Search

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 modeling. Transcriptomic data identified uptake systems for the four carbon sources, and candidates were subjected to preliminary experimental characterization by mutant strain growth to test their involvement in substrate assimilation. The OpdH and BenF-like porins were involved in citrate and ferulic acid uptake, respectively. The citrate transporter (encoded by PP_0147) and the TctABC system were important for supporting cell growth in citrate; PcaT and VanK were associated with ferulic acid uptake; and the ABC transporter AapJPQM was involved in serine transport. A genome-scale metabolic model of P. putida KT2440 was used to integrate and analyze the transcriptomic data, identifying and confirming the active catabolic pathways for each carbon source. This study reveals novel information about transporters that are essential for understanding bacterial adaptation to different environments.

Summary Small proteins of fifty 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 P. putida, bioinformatic and proteomic approaches were used to identify putative small open reading frames (sORFs) in the well-characterized strain KT2440. A plasmid-based system was established for sORF validation, enabling expression of C-terminal sequential peptide affinity (SPA) tagged variants and their detection via protein immunoblotting. Out of 22 tested putative sORFs, the expression of fourteen sORFs was confirmed, where all except one are novel. All of the validated sORFs except one are located adjacent to annotated genes on the same strand and three are in close proximity to genes with known functions. These include an ABC transporter operon and the two transcriptional regulators Fis and CysB involved in biofilm formation and cysteine biosynthesis, respectively. The work sheds light on the P. putida small proteome and small protein identification, a necessary first step towards gaining insights into their functions and possible evolutionary implications. This article is protected by copyright. All rights reserved.

The human-specific gene ARHGAP11B is preferentially expressed in neural progenitors of fetal human neocortex and increases abundance and proliferation of basal progenitors (BPs), which have a key role in neocortex expansion. ARHGAP11B has therefore been implicated in the evolutionary expansion of the human neocortex, but its mode of action has been unknown. Here, we show that ARHGAP11B is imported into mitochondria, where it interacts with the adenine nucleotide translocase (ANT) and inhibits the mitochondrial permeability transition pore (mPTP). BP expansion by ARHGAP11B requires its presence in mitochondria, and pharmacological inhibition of ANT function or mPTP opening mimic BP expansion by ARHGAP11B. Searching for the underlying metabolic basis, we find that BP expansion by ARHGAP11B requires glutaminolysis, the conversion of glutamine to glutamate for the tricarboxylic acid (TCA) cycle. Hence, an ARHGAP11B-induced, mitochondria-based effect on BP metabolism that is a hallmark of highly mitotically active cells appears to underlie its role in neocortex expansion.

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. In addition, Bio-layer interferometry is used to screen clonal libraries of aptamer candidates and is used to interrogate aptamer affinity. The RNA-based Capture-SELEX strategy described here simplifies selection of RNA aptamers against small molecules by avoiding ligand immobilization, while also allowing selection against multiple candidate targets in a single experiment. This makes RNA Capture-SELEX particularly attractive for accelerated development of RNA aptamers targeting small metabolites for incorporation into synthetic riboswitches and for analytical biosensors.

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. In addition, Bio-layer interferometry is used to screen clonal libraries of aptamer candidates and is used to interrogate aptamer affinity. The RNA-based Capture-SELEX strategy described here simplifies selection of RNA aptamers against small molecules by avoiding ligand immobilization, while also allowing selection against multiple candidate targets in a single experiment. This makes RNA Capture-SELEX particularly attractive for accelerated development of RNA aptamers targeting small metabolites for incorporation into synthetic riboswitches and for analytical biosensors.

Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida 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. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings. IMPORTANCE This study maps the complete transcriptional response of P. putida KT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one condition, suggesting their involvement in adaptation to stress conditions and identifying interesting candidates for further functional characterization.

The unique cell biological features of neural stem and progenitor cells contribute to the evolutionary expansion of the neocortex. The major types of progenitor cells responsible for this are the proliferative basal progenitors (BPs), which mainly reside within the outer subventricular zone (oSVZ) of gyrencephalic species, such as primates. Primate BPs are able to generate their own niche microenvironment in the oSVZ, sustaining their increased proliferative potential. Recent advances in transcriptional profiling have led to the identification of (1) human-specific expression patterns underlying the generation of this niche and (2) human-specific genes involved in cortical development such as ARHGAP11B.

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. Out of 22 tested putative sORFs, the expression of 14 sORFs was confirmed, where all except one are novel. All of the validated sORFs except one are located adjacent to annotated genes on the same strand and three are in close proximity to genes with known functions. These include an ABC transporter operon and the two transcriptional regulators Fis and CysB involved in biofilm formation and cysteine biosynthesis respectively. The work sheds light on the P. putida small proteome and small protein identification, a necessary first step towards gaining insights into their functions and possible evolutionary implications.

The environmental bacterium Pseudomonas putida is an organism endowed with a versatile metabolism and stress tolerance traits that are desirable in an efficient production organism. In this work, differential RNA sequencing was used to investigate the primary transcriptome and RNA regulatory elements of P. putida strain KT2440. A total of 7937 putative transcription start sites (TSSs) were identified, where over two-thirds were located either on the opposite strand or internal to annotated genes. For TSSs associated with mRNAs, sequence analysis revealed a clear Shine-Dalgarno sequence but a lack of conserved overrepresented promoter motifs. These TSSs defined approximately 50 leaderless transcripts and an abundance of mRNAs with long leader regions of which 18 contain RNA regulatory elements from the Rfam database. The thiamine pyrophosphate riboswitch upstream of the thiC gene was examined using an in vivo assay with GFP-fusion vectors and shown to function via a translational repression mechanism. Furthermore, 56 novel intergenic small RNAs and 8 putative actuaton transcripts were detected, as well as 8 novel open reading frames (ORFs). This study illustrates how global mapping of TSSs can yield novel insights into the transcriptional features and RNA output of bacterial genomes.

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. Three cfr -like genes from the order Bacillales were identified from BLAST searches and cloned into plasmids under the control of an inducible promoter. Expression of the genes was induced in Escherichia coli , and MICs for selected antibiotics indicate that the cfr -like genes confer resistance to PhLOPSa (phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A) antibiotics in the same way as the cfr gene. In addition, modification at A2503 on 23S rRNA was confirmed by primer extension. Finally, expression of the Cfr-like proteins was verified by SDS gel electrophoresis of whole-cell extracts. The work shows that cfr -like genes exist in the environment and that Bacillales are natural residences of cfr -like genes.

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.aeruginosa involving a combination of three different sequencing libraries. Almost all known sRNAs and over 500 novel intergenic sRNAs are identified with this approach. Although the use of three libraries increased the number of novel transcripts identified, there were significant differences in the subset of transcripts detected in each library, underscoring the importance of library preparation strategy and relative sRNA abundance for successful sRNA detection. Nearly 90% of the novel sRNAs have no orthologous bacterial sequences outside of P.aeruginosa, supporting a limited degree of sequence conservation and rapid evolution of sRNAs at the species level. We anticipate that the data will be useful for the study of regulatory sRNAs in bacteria and that the approach described here may be applied to identify sRNAs in any bacterium under different growth and stress conditions.

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. Phylogenetic analysis of the Cfr/RlmN family suggests that the RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer. A structural model of Cfr has been calculated and used as a guide for alanine mutagenesis studies that corroborate the model-based predictions of a 4Fe-4S cluster, a SAM molecule coordinated to the iron-sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis. The investigation has identified essential amino acids and Cfr variants with altered reaction mechanisms and represents a first step towards understanding the structural basis of Cfr activity.

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. isolates, but as multiple mutations were isolated together, the roles of the individual mutations are unclear. In this work, individual 23S rRNA mutations associated with pleuromutilin resistance at positions 2055, 2447, 2504 and 2572 (Escherichia coli numbering) are introduced into a Mycobacterium smegmatis strain with a single rRNA operon. The single mutations each confer a significant and similar degree of valnemulin resistance and those at 2447 and 2504 also confer cross-resistance to other antibiotics that bind to the PTC in M. smegmatis. Antibiotic footprinting experiments on mutant ribosomes show that the introduced mutations cause structural perturbations at the PTC and reduced binding of pleuromutilin antibiotics. This work underscores the fact that mutations at nucleotides distant from the pleuromutilin binding site can confer the same level of valnemulin resistance as those at nucleotides abutting the bound drug, and suggests that the former function indirectly by altering local structure and flexibility at the drug binding pocket.

The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding to an additional methyl group, but its specific identity and position remained to be elucidated. A novel tandem mass spectrometry approach has been developed to further characterize the Cfr-catalyzed modification. Comparison of nucleoside fragmentation patterns of A2503 from Escherichia coli cfr+ and cfr− strains with those of a chemically synthesized nucleoside standard shows that Cfr catalyzes formation of 8-methyladenosine. In addition, analysis of RNA derived from E. coli strains lacking the m2A2503 methyltransferase reveals that Cfr also has the ability to catalyze methylation at position 2 to form 2,8-dimethyladenosine. The mutation of single conserved cysteine residues in the radical SAM motif CxxxCxxC of Cfr abolishes its activity, lending support to the notion that the Cfr modification reaction occurs via a radical-based mechanism. Antibiotic susceptibility data confirm that the antibiotic resistance conferred by Cfr is provided by methylation at the 8 position and is independent of methylation at the 2 position of A2503. This investigation is, to our knowledge, the first instance where the 8-methyladenosine modification has been described in natural RNA molecules.

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. Antimicrobial susceptibility testing shows that S. aureus and E. coli strains expressing the cfr gene exhibit elevated MICs to a number of chemically unrelated drugs. The phenotype is named PhLOPS A for resistance to the following drug classes: Ph enicols, L incosamides, O xazolidinones, P leuromutilins, and S treptogramin A antibiotics. Each of these five drug classes contains important antimicrobial agents that are currently used in human and/or veterinary medicine. We find that binding of the PhLOPS A drugs, which bind to overlapping sites at the peptidyl transferase center that abut nucleotide A2503, is perturbed upon Cfr-mediated methylation. Decreased drug binding to Cfr-methylated ribosomes has been confirmed by footprinting analysis. No other rRNA methyltransferase is known to confer resistance to five chemically distinct classes of antimicrobials. In addition, the findings described in this study represent the first report of a gene conferring transferable resistance to pleuromutilins and oxazolidinones.

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. Mutations were identified in genes encoding ribosomal protein L3 and 23S rRNA at positions proximal to the peptidyl transferase centre. In two groups of laboratory-selected mutants, mutations were found at nucleotide positions 2032, 2055, 2447, 2499, 2504 and 2572 of 23S rRNA (Escherichia coli numbering) and at amino acid positions 148 and 149 of ribosomal protein L3 (Brachyspira pilosicoli numbering). In a third group of clinical B. hyodysenteriae isolates, only a single mutation at amino acid 148 of ribosomal protein L3 was detected. Chemical footprinting experiments show a reduced binding of tiamulin to ribosomal subunits from mutants with decreased susceptibility to the drug. This reduction in drug binding is likely the resistance mechanism for these strains. Hence, the identified mutations located near the tiamulin binding site are predicted to be responsible for the resistance phenotype. The positions of the mutated residues relative to the bound drug advocate a model where the mutations affect tiamulin binding indirectly through perturbation of nucleotide U2504.

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 approach may be applied to identify sRNAs in any bacterium under different growth and stress conditions.

Basic peptides from the carboxy terminus of the HIV-1 Tat protein bind to the apical stem-loop region of TAR RNA with high affinity and moderate specificity. The conformations of the unbound and 24 residue Tat peptide (Tfr24)-bound forms of TAR RNA have been characterized by NMR spectroscopy. The unbound form of TAR exists in major and minor forms having different trinucleotide bulge conformations. A specific TAR RNA conformational change is observed upon complex formation with Tfr24, consisting of coaxial stacking of helical stems and base triple formation. A U23-A27-U38 base triple is proposed based on exchangeable proton NMR data, where U23 forms a base pair with A27 in the major groove. No evidence for base triple formation was found for Tat peptides in which lysine residues are extensively substituted for arginine.

A hydrological analysis of historic stream data collected with the gauge on the Cache River at Patterson, Arkansas, USA, is presented as the basis for cumulative impact analysis of riverine wetlands. Subtle, long-term changes in hydroperiod, which could collectively have major effects on wetland function, are quantified. Harmonic analysis, time-scale analysis, and conventional methods of hydrological analysis of gauge data, at decade intervals, are employed, showing a steady decline in the magnitude and predictability of the baseflow during low flow periods, beginning with the 1920s and becoming increasingly more pronounced into the 1980s. Complementary information suggests that hydroperiod alterations are associated with increased groundwater pumping to support rice agriculture in the basin. These hydrological methods are simple enough for routine application (when adequate data are available) but sufficiently sophisticated to identify subtle changes in hydroperiod associated with cumulative effects. The changes in hydroperiod identified using these methods may have potential to explain changes in biotic communities or wetlands structure as part of comprehensive wetlands studies. © 1997 John Wiley & Sons, Ltd.

A hydrologic analysis of historic stream gage data collected on the Cache River at Patterson, Arkansas is presented as a basis for impact analysis of riverine wetlands. Subtle, long-term changes in hydroperiod that could collectively have major impacts on wetland functions are quantified. Harmonic analysis, time-scale analysis, and conventional methods of hydrologic analysis of gage data at decade intervals are employed. These various techniques indicate a steady decline in the magnitude and predictability of the base flow during low flow periods, beginning with the 1920s and becoming increasingly more pronounced into the 1980s. Complementary information suggests that hydroperiod alterations can be associated with increased ground-water pumping and associated land use changes in the Cache River basin. Hydrologic indices wer developed that range from intuitively simple but generally insensitive indices based on means, medians, ranges, and discharge-duration curves to indices that are based on methods sensitive to subtle changes in hydrologic patterns. One set of sensitive indices generated by harmonic analysis is relatively simple to obtain and useful to explore changes in the pattern of discharges or stages in wetlands. The timescale analysis can be employed to provide a relatively high resolution quantification of changes in hydrology that can be related to long-term changes in land- or water-use patterns. The hydrologic methods presented in these analyses are simple enough for routine application (when adequate data are available) but sufficiently sophisticated to identify subtle changes in hydroperiod associated with impacts and passage of time. Changes in hydroperiod identified by these methods may have the potential to explain changes in biotic communities or wetland structure as part of comprehensive wetland studies.

Basic peptides from the carboxy terminus of the human immunodeficiency virus type 1 (HIV-1) Tat protein bind to the stem-loop region of TAR RNA, spanning a trinucleotide bulge, with high affinity and moderate specificity. Previous studies have demonstrated that TAR RNA contains a specific arginine binding pocket. A series of 24 amino acid Tat-derived peptides with one or two arginines has been evaluated as possible structural models of the wild-type peptide in its interaction with TAR RNA, using gel electrophoretic methods and circular dichroism (CD) spectroscopy. Dissociation rate measurements indicate that these peptides form complexes with TAR RNA that are significantly less stable kinetically than the wild-type complex. Through a combination of dissociation and association rate measurements, we estimate that wild-type Tat peptide and TAR RNA interact with a Kd of about 16 pM. Together with competition experiments, these results confirm that band shift gel titration methods significantly underestimate absolute peptide-RNA binding affinities in the subnanomolar range. Through competition experiments with bulge mutants of TAR RNA, we demonstrate that peptides that form longer lived complexes with wild-type TAR RNA also show greater discrimination over TAR RNA bulge mutants. Difference CD spectra show that the Tat-derived peptides do not induce the same changes in TAR RNA as the wild-type peptide. The difference CD spectrum of argininamide bound to TAR RNA is most similar to that of the wild-type peptide-TAR RNA complex, implying that the differences in CD spectra upon complex formation are mostly due to changes in TAR RNA conformation.

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.aeruginosa involving a combination of three different sequencing libraries. Almost all known sRNAs and over 500 novel intergenic sRNAs are identified with this approach. Although the use of three libraries increased the number of novel transcripts identified, there were significant differences in the subset of transcripts detected in each library, underscoring the importance of library preparation strategy and relative sRNA abundance for successful sRNA detection. Nearly 90% of the novel sRNAs have no orthologous bacterial sequences outside of P.aeruginosa, supporting a limited degree of sequence conservation and rapid evolution of sRNAs at the species level. We anticipate that the data will be useful for the study of regulatory sRNAs in bacteria and that the approach described here may be applied to identify sRNAs in any bacterium under different growth and stress conditions.

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. Once inside the cell, most antibiotics affect all bacteria similarly. The ribosome is a major site of antibiotic action and is targeted by a large and chemically diverse group of antibiotics. A number of these antibiotics have important applications in human and veterinary medicine in the treatment of bacterial infections. The antibiotic binding sites are clustered at functional centers of the ribosome, such as the decoding center, the peptidyl transferase center, the GTPase center, the peptide exit tunnel, and the subunit interface spanning both subunits on the ribosome. Upon binding, the drugs interfere with the positioning and movement of substrates, products, and ribosomal components that are essential for protein synthesis. Ribosomal antibiotic resistance is due to the alteration of the antibiotic binding sites through either mutation or methylation. Our knowledge of antibiotic resistance mechanisms has increased, in particular due to the elucidation of the detailed structures of antibiotic-ribosome complexes and the components of the efflux systems. A number of mutations and methyltransferases conferring antibiotic resistance have been characterized. These developments are important for understanding and approaching the problems associated with antibiotic resistance, including design of antimicrobials that are impervious to known bacterial resistance mechanisms.

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. Mutations were identified in genes encoding ribosomal protein L3 and 23S rRNA at positions proximal to the peptidyl transferase centre. In two groups of laboratory-selected mutants, mutations were found at nucleotide positions 2032, 2055, 2447, 2499, 2504 and 2572 of 23S rRNA (Escherichia coli numbering) and at amino acid positions 148 and 149 of ribosomal protein L3 (Brachyspira pilosicoli numbering). In a third group of clinical B. hyodysenteriae isolates, only a single mutation at amino acid 148 of ribosomal protein L3 was detected. Chemical footprinting experiments show a reduced binding of tiamulin to ribosomal subunits from mutants with decreased susceptibility to the drug. This reduction in drug binding is likely the resistance mechanism for these strains. Hence, the identified mutations located near the tiamulin binding site are predicted to be responsible for the resistance phenotype. The positions of the mutated residues relative to the bound drug advocate a model where the mutations affect tiamulin binding indirectly through perturbation of nucleotide U2504.