Lesions induced by complement in vitro on the protoscoleces of Echinococcus multilocularis: a study by electron microscopy.

Changes in the ultrastructure of the tegument of Echinococcus multilocularis protoscoleces during complement-mediated lysis in vitro was studied using transmission and scanning electron microscopy. It was found that the total disintegration of protoscoleces by complement proceeds through formation of ‘tegumental bubbles’ and disruption of the external plasma membrane. This sequence of events was evident in the appearance of numerous loose membrane fragments and vesicles, the lifting of the external unit membrane of the microtriches and the release of organelles from the distal cytoplasm. Subsequent events, such as the appearance of a ‘fuzzy’ coat and disruption of the basement membrane, were probably due to autolysis.     

Continuous production of glucose oxidase with Aspergillus sp. under ultrasound waves

Glucose oxidase (β-d-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4, GOD) was continuously released from Aspergillus sp. under mild ultrasound waves (20 kHz, 15 W). However, GOD was not released from the cells under normal conditions because of their thick wall. GOD production under ultrasound waves was optimum at pH 7.5 and 30°C and decreased with increasing ultrasonic frequency. Ultrasonic cavitation accelerated GOD release from the cells. Microscopic observation and determination of ATP and nucleic acids in the broth revealed that the mycelia were not broken during a 5 h reaction under ultrasound waves (15 W). About 10% of GOD produced in cells was released during the reaction.     

The hydrophilic C-terminal part of the lambda S holin is non-essential for intermolecular interactions

Available evidence indicates that oligomerization of the bacteriophage lambda S holin leads to a non-specific lesion in the cytoplasmic membrane which permits transit of the phage encoded transglycosylase to the periplasm. In an attempt to locate an intermolecular interaction domain in S a chimeric protein comprising the N-terminal 32 aa of phage PhiX174 lysis protein E and the last 75 aa of lambda S has been constructed. We report that the EΦS fusion protein is stable, membrane bound, and inhibits S-mediated lysis in trans. C-terminal truncations of the EΦS fusion protein indicated that the hydrophilic C-terminal end of S (i.e. the last 15 aa) is non-essential for oligomerization.     

Plasmid DNA production combining antibiotic‐free selection,inducible high yield fermentation,and novel autolytic purification

DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. However, the challenges of performing cell lysis processes for plasmid DNA purification at an industrial scale are well known. To address downstream purification challenges, we have developed autolytic Escherichia coli host strains that express endolysin (phage λR) in the cytoplasm. Expression of the endolysin is induced during fermentation by a heat inducible promoter. The endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall; hence the cells remain alive and intact and can be harvested by the usual methods. The plasmid DNA is then recovered by autolytic extraction under slightly acidic, low salt buffer conditions and treatment with a low concentration of non‐ionic detergent. Under these conditions the E. coli genomic DNA remains associated with the insoluble cell debris and is removed by a solid–liquid separation. Here, we report fermentation, lysis methods, and plasmid purification using autolytic hosts. Biotechnol. Bioeng. 2009; 104: 505–515 © 2009 Wiley Periodicals, Inc.     

Suppression of fusarium wilts by fluorescent pseudomonads: Mechanisms and applications

Fluorescent pseudomonads are involved in the natural suppressiveness of some soils to fusarium wilts. These bacteria have been applied successfully to suppress fusarium wilts of various plant species grown in conducive soils and growing substrates. Suppression of fusarium wilts by fluorescent pseudomonads can be ascribed to direct and indirect effects against pathogenic Fusarium oxysporum. Direct effects are expressed by a reduction of the saprophytic growth of the pathogen leading to delay and reduction of root infections. This antagonism was demonstrated to be related to siderophore‐mediated iron competition. Iron competition was also shown to enhance the antagonistic effect of non‐pathogenic F. oxysporum by making the pathogen more susceptible to fungal competition for carbon. Indirect effects of fluorescent pseudomonads against pathogenic F. oxysporum are mainly related to the induction of host plant resistance which can be associated with the bacterial lipopolysaccharides. Suppression of fusarium wilts by some fluorescent pseudomonads could also be related to their ability to detoxify fungal metabolites such as fusaric acid. Association of different mechanisms of suppression increases the efficacy and consistency of the biological control under various experimental conditions. Increased knowledge of mechanisms of suppression now enables management of the environment, to some extent, to favour expression of the beneficial activities. These activities are only expressed if the bacterial density is sufficiently high.     

Extraction of geminiviral DNA from a highly mucilaginous plant (Abelmoschus esculentus)

A protocol is described for the extraction of geminiviral DNA from bhendi yellow vein mosaic virus-infectedAbelmoschus esculentus (known as bhendi or okra) containing high amounts of mucilage and other phenolic compounds. This method involves extraction with a buffer containing sodium citrate at pH 6 and PEG precipitation of the virus followed by alkali lysis. The extraction buffer eliminates the mucilage and other polyphenols, PEG precipitates the viral particles and DNA and the alkali lysis enriches the replicative forms of the viral DNA. The extracted DNA could be digested with restriction enzymes and cloned without any interference from chromosomal DNA. The quality of the DNA extracted by this method was compared to three other common plant DNA extraction protocols and was found superior. This method was used for PCR amplification and cloning of the 2.7 kbp DNA-A of BYVMV.     

Effect of glucose fermentation of the functioning of protein H in the excretion of cloacin DF13 by Escherichia coli

Abstract The inhibitory effect of glucose on the excretion of cloacin DF13 by mitomycin C-induced cloacinogenic Escherichia coli cells (Van Tiel-Menkveld et al. (1981) J. Bacteriol. 146, 41–48) was investigated. Cells grown and induced under the same conditions as reported by Van Tiel-Menkveld et al. fermented the glucose added to the culture. The pH of the culture decreased to about 5.2 and fermentation products such as formate, acetate, and lactate accumulated in the culture medium. This resulted in the inhibition of the proteolytic processing of the precursor of the pCloDF13-encoded protein H to its mature form, which is essential for the release of cloacin DF13. In cultures buffered at pH 7.0 these effects were not observed.     

Bacterial communication through membrane vesicles

ABSTRACT

Bacteria can communicate through diffusible signaling molecules that are perceived by cognate receptors. It is now well established that bacterial communication regulates hundreds of genes. Hydrophobic molecules which do not diffuse in aqueous environments alone have been identified in bacterial communication, that raised the question on how these molecules are transported between cells and trigger gene expressions. Recent studies show that these hydrophobic signaling molecules, including a long-chain N-acyl homoserine lactone signal produced in Paracoccus denitrificans, are carried by membrane vesicles (MVs). MVs were thought to be formed only through the blebbing of the cell membrane, but new findings in Pseudomonas aeruginosa and Bacillus subtilis revealed that different types of MVs can be formed through explosive cell lysis or bubbling cell death, which findings have certain implications on our view of bacterial interactions.