Fig 1.
iIhtA targets conserved sequences of hctA.
(A) Mutations made across the conserved region of hctA resulted in the mutant hctA6–27. Each mutation was designed to maintain the amino acid structure whilst disrupting the linear RNA sequence of the potential target region for IhtA. (B) Mutant hctA constructs were co-transformed with empty pLac vector or wild type ihtA into E. coli and assayed for growth upon induction of HctA expression. Wild type hctA co-transformed with pLac or ihtA served as controls. Cell viability was expressed as a percentage of the ratio between the induced and uninduced samples. Each condition was performed in triplicate over at least three separate experiments. The bars represent the mean ± SEM of the triplicates in all experiments combined. * indicates P value < 0.01 using t-test statistical analysis.
Fig 2.
iLoop 1 of IhtA targets hctA mRNA.
m(A) Schematic of the structure of IhtA and color coded base pair probability (color coded 1–0) as predicted by the RNAfold web server. The three stem:loops are indicated, stem:loop 3 is the rho-independent terminator. (B) TargetRNA prediction of interacting nucleotides between IhtA and hctA. Indicated are location of loop 1 and 2 of IhtA and the Shine-Dalgarno and start site of hctA. (C) Wild type hctA constructs were co-expressed with IhtA loop mutants ihtAL1, ihtAL2 and ihtAL3. Cell viability was expressed as a percentage of the ratio between the induced and uninduced samples. Each condition was performed in triplicate over at least three separate experiments. The bars represent mean ± SEM of the triplicates in all experiments combined. Statistical analysis performed using t-test, * indicates P value < 0.01 when compared to ihtA/hctA.
Fig 3.
iIhtA occludes the start site of hctA.
(A) Schematic of the mutations made in IhtA and the corresponding compensatory mutations made in hctA. The start site of hctA is underlined. (B) E. coli were co-transformed with A/T rich mutant construct pairs ihtA#21 and hctA#22 and with G/C rich mutant pairs ihtA#9 and hctA#14 and cell viability was assayed. Wild type hctA co-transformed with pLac, ihtA, ihtA#21 or ihtA#9, and mutant hctAs co-transformed with pLac served as controls. (C) To determine importance of the G/C pairing 3’ of the hctA AUG, E. coli were co-transformed with A/T rich mutant construct ihtA#10 and its compensatory hctA partner hctA#16 or with G/C rich mutant ihtA#26 and the compensatory mutant hctA#25. Wild type hctA co-transformed with pLac, ihtA, ihtA#10 or ihtA#26, and mutant hctAs co-transformed with pLac served as controls. Cell viability in graphs B and C were expressed as a percentage of the ratio between the induced and uninduced samples. Each condition was performed in triplicate over at least three separate experiments. The bars represent the mean ± SEM of all samples. Statistical analysis using t-test, * indicates P value < 0.01 when compared to the relevant hctA control and ** indicates P value < 0.01 when compared to ihtA/hctA control.
Fig 4.
Integrity of stem:loop 1 structure.
(A) Schematic of ihtA mutants and the corresponding compensatory hctA mutant. (B) Wt hctA was co-transformed with ihtA mutants #11 and #16–19 and ability to rescue growth was assayed. Wt hctA co-transformed with pLac and ihtA served as controls. Compensatory hctA mutants to ihtA#16 and ihtA#17 were co-transformed with the appropriate ihtA mutant and assayed for rescue of growth repression. hctA#24 and hctA#23 co-transformed with pLac served as baseline controls. (C) Predicted structure of mutants IhtA#16 and IhtA#17 compared to their corresponding intramolecular compensatory mutants IhtA#27 and IhtA#28 respectively. Structures and base pair probabilities (color coded 0–1) were predicted by the RNAfold web server. The location of the G/C rich binding site that occludes the start site of hctA is circled. The * marks the mutated nucleotide/s. (D) Wt hctA was co-transformed with the intramolecular compensatory mutants ihtA#27 and ihtA#28 and assayed for rescue of growth. Wt hctA co-transformed with pLac or ihtA served as controls. Cell growth in graphs B and D were expressed as a percentage of the ratio between the induced and uninduced samples. Each condition was performed in triplicate over at least three separate experiments. The bars represent the mean ± SEM of all samples. Statistical analysis was performed using t-test, * indicates P value < 0.01 when compared to the relevant hctA or mutant hctA control and ** indicates P value < 0.01 when compared to ihtA/hctA control.
Fig 5.
iIhtA does not interact with the Shine-Dalgarno site.
(A) Schematic of the UTR of pTet compared to the 5’ UTR, starting at the TSS of C. trachomatis serovar L2. The sequences in common are boxed and the predicted serovar L2 SD site is underlined. (B) Schematic of mutations made in the anti-SD of IhtA. (C) Wild type hctA was co-transformed with ihtA mutants #1–7 and assayed for growth upon induction of HctA expression. Co-transformation with pLac and wt ihtA served as controls. Statistical analysis was performed using t-test, * indicates P value < 0.01 when compared to the ihtA/hctA control (D) sRNA structure and base pair probabilities (color coded 0–1) predicted by the RNAfold web server of IhtA#3 and its intramolecular mutant IhtA#8. The * indicates mutated nucleotide/s. (E) Cell viability of strains co-transformed with intramolecular mutant ihtA#8 and hctA. Co-transformation of hctA with pLac, ihtA or ihtA#3 served as controls. * indicates P value < 0.01 when compared to hctA control and ** indicates P value < 0.01 when compared to ihtA/hctA. For graphs C and E, cell viability was expressed as a percentage of the ratio between the induced and uninduced samples. Each condition was performed in triplicate over at least three separate experiments. Bars represent the mean ± SEM of all experiments combined.
Fig 6.
iInteraction of IhtA with predicted mRNA targets CTL0097 and CTL0322.
(A) Predicted base-pairing by TargetRNA of additional IhtA target mRNAs which encode at least a partial G/C rich IhtA binding region (underlined). The location of loop 1 of IhtA is also indicated. (B) In vitro transcribed IhtA was incubated with CTL0322 and CTL0097 immobilized on a biosensor tip. IhtA incubated with in vitro transcribed hctA served as a positive control for binding and in vitro transcribed hctB served as a negative control. Binding was measured as a change in internally reflected light through the tip over time.
Fig 7.
iIhtA represses the translation of CTL0322 in vitro.
(A) Schematic of cheZ constructs. Blue and red are chlamydial specific sequences, red indicates sequence that is translated. Green indicates E. coli cheZ. (B) E. coli lacking motility (MG1655 ΔcheZ) were co-transformed with either ihtAL1 (deficient in hctA translation repression) and cheZ, hctBcheZ or hctAcheZ (panels A,C and E) or ihtA and the aforementioned constructs (panels B, D and F). (C) E. coli lacking motility (MG1655 ΔcheZ) were co-transformed with ihtAL1 and CTL0097cheZ or CTL0322cheZ (panel A and C) and ihtA and CTL0097cheZ or CTL0322cheZ (panels B and D). All mobility assays were performed at least 3 times per strain.