The calcium-stimulated lipid A 3-O deacylase from Rhizobium etli is not essential for plant nodulation |
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Authors: | Christian Sohlenkamp Christian R.H. Raetz Brian O. Ingram |
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Affiliation: | 1. Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Av. Universidad s/n, Apdo. Postal 565-A, Cuernavaca, Morelos, CP62210, Mexico;2. Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA |
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Abstract: | The lipid A component of lipopolysaccharide from the nitrogen-fixing plant endosymbiont, Rhizobium etli, is structurally very different from that found in most enteric bacteria. The lipid A from free-living R. etli is structurally heterogeneous and exists as a mixture of species which are either pentaacylated or tetraacylated. In contrast, the lipid A from R. etli bacteroids is reported to consist exclusively of tetraacylated lipid A species. The tetraacylated lipid A species in both cases lack a β-hydroxymyristoyl chain at the 3-position of lipid A. Here, we show that the lipid A modification enzyme responsible for 3-O deacylation in R. etli is a homolog of the PagL protein originally described in Salmonella enterica sv. typhimurium. In contrast to the PagL proteins described from other species, R. etli PagL displays a calcium dependency. To determine the importance of the lipid A modification catalyzed by PagL, we isolated and characterized a R. etli mutant deficient in the pagL gene. Mass spectrometric analysis confirmed that the mutant strain was exclusively tetraacylated and radiochemical analysis revealed that 3-O deacylase activity was absent in membranes prepared from the mutant. The R. etli mutant was not impaired in its ability to form nitrogen-fixing nodules on Phaseolus vulgaris but it displayed slower nodulation kinetics relative to the wild-type strain. The lipid A modification catalyzed by R. etli PagL, therefore, is not required for nodulation but may play other roles such as protecting bacterial endosymbionts from plant immune responses during infection. |
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Keywords: | BCA, bicinchoninic acid EDTA, ethylenediaminetetraacetic acid ESI, electrospray ionization Kdo, 3-deoxy-d-manno-oct-2-ulosonic acid LC, liquid chromatography LPS, lipopolysaccharide MES, 2-(N-morpholino)-ethanesulfonic acid MS, mass spectrometry PBS, phosphate-buffered saline TLC, thin layer chromatography TLR-4, Toll-like receptor-4 |
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