Byoung-Mo Koo

Purification of Mlc and analysis of its effects on the pts expression in Escherichia coli.

Products of the pts operon ofEscherichia coli have multiple physiological roles such as sugar transport, and the operon is controlled by two promoters, P0 and P1. Expression of the pts P0 promoter that is increased during growth in the presence of glucose is also activated by cAMP receptor protein·cAMP. Based on the existence of a sequence that has a high similarity with the known Mlc binding site in the promoter, the effects of the Mlc protein on the pts P0 promoter expression were studied. In vivo transcription assays using wild type and mlc-negative E. coli strains grown in the presence and absence of glucose indicate that Mlc negatively regulates expression of the P0 promoter, and Mlc-dependent repression is relieved by glucose in the growth medium. In vitro transcription assay using purified recombinant Mlc showed that Mlc repressed transcription from the P0 but did not affect the activity of the P1. DNase I footprinting experiments revealed that a Mlc binding site was located around +1 to +25 of the promoter and that Mlc inhibited the binding of RNA polymerase to the P0 promoter. Cells overexpressing Mlc showed a very slow fermentation rate compared with the wild type when grown in the presence of various phosphoenolpyruvate-carbohydrate phosphotransferase system sugars but few differences in the presence of non-phosphoenolpyruvate-carbohydrate phosphotransferase system sugars except maltose. These results suggest that the pts operon is one of major targets for the negative regulation by Mlc, and thus Mlc regulates the utilization of various sugars as well as glucose inE. coli. The possibility that the inducer of Mlc may not be sugar or its derivative but an unknown factor is proposed to explain the Mlc induction mechanism by various sugars.

Requirement of the dephospho-form of enzyme IIANtr for derepression of Escherichia coli K-12 ilvBN expression.

While the proteins of the phosphoenolpyruvate:carbohydrate phosphotransferase system (carbohydrate PTS) have been shown to regulate numerous targets, little such information is available for the nitrogen‐metabolic phosphotransferase system (nitrogen‐metabolic PTS). To elucidate the physiological role of the nitrogen‐metabolic PTS, we carried out phenotype microarray (PM) analysis with Escherichia coli K‐12 strain MG1655 deleted for the ptsP gene encoding the first enzyme of the nitrogen‐metabolic PTS. Together with the PM data, growth studies revealed that a ptsN (encoding enzyme IIANtr) mutant became extremely sensitive to leucine‐containing peptides (LCPs), while both ptsP (encoding enzyme INtr) and ptsO (encoding NPr) mutants were more resistant than wild type. The toxicity of LCPs was found to be due to leucine and the dephospho‐form of enzyme IIANtr was found to be necessary to neutralize leucine toxicity. Further studies showed that the dephospho‐form of enzyme IIANtr is required for derepression of the ilvBN operon encoding acetohydroxy acid synthase I catalysing the first step common to the biosynthesis of the branched‐chain amino acids.

Dissection of recognition determinants of Escherichia coli σ32 suggests a composite −10 region with an ‘extended −10’ motif and a core −10 element

σ32 controls expression of heat shock genes in Escherichia coli and is widely distributed in proteobacteria. The distinguishing feature of σ32 promoters is a long −10 region (CCCCATNT) whose tetra‐C motif is important for promoter activity. Using alanine‐scanning mutagenesis of σ32 and in vivo and in vitro assays, we identified promoter recognition determinants of this motif. The most downstream C (−13) is part of the −10 motif; our work confirms and extends recognition determinants of −13C. Most importantly, our work suggests that the two upstream Cs (−16, −15) constitute an ‘extended −10’ recognition motif that is recognized by K130, a residue universally conserved in β‐ and γ‐proteobacteria. This residue is located in the α‐helix of σDomain 3 that mediates recognition of the extended −10 promoter motif in other σs. K130 is not conserved in α‐ and δ‐/ε‐proteobacteria and we found that σ32 from the α‐proteobacterium Caulobacter crescentus does not need the extended −10 motif for high promoter activity. This result supports the idea that K130 mediates extended −10 recognition. σ32 is the first Group 3 σ shown to use the ‘extended −10’ recognition motif.

Mutational analysis of Escherichia coli σ28 and its target promoters reveals recognition of a composite −10 region, comprised of an ‘extended −10’ motif and a core −10 element

σ28 controls the expression of flagella‐related genes and is the most widely distributed alternative σ factor, present in motile Gram‐positive and Gram‐negative bacteria. The distinguishing feature of σ28 promoters is a long −10 region (GCCGATAA). Despite the fact that the upstream GC is highly conserved, previous studies have not indicated a functional role for this motif. Here we examine the functional relevance of the GCCG motif and determine which residues in σ28 participate in its recognition. We find that the GCCG motif is a functionally important composite element. The upstream GC constitutes an extended −10 motif and is recognized by R91, a residue in Domain 3 of σ28. The downstream CG is the upstream edge of −10 region of the promoter; two residues in Region 2.4, D81 and R84, participate in its recognition. Consistent with their role in base‐specific recognition of the promoter, R91, D81 and D84 are universally conserved in σ28 orthologues. σ28 is the second Group 3 σ shown to use an extended −10 region in promoter recognition, raising the possibility that other Group 3 σs will do so as well.

A mammalian insulysin homolog is regulated by enzyme IIAGlc of the glucose transport system in Vibrio vulnificus

MINT‐8045817, MINT‐8045967: IIA glu (uniprotkb:Q7MBY2) physically interacts (MI:0915) with vIDE (uniprotkb:Q7MIS6) by pull down (MI:0096)