Di-adenosine tetraphosphate (Ap4A) metabolism is ubiquitous in nature, yet its biological roles are still poorly understood. In this study, we explored a novel role for Ap4A metabolism in transcriptional control of Pho regulon expression and regulation of biofilm formation by P. fluorescens. Our results are consistent with the hypothesis that Ap4A metabolism has a role as an intracellular regulator; however, they also support the hypothesis that perturbations in Ap4A metabolism can impact global cellular traits, such as biofilm formation, through more general disruption of purine-based nucleotide dynamics.
We rigorously demonstrated that Pfl_5137 of P. fluorescens Pf0-1 encodes a di-nucleotide tetraphosphatase similar to ApaH from E. coli. Loss of apaH resulted in accumulation of high levels of di-adenosine tetraphosphate (Ap4A) and was phenotypically pleiotropic.
In the current work we focused on substantiating the relationship between Ap4A metabolism and its novel regulation of P. fluorescens
biofilm formation. Ap4A metabolism affects biofilm formation via two separate yet related pathways. First, high levels of Ap4A due to mutation of apaH
prevent loss of biofilm formation in response to low levels of extracellular Pi
. In the wild type, activation of the Pho regulon in low-Pi
environments results in expression of a c-di-GMP phosphodiesterase, RapA (24
). Subsequent RapA-mediated reductions in the c-di-GMP concentration inhibit secretion and localization of the adhesin LapA to the outer membrane. LapA is required for proper colonization of surfaces and subsequent biofilm formation. An apaH
mutant circumvents this regulatory response to low Pi
by preventing efficient activation of the Pho regulon. We do not yet know the mechanism by which the level of Ap4A impacts Pho regulon activation (Fig. ).
FIG. 9. Summary of the current model for the role of ApaH in biofilm formation by P. fluorescens. Loss of the ApaH function and subsequent accumulation of Ap4A promote biofilm formation by two mechanisms. (i) Accumulation of Ap4A prevents efficient recycling (more ...)
A second, more general mechanism by which Ap4A metabolism affects biofilm formation was also observed. In Pi-replete conditions, when Pho regulon expression was repressed, we observed that the apaH mutant produced approximately 2-fold more biofilm than the wild type produced. The increased propensity for surface attachment is explained by substantial increases in the amount of LapA attached to the outer membrane and the concurrent increases in the level of intracellular c-di-GMP.
In both cases, Ap4A modulates biofilm formation by altering the concentration of c-di-GMP. In low-Pi conditions this connection is mediated through the Pho regulon and RapA; however, mechanisms connecting increases in the Ap4A level with Pho-independent increases in the c-di-GMP level are less obvious. One possibility that we have begun to explore is that imbalances in general nucleotide pools due to a block in Ap4A turnover result in changes in c-di-GMP pools in the cell. Whole-cell nucleotide analysis indicated that, in addition to increased c-di-GMP levels, GTP levels were elevated 3-fold in the apaH mutant. Furthermore, the ATP levels did not change even though large amounts of ADP were sequestered in the cell as Ap4A. Together, these observations raise the possibility that de novo purine biosynthetic pathways might be activated to a greater degree in the apaH mutant, resulting in higher GTP concentrations and more synthesis of c-di-GMP. This hypothesis is supported by the fact that addition of the purine adenine led to significant reductions in biofilm formation by the apaH mutant but had no effect on wild-type biofilm formation. Also consistent with our hypothesis, we observed a small (36%) yet statistically significant decrease in the c-di-GMP level when the apaH mutant was treated exogenously with adenine. Although addition of adenine could not fully restore wild-type biofilm dynamics or c-di-GMP profiles to the apaH mutant, the partial rescue of both phenotypes implies that perturbation of purine metabolism is an important component of the mechanisms connecting Ap4A metabolism to the c-di-GMP level and the regulation of biofilm formation by P. fluorescens. Nucleotide biosynthesis is a complicated biological process. Thus, a more in-depth analysis is required to understand exactly how Ap4A levels impact purine metabolism and to what extent the perturbations explain increases in c-di-GMP levels and biofilm formation in Pi-replete conditions.
The current view is that c-di-GMP levels are tightly controlled through the opposing actions of diguanylate cyclase (DGC) and PDE domain-containing proteins (34
), and the regulation of PDE and DGC activity, rather than substrate availability, is considered a major control point for fine-tuning c-di-GMP levels. In contrast to this view, our data suggest that the level of c-di-GMP may also reflect the general metabolic status of the cell and respond to changes in the flux of nucleotides and their precursors. We feel that our results provide an important reminder that although c-di-GMP acts as a signaling molecule, its biosynthesis is intimately connected to the core metabolic networks of the cell and therefore must be understood in this context.
In these studies we demonstrated that overexpression of the adhesin LapA or the transporter LapEBC was not sufficient to appreciably increase biofilm formation by the wild type. Interestingly, overexpression of both the adhesin and its transporter allowed export of large quantities of LapA from the cytoplasm but resulted in only small increases in the CA LapA level and biofilm formation. These findings confirmed that increases in transcription of lapA
were not sufficient to explain the increased biofilm formation by the apaH
mutant. In addition to answering a specific question, these results reinforce the critical role of posttranslational regulation in mediating efficient localization of LapA to the cell surface. A strong candidate for mediating such interactions is c-di-GMP, especially considering a recent report that identified LapD as a c-di-GMP receptor protein that regulates localization of LapA to the outer membrane (27
). We hypothesize that increases in lapA
expression, like those seen in the apaH
mutant, can contribute to upregulation of biofilm formation, but only when they are accompanied by activation of c-di-GMP-dependent pathways that facilitate localization of LapA to the cell surface.
The studies that we describe here utilized a mutation in apaH
to increase the levels of Ap4A in the cell. Ultimately, we would like to know whether physiological conditions can promote increases in the Ap4A concentration that are sufficient to impact regulation of biofilm formation. Studies of E. coli
have shown that treatment with a range of oxidizing agents or a heat shock can stimulate production of Ap4A so that the levels in the cell are comparable to those seen in an apaH
). These studies formed the basis of the hypothesis that Ap4A is an alarmone that regulates cellular responses to stress resulting from oxidation or temperature. In contrast to results obtained with E. coli
, we did not detect increases in Ap4A levels when P. fluorescens
wild-type cells were treated with the oxidizing agent hydrogen peroxide (data not shown). Further studies are required to rigorously determine what physiological conditions promote Ap4A formation in P. fluorescens
and how these conditions affect c-di-GMP levels and biofilm formation.
The biological effects of disruptions in Ap4A metabolism are complicated and diverse, and such effects have been found in different species, phyla, and domains. It is clear that a greater understanding of the mechanism is required if we are to move beyond phenomenological descriptions of diverse functions ascribed to Ap4A and its related nucleotides. We believe that our work speaks to the more general concept that metabolic networks can play central roles in the regulation of complex cellular traits, rather than simply being confined to management of the energy and biosynthetic needs of the cell.