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Different phenotypic classes of Sinorhizobium meliloti mutants defective in synthesis of K antigen
G R Campbell1, B L Reuhs, G C Walker
1Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Abstract:
For Sinorhizobium meliloti (also known as Rhizobium meliloti) AK631 to establish effective symbiosis with alfalfa, it must be able to synthesize a symbiotically active form of its K antigen, a capsular polysaccharide containing a Kdo (3-deoxy-D-manno-octulosonic acid) derivative. Previously isolated mutants defective in the synthesis of K antigen are resistant to bacteriophage phi16-3. By screening ca. 100,000 Tn5-mutagenized R. meliloti bacteria for resistance to bacteriophage phi16-3, we isolated 119 mutants, 31 of which could not be complemented by genes previously identified as being required for K-antigen synthesis. Of these 31 new mutants, 13 were symbiotically defective and lacked the K antigen. Through genetic and phenotypic analyses, we have grouped these mutants into four distinct classes. Although all of these mutants lack the K antigen, many also have altered lipopolysaccharides (LPS), suggesting that the biochemical pathways for the synthesis of K antigen and LPS have common enzymatic steps. In addition, we have found that these and other classes of K-antigen-defective mutants of S. meliloti AK631 exhibit unique patterns of sensitivities to phage strains to which the parental strain was resistant. Our studies have identified new classes of genes required for both the synthesis of K antigen and the symbiotic proficiency of S. meliloti AK631. Some of these classes of genes also play a role in LPS synthesis.
Insights
New genes essential for Sinorhizobium meliloti
Area of Science:
- Microbiology
- Molecular Biology
- Plant-Bacterial Interactions
Background:
- Sinorhizobium meliloti (Rhizobium meliloti) AK631 requires K antigen synthesis for effective alfalfa symbiosis.
- K antigen is a capsular polysaccharide containing a Kdo derivative.
- Mutants lacking K antigen are often resistant to bacteriophage phi16-3.
Purpose of the Study:
- To identify novel genes involved in K antigen synthesis in S. meliloti AK631.
- To understand the relationship between K antigen, lipopolysaccharides (LPS), and symbiotic proficiency.
- To discover new phage sensitivities associated with K-antigen defective mutants.
Main Methods:
- Large-scale screening of Tn5-mutagenized S. meliloti for resistance to bacteriophage phi16-3.
- Genetic complementation to identify novel mutants.
- Phenotypic analysis including symbiotic defect and K antigen/LPS presence.
- Phage sensitivity profiling.
Main Results:
- 119 bacteriophage-resistant mutants were isolated, with 31 representing new genes for K antigen synthesis.
- 13 of these new mutants were symbiotically defective and lacked K antigen.
- Mutants lacking K antigen often exhibited altered lipopolysaccharides (LPS), indicating shared biosynthetic pathways.
- New classes of K-antigen defective mutants displayed unique phage sensitivity patterns.
Conclusions:
- Identified new gene classes crucial for K antigen synthesis and symbiotic effectiveness in S. meliloti.
- Demonstrated overlap in enzymatic steps between K antigen and LPS biosynthesis.
- Highlighted the potential of phage resistance screening for uncovering essential bacterial genes.