The glycerolipid and protein compositions of the outer and inner membranes of Rhizobium meliloti were studied. The wild-type R. meliloti strain Rm41 was shown to contain three phospholipids characteristic for most Gram-negative bacteria, phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol, this last compound being concentrated into the inner membrane. As in several bacteria interacting with plants, the presence of phosphatidylcholine, phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine was also demonstrated. Induction of the nod genes by luteolin did not affect the lipid composition and no difference in lipid composition was found between the wild-type strain and a number of Nod−and Fix−mutants tested. Protein analysis of the inner and outer membranes showed that they exhibit very different patterns with several bands specific for one or the other membrane. A Nod−mutant carrying a large deletion in the symbiotic megaplasmid pRme41b showed differences in the protein patterns even before induction by luteolin, indicating that this megaplasmid codes for several membrane proteins. When the nod genes of strain Rm41 were induced by luteolin, two new bands at around 60 kDa and 44 kDa appeared in both the outer and the inner membranes. By using a strain overexpressing the nod genes and the technique of immunoblotting with antibodies against NodC, it was confirmed that the 44 kDa band corresponded to the NodC protein. This protein was not found in a nodC:: Tn5 mutant. This work represents the first step in the characterization of modifications induced by luteolin treatment at the membrane level.
BanfalviZ.,
SakanyanV.,
KonczC.,
KissA.,
DushaI.,
KondorosiA.1981; Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti. Molecular and General Genetics 184:318–325
BaevN.,
EndreG.,
PetrovicsG.,
BanfalviZ.,
KondorosiA.1991; Six nodulation genes of nod box locus 4 in Rhizobium meliloti are involved in nodulation signal production: nodM codes for d-glucosamine synthetase. Molecular and General Genetics 228:113–124
BradfordM. M.1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
GersonT.,
PatelJ. J.1975; Neutral lipids and phospholipids of free-living and bacteroid forms of 2 strains of Rhizobium infective on Lotus pedunculatus. Applied Microbiology 30:193–198
JohnM.,
SchmidtJ.,
WienekeU.,
KondorosiE.,
KondorosiA.,
SchellJ.1985; Expression of the nodulation gene nodC of Rhizobium meliloti in Escherichia coli: role of the nodC gene product in nodulation. EMBO Journal 4:2425–2430
JohnM.,
SchmidtJ.,
WienekeL.,
KrussmannH. D.,
SchellJ.1988; Transmembrane orientation and receptor like structure of the Rhizobium meliloti common nodulation protein NodC. EMBO Journal 7:583–588
KatesM.1986; Isolation, analysis, identification of lipids. In Techniques in Lipidology, 2nd edn. p. 464 Edited by
BurdonR. H.,
KnippenbergP. H.
Amsterdam: Elsevier;
KondorosiA.1990; Rhizobium–legume interactions: nodulation genes. In Plant–Microbe Interactions. Molecular and Genetic Perspectives vol. 3 pp. 383–420 Edited by
KosugeT.,
NesterE. W.
New York: McGraw-Hill;
KondorosiA.,
SvabZ.,
KissG. B.,
DixonR. A.1977; Ammonia assimilation and nitrogen fixation in Rhizobium meliloti. Molecular and General Genetics 151:221–226
KondorosiE.,
GyurisJ.,
SchmidtJ.,
JohnM.,
DudaE.,
HoffmannB.,
SchellJ.,
KondorosiA.1989; Positive and negative control of nod gene expression in Rhizobium meliloti is required for optimal nodulation. EMBO Journal 5:1331–1340
LerougeP.,
RocheP.,
FaucherC.,
MailletF.,
TruchetG.,
PromeJ. C.,
DenarieJ.1990; Symbiotic host specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature, London 344:781–784
LugtenbergB. J. J.,
van AlphenL.1983; Molecular architecture of the outer membrane of E. coli and other Gram-negative bacteria. Biochimica et Biophysica Acta 737:51–115
de MaagdR.,
LugtenbergB.1986; Fractionation of Rhizobium leguminosarum cells into outer membrane, cytoplasmic membrane, periplasmic and cytoplasmic components. Journal of Bacteriology 167:1083–1985
de MaagdR.,
van RossumA. C.,
LugtenbergB. J. J.1988; Recognition of individual strains of fast-growing rhizobia by using profiles of membrane proteins and lipopolysaccharides. Journal of Bacteriology 170:3782–3785
MetcalfeL. D.,
SchmitzA. A.,
PelkaJ. R.1966; Rapid preparation of fatty acids esters from lipids for gas chromatographic analysis. Analytical Chemistry 38:514–515
MillerR. W.,
TremblayP. A.1983; Cytoplasmic membrane of Rhizobium meliloti bacteroids. I. Alterations in lipid composition, physical properties, and respiratory proteins. Canadian Journal of Biochemical Cell Biology 61:1334–1340
OsbornM. J.,
GanderJ. E.,
ParisiE.,
CarsonJ.1972; Mechanism of assembly of the outer membrane of Salmonella typhimurium. Journal of Biological Chemistry 247:3962–3972
PowelG. L.,
AbramovitchD. A.1985; Function of cardiolipin in the cytochrome C oxidase. Federation Proceedings of the American Society of Experimental Biology 44:478–485
SchmidtJ.,
JohnM.,
KondorosiE.,
KondorosiA.,
WienekeU.,
SchroderG.,
SchroderJ.,
SchellJ.1984; Mapping of the protein-coding regions of Rhizobium meliloti common nodulation genes. EMBO Journal 3:1705–1711
SchultzeM.,
Quiclet-SireJ. E.,
KondorosiE.,
VirelizierH.,
GlushkaJ. N.,
EndreG.,
GeroS. D.,
KondorosiA.1992; Rhizobium meliloti produces a family of sulfated lipooligosaccharides exhibiting different degrees of plant host specificity. Proceedings of the National Academy of Sciences of the United States of America 89:192–196
ThompsonE. A.,
KaupmanA. E.,
JohnstonN. C.,
GoldfineH.1983; Phospholipids of Rhizobium meliloti and Agrobacterium tumefaciens: lack of effect of Ti plasmid. Lipids 18:602–606
TowbinH.,
StaehelinT.,
GordonJ.1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76:4350–4354
VarenneP.,
DasB. C.,
PolonskyJ.,
TenceM.1985; Chemical ionization and fast atom bombardment mass spectometry of platelet activating factor (PAF-acether) and related phospholipids. Biomedical Mass Spectrometry 12:6–10