Jean-Pierre Hernalsteens

Gene tagging in plants by a T-DNA insertion mutagen that generates APH(3′)II-plant gene fusions

SummaryA new method is presented for gene tagging in higher plants. Its advantage compared with transposable elements is to provide information on the regulation of the mutated genes at the same time. The mutagenesis described here relies on the integration of the T-DNA into the plant genome. A Ti-plasmid vector was constructed for the selection of plant gene fusions with a promoterless APH(3′)II reporter sequence. Infection of haploid and diploid Nicotiana plumbaginifolia protoplasts with this vector allowed the isolation of kanamycin-resistant cell clones at a high frequency. The characteristics of the APH(3′)II fusion proteins in these lines showed that both transcriptional and translational fusions had been obtained. Some of the regenerated plants had a particular morphology which could have resulted from the inactivation of plant genes by T-DNA insertion and others showed tissue-specific expression of the gene fusions.

Petunia plants escape from negative selection against a transgene by silencing the foreign DNA via methylation

SummaryTransgenicPetunia hybrida clones harbouring the T-DNA gene2 ofAgrobacterium tumefaciens were used to test a strategy for the trapping of plant transposable elements. In thePetunia line used, floral variegation is due to the presence of the non-autonomous transposable elementdTph1 at theAn1 locus. The gene2 product converts the auxin precursor indole-3-acetamide and its analogue 1-naphthalene acetamide into the active auxins indole-3-acetic acid and 1-naphthalene acetic acid. Plant cells that express gene2 can use a low concentration of the precursors as auxins and become sensitive to the toxicity of high concentrations of these compounds. By selecting protoplast-derived microcalli or seedlings able to grow on medium with high precursor concentrations, variant plants were obtained in which gene2 was no longer expressed. Southern analysis, using gene2-specific probes, revealed that in one variant the T-DNA was deleted. For 30 other variants no alteration in gene2 structure was observed, indicating that transposable element insertion was not responsible for the inactivation of gene2. Analysis with restriction enzymes allowing discrimination between methylated or non-methylated DNA sequences showed that the inactivated gene2 sequences were methylated. Addition of the in vivo methylation inhibitor 5-azacytidine to the medium led to reactivation of gene2 expression in some of the variants. These observations demonstrated that reversible DNA methylation was the main cause of silencing of gene2 in this system.

Identification and molecular characterization of a novel Salmonella enteritidis pathogenicity islet encoding an ABC transporter

Using a newly constructed minitransposon with a phoA reporter gene in a Salmonella enteritidis phoN mutant, we have identified an iron‐ and pH‐inducible lipoprotein gene sfbA, which is a component of a novel ABC‐type transporter system required for virulence. This gene is located on a 4 kb Salmonella‐specific chromosomal segment, which constitutes a new pathogenicity islet. This islet encodes an outer membrane protein, OmpX, and contains the operon designated sfbABC (Salmonella ferric binding) encoding a putative periplasmic iron‐binding lipoprotein SfbA, a nucleotide‐binding ATPase SfbB and a cytoplasmic permease SfbC, as predicted by their characteristic signature sequences. Inactivation of the sfbA gene resulted in a mutant that is avirulent and induces protective immunity in BALB/c mice. The wild‐type phenotype could be restored by in vivo complementation with the sfbABC operon. This novel transporter might be involved in iron uptake in Salmonella.

Introduction and expression of the octopine T-DNA oncogenes in tobacco plants and their progeny

Abstract The tumour-inducing T-DNA genes 1, 2 and 4 of the octopine Ti-plasmid pTiAch5 were cloned and introduced into tobacco cells by cocultivation or leaf disk transformation using pTi derived vectors. When a selectable marker was needed, we used a aminoglycoside phosphotransferase II (nos-APH(3′)II) chimeric gene conferring kanamycin resistance to plant cells. The expression of gene 4 in transformed tissue cultures precluded the regeneration of normal transformed plants. Normal transformed plants were obtained with the construction carrying genes 1 or 2. We report in vivo complementation of genes 1 and 2 after crosses of transformed plants. Strategies are described for the use of genes 1 and 2 as selection or screening markers in plant cells or regenerated plants.

Genetic typing of shiga toxin 2 variants of Escherichia coli by PCR-restriction fragment length polymorphism analysis.

ABSTRACT Shiga toxins Stx1 and Stx2 play a prominent role in the pathogenesis of Shiga toxin-producing Escherichia coli (STEC) infections. Several variants of the stx2 gene, encoding Stx2, have been described. In this study, we developed a PCR-restriction fragment length polymorphism system for typing stx2 genes of STEC strains. The typing system discriminates eight described variants and allows the identification of new stx2 variants and STEC isolates carrying multiple stx2 genes. A phylogenetic tree, based on the nucleotide sequences of the toxin-encoding genes, demonstrates that stx2 sequences with the same PvuII HaeIII HincII AccI type generally cluster together.

A live Salmonella enterica serovar Enteritidis vaccine allows serological differentiation between vaccinated and infected animals.

ABSTRACT Three precisely defined deletion mutants of Salmonella enterica serovar Enteritidis were constructed, a guanine auxotrophic ΔguaB mutant, a nonflagellated ΔfliC mutant, and an auxotrophic and nonflagellated ΔguaB ΔfliC double mutant. All three mutants were less invasive than the wild-type strain in primary chicken cecal epithelial cells and the human epithelial cell line T84 and less efficiently internalized in the chicken macrophage cell line HD11. The ΔfliC mutant was pathogenic in orally infected BALB/c mice, while the ΔguaB mutant was attenuated and conferred protection against a challenge with the pathogenic parent strain. The ΔguaB ΔfliC double mutant was totally asymptomatic and conferred better protection than the ΔguaB mutant. This indicates that the major flagellar protein flagellin is not required for efficient vaccination of BALB/c mice against Salmonella infection. The ΔguaB ΔfliC mutant was also safe for vaccination of 1-day-old chickens. After two immunizations, it induced statistically significant protection against infection of the internal organs of the birds by a virulent S. enterica serovar Enteritidis challenge strain but not against intestinal colonization. These data demonstrate that nonflagellated attenuated Salmonella mutants can be used as marker vaccines.

Selectable and screenable markers

The transfer of foreign DNA to plant cells can be achieved in a variety of ways that are described in several chapters of this manual. This chapter describes the various selectable and screenable marker genes that are presently used in plant transformation experiments. Screenable markers have been widely used in gene constructs to study the regulation of plant gene expression.

Surface display of the receptor-binding domain of the F17a-G fimbrial adhesin through the autotransporter AIDA-I leads to permeability of bacterial cells

Surface exposure of antigens on bacterial cells can be critical for eliciting an effective antibody response. Therefore, we investigated the cellular localization of the fimbrial F17a-G receptor-binding domain, fused to the translocator domain of the AIDA-I autotransporter. Synthesis of the fusion protein, under the control of the L-arabinose-inducible PBAD promoter, was shown to permeabilize Escherichia coli K-12 and Salmonella enterica serovar Typhimurium cells. The presence of permeable cells interfered with several methods that are typically used to determine surface exposure of proteins, such as protease treatment and whole-cell ELISA. Double immunofluorescence microscopy, using a second antibody directed against beta-galactosidase, a bacterial protein expressed in the cytoplasm, allowed the simultaneous detection of antigen expression and permeability in individual cells.

The Shiga-toxin VT2-encoding bacteriophage ϕ297 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.

Insertional mutagenesis in Arabidopsis thaliana

Abstract Recently, a number of mutant gene loci in the Arabidopsis thaliana plant genome have been identified through insertional mutagenesis. In this review, we evaluate different methods used for Agrobacterium tumefaciens -mediated T-DNA insertional mutagenesis with regard to their mutation frequencies and conclude that a major breakthrough in the isolation of genes involved in plant development has been acheived. To provide a specific example, we summarize recent progress made in the understanding of flower morphogenesis at the molecular level through the study of homeotic genes obtained via gene tagging. T-DNA gene fusion vectors are being discussed that will allow the isolation of plant regulatory sequences with particular cell or tissue specificity, or that are controlled by specific external stimuli. Finally, we report on the approaches followed to convert the maize transposons Ac/Ds into valuable gene tags for use in a heterologous host such as Arabidopsis .