Abstract
Pseudomonas species infect a variety of organisms, including mammals and plants. Mammalian pathogens of the Pseudomonas family modify their lipid A during host entry to evade immune responses and to create an effective barrier against different environments, for example by removal of primary acyl chains, addition of phosphoethanolamine (P-EtN) to primary phosphates, and hydroxylation of secondary acyl chains. For Pseudomonas syringae pv. phaseolicola (Pph) 1448A, an economically important pathogen of beans, we observed similar lipid A modifications by mass spectrometric analysis. Therefore, we investigated predicted proteomes of various plant-associated Pseudomonas spp. for putative lipid A-modifying proteins using the well-studied mammalian pathogen Pseudomonas aeruginosa as a reference. We generated isogenic mutant strains of candidate genes and analyzed their lipid A. We show that the function of PagL, LpxO, and EptA is generally conserved in Pph 1448A. PagL-mediated de-acylation occurs at the distal glucosamine, whereas LpxO hydroxylates the secondary acyl chain on the distal glucosamine. The addition of P-EtN catalyzed by EptA occurs at both phosphates of lipid A. Our study characterizes lipid A modifications in vitro and provides a useful set of mutant strains relevant for further functional studies on lipid A modifications in Pph 1448A.
Highlights
The Gram-negative plant pathogen Pseudomonas syringae infects a wide range of economically important crop species
Since structural features and dynamic adaption of the lipid A moiety play a crucial role in the host colonization process of P. aeruginosa, we were interested in whether orthologous genes are present in different plant-associated Pseudomonas spp., which fulfill a similar role
In all analyzed Pseudomonas spp., proteins involved in lipid A biosynthesis (LpxA, LpxC, LpxD, LpxH, LpxB, LpxK, LpxL, and LpxM) were identified with sequence identities of at least 72% compared to P. aeruginosa PAO1, suggesting that the basic lipid A synthesis is conserved in Pseudomonas
Summary
The Gram-negative plant pathogen Pseudomonas syringae infects a wide range of economically important crop species. Worldwide disease outbreaks caused by P. syringae have long prompted researchers to study this pathogen. P. syringae is one of the best-studied plant pathogens for investigating molecular mechanisms of pathogenicity and pathogen–host interactions [1]. A major characteristic of Gram-negative bacteria is the presence of lipopolysaccharide (LPS) in the cell envelope. The LPS structure can be divided into three parts: the Opolysaccharide (OPS), a core oligosaccharide, and the lipid A, which anchors the molecule in the outer leaflet of the outer cell membrane. The interplay of all three parts of the LPS provides Gram-negative bacteria with an effective barrier against a variety of harmful compounds [5–8]
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