紫云英根瘤菌由种子浸提物诱导的基因调控片段的克隆和分析

The large plasmids of strain 7653R were digested with restriction enzyme EcoRI. Their DNA fragments were cloned into the expression vector pMP220 to construct a lacZ fusion pool, which were transferred into the recipient strain 7653R Tri-transconjugants were selected onto plates containing X-gal and seed extract Five blue colonies were assayed of their β-galactosidase activity after incubation with or without seed extract. A positive induced strain HN18 was obtained. Hybridization of nodDABC probe on the re…     

紫云英根瘤菌糖基转移酶基因exoL的定位突变及序列分析

利用转座子Tn5对质粒pJB-B6既定位诱变,经同源交换,筛选获得一株Rhizobiumhuakuii107胞外糖合成缺陷（Exo－）变种RH983。三亲本杂交实验显示,pJB-B6及其亚克隆pJB-B601均可纠正变种RH983的胞外多糖合成缺陷。pJB-B601的2．3kbDNA片段的核苷酸顺序表明,该片段内存在一个完整的开放阅读框架（ORF）。ORF全长1170bp,编码390个氨基酸的蛋白,该蛋白与Rhi－zobiummeliloti的糖基转移酶ExoL有56．7％．的同源性,称为RhexoL。利用启动子探测质粒,构建了RhexoL-lacZ转录融合子,发现RhexoL基因5’上游有较强的启动子活性。     

Enhanced remediation of chlorpyrifos by ryegrass (Lolium multiflorum) and a chlorpyrifos degrading bacterial endophyte Mezorhizobium sp. HN3

For effective remediation of contaminants, plant-endophyte partnership is a promising field to be explored. Generally endophytic bacteria assist their host plant by withstanding the stress induced by the contaminants. The objective of this study was to explore the suitability of plant-bacterial partnership for chlorpyrifos (CP) remediation using ryegrass and a CP degrading endophyte, Mesorhizobium sp. HN3 which belongs to plant growth promoting rhizobia. The inoculated yfp-tagged Mesorhizobium sp. HN3 efficiently colonized in the rhizosphere, enhanced plant growth and degradation of CP and its metabolite 3,5,6 trichloro-2-pyridinol (TCP). Significantly lower CP residues were observed in the roots and shoots of plants vegetated in inoculated soil which might be attributed to the efficient root colonization of HN3yfp. These results suggest the involvement of Mesorhizobium sp. HN3yfp in CP degradation inside the roots and rhizosphere of plants and further emphasize on the effectiveness of endophytic bacteria in stimulating the remediation of pesticide contaminants. This is the first report which demonstrates the efficacy of bacterial endophyte for degradation of CP residues taken up by the plant and enhanced remediation of chlorpyrifos contaminated soil.     

百脉根根瘤菌群体感应相关基因的克隆与表达

在实验室的纯培养条件下,检测不到百脉根根瘤菌自体诱导物的产生,但是通过基因序列同源性比对分析发现,百脉根根瘤菌基因组中至少含有4种自体诱导物合成酶基因;将其中一个自体诱导物合成酶基因ml4543克隆到大肠杆菌表达载体pET30a,构建得到能异源表达该基因的大肠杆菌重组菌株;对该大肠杆菌重组菌株进行自体诱导物检测发现,该合成酶基因在大肠杆菌中能合成至少3种自体诱导物因子。     

Characterization of a periplasmic S1-like nuclease coded by the Mesorhizobium loti symbiosis island

DNA sequences encoding hypothetical proteins homologous to S1 nuclease from Aspergillus oryzae are found in many organisms including fungi, plants, pathogenic bacteria, and eukaryotic parasites. One of these is the M1 nuclease of Mesorhizobium loti which we demonstrate herein to be an enzymatically active, soluble, and stable S1 homolog that lacks the extensive mannosyl-glycosylation found in eukaryotic S1 nuclease homologs. We have expressed the cloned M1 protein in M. loti and purified recombinant native M1 to near homogeneity and have also isolated a homogeneous M1 carboxy-terminal hexahistidine tag fusion protein. Mass spectrometry and N-terminal Edman degradation sequencing confirmed the protein identity. The enzymatic properties of the purified M1 nuclease are similar to those of S1. At acidic pH M1 is 25 times more active on single-stranded DNA than on double-stranded DNA and 3 times more active on single-stranded DNA than on single-stranded RNA. At neutral pH the RNase activity of M1 exceeds the DNase activity. M1 nicks supercoiled RF-I plasmid DNA and rapidly cuts the phosphodiester bond across from the nick in the resultant relaxed RF-II plasmid DNA. Therefore, M1 represents an active bacterial S1 homolog in spite of great sequence divergence. The biochemical characterization of M1 nuclease supports our sequence alignment that reveals the minimal 21 amino acid residues that are necessarily conserved for the structure and functions of this enzyme family. The ability of M1 to degrade RNA at neutral pH implies previously unappreciated roles of these nucleases in biological systems.     

The conserved glycine/alanine residue of the active-site loop containing the putative acetylCoA-binding motif is essential for the overall structural integrity of Mesorhizobium loti arylamine N-acetyltransferase 1

The arylamine N-acetyltransferases are important xenobiotic-metabolizing enzymes that catalyze an acetyl group transfer from acetylCoA to arylamine substrates. NAT enzymes possess an active-site loop (the active-site P-loop) involved in substrate binding and selectivity. The Gly/Ala residue present at the start of the active-site P-loop, although conserved in all NAT enzymes, is not involved in the catalytic mechanism or substrate binding. Here we show that a small amino acid (such as Gly or Ala) at this position is important not only for maintaining the functions of the active-site P-loop but, more surprisingly, also important for maintaining the overall structural integrity of NAT enzymes. Our data thus suggest that in addition to its role in substrate binding and selectivity, the active-site P-loop could play a wider structural role in NAT enzymes.     

Different Mesorhizobium species sharing the same symbiotic genes nodulate the shrub legume Anagyris latifolia

The isolation and characterization of six rhizobial strains isolated from Anagyris latifolia, a shrub legume endemic to the Canary Islands, is reported in this study. The isolates were characterized by 16S-ARDRA, and sequencing of the ribosomal 16S rRNA gene, the 16S–23S rDNA intergenic spacer region, and the housekeeping gene for glutamine synthetase II (glnII). The phylogenies based on the three types of sequences matched, showing that the isolates belonged to three distinct lineages within the genus Mesorhizobium that could represent different species. However, the ribosomal and glnII phylogenies revealed some discrepancies in the relationships between the isolates and the named species in this genus. Despite their different taxonomic affiliation, all the isolates showed identical nodC sequences which were closely related (95% similarity) to that of the Mesorhizobium tianshanense type strain, indicating that they must have acquired the nodulation genes by a phenomenon of lateral gene transfer.