Interactions ofRhizobium japonicum strains in soybean nodules and their contribution to nitrogen fixation

Summary Rhizobium japonicum strain 8-0 Str^R applied as inoculum to Clark 63 soybeans formed small ineffective nodules which had very low nitrogenase activity compared to nodules formed by two effective strains, 110 Tet^R and 138 Kan^R. Mean numbers of cells per milligram of nodule tissue for plants up to 34 days old were 7.7×10⁶ for 8-0 Str^R, 4.1×10⁸ for 110 Tet^R and 7.6×10⁸ for 138 Kan^R. Cell counts per unit mass of nodule were independent of plant age for strains 110 Tet^R and 138 Kan^R, however, for strain 8-0 Str^R, 25 and 34 days old plants had fewer viable cells per nodule mass than 18 day old plants. When a mixture of two effective strains was used, the nodules of individual plants were predominantly caused by either 110 Tet^R or 138 Kan^R. In one experiment the predominance was random, but in another, strain 110 Tet^R clearly dominated. Strain 138 Kan^R was absent in some nodules on 18 day old plants, and in others, less than 10² cells per nodule were found. When strains 8-0 Str^R and 138 Kan^R were used as mixed inoculum, most of the nodules had strain 8-0 Str^R but strain 138 Kan^R was detected in many nodules and was generally evident in the largest nodules. Nitrogenase activity by many individual nodules was low except for nodules which had cells of 138 Kan^R. Nitrogenase activity by whole root systems of these plants was relatively high and similar to plants that had only nodules of strain 138 Kan^R. Similar relationships were observed for a mixed inoculum of 8-0 Str^R and 110 Tet^R. In general, mixed inoculations resulted in nodules with a particular strain being dominant for each individual plant. Double infections within individual nodules were not uncommon and such nodules often had disproportionate numbers of cells of two competingR. japonicum strains.Contribution from the Laboratory of Soil Microbiology, Department of Agronomy, Missouri Agricultural Experiment Station. Missouri Journal Series Number 7967.     

Chemical and radiation induced late dominant lethal effects in mice.

Although theoretically expected, experimental data to date have not shown dominant lethal expression to occur throughout the developmental period. Specifically, late post-implantation effects have not been demonstrated. We routinely use an experimental technique in which parental females mated to mutagenically treated males are allowed to give birth and wean their litter, and their uterine horns are then inspected for uterine scars indicative of live and dead embryos. In a number of experiments in which males were mutagenically treated with either chemicals or X-irradiation, a discrepancy was observed between the number of live embryos as determined by the scar technique and the number of live observed at birth, suggesting the possibility of embryonic losses at a late stage in development. Initial analyses showed that mutagenic treatment increased the percentage of these late losses. These differences were statistically significant in 2 of 3 analyses. Factors affecting statistical significance and an understanding of dominant lethal mutations are discussed.     

Strain identification inRhizobium meliloti using the antibiotic disk susceptibility test

Summary The antibiotic disk susceptibility test was used to measure the variation in the intrinsic resistance of 49 strains ofRhizobium meliloti to 9 antibiotics. Several strains had unique patterns of resistance. However, during cluster analysis, when a minimum Euclidean distance equal to 4 was used as a discriminating tool, the strains were grouped in 12 groups. The largest group contained 74% of the strains but 9 strains (2 very effective, 4 effective and 3 ineffective) showed very unique patterns of resistance and formed 9 distinct groups.R. meliloti strains in general showed high intrinsic resistance to the 9 antibiotics tested.Contribution no. 189 Station de Recherches, Agriculture Canada.     

耐碳青霉烯类肠杆菌的流行病学特点与治疗现状

耐碳青霉烯类肠杆菌(carbapenem-resistant Enterobacteriaceae,CRE)是近年来检出率不断升高的一种临床常见耐药菌,是导致患者死亡的独立危险因素。CRE的出现主要是由于细菌产碳青霉烯酶,包括肺炎克雷伯菌产生的碳青霉烯酶(Klebsiella pneumoniae carbapenemase,KPC)、金属β-内酰胺酶(metallo-β-lactamase,MBL)和 苯唑西林酶(oxacillinase,OXA),少数是由于细菌外膜蛋白改变以及外排泵高表达。临床上最常见的CRE是肺炎克雷伯菌,最常暴发CRE感染的科室是重症监护室(intensive care unit,ICU),感染高危因素包括接触医疗机构、各种侵入性操作以及抗菌药物使用史。由于缺乏前瞻性临床试验数据,目前在治疗CRE感染的高危患者时多采用多药联合的经验性治疗措施,一些经典药物如多黏菌素、替加环素、磷霉素等起到了意想不到的效果,一些新药如头孢他啶-阿维巴坦也投入使用并发挥了一定作用。本文就近年来CRE感染的流行病学特点以及目前临床上主要使用的药物进行综述。     

Independent breakdown events of the Brassica napus Rlm7 resistance gene including via the off-target impact of a dual-specificity avirulence interaction

Protection of many crops is achieved through the use of genetic resistance. Leptosphaeria maculans, the causal agent of blackleg disease of Brassica napus, has emerged as a model for understanding gene-for-gene interactions that occur between plants and pathogens. Whilst many of the characterized avirulence effector genes interact with a single resistance gene in the host, the AvrLm4-7 avirulence gene is recognized by two resistance genes, Rlm4 and Rlm7. Here, we report the “breakdown” of the Rlm7 resistance gene in Australia, under two different field conditions. The first, and more typical, breakdown probably resulted from widescale use of Rlm7-containing cultivars whereby selection has led to an increase of individuals in the L. maculans population that have undergone repeat-induced point (RIP) mutations at the AvrLm4-7 locus. This has rendered the AvrLm4-7 gene ineffective and therefore these isolates have become virulent towards both Rlm4 and Rlm7. The second, more atypical, situation was the widescale use of Rlm4 cultivars. Whilst a single-nucleotide polymorphism is the more common mechanism of virulence towards Rlm4, in this field situation, RIP mutations have been selected leading to the breakdown of resistance for both Rlm4 and Rlm7. This is an example of a resistance gene being rendered ineffective without having grown cultivars with the corresponding resistance gene due to the dual specificity of the avirulence gene. These findings highlight the value of pathogen surveillance in the context of expanded knowledge about potential complexities for Avr–R interactions for the deployment of appropriate resistance gene strategies.     

Probing the “fingers” domain binding pocket of Hepatitis C virus NS5B RdRp and D559G resistance mutation via molecular docking,molecular dynamics simulation and binding free energy calculations

The NS5B RdRp polymerase is a prominent enzyme for the replication of Hepatitis C virus (HCV). During the HCV replication, the template RNA binding takes place in the “fingers” sub-domain of NS5B. The “fingers” domain is a new emerging allosteric site for the HCV drug development. The inhibitors of the “fingers” sub-domain adopt a new antiviral mechanism called RNA intervention. The details of essential amino acid residues, binding mode of the ligand, and the active site intermolecular interactions of RNA intervention reflect that this mechanism is ambiguous in the experimental study. To elucidate these details, we performed molecular docking analysis of the fingers domain inhibitor quercetagetin (QGN) with NS5B polymerase. The detailed analysis of QGN-NS5B intermolecular interactions was carried out and found that QGN interacts with the binding pocket amino acid residues Ala97, Ala140, Ile160, Phe162, Gly283, Gly557, and Asp559; and also forms π?π stacking interaction with Phe162 and hydrogen bonding interaction with Gly283. These are found to be the essential interactions for the RNA intervention mechanism. Among the strong hydrogen bonding interactions, the QGN?Ala140 is a newly identified important hydrogen bonding interaction by the present work and this interaction was not resolved by the previously reported crystal structure. Since D559G mutation at the fingers domain was reported for reducing the inhibition percentage of QGN to sevenfold, we carried out molecular dynamics (MD) simulation for wild and D559G mutated complexes to study the stability of protein conformation and intermolecular interactions. At the end of 50?ns MD simulation, the π?π stacking interaction of Phe162 with QGN found in the wild-type complex is altered into T-shaped π stacking interaction, which reduces the inhibition strength. The origin of the D559G resistance mutation was studied using combined MD simulation, binding free energy calculations and principal component analysis. The results were compared with the wild-type complex. The mutation D559G reduces the binding affinity of the QGN molecule to the fingers domain. The free energy decomposition analysis of each residue of wild-type and mutated complexes revealed that the loss of non-polar energy contribution is the origin of the resistance.

Communicated by Ramaswamy H. Sarma     

Potential of bioinsecticidal Bacillus thuringiensis inoculum to suppress gray mold in tomato based on induced systemic resistance

The potential of the active ingredient of a commercial bioinsecticide, XenTari^® (Bacillus thuringiensis [BT] serovar aizawai strain ABTS‐1857), to suppress gray mold in tomato plants was elucidated. First, a suspension of the active ingredient of XenTari^® and a liquid culture of the bacterial strain as BT inocula were sprayed onto detached leaves or drenched into pots of tomato seedlings, and then, propagules of the gray mold fungus, Botrytis cinerea, were inoculated onto the leaves. The gray mold disease was significantly suppressed when rhizospheres were drenched with either inoculum, but not when inocula were sprayed onto detached leaves of seedlings. Both BT inocula were verified not to directly inhibit the mycelial growth of B. cinerea based on in vitro culture plate assays. Additionally, real‐time RT‐PCR analysis verified that the active ingredient increased the expression levels of defence‐related genes, such as PR‐1(P6) and P4, in the leaves of tomato seedlings. These results suggest that the active ingredient has the potential to suppress gray mold disease in tomato, not through direct antagonistic interactions with B. cinerea, but rather through systemic activation of the plant defence system by increased expression of several defence‐related genes.     

Profiling interactions of vaborbactam with metallo-β-lactamases

β-Lactams are the most successful antibacterials, yet their use is threatened by resistance, importantly as caused by β-lactamases. β-Lactamases fall into two mechanistic groups: the serine β-lactamases that utilise a covalent acyl-enzyme mechanism and the metallo β-lactamases that utilise a zinc-bound water nucleophile. Achieving simultaneous inhibition of both β-lactamase classes remains a challenge in the field. Vaborbactam is a boronate-based inhibitor that reacts with serine-β-lactamases to form covalent complexes that mimic tetrahedral intermediates in catalysis. Vaborbactam has recently been approved for clinical use in combination with the carbapenem meropenem. Here we show that vaborbactam moderately inhibits metallo-β-lactamases from all 3 subclasses (B1, B2 and B3), with a potency of around 20–100 fold below that by which it inhibits its current clinical targets, the Class A serine β-lactamases. This result contrasts with recent investigations of bicyclic boronate inhibitors, which potently inhibit subclass B1 MBLs but which presently lack activity against B2 and B3 enzymes. These findings indicate that cyclic boronate scaffolds have the potential to inhibit the full range of β-lactamases and justify further work on the development of boronates as broad-spectrum β-lactamase inhibitors.     

枯草杆菌二联活菌制剂鼻饲给药治疗昏迷患者抗生素相关性腹泻的疗效观察

目的：研究枯草杆菌二联活菌制剂经鼻饲给药治疗昏迷患者抗生素相关性腹泻的临床疗效。方法：收集以2011 年1 月至 2014 年1 月本院确诊的76 例患抗生素相关性腹泻的昏迷患者作为研究对象,按随机数字表法分为对照组(38 例)和试验组(38 例),对照组在基础治疗的基础上给予鼻饲蒙脱石散收敛止泻治疗,而试验组在对照组的基础上,同时给予枯草杆菌二联活菌胶囊 （美常安）鼻饲给药治疗。对比治疗前后两组患者的症状改善情况以及粪便实验室检查指标,记录两组不良反应。结果：除治疗过 程中死亡4 例、意识转清停止鼻饲8 例外,试验组33 例,对照组31 例完成试验。试验组治疗有效率明显高于对照组的(X2=2.627, P <0.05)。给药第6d 试验组的白细胞转阴率为81.8%,明显高于对照组的30%(P<0.05),给药第6 d 及治疗后试验组的肠道菌群纠 正率分别为84.2%、94.7%,明显高于对照组的41.2%、64.7%,差异均有统计学意义(P<0.05)。对照组患者不良反应的发生率明显高 于试验组（P<0.05）。结论：枯草杆菌二联活菌制剂鼻饲给药可显著改善昏迷患者抗生素相关性腹泻的症状,使肠道菌群更快得以 纠正、缓解使肠道炎症,且安全性高,值得临床推广使用。