Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 10, 1847
eCollection

AmrZ Regulates Swarming Motility Through Cyclic di-GMP-Dependent Motility Inhibition and Controlling Pel Polysaccharide Production in Pseudomonas aeruginosa PA14

Affiliations

AmrZ Regulates Swarming Motility Through Cyclic di-GMP-Dependent Motility Inhibition and Controlling Pel Polysaccharide Production in Pseudomonas aeruginosa PA14

Lingli Hou et al. Front Microbiol.

Abstract

Swarming is a surface-associated motile behavior that plays an important role in the rapid spread, colonization, and subsequent establishment of bacterial communities. In Pseudomonas aeruginosa, swarming is dependent upon a functional flagella and aided by the production of biosurfactants. AmrZ, a conserved transcription factor across pseudomonads, has been shown to be a global regulator of multiple genes important for virulence and ecological fitness. In this study, we expand this concept of global control to swarming motility by showing that deletion of amrZ results in a severe defect in swarming, while multicopy expression of this gene stimulates swarming of P. aeruginosa. Mechanistic studies showed that the swarming defect of an amrZ mutant does not involve changes of biosurfactant production but is associated with flagellar malfunction. The ∆amrZ mutant exhibits increased levels of the second messenger cyclic di-GMP (c-di-GMP) compared to the wild-type strain, under swarming conditions. We found that the diguanylate cyclase GcbA was the main contributor to the increased accumulation of c-di-GMP observed in the ∆amrZ mutant and was a strong inhibitor of flagellar-dependent motility. Our results revealed that the GcbA-dependent inhibition of motility required the presence of two c-di-GMP receptors containing a PilZ domain: FlgZ and PA14_56180. Furthermore, the ∆amrZ mutant exhibits enhanced production of Pel polysaccharide. Epistasis analysis revealed that GcbA and the Pel polysaccharide act independently to limit swarming in ΔamrZ. Our results support a role for AmrZ in controlling swarming motility, yet another social behavior besides biofilm formation that is crucial for the ability of P. aeruginosa to colonize a variety of surfaces. The central role of AmrZ in controlling these behaviors makes it a good target for the development of treatments directed to combat P. aeruginosa infections.

Keywords: AmrZ; FlgZ; GcbA; PA14_56180; Pseudomonas aeruginosa; cyclic di-GMP; exopolysaccharide; swarming motility.

Figures

Figure 1
Figure 1
AmrZ plays a role in the regulation of swarming motility in P. aeruginosa PA14. (A) Swarming motility and average percentage of the surface coverage of WT PA14, the ΔamrZ mutant, and the single-copy complemented strain of the ΔamrZ mutant carrying an amrZ gene inserted into chromosome by mini-Tn7T. Error bars represent the standard deviations. **p < 0.01 compared with WT or complemented strains by Student’s t-test. (B) Swarm analysis and quantification of percent coverage of swarm plates for the WT strain carrying vector control (PA14/pUCP) or a multicopy amrZ-containing plasmid (PA14/pUCP-amrZ). (C) RT-qPCR analysis of amrZ mRNA levels in WT PA14 from actively swarming tendril tips, cells localized in the center of swarming colonies, free-swimming planktonic cells, and cells grown as biofilm at 6 h (initial attachment) and 24 h (developed biofilm). The relative gene expression was reported relative to the levels in swarm center cells. Significance was determined by Students’ t-test (**p < 0.01).
Figure 2
Figure 2
The swarming defect of the amrZ mutant is independent of changes of biosurfactant production or flagellar formation but is associated with flagellar malfunction. (A) Methylene blue-rhamnolipid plate assay. The presence of surfactant is indicated by the formation of a dark ring surrounding a colony and the non-rhamnolipid-producing rhlA mutant served as a negative control. (B) Drop collapse analysis of PA14, ∆amrZ, and ∆rhlA mutants to detect the presence of the rhamnolipid precursor HAA. Samples were diluted in dH2O, spotted on the lid of a 96-well plate, and assayed for bead formation. The dilution factors are shown at the top. U, undiluted. (C) Morphology of PA14 and amrZ mutant cells by TEM of bacteria taken directly from swarm plates. Bar, 2 μm. (D) Swimming motility assay of the indicated strains. (E) Flagellar reversal rates under low (3% Ficoll) and high-viscosity (15% Ficoll) conditions, representing swimming and swarming conditions, respectively, were measured as changes in direction of movement of cells. Rates are expressed as number of reversals per cell per minute. Error bars represent the standard deviations. *p < 0.05; **p < 0.01 compared with WT by Student’s t-test.
Figure 3
Figure 3
c-di-GMP contributes to the swarming defects of the amrZ mutant. (A) Measurements of c-di-GMP by LC-MS/MS for the indicated strains grown on swarm plates. Data are expressed as pmol of c-di-GMP/mg of dry weight of the cell pellets from which the nucleotides were extracted. (B) Representative swarming images of the WT or amrZ mutant carrying either the empty vector or pUCP-2133 for overproducing the PA2133 phosphodiesterase as indicated. (C) Percent coverage of swarm plates for the strains shown in (B). Statistical analysis is based on three replicates and significance was determined with Student’s t-test (*p < 0.05; **p < 0.01).
Figure 4
Figure 4
GcbA is required for increased c-di-GMP accumulation in ΔamrZ and partially responsible for its swarming phenotype. (A) Relative mRNA levels of gcbA in indicated strains under swarming conditions as determined by real-time PCR. (B) Representative images of swarming motilities of indicated strains. (C) Percent coverage of swarm plates by the respective swarms. (D) Quantification of c-di-GMP levels of the indicated strains grown on swarm plates. **p < 0.01 as determined by Student’s t-test.
Figure 5
Figure 5
FlgZ and PA14_56180 are required for GcbA c-di-GMP-mediated swarming repression. (A) Swarming phenotype and quantification of the percentages of plate surface coverage of the indicated strains carrying either the empty vector (pUCP) or the GcbA overproducing plasmid pUCP-gcbA. **p < 0.01 (Student’s t-test, compared to PA14/pUCP-gcbA). (B) Representative swarming plates (upper row) and average percentage of the plate coverage (middle row) by ΔflgZ/pUCP-gcbA carrying the chromosomally encoded C-terminally FLAG-tagged WT FlgZ or FlgZ (R140A) under the control of the PBAD promoter at the attTn7 site. Significance was determined by Student’s t-test (**p < 0.01) relative to the vector control. Lower row: Western analysis of the FLAG-tagged FlgZ derivatives in indicated strains. Samples from equivalent number of bacterial cells were loaded onto SDS-PAGE gels and probed with anti-FLAG antibody. (C) Swarm analysis (upper row), quantification of percent coverage of swarm plates (middle row), and protein expression levels of FLAG-tagged derivatives of PA14_56180 (lower row) in ΔPA14_56180/pUCP-gcbA harboring the chromosomally encoded C-terminally FLAG-tagged WT PA14_56180 or PA14_56180 (R5A) as under panel (B).
Figure 6
Figure 6
FlgZ and PA14_56180 contribute to swarming motility repression of the amrZ mutant. (A) Representative swarm plates of the indicated strains. (B) Percentage of the plate surface occupied by the respective swarms shown in (A). Significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test (*p < 0.05; **p < 0.01).
Figure 7
Figure 7
The pel-derived polysaccharide collaborates with GcbA to contribute to the swarming defect of ΔamrZ. (A) CR binding (upper row) and representative swarm plates (lower row) of the indicated strains. (B) Quantification of the extent of swarming for the strains indicated in (A). (C) CR-binding phenotypes of the indicated strains. The name of the corresponding strain is indicated on the left. In PA14 and ΔgcbA additional amrZ mutation was introduced as indicated at the top of the second column. Significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test (**p < 0.01).
Figure 8
Figure 8
Model for AmrZ-mediated swarming motility control. AmrZ affects swarming by negatively regulating the production of Pel polysaccharide and a GcbA-dependent c-di-GMP signaling. The Pel polysaccharide inhibits swarming motility by promoting the transition from reversible to irreversible attachment (Caiazza et al., 2007). The GcbA c-di-GMP signal can be further sensed by two PilZ-containing proteins, FlgZ and PA14_56180. Binding of c-di-GMP to FlgZ induces its interaction with the MotC stator and impairs flagellar function probably by preventing the engagement of MotCD with the rotor (Baker et al., 2016). PA14_56180 regulates swarming in a c-di-GMP-dependent manner, but the downstream target(s) of PA14_56180 remains unknown. The solid lines represent direct regulations, and the dashed lines indicate probable indirect regulations.

Similar articles

See all similar articles

Cited by 1 article

References

    1. Allsopp L. P., Wood T. E., Howard S. A., Maggiorelli F., Nolan L. M., Wettstadt S., et al. . (2017). RsmA and AmrZ orchestrate the assembly of all three type VI secretion systems in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 114, 7707–7712. 10.1073/pnas.1700286114, PMID: - DOI - PMC - PubMed
    1. Amikam D., Galperin M. Y. (2006). PilZ domain is part of the bacterial c-di-GMP binding protein. Bioinformatics 22, 3–6. 10.1093/bioinformatics/bti739, PMID: - DOI - PubMed
    1. Andrews G. P., Maurelli A. T. (1992). mxiA of Shigella flexneri 2a, which facilitates export of invasion plasmid antigens, encodes a homolog of the low-calcium-response protein, LcrD, of Yersinia pestis. Infect. Immun. 60, 3287–3295. 10.1007/BF01989988, PMID: - DOI - PMC - PubMed
    1. Baker A. E., Diepold A., Kuchma S. L., Scott J. E., Ha D. G., Orazi G., et al. . (2016). PilZ domain protein FlgZ mediates cyclic di-GMP-dependent swarming motility control in Pseudomonas aeruginosa. J. Bacteriol. 198, 1837–1846. 10.1128/JB.00196-16, PMID: - DOI - PMC - PubMed
    1. Baker A. E., Webster S. S., Diepold A., Kuchma S. L., Bordeleau E., Armitage J. P., et al. . (2019). Flagellar stators stimulate c-di-GMP production by Pseudomonas aeruginosa. J. Bacteriol. [Preprint]. 10.1128/JB.00741-18, PMID: - DOI - PMC - PubMed

LinkOut - more resources

Feedback