In Escherichia coli K-12, the major glucose transporter with a central role in carbon catabolite repression and in inducer exclusion is the phosphoenolpyruvate-dependent glucose:phosphotransferase system (PTS). Its membrane-bound subunit, IICB Glc , is encoded by the gene ptsG; its soluble domain, IIA Glc, is encoded by crr, which is a member of the pts operon. The system is inducible by D-glucose and, to a lesser degree, by L-sorbose. The regulation of ptsG transcription was analyzed by testing the induction of IICB Glc transporter activity and of a single-copy ⌽(ptsGop-lacZ) fusion. Among mutations found to affect directly ptsG expression were those altering the activity of adenylate cyclase (cyaA), the repressor DgsA (dgsA; also called Mlc), the general PTS proteins enzyme I (ptsI) and histidine carrier protein HPr (ptsH), and the IIA Glc and IIB Glc domains, as well as several authentic and newly isolated UmgC mutations. The latter, originally thought to map in the repressor gene umgC outside the ptsG locus, were found to represent ptsG alleles. These affected invariably the substrate specificity of the IICB Glc domain, thus allowing efficient transport and phosphorylation of substrates normally transported very poorly or not at all by this PTS. Simultaneously, all of these substrates became inducers for ptsG. From the analysis of the mutants, from cis-trans dominance tests, and from the identification of the amino acid residues mutated in the UmgC mutants, a new regulatory mechanism involved in ptsG induction is postulated. According to this model, the phosphorylation state of IIB Glc modulates IIC Glc which, directly or indirectly, controls the repressor DgsA and hence ptsG expression. By the same mechanism, glucose uptake and phosphorylation also control the expression of the pts operon and probably of all operons controlled by the repressor DgsA.In Escherichia coli K-12, D-glucose (Glc) is taken up and concomitantly phosphorylated either by the glucose-specific enzyme II (EII) transporter (II Glc ) or the mannose-specific EII transporter (II Man ) (genes manXYZ) of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) (for reviews, see references 10 and 21). As for most other PTS carbohydrates, the phosphoryl groups are sequentially transferred from PEP through two common intermediates, enzyme I (EI; gene: ptsI) and the phosphohistidine carrier protein (HPr; gene: ptsH), to sugar-specific EII (IICB Glc ; see below) and to glucose (for a review see reference 41). II Glc consists of two subunits, IIA Glc (crr [catabolite repression resistance]) and membrane-bound IICB Glc (ptsG) (8). The crr gene is part of the ptsHI crr operon (46) separated from the ptsG gene, which maps at 25.0 min (4). IIA Glc is a small hydrophilic protein which has, in addition to its transport function, a central regulatory role in carbon catabolite repression and inducer exclusion (for a review, see reference 22). The IICB Glc subunit is composed of an amino-terminal, hydrophobic IIC Glc domain, which large...

BackgroundIntroducing 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.ResultsHere 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®/ET® 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.ConclusionChromosomal modifications performed by rpsL counter-selection may also be used for other bacteria that contain an rpsL homologue, since Red®/ET® Recombination has been applied to several enteric bacteria before.

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 glucosephosphotransferase 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-␣-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.In Escherichia coli K-12, as in many other gram-positive and gram-negative bacteria, the phosphoenolpyruvate-dependent carbohydrate phosphotransferase systems (PTSs) are the major transport and sensor systems for carbohydrates. All of the PTSs except the mannose-specific PTS consist of five conserved functional domains, designated enzyme I (EI) (gene, ptsI), the histidine-containing phosphoryl carrier protein HPr (gene, ptsH), enzyme IIA (EIIA), enzyme IIB (EIIB), and enzyme IIC (EIIC) (for reviews see references 30 and 31). Depending on the organism or system, these functional PTS domains exist as single or multidomain proteins. The two cytoplasmic proteins, EI and HPr, are the general components of all PTS, whereas the EII complexes are carbohydrate specific. The protein kinase EI uses phosphoenolpyruvate in an autophosphorylation reaction, and the phosphoryl group is subsequently transferred to HPr, EIIA, and EIIB. Finally, the carbohydrate substrate, which is bound by the integral membrane domain of EIIC, is phosphorylated and concomitantly translocated across the membrane. The preferred carbon source of E. coli, D-glucose, is taken up by two different EIIs, the highaffinity glucose-specific molecule EII Glc (Glc-PTS) and the low-affinity mannose-specific molecule EII Man (Man-PTS).The Glc-PTS consists of the cytoplasmic protein EIIA Glc , encoded by the crr g...

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.

No abstract