Transgenic ice nucleation-active Enterobacter cloacae reduces cold hardiness of corn borer and cotton bollworm larvae

The ice nucleation (IN) gene iceA of Erwinia ananas 110 was integrated into the chromosomes of two Enterobacter cloacae strains (Enc1.2022 and Enc1.181). These two newly derived transgenic strains, designated Enc2022-I and Enc181-I, respectively, possessed ice nucleation activity at -2.5 degrees C, significantly higher than their parent strains (active at approx -10 degrees C or lower). After ingesting these transgenic bacteria, the mean supercooling points (SCPs) of corn borer and cotton bollworm larvae were -3 to -4 degrees C, significantly higher than those of untreated controls. The SCPs remained significantly elevated over the 9-day period after ingestion, which matched well with the efficient gut colonization of the bacteria during this period. All treated larvae froze and eventually died after exposure for 6 h to a temperature of -7 degrees C, and more than 95% died after 12 h at -5 degrees C. In contrast, few or none of the untreated control larvae froze and died under the same conditions. Furthermore, the growth ability of these transgenic ice nucleation-active (INA) En. cloacae strains on corn leaves was reduced, compared to that of wild-type epiphytic E. ananas, as revealed by pot tests conducted in both greenhouse and outdoor conditions. The stable colonization in insect guts and their lower affinity to plants would make these transgenic INA bacteria useful as a novel tool for biological control of insect pests in agricultural fields.     

Development of a fast DNA-DNA hybridization method based on melting profiles in microplates

DNA-DNA hybridization is still the “gold standard” for the genotypic delineation of bacterial species. However, it is not widely used because traditional DNA-DNA hybridization techniques are rather time-consuming and not easy to perform in routine laboratories. In the present study, DNA of reference strains was digested with Sau3A, ligated with linker oligonucleotides S1/2 and in vitro amplified. The amplified DNA fragments were immobilized on MaxiSorb 96-well plates. DNA isolated from target strains was also digested with Sau3A, ligated with linker oligonuleotides P1/2 and in vitro amplified in the presence of digoxygenin modified dUTP. The labeled amplificate was hybridized to the immobilized reference DNA under isothermal conditions. Thermal denaturation curves of the DNA-DNA hybrids were obtained by using washing solutions of increasing stringency. Remaining hybrids were colorimetrically detected with anti-digoxygenin-horseradish peroxidase anti-bodies. The new method was validated with strains of the genus Pedioccocus for which DNA-DNA similarities have also been determined by the filter hybridization method. In addition, DNA-DNA hybridizations were performed with genotypically defined Enterobacter species.     

Sensitivity to pH, product inhibition, and inhibition by NAD+ of 1,3-propanediol dehydrogenase purified from Enterobacter agglomerans CNCM 1210

Because of its key role in the metabolism of glycerol during fermentation, 1,3-propanediol dehydrogenase (EC 1.1.1.202) of Enterobacter agglomerans CNCM 1210 was purified to homogeneity and studied with respect to its sensitivity to pH and to nucleotide and 1,3-propanediol concentrations. Enzyme activity was optimal at pH 7.8. The enzyme was competitively inhibited by NAD⁺ (K_i of 0.29 mM), and 1,3-propanediol exerted a strong inhibitory effect according to a mixed-type inhibition with a K_i of 13.7 mM and an a-factor of 9.0. It is proposed that these dehydrogenase properties be extended to the dehydrogenases of Citrobacter freundii and Klebsiella pneumoniae, which exhibited numerous similar physical properties. Received: 4 December 1996 / Accepted: 24 March 1997     

Root colonization by symplasmata-forming Enterobacter agglomerans

Abstract Enterobacter agglomerans strains are able to form cell aggregates called symplasmata when grown in a liquid medium. The nitrogen-fixing E. agglomerans strain NO30, isolated from the rhizosphere soil of rice, was inoculated onto roots of axenically grown wheat and rice seedlings and could colonize the roots of both plants. The ability of NO30 cells to colonize the plant roots seemed comparable in the host and non-host plants, as far as colony forming units (cfu) measurements were concerned. Nevertheless, electron microscopy (SEM, TEM) revealed that, in the case of rice, the normal host plant for NO30, the colonization was characterized by the formation of symplasmata, whereas only individual cells were found on wheat roots. Symplasmata formation seems to be specific for colonization of the host plant, rice. This finding also means that colonization of the host plant may be largely underestimated when measured by conventional techniques. Symplasmata formed in liquid medium or on the roots of rice were stained using Thiery's and Swift's technique, and the presence of polysaccharides and proteins was revealed in the extracellular matrix as well as in fibrils anchoring symplasmata to other symplasmata or to plant cells.     

Management of common scab of potato in the field using biopesticides,fungicides, soil additives,or soil fumigants

Common scab, caused by Streptomyces scabiei is an economically important potato disease worldwide. The potato industry in New Brunswick, Canada experience $1.2 million loss every year due to this disease. Superficial, raised, or deep-pitted brownish lesions on infected tubers reduce the quality and marketability of both fresh-market and processing potatoes, and hence, common scab is considered a priority disease for which adequate control measures are lacking. The objective of this research was to compare various potential treatments in suppressing the disease. Two field experiments were conducted at McCain's Research Farm, Florenceville-Bristol, New Brunswick, Canada, in 2008 and 2009 to assess the efficacy of Bacillus subtilis, Enterobacter cloacae, Chloropicrin, Pic-Plus, manganese sulphate and mustard meal in comparison with the chemical controls fludioxonil and mancozeb against common scab of potato. The disease incidence was significantly reduced by 36% due to the addition of mustard meal to the soil; 35.4% due to fludioxonil seed treatment; 30.0% due to soil fumigation with Pic-Plus; or by 27.2% due to soil fumigation with Chloropicrin. Potato tubers with scab severity ≥5% which are considered unmarketable in Canada were significantly reduced by 56.1% due to seed treatments with B. subtilis; by 57.8% due to fludioxonil; or by 63.1% due to the soil addition of mustard meal. The same treatments significantly increased marketable yield by 32.5%, 24.6%, and 24.6%, respectively. Soil fumigation with Chloropicrin or Pic-Plus increased marketable yield by 9.5% or 7.1%, respectively. These findings indicate that, in addition to the fludioxonil seed treatment, the seed treatment with the biopesticide containing B. subtilis and the soil addition of mustard meal treatments are potential alternatives for managing common scab of potatoes.     

Chemosensory Response in Blepharisma. I. Accumulation of Cells in Products of Bacterial Metabolism

ABSTRACT. Blepharisma cells were attracted by a pellet of live bacteria (Enterobacter) which was separated from the Blepharisma suspension by a cellulose membrane (fractionation: M.W. 14,000). The cells, however, were not attracted by killed bacteria. Crude and heat-treated supernatants obtained from bacterial suspension also induced chemoaccumulation of cells. These results suggest that the cells of Blepharisma detect certain small molecules, produced by live bacteria, that can pass through the cellulose membrane and are stable to heat. From the live bacteria supernatant, several ninhydrin-positive substances were isolated by means of two-dimensional thin-layer chromatography. Several of the spots contained substances that attracted the cells, indicating that certain ninhydrin-positive components, such as peptides or free amino acids (probably products of bacterial metabolism), may serve as a signal for food.     

Cloning and sequencing of genes encoding the beta subunits of the ATP-synthases from Enterobacter aerogenes and Flavobacterium ferrugineum

Abstract The genes encoding the β-subunit of the ATPase from Enterobacter aerogenes and Flavobacterium ferrugineum were cloned and their sequences determined. The predicted amino acid sequences were compared with the corresponding proteins from other eubacteria. Homology values of 58–98% confirmed the highly conserved character of the ATPase β-subunit. The enterobacterial ( Escherichia coli, E. aerogenes ) β-subunits represent the shortest sequences, whereas the corresponding F. ferrugineum protein exhibits an additional 33 amino acid residues as insertions at three different locations.     

阪崎肠杆菌显色培养基的应用研究

阪崎肠杆菌(Enterobacter sakazakii)是新近引起广泛关注的一种危险的条件致病菌, 主要存在于婴幼儿奶粉、婴幼儿补充食品中。由于目前日常使用的传统检验方法存在检测周期长等方面的不足之处, 本实验室研究设计出一种新的显色培养基(HKMCES), 通过与OXOID公司的同类产品(OXCES)比较, 分别应用于质控菌株、污染样品和实际样品的测试, 对这2种显色培养基的灵敏度、特异性、检测效果以及前增菌方法进行了初步评价。结果表明, 合适的增菌方法更有利于样品中阪崎肠杆菌的检出, 本实验室研制的显色培养基和OXOID公司的显色培养基均具有较好的选择性和特异性, 检测效果相当。这种新的显色培养基能使检测周期缩短, 具有较好的应用价值。     

皂角发酵物对红托竹荪病害防治及土壤主要微生物群落影响

为了研究皂角发酵物对贵州当地特色产业红托竹荪的病害防治及促生长作用,本研究在室内分离病原菌并进行平板对峙实验,田间试验设计4个处理：不施用药剂的常规处理、解淀粉芽孢杆菌HN11菌液、皂角粉末、皂角发酵物,调查防治效果,测量菌蛋大小及个数,检测土壤微生物群落变化。从发病组织中分离出一株病原菌,鉴定为阴沟肠杆菌Enterobacter cloacae。皂角发酵物对竹荪病害田间防治效果达77.86%,生长面积提高61.22%。土壤微生物群落中细菌和真菌分析结果显示,相较于其他处理组,皂角发酵物处理组中竹荪相对丰度占比最大,达25.83%。皂角发酵物能有效防治红托竹荪病害,促进红托竹荪菌蛋生长,减少化学农药的用量,促进生态循环,保障农产品食用安全,提升皂角和竹荪产业综合效益。