Bacterial cyclic diguanylate signaling networks sense temperature

Abstract

Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria.

Fig. 1. Genomics and genetic linkage analysis identify a gene for the thermosensory diguanylate cyclase (tdcA) in P. aeruginosa CF39S.

a P. aeruginosa CF39S displays temperature-dependent rugose colony morphology on agar. b P. aeruginosa CF39S exhibits temperature-dependent biofilm formation in microplates. Datum points represent 16 technical replicates from each of three biological replicates, and lines and bars represent means and standard deviations, respectively. c Liquid chromatography tandem mass spectrometry (LC–MS/MS) measurements of cellular nucleotide pools revealed that P. aeruginosa CF39S displays temperature-dependent increases in cyclic diguanylate (c-di-GMP). Each datum point represents an independent biological replicate, and lines and bars represent means and standard deviations, respectively, for 3–5 independent biological replicates each. d Schematic illustrating the Tn7-like transposon in the P. aeruginosa CF39S chromosome encoding tdcA and its synteny to the E. coli AW1.7 locus of heat resistance (LHR). Annotations for the E. coli AW1.7 genome were taken from Mercer and colleagues. Open reading frames are color coded by putative functions as illustrated in the legend: gray arrows denote genes of unknown function or functions other than those illustrated here; bluish green arrows denote genes for putative proteases or chaperones; yellow arrows denote genes for thioredoxins; orange arrow denotes a diguanylate cyclase; blue arrows denote genes encoding functions for transposition; and reddish purple segments denote inverted repeats. e Acquisition of tdcA confers thermal control of P. aeruginosa biofilm formation in microplates. Datum points represent six technical replicates from each of three biological replicates, and lines and bars represent means and standard deviations, respectively. In b, c, and e, tdcA⁻ and tdcA⁺ strains are represented by sky blue and orange dots, respectively. Strains denoted tdcA⁻ have the tdcA_162ΔG allele. OD_595nm denotes the optical density measured at a wavelength of 595 nm.

Fig. 2. Cyclic diguanylate (c-di-GMP)-dependent thermotransduction.

a Schematic of the heat-activated c-di-GMP pathway that drives biofilm development in P. aeruginosa CF39S. b Western blots for c-di-GMP-regulated Pel and Psl proteins. Measurements were performed in biological triplicate and one representative blot is shown (original, unedited blot images are provided in Supplementary Information). Lanes were loaded with a normalized quantity of total cellular protein. The ΔwspF strain, which has constitutively high c-di-GMP levels^, , was used as a positive control. c Expression of the P_cdrA::gfp reporter. Data represent means and standard deviations of measurements each performed in biological and technical triplicate. The tdcA⁻ and tdcA⁺ strains are represented by sky blue and orange lines, respectively. OD_600nm denotes the optical density measured at a wavelength of 600 nm, and RFU denotes relative fluorescence units. d Thermal control of swimming motility in P. aeruginosa CF39S. Measurements were made in ΔpelF ΔpslD backgrounds to limit interference from extracellular polymers in motility phenotypes. Each datum point represents an independent biological replicate, and lines and bars represent means and standard deviations, respectively, for 4–6 independent biological replicates; tdcA⁻ and tdcA⁺ strains are represented by sky blue and orange dots, respectively. e Biofilm assays indicate that TdcA integrates into the P. aeruginosa c-di-GMP regulatory network, in part, via the c-di-GMP-binding transcription factor, FleQ. Datum points represent 12 technical replicates from each of three biological replicates, and lines and bars represent means and standard deviations, respectively. Temperatures of 25 °C and 37 °C are represented by sky blue and orange bars, respectively. OD_595nm denotes the optical density measured at a wavelength of 595 nm. Significant difference via two-tailed Student’s t-test (*** denotes P < 0.001). Strains denoted tdcA⁻ have the tdcA_162ΔG allele.

Fig. 3. TdcA displays thermosensitive catalytic activity.

a Schematic illustrating the predicted domain organization of TdcA. b Substrate-dependent enzyme kinetics of His₆-NusA-TdcA. Measurements were performed in triplicate and one independent replicate is shown. c GTP is the substrate for His₆-MBP-TdcA. Each bar represents the mean and standard deviation of three independent replicates. ATP, adenosine-5ʹ-triphosphate; GDP, guanosine-5ʹ-diphosphate; GTP, guanosine-5ʹ-triphosphate. d TdcA is an outlier to theory for universal enzymatic rate–temperature dependencies in bacteria. Each datum point represents a Q₁₀ value for an enzyme: Q₁₀ values for WspR and TdcA were calculated in the present work, whereas all other temperature coefficients are derived from published literature values and grouped according to the temperature-dependent activity maximum of the enzyme (psychrophilic, mesophilic, thermophilic). Lines and bars represent means and standard deviations, respectively, for 11–16 literature values in each category. e Temperature-dependent enzyme kinetics of His₆-MBP-TdcA. Measurements were performed in technical triplicate and datum points and lines represent individual replicates. f LC–MS/MS measurements of temperature-dependent c-di-GMP production by recombinant His₆-MBP-TdcA in vitro. Each datum point represents an independent replicate, and lines and bars represent means and standard deviations, respectively.

Fig. 4. Engineered protein chimeras identify a thermosensitive Per-Arnt-SIM (thermoPAS) domain.

a CLUSTALW sequence alignment of WspR (blue) and TdcA (orange) protein sequences. The TdcA thermoPAS domain was fused to the GGDEF domain of WspR via the region of identity at the N-terminus of the GGDEF domains (black), yielding the chimeric synthetic thermosensory protein 1 (Stp1). b LC–MS/MS measurements of c-di-GMP levels in cells expressing WspR, TdcA, and Stp1 from an arabinose-inducible expression cassette (araC-P_BAD). Each datum point represents an independent biological replicate, and lines and bars represent means and standard deviations, respectively, for 6–12 independent biological replicates. Temperatures of 25 °C and 37 °C are represented by sky blue and orange bars, respectively. c Internal fusions of the N-terminal portion of TdcA to LacZ (β-galactosidase) produce an enzyme, thermo-gal, which displays temperature-sensitive catalytic activity when expressed in E. coli LMG194. Bacteria were grown on LB agar containing X-gal and 1% l-(+)-arabinose.