PdeB, a cyclic Di-GMP-specific phosphodiesterase that regulates Shewanella oneidensis MR-1 motility and biofilm formation

Abstract

Shewanella oneidensis MR-1, a gammaproteobacterium with respiratory versatility, forms biofilms on mineral surfaces through a process controlled by the cyclic dinucleotide messenger c-di-GMP. Cellular concentrations of c-di-GMP are maintained by proteins containing GGDEF and EAL domains, which encode diguanylate cyclases for c-di-GMP synthesis and phosphodiesterases for c-di-GMP hydrolysis, respectively. The S. oneidensis MR-1 genome encodes several GGDEF and EAL domain proteins (50 and 31, respectively), with a significant fraction (∼10) predicted to be multidomain (e.g., GGDEF-EAL) enzymes containing an additional Per-Arnt-Sim (PAS) sensor domain. However, the biochemical activities and physiological functions of these multidomain enzymes remain largely unknown. Here, we present genetic and biochemical analyses of a predicted PAS-GGDEF-EAL domain-containing protein, SO0437, here named PdeB. A pdeB deletion mutant exhibited decreased swimming motility and increased biofilm formation under rich growth medium conditions, which was consistent with an increase in intracellular c-di-GMP. A mutation inactivating the EAL domain also produced similar swimming and biofilm phenotypes, indicating that the increase in c-di-GMP was likely due to a loss in phosphodiesterase activity. Therefore, we also examined the enzymatic activity of purified PdeB and found that the protein exhibited phosphodiesterase activity via the EAL domain. No diguanylate cyclase activity was observed. In addition to the motility and biofilm phenotypes, transcriptional profiling by DNA microarray analysis of biofilms of pdeB (in-frame deletion and EAL) mutant cells revealed that expression of genes involved in sulfate uptake and assimilation were repressed. Addition of sulfate to the growth medium resulted in significantly less motile pdeB mutants. Together, these results indicate a link between c-di-GMP metabolism, S. oneidensis MR-1 biofilm development, and sulfate uptake/assimilation.

Fig 1

Forms of PdeB used in this study. (A) Predicted domain structure of PdeB (Swiss-Prot). The small black rectangles represent the predicted transmembrane regions (THMM). (B) The PdeB(260-856) purified protein (MBP, maltose-binding protein). (C) Mutant version of PdeB with PDE activity inactivated (PdeB^eal, E634A). The black triangle represents a mutated amino acid residue.

Fig 2

Swimming motility patterns for pdeB mutants and the wild type. Swimming motility of WT (top) and ΔpdeB mutant (bottom) cultures were assessed in LB (A) or 4M plus 20 mM fumarate (B). Plates contained 0.25% agar. LB cultures were incubated at 30°C for 48 h, and 4M cultures were incubated anaerobically at room temperature for 48 h. (C) LM swim plate incubated at 30°C for 48 h.

Fig 3

Biofilms of ΔpdeB and pdeB^eal mutants. Biofilms were grown in hydrodynamic flow chambers with LM for 15 to 16 h at 30°C before imaging. White bars represent 100 µm. Quantification of biofilms is shown in Table 2.

Fig 4

Cyclic di-GMP-specific phosphodiesterase activity of PdeB. [³²P]c-di-GMP was incubated with (1 μM) or without MBP-PdeB(260-856)-His₆ at 30°C for 30 min before 1 μl of sample was resolved on a PEI-cellulose TLC plate. −, no enzyme; PdeB, MBP-PdeB(260-856)-His₆; PdeB^eal, E634A mutant.

Fig 5

Effect of sulfate addition on swimming motility of ΔpdeB and pdeB^eal mutants. Swimming motility was tested on LM (A) and LM supplemented with 40 mM Na₂SO₄ (B). (C) The relative difference in diameter compared to that of the WT was calculated. Relative difference is the diameter of the WT minus the diameter of the mutant, divided by the average diameter of the mutant and WT. Black, no sulfate; gray, 40 mM NH₄SO₄; white, 40 mM Na₂SO₄.