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Decision letter: Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases

Carter, Andrew P ; Pfeffer, Suzanne R

2022

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Editor's evaluation: Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases

Carter, Andrew P

2022

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Author response: Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases

Mehta, Ved ; Khanppnavar, Basavraj ; et al.

2022

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Figure 3. Bacterial glycogen metabolism enzymes are secreted in the inclusion lumen. ; (A) HeLa cells were infected for 24 hr or 48 hr with GFP expressing L2 (GFP-Chlam, green) and stained with an anti-GlgX antibody (red) and Hoechst (blue). Insets to the right show enlargements of the boxed areas. Scale bar: 5 µm. (B) Heterologous test of secretion: the N-terminal 20 amino acids of the indicated proteins were fused to the reporter Cya, and constructs were transformed into the S. flexneri strains ipaB (T3S+) and mxiD (T3S-). Liquid cultures were fractionated into pellet (P) and supernatant (S). All chimeras except GlgC/cya were detected in the supernatant in T3S competent bacteria and not in T3S defective bacteria, indicative of a functional T3S signal. The endogenous T3S substrate of S. flexneri, IpaD, serves as a positive control, while the non-secreted cAMP receptor protein (CRP) controls for non-specific leakage into the supernatant.

2016

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Figure 4. CleA localizes to the flagellated cell pole to interact with FliM. ; Examples of the subcellular localization of CleA-mGFPmut3 are shown in (a) and quantified as shown in (b). Variants of CleA fused to mGFPmut3 are indicated on the left with the three domains highlighted as in Figures 2 and 3. Individual amino acid substitutions are indicated. CleA fusion proteins were expressed in a ΔcleA deletion strain and additional genetic alterations as indicated on the right of each panel. Cells were classified according to the localization patterns shown in (a): delocalized cytosolic (blue); foci at flagellated pole of PD cells (red); polar foci in small SW cells (green); other localization patterns (purple), including PD cells with foci at both poles, PD cells with foci at stalked pole, cells with no GFP signal. Number of cells analyzed (top to bottom): 3452, 1450, 637, 2357, 2603, 795, 1148, 1347. (c) CleA interacts with the FliM flagellar switch protein. Binding of CleA to the N-terminus of FliM was determined by bacterial two-hybrid analysis. Fusion proteins between the T25 and T18 fragments of adenylate cyclase (Karimova et al., 1998) and the N-terminal peptide of FliM or FliM_ID57WA (aa 2–65), or full-length CleA were constructed and expressed in the E. coli cya mutant strain AB1770. Strains harboring combinations of FliM and CleA fusions were spotted on McConkey agar plates to score for interaction. Red color indicates reporter gene expression from a cAMP-dependent promoter. The spotting order is indicated in the grids on the right. A positive control with the adenylate cyclase fragments fused to the leucine zipper region of the yeast GCN4 protein (zip) is shown (lower right spots).

2017

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Figure 1—figure supplement 2. Abrogation of Shh signaling abolishes vegfaa expression, but does not influence angioblast migration. ; (A, B) Embryos treated with 100 μM Cyclopamine (CyA) from 11 hpf to 18 hpf to block Sonic hedgehog signaling showed no defects in medial angioblast migration. Confocal projections of Tg(fli1a:EGFP)y1 embryos in dorsal views at 18 hpf. c-d) vegfaa expression in the somites is abolished in embryos treated with 100 μM Cyclopamine (CyA) from 11 hpf to 18 hpf.

2015

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Figure 1—figure supplement 1. Multiple sequence alignments of recently amplified TPR repeat units. ; (a) Alignments of the TPR units used for the phylogeny in Figure 1a. Residues different from the most common one in each column are shown in bold face and highlighted in yellow. Abbreviations: Ana: Anabaena sp. 90 (gi: 752818954, accession: WP_041458168.1); Cal: Calothrix sp. 336/3 (gi: 821031795, accession: WP_046815017.1); Cya: Cyanothece sp. PCC 8801 (gi: 501590504, accession: WP_012594639.1); fil: filamentous cyanobacterium ESFC-1 (gi: 740500649, accession: WP_038331513.1); Mic: Microcystis aeruginosa SPC777 (gi: 513477764, accession: EPF24195.1). (b) The corresponding alignment of the DNA sequences for the most recently amplified TPR units, Cal4-Cal18, of which the central repeats, Cal9-Cal16, are fully identical. Synonymous mutations (highlighted in gray) are found at less than 1% of the nucleotides, illustrating the recent time point of the amplification. Non-synonymous mutations (highlighted in yellow) are about 2.5 times as frequent as synonymous ones.

2016

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