A purL mutant of Sinorhizobium fredii HH103 is symbiotically defective and altered in its lipopolysaccharide

Abstract

The pleiotropic phenotype of an auxotrophic purL mutant (SVQ295) of Sinorhizobium fredii HH103 has been investigated. SVQ295 forms colonies that are translucent, produce more slime and absorb less Congo red than those of wild-type strain HH103. SVQ295 did not grow in minimal medium unless the culture was supplemented with thiamin and adenine or with thiamin and AICA-riboside (5-aminoimidazole-4-carboxamide 1-b-D-ribofuranoside), an intermediate of purine biosynthesis. Bacterial cultures supplemented with AICA-riboside or adenine reached the same culture density, although the doubling time of SVQ295 cultures containing AICA-riboside was clearly longer. S. fredii SVQ295 induced pseudonodules on Glycine max and failed to nodulate six different legumes. On Glycyrrhiza uralensis, however, nodules showing nitrogenase activity and containing infected plant cells were formed. SVQ295 showed auto-agglutination when grown in liquid TY medium and its lipopolysaccharide (LPS) electrophoretic profile differed from that of its parental strain HH103-1. In addition, four monoclonal antibodies that recognize the LPS of S. fredii HH103 failed to recognize the LPS produced by SVQ295. In contrast, 1 H-NMR spectra of K-antigen capsular polysaccharides (KPS) produced by SVQ295 and the wild-type strain HH103 were similar. Co-inoculation of soybean plants with SVQ295 and SVQ116 (a nodA mutant derivative of HH103) produced nitrogen-fixing nodules that were only occupied by SVQ116.

Glycolysis and pyrimidine biosynthesis are required for replication of adherent–invasive Escherichia coli in macrophages

“…The purM gene encodes an enzyme that catalyses the fifth step in the de novo biosynthesis of purines. Whilst there are no reports of any requirement for purine biosynthesis during bacterial intramacrophagic replication, it has been shown that purine biosynthesis-deficient mutants of Burkholderia, Sinorhizobium and Photorhabdus are defective in their ability to form symbiotic associations with insects, plants and nematodes, respectively (Ruisheng & Grewal, 2011;Buendía-Clavería et al, 2003;Kim et al, 2014). Moreover Pseudomonas aeruginosa must synthesize purines during both chronic and acute infections of animals (Turner et al, 2014).…”

Sinorhizobium fredii HH103 cgs Mutants Are Unable to Nodulate Determinate- and Indeterminate Nodule–Forming Legumes and Overproduce an Altered EPS

“…The cgs mutation did not apparently affect bacterial LPS because S. fredii HH103-Rif r and SVQ562 showed the same LPS electrophoretic profile and the same pattern of LPS bands recognized by the monoclonal antibody NB6-228.22 (data not shown). This monoclonal antibody specifically recognizes the LPS of S. fredii HH103 (Buendía-Clavería et al 2003).…”

“…SVQ295 is an auxotrophic purL mutant unable to invade soybean roots (it secretes LCO but only pseudonodules are formed) that, in co-inoculation experiments, can complement S. fredii SVQ116 (a nodA mutant, unable to produce LCO) to form nitrogen-fixing nodules on soybean plants (Buendía-Clavería et al 2003). Mutant SVQ562 was chosen for these studies because any nodule formed could be investigated for its β-galactosidase activity (presence of SVQ562).…”

“…To investigate EPS production on solid YM medium, rhizobial strains were grown for 120 h at 28°C followed by 48 h at room temperature. LPS extraction, separation on SDS polyacrylamide gel electrophoresis, and silver staining were performed as previously described (Buendía-Clavería et al 2003). Immunostaining procedures and the monoclonal antibody NB6-228.22 were described by Buendía-Clavería and associates (2003).…”

Transcriptional Regulator LsrB of Sinorhizobium meliloti Positively Regulates the Expression of Genes Involved in Lipopolysaccharide Biosynthesis

“…S. meliloti cells were collected from LB/MC cultures (optical density at 600 nm [OD 600 ], Ϸ0.8). The crude LPS was extracted with both hot phenol and boiled methods and then treated by RNase, DNase I, and protease K (4,26). The sulfate-anthrone method was performed to quantify the total sugar content of dialyzed LPS extract (27).…”