Journal: bioRxiv

Article Title: Spatial pattern formation of bacterial colonies with social antibiotic resistance

doi: 10.1101/2024.08.21.608973

Figure Lengend Snippet: (A) Experimental setup to investigate homeland spatial organization. Left: A mixture of defined density and ratio of fluorescently labeled ampicillin-resistant E. faecalis bacteria expressing β-lactamase and ampicillin sensitive E. faecalis bacteria is added in a 2µl droplet to an agar plate containing growth media (Brain Heart Infusion), ampicillin, and spectinomycin to select for the fluorescent plasmids. Approximate scales of the plate diameter, droplet diameter and cell diameter are shown. Center left: The seeded colony grows overnight at optimal growth temperature, and is then imaged on a confocal fluorescence microscope, using a cover glass spacer to leave the colony at the agar-air interface unperturbed. The spatial arrangement inside the seeded colony area (the homeland) is analyzed. Center right: Overall density and ampicillin-resistant fraction of the colony, as well as ampicillin concentration were the input variables that were varied across different initial conditions to test their impact on spatial pattern formation. Right: Ampicillin-resistant and sensitive subpopulations are expected to have the following ecological interactions: Competition for space or nutrients within the colony area and ampicillin-induced death that is alleviated by degradation of antibiotic by the β-lactamase expressed by resistant bacteria. As these immotile bacteria maintain relatively fixed position in the spatially structured colony apart from cell-division and death processes, these ecological interactions are expected to be heterogeneous and neighborhood-dependent. Resistant cells are shown in green, sensitive cells in magenta and antibiotic concentration is encoded in blue. (B) Resistant and sensitive E. faecalis subpopulations form a diverse array of spatial patterns on agar. Representative images (maximum intensity z-projections) of spatial homeland organization of ampicillin-resistant (green, fluorophore mDasher) and ampicillin-sensitive (magenta, fluorophore mRudolph) bacteria after overnight growth. Scale bars = 500 µm; unless otherwise noted, all images in the same panel are at the same scale. “Rings” of varied intensity of the green signal at high ampicillin concentrations and low initial resistant fractions are imaging artifacts arising from the maximum intensity projection in combination with non-uniform illumination across angled surfaces.

Article Snippet: Single colonies of relevant strains were inoculated into ∼3ml Brain Heart Infusion (BHI) media (Remel) with 120 μg/ml spectinomycin sulfate (Thermo Fisher Scientific, 430.43 g/mol) to select for pBSU101 plasmids over night at 37C.

Techniques: Labeling, Bacteria, Expressing, Fluorescence, Microscopy, Concentration Assay, Imaging

Journal: iScience

Article Title: An improved temperature-sensitive shuttle vector system for scarless gene deletion in human-gut-associated Bifidobacterium species

doi: 10.1016/j.isci.2024.111080

Figure Lengend Snippet: Replication initiation protein RepB with S139W substitution leads to an enhanced Ts phenotype in B. longum JCM 31944 (A and B) Effect of the 139 th amino acid residue of RepB on plasmid stability at 30°C (A) or 38.5°C (B). Ser-139 of the E. coli-Bifidobacterium shuttle vector, pMSK162 (blue bar; Figure S1 ), was replaced with the remaining 19 amino acid residues by site-directed mutagenesis. The resulting plasmids and parental plasmid pMSK162 were introduced into B. longum , and the obtained 14 transformant cells were then spread onto GAM and GAM-Sp plates. The plates were incubated at 30°C or 38.5°C to determine the CFU. Plasmid stability is shown as the percentage of Sp R cells in the total number of cells. Similar experiments were performed for pKO403 and its derivatives (red bar; Figure S1 ). Data are represented as dot plots with mean of at least biological duplicates. (C) Schematic maps of the E . coli - Bifidobacterium shuttle vectors, pMSK183 and pMSK197. pMSK183 and pMSK197, which are derivatives of pMSK162 and pKO403 ( Figure S1 ), respectively, harbor mutated repB (S139W). pTB6 replicon, replicon for bifidobacteria; sso , single-strand origin for replication; dso , double-strand origin for replication; repB , replication initiation protein gene; T hup , transcription terminator of hup gene from B. longum ; pUC replicon, replicon for E. coli ; Sp R gene, spectinomycin resistance gene. Open-reading frames are indicated by arrows. Plasmid maps were drawn using SnapGene Viewer 7.0.1. See also Figures S1 and .

Article Snippet: Spectinomycin dihydrochloride , LKT Laboratories , Cat#S6018.

Techniques: Residue, Plasmid Preparation, Mutagenesis, Incubation

Journal: iScience

Article Title: An improved temperature-sensitive shuttle vector system for scarless gene deletion in human-gut-associated Bifidobacterium species

doi: 10.1016/j.isci.2024.111080

Figure Lengend Snippet:

Article Snippet: Spectinomycin dihydrochloride , LKT Laboratories , Cat#S6018.

Techniques: Virus, Recombinant, Starch, DNA Purification, Staining, Sequencing, Software

Journal: Nature Structural & Molecular Biology

Article Title: Structural conservation of antibiotic interaction with ribosomes

doi: 10.1038/s41594-023-01047-y

Figure Lengend Snippet: a-k , Superimposition of previous structures of diverse antibiotic-ribosome complexes, including ( a ) gentamicin (yellow) on the E. coli 70S ribosome at 3.5 Å (PDB ID 4V53) with gentamicin (green) on the T. thermophilus 30S at 3.3 Å (PDB ID 4LF9), ( b ) kasugamycin (yellow) on the E. coli 70S ribosome at 3.5 Å (PDB ID 4V4H) with kasugamycin (red) on the T. thermophilus 30S at 3.4 Å (PDB ID 2HHH) , ( c ) streptomycin on the T. thermophilus 30S at 3.0 Å (yellow; PDB ID 1FJG) at 3.3 Å (green, PDB ID 4DR6) at 3.35 Å (blue, PDB ID 4JI1) with streptomycin (red) on the human mitochondrial small subunit at 2.4 Å (PDB ID 7P2E) , ( d ) apramycin (yellow) on the T. thermophilus 30S at 3.5 Å (PDB ID 4AQY) with apramycin on the E. coli 70S ribosome at 2.4 Å (red, PDB ID 7PJS) and 3.1 Å (green, PDB ID 7PJV) , ( e ) hygromycin B on the T. thermophilus 30S at 3.3 Å (yellow, PDB ID 1HNZ) and 3.7 Å (blue, PDB ID 4LFA) with hygromycin B on the E. coli 70S ribosome at 3.5 Å (red, PDB ID 4V64) , ( f ) spectinomycin (red) on the T. thermophilus 30S at 3.0 Å (PDB ID 1FJG) with spectinomycin on E. coli 70S ribosome at 3.5 Å (green, PDB ID 4V57) , E. faecalis 70S ribosome at 2.4 Å (yellow, PDB ID 7P7Q) and within an E. coli 70S translocation intermediate at 2.5 Å (blue, PDB ID 7N2V) , ( g ) tetracycline on the T. thermophilus 30S at 3.4 Å (red, PDB ID 1HNW) and 4.5 Å (yellow, PDB ID 1I97) with tetracycline (blue) on the T. thermophilus 70S at 3.3 Å (PDB ID 4V9A) and tetracycline (green) on the E. coli 70S at 2.8 Å (PDB ID 5J5B) , ( h ) the pleuromutilins tiamulin (yellow) on the D. radiodurans 50S at 3.5 Å (PDB ID 1XBP) , retapamulin (blue) on the D. radiodurans 50S at 3.7 Å (PDB ID 2OGO) , tiamulin (red) on the archaeal H. marismortui 50S at 3.2 Å (PDB ID 3G4S) and lefamulin (green) on the S. aureus 50S at 3.6 Å (PDB ID 5HL7) , ( i ) capreomycin (yellow) on the T. thermophilus 70S at 3.5 Å (PDB ID 4V7M) with capreomycin (red) on the M. tuberculosis 70S at 4.0 Å (PDB ID 5V93) , ( j ) the orthosomycins avilamycin (blue, PDB ID 5KCR) and evernimicin (red, PDB ID 5KCS) on the E. coli 70S at 3.9 Å with avilamycin (green, PDB ID 5JVG) and evernimicin (yellow, PDB ID 5JVH) on the D. radiodurans 50S at at 3.6 Å and 3.4 Å, respectively , ( k ) the lincosamide clindamycin on the D. radiodurans 50S at 3.1 Å (yellow, PDB ID 1JZY) , on the H. marismortui 50S at 3.0 Å (red, PDB ID 1YJN) and on the E. coli 70S at 3.3 Å (green, PDB ID 4V7V) with lincomycin on the S. aureus 50S at 3.7 Å (blue, PDB ID 5HKV) . Alignments were made using the rRNA within 10 Å of the antibiotic. rRNA and r-proteins comprising the binding site are colored grey, whereas antibiotics (including waters and ions if present) are color-coded as indicated.

Article Snippet: Final antibiotic concentrations for complexes formed with each cocktail was: cocktail 1 contained 200 μM omadacycline (MedChemExpress), 200 μM spectinomycin (Santa Cruz Biotechnology), 200 μM streptomycin (Santa Cruz Biotechnology), 200 μM evernimicin, 200 μM hygromycin B (Cayman Chemical); cocktail 2 contained 100 μM capreomycin (Sigma Aldrich), 100 μM kasugamycin (Sigma Aldrich) and 100 μM retapamulin (Sigma Aldrich); cocktail 3 contained 100 μM tetracycline (Sigma Aldrich), 100 μM viomycin (Sigma Aldrich), 100 μM streptomycin (Santa Cruz Biotechnology), 100 μM lincomyin (Sigma Aldrich) and 100 μM avilamycin (Cayman Chemical); cocktail 4 contained 10 μM apramycin (Sigma Aldrich), 10 μM eravacycline (MedChemExpress) and 100 μM clindamycin (Santa Cruz Biotechnology); cocktail 5 contained 100 μM pentacycline (Tetraphase), 10 μM gentamicin (Carl Roth) and 100 μM tiamulin (Sigma Aldrich).

Techniques: Translocation Assay, Binding Assay

Journal: Nature Structural & Molecular Biology

Article Title: Structural conservation of antibiotic interaction with ribosomes

doi: 10.1038/s41594-023-01047-y

Figure Lengend Snippet: The central ring shows a superimposition of the binding sites on the SSU (gray) of the antibiotics tetracycline (blue), spectinomycin (yellow), hygromycin B (pink), kasugamycin (red), apramycin (green), gentamicin (cyan) and streptomycin (orange), which is surrounded by insets highlighting the interactions between the drug and the 16S rRNA (gray), waters (red spheres with gray transparent density), magnesium ions (green spheres), putative K + ions (purple sphere with transparent gray density) and uS12 (orange). Potential hydrogen bonds are indicated as dashed lines, colored orange for direct interaction between the drug and the small subunit, cyan for water-mediated interactions, green for Mg 2+ ion coordination and purple for K + coordination.

Article Snippet: Final antibiotic concentrations for complexes formed with each cocktail was: cocktail 1 contained 200 μM omadacycline (MedChemExpress), 200 μM spectinomycin (Santa Cruz Biotechnology), 200 μM streptomycin (Santa Cruz Biotechnology), 200 μM evernimicin, 200 μM hygromycin B (Cayman Chemical); cocktail 2 contained 100 μM capreomycin (Sigma Aldrich), 100 μM kasugamycin (Sigma Aldrich) and 100 μM retapamulin (Sigma Aldrich); cocktail 3 contained 100 μM tetracycline (Sigma Aldrich), 100 μM viomycin (Sigma Aldrich), 100 μM streptomycin (Santa Cruz Biotechnology), 100 μM lincomyin (Sigma Aldrich) and 100 μM avilamycin (Cayman Chemical); cocktail 4 contained 10 μM apramycin (Sigma Aldrich), 10 μM eravacycline (MedChemExpress) and 100 μM clindamycin (Santa Cruz Biotechnology); cocktail 5 contained 100 μM pentacycline (Tetraphase), 10 μM gentamicin (Carl Roth) and 100 μM tiamulin (Sigma Aldrich).

Techniques: Binding Assay

Journal: Nature Structural & Molecular Biology

Article Title: Structural conservation of antibiotic interaction with ribosomes

doi: 10.1038/s41594-023-01047-y

Figure Lengend Snippet: Segmented cryo-EM map densities (transparent gray) and molecule models (colored by atom) are shown for tetracyclines (tetracycline, omadacycline, eravacycline and pentacycline), aminoglycosides (hygromycin B, gentamicin, spectinomycin, streptomycin, apramycin and kasugamycin), tuberactinomycin (capreomycin), orthosomycin (avilamycin and evernimicin), pleuromutilin (tiamulin and retapamulin) and lincosamide (lincomycin and clindamycin) antibiotics.

Article Snippet: Final antibiotic concentrations for complexes formed with each cocktail was: cocktail 1 contained 200 μM omadacycline (MedChemExpress), 200 μM spectinomycin (Santa Cruz Biotechnology), 200 μM streptomycin (Santa Cruz Biotechnology), 200 μM evernimicin, 200 μM hygromycin B (Cayman Chemical); cocktail 2 contained 100 μM capreomycin (Sigma Aldrich), 100 μM kasugamycin (Sigma Aldrich) and 100 μM retapamulin (Sigma Aldrich); cocktail 3 contained 100 μM tetracycline (Sigma Aldrich), 100 μM viomycin (Sigma Aldrich), 100 μM streptomycin (Santa Cruz Biotechnology), 100 μM lincomyin (Sigma Aldrich) and 100 μM avilamycin (Cayman Chemical); cocktail 4 contained 10 μM apramycin (Sigma Aldrich), 10 μM eravacycline (MedChemExpress) and 100 μM clindamycin (Santa Cruz Biotechnology); cocktail 5 contained 100 μM pentacycline (Tetraphase), 10 μM gentamicin (Carl Roth) and 100 μM tiamulin (Sigma Aldrich).

Techniques: Cryo-EM Sample Prep

Journal: Nature Structural & Molecular Biology

Article Title: Structural conservation of antibiotic interaction with ribosomes

doi: 10.1038/s41594-023-01047-y

Figure Lengend Snippet: a , View of tetracycline binding site (16S rRNA, grey) in the absence (left panel) and presence of tetracycline (right panel). Waters (spheres) that are displaced upon tetracycline (Tet, blue) binding are colored red. b , View of streptomycin binding site (16S rRNA, grey) in the absence (left panel) and presence of streptomycin (right panel). Waters (spheres) that are displaced upon streptomycin (Str, blue) binding are colored red. c , View of spectinomycin binding site (16S rRNA, grey) in the absence (left panel) and presence of spectinomycin (right panel). Waters (spheres) that are displaced upon spectinomycin (Spc, blue) binding are colored red. d , View of kasugamycin binding site (16S rRNA, grey) in the absence (left panel) and presence of kasugamycin (right panel). Waters (spheres) that are displaced upon kasugamycin (Ksg, blue) binding are colored red. e-h , View of ( e ) lincomycin, ( f ) clindamycin, ( g ) tiamulin and ( h ) retapamulin binding site (23S rRNA, grey) in the absence (left panel) and presence of the drug (right panel). Waters (spheres) that are displaced upon binding of the antibiotic are colored red. In ( a )-( h ), waters (spheres) that are stabilized upon drug binding are colored green.

Article Snippet: Final antibiotic concentrations for complexes formed with each cocktail was: cocktail 1 contained 200 μM omadacycline (MedChemExpress), 200 μM spectinomycin (Santa Cruz Biotechnology), 200 μM streptomycin (Santa Cruz Biotechnology), 200 μM evernimicin, 200 μM hygromycin B (Cayman Chemical); cocktail 2 contained 100 μM capreomycin (Sigma Aldrich), 100 μM kasugamycin (Sigma Aldrich) and 100 μM retapamulin (Sigma Aldrich); cocktail 3 contained 100 μM tetracycline (Sigma Aldrich), 100 μM viomycin (Sigma Aldrich), 100 μM streptomycin (Santa Cruz Biotechnology), 100 μM lincomyin (Sigma Aldrich) and 100 μM avilamycin (Cayman Chemical); cocktail 4 contained 10 μM apramycin (Sigma Aldrich), 10 μM eravacycline (MedChemExpress) and 100 μM clindamycin (Santa Cruz Biotechnology); cocktail 5 contained 100 μM pentacycline (Tetraphase), 10 μM gentamicin (Carl Roth) and 100 μM tiamulin (Sigma Aldrich).

Techniques: Binding Assay

Journal: Nature Structural & Molecular Biology

Article Title: Structural conservation of antibiotic interaction with ribosomes

doi: 10.1038/s41594-023-01047-y

Figure Lengend Snippet: a-b , Interaction of spectinomycin (Spc) determined ( a ) here on E. coli (Spc E.coli ) at 1.8 Å (from Fig. ) and (b) on the E. faecalis 70S ribosome (Spc E.fae . ) at 2.4 Å . c , Superimposition of ( a ) and ( b ). d-e , Interaction of spectinomycin (Spc) determined ( d ) here on E. coli (Spc E.coli ) at 1.8 Å (from Fig. ) and ( e ) within an E. coli translocation intermate (Spc Trans ) at 2.54 Å . f , Superimposition of ( d ) and ( e ). g-h , Interaction of apramycin (Apr) determined ( g ) here on E. coli (Apr E.coli ) (from Fig. ) and (h) within an E. coli translocation intermediate (Apr Trans ) at 2.35 Å . i , Superimposition of ( g ) and ( h ). j-k , Interaction of ( j ) gentamicin (Gen) determined here on E. coli (Gen) (from Fig. ) and ( k ) paromomycin (Par) on an E. coli bearing mRNA, A- and P-site tRNAs at 1.98 Å . l , Superimposition of ( i ) and ( j ).

Article Snippet: Final antibiotic concentrations for complexes formed with each cocktail was: cocktail 1 contained 200 μM omadacycline (MedChemExpress), 200 μM spectinomycin (Santa Cruz Biotechnology), 200 μM streptomycin (Santa Cruz Biotechnology), 200 μM evernimicin, 200 μM hygromycin B (Cayman Chemical); cocktail 2 contained 100 μM capreomycin (Sigma Aldrich), 100 μM kasugamycin (Sigma Aldrich) and 100 μM retapamulin (Sigma Aldrich); cocktail 3 contained 100 μM tetracycline (Sigma Aldrich), 100 μM viomycin (Sigma Aldrich), 100 μM streptomycin (Santa Cruz Biotechnology), 100 μM lincomyin (Sigma Aldrich) and 100 μM avilamycin (Cayman Chemical); cocktail 4 contained 10 μM apramycin (Sigma Aldrich), 10 μM eravacycline (MedChemExpress) and 100 μM clindamycin (Santa Cruz Biotechnology); cocktail 5 contained 100 μM pentacycline (Tetraphase), 10 μM gentamicin (Carl Roth) and 100 μM tiamulin (Sigma Aldrich).

Techniques: Translocation Assay