In V
 itro
 and
 In V
 ivo
 Characterization of the
 Pseudomonas aeruginosa
 Cyclic AMP (cAMP) Phosphodiesterase CpdA, Required for cAMP Homeostasis and Virulence Factor Regulation

Fuchs, Erin L.; Brutinel, Evan D.; Klem, Erich; Fehr, Anthony R.; Yahr, Timothy L.; Wolfgang, Matthew C.

doi:10.1128/jb.00168-10

Cited by 79 publications

(111 citation statements)

References 58 publications

(67 reference statements)

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“…Regardless of the mechanism, it is clear that the CTE of Rv0805 reduced the extent of hydrolysis of cAMP in cells. Indeed, the extent of reduction of intracellular cAMP levels upon Rv0805⌬40 expression is similar to that observed when other bacterial cAMP phosphodiesterases such as CpdA from E. coli (42) or Pseudomonas aeruginosa (43) are overexpressed in their respective systems, supporting the above argument.…”

Section: Discussionsupporting

confidence: 69%

The Non-catalytic “Cap Domain” of a Mycobacterial Metallophosphoesterase Regulates Its Expression and Localization in the Cell

Matange

Podobnik

Visweswariah

2014

Journal of Biological Chemistry

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Background: Metallophosphoesterases possess unique, poorly characterized cap domains located at the entrance to the active site. Results: The C terminus of the cap domain of the Rv0805 metallophosphoesterase modulates its expression, cell envelope localization, and biological functions. Conclusion:The functions of the Rv0805 metallophosphoesterase are regulated by the cap domain. Significance: Cap domains may provide catalysis-independent features to metallophosphoesterases that shape their cellular functions.

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Section: Discussionsupporting

confidence: 69%

The Non-catalytic “Cap Domain” of a Mycobacterial Metallophosphoesterase Regulates Its Expression and Localization in the Cell

Matange

Podobnik

Visweswariah

2014

Journal of Biological Chemistry

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“…Deletion of the cpdA phosphodiesterase activity resulted in a low induction of maltose utilization. That the cpdA mutation leads to increased internal cAMP levels has been documented in different bacteria such as E. coli (9), Salmonella enterica serovar Typhimurium (3), P. aeruginosa (6), Bradyrhizobium japonicum (4), and Myxococcus xanthus (11).…”

Section: Discussionmentioning

confidence: 99%

“…Another possibility to reset the cAMP system in E. coli may be degradation by a known cAMP phosphodiesterase activity (CpdA). However, the K m of this enzyme was reported to be 47 M or even 500 M cAMP (6,9). Since CRP has an apparent dissociation constant of about 400 nM for cAMP (15), it is unlikely that CpdA with its low substrate affinity has a major impact in setting intracellular cAMP concentrations in E. coli.…”

mentioning

confidence: 99%

Escherichia coli Exports Cyclic AMP via TolC

2011

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In Escherichia coli more than 180 genes are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. However, more than 90% of cAMP that is made by intracellular adenylyl cyclases is found in the culture medium. How is cAMP exported from E. coli? In a tolC mutant, 0.03 mM IPTG (isopropyl-␤-Dthiogalactopyranoside) was sufficient to induce ␤-galactosidase compared to 0.1 mM IPTG in the parent strain. In a cya mutant unable to produce cAMP about 1 mM extracellular cAMP was required to induce ␤-galactosidase, whereas in a cya tolC mutant 0.1 mM cAMP was sufficient. When cAMP in E. coli cya was generated intracellularly by a recombinant, weakly active adenylyl cyclase from Corynebacterium glutamicum, the critical level of cAMP necessary for induction of maltose degradation was only achieved in a tolC mutant and not in the parent strain. Deletion of a putative cAMP phosphodiesterase of E. coli, CpdA, resulted in a slightly similar, yet more diffuse phenotype. The data demonstrate that export of cAMP via TolC is a most efficient way of E. coli to lower high concentrations of cAMP in the cell and maintain its sensitivity in changing metabolic environments.In Escherichia coli the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex affects the expression of more than 180 genes (16,18). Hence, the intracellular levels of cAMP need to be tightly controlled (5). Recently, a critical review of published data concluded that the rate of cAMP biosynthesis in E. coli is determined mainly by the cellular energy charge (13). At low ATP levels cAMP formation is enhanced. cAMP-CRP then stimulates transcription of catabolic enzymes and inhibits transcription of anabolic enzymes to ensure that the energy demands required for continual proliferation are met (13). The high affinity between cAMP and CRP requires E. coli to keep intracellular cAMP levels very low, which makes determination of intracellular cAMP levels in E. coli difficult and variable. Actually, whenever investigated thoroughly, most of the cAMP produced by E. coli was detected in the medium (2). Using bacterial membrane vesicles it was demonstrated that cAMP export is an active, energy-dependent process, whereas cAMP uptake appeared to be controlled by diffusion. However, the data further indicated that efflux and uptake may use identical protein components for cAMP trafficking (8). Another possibility to reset the cAMP system in E. coli may be degradation by a known cAMP phosphodiesterase activity (CpdA). However, the K m of this enzyme was reported to be 47 M or even 500 M cAMP (6, 9). Since CRP has an apparent dissociation constant of about 400 nM for cAMP (15), it is unlikely that CpdA with its low substrate affinity has a major impact in setting intracellular cAMP concentrations in E. coli.Thus, E. coli appears to rapidly quench the intracellular cAMP levels mainly by export and to a much smaller extent by degradation to reset the cellular regulatory systems. Thus far, the mechanism of cAMP export from E. coli that results in considerable cAMP concen...

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“…Based on these observations, it has been proposed that Vfr may respond to cAMP, cGMP and/or other allosteric regulators (4,55). While there is currently no evidence that P. aeruginosa has the ability to synthesize cGMP (16), it does produce cyclic diguanosine monophosphate (c-di-GMP) and possibly cyclic diadenosine monophosphate (c-di-AMP) (22,25,56). Recent findings indicate that some members of the CRP family bind c-di-GMP, which acts as a negative allosteric regulator (27,51).…”

mentioning

confidence: 99%

“…In addition, Vfr negatively regulates flagellar gene expression (10). A consensus Vfr binding sequence has been proposed (24), and direct binding of Vfr to target promoters has been demonstrated for several genes, including those encoding ToxA (toxA), LasR (the las quorum-sensing regulator), FleQ (the master regulator of flagellar biogenesis), RegA and PtxR (regulators of toxA expression), and CpdA (a cyclic AMP [cAMP] phosphodiesterase) (2,10,14,16,24). While the global role of Vfr in regulating virulence gene expression has been established, the molecular mechanisms that control Vfr activity and expression are not well understood.…”

mentioning

confidence: 99%

The Pseudomonas aeruginosa Vfr Regulator Controls Global Virulence Factor Expression through Cyclic AMP-Dependent and -Independent Mechanisms

et al. 2010

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Vfr is a global regulator of virulence factor expression in the human pathogen Pseudomonas aeruginosa. Although indirect evidence suggests that Vfr activity is controlled by cyclic AMP (cAMP), it has been hypothesized that the putative cAMP binding pocket of Vfr may accommodate additional cyclic nucleotides. In this study, we used two different approaches to generate apo-Vfr and examined its ability to bind a representative set of virulence gene promoters in the absence and presence of different allosteric effectors. Of the cyclic nucleotides tested, only cAMP was able to restore DNA binding activity to apo-Vfr. In contrast, cGMP was capable of inhibiting cAMP-Vfr DNA binding. Further, we demonstrate that vfr expression is autoregulated and cAMP dependent and involves Vfr binding to a previously unidentified site within the vfr promoter region. Using a combination of in vitro and in vivo approaches, we show that cAMP is required for Vfr-dependent regulation of a specific subset of virulence genes. In contrast, we discovered that Vfr controls expression of the lasR promoter in a cAMP-independent manner. In summary, our data support a model in which Vfr controls virulence gene expression by distinct (cAMP-dependent and -independent) mechanisms, which may allow P. aeruginosa to fine-tune its virulence program in response to specific host cues or environments.

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In V itro and In V ivo Characterization of the Pseudomonas aeruginosa Cyclic AMP (cAMP) Phosphodiesterase CpdA, Required for cAMP Homeostasis and Virulence Factor Regulation

Cited by 79 publications

References 58 publications

The Non-catalytic “Cap Domain” of a Mycobacterial Metallophosphoesterase Regulates Its Expression and Localization in the Cell

The Non-catalytic “Cap Domain” of a Mycobacterial Metallophosphoesterase Regulates Its Expression and Localization in the Cell

Escherichia coli Exports Cyclic AMP via TolC

The Pseudomonas aeruginosa Vfr Regulator Controls Global Virulence Factor Expression through Cyclic AMP-Dependent and -Independent Mechanisms

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