Mutation in the pssM gene encoding ketal pyruvate transferase leads to disruption of Rhizobium leguminosarum bv. viciae-Pisum sativum symbiosis.
OBJECTIVE
To study the question whether acidic exopolysaccharide (EPS) modification, e.g. pyruvylation, plays any role in the development of Rhizobium leguminosarum/Pisum sativum symbiosis.


RESULTS
The amino acid sequence deduced from the pssM gene, localized within the pss (polysaccharide synthesis) gene locus, was shown to be homologous to several known and putative ketal pyruvate transferases, including ExoV from Sinorhizobium meliloti and GumL from Xanthomonas campestris. Rh. l. bv. viciae strain VF39 carrying a Km-cassette insertion into the pssM gene was obtained by the gene replacement technique. Knock-out of pssM led to the absence of the pyruvic acid ketal group at the subterminal glucose in the repeating unit of EPS as it was shown by (13)C and (1)H nuclear magnetic resonance (NMR) analysis. Complementation in trans restored the EPS modification in the pssM mutant. Disruption of the pssM gene resulted also in the formation of aberrant non-nitrogen-fixing nodules on peas. Ultrastructural studies of mutant nodules revealed normal nodule invasion and release of bacteria into the plant cell cytoplasm, but further differentiation of bacteroids was impaired, and the existing symbiosomes underwent lysis.


CONCLUSIONS
PssM encodes ketal pyruvate transferase involved in the modification of the Rh. l. bv. viciae EPS. The absence of subterminal glucose pyruvylation in the EPS repeating units negatively influences (directly or indirectly) the formation of the nitrogen-fixing symbiosis with peas.


CONCLUSIONS
Our finding that the absence of modification even at the single position of EPS is likely to be crucial for establishment of nitrogen-fixing symbiosis argues in favour of the idea concerning their specific signalling role in this process.
 Skryabin Institute of Biochemistry and Physiology of Micro-organisms, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.| Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
T V
TV
Ivashina
 Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia.
E E
EE
Fedorova
 Skryabin Institute of Biochemistry and Physiology of Micro-organisms, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
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NP
Ashina
 Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia.
N A
NA
Kalinchuk
 Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia.
T N
TN
Druzhinina
 Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia.
A S
AS
Shashkov
 Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia.
V N
VN
Shibaev
 Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
V N
VN
Ksenzenko
D000217:Acyltransferases / Q000235:genetics*; D002240:Carbohydrate Sequence; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009586:Nitrogen Fixation / Q000235:genetics; D018532:Pisum sativum / Q000382:microbiology* / Q000648:ultrastructure; D011135:Polysaccharides, Bacterial / Q000737:chemistry / Q000378:metabolism; D016961:Rhizobium leguminosarum / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D013559:Symbiosis / Q000235:genetics
D011135:Polysaccharides, Bacterial; D000217:Acyltransferases; C071727:ketal pyruvate transferase