The second messenger bis‐(3′‐5′)‐cyclic‐GMP and its PilZ domain‐containing receptor Alg44 are required for alginate biosynthesis in <i>Pseudomonas aeruginosa</i>

Merighi, Massimo; Lee, Vincent T.; Hyodo, Mamoru; Hayakawa, Yoshihiro; Lory, Stephen

doi:10.1111/j.1365-2958.2007.05817.x

Cited by 315 publications

(367 citation statements)

References 57 publications

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“…Gel loading was normalized per number of cells. For total membrane purification, bacteria were grown as above, lysed, and total membrane purified as described previously (Merighi et al 2007). …”

Section: Thin-layer Chromatographymentioning

confidence: 99%

Direct interaction between sensor kinase proteins mediates acute and chronic disease phenotypes in a bacterial pathogen

Goodman¹,

Merighi²,

Hyodo³

et al. 2009

Genes Dev.

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271

297

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The genome of the opportunistic pathogen Pseudomonas aeruginosa encodes over 60 two-component sensor kinases and uses several (including RetS and GacS) to reciprocally regulate the production of virulence factors involved in the development of acute or chronic infections. We demonstrate that RetS modulates the phosphorylation state of GacS by a direct and specific interaction between these two membrane-bound sensors. The RetS-GacS interaction can be observed in vitro, in heterologous systems in vivo, and in P. aeruginosa. This function does not require the predicted RetS phosphorelay residues and provides a mechanism for integrating multiple signals without cross-phosphorylation from sensors to noncognate response regulators. These results suggest that multiple two-component systems found in a single bacterium can form multisensor signaling networks while maintaining specific phosphorelay pathways that remain insulated from detrimental cross-talk. Two-component system (TCS) signaling pathways are a major signaling mechanism in bacteria and archaea, and are also found in simple eukaryota and higher plants (Wolanin et al. 2002). These diverse organisms capitalize on TCS pathways to monitor critical external and internal stimuli (including levels of nutrients, concentration of ions and gases, temperature, redox states, and cell density) and translate these signals into adaptive responses. Classical TCS pathways share a conserved core architecture: a homodimerizing histidine kinase protein domain (the ''sensor'') and a cognate receiver domain (the ''response regulator''), coupled mechanistically through a histidine-aspartic acid phosphorelay (Stock et al. 2000). Most cognate sensor response regulator pairs are also linked genetically, encoded by adjacent loci in the chromosome (Alm et al. 2006). Although a single bacterial species can encode up to hundreds of genes specifying TCS pathways, it appears that these systems are insulated against detrimental cross-phosphorylation between sensors and noncognate response regulators (Bijlsma and Groisman 2003;Baker and Stock 2007;Laub and Goulian 2007). The identification of multistep phosphorelays (with intermediary proteins between sensor and regulator) and branched pathways (phosphotransfer between one sensor and multiple response regulators and vice versa) (Laub and Goulian 2007) suggests that TCS pathways have the capacity to form sensitive and complex signaling networks.In contrast to microorganisms with restricted habitats, the genomes of bacteria capable of occupying a number of diverse environments typically contain a disproportionately large number of genes encoding signal transduction and regulatory systems, including TCSs that allow them to sense and respond to a wide range of environmental signals. For opportunistic bacterial pathogens, a number of these systems regulate the expression of genes necessary for transitioning from the environmental reservoir to the host, overcoming innate defense mechanisms and initiating the disease process. The bacterial pathogen ...

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Section: Thin-layer Chromatographymentioning

confidence: 99%

Direct interaction between sensor kinase proteins mediates acute and chronic disease phenotypes in a bacterial pathogen

Goodman¹,

Merighi²,

Hyodo³

et al. 2009

Genes Dev.

Self Cite

271

297

View full text Add to dashboard Cite

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“…Several binding devices for c-di-GMP, apart from c-di-GMP metabolic enzymes, have been identified recently. These binding devices are the proteins that contain the PilZ domain (16)(17)(18)(19)(20)(21)(22), the PelD protein (23), which contains the RxxD motif, the c-di-GMPresponsive transcriptional regulator FleQ (24), and a first class of widely distributed riboswitches (25).…”

mentioning

confidence: 99%

Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella

Solano

García

Latasa

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

116

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Bacteria have developed an exclusive signal transduction system involving multiple diguanylate cyclase and phosphodiesterase domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) that modulate the levels of the same diffusible molecule, 3 -5 -cyclic diguanylic acid (c-di-GMP), to transmit signals and obtain specific cellular responses. Current knowledge about c-di-GMP signaling has been inferred mainly from the analysis of recombinant bacteria that either lack or overproduce individual members of the pathway, without addressing potential compensatory effects or interferences between them. Here, we dissected c-di-GMP signaling by constructing a Salmonella strain lacking all GGDEF-domain proteins and then producing derivatives, each restoring 1 protein. Our analysis showed that most GGDEF proteins are constitutively expressed and that their expression levels are not interdependent. Complete deletion of genes encoding GGDEFdomain proteins abrogated virulence, motility, long-term survival, and cellulose and fimbriae synthesis. Separate restoration revealed that 4 proteins from Salmonella and 1 from Yersinia pestis exclusively restored cellulose synthesis in a c-di-GMP-dependent manner, indicating that c-di-GMP produced by different GGDEF proteins can activate the same target. However, the restored strain containing the STM4551-encoding gene recovered all other phenotypes by means of gene expression modulation independently of c-di-GMP. Specifically, fimbriae synthesis and virulence were recovered through regulation of csgD and the plasmid-encoded spvAB mRNA levels, respectively. This study provides evidence that the regulation of the GGDEF-domain proteins network occurs at 2 levels: a level that strictly requires c-di-GMP to control enzymatic activities directly, restricted to cellulose synthesis in our experimental conditions, and another that involves gene regulation for which c-di-GMP synthesis can be dispensable.biofilm formation ͉ Salmonella virulence ͉ signal transduction system cellulose ͉ STM4551

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“…PilZ domains function as receptors for c-di-GMP, which can act as a cofactor to regulate the activity of the protein (Romling et al, 2005). Alg44's PilZ domain has recently been shown to bind c-di-GMP, and that this is critical for alginate biosynthesis (Merighi et al, 2007). In addition to PilZ in the N terminus, the C terminus of Alg44 (residues 220-364) shares homology with proteins in multi-drug efflux pumps, and in particular those known as membrane fusion proteins (labelled MFP in Fig.…”

Section: Alg44 Shares Homology With Efflux Proteinsmentioning

confidence: 99%

“…Constructs are designated by their terminal amino acid in Alg8 before the PhoA fusion. residues 42 and 62, but this is unlikely considering its weak hydrophobicity and the presence of a c-di-GMP-binding PilZ domain (residues 7-104), which would have a cytoplasmic localization (Merighi et al, 2007). SignalP predicts that Alg44 does not contain a cleavable signal sequence.…”

Section: Construction Of a Topological Model For Alg44 By Phoa Fusionmentioning

confidence: 99%

“…Another protein required for alginate biosynthesis is Alg44 (Maharaj et al, 1993;Remminghorst & Rehm, 2006b). Alg44 has been shown to have a PilZ domain for the binding of bis-(39,59)-cyclic dimeric GMP (c-di-GMP), a cofactor that is essential for Alg44 function and alginate production (Merighi et al, 2007). The polymer undergoes further modification by periplasmic AlgG, a C-5 epimerase that converts some D-mannuronates to L-guluronates (Franklin et al, 1994), and by AlgIJF, which O-acetylates the mannuronates at the polymer level (Franklin & Ohman, 2002).…”

Section: Introductionmentioning

confidence: 99%

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Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization

2008

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Mucoid strains of Pseudomonas aeruginosa that overproduce alginate are associated with chronic pulmonary disease (e.g. cystic fibrosis). Mutants defective in one of several periplasmic proteins (AlgKGX) for alginate secretion release alginate fragments due to the activity of an alginate lyase (AlgL) in the periplasm, which cleaves the newly formed polymers. However, mutants defective in Alg8 or Alg44 did not secrete polymer or alginate fragments, suggesting that both these membrane proteins have a role in the polymerization reaction. A model for the membrane topology of Alg8, a glycosyltransferase (GT), was constructed using PhoA fusions. This provided evidence for a large cytoplasmic loop containing the active domains predicted for bGTs such as Alg8 and five transmembrane (TM) domains, one of which resembles a cleavable signal peptide. The C-terminal TM domain of Alg8 was critical for the polymerization reaction in vivo. Alanine substitution mutagenesis showed that all of the predicted active site residues in the widely spaced D, DxD, D, LxxRW motif were required for polymerization activity in vivo, and two of these substitutions also affected Alg8 protein stability. A membrane topology model for Alg44 was also constructed using PhoA fusions, and this showed a central TM domain and predicted an N-terminal TM domain that may be a membrane anchor. An N-terminal PilZ domain in Alg44 for cdi-GMP [bis-(39,59)-cyclic dimeric GMP] binding, which is required for alginate synthesis, was localized to the cytoplasmic loop. The long periplasmic C terminus of Alg44 contains a region similar to membrane fusion proteins (MFPs) of multi-drug efflux systems, which predicts the possibility of its interaction with another protein in this compartment. A Western blot analysis of the outer-membrane porin AlgE showed reduced AlgE levels in the alg44 mutant, whereas expression of Alg44 in trans restored AlgE within the cell. C-terminal truncations of Alg44 as small as 24 amino acids blocked alginate polymerization in vivo, indicating a critical role for the MFP domain. These studies suggest that Alg44 may act as a co-polymerase in concert with Alg8, the major GT, and that both inner-membrane proteins are required in vivo for the polymerization reaction leading to alginate production.

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The second messenger bis‐(3′‐5′)‐cyclic‐GMP and its PilZ domain‐containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa

Cited by 315 publications

References 57 publications

Direct interaction between sensor kinase proteins mediates acute and chronic disease phenotypes in a bacterial pathogen

Direct interaction between sensor kinase proteins mediates acute and chronic disease phenotypes in a bacterial pathogen

Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella

Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization

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