Overexpression of hns in the plant growth‐promoting bacterium Enterobacter cloacae UW5 increases root colonization

Aims: Plant growth‐promoting rhizobacteria (PGPR) introduced into soil often do not compete effectively with indigenous micro‐organisms for plant colonization. The aim of this study was to identify novel genes that are important for root colonization by the PGPR Enterobacter cloacae UW5. Methods and Results: A library of transposon mutants of Ent. cloacae UW5 was screened for mutants with altered ability to colonize canola roots using a thermal asymmetric interlaced (TAIL)‐PCR‐based approach. A PCR fragment from one mutant was reproducibly amplified at greater levels from genomic DNA extracted from mutant pools recovered from seedling roots 6 days after seed inoculation compared to that from the cognate inoculum cultures. Competition assays confirmed that the purified mutant designated Ent. cloacae J28 outcompetes the wild‐type strain on roots but not in liquid cultures. In Ent. cloacae J28, the transposon is inserted upstream of the hns gene. Quantitative RT‐PCR showed that transposon insertion increased expression of hns on roots. Conclusions: These results indicate that increased expression of hns in Ent. cloacae enhances competitive colonization of roots. Significance and Impact of the Study: A better understanding of the genes involved in plant colonization will contribute to the development of PGPR that can compete more effectively in agricultural soils.     

An ACC Deaminase Minus Mutant of Enterobacter cloacae UW4No Longer Promotes Root Elongation

The ACC deaminase gene (acdS) from Enterobacter cloacae UW4 was replaced by homologous recombination with the acdS gene with a tetracycline resistance gene inserted within the coding region. Upon characterization of this AcdS minus mutant, it was determined that both ACC deaminase activity and the ability to promote the elongation of canola roots under gnotobiotic conditions were greatly diminished. This result is consistent with a previously postulated model that suggests that a major mechanism utilized by plant growth-promoting bacteria involves the lowering of plant ethylene levels, and hence ethylene inhibition of root elongation, by bacterial ACC deaminase. Received: 20 January 2000 / Accepted: 22 February 2000     

阴沟肠杆菌nifL对nifA蛋白激活固氮基因表达的影响

通过基因定位突变的方法 ,构建了阴沟肠杆菌E2 6染色体上基因nifL突变菌E11和E12 ,并对其固氮特性进行分析。结果显示 ,当细菌在有NH 4 的条件下 ,nifL作为固氮基因的负调节因子 ,可能是通过nifL蛋白与nifA蛋白之间的相互作用 ,形成某种复合物 ,导致NifA的失活 ,从而关闭nif基因的表达。高温条件下 (37℃ ) ,nifL并不参与对nif基因表达的阻遏作用 ,但是nifL的突变影响NifA激活nif基因表达对热的稳定性     

The Arabidopsis MYB5 Transcription Factor Regulates Mucilage Synthesis,Seed Coat Development,and Trichome Morphogenesis

The Arabidopsis thaliana MYB5 gene is expressed in trichomes and seeds, including the seed coat. Constitutive expression of MYB5 resulted in the formation of more small trichomes and ectopic trichomes and a reduction in total leaf trichome numbers and branching. A myb5 mutant displayed minimal changes in trichome morphology, while a myb23 mutant produced increased numbers of small trichomes and two-branched trichomes. A myb5 myb23 double mutant developed more small rosette trichomes and two-branched trichomes than the single mutants. These results indicate that MYB5 and MYB23 regulate trichome extension and branching. The seed coat epidermal cells of myb5 and myb5 myb23 were irregular in shape, developed flattened columellae, and produced less mucilage than those of the wild type. Among the downregulated genes identified in the myb5 seeds using microarray analysis were ABE1 and ABE4 (α/β fold hydrolase/esterase genes), MYBL2, and GLABRA2. The same genes were also downregulated in transparent testa glabra1 (ttg1) seeds, suggesting that MYB5 collaborates with TTG1 in seed coat development. These genes were upregulated in leaves and roots by ectopically expressed MYB5. The MYBL2, ABE1, and ABE4 promoters were active in seeds, including seed coats, and the latter two also in trichomes. Models of the MYB5 regulatory networks involved in seed coat and trichome development are presented.     

Population genetics of the nomenspecies Enterobacter cloacae

The genetic heterogeneity of the nomenspecies Enterobacter cloacae is well known. Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter dissolvens, Enterobacter hormaechei, Enterobacter kobei, and Enterobacter nimipressuralis are closely related to it and are subsumed in the so-called E. cloacae complex. DNA-DNA hybridization studies performed previously identified at least five DNA-relatedness groups of this complex. In order to analyze the genetic structure and the phylogenetic relationships between the clusters of the nomenspecies E. cloacae, 206 strains collected from 22 hospitals, a veterinarian, and an agricultural center in 11 countries plus all 13 type strains of the genus and reference strain CDC 1347-71(R) were examined with a combination of sequence and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses of the three housekeeping genes hsp60, rpoB, and hemB as well as ampC, the gene of a class C beta-lactamase. Based on the neighbor-joining tree of the hsp60 sequences, 12 genetic clusters (I to XII) and an unstable sequence crowd (xiii) were identified. The robustness of the genetic clusters was confirmed by analyses of rpoB and hemB sequences and ampC PCR-RFLPs. Sequence crowd xiii split into two groups after rpoB analysis. Only three strains formed a cluster with the type strain of E. cloacae, indicating that the minority of isolates identified as E. cloacae truly belong to the species; 13% of strains grouped with other type strains of the genus, suggesting that the phenotypes of these species seem to be more heterogeneous than so far believed. Three clusters represented 70% of strains, but none of them included a type or reference strain. The genetic clustering presented in this study might serve as a framework for future studies dealing with taxonomic, evolutionary, epidemiological, or pathogenetic characteristics of bacteria belonging to the E. cloacae complex.     

Involvement of gacS and rpoS in enhancement of the plant growth-promoting capabilities of Enterobacter cloacae CAL2 and UW4

The plant growth-promoting bacteria Enterobacter cloacae CAL2 and UW4 were genetically transformed with a multicopy plasmid containing an rpoS or gacS gene from Pseudomonas fluorescens. The transformed strains were compared with the nontransformed strains for growth, indoleacetic acid (IAA) production, antibiotic production, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, siderophore production, cell morphology, and the ability to promote canola root elongation. All transformed strains had a longer lag phase, were slower in reaching stationary phase, and attained a higher cell density than the nontransformed strains. Transformation resulted in cells that were significantly shorter than the nontransformed cells. The transformed strains also produced significantly more IAA than the nontransformed strains. Introduction of rpoS or gacS from Pseudomonas fluorescens was associated with a reduction in the production of both antibiotics, 2,4-diacetylphloroglucinol and mono-acetylphloroglucinol, produced by Enterobacter cloacae CAL2. With Enterobacter cloacae CAL2, plasmid-borne rpoS, but not gacS, increased the level of ACC deaminase activity, while introduction of rpoS in Enterobacter cloacae UW4 caused a decrease in ACC deaminase activity. Neither gacS nor rpoS significantly affected the level of siderophores synthesized by either bacterial strain. Overproduction of either GacA or RpoS in Enterobacter cloacae CAL2 resulted in a significant increase in the root lengths of canola seedlings when seeds were treated with the bacteria, and overproduction of RpoS caused an increase in canola shoot as well as root lengths.     

玉米转录因子 ZmNAC59 调控植物抗盐性

【目的】NAC转录因子家族是植物中研究较多的一类转录因子,在调控植物生长发育和响应非生物胁迫过程中起重要作用。玉米是三大粮食作物之一,其生长过程常会面临各种逆境胁迫,盐害被认为是限制作物生长和生产的主要环境因素之一。因此鉴定玉米抗盐基因,解析其抗盐机制对培育玉米抗逆品种具有重要意义。【方法】研究克隆了玉米转录因子ZmNAC59,并用生物信息学手段分析其保守结构域和系统进化关系,用实时荧光定量PCR方法分析该基因在NaCl和MeJA处理下的表达模式,并用稳定转基因体系将该基因异源表达拟南芥观察表型,同时还用病毒诱导沉默技术将该基因在玉米中沉默后进行盐处理表型观察,并进行酶活性检测。【结果】ZmNAC59能够被NaCl和MeJA诱导上调。病毒诱导沉默ZmNAC59后进行盐胁迫,沉默株系对盐胁迫更敏感,ROS积累更多;而ZmNAC59过表达拟南芥后,过表达株系在盐胁迫处理下存活率更高,活性氧积累更少,Na⁺/K⁺比率低,表明ZmNAC59作为盐胁迫中的正调控因子可以通过调节离子流动提高植物抗盐性。实时荧光定量PCR结果表明拟南芥过表达株系中Na