Complete Genome Sequence of the Novel Escherichia coli Phage phAPEC8

Bacteriophage phAPEC8 is an Escherichia coli-infecting myovirus, isolated on an avian pathogenic Escherichia coli (APEC) strain. APEC strains cause colibacillosis in poultry, resulting in high mortality levels and important economic losses. Genomic analysis of the 147,737-bp double-stranded DNA phAPEC8 genome revealed that 53% of the 269 encoded proteins are unique to this phage. Its closest relatives include the Salmonella phage PVP-SE1 and the coliphage rv5, with 19% and 18% similar proteins, respectively. As such, phAPEC8 represents a novel, phylogenetically distinct clade within the Myoviridae, with molecular properties suitable for phage therapy applications.     

Screening of yeast strains for transfructosylating activity

Fructooligosaccharides (FOSs) are functional food ingredients with prebiotic properties, and a recent increase in the use of oligosaccharides in the food industry has led to the search for “new” microorganisms and enzymes for the production of oligosaccharides. This paper focuses on the screening of yeasts obtained from fruits and flowers (from Brazilian tropical forests), and capable of secreting extra-cellular enzymes with high fructosyl transferase activity (FTA). The screening and isolation procedures resulted in four potentially interesting yeast strains: Candida sp. (LEB-I3), Rhodotorula sp. (LEB-U5.), Cryptococcus sp. (LEB-V2) and Rhodotorula sp. LEB-V10. All were able to produce more then 100 g l⁻¹ of FOS from a 500 g l⁻¹ sucrose solution, but only the last one, (LEB-V10), showed no hydrolytic activity with respect to the FOS produced, giving a continuous increase in FOS content up to the end of the reaction, when it was about 50% of the total carbohydrates.     

Expression of in vivo-inducible Salmonella enterica promoters during infection of Caenorhabditis elegans

In vitro mimicking of the stimuli controlling in vivo-inducible bacterial promoters during infection of the host can be complex. Therefore, the use of the nematode Caenorhabditis elegans was evaluated, as a surrogate host to examine the expression of Salmonella enterica promoters. Green fluorescent protein (GFP+) was put under the control of the promoters of the pagC, mgtB, sseA, pgtE and fur genes of S. enterica. After infection of C. elegans with an S. enterica serovar Typhimurium vaccine strain expressing these constructs, clear bacterial expression of GFP+ was observed under the control of all five promoters, although significant expression was not always obtained in vitro. It is concluded that C. elegans constitutes a useful model system for the study of the in vivo expression of Salmonella promoters.     

The Shiga-toxin VT2-encoding bacteriophage varphi297 integrates at a distinct position in the Escherichia coli genome

The plaque-forming VT2-encoding lambdoid bacteriophage varphi297 was isolated from a Belgian clinical Escherichia coli O157:H7 isolate. PCR walking, starting from the int gene of phage varphi297, demonstrated that the varphi297 prophage integrated in the yecE gene of a lysogenic E. coli K12 strain. This integration site, in E. coli K12 and in the original clinical O157:H7 isolate, was confirmed by PCR using primers flanking this site. The excisionase protein of phage varphi297 is identical to the excisionase of VT1-encoding phage VT1-Sakai, while the integrases, which are 82% identical, show significant sequence divergence in the central and C-terminal region. This can explain the different integration sites of both prophages. The activity of the integrase was proven by its ability to mediate the integration of a suicide plasmid, carrying the attachment site of varphi297, at the appropriate position in the E. coli chromosome.     

Presentation of the functional receptor-binding domain of the bacterial adhesin F17a-G on bacteriophage M13

Bovine enterotoxigenic Escherichia coli (ETEC) carrying F17a fimbriae attach to the intestinal epithelium by means of the F17a-G adhesin. Since filamentous bacteriophages can be employed for the display of foreign peptides, we tested the applicability of this system to F17a-G. The receptor-binding domain of the F17a-G adhesin was expressed on bacteriophage M13, as an amino-terminal fusion with the phage protein pIII. This domain retained its N-acetyl-beta-d-glucosamine binding activity. The phage presenting the fimbrial receptor-binding domain elicited an IgG response against F17a-G after intraperitoneal immunisation of mice.     

Purification and characterisation of a fructosyltransferase from Rhodotorula sp

The present work was devoted to investigations concerning the purification and characterisation of the fructooligosaccharide (FOS)-producing extracellular enzyme of Rhodotorula sp. LEB-V10. FOS are functional food ingredients showing prebiotic properties, meaning that it could stimulate selectively the growth and/or activity of probiotic bacteria in the gut. The purification of the enzyme was carried out according to the following sequential procedure: cell separation by centrifugation, recovering by ethanol precipitation and purification by anion exchange chromatography. The molecular weight was estimated to be 170 kDa by preparative gel filtration and 77 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, signifying that the native enzyme exists as a dimer. With sucrose as substrate, the data failed to fit the Michaelis-Menten behaviour, rather showing a sigmoid shape similar to that of the allosteric enzymes (cooperative behaviour), requiring high sucrose concentrations to obtain high reaction rates. The enzyme showed both fructofuranosidase (FA) and fructosyl-transferase (FTA) activities. The optimum pH and temperature for FA activity were found to be around 4.0 and 72-75 degrees C, respectively, while FTA showed optimum activity at pH 4.5 and 65-70 degrees C. Both activities were very stable at temperatures below 66 degrees C, while for FA, the enzyme was more stable at pH 4.0 and for FTA at pH 5.0.     

Functional Expression of Agrobacterium tumefaciens T-DNA onc-genes in Asparagus Crown Gall Tissues

We analyzed Asparagus crown gall tissues transformed with A.tumefaciens C58 and C58C1 (pTiB6S3) and selected for hormoneautotrophic growth. No increased IAA levels were observed inthe Asparagus tumor lines notwithstanding the presence of allthree T-DNA onc genes. The endogenous cytokinin levels indicatethat Asparagus crown gall is dependent on enhanced zeatin ribosideequivalent levels for its growth. We conclude that phytohormone autotrophic growth of Asparaguscrown gall tissue seems only to be dependent upon an activegene 4, inducing enhanced cytokinin levels. Moreover, the presenceof an active gene 1 seems to be lethal as was indicated by theabsence of tryptophan-2-mono-oxygenase activity in transformedtissues and the toxicity of exogenously supplied indole-3-acetamide(IAM) or naphthalene-1-acetamide (NAM) as a substitute for anactive gene 1. (Received August 7, 1989; Accepted October 31, 1989)     

Mendelian transcmission of genes introduced into plants by the Ti plasmids of Agrobacterium tumefaciens

Summary Insertion of the bacterial transposon Tn7 was used to obtain mutants of an octopine Ti plasmid. Crown gall tumours induced on tobacco by an Agrobacterium tumefaciens strain carrying a particular mutant Ti plasmid (pGV2100) were found to give rise to shoots. These shoots were grown in vitro and one of them (rGV-1) was found to contain the T-DNA specific enzyme lysopine dehydrogenase (LpDH) and to form roots. After transfer to soil, rGV-1 developed into a morphologically and functionally normal tobacco plant. All cells of the regenerant and of vegetatively produced offspring were shown, by cloning of leaf protoplasts, to contain T-DNA and LpDH activity. rGV-1 and vegetatively produced offspring flowered normally. Plantlets obtained from haploid anther cultures were tested for LpDH activity Forty-one percent of these plantlets were LpDH positive. Moreover, both self-pollination of rGV-1 and crosses between rGV-1 and normal tobacco plants showed that the LpDH character was transmitted both through the pollen and through the eggs of rGV-1 as a single dominant factor with Mendelian segregation ratios typical for monohybrid crosses. By repeated selfing, homozygous plants were obtained which bred true with respect to LpDH. The importance of these findings with respect to the use of Agrobacterium tumefaciens and Ti plasmids for genetic engineering in plants is discussed.This paper is dedicated to Prof. Georg Melchers on the occasion of his 75th birthday, in recognition and gratitude for his relentless and enthousiastic pioneering efforts in the field of experimental plant genetics and cell biology