Identification and Partial Characterization of Potential FtsL and FtsQ Homologs of Chlamydia

Abstract

Chlamydia is amongst the rare bacteria that lack the critical cell division protein FtsZ. By annotation, Chlamydia also lacks several other essential cell division proteins including the FtsLBQ complex that links the early (e.g., FtsZ) and late (e.g., FtsI/Pbp3) components of the division machinery. Here, we report chlamydial FtsL and FtsQ homologs. Ct271 aligned well with Escherichia coli FtsL and shared sequence homology with it, including a predicted leucine-zipper like motif. Based on in silico modeling, we show that Ct764 has structural homology to FtsQ in spite of little sequence similarity. Importantly, ct271/ftsL and ct764/ftsQ are present within all sequenced chlamydial genomes and are expressed during the replicative phase of the chlamydial developmental cycle, two key characteristics for a chlamydial cell division gene. GFP-Ct764 localized to the division septum of dividing transformed chlamydiae, and, importantly, over-expression inhibited chlamydial development. Using a bacterial two-hybrid approach, we show that Ct764 interacted with other components of the chlamydial division apparatus. However, Ct764 was not capable of complementing an E. coli FtsQ depletion strain in spite of its ability to interact with many of the same division proteins as E. coli FtsQ, suggesting that chlamydial FtsQ may function differently. We previously proposed that Chlamydia uses MreB and other rod-shape determining proteins as an alternative system for organizing the division site and its apparatus. Chlamydial FtsL and FtsQ homologs expand the number of identified chlamydial cell division proteins and suggest that Chlamydia has likely kept the late components of the division machinery while substituting the Mre system for the early components.

Keywords: Chlamydia; bacterial two-hybrid system; cell division; ftsL; ftsQ.

FIGURE 1

A bacterial cell division pathway based on Escherichia coli. The cell division machinery is recruited and organized in an hierarchical and temporal manner beginning with the recruitment of FtsZ to the division site and ending with the separation of daughter cells by the amidases. The boxed proteins are those for which Chlamydia has an annotated homolog.

FIGURE 2

Identification of ftsL and ftsQ homologs in Chlamydia. (A) Arrangement of the dcw gene cluster from E. coli encoding cell division and peptidoglycan synthesis genes. Note the presence of ftsL upstream of ftsI and ftsQ downstream of ddlB. (B) Arrangement of cell division and peptidoglycan synthesis genes in Chlamydia. The bar indicates that the clusters are separated on the chromosome. Note the presence of ct271 upstream of ftsI. For (A) and (B), gray boxes are intergenic regions. (C) A BLAST alignment of E. coli FtsL (EcFtsL; 121 amino acids) vs. chlamydial Ct271 (95 amino acids) and the corresponding sequence alignment of the indicated amino acids. Bolded, italicized Leu/Iso residues indicate the leucine zipper-like motifs within the FtsL homologs. (D) Phyre2 structural prediction of Ct764 and the structure of E. coli FtsQ. The Ct764 model had a 98.8% confidence match to E. coli FtsQ. Ribbon diagrams are color-coded red to violet (Nter to Cter). (E) Clustal Omega alignment of Ct764 and E. coli FtsQ (EcFtsQ). Key residues in the POTRA domain of E. coli FtsQ are bolded and italicized with the domain itself boxed (amino acids 55–126). For (C) and (E), ^∗ = identical residues; + = similar residues.

FIGURE 3

Transcriptional analysis of ct271/ftsL and ct764/ftsQ in Chlamydia. RNA was collected from infected cells at the indicated timepoints and assayed for transcripts by RT-qPCR. Transcript levels were normalized to genomic DNA (ng cDNA/gDNA). (A) Representative early stage (euo), (B) mid cycle division-related (mreB, rodZ, ftsK), and (C) late-stage (omcB) transcripts are shown for comparison. (D) ct271 and ct764 transcription are consistent with a mid-cycle profile. Data are representative of a minimum of two experiments. Standard deviations were typically less than 5% of the mean.

FIGURE 4

Localization of GFP-Ct764 in Chlamydia. Chlamydia trachomatis L2 was transformed with a tetracycline-inducible expression vector encoding GFP-Ct764. (A) After 18 hpi, 1 nM anhydrotetracycline (aTc) was added for 6 h. Cells were fixed and processed as described in section “Materials and Methods.” Confocal images of inclusions with multiple organisms were acquired with a 60x objective and a 2x digital zoom. The left panel shows the localization of GFP-Ct764, the middle panel an outer membrane labeling (anti-Ctr), and the right panel a merged image. Small arrows indicate crescent-like structures whereas asterisks indicate puncta. Scalebar = 5μm. (B) After 9 hpi, 0.06 nM aTc was added for 3 h, and cells were subsequently fixed and processed as described with the GFP-Ct764 fusion protein visualized using an anti-GFP antibody. Representative images of Ct764 localization from four pairs of dividing RBs are shown. Deconvolved images were acquired with a 100x objective.

FIGURE 5

Effect of over-expression of GFP-Ct764 on chlamydial growth and development. Cells were infected with wild-type C. trachomatis L2 (Ctr L2), Ctr L2 transformed with a plasmid encoding an inducible GFP (pASK e.v.), or Ctr L2 transformed with a plasmid encoding an inducible GFP-Ct764. (A) At 16 hpi, infected cells were treated or not with 10 nM aTc. At 40 hpi, cells were lysed and recoverable inclusion forming units (IFUs) were quantified on fresh cell layers without aTc treatment. Data are expressed as a percentage IFUs recovered compared to the untreated (UTD) control for the given strain. Data are the average of two experiments. ^∗indicates p < 0.05 by Student’s one-tailed t-test. See also Supplemental Figure S2. (B) Representative images of inclusions at 40 hpi after treatment with 10 nM aTc at 16 hpi. Note the presence of large, aberrant RBs in the GFP-Ct764-expressing bacteria compared to those expressing GFP alone (pASK e.v.). The left panels show an outer membrane labeling (anti-Ctr), the middle panels GFP fluorescence, and the right panels the merged images. The width of each panel is 10 μm.

FIGURE 6

Interactions between Ct764 and chlamydial cell division proteins. Bacterial adenylate cyclase-based two hybrid (BACTH) assays were performed to test interactions. DHT1 E. coli were co-transformed with plasmids expressing the indicated fusion proteins and plated on minimal medium with maltose. Only positive interactions result in growth on maltose and dark colonies. A positive control of T25-RodZ and T18-MreB, and a negative control of T18-Ct764 and T25-Ct471 are shown. These tests were performed a minimum of two times.

FIGURE 7

Interactions of Ct764 with E. coli cell division proteins and functional complementation of an FtsQ mutant. (A) DHT1 E. coli were co-transformed with T25-Ct764 (red bars) or T18-Ct764 (blue bars) and an E. coli cell division (Fts) protein designated by its letter (e.g., A = FtsA). Y^∗ = YmgF. The positive control was T25-Zip vs. T18-Zip, and the negative control was T18-Ct764 vs. T25-Ct471, an unrelated membrane protein. Beta-galactosidase activity was quantified from overnight cultures as described in section “Materials and Methods.” A positive interaction is defined as having fivefold greater activity than the negative control. Here, the cutoff is approximately 50 units, indicated by the horizontal black line at this value. Data are representative of at least two experiments. (B) The FtsQ depletion strain, JOE417, was transformed with tetracycline-inducible plasmids expressing Ct764, E. coli FtsQ, or nothing (empty). Overnight cultures were subcultured and normalized for cell density, and serial 1:10 dilutions (e.g., -1 = 10^-1) were spotted onto permissive or non-permissive agar. The same results were obtained using two different clones from different transformations of JOE417.

FIGURE 8

A possible chlamydial cell division pathway. Chlamydia lacks FtsZ and the early division proteins. The Mre system has been proposed to substitute for these early proteins. Chlamydia has maintained the late division proteins with the exception of FtsN (see text). The pathway contains known and predicted interactions.