The diguanylate cyclase SadC is a central player in Gac/Rsm-mediated biofilm formation in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen and a threat for immunocompromised and cystic fibrosis patients. It is responsible for acute and chronic infections and can switch between these lifestyles upon taking an informed decision involving complex regulatory networks. The RetS/LadS/Gac/Rsm network and the cyclic-di-GMP (c-di-GMP) signaling pathways are both central to this phenomenon redirecting the P. aeruginosa population toward a biofilm mode of growth, which is associated with chronic infections. While these two pathways were traditionally studied independently from each other, we recently showed that cellular levels of c-di-GMP are increased in the hyperbiofilm retS mutant. Here, we have formally established the link between the two networks by showing that the SadC diguanylate cyclase is central to the Gac/Rsm-associated phenotypes, notably, biofilm formation. Importantly, SadC is involved in the signaling that converges onto the RsmA translational repressor either via RetS/LadS or via HptB/HsbR. Although the level of expression of the sadC gene does not seem to be impacted by the regulatory cascade, the production of the SadC protein is tightly repressed by RsmA. This adds to the growing complexity of the signaling network associated with c-di-GMP in P. aeruginosa. While this organism possesses more than 40 c-di-GMP-related enzymes, it remains unclear how signaling specificity is maintained within the c-di-GMP network. The finding that SadC but no other diguanylate cyclase is related to the formation of biofilm governed by the Gac/Rsm pathway further contributes to understanding of this insulation mechanism.

FIG 1

PA0338 is upregulated in a retS mutant background. (A) Transcript levels of hsiA1 (positive control), pcrV (negative control), and PA0338 measured by qRT-PCR and normalized to those of gyrA. Statistical Student's t-test analysis was based on three replicates, and significant changes are indicated (**, P < 0.001; ***, P < 0.0001). (B) Congo red binding of the indicated strains after 2 days of incubation. (C) Intracellular levels of c-di-GMP measured with the transcriptional PcdrA-gfp reporter. Relative fluorescence units (RFU) are arbitrary fluorescence intensity units corrected for cell density. At least three independent experiments were performed. Statistically significant changes were calculated using the Student t test and are indicated (***, P < 0.0001).

FIG 2

SadC is required for the retS mutant hyperbiofilm phenotype. (A) Quantification of the crystal violet staining of biofilms grown in microtiter plates for 14 h. A photo of the test tubes was taken prior to the addition of ethanol for quantification purposes. (B) Crystal violet staining of biofilms grown in test tubes for 6 h. The empty vector pBBR1MCS-4 (pvector) and pBBR1MCS-4-sadC (psadC) were conjugated into PAK ΔretS or PAK ΔretS ΔsadC. (C) Results of a swimming motility assay performed in 0.3% LB agar plates. (D) Intracellular levels of c-di-GMP measured with the transcriptional PcdrA-gfp reporter. Relative fluorescence units (RFU) are arbitrary fluorescence intensity units corrected for cell density. At least three independent experiments were performed. (A, C, and D) Statistically significant changes were calculated using the Student t test and are indicated (**, P < 0.001; ***, P < 0.0001).

FIG 3

SadC is required for the hptB mutant hyperbiofilm phenotype. (A) Congo red binding of the indicated strains after 2 days of incubation. (B) Intracellular levels of c-di-GMP measured with the transcriptional PcdrA-gfp reporter. Relative fluorescence units (RFU) are arbitrary fluorescence intensity units corrected for cell density. At least three independent experiments were performed. Statistically significant changes are indicated (***, P < 0.0001, Student's t test). (C) PAK and PAK ΔsadC were conjugated with pBBR1-MCS4-ladS (pladS) or pBBR1-MCS4-hsbR (phsbR). Congo red binding is from day 2 of incubation.

FIG 4

Deletion of sadC abrogates the hyperbiofilm and high c-di-GMP phenotypes of an rsmA mutant. (A) Congo red binding phenotypes visualized on day 2 of incubation. (B) Intracellular levels of c-di-GMP measured with the transcriptional PcdrA-gfp reporter. Relative fluorescence units (RFU) are arbitrary fluorescence intensity units corrected for cell density. Statistically significant changes are indicated (***, P < 0.0001, Student's t test).

FIG 5

SadC production is controlled by RsmA. (A) Quantification of the crystal violet staining of biofilms grown in microtiter plates for 6 h. Statistically significant changes are indicated (**, P < 0.001, Student's t test). (B) Immunoblots with M2 antiserum showing the levels of DGC-Flag variants in whole-cell lysates. The PAO1 DGC-Flag strains indicated on the left contain different plasmids: pvector (empty vector), prsmY (IPTG-inducible rsmY), and prsmA (IPTG-inducible rsmA). Cells were harvested after 1 h induction with 1 mM IPTG. Shown here is a representative immunoblot from at least three independent experiments.

FIG 6

SadB is central to the RetS- and HptB-dependent signaling pathways. Crystal violet staining of biofilms grown under shaking conditions for 8 h (A) and 18 h (B) is shown.

FIG 7

The SadC and the Gac/Rsm pathways are interlinked. The Gac/Rsm system is a complex signaling cascade that regulates several biological functions, including biofilm formation. The proteins belonging to this cascade that negatively impact the formation of biofilms are indicated in red. All the proteins of this cascade indicated in green have a positive impact on biofilm formation. The hyperbiofilm phenotype induced by the deletion of the retS, hptB, or rsmA gene is dependent on an intact sadC gene product to produce c-di-GMP (represented by stars). SadC levels are under the control of RsmA, but the exact mechanism by which it occurs is still unknown. Downstream of SadC is SadB, whose function remains obscure.