Reeve Wayne G, Dilworth Michael J, Tiwari Ravi P, Glenn Andrew R
Nitrogen Fixation Research Group, School of Biological and Environmental Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia.
Microbiology (Reading). 1997 Jun;143 ( Pt 6):1951-1958. doi: 10.1099/00221287-143-6-1951.
A mildly acid-sensitive mutant of Rhizobium leguminosarum bv. viciae WSM710 (WR6-35) produced colonies which were more mucoid in phenotype than the wild-type. Strain WR6-35 contained a single copy of Tn5 and the observed mucoid phenotype, acid sensitivity and Tn5-induced kanamycin resistance were 100% co-transducible using phage RL38. WR6-35 produced threefold more exopolysaccharide (EPS) than the wild-type in minimal medium devoid of a nitrogen source. EPS produced by the mutant and the wild-type was identical as determined by proton NMR spectra. An EcoRI rhizobial fragment containing Tn5 and flanking rhizobial sequences was cloned from the mutant, restriction mapped and sequenced. There was extensive similarity between the ORF disrupted by Tn5 in R. leguminosarum bv. viciae WR6-35 and the exoR gene of Rhizobium (Sinorhizobium) meliloti Rm1021 (71.3% identity over 892 bp). At the protein level there was 70% identity and 93.3% similarity over 267 amino acids with the ExoR protein of R. meliloti Rm1021. Hydrophilicity profiles of the two proteins from these two rhizobia are superimposable. This gene in R. leguminosarum bv. viciae was thus designated exoR. The data suggest that Tn5 has disrupted a regulatory gene encoding a protein that negatively modulates EPS biosynthesis in R. leguminosarum bv. viciae WSM710. Despite earlier suggestions that EPS production and acid tolerance might be positively correlated, disruption of exoR in either R. leguminosarum bv. viciae or R. meliloti and its associated overproduction of EPS does not result in a more acid-tolerant phenotype than the wild-type when cultures are screened on conventional laboratory agar.
豌豆根瘤菌蚕豆生物变种WSM710（WR6 - 35）的一个轻度酸敏感突变体产生的菌落，其表型比野生型更呈黏液状。菌株WR6 - 35含有单拷贝的Tn5，观察到的黏液状表型、酸敏感性和Tn5诱导的卡那霉素抗性使用噬菌体RL38共转导率为100%。在无氮源的基本培养基中，WR6 - 35产生的胞外多糖（EPS）比野生型多三倍。通过质子核磁共振光谱测定，突变体和野生型产生的EPS是相同的。从突变体中克隆出一个含有Tn5和侧翼根瘤菌序列的EcoRI根瘤菌片段，进行了限制性图谱分析和测序。在豌豆根瘤菌蚕豆生物变种WR6 - 35中被Tn5破坏的开放阅读框与苜蓿根瘤菌（中华根瘤菌）Rm1021的exoR基因有广泛的相似性（在892 bp上有71.3%的同一性）。在蛋白质水平上，与苜蓿根瘤菌Rm1021的ExoR蛋白在267个氨基酸上有70%的同一性和93.3%的相似性。这两种来自这两种根瘤菌的蛋白质的亲水性图谱是可叠加的。因此，豌豆根瘤菌蚕豆生物变种中的这个基因被命名为exoR。数据表明，Tn5破坏了一个编码蛋白质的调控基因，该蛋白质在豌豆根瘤菌蚕豆生物变种WSM710中对EPS生物合成起负调控作用。尽管早期有观点认为EPS产生和耐酸性可能呈正相关，但当在传统实验室琼脂上筛选培养物时，豌豆根瘤菌蚕豆生物变种或苜蓿根瘤菌中exoR的破坏及其相关的EPS过量产生并没有导致比野生型更耐酸的表型。
