The role of LRP and H-NS in transcription regulation: involvement of synergism, allostery and macromolecular crowding.

LRP has recently been shown to interact with the regulatory regions of bacterial ribosomal RNA promoters. Here we study details of the LRP-rDNA interaction by gel retardation and high-resolution footprinting techniques. We show that a second regulator for rRNA transcription, H-NS, facilitates the formation of a higher-order LRP-nucleoprotein complex, probably acting transiently as a DNA chaperone. The macromolecular crowding substance ectoine stabilizes the formation of this dynamic complex, while the amino acid leucine, as a metabolic effector, has the opposite effect. DNase I and hydroxyl radical footprint experiments with LRP-DNA complexes reveal a periodic change of the target DNA structure, which implies extensive DNA wrapping reaching into the promoter core region. We show furthermore that LRP binding is able to constrain supercoils, providing a link between DNA topology and regulation. The results support the conclusion that the bacterial DNA-binding protein LRP, assisted by H-NS, forms a repressive nucleoprotein structure involved in regulation of rRNA transcription. The formation of this regulatory structure appears to be directly affected by environmental changes.

Ribosomal and non-ribosomal resistance to oxazolidinones: species-specific idiosyncrasy of ribosomal alterations.

A derivative of Mycobacterium smegmatis, which carries only one functional rRNA (rrn) operon, was used to isolate mutants resistant to the ribosome-targeted antibiotic linezolid. Isolation and characterization of linezolid-resistant clones revealed two classes of mutants. Ribosomes from class I mutants are resistant to oxazolidinones in an in vitro peptidyl transferase assay, indicating that resistance maps to the ribosome component. In contrast, ribosomes from class II mutants show wild-type susceptibility to a linezolid derivative in vitro, pointing to a non-ribosomal mechanism of resistance. Introduction of a wild-type ribosomal RNA operon into linezolid-resistant strains restored linezolid sensitivity in class I mutants, indicating that resistance (i) maps to the rRNA and (ii) is recessive. Sequencing of the entire rrn operon identified a single nucleotide alteration in 23S rRNA of class I mutant strains, 2447G --> T (Escherichia coli numbering). Introduction of mutant rrl2447T into M. smegmatis rrn- resulted in a linezolid-resistant phenotype, demonstrating a cause-effect relationship of the 2447G --> T alteration. The 2447G --> T mutation, which renders M. smegmatis linezolid resistant, confers lethality in E. coli. This finding is strong evidence of structural and pos-sibly functional differences between the ribosomes of Gram-positive and Gram-negative bacteria. In agreement with the results of the in vitro assay, class II mutants show a wild-type sequence of the complete rRNA operon. The lack of cross-resistance of the class II mutants to other antibiotics suggests a resistance mechanism other than activation of a broad-spectrum multidrug transporter.

Interaction of avilamycin with ribosomes and resistance caused by mutations in 23S rRNA.

The antibiotic growth promoter avilamycin inhibits protein synthesis by binding to bacterial ribosomes. Here the binding site is further characterized on Escherichia coli ribosomes. The drug interacts with domain V of 23S rRNA, giving a chemical footprint at nucleotides A2482 and A2534. Selection of avilamycin-resistant Halobacterium halobium cells revealed mutations in helix 89 of 23S rRNA. Furthermore, mutations in helices 89 and 91, which have previously been shown to confer resistance to evernimicin, give cross-resistance to avilamycin. These data place the binding site of avilamycin on 23S rRNA close to the elbow of A-site tRNA. It is inferred that avilamycin interacts with the ribosomes at the ribosomal A-site interfering with initiation factor IF2 and tRNA binding in a manner similar to evernimicin.

Antisignature oligonucleotides and their analogs as inhibitors of mollicutes-cofactors of HIV.

Inhibition of mollicutes by synthetic oligonucleotides and their analogs complementary to specific "signature" regions of 16S rRNA and corresponding sequences of ribosomal operon DNA was studied. It was shown that antisignature oligonucleotides inhibited transcription in vitro for above 79% interacting specifically with ribosomal operon and non-specific with DNA-dependent RNA-polymerase. The inhibition efficiency depended on oligonucleotide sequence and type of modification. Translation in vitro was suppressed most efficiently (up to 60%) by oligonucleotides complementary to 3'-end region of 16S rRNA, also depending on their modification. Translation in vivo was inhibited most efficiently (up to 73%) by thiophosphate analogs of oligonucleotides complementary to sequences 499-507 and 523-532 of 16S rRNA responsible for binding of ribosomal "core" protein S4 starting the assembly of 30S ribosome subunit. With the simultaneous use of the last two oligonucleotides, the growth of mollicutes in SM IMV-72 medium rich in exogenous sources of nucleosides was suppressed for over 90%. It is supposed that under conditions where mollicutes have no free access to starting materials for their own synthesis of nucleic acid these nucleotides could suppress microorganisms completely. Antisignature oligonucleotides are considered as superspecific agents not leading to the development of resistance of mollicutes and believed to be the main future remedy against diseased caused by microorganisms lacking the system of nucleoside synthesis.