Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red(R)/ET(R) Recombination.

BACKGROUND: Introducing point mutations into bacterial chromosomes is important for further progress in studies relying on functional genomics, systems- and synthetic biology, and for metabolic engineering. For many investigations, chromosomal systems are required rather than artificial plasmid based systems. RESULTS: Here we describe the introduction of a single point mutation into the Escherichia coli chromosome by site-directed mutagenesis without leaving any selection marker. We used Red(R)/ET(R) Recombination in combination with rpsL counter-selection to introduce a single point mutation into the E. coli MG1655 genome, one of the widely used bacterial model strains in systems biology. The method we present is rapid and highly efficient. Since single-stranded synthetic oligonucleotides can be used for recombination, any chromosomal modification can be designed. CONCLUSION: Chromosomal modifications performed by rpsL counter-selection may also be used for other bacteria that contain an rpsL homologue, since Red(R)/ET(R) Recombination has been applied to several enteric bacteria before.

iGentifier: indexing and large-scale profiling of unknown transcriptomes.

Development and refinement of methods to analyse differential gene expression has been essential in the progress of molecular biology. A novel approach called iGentifier is presented for profiling known and unknown transcriptomes, thus bypassing a major limitation in microarray analysis. The iGentifier technology combines elements of fragment display (e.g. Differential Display or RMDD) and tag sequencing (e.g. SAGE, MPSS) and allows for analysis of samples in high throughput using current capillary electrophoresis equipment. Application to epidermal tissue of wild-type and mlo5 barley (Hordeum vulgare) plants, infected with powdery mildew [Blumeria graminis (DC.) E.O. Speer f.sp.hordei], led to the identification of several 100 genes induced or repressed upon infection with many well known for their response to fungal pathogens or other stressors. Ten of these genes are suggested to be classified as marker genes for durable resistance mediated by the mlo5 resistance gene.

YeeI, a novel protein involved in modulation of the activity of the glucose-phosphotransferase system in Escherichia coli K-12.

The membrane-bound protein EIICB(Glc) encoded by the ptsG gene is the major glucose transporter in Escherichia coli. This protein is part of the phosphoenolpyruvate:glucose-phosphotransferase system, a very important transport and signal transduction system in bacteria. The regulation of ptsG expression is very complex. Among others, two major regulators, the repressor Mlc and the cyclic AMP-cyclic AMP receptor protein activator complex, have been identified. Here we report identification of a novel protein, YeeI, that is involved in the regulation of ptsG by interacting with Mlc. Mutants with reduced activity of the glucose-phosphotransferase system were isolated by transposon mutagenesis. One class of mutations was located in the open reading frame yeeI at 44.1 min on the E. coli K-12 chromosome. The yeeI mutants exhibited increased generation times during growth on glucose, reduced transport of methyl-alpha-d-glucopyranoside, a substrate of EIICB(Glc), reduced induction of a ptsG-lacZ operon fusion, and reduced catabolite repression in lactose/glucose diauxic growth experiments. These observations were the result of decreased ptsG expression and a decrease in the amount of EIICB(Glc). In contrast, overexpression of yeeI resulted in higher expression of ptsG, of a ptsG-lacZ operon fusion, and of the autoregulated dgsA gene. The effect of a yeeI mutation could be suppressed by introducing a dgsA deletion, implying that the two proteins belong to the same signal transduction pathway and that Mlc is epistatic to YeeI. By measuring the surface plasmon resonance, we found that YeeI (proposed gene designation, mtfA) directly interacts with Mlc with high affinity.