2 EPS. Only after introducing full-length copies of rosR into Rt24.2 (especially under its own promoter, on plasmid pBR24), the negative dominant effect had been overcome, with the increase of EPS synthesis up to 183% of the control. These results suggested that additional copies of the rosR upstream region with the RosR-box sequence, rather than RosR protein deprived of the C-terminal DNA binding domain, affected the level of EPS production. Most likely, the positive regulation of EPS synthesis by RosR depends
on an equilibrium between rosR regulatory sequences and the amount of RosR. These results explain, to some extent, the phenotype of the Rt2441 mutant. Figure 2 The effect of additional copies of different regulatory rosR sequences on the EPS production by R. leguminosarum. Data shown are the means of three replicates ± SD. EPSs isolated from the Rt24.2 wild type and Rt2440 and Rt2441 rosR mutants were fractionated by Rabusertib manufacturer gel permeation chromatography on a Bio-Gel A-5m column, and two fractions of EPS with significantly different molecular weights were obtained (Figure 3A). The ratio of high-molecular-weight (HMW) to low-molecular-weight (LMW) fractions was 68%:32% in the EPS of Rt24.2 wild type. In the Rt2440 and Rt2441 rosR mutants, a considerable change was observed in the HMW to LMW EPS ratio in favor CX-6258 ic50 of HMW, i.e., 79%:21% and 76%:24%, respectively. To
establish the sugar composition of EPS Adenosine triphosphate of the wild type and the rosR mutant, peak samples from Bio-Gel A-5m chromatography (Figure 3A) were evaluated for monosaccharide composition by GC-MS. The glucose/glucuronic acid/galactose ratio was found to be approximately
5:2:1, which is characteristic of the acidic EPS of R. leguminosarum (Figure 3C). Additionally, non-carbohydrate substituents in the EPS of Rt2440 and Rt24.2 wild type were determined (Figure 3B-C). EPS secreted by the rosR mutant had a lower level of O-acetyl and 3-hydroxybutyryl substitutions and slightly more pyruvyl substitutions in relation to the wild type EPS (Figure 3B). Figure 3 Gel filtration chromatography of exopolysaccharides (EPS) produced by the R. leguminosarum bv. trifolii 24.2 wild type and the rosR mutants (Rt2440 and Rt2441). (A) EPS was fractionated on a Bio-Gel A-5m column, as described in the Methods. The retention times of molecular mass markers: dextran blue (2 MDa), dextran T250 (250 kDa), and dextran T10 (10 kDa) are indicated by arrows. (B) A 500 MHz 1H-NMR spectrometry analysis of the R. leguminosarum wild type and the rosR mutant (Rt2440). (C) The glycosyl components and non-carbohydrate substituents of EPS from the wild type and the mutant Rt2440. (D) Silver-stained Tricine SDS-PAGE profiles of LPS from the wild type and the rosR mutants. LPSs (2 μg) were loaded in 2 μl sample buffer. Lanes: 1- Salmonella enterica sv. Typhimurium (Sigma), 2- wild type Rt24.2, 3- Rt2440, 4- Rt2441. LPS I, high-molecular-weight LPS; LPS II, low-molecular-weight LPS.