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1.
Antimicrob Agents Chemother ; 67(8): e0039523, 2023 08 17.
Article in English | MEDLINE | ID: mdl-37409958

ABSTRACT

Two strains of Streptococcus pneumoniae, one expressing the methyltransferase Erm(B) and the other negative for erm(B), were selected for solithromycin resistance in vitro either with direct drug selection or with chemical mutagenesis followed by drug selection. We obtained a series of mutants that we characterized by next-generation sequencing. We found mutations in various ribosomal proteins (L3, L4, L22, L32, and S4) and in the 23S rRNA. We also found mutations in subunits of the phosphate transporter, in the DEAD box helicase CshB, and in the erm(B)L leader peptide. All mutations were shown to decrease solithromycin susceptibility when transformed into sensitive isolates. Some of the genes derived from our in vitro screens were found to be mutated also in clinical isolates with decreased susceptibility to solithromycin. While many mutations were in coding sequences, some were found in regulatory regions. These included novel phenotypic mutations in the intergenic regions of the macrolide resistance locus mef(E)/mel and in the vicinity of the ribosome binding site of erm(B). Our screens highlighted that macrolide-resistant S. pneumoniae can easily acquire resistance to solithromycin, and they revealed many new phenotypic mutations.


Subject(s)
Anti-Bacterial Agents , Macrolides , Macrolides/pharmacology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Streptococcus pneumoniae , Drug Resistance, Bacterial/genetics , Microbial Sensitivity Tests , Mutation
2.
Nat Commun ; 13(1): 7076, 2022 11 18.
Article in English | MEDLINE | ID: mdl-36400772

ABSTRACT

The ProQ/FinO family of RNA binding proteins mediate sRNA-directed gene regulation throughout gram-negative bacteria. Here, we investigate the structural basis for RNA recognition by ProQ/FinO proteins, through the crystal structure of the ProQ/FinO domain of the Legionella pneumophila DNA uptake regulator, RocC, bound to the transcriptional terminator of its primary partner, the sRNA RocR. The structure reveals specific recognition of the 3' nucleotide of the terminator by a conserved pocket involving a ß-turn-α-helix motif, while the hairpin portion of the terminator is recognized by a conserved α-helical N-cap motif. Structure-guided mutagenesis reveals key RNA contact residues that are critical for RocC/RocR to repress the uptake of environmental DNA in L. pneumophila. Structural analysis and RNA binding studies reveal that other ProQ/FinO domains also recognize related transcriptional terminators with different specificities for the length of the 3' ssRNA tail.


Subject(s)
RNA, Small Untranslated , RNA-Binding Proteins , RNA-Binding Proteins/metabolism , RNA, Small Untranslated/genetics
3.
Proc Natl Acad Sci U S A ; 113(31): 8813-8, 2016 08 02.
Article in English | MEDLINE | ID: mdl-27432973

ABSTRACT

A highly conserved DNA uptake system allows many bacteria to actively import and integrate exogenous DNA. This process, called natural transformation, represents a major mechanism of horizontal gene transfer (HGT) involved in the acquisition of virulence and antibiotic resistance determinants. Despite evidence of HGT and the high level of conservation of the genes coding the DNA uptake system, most bacterial species appear non-transformable under laboratory conditions. In naturally transformable species, the DNA uptake system is only expressed when bacteria enter a physiological state called competence, which develops under specific conditions. Here, we investigated the mechanism that controls expression of the DNA uptake system in the human pathogen Legionella pneumophila We found that a repressor of this system displays a conserved ProQ/FinO domain and interacts with a newly characterized trans-acting sRNA, RocR. Together, they target mRNAs of the genes coding the DNA uptake system to control natural transformation. This RNA-based silencing represents a previously unknown regulatory means to control this major mechanism of HGT. Importantly, these findings also show that chromosome-encoded ProQ/FinO domain-containing proteins can assist trans-acting sRNAs and that this class of RNA chaperones could play key roles in post-transcriptional gene regulation throughout bacterial species.


Subject(s)
Gene Expression Regulation, Bacterial , Gene Transfer, Horizontal , Legionella pneumophila/genetics , RNA, Bacterial/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA, Bacterial/genetics , Gene Expression Profiling/methods , Humans , Legionella pneumophila/metabolism , Legionnaires' Disease/microbiology , Models, Genetic , Regulon/genetics , Transformation, Bacterial
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