Sodium Dodecyl Sulfate Hypersensitivity of \u3ci\u3eclpP\u3c/i\u3e and \u3ci\u3eclpB\u3c/i\u3e Mutants of \u3ci\u3eEscherichia coli\u3c/i\u3e

We studied the hypersensitivity of clpP and clpB mutants of Escherichia coli to sodium dodecyl sulfate (SDS). Both wild-type E. coli MC4100 and lon mutants grew in the presence of 10% SDS, whereas isogenic clpP and clpB single mutants could not grow above 0.5% SDS and clpA and clpX single mutants could not grow above 5.0% SDS. For wild-type E. coli, cellular ClpP levels as determined by Western immunoblot analysis increased ca. sixfold as the levels of added SDS increased from 0 to 2%. Capsular colanic acid, measured as uronic acid, increased ca. sixfold as the levels of added SDS increased from 2 to 10%. Based on these findings, 3 of the 19 previously identified SDS shock proteins (M. Adamowicz, P. M. Kelley, and K. W. Nickerson, J. Bacteriol. 173:229-233, 1991) are tentatively identified as ClpP, ClpX, and ClpB

Thiamine Pyrophosphate Riboswitches Are Targets for the Antimicrobial Compound Pyrithiamine

SummaryThiamine metabolism genes are regulated in numerous bacteria by a riboswitch class that binds the coenzyme thiamine pyrophosphate (TPP). We demonstrate that the antimicrobial action of the thiamine analog pyrithiamine (PT) is mediated by interaction with TPP riboswitches in bacteria and fungi. For example, pyrithiamine pyrophosphate (PTPP) binds the TPP riboswitch controlling the tenA operon in Bacillus subtilis. Expression of a TPP riboswitch-regulated reporter gene is reduced in transgenic B. subtilis or Escherichia coli when grown in the presence of thiamine or PT, while mutant riboswitches in these organisms are unresponsive to these ligands. Bacteria selected for PT resistance bear specific mutations that disrupt ligand binding to TPP riboswitches and derepress certain TPP metabolic genes. Our findings demonstrate that riboswitches can serve as antimicrobial drug targets and expand our understanding of thiamine metabolism in bacteria

Sodium Dodecyl Sulfate Hypersensitivity of clpP and clpB Mutants of Escherichia coli

We studied the hypersensitivity of clpP and clpB mutants of Escherichia coli to sodium dodecyl sulfate (SDS). Both wild-type E. coli MC4100 and lon mutants grew in the presence of 10% SDS, whereas isogenic clpP and clpB single mutants could not grow above 0.5% SDS and clpA and clpX single mutants could not grow above 5.0% SDS. For wild-type E. coli, cellular ClpP levels as determined by Western immunoblot analysis increased ca. sixfold as the levels of added SDS increased from 0 to 2%. Capsular colanic acid, measured as uronic acid, increased ca. sixfold as the levels of added SDS increased from 2 to 10%. Based on these findings, 3 of the 19 previously identified SDS shock proteins (M. Adamowicz, P. M. Kelley, and K. W. Nickerson, J. Bacteriol. 173:229-233, 1991) are tentatively identified as ClpP, ClpX, and ClpB

Mechanism for gene control by a natural allosteric group I ribozyme

An allosteric ribozyme consisting of a metabolite-sensing riboswitch and a group I self-splicing ribozyme was recently found in the pathogenic bacterium Clostridium difficile. The riboswitch senses the bacterial second messenger c-di-GMP, thereby controlling 5′ splice site choice by the downstream ribozyme. In the presence of c-di-GMP, the allosteric ribozyme in the CD3246 precursor transcript generates a spliced transcript that retains the riboswitch aptamer. This architecture provides the riboswitch with a mechanism for extended regulation after splicing has occurred, or as a backup mechanism for suppression of translation in the event of misregulated splicing

An mRNA structure in bacteria that controls gene expression by binding lysine

Riboswitches are metabolite-responsive genetic control elements that reside in the untranslated regions (UTRs) of certain messenger RNAs. Herein, we report that the 5′-UTR of the lysC gene of Bacillus subtilis carries a conserved RNA element that serves as a lysine-responsive riboswitch. The ligand-binding domain of the riboswitch binds to L-lysine with an apparent dissociation constant (K(D)) of ∼1 µM, and exhibits a high level of molecular discrimination against closely related analogs, including D-lysine and ornithine. Furthermore, we provide evidence that this widespread class of riboswitches serves as a target for the antimetabolite S-(2-aminoethyl)-L-cysteine (AEC). These findings add support to the hypotheses that direct sensing of metabolites by messenger RNAs is a fundamental form of genetic control and that riboswitches represent a new class of antimicrobial drug targets

An mRNA structure that controls gene expression by binding FMN.

Riboswitches are structural domains embedded within the noncoding sequences of certain mRNAs that serve as metabolite-responsive genetic control elements Three distinct classes of riboswitches have been identified previously, and were shown to selectively recognize coenzyme B 12 (ref. 1), thiamine pyrophosphate (TPP) 2,4,5 or flavin mononucleotide (FMN) We report here that a highly conserved RNA domain termed the S box serves as a selective and high-affinity aptamer for SAM. Allosteric modulation of secondary and tertiary structures is induced upon SAM binding to the aptamer domain, and these structural changes are responsible for inducing termination of mRNA transcription. These findings provide additional support that RNA structures can adopt a wide range of sophisticated structures that can function as precision genetic switches. RESULTS Identification of a SAM-responsive riboswitch Each of the compounds sensed by previously identified riboswitches (coenzyme B 12 , TPP and FMN) is used as a coenzyme by modern protein enzymes. Notably, these coenzymes have substantial structural similarity to RNA and this has been used to support speculation that they might also have been used as coenzymes by ancient ribozymes in an RNA world In this effort we have examined the S box 14 , a highly conserved sequence domai

6S RNA is a widespread regulator of eubacterial RNA polymerase that resembles an open promoter

6S RNA is an abundant noncoding RNA in Escherichia coli that binds to σ(70) RNA polymerase holoenzyme to globally regulate gene expression in response to the shift from exponential growth to stationary phase. We have computationally identified >100 new 6S RNA homologs in diverse eubacterial lineages. Two abundant Bacillus subtilis RNAs of unknown function (BsrA and BsrB) and cyanobacterial 6Sa RNAs are now recognized as 6S homologs. Structural probing of E. coli 6S RNA and a B. subtilis homolog supports a common secondary structure derived from comparative sequence analysis. The conserved features of 6S RNA suggest that it binds RNA polymerase by mimicking the structure of DNA template in an open promoter complex. Interestingly, the two B. subtilis 6S RNAs are discoordinately expressed during growth, and many proteobacterial 6S RNAs could be cotranscribed with downstream homologs of the E. coli ygfA gene encoding a putative methenyltetrahydrofolate synthetase. The prevalence and robust expression of 6S RNAs emphasize their critical role in bacterial adaptation