Analysis of Pseudomonas putida growth on non-trivial carbon sources using transcriptomics and genome-scale modelling.

Pseudomonas putida is characterized by a versatile metabolism and stress tolerance traits that allow the bacterium to cope with different environmental conditions. In this work, the mechanisms that allow P. putida KT2440 to grow in the presence of four sole carbon sources (glucose, citrate, ferulic acid, serine) were investigated by RNA sequencing (RNA-seq) and genome-scale metabolic modelling.

A Capture-SELEX Strategy for Multiplexed Selection of RNA Aptamers Against Small Molecules.

In vitro selection of aptamers that recognize small organic molecules has proven difficult, in part due to the challenge of immobilizing small molecules on solid supports for SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This study describes the implementation of RNA Capture-SELEX, a selection strategy that uses an RNA library to yield ligand-responsive RNA aptamers targeting small organic molecules in solution. To demonstrate the power of this method we selected several aptamers with specificity towards either the natural sweetener rebaudioside A or the food-coloring agent carminic acid.

Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida.

Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes.

Identification and validation of novel small proteins in Pseudomonas putida.

Small proteins of 50 amino acids or less have been understudied due to difficulties that impede their annotation and detection. In order to obtain information on small open reading frames (sORFs) in Pseudomonas putida, bioinformatic and proteomic approaches were used to identify putative sORFs in the well-characterized strain KT2440. A plasmid-based system was established for sORF validation, enabling expression of C-terminal sequential peptide affinity tagged variants and their detection via protein immunoblotting.

Differential expression of small RNAs under chemical stress and fed-batch fermentation in E. coli.

Background: Bacterial small RNAs (sRNAs) are recognized as posttranscriptional regulators involved in the control of bacterial lifestyle and adaptation to stressful conditions. Although chemical stress due to the toxicity of precursor and product compounds is frequently encountered in microbial bioprocessing applications, the involvement of sRNAs in this process is not well understood. We have used RNA sequencing to map sRNA expression in E.

Identification of bacterial small RNAs by RNA sequencing.

Small regulatory RNAs (sRNAs) in bacteria are known to modulate gene expression and control a variety of processes including metabolic reactions, stress responses, and pathogenesis in response to environmental signals. A method to identify bacterial sRNAs on a genome-wide scale based on RNA sequencing (RNA-seq) is described that involves the preparation and analysis of three different sequencing libraries. As a significant number of unique sRNAs are identified in each library, the libraries can be used either alone or in combination to increase the number of sRNAs identified.

The order Bacillales hosts functional homologs of the worrisome cfr antibiotic resistance gene.

The cfr gene encodes the Cfr methyltransferase that methylates a single adenine in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to several classes of antibiotics that include drugs of clinical and veterinary importance. This paper describes a first step toward elucidating natural residences of the worrisome cfr gene and functionally similar genes.

Genome-wide identification of novel small RNAs in Pseudomonas aeruginosa.

Bacterial small regulatory RNAs (sRNAs) function in post-transcriptional control of gene expression and control a variety of processes including metabolic reactions, stress responses and pathogenesis in response to environmental signals. A variety of approaches have been used previously to identify 44 sRNAs in the opportunistic human pathogen Pseudomonas aeruginosa. In this work, RNA sequencing (RNA-seq) is used to identify novel transcripts in P.

Resistance to linezolid caused by modifications at its binding site on the ribosome.

Linezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance.

Mutations in 23S rRNA at the peptidyl transferase center and their relationship to linezolid binding and cross-resistance.

The oxazolidinone antibiotic linezolid targets the peptidyl transferase center (PTC) on the bacterial ribosome. Thirteen single and four double 23S rRNA mutations were introduced into a Mycobacterium smegmatis strain with a single rRNA operon. Converting bacterial base identity by single mutations at positions 2032, 2453, and 2499 to human cytosolic base identity did not confer significantly reduced susceptibility to linezolid.

Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria.

The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase RlmN that methylates the C-2 position. Database searches with the Cfr sequence have revealed a large group of closely related sequences from all domains of life that contain the conserved CX(3)CX(2)C motif characteristic of radical S-adenosyl-l-methionine (SAM) enzymes.

Single 23S rRNA mutations at the ribosomal peptidyl transferase centre confer resistance to valnemulin and other antibiotics in Mycobacterium smegmatis by perturbation of the drug binding pocket.

Tiamulin and valnemulin target the peptidyl transferase centre (PTC) on the bacterial ribosome. They are used in veterinary medicine to treat infections caused by a variety of bacterial pathogens, including the intestinal spirochetes Brachyspira spp. Mutations in ribosomal protein L3 and 23S rRNA have previously been associated with tiamulin resistance in Brachyspira spp.

Antibiotic Resistance Mechanisms, with an Emphasis on Those Related to the Ribosome.

Antibiotic resistance is a fundamental aspect of microbiology, but it is also a phenomenon of vital importance in the treatment of diseases caused by pathogenic microorganisms. A resistance mechanism can involve an inherent trait or the acquisition of a new characteristic through either mutation or horizontal gene transfer. The natural susceptibilities of bacteria to a certain drug vary significantly from one species of bacteria to another and even from one strain to another.

The Cfr rRNA methyltransferase confers resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics.

A novel multidrug resistance phenotype mediated by the Cfr rRNA methyltransferase is observed in Staphylococcus aureus and Escherichia coli. The cfr gene has previously been identified as a phenicol and lincosamide resistance gene on plasmids isolated from Staphylococcus spp. of animal origin and recently shown to encode a methyltransferase that modifies 23S rRNA at A2503.

Interaction of pleuromutilin derivatives with the ribosomal peptidyl transferase center.

Tiamulin is a pleuromutilin antibiotic that is used in veterinary medicine. The recently published crystal structure of a tiamulin-50S ribosomal subunit complex provides detailed information about how this drug targets the peptidyl transferase center of the ribosome. To promote rational design of pleuromutilin-based drugs, the binding of the antibiotic pleuromutilin and three semisynthetic derivatives with different side chain extensions has been investigated using chemical footprinting.

Mutations in ribosomal protein L3 and 23S ribosomal RNA at the peptidyl transferase centre are associated with reduced susceptibility to tiamulin in Brachyspira spp. isolates.

The pleuromutilin antibiotic tiamulin binds to the ribosomal peptidyl transferase centre. Three groups of Brachyspira spp. isolates with reduced tiamulin susceptibility were analysed to define resistance mechanisms to the drug.

A conserved chloramphenicol binding site at the entrance to the ribosomal peptide exit tunnel.

The antibiotic chloramphenicol produces modifications in 23S rRNA when bound to ribosomes from the bacterium Escherichia coli and the archaeon Halobacterium halobium and irradiated with 365 nm light. The modifications map to nucleotides m(5)U747 and C2611/C2612, in domains II and V, respectively, of E.coli 23S rRNA and G2084 (2058 in E.

Resistance to the peptidyl transferase inhibitor tiamulin caused by mutation of ribosomal protein l3.

The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the peptidyl transferase center. Tiamulin-resistant Escherichia coli mutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon.