The Sinorhizobium meliloti Lon protease is involved in regulating exopolysaccharide synthesis and is required for nodulation of alfalfa

While screening for Sinorhizobium meliloti Pho regulatory mutants, a transp oson mutant was isolated that constitutively expressed higher levels of aci d and alkaline phosphatase enzymes. This mutant was also found to form pseu donodules on alfalfa that were delayed in appearance relative to those form ed by the wild-type strain, it contained few bacteroids, and it did not fix nitrogen. Sequence analysis of the transposon insertion site revealed the affected gene to have high homology to Lon proteases from a number of organ isms. In minimal succinate medium, the mutant strain was found to grow more slowly, reach lower maximal optical density, and produce more extracellula r polysaccharide (EPS) than the wild-type strain. The mutant fluoresced bri ghtly on minimal succinate agar containing calcofluor (which binds to EPSI, a constitutively expressed succinoglycan), and gas chromotographic analysi s of purified total EPS showed that the glucose-to-galactose ratio in the I on mutant total EPS was 5.0 +/- 0.2 (mean +/- standard error), whereas the glucose-to-galactose ratio in the wild-type strain was 7.1 +/- 0.5. These d ata suggested that in addition to EPSI, the lon mutant also constitutively synthesized EPSII, a galactoglucan which is the second major EPS known to b e produced by S. meliloti, but typically is expressed only under conditions of phosphate limitation, C-13 nuclear magnetic resonance analysis showed n o major differences between EPS purified from the mutant and wild-type stra ins. Normal growth, EPS production, and the symbiotic phenotype were restor ed in the mutant strain when the wild-type Ion gene was present in trans. T he results of this study suggest that the S. meliloti Lon protease is impor tant for controlling turnover of a constitutively expressed protein(s) that , when unregulated, disrupts normal nodule formation and normal growth.