Vibrio vulnificus is an opportunistic human pathogen that causes severe infections in susceptible individuals. While the components of the
Escherichia coli phosphoenolpyruvate: sugar ...phosphotransferase system (PTS) have been shown to regulate numerous targets, little such information is available for the
V. vulnificus PTS. Here we show that enzyme IIA
Glc of the PTS regulates the peptidase activity of a mammalian insulysin homolog in
V. vulnificus. While interaction of IIA
Glc with the insulysin homolog is independent of the phosphorylation state of IIA
Glc, only unphosphorylated IIA
Glc activates the insulysin homolog. Taken together, our results suggest that the
V. vulnificus insulysin-IIA
Glc complex plays a role in survival in the host by sensing glucose.
MINT-
8045996:
IIA glu (uniprotkb:Q7MBY2)
binds (MI:
0407) to
vIDE (uniprotkb:
Q7MIS6) by
pull down (MI:
0096)
MINT-
8045817, MINT-
8045967:
IIA glu (uniprotkb:Q7MBY2)
physically interacts (MI:
0915) with
vIDE (uniprotkb:
Q7MIS6) by
pull down (MI:
0096)
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 IIA^sup Ntr^) mutant became extremely sensitive to leucine-containing peptides (LCPs), while both ptsP (encoding enzyme I^sup Ntr^) 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 IIA^sup Ntr^ was found to be necessary to neutralize leucine toxicity. Further studies showed that the dephospho-form of enzyme IIA^sup Ntr^ 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. PUBLICATION ABSTRACT
Summary
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 IIA
Ntr
) mutant became extremely sensitive to leucine‐containing peptides (LCPs), while both
ptsP
(encoding enzyme I
Ntr
) 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 IIA
Ntr
was found to be necessary to neutralize leucine toxicity. Further studies showed that the dephospho‐form of enzyme IIA
Ntr
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.
In bacteria, multiple sigmas direct RNA polymerase to distinct sets of promoters. Housekeeping sigmas direct transcription from thousands of promoters, whereas most alternative sigmas are more ...selective, recognizing more highly conserved promoter motifs. For sigma(32) and sigma(28), two Escherichia coli Group 3 sigmas, altering a few residues in Region 2.3, the portion of sigma implicated in promoter melting, to those universally conserved in housekeeping sigmas relaxed their stringent promoter requirements and significantly enhanced melting of suboptimal promoters. All Group 3 sigmas and the more divergent Group 4 sigmas have nonconserved amino acids at these positions and rarely transcribe >100 promoters. We suggest that the balance of "melting" and "recognition" functions of sigmas is critical to setting the stringency of promoter recognition. Divergent sigmas may generally use a nonoptimal Region 2.3 to increase promoter stringency, enabling them to mount a focused response to altered conditions.
Sigma28 controls the expression of flagella-related genes and is the most widely distributed alternative sigma factor, present in motile Gram-positive and Gram-negative bacteria. The distinguishing ...feature of sigma28 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 sigma28 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 sigma28. 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 sigma28 orthologues. Sigma28 is the second Group 3 sigma shown to use an extended -10 region in promoter recognition, raising the possibility that other Group 3 sigmas will do so as well.
Sigma32 controls expression of heat shock genes in Escherichia coli and is widely distributed in proteobacteria. The distinguishing feature of sigma32 promoters is a long -10 region (CCCCATNT) whose ...tetra-C motif is important for promoter activity. Using alanine-scanning mutagenesis of sigma32 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 beta- and gamma-proteobacteria. This residue is located in the alpha-helix of sigmaDomain 3 that mediates recognition of the extended -10 promoter motif in other sigmas. K130 is not conserved in alpha- and delta-/epsilon-proteobacteria and we found that sigma32 from the alpha-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. Sigma32 is the first Group 3 sigma shown to use the 'extended -10' recognition motif.
Enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system undergoes a slow monomer-dimer transition. In vitro autophosphorylation of Enzyme I by PEP was studied at limiting ...concentrations of the protein. Addition to incubation mixtures containing wild-type Enzyme I of inactive or low-activity mutant forms of Enzyme I resulted in stimulation of autophosphorylation activity. The kinetics of the activation fit well to a model in which the active form of Enzyme I is the dimer. These experiments provide support for the argument that only the dimeric form of Enzyme I can be autophosphorylated.
The unphosphorylated form of enzyme IIA super(glc) of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system inhibits transport catalyzed by lactose permease. We previously ...characterized the area on the cytoplasmic face of lactose permease that interacts with enzyme IIA super(glc), using radioactive enzyme IIA super(glc). Subsequent studies suggested consensus binding sequences on proteins that interact with enzyme IIA super(glc). The present study characterizes a region on the surface of enzyme IIA super(glc) that interfaces with lactose permease. Acetylation of lysine residues by sulfosuccinimidyl acetate treatment of enzyme IIA super(glc), but not lactose permease, reduced the degree of interaction between the two proteins. To localize the lysine residue(s) on enzyme IIA super(glc) that is(are) involved in the regulatory interaction, selected lysine residues were mutagenized. Conversion of nine separate lysines to glutamic acid resulted in proteins that were still capable of phosphoryl acceptance from HPr. Except for Lys69, all the modified proteins were as effective as the wild-type enzyme IIA super(glc) in a test for binding to lactose permease. The Lys69 mutant was also defective in phosphoryl transfer to glucose permease. To derive further information concerning the contact surface, additional selected residues in the vicinity of Lys69 were mutagenized and tested for binding to lactose permease. On the basis of these studies, a model for the region of the surface of enzyme IIA super(glc) that interacts with lactose permease is proposed.
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