Regulation of urease activity in Rhizobium meliloti

Abstract Using an ureC-lacZ fusion, the expression of urease structural genes of the soil bacterium Rhizobium meliloti strain AK631 was studied in response to different nitrogen sources and nickel contents in the growth medium. The expression of urease genes is repressed by ammonia and is not inducible by urea. Urease activity depends on the nickel concentration of the medium. Nickel uptake is repressed in medium containing ammonia and is not affected by the genes located in the urease operon investigated.     

Cloning of the second adenylate cyclase gene (cya2) from Rhizobium meliloti F34: Sequence similarity to eukaryotic cyclases

Abstract A second adenylate cyclase ( cya2 ) gene was isolated from a Rhizobium meliloti F34 gene bank. Complemented E. coli Acya mutants were capable of utilizing a number of, but not all, carbon sources known to be regulated by cAMP. DNA hybridization studies showed cya2 to be unique to R. meliloti strains. The cya2 nucleotide sequence was determined and found to encode a protein of 363 amino acids. Residues were identified within the C-terminal domain which are conserved in both eukaryotic adenylate and guanylate cyclases, including a putative ATP binding site. Similiar residues were also found in the prokaryotic R. meliloti Cya1 protein. A R. meliloti cyal/cya2 double mutant was constructed and characterized; however, cAMP production was still observed in this strain indicating the presence of a third cya gene.     

Legacy of prior host and soil selection on rhizobial fitness in planta

Measuring selection acting on microbial populations in natural or even seminatural environments is challenging because many microbial populations experience variable selection. The majority of rhizobial bacteria are found in the soil. However, they also live symbiotically inside nodules of legume hosts and each nodule can release thousands of daughter cells back into the soil. We tested how past selection (i.e., legacies) by two plant genotypes and by the soil alone affected selection and genetic diversity within a population of 101 strains of Ensifer meliloti. We also identified allelic variants most strongly associated with soil‐ and host‐dependent fitness. In addition to imposing direct selection on rhizobia populations, soil and host environments had lasting effects across host generations. Host presence and genotype during the legacy period explained 22% and 12% of the variance in the strain composition of nodule communities in the second cohort, respectively. Although strains with high host fitness in the legacy cohort tended to be enriched in the second cohort, the diversity of the strain community was greater when the second cohort was preceded by host rather than soil legacies. Our results indicate the potential importance of soil selection driving the evolution of these plant‐associated microbes.     

苜蓿中华根瘤菌desA基因功能的鉴定

为研究苜蓿中华根瘤菌脂肪酸脱饱和酶desA基因在不饱和脂肪酸合成、共生结瘤固氮以及应对逆境胁迫中的功能,为高效利用苜蓿中华根瘤菌提供理论依据,本文通过异体遗传互补和脂肪酸组成薄层层析,分析SmdesA编码蛋白是否具有脱饱和酶的活性并参与不饱和脂肪酸的合成,构建SmdesA的缺失突变株和互补菌株,比较各菌株在不同逆境胁迫条件下的生长速率以及回接宿主植物后与紫花苜蓿共生结瘤的能力.结果表明SmdesA不能互补大肠杆菌CY57中EcfabA的突变,但具有将饱和脂肪酸脱饱和形成不饱和的棕榈油酸和十八碳烯酸的能力.另外,SmdesA缺失突变对苜蓿中华根瘤菌的脂肪酸组成影响不大,但会显著影响低温和高盐条件下菌株的生长速率以及与紫花苜蓿共生结瘤的能力.我们推测,SmdesA参与的脱饱和途径可能是苜蓿中华根瘤菌不饱和脂肪酸合成的补偿途径,其编码的蛋白DesA不是不饱和脂肪酸合成的关键酶,但在应对逆境胁迫和共生结瘤中具有重要的生物学功能.     

Colonization behaviour of Pseudomonas fluorescens and Sinorhizobium meliloti in the alfalfa (Medicago sativa) rhizosphere

The colonization ability of Pseudomonas fluorescens F113rif in alfalfa rhizosphere and its interactions with the alfalfa microsymbiont Sinorhizobium meliloti EFB1 has been analyzed. Both strains efficiently colonize the alfalfa rhizosphere in gnotobiotic systems and soil microcosms. Colonization dynamics of F113rif on alfalfa were similar to other plant systems previously studied but it is displaced by S. meliloti EFB1, lowering its population by one order of magnitude in co-inoculation experiments. GFP tagged strains used to study the colonization patterns by both strains indicated that P. fluorescens F113rif did not colonize root hairs while S. meliloti EFB1 extensively colonized this niche. Inoculation of F113rif had a deleterious effect on plants grown in gnotobiotic systems, possibly because of the production of HCN and the high populations reached in these systems. This effect was reversed by co-inoculation. Pseudomonas fluorescens F113 derivatives with biocontrol and bioremediation abilities have been developed in recent years. The results obtained support the possibility of using this bacterium in conjunction with alfalfa for biocontrol or rhizoremediation technologies.     

费氏中华根瘤菌HN01DL在大豆根圈的定殖动态与结瘤研究

以发光酶基因luxAB为标记,在根盒缩影条件下研究了费氏中华根瘤菌HN01DL在大豆根圈的定殖动态、分布范围及其结瘤情况.结果表明,HN01DL在根盒灭菌土壤和非灭菌土壤缩影中的大豆全根系定殖动态与水平明显不同,前者在第12d时达到最高定殖密度(8.65logcfu·g^-1),而后者的早期定殖数量下降较快,且于第15d时达到最高定殖密度(6.88logcfu·g^-1).HN01DL在大豆播种5d后在大豆根部的A(0～4cm)区根段上达到最高定殖密度(7.05logcfu·g^-1),然后开始缓慢下降,至第19d时仍维持相对稳定,在第33d时又开始回升.至播种后第46d时HN01DL可散布至种子下方16cm处的根段部位.HN01DL在A区根段的定殖密度持续较高,所形成的发光根瘤总数(16.3个)及发光比例(68.8%)最高,且发光根瘤主要集中于该区段主根上.发光根瘤比例沿A-E区段逐渐下降,在E区段未检测到发光根瘤.     

苜蓿中华根瘤菌(Sinorhizobium meliloti)LuxR家族转录因子ExpR调节motC操纵子的表达

目前已知苜蓿中华根瘤菌(S.meliloti)Rm1021 ExpR 突变导致胞外多糖Ⅱ(EPSⅡ)的过量表达,而胞外多糖是根瘤菌成功侵染宿主植物形成有效根瘤必需的物质。软琼脂板实验发现ExpR 突变株运动能力有缺陷。但是鞭毛染色实验并没有检测到突变株的鞭毛与野生型有什么不同。通过启动子-lacZ融合子进一步研究突变株中基因表达的差异发现,ExpR以细胞密度依赖的方式调节motC操纵子的表达。由此可见,在苜蓿中华根瘤菌中,ExpR同时参与了胞外多糖Ⅱ的合成和细胞运动能力的调节。     

Sinorhizobium morelens S-5中N-氨甲酰基水解酶基因的克隆、表达和纯化

N-氨甲酰基水解酶是一种非常具有工业应用价值的水解酶,可用于制备光学纯氨基酸。通过LA PCR从Sinorhizobium morelensS-5菌中克隆到1.3kb的DNA片段,测序表明该片段上含有一个完整的N-氨甲酰基水解酶的基因(hyuC)序列。将hyuC基因克隆到表达载体pET30a上,重组质粒pET30a-HyuC在大肠杆菌中获得了高水平表达。重组的N-氨甲酰基水解酶经过热处理和三步柱色谱分离而纯化。纯化倍数为16.1倍,收率21.2%。该酶为同源四聚体,亚基分子量是38kDa。最适温度是60℃,最适pH为7.0。该酶有较高的热稳定性和氧化稳定性。Fe2 和Ca2 对酶的活性有一定的促进作用,而金属螯合剂和巯基试剂对酶活无明显影响。     

Charcoal and shrubs modify soil processes in ponderosa pine forests of western Montana

In split-root systems of alfalfa (Medicago sativa L.), already existing nodules or arbuscular mycorrhizal roots suppress further establishment of symbiosis in other root parts, a phenomenon named autoregulation. Roots treated with rhizobial nodulation signals (Nod factors) induce a similar systemic suppression of symbiosis.In order to test the hypothesis that flavonoids play a role in this systemic suppression, split-root systems of alfalfa plants were inoculated on one side of the split-root system with Sinorhizobium meliloti or Glomus mosseae or were treated with Nod factor. HPLC-analysis of alfalfa root extracts from both sides of the split-root system revealed a persistent local and systemic accumulation pattern of some flavonoids associated with the different treatments. The two flavonoids, formononetin and ononin, could be identified to be similarily altered after rhizobial or mycorrhizal inoculation or when treated with Nod factor.Exogenous application of formononetin and ononin partially restored nodulation and mycorrhization pointing towards the involvement of these two secondary compounds in the autoregulation of both symbioses.