Chitinolytic and cellulolytic Pseudomonas sp. antagonistic to fungal pathogens enhances nodulation by Mesorhizobium sp. Cicer in chickpea.

Pseudomonas strains isolated from the rhizosphere of chickpea (Cicer arietinum L.) and green gram (Vigna radiata L.) were screened for the production of chitinases and cellulases. Five Pseudomonas strains were found to produce appreciable amounts of both enzymes in culture-free supernatants and showed growth inhibition of the two fungi Pythium aphanidermatum (Oomycete) and Rhizoctonia solani (Basidiomycete) in plates on potato dextrose agar medium. The fungal growth inhibition was not correlated with cell wall-degrading enzyme activity, which suggested that other antifungal compounds produced by these rhizobacteria were also involved in antagonism. Coinoculation of the Pseudomonas strains with the Mesorhizobium sp. Cicer strain Ca181 resulted in a significant increase in nodule biomass when grown under sterilized chillum jar conditions. The results suggest that hydrolytic enzymes produced by Pseudomonas sp. contribute to suppression of plant diseases by inhibiting growth of phytopathogenic fungi and also promote nodulation of legumes by rhizobia.     

Biochemical,physiological and molecular characteristics of chickpea rhizobacteria from Kurdistan province,Iran

Kurdistan province of Iran is one of the main places for producing chickpea, and there is no published research on root-nodulating bacteria of this crop. Plant samples were collected and a total of 73 Rhizobium strains were isolated from root nodules. Nodulation test was done on chickpea plants. Phenotypic characteristics of the 16 representative strains were determined based on the standard bacteriological methods. Total soluble cell protein patterns by electrophoresis approach (SDS-PAGE) showed heterogeneity among the tested rhizobia strains. Based on the phenotypic features, Rhizobium strains of three groups belong to different species of the genus Mesorhizobioum including M. ciceri and M. mediterraneum and Mesorhizobium sp. The PCR technique was employed for amplification of 16S rDNA and atpD genes. For further characterisation, amplified fragment of 16S rDNA gene from a representative strain (AK21) using primers 41F and 1488R was subjected to sequencing. Sequences were aligned by BLAST software at NCBI GenBank and results showed 99% similarity with M. mediterraneum strain BKBCF3q.     

Fatty acid composition of Mesorhizobium huakuii lipopolysaccharides. Identification of 27-oxooctacosanoic acid

Lipopolysaccharides of three Mesorhizobium huakuii strains carried a number of amide-linked 3-hydroxylated fatty acids including: 3-OH-12:0, 3-OH-i-13:0, 3-OH-20:0, 3-OH-i-21:0, 3-OH-22:0, 3-OH-23:0 and unsaturated 3-OH-22:1. The first three of the above mentioned acids are the main amide-linked fatty acids in the LPS preparations. The main ester-bound fatty acids comprise 16:0, i-17:0, 18:0, 20:0 and 27-OH-28:0. Among minor constituents of lipid A 25-OH-26:0 and 29-OH-30:0 together with some non-polar fatty acids were found. Additionally, the presence of 4-oxo-20:0, 4-oxo-i-21:0 and 4-oxo-22:0 amide-bound fatty acids as well as the 27-oxo-28:0 ester-linked fatty acid were proved. To our knowledge oxo fatty acids are rare constituents of lipopolysaccharides and 27-oxo-28:0 was found for the first time in the LPS preparations from members of Rhizobiaceae.     

A Large Scale Analysis of Protein-Protein Interactions in the Nitrogen-fixing Bacterium Mesorhizobium loti

Global viewing of protein–protein interactions (PPIs)is a useful way to assign biological roles to large numbersof proteins predicted by complete genome sequence. Here, wesystematically analyzed PPIs in the nitrogen-fixing soil bacteriumMesorhizobium loti using a modified high-throughput yeast two-hybridsystem. The aims of this study are primarily on the providingfunctional clues to M. loti proteins that are relevant to symbioticnitrogen fixation and conserved in other rhizobium species,especially proteins with regulatory functions and unannotatedproteins. By the screening of 1542 genes as bait, 3121 independentinteractions involving 1804 proteins (24% of the total proteincoding genes) were identified and each interaction was evaluatedusing an interaction generality (IG) measure and the generalfeatures of the interacting partners. Most PPIs detected inthis study are novel interactions revealing potential functionalrelationships between genes for symbiotic nitrogen fixationand signal transduction. Furthermore, we have predicted theputative functions of unannotated proteins through their interactionswith known proteins. The results described here represent newinsight into protein network of M. loti and provide useful experimentalclues to elucidate the biological function of rhizobial genesthat can not be assigned directly from their genomic sequence.     

Characterization of novel 2-oxoglutarate dependent dioxygenases converting l-proline to cis-4-hydroxy-l-proline

Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing l-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-l-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-l-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel l-proline cis-4-hydroxylases converting free l-proline to cis-4-hydroxy-l-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected l-proline cis-4-hydroxylase activity in both proteins. Likewise l-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).     

华癸根瘤菌7653R hfq基因突变株的构建及其生物学特性

【目的】研究hfq基因在Mesorhizobium huakuii 7653R抵抗外界不利环境和共生固氮中的功能特性。【方法】利用pK19mob同源重组方法构建7653R hfq基因的插入失活突变株7653RΔhfq,并构建互补菌株7653RΔhfq-C,对hfq在压力胁迫和共生固氮中的功能特性进行研究。【结果】与野生型7653R相比,突变株7653RΔhfq的生长速率降低,热激处理后致死率升高;hfq突变影响了7653R中部分sRNA的表达;在4.5%乙醇和50 mmol H_2O_2生长胁迫下,突变株适应性明显较野生型差。另外,接种突变株的紫云英结瘤能力和固氮酶活性都明显降低。【结论】hfq基因作为重要的转录后调控因子,在7653R抵御外界胁迫环境和与宿主紫云英的共生固氮中发挥了重要作用。     

Approaches for enhancement of N2 fixation efficiency of chickpea (Cicer arietinum L.) under limiting nitrogen conditions

Chickpea (Cicer arietinum) is an important pulse crop in many countries in the world. The symbioses between chickpea and Mesorhizobia, which fix N₂ inside the root nodules, are of particular importance for chickpea's productivity. With the aim of enhancing symbiotic efficiency in chickpea, we compared the symbiotic efficiency of C‐15, Ch‐191 and CP‐36 strains of Mesorhizobium ciceri in association with the local elite chickpea cultivar ‘Bivanij’ as well as studied the mechanism underlying the improvement of N₂ fixation efficiency. Our data revealed that C‐15 strain manifested the most efficient N₂ fixation in comparison with Ch‐191 or CP‐36. This finding was supported by higher plant productivity and expression levels of the nifHDK genes in C‐15 nodules. Nodule specific activity was significantly higher in C‐15 combination, partially as a result of higher electron allocation to N₂ versus H⁺. Interestingly, a striking difference in nodule carbon and nitrogen composition was observed. Sucrose cleavage enzymes displayed comparatively lower activity in nodules established by either Ch‐191 or CP‐36. Organic acid formation, particularly that of malate, was remarkably higher in nodules induced by C‐15 strain. As a result, the best symbiotic efficiency observed with C‐15‐induced nodules was reflected in a higher concentration of the total and several major amino metabolites, namely asparagine, glutamine, glutamate and aspartate. Collectively, our findings demonstrated that the improved efficiency in chickpea symbiotic system, established with C‐15, was associated with the enhanced capacity of organic acid formation and the activities of the key enzymes connected to the nodule carbon and nitrogen metabolism.     

紫云英根瘤菌共同结瘤基因nodA和nodBC的核苷酸序列

以~(32)p标记的苜蓿根瘤菌(Rhizobium meliloti)2.3kb nod DNA作探针,从紫云英根瘤菌(Rhizobium huakuii即R. astragali)159基因文库中分离到一株能与探针DNA呈阳性反应的克隆pRaN109。同源DNA-DNA杂交及DNA序列分析表明:pRaN109DNA的9kb EcoRI片段上携带了nodD_1BC基因,pRaN109 NDA的18kb EcoRI片段上携带了nodD_2A基因。共同结瘤基因nodA与nodBC两者相距6.7kb。在nodA基因和nodBC基因的上游都存在有结瘤盒(nod box)。与来自不同种属的菌株所报告的结果相比较,紫云英根瘤菌159中的共同结瘤基因有着明显不同的组合。     

紫云英根瘤菌胞外多糖合成基因exo1的序列分析

对互补紫云英根瘤菌Ｅｘｏ－Ｎｄｖ－Ｆｉｘ－变种的２．６ｋｂＤＮＡ片段进行序列测定。分析结果表明,该片段内存在一个完整的阅读框架（ＯＲＦ）ｅｘｏ１。ｅｘｏ１编码３４０个氨基酸,为细胞质蛋白。Ｅｘｏ１蛋白序列与苜蓿根瘤菌的糖基转移酶ＥｘｏＵ有较强的同源性。利用启动子检测质粒,分析ｅｘｏ１基因的表达,发现在ｅｘｏ１基因上有两个启动子活性区段,Ｐｅｘｏ１ａ位于基因前端,Ｐｅｘｏ１ｂ位于基因中间。Ｐｅｘｏ１ａ很可能包含了ｅｘｏ１基因的启动子。     

Isolation of the PCB-degrading bacteria Mesorhizobium sp. ZY1 and its combined remediation with Astragalus sinicus L. for contaminated soil

A bacterial strain ZY1 capable of utilizing PCBs as its carbon source was isolated from the root nodules of Chinese milk vetch (Astragalus sinicus L.). The strain was identified as Mesorhizobium sp. according to its physiological-biochemical properties and the analysis of its 16S rRNA gene sequence. When the initial OD600 was 0.15, 62.7% of 15 mg L^?1 3,3′,4,4′-TCB in a liquid culture was degraded by Mesorhizobium sp. ZY1 within 10 days. Mesorhizobium sp. ZY1 also greatly increased the biotransformation of soil PCBs. Pot experiments indicated that the soil PCB concentrations of a single incubation of strain ZY1 (R) and a single planting of A. sinicus (P) decreased by 20.5% and 23.0%, respectively, and the concentration of PCBs in soil treated with A. sinicus and strain ZY1 decreased by 53.1%. We also observed that A. sinicus-Mesorhizobium sp. ZY1 treatment (PR) improved plant biomass and the concentration of PCBs in plants compared with a single A. sinicus planting treatment (P). The results suggest that the synergistic association between A. sinicus and PCBs-degrading Mesorhizobium sp. ZY1 can stimulate the phytoextraction of PCBs and the rhizosphere microflora to degrade PCBs, and might be a promising bioremediation strategy for PCB-contaminated soil.