Bacterial Cyclic Diguanylate Signaling Networks Sense Temperature

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

Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus

International audienceThe WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cis-tronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcel-lular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene cluster, walRKJ

The Cyclic AMP-Vfr Signaling Pathway in <i>Pseudomonas aeruginosa</i> Is Inhibited by Cyclic Di-GMP

The opportunistic human pathogen Pseudomonas aeruginosa expresses numerous acute virulence factors in the initial phase of infection, and during long-term colonization it undergoes adaptations that optimize survival in the human host. Adaptive changes that often occur during chronic infection give rise to rugose small colony variants (RSCVs), which are hyper-biofilm-forming mutants that commonly possess mutations that increase production of the biofilm-promoting secondary messenger cyclic di-GMP (c-di-GMP). We show that RSCVs display a decreased production of acute virulence factors as a direct result of elevated c-di-GMP content. Overproduction of c-di-GMP causes a decrease in the transcription of virulence factor genes that are regulated by the global virulence regulator Vfr. The low level of Vfr-dependent transcription is caused by a low level of its coactivator, cyclic AMP (cAMP), which is decreased in response to a high level of c-di-GMP. Mutations that cause reversion of the RSCV phenotype concomitantly reactivate Vfr-cAMP signaling. Attempts to uncover the mechanism underlying the observed c-di-GMP-mediated lowering of cAMP content provided evidence that it is not caused by inhibition of adenylate cyclase production or activity and that it is not caused by activation of cAMP phosphodiesterase activity. In addition to the studies of the RSCVs, we present evidence that the deeper layers of wild-type P. aeruginosa biofilms have high c-di-GMP levels and low cAMP levels.Published versio

WalH and WalI are membrane-anchored extracellular proteins.

<p>(A) <i>E</i>. <i>coli</i> DH5α cells producing ‘PhoA’-‘LacZ’ fusion proteins (WalH_1-40-PhoA_21-471-LacZ_4-60, WalI_1-40-PhoA_21-471-LacZ_4-60, or WalJ_1-40-PhoA_21-471-LacZ_4-60) were plated on indicator medium with two chromogenic substrates, Red-Gal (for β-galactosidase activity) and X-Pho (for phosphatase activity). Blue coloring of the colonies (high phosphatase activity) indicates a membrane or extracellular localization of the fusion point. Red coloring of the colonies (high β-galactosidase activity) indicates cytosolic localization of the fusion point. (B) Schematic representation of Wal protein localization and topology with respect to the cell membrane. In the case of WalK and WalR, topology and localization were deduced from primary sequence analysis using the Phobius Hidden Markov Model (indicated by stars).</p

Phylogenetic relationships within the Bacilli class and inferred losses of <i>wal</i> genes.

<p>(A) Maximum likelihood phylogeny of Bacilli based on a concatenation of 47 ribosomal proteins comprising 5,945 amino acid positions. The conservation of <i>wal</i> genes in each genome is indicated by circles: <i>walR</i> (red), <i>walK</i> (green), <i>walH</i> (blue), <i>walI</i> (yellow), <i>walJ</i> (purple). Red crosses indicate the loss of <i>walH</i> and <i>walI</i> in Streptococcaceae and of <i>walJ</i> in <i>Leuconostoc</i>. (B) Maximum likelihood phylogeny based on a concatenation of WalR, WalK, WalH, WalI and WalJ protein sequences comprising 1,102 amino acid positions. For both analyses, values at nodes represent bootstrap proportions calculated on 1,000 resamplings of the original data set. For clarity, only the values corresponding to monophyly of families and their evolutionary relationships are shown. The scale bar represents the average number of substitutions per site. For details on analyses, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151449#sec002" target="_blank">Materials and Methods</a>.</p

Biofilm formation is decreased in the Δ<i>walH</i>, Δ<i>walI</i> and Δ<i>walHI</i> mutants.

<p>Biofilm assays were performed in microtiter plates after growth at 37°C for 24 h. Adherent biomass was quantified, normalized to the OD_{600 nm} of each cell culture and represented as n-fold variation compared to the parental strain. Dark grey bars indicate biomass levels in the parental and mutant strains and light grey bars correspond to values in the complemented strains carrying pMK4-Pprot<i>walHI</i>. Experiments were carried out in quadruplicate and standard deviations are indicated. ** <i>P</i><0.01 as determined using Student’s <i>t</i>-test.</p

Triton-induced autolysis is decreased in the absence of WalH.

<p>Bacteria were grown in TSB at 37°C with shaking until OD_{600 nm} ≈ 1, pelleted (10 min; 5,400 x <i>g</i>), resuspended in phosphate buffered saline (PBS) with Triton X-100 (0.1%), and incubated at 37°C with shaking. Lysis was determined as the decrease in OD_{600 nm} over time and indicated as a percentage of the initial OD (measured OD_{600 nm} / initial OD_{600 nm}). Results are shown as the mean and standard deviation of three independent experiments. Strains: HG001 (■); ST1397 Δ<i>walH</i> (○); ST1410 Δ<i>walHI</i> (△); ST1415 Δ<i>walH</i> pMK4Pprot-<i>walHI</i> (●); ST1417 Δ<i>walHI</i> pMK4Pprot-<i>walHI</i> (▲).</p