingly, sequence similarity searches reveal that this protein contains a predicted helix‐turn‐helix motif within a conserved xenobiotic response element (XRE) domain superfamily of DNA‐binding transcription factors. However, a recent study (Ouellette et al ., ) using bacterial two‐hybrid analysis indicated that CT009 interacts with MreB and supported a potential role as RodZ homolog. As described herein, in‐depth computational and experimental protein structural analyses were performed to provide more comprehensive support for CT009 as a RodZ homolog.…”

Chlamydia cell biology and pathogenesis

“…Second, using a new technique that is based on the incorporation of click-chemistry-modified d -amino acid probes, newly assembled peptidoglycan was visualized at the septum of dividing reticulate bodies in a ring-like structure, which indicated that peptidoglycan may have a role in cell division 170 . Third, MreB, an actin homologue, was found to localize at the septum in a manner that was dependent on peptidoglycan precursors and its conserved regulator RodZ (also known as CT009) 171–173 , where it is thought to functionally compensate for the lack of FtsZ. Fourth, muramyl peptides and larger muropeptide fragments in Chlamydia trachomatis were isolated from fractionated infected cell lysates using a NOD-activated reporter cell line as a sensitive read-out 174 .…”