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Chapter 11 : Role of Cyclic Di-GMP in Pseudomonas aeruginosa Biofilm Development
Author:
Caroline S. Harwood1
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Affiliations:
1: Department of Microbiology, The University of Washington, Seattle, WA 98195
Content Type: Monograph
Publication Year:
2010
Category: Microbial Genetics and Molecular Biology
Book DOI:
10.1128/9781555816667
Chapter DOI:
10.1128/9781555816667.ch11
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Abstract:
This chapter reviews cyclic di-GMP (c-di-GMP) metabolism and activity in Pseudomonas aeruginosa with a focus on how c-di-GMP modulates the expression and activities of genes and proteins required for biofilm formation. P. aeruginosa is an opportunistic human pathogen that persistently colonizes the lungs of people with cystic fibrosis (CF) and eventually kills them. The domain architectures of many of the proteins are discussed in the chapter. In P. aeruginosa, as in other bacteria, a high percentage of GGDEF and EAL domains are present as modules in proteins that have N-terminal domains implicated in ligand binding. The chapter describes three different assays used to visualize different but overlapping aspects of biofilms and then how they have been used to assess the effects of high and low intracellular c-di-GMP. The first and most commonly used assay is often referred to as the microtiter dish attachment assay. A second assay, often referred to as a continuous flow assay, involves growing biofilms on a surface of a small chamber through which growth medium is continuously flowed. A third measure of biofilm formation is colony morphology and dye binding. A systematic analysis of a P. aeruginosa transposon mutant library by the Lory laboratory and other more directed studies carried out by other investigators have identified just 8 of the 40 diguanylate cyclase (DGC) and phosphodiesterase (PDE) genes present in strain PAO1 as influencing biofilm formation. The chapter describes each of these genes and studies that have addressed their functions.
Citation:
Harwood C.
2010.
Role of Cyclic Di-GMP in Pseudomonas aeruginosa Biofilm Development,
p 156-172.
In
Wolfe A, Visick K (ed),
The Second Messenger Cyclic Di-GMP.
ASM Press, Washington, DC.
doi: 10.1128/9781555816667.ch11
Key Concept Ranking
- Gene Expression and Regulation
- 0.5285651
- Bacterial Proteins
- 0.48015127
- Urinary Tract Infections
- 0.4236446
0.5285651
Figures
Role of Cyclic Di-GMP in Pseudomonas aeruginosa Biofilm Development
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Citation:
Harwood C.
2010.
Role of Cyclic Di-GMP in Pseudomonas aeruginosa Biofilm Development,
p 156-172.
In
Wolfe A, Visick K (ed),
The Second Messenger Cyclic Di-GMP.
ASM Press, Washington, DC.
doi: 10.1128/9781555816667.ch11
/content/10.1128/9781555816667.ch11.ch11fig01
Figure 1.
Domain architectures of selected DGC and PDE proteins from P. aeruginosa.
10.1128/9781555816667/f0158-01_thmb.gif
10.1128/9781555816667/f0158-01.gif
Figure 1.
Domain architectures of selected DGC and PDE proteins from P. aeruginosa.
Citation:
Harwood C.
2010.
Role of Cyclic Di-GMP in Pseudomonas aeruginosa Biofilm Development,
p 156-172.
In
Wolfe A, Visick K (ed),
The Second Messenger Cyclic Di-GMP.
ASM Press, Washington, DC.
doi: 10.1128/9781555816667.ch11
/content/10.1128/9781555816667.ch11.ch11fig02
Figure 2.
Loss of EPS production reverses the colony and attachment phenotypes of a wspF mutant. EPS is encoded by psl and pel genes in P. aeruginosa. (Top) Colony morphologies. (Bottom) Attachment assays were carried out in microtiter dish wells (J. W. Hickman and C. S. Harwood, unpublished data). OD, optical density.
10.1128/9781555816667/f0159-01_thmb.gif
10.1128/9781555816667/f0159-01.gif
Figure 2.
Loss of EPS production reverses the colony and attachment phenotypes of a wspF mutant. EPS is encoded by psl and pel genes in P. aeruginosa. (Top) Colony morphologies. (Bottom) Attachment assays were carried out in microtiter dish wells (J. W. Hickman and C. S. Harwood, unpublished data). OD, optical density.
Citation:
Harwood C.
2010.
Role of Cyclic Di-GMP in Pseudomonas aeruginosa Biofilm Development,
p 156-172.
In
Wolfe A, Visick K (ed),
The Second Messenger Cyclic Di-GMP.
ASM Press, Washington, DC.
doi: 10.1128/9781555816667.ch11
/content/10.1128/9781555816667.ch11.ch11fig03
Figure 3.
Characteristics of the Wsp signal transduction system. (A) Organization of the genes encoding the Wsp system. (B) The Wsp proteins are predicted to be a membrane-bound methyl-accepting chemotaxis protein (WspA), a CheR-like methyltransferase (WspC), a CheB-like methylesterase (WspF), and two CheW homologues (WspB and WspD) that are predicted to serve as adapters between WspA and a hybrid histidine kinase response regulator (WspE). The response regulator protein, WspR, has a GGDEF domain and catalyzes the synthesis of c-di-GMP when phosphorylated. As described in the text, a wspF mutation is predicted to lock the Wsp system into a configuration where WspR is constantly phosphorylated and thus constantly producing c-di-GMP. (C) Physical organization of homologous Che proteins. (D) Colony morphologies of P. aeruginosa PAO1 wild-type and wsp deletion strains. wspF mutants have high levels of intracellular c-di-GMP relative to wild-type cells. Reprinted from Güvener and Harwood (
23
) with permission.
10.1128/9781555816667/f0162-01_thmb.gif
10.1128/9781555816667/f0162-01.gif
Figure 3.
Characteristics of the Wsp signal transduction system. (A) Organization of the genes encoding the Wsp system. (B) The Wsp proteins are predicted to be a membrane-bound methyl-accepting chemotaxis protein (WspA), a CheR-like methyltransferase (WspC), a CheB-like methylesterase (WspF), and two CheW homologues (WspB and WspD) that are predicted to serve as adapters between WspA and a hybrid histidine kinase response regulator (WspE). The response regulator protein, WspR, has a GGDEF domain and catalyzes the synthesis of c-di-GMP when phosphorylated. As described in the text, a wspF mutation is predicted to lock the Wsp system into a configuration where WspR is constantly phosphorylated and thus constantly producing c-di-GMP. (C) Physical organization of homologous Che proteins. (D) Colony morphologies of P. aeruginosa PAO1 wild-type and wsp deletion strains. wspF mutants have high levels of intracellular c-di-GMP relative to wild-type cells. Reprinted from Güvener and Harwood ( 23 ) with permission.
Citation:
Harwood C.
2010.
Role of Cyclic Di-GMP in Pseudomonas aeruginosa Biofilm Development,
p 156-172.
In
Wolfe A, Visick K (ed),
The Second Messenger Cyclic Di-GMP.
ASM Press, Washington, DC.
doi: 10.1128/9781555816667.ch11
/content/10.1128/9781555816667.ch11.ch11fig04
Figure 4.
Phosphorylation is required for WspR cluster formation. The localization of WspR-YFP in ΔwspF wspR-yfp or ΔwspF ΔwspA wspR-yfp mutant cells are shown in the top panels, and the localization of two WspR-YFP mutant proteins in ΔwspF cells are shown in the bottom panels. Phase-contrast images of cells are shown on the left. Bar, 1 μm. Reprinted from Güvener and Harwood (
23
) with permission.
10.1128/9781555816667/f0163-01_thmb.gif
10.1128/9781555816667/f0163-01.gif
Figure 4.
Phosphorylation is required for WspR cluster formation. The localization of WspR-YFP in ΔwspF wspR-yfp or ΔwspF ΔwspA wspR-yfp mutant cells are shown in the top panels, and the localization of two WspR-YFP mutant proteins in ΔwspF cells are shown in the bottom panels. Phase-contrast images of cells are shown on the left. Bar, 1 μm. Reprinted from Güvener and Harwood ( 23 ) with permission.
Citation:
Harwood C.
2010.
Role of Cyclic Di-GMP in Pseudomonas aeruginosa Biofilm Development,
p 156-172.
In
Wolfe A, Visick K (ed),
The Second Messenger Cyclic Di-GMP.
ASM Press, Washington, DC.
doi: 10.1128/9781555816667.ch11
/content/10.1128/9781555816667.ch11.ch11fig05
Figure 5.
Surface growth stimulates Wsp signal transduction as assayed by cluster formation of WspR-YFP. WspR-YFP localization in wild-type cells grown in liquid or on agar is shown. The phase-contrast (left) and fluorescence (right) images of cells expressing wspR-yfp are shown. Bar, 1 µm. Reprinted from Güvener and Harwood (
23
) with permission.
10.1128/9781555816667/f0163-02_thmb.gif
10.1128/9781555816667/f0163-02.gif
Figure 5.
Surface growth stimulates Wsp signal transduction as assayed by cluster formation of WspR-YFP. WspR-YFP localization in wild-type cells grown in liquid or on agar is shown. The phase-contrast (left) and fluorescence (right) images of cells expressing wspR-yfp are shown. Bar, 1 µm. Reprinted from Güvener and Harwood ( 23 ) with permission.
Citation:
Harwood C.
2010.
Role of Cyclic Di-GMP in Pseudomonas aeruginosa Biofilm Development,
p 156-172.
In
Wolfe A, Visick K (ed),
The Second Messenger Cyclic Di-GMP.
ASM Press, Washington, DC.
doi: 10.1128/9781555816667.ch11
/content/10.1128/9781555816667.ch11.ch11fig06
Figure 6.
Structure of WspR in its active conformation. The site of c-di-GMP binding that leads to an inhibited conformation is also shown. From De et al. (
14
).
10.1128/9781555816667/f0164-01_thmb.gif
10.1128/9781555816667/f0164-01.gif
Figure 6.
Structure of WspR in its active conformation. The site of c-di-GMP binding that leads to an inhibited conformation is also shown. From De et al. ( 14 ).
Citation:
Harwood C.
2010.
Role of Cyclic Di-GMP in Pseudomonas aeruginosa Biofilm Development,
p 156-172.
In
Wolfe A, Visick K (ed),
The Second Messenger Cyclic Di-GMP.
ASM Press, Washington, DC.
doi: 10.1128/9781555816667.ch11
/content/10.1128/9781555816667.ch11.ch11fig07
Figure 7.
Predicted organization of the alginate biosynthesis complex. OM, outer membrane; PG, peptidoglycan; CM, cell membrane. Adapted from Ramsey and Wozniak (
63
) by J. Weadge, J. Whitney, C.-K. Keiski, A. N. Neculai, and P. L. Howell.
10.1128/9781555816667/f0164-02_thmb.gif
10.1128/9781555816667/f0164-02.gif
Figure 7.
Predicted organization of the alginate biosynthesis complex. OM, outer membrane; PG, peptidoglycan; CM, cell membrane. Adapted from Ramsey and Wozniak ( 63 ) by J. Weadge, J. Whitney, C.-K. Keiski, A. N. Neculai, and P. L. Howell.
Citation:
Harwood C.
2010.
Role of Cyclic Di-GMP in Pseudomonas aeruginosa Biofilm Development,
p 156-172.
In
Wolfe A, Visick K (ed),
The Second Messenger Cyclic Di-GMP.
ASM Press, Washington, DC.
doi: 10.1128/9781555816667.ch11
/content/10.1128/9781555816667.ch11.ch11fig08
Figure 8.
Predicted organization of the Pel biosynthesis complex. OM, outer membrane; PG, peptidoglycan; CM, cell membrane. Courtesy of J. Weadge, J. Whitney, C.-K. Keiski, A. N. Neculai, and P. L. Howell.
10.1128/9781555816667/f0166-01_thmb.gif
10.1128/9781555816667/f0166-01.gif
Figure 8.
Predicted organization of the Pel biosynthesis complex. OM, outer membrane; PG, peptidoglycan; CM, cell membrane. Courtesy of J. Weadge, J. Whitney, C.-K. Keiski, A. N. Neculai, and P. L. Howell.
Citation:
Harwood C.
2010.
Role of Cyclic Di-GMP in Pseudomonas aeruginosa Biofilm Development,
p 156-172.
In
Wolfe A, Visick K (ed),
The Second Messenger Cyclic Di-GMP.
ASM Press, Washington, DC.
doi: 10.1128/9781555816667.ch11
/content/10.1128/9781555816667.ch11.ch11fig09
Figure 9.
Model for the regulation of gene expression by FleQ, FleN, and c-di-GMP. (A) FleQ in the absence of FleN or c-di-GMP maximally represses pel transcription. (B) Situation in wild-type cells. FleQ binding at the pelA promoter is reduced by FleN and ATP/ADP, resulting in less pel repression than the situation in panel A. (C) c-di-GMP binds to FleQ to cause it to dissociate from DNA, thereby causing derepression of transcription from the pel promoter. Reprinted from Hickman and Harwood (
29
) with permission.
10.1128/9781555816667/f0167-01_thmb.gif
10.1128/9781555816667/f0167-01.gif
Figure 9.
Model for the regulation of gene expression by FleQ, FleN, and c-di-GMP. (A) FleQ in the absence of FleN or c-di-GMP maximally represses pel transcription. (B) Situation in wild-type cells. FleQ binding at the pelA promoter is reduced by FleN and ATP/ADP, resulting in less pel repression than the situation in panel A. (C) c-di-GMP binds to FleQ to cause it to dissociate from DNA, thereby causing derepression of transcription from the pel promoter. Reprinted from Hickman and Harwood ( 29 ) with permission.
Citation:
Harwood C.
2010.
Role of Cyclic Di-GMP in Pseudomonas aeruginosa Biofilm Development,
p 156-172.
In
Wolfe A, Visick K (ed),
The Second Messenger Cyclic Di-GMP.
ASM Press, Washington, DC.
doi: 10.1128/9781555816667.ch11
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