Constitutive versus thermoinducible expression of heterologous proteins in Escherichia coli based on strong PR,PL promoters from phage lambda

Constitutive and thermoinducible expression plasmids based on strong P(R),P(L) promoters from phage lambda were compared for production of TNF-alpha and its analogs under various conditions. Much higher accumulation of TNF was obtained in a constitutive system, so the wider applicability of such systems was studied. In constitutive systems, proteolytically susceptible proteins can be produced easily at low cultivation temperatures and the addition of expensive or toxic chemical inducers is not required. On the other hand, toxic proteins cannot be produced and selection pressure must be strictly maintained to ensure segregational stability of plasmids. Accumulation of TNF-alpha and various analogs at levels up to 25% of total soluble protein was repeatedly achieved, which was 2-3-fold higher than in a thermoinducible system. The stable behavior of the constitutive system in laboratory fermentors was also confirmed. We propose the constitutive system described here as a general model for many currently used expression systems containing strong but not completely repressed promoters. Such systems may be considered as constitutive ones with reduced promoter strengths, but still exhibiting all the intrinsic properties of constitutive expression systems. Although all modern expression systems are inducible, wider use of a constitutive system is evidently possible.     

Bacteriophage lambda-based expression vectors

Bacteriophage lambda has been in use as a cloning vector for over 25 years, and has been used extensively as an expression vector. The efficiency of packaging and infection, and the simplicity of plaque screening are advantages of lambda as a cloning vector. A number of ingenious modifications help overcome the disadvantages associated with its mode of growth and its size. Some lambda vectors have been designed to be readily converted into plasmids or phagemids, and there are a variety of promoters and fusions that can be used to drive expression of foreign genes. Screening lambda libraries with antibodies or ligands is a powerful way of identifying novel genes.     

Rapid and sensitive detection method of a bacterium by using a GFP reporter phage

A rapid, sensitive, and convenient method for detecting a specific bacterium was developed by using a GFP phage. Here we describe a model system that utilizes the temperate Escherichia coli-restricted bacteriophage lambda, which was genetically modified to express a reporter gene for GFP to identify the colon bacillus E. coli in the specimen. E. coli infected with GFP phage was detected by GFP fluorescence after 4-6 hr of incubation. The results show that a few bacteria in a specimen can be detected under fluorescence microscopy equipped with a sensitive cooled CCD camera. When E. coli and Mycobacterium smegmatis were mixed in a solution containing GFP phage, only E. coli was infected, indicating the specificity of this method. The method has the following advantages: 1) Bacteria from biological samples need not be purified unless they contain fluorescent impurities; 2) The infection of GFP phage to bacteria is specific; 3) The fluorescence of GFP within infected bacteria enables highly sensitive detection; 4) Exogenous substrates and cofactors are not required for fluorescence. Therefore this method is suitable for any phage-bacterium system when bacteria-specific phages are available.     

A generalized heterozygote deficiency assessed with microsatellites in French common ash populations

Common ash is a temperate forest tree with a colonizing behaviour, a discontinuous spatial distribution and a peculiar and poorly known mating system. Microsatellite markers were used to study the genetic structure in natural populations of common ash. Twelve populations located in northeastern France were analysed at five loci. Levels of genetic variability within and among stands were estimated for the seedling and adult stages. As expected for a forest tree, our results reveal high levels of intrapopulation diversity and a low genetic differentiation between stands. However, a general and significant heterozygote deficiency was found, with a mean F(IS) of 0.163 for the seedlings and of 0.292 for the adult trees. The different explanations for such an excess homozygosity are discussed: a nonMendelian inheritance of alleles, the presence of null alleles, a Wahlund effect and assortative mating.     

Bacteriophages: evolution of the majority

The dsDNA-tailed bacteriophages are probably the largest evolving group in the Biosphere and they are arguably very ancient. Comparative examination of genomes indicates that the hallmark of phage evolution is horizontal exchange of sequences. This is accomplished, first, by rampant non-homologous recombination between different genomes and, second, by reassortment of the variant sequences so created through homologous recombination. The comparative analysis suggests mechanisms by which new genes can be added to phage genomes and by which genes with novel functions may be assembled from parts. Horizontal exchange of sequences occurs most frequently among closely related phages, but it also extends across the entire global population at lower frequency. Bacteriophages also have probable ancestral connections with viruses of eukaryotes and archaea.     

Mutation in the Xanthomonas campestris xanA gene required for synthesis of xanthan and lipopolysaccharide drastically reduces the efficiency of bacteriophage (phi)L7 adsorption

(Phi)L7 is a lytic phage infecting the gram-negative Xanthomonas campestis pv. campestris, a plant pathogen. To study phage-host interaction, a (phi)L7-resistant mutant was isolated from strain Xc17 by mini-Tn5 transposition and designated CH7LR. CH7LR could not plate (phi)L7 in double-layered assay and formed turbid clearing zones when the cell lawn was dropped with a high titer of (phi)L7. Sequence analysis showed that the mutated gene is xanA coding for phosphoglucomutase/phosphomannomutase, required for the synthesis of lipopolysaccharide and exopolysaccharide (xanthan). The involvement of xanA was confirmed by isolating another mutant with interrupted xanA and complementing with the cloned wild-type gene. Nonmucoid mutants are still sensitive to (phi)L7, indicating that xanthan is not involved in (phi)L7 adsorption. Since the mutants still exhibited low efficiencies of phage adsorption, we predict, by analogy with the cases in other bacteriophages of gram-negative bacteria, that other outer membrane components such as a protein are required for the formation of a complex receptor.     

Recognition of diverse RNAs by a single protein structural framework

The coat proteins of different single-strand RNA phages utilize a common structural framework to recognize different RNA targets, making them suitable models for studies of RNA-protein recognition generally, especially for the class of proteins that bind RNA on a beta-sheet surface. Here we show that structurally distinct molecules are capable of satisfying the requirements for binding to Qbeta coat protein. Although the predicted secondary structures of the RNAs differ markedly, we contend that they are approximately equivalent structurally in their complexes with coat protein. Based on our prior observations that the RNA-binding specificities of Qbeta and MS2 coat proteins can be interconverted with as few as one amino acid substitution each, and taking into account details of the structures of complexes of MS2 coat protein with wild-type and aptamer RNAs, we propose a model for the Qbeta coat protein-RNA complex.     

Combinatorial discovery of tumor targeting peptides using phage display

Peptides possess appropriate pharmacokinetic properties to serve as cancer imaging or therapeutic targeting agents. Currently, only a small number of rationally-derived, labeled peptide analogues that target only a limited subset of antigens are available. Thus, finding new cancer targeting peptides is a central goal in the field of molecular targeting. Novel tumor-avid peptides can be efficiently identified via affinity selections using complex random peptide libraries containing millions of peptides that are displayed on bacteriophage. In vitro and in situ affinity selections may be used to identify peptides with high affinity for the target antigen in vitro. Unfortunately, it has been found that peptides selected in vitro or in situ may not effectively target tumors in vivo due to poor peptide stability and other problems. To improve in vivo targeting, methodological combinatorial chemistry innovations allow selections to be conducted in the environment of the whole animal. Thus, new targeting peptides with optimal in vivo properties can be selected in vivo in tumor-bearing animals. In vivo selections have been proven successful in identifying peptides that target the vasculature of specific organs. In addition, in vivo selections have identified peptides that bind specifically to the surface of or are internalized into tumor cells. In the future, direct selection of peptides for cancer imaging may be expedited using genetically engineered bacteriophage libraries that encode peptides with intrinsic radiometal-chelation or fluorescent sequences.     

Evaluation of biotracers to monitor effluent retention time in constructed wetlands

AIMS: With concern surrounding the environmental impact of chemical tracers on the aquatic environment, this paper presents the initial evaluation of biotracers used to determine the effluent retention time, an important performance indicator, in a Free Water Surface Constructed Wetland. METHODS AND RESULTS: Production of the biotracers, coliphage MS2, and the bacteriophage of Enterobacter cloacae and antibiotic resistant endospores of Bacillus globigii is described in detail. Their subsequent use in three separate tracer experiments - January, March and June (2000) - revealed the variability of retention time with respect to effluent flow. The biotracer MS2 showed the constructed wetland had a retention time of 8-9 h at a mean discharge of 0.9 l s-1, increasing to 10-12 h at a mean discharge 0.3 l s-1. A similar retention of 9-10 h at a mean discharge of 0.3 l s-1 was calculated for the Ent. cloacae phage. In contrast, use of endospores revealed considerably longer retention times at these mean discharge rates; 12-24 h and 36-48 h, respectively. CONCLUSION: Biotracers could provide a useful and environmentally friendly technique to monitor effluent retention in constructed wetlands. At this stage the phage tracers appear particularly promising due to ease of isolation and recovery. SIGNIFICANCE AND IMPACT OF THE STUDY: Initial results are encouraging and have highlighted the potential of biotracers as alternatives to chemical tracers, even in microbially-rich waters.     

Improvement of phage defence in Lactococcus lactis by introduction of the plasmid encoded restriction and modification system LlaAI

AIMS: To study the ability of the plasmid-encoded restriction and modification (R/M) system LlaAI to function as a bacteriophage resistance mechanism in Lactococcus lactis during milk fermentations. METHODS AND RESULTS: Plasmid pAIcat4, carrying the R/M system LlaAI and a chloramphenicol resistance cassette, was introduced into the plasmid-free strain L. lactis MG1614 and the industrial strain L. lactis 964. By measuring changes in conductivity the influence of different phage on the growth was determined. CONCLUSIONS: The plasmid-encoded R/M system LlaAI significantly improves the bacteriophage resistance of L. lactis during milk fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: It is essential to determine the potential of a phage defence mechanism in L. lactis starter culture strains during growth in milk before steps are taken to improve starter cultures. This study shows that LlaAI is useful for improvement of starter cultures.