Identification and Characterization of Rhizosphere-Competent Bacteria of Wheat

To obtain rhizosphere-competent bacteria which could subsequently be modified for the development of biological control agents, bacteria were isolated from the rhizosphere and rhizoplane of wheat and barley plants by standard techniques. Of these isolates, 60 were selected for field testing as spring wheat seed inoculants in 1985. Isolates were marked genetically for resistance to antibiotics via selection of spontaneous mutants to detect and monitor isolates in the field. Forty-three days after planting, the average log₁₀ CFU/mg (dry weight) of roots and rhizosphere soil for the mutant isolates sampled ranged from 0 to 3.4. Twenty mutant isolates were retested in 1986. A total of 4 isolates were not detected, but the other 16 had an average root colonization value of log₁₀ 2.1 CFU and a range of log₁₀ 0.9 CFU to log₁₀ 3.2 CFU when sampled 32 days after planting. The average colonization value dropped to log₁₀ 1.1 CFU 51 days later. Some isolates detected previously were not detected in the second sampling; others had root colonization values similar to those obtained in the first sampling. Mutant isolates of rhizosphere bacteria included Bacillus pumilus, Bacillus subtilis, Pseudomonas fluorescens, Streptomyces spp., Xanthomonas maltophilia, and a saprophytic coryneform. Mixtures of isolates from different genera and species were compatible on seeds and roots.     

Selection and Characterization of Biofuel-Producing Environmental Bacteria Isolated from Vegetable Oil-Rich Wastes

Fossil fuels are consumed so rapidly that it is expected that the planet resources will be soon exhausted. Therefore, it is imperative to develop alternative and inexpensive new technologies to produce sustainable fuels, for example biodiesel. In addition to hydrolytic and esterification reactions, lipases are capable of performing transesterification reactions useful for the production of biodiesel. However selection of the lipases capable of performing transesterification reactions is not easy and consequently very few biodiesel producing lipases are currently available. In this work we first isolated 1,016 lipolytic microorganisms by a qualitative plate assay. In a second step, lipolytic bacteria were analyzed using a colorimetric assay to detect the transesterification activity. Thirty of the initial lipolytic strains were selected for further characterization. Phylogenetic analysis revealed that 23 of the bacterial isolates were Gram negative and 7 were Gram positive, belonging to different clades. Biofuel production was analyzed and quantified by gas chromatography and revealed that 5 of the isolates produced biofuel with yields higher than 80% at benchtop scale. Chemical and viscosity analysis of the produced biofuel revealed that it differed from biodiesel. This bacterial-derived biofuel does not require any further downstream processing and it can be used directly in engines. The freeze-dried bacterial culture supernatants could be used at least five times for biofuel production without diminishing their activity. Therefore, these 5 isolates represent excellent candidates for testing biofuel production at industrial scale.     

A Method for Staining Certain Bacteria and Antherozoids

A modification of Loeffler's method for staining the flagella of bacteria was employed in staining large forms of bacteria and antherozoids. The bacteria or the antherozoids are killed and fixed in a drop of water on a slide and set aside to dry, before the next step is undertaken. The slide is treated for a period of time, varying from about ten minutes to several hours, in a practically saturated solution of tannic acid. After the slide is thoroly rinsed in water, it is stained with either a single stain or a combination of stains. The slide is then dehydrated with absolute alcohol, cleared, with clove oil, and completed in the usual manner.

The body of the bacterium and that of the antherozoid are well differentiated and the cilia are distinctly brought out by means of the method herein described.

The technic is of especial value in staining the antherozoids of mosses, liverworts, and ferns.     

鹰嘴豆种子胰蛋白酶抑制剂的分离纯化与鉴定

为了寻找具有药物作用的天然胰蛋白酶抑制物,采用硫酸铵分级沉淀、离子交换层析(DEAE-纤维素52)及Sephadex G-100凝胶层析等方法, 从鹰嘴豆种子中分离出一种鹰嘴豆胰蛋白酶抑制剂（CPTI）. 研究表明：CPTI对胰蛋白酶有较强的抑制作用,抑制率达80%,而对胰凝乳蛋白酶抑制作用较弱,抑制率为32%, 对胃蛋白酶、木瓜蛋白酶及枯草杆菌蛋白酶均无抑制作用; 用SDS-PAGE测得CPTI近似分子质量为25.7 kD; CPTI具有较高的热稳定性,在100 ℃下加热60 min,对胰蛋白酶活性仍保持78%抑制率; Lineveaer-Burk作图得知该抑制剂属竞争性抑制类型. 动力学测定显示,来自鹰嘴豆中的CPTI对胰蛋白酶的抑制作用常数(Ki)为3.99×10^-7 mol/L.     

Selection for Chromosome Architecture in Bacteria

Bacterial chromosomes are immense polymers whose faithful replication and segregation are crucial to cell survival. The ability of proteins such as FtsK to move unidirectionally toward the replication terminus, and direct DNA translocation into the appropriate daughter cell during cell division, requires that bacterial genomes maintain an architecture for the orderly replication and segregation of chromosomes. We suggest that proteins that locate the replication terminus exploit strand-biased sequences that are overrepresented on one DNA strand, and that selection increases with decreased distance to the replication terminus. We report a generalized method for detecting these architecture imparting sequences (AIMS) and have identified AIMS in nearly all bacterial genomes. Their increased abundance on leading strands and decreased abundance on lagging strands toward replication termini are not the result of changes in mutational bias; rather, they reflect a gradient of long-term positive selection for AIMS. The maintenance of the pattern of AIMS across the genomes of related bacteria independent of their positions within individual genes suggests a well-conserved role in genome biology. The stable gradient of AIMS abundance from replication origin to terminus suggests that the replicore acts as a target of selection, where selection for chromosome architecture results in the maintenance of gene order and in the lack of high-frequency DNA inversion within replicores. [Reviewing Editor: Dr. Martin Kreitman]     

A Reliable Method for Demonstrating Gram-Positive and Gram-Negative Bacteria

Combined Gram techniques have been reviewed in the interest of improving technical safety and reliability in the demonstration of bacteria, particularly the Gram-negative type. The many modifications of the technique present various difficulties (Brown and Brenn 193 1, Humberstone 1963, Taylor 1966, Luna 1968, Brown and Hopps 1973, Engbaek et al. 1979, Bancroft and Stevens 1982, Churukian and Schenk 1982).     

Biological Control of Chickpea Collar Rot by Co-inoculation of Antagonistic Bacteria and Compatible Rhizobia

Two hundred and seven bacteria were isolated from composts and macrofauna and screened for plant growth promoting and antagonistic traits. Seven of the 207 isolates showed antagonistic activity against Sclerotium rolfsii in plate culture. Inhibition of S. rolfsii by the bacterial isolates ranged between 61 and 84%. Two of the seven isolates were Bacillus sp. and rest belonged to Pseudomonas sp. Two isolates, Pseudomonas sp. CDB 35 and Pseudomonas sp. BWB 21 was compatible with chickpea Rhizobium sp. IC 59 and IC 76 in plate culture conditions. Increase in plant biomass (dry weight) ranged between 18 and 30% on application of these bacteria by seed coating and seed priming methods. However, by seed-priming there was an increase in plant biomass by 5–7% compared to seed coating. Number of nodules and the nodule weight was similar by both seed coating and seed priming methods. Disease incidence was reduced up to 47% in treatments where captan (fungicide) or antagonistic Pseudomonas sp. CDB 35 was applied. Increase in shoot weight was 36% by seed coating with Rhizobium sp. IC 59 and Pseudomonas sp. CDB 35 when compared to captan application. Whereas by seed priming with IC 59 and CDB 35 increased shoot weight by 3 and 39% increase in nodulation was observed.     

细菌巨大质粒的快速检测

本文报道了一种快速检测微生物巨大质粒的方法．该方法是通过对Ｅｃｋｈａｒｄｔ所报道的方法加以改进,使之能对根瘤菌、大肠杆菌、甚至链霉菌的大质粒进行快速检测．     

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.     

A Method for the Detection of Starch Hydrolysis by Bacteria

S ummary . A starch agar medium for the detection of starch hydrolysis is described. The development of a cloudy zone round the colony indicates starch hydrolysis without the use of iodine or 95% (v/v) ethanol.     

厌氧菌预还原琼脂平板培养方法

为简化厌氧菌分离培养方法,使其在普通实验条件下于固体培养基上形成单菌落,本研究增加庖肉培养基无氧溶液体积,用作无氧倍比稀释液,在琼脂柱下进行倍比稀释,将皿盖带有胶塞孔的厌氧琼脂平板进行预还原,注射接种倍比稀释菌液,通过厌氧指示剂监测无氧效果,初步试用于肠道厌氧菌分离培养。结果显示,该方法整个操作过程厌氧效果良好,无需专门厌氧设备即可以分离纯化培养肠道乳酸杆菌,甚至无芽胞专性厌氧菌,如双歧杆菌和韦荣球菌。     

Characterisation of Phosphate Solubilising Bacteria in Sandy Loam Soil Under Chickpea Cropping System

With the aim to explore the possible role of phosphate-solubilizing bacteria (PSB) in phosphorus (P) cycling in agricultural soils, we isolated PSB inhabiting naturally in the sandy loam soils under chickpea cropping of Patiala (Punjab State). A total of 31 bacterial isolates showing solubilizing activities were isolated on Pikovskaya agar plates. The potent phosphate solubilizers were selected for further characterization. These isolates were shown to belong to the genera Pseudomonas and Serratia by partial sequencing analysis of their respective 16S rDNA genes. ERIC-PCR based fingerprinting was done for tracking the survival of introduced populations of the PSB during mass inoculation of these strains under chickpea plots. The results showed positive correlation (r² = 0.853) among soil phosphatase activity and phosphate solubilizers population, which was also positively correlated (r² = 0.730) to available phosphorus. Identification and characterization of soil PSB for the effective plant growth-promotion broadens the spectrum of phosphate solubilizers available for field application.     

一种产甲烷优势菌群的筛选方法

目的:用简便易行的产甲烷优势菌群的筛选方法,筛选出能够人工培养的且高效产甲烷的复合微生物.方法:用乙酸钠除氧培养基驯化富含产甲烷菌的厌氧活性污泥,逐渐增加驯化体系中培养基和菌液的比率,使驯化体系中微生物菌群适应人工培养条件.结果:筛选出4个产甲烷优势菌群.筛选出的1号、2号、3号、5号菌群在培养7d后均产甲烷量分别达到46mL、38mL、51mL、38mL,在短时间内4个菌群产甲烷量能持续稳定上升.结论:验证表明建立的方法筛选高效产甲烷菌群简便易行,为沼气工作者提供了一定的参考.     

Mutator Genes and Selection for the Mutation Rate in Bacteria

Gene frequencies in populations of haploid, asexual organisms are described by linear recurrence equations. Several models in which the mutation rate is controlled by one locus and the fitness is controlled at one or more other loci are developed. It is shown that good approximations can be introduced to give explicit solutions for the course of selection in these models. It is shown that a strong non-equilibrium selection for mutator genes is possible even when the presence of such a gene decreases the fitness of an individual. Experiments that corroborate these conclusions are discussed along with the effects of population size that determine the applicability of these results to natural populations.