Abstract
BackgroundThe phenazines are redox-active secondary metabolites that a large number of bacterial strains produce and excrete into the environment. They possess antibiotic activity owing to the fact that they can reduce molecular oxygen to toxic reactive oxygen species. In order to take advantage of this activity, phenazine producers need to protect themselves against phenazine toxicity. Whereas it is believed that phenazine-producing pseudomonads possess highly active superoxide dismutases and catalases, it has recently been found that the plant-colonizing bacterium Enterobacter agglomerans expresses a small gene ehpR to render itself resistant towards D-alanyl-griseoluteic acid, the phenazine antibiotic produced by this strain.ResultsTo understand the resistance mechanism installed by EhpR we have determined its crystal structure in the apo form at 2.15 Å resolution and in complex with griseoluteic acid at 1.01 Å, respectively. While EhpR shares a common fold with glyoxalase-I/bleomycin resistance proteins, the ligand binding site does not contain residues that some related proteins employ to chemically alter their substrates. Binding of the antibiotic is mediated by π-stacking interactions of the aromatic moiety with the side chains of aromatic amino acids and by a few polar interactions. The dissociation constant KD between EhpR and griseoluteic acid was quantified as 244 ± 45 μM by microscale thermophoresis measurements.ConclusionsThe data accumulated here suggest that EhpR confers resistance by binding D-alanyl-griseoluteic acid and acting as a chaperone involved in exporting the antibiotic rather than by altering it chemically. It is tempting to speculate that EhpR acts in concert with EhpJ, a transport protein of the major facilitator superfamily that is also encoded in the phenazine biosynthesis operon of E. agglomerans. The low affinity of EhpR for griseoluteic acid may be required for its physiological function.
Highlights
The phenazines are redox-active secondary metabolites that a large number of bacterial strains produce and excrete into the environment
The blue phenazine derivative pyocyanin (5-N-methyl-1-hydroxyphenazium betaine) induces tissue damage in patients infected with a well-studied Gram-negative phenazine producer Pseudomonas aeruginosa [6,7] and it has been demonstrated that the immune system can clear P. aeruginosa infections significantly more if phenazine biosynthesis is impaired [8]
Enterobacter agglomerans strain Eh1087 generates the phenazine antibiotic D-alanyl griseoluteic acid to compete with other microorganisms in its habitat
Summary
The phenazines are redox-active secondary metabolites that a large number of bacterial strains produce and excrete into the environment. In the case of compounds with nonspecific toxicity, such as those that give rise to reactive oxygen species (ROS), the producing strain is faced with the problem of having to avoid selfpoisoning This cannot be resolved by destroying the antibiotic as this would contradict the purpose of synthesizing these toxins in the first place and would lead to a waste of metabolic energy instead. Whereas the reoxidation of NADH may play an important role in the survival of phenazine producers in anoxic environments, like those found in the deeper layers of biofilms, the reduction of ferric iron or O2 directly or indirectly leads to the generation of toxic ROS This explains the broad specificity antibiotic activity of phenazines and their function as virulence factors in infectious disease. The blue phenazine derivative pyocyanin (5-N-methyl-1-hydroxyphenazium betaine) induces tissue damage in patients infected with a well-studied Gram-negative phenazine producer Pseudomonas aeruginosa [6,7] and it has been demonstrated that the immune system can clear P. aeruginosa infections significantly more if phenazine biosynthesis is impaired [8]
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