Dataset Information

A new suite of tnaA mutants suggests that Escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site.

ABSTRACT: The Escherichia coli enzyme tryptophanase (TnaA) converts tryptophan to indole, which triggers physiological changes and regulates interactions between bacteria and their mammalian hosts. Tryptophanase production is induced by external tryptophan, but the activity of TnaA is also regulated by other, more poorly understood mechanisms. For example, the enzyme accumulates as a spherical inclusion (focus) at midcell or at one pole, but how or why this localization occurs is unknown.TnaA activity is low when the protein forms foci during mid-logarithmic growth but its activity increases as the protein becomes more diffuse, suggesting that foci may represent clusters of inactive (or less active) enzyme. To determine what protein characteristics might mediate these localization effects, we constructed 42 TnaA variants: 6 truncated forms and 36 missense mutants in which different combinations of 83 surface-exposed residues were converted to alanine. A truncated TnaA protein containing only domains D1 and D3 (D1D3) localized to the pole. Mutations affecting the D1D3-to-D1D3 interface did not affect polar localization of D1D3 but did delay assembly of wild type TnaA foci. In contrast, alterations to the D1D3-to-D2 domain interface produced diffuse localization of the D1D3 variant but did not affect the wild type protein. Altering several surface-exposed residues decreased TnaA activity, implying that tetramer assembly may depend on interactions involving these sites. Interestingly, changing any of three amino acids at the base of a loop near the catalytic pocket decreased TnaA activity and caused it to form elongated ovoid foci in vivo, indicating that the alterations affect focus formation and may regulate how frequently tryptophan reaches the active site.The results suggest that TnaA activity is regulated by subcellular localization and by a loop-associated occlusion of its active site. Equally important, these new TnaA variants are immediately available to the research community and should be useful for investigating how tryptophanase is localized and assembled, how substrate accesses its active site, the functional role of acetylation, and other structural and functional questions.

SUBMITTER: Li G

PROVIDER: S-EPMC4323232 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A new suite of tnaA mutants suggests that Escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site.

Li Gang G Young Kevin D KD

BMC microbiology 20150204

<h4>Background</h4>The Escherichia coli enzyme tryptophanase (TnaA) converts tryptophan to indole, which triggers physiological changes and regulates interactions between bacteria and their mammalian hosts. Tryptophanase production is induced by external tryptophan, but the activity of TnaA is also regulated by other, more poorly understood mechanisms. For example, the enzyme accumulates as a spherical inclusion (focus) at midcell or at one pole, but how or why this localization occurs is unknow ...[more]

PMID: 25650045

Similar Datasets

Project description:Escherichia coli ribosomal protein (r-protein) L4 has extraribosomal biological functions. Previously, we described L4 as inhibiting RNase E activity through protein-protein interactions. Here, we report that from stabilized transcripts regulated by L4-RNase E, mRNA levels of tnaA (encoding tryptophanase from the tnaCAB operon) increased upon ectopic L4 expression, whereas TnaA protein levels decreased. However, at nonpermissive temperatures (to inactivate RNase E), tnaA mRNA and protein levels both increased in an rne temperature-sensitive [rne(Ts)] mutant strain. Thus, L4 protein fine-tunes TnaA protein levels independently of its inhibition of RNase E. We demonstrate that ectopically expressed L4 binds with transcribed spacer RNA between tnaC and tnaA and downregulates TnaA translation. We found that deletion of the 5' or 3' half of the spacer compared to the wild type resulted in a similar reduction in TnaA translation in the presence of L4. In vitro binding of L4 to the tnaC-tnaA transcribed spacer RNA results in changes to its secondary structure. We reveal that during early stationary-phase bacterial growth, steady-state levels of tnaA mRNA increased but TnaA protein levels decreased. We further confirm that endogenous L4 binds to tnaC-tnaA transcribed spacer RNA in cells at early stationary phase. Our results reveal the novel function of L4 in fine-tuning TnaA protein levels during cell growth and demonstrate that r-protein L4 acts as a translation regulator outside the ribosome and its own operon.IMPORTANCE Some ribosomal proteins have extraribosomal functions in addition to ribosome translation function. The extraribosomal functions of several r-proteins control operon expression by binding to own-operon transcripts. Previously, we discovered a posttranscriptional, RNase E-dependent regulatory role for r-protein L4 in the stabilization of stress-responsive transcripts. Here, we found an additional extraribosomal function for L4 in regulating the tna operon by L4-intergenic spacer mRNA interactions. L4 binds to the transcribed spacer RNA between tnaC and tnaA and alters the structural conformation of the spacer RNA, thereby reducing the translation of TnaA. Our study establishes a previously unknown L4-mediated mechanism for regulating gene expression, suggesting that bacterial cells have multiple strategies for controlling levels of tryptophanase in response to varied cell growth conditions.

Dataset Information

A new suite of tnaA mutants suggests that Escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site.

Publications

A new suite of tnaA mutants suggests that Escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets