Abstract
In this work we have characterised the Sinorhizobium fredii HH103 greA lpsB lpsCDE genetic region and analysed for the first time the symbiotic performance of Sinorhizobium fredii lps mutants on soybean. The organization of the S. fredii HH103 greA, lpsB, and lpsCDE genes was equal to that of Sinorhizobium meliloti 1021. S. fredii HH103 greA, lpsB, and lpsE mutant derivatives produced altered LPS profiles that were characteristic of the gene mutated. In addition, S. fredii HH103 greA mutants showed a reduction in bacterial mobility and an increase of auto-agglutination in liquid cultures. RT-PCR and qPCR experiments demonstrated that the HH103 greA gene has a positive effect on the transcription of lpsB. Soybean plants inoculated with HH103 greA, lpsB or lpsE mutants formed numerous ineffective pseudonodules and showed severe symptoms of nitrogen starvation. However, HH103 greA and lps mutants were also able to induce the formation of a reduced number of soybean nodules of normal external morphology, allowing the possibility of studying the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis. The infected cells of these nodules showed signs of early termination of symbiosis and lytical clearance of bacteroids. These cells also had very thick walls and accumulation of phenolic-like compounds, pointing to induced defense reactions. Our results show the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis and their role in preventing host cell defense reactions. S. fredii HH103 lpsB mutants also showed reduced nodulation with Vigna unguiculata, although the symbiotic impairment was less pronounced than in soybean.
Highlights
The lipopolysaccharide (LPS) is a complex glycolipid component present in the outer leaflet of the Gram-negative bacteria, including rhizobia [1,2,3]
In silico analysis of the sequenced fragment revealed that the S. fredii HH103 greA lpsB lpsCDE genes show the same genetic organization that in S. meliloti 1021 (Figure 1)
PAGE and/or Nuclear Magnetic Resonance (NMR) studies showed that Kantigen polysaccharides (KPS) and cyclic glucans (CG) produced by SVQ613, SVQ642, SVQ655, and SVQ656 appear to be identical to that produced by the parental strain HH103 RifR (Figures S1 and S2)
Summary
The lipopolysaccharide (LPS) is a complex glycolipid component present in the outer leaflet of the Gram-negative bacteria, including rhizobia [1,2,3]. The LPS of rhizobia shows the same general architecture as that from LPS of enteric and animal Gramnegative pathogens It can be conceptually divided into three different structural regions: an inner acylated saccharide known as lipid A that is linked to a central core oligosaccharide which is attached to an outer O-chain polysaccharide [4]. PMUS908, which contains the greA lpsB lpsCDE genes, into S. fredii greA mutants restored the wild type LPS electrophoretic and NB6-228.22 recognition patterns (Figure 2A, B). This approach failed to complement both lpsE and lpsB mutants. All the greA, lpsB, and lpsE mutants formed colonies in TY and YMA media that were mucous to those formed by HH103 RifR (data not shown), suggesting that EPS production was not altered in these mutants
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