Search

Glycyrrhiza uralensis Fisch is a widely cultivated traditional Chinese medicine plant. In the present study, culture-independent microbial diversity analysis and functional prediction of rhizosphere microbes associated with wild and cultivated G. uralensis Fisch plant (collected from two locations) were carried. Soil physicochemical parameters were tested to assess their impact on microbial communities. A total of 4428 OTUs belonging to 41 bacterial phyla were identified. In general, cultivated sample sites were dominated by Actinobacteria whereas wild sample sites were dominated by Proteobacteria. The alpha diversity analysis showed the observed species number was higher in cultivated soil samples when compared with wild soil samples. In beta diversity analysis, it was noticed that the weighted-unifrac distance of two cultivated samples was closer although the samples were collected from different regions. Functional annotation based on PICRUST and FAPROTAX showed that the nitrogen metabolism pathway such as nitrate reduction, nitrogen fixation, nitrite ammonification, and nitrite respiration were more abundant in rhizosphere microorganisms of wild G. uralensis Fisch. These results also correlate in redundancy analysis results which show correlation between NO 3- -N and wild samples, which indicated that nitrogen nutrition conditions might be related to the quality of G. uralensis Fisch., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A novel Gram-staining negative, motile, rod-shaped and aerobic bacterial strain, designated EGI 60010(T), was isolated from healthy roots of Glycyrrhiza uralensis F. collected from Yili County, Xinjiang Province, North-West China. The 16S rRNA gene sequence of strain EGI 60010(T) showed 97.2 % sequence similarities with Ochrobactrum anthropi ATCC 49188(T) and Ochrobactrum cytisi ESC1(T), and 97.1 % with Ochrobactrum lupini LUP21(T). The phylogenetic analysis based on 16S rRNA gene sequences showed that the new isolate clustered with members of the genera Ochrobactrum, and formed a distinct clade in the neighbour-joining tree. Q-10 was identified as the respiratory quinone for strain EGI 60010(T). The major fatty acids were summed feature 8 (C18:1 ω6c and/or C18:1 ω7c), C19:0 cyclo ω8c, summed feature 4 (C17:1 iso I/anteiso B) and C16:0. The polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol and phosphatidylcholine. The DNA G+C content of strain EGI 60010(T) was determined to be 60.4 mol%. The genomic DNA relatedness values determined between strain EGI 60010(T) and the closely related strains O. anthropi JCM 21032(T), O. cytisi CCTCC AB2014258(T) and O. lupini NBRC 102587(T) were 50.3, 50.0 and 41.6 %, respectively. Based on the results of the molecular studies supported by its differentiating phenotypic characteristics, strain EGI 60010(T) was considered to represent a novel species within the genus Ochrobactrum, for which the name Ochrobactrum endophyticum sp. nov., is proposed. The type strain is EGI 60010(T) (=CGMCC 1.15082(T) = KCTC 42485(T) = DSM 29930(T)).

A novel Gram-staining-negative, non-motile, rod-shaped and aerobic bacterial strain, designated EGI 60015(T), was isolated from healthy roots of Glycyrrhiza uralensis F. collected from Yili County, Xinjiang Province, Northwest China. The 16S rRNA gene sequence of strain EGI 60015(T) was found to show 97.6% sequence similarity with Novosphingobium pentaromativorans US6-1(T). The phylogenetic analysis based on 16S rRNA gene sequences showed that the strain formed a clade with N. pentaromativorans US6-1(T) in the neighbor-joining tree. Q-10 was identified as the respiratory quinone of strain EGI 60015(T). The major fatty acids were summed feature 8 (C18:1 ω6c and/or C18:1 ω7c; 55.04%), summed feature 4 (C17:1 anteiso B and/or iso I; 18.34%) and summed feature 3 (C16:1 ω6c and/or C16:1 ω7c; 8.53%). The polar lipids detected were phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and sphingoglycolipids. The DNA G+C content of strain EGI 60015(T) was determined to be 66.6 mol%. The genomic DNA relatedness value between EGI 60015(T) and N. pentaromativorans US6-1(T) (54%) was below the 70% limit for species identification. Based on the result of the molecular studies supported by its morphological, physiological, chemotaxonomic and other differentiating phenotypic characteristics, strain EGI 60015(T) was considered to represent a novel species within the genus Novosphingobium, for which the name Novosphingobium endophyticum sp. nov. is proposed. The type strain is EGI 60015(T) (=CGMCC 1.15095(T) = KCTC 42486(T) = DSM 29948(T)).

Mesorhizobium tianshanense employs MsiA as canavanine exporter, which is upregulated by MsiR, to successfully form a symbiosis with the legume Glycyrrhiza uralensis. In this research, through gel-shift and bacterial two-hybrid examination, MsiR was found to spontaneously form dimers and bind to msiA promoter without additional canavanine. Six truncated forms of MsiR were constructed, and the conserved helix-turn-helix (HTH), substrate-binding, and surface-loop domains were found essential for MsiR functions. Random mutagenesis was used to study the functional sites of MsiR. Seven point mutants were selected, in which three mutants constitutively induced msiA expression without additional canavanine, two mutants partially changed substrate specificity, and the other two were almost null mutants. Results from the site mutation show that the functional subunits (HTH domain, dimerization interface domains, and C-terminal) are important in the conformation and induction ability of MsiR.

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharide (KPS) biosynthesis, is constituted by the rkpU, rkpAGHIJ, and kpsF3 genes. Two mutants in this region affecting the rkpA (SVQ536) and rkpI (SVQ538) genes were constructed. Polyacrylamide gel electrophoresis and (1)H-NMR analyses did not detect KPS in these mutants. RT-PCR experiments indicated that, most probably, the rkpAGHI genes are cotranscribed. Glycine max cultivars (cvs.) Williams and Peking inoculated with mutants SVQ536 and SVQ538 showed reduced nodulation and symptoms of nitrogen starvation. Many pseudonodules were also formed on the American cv. Williams but not on the Asiatic cv. Peking, suggesting that in the determinate nodule-forming S. fredii-soybean symbiosis, bacterial KPS might be involved in determining cultivar-strain specificity. S. fredii HH103 mutants unable to produce KPS or exopolysaccharide (EPS) also showed reduced symbiotic capacity with Glycyrrhiza uralensis, an indeterminate nodule-forming legume. A HH103 exoA-rkpH double mutant unable to produce KPS and EPS was still able to form some nitrogen-fixing nodules on G. uralensis. Thus, here we describe for the first time a Sinorhizobium mutant strain, which produces neither KPS nor EPS is able to induce the formation of functional nodules in an indeterminate nodule-forming legume.

LuxR/LuxI-type quorum-sensing systems have been shown to be important for symbiotic interactions between a number of rhizobium species and host legumes. In this study, we found that different isolates of Mesorhizobium tianshanense, a moderately-growing Rhizobium that forms nodules on a number of types of licorice plants, produces several different N-acyl homoserine lactone-like molecules. In M. tianshanense CCBAU060A, we performed a genetic screen and identified a network of regulatory components including a set of LuxI/LuxR-family regulators as well as a MarR-family regulator that is required for quorum-sensing regulation. Furthermore, compared with the wild-type strains, quorum-sensing deficient mutants showed a reduced growth rate and were defective in nodule formation on their host plant Glycyrrhiza uralensis. These data suggest that different M. tianshanense strains may use diverse quorum-sensing systems to regulate symbiotic process.

Mesorhizobium tianshanense is a nitrogen-fixing bacterium that can establish symbiotic associations with Glycyrrhiza uralensis in the form of root nodules. Nodule formation in rhizobia often requires various secreted carbohydrates. To investigate exopolysaccharide (EPS) production and function in M. tianshanense, we performed a genome-wide screen using transposon mutagenesis to identify genes involved in EPS production. We identified seven mutants that produced significantly lower amounts of EPS as well as a two-component sensor kinase/response regulator system that is involved in the activation of EPS synthesis. EPS mutants formed significantly less biofilm and displayed severely reduced nodulation capacity than wild type bacteria, suggesting that EPS synthesis can play important roles in the symbiosis process.