Journal: Journal of molecular biology

Article Title: DafA cycles between the DnaK chaperone system and translational machinery.

doi: 10.1016/j.jmb.2004.04.052

Figure Lengend Snippet: Figure 3. Sucrose cushion assay: in vitro binding of DafATth and Alexa488-labeled DafATth–S14C to T. thermo- philus 70 S ribosomes. (A) Immunoblotting. 30 mM DafATth was incubated at 30 8C or 70 8C with 2 mM 70 S ribosomes for 15 minutes and applied on two volumes of 30% sucrose cushion followed by ultracentrifugation to re-isolate the ribosomal particles. Equal volumes of supernatants (S) and ribosomal pellets (P) were separ- ated by SDS-PAGE and subjected to Western blotting using rabbit anti-DafATth antibodies. DafATth (2 mM) was applied as a standard (std). (B) Fluoroimaging. Alexa488-labeled DafATth–S14C at 5 mM final concen- tration was incubated 15 minutes with 1 mM 70 S ribo- somes at 37 8C. After ultracentrifugation on 30% sucrose cushion the pellets (P) and the supernatants (S) were TCA-precipitated and subsequently applied to SDS- PAGE. The fluorescence of the bands was detected directly from the gel using a fluorescence scanner. Figure 4. Interaction of DafATth–S14C–Alexa488 with T. thermophilus 70 S ribosomes assayed by analytical gel filtration. (A) Binding of Alexa488-labeled DafATth–S14C to the ribosomes and its displacement by an excess of unlabeled protein. Ribosomes (1 mM) (large dots) were incubated with 5 mM labeled protein for 15 minutes at 30 8C in the absence (continuous line) or in the presence (small dots) of a tenfold excess of unlabeled protein. The influence of large RNA impurities present in the samples was analyzed by incubation of the ribosomes with DafATth–S14C–Alexa488 in the presence of RNaseA (dashed-dot). The samples were briefly centrifuged and then applied to a Superdex S-200 analytical gel filtration column at a flow rate of 0.75 ml/minute. (B) Incubation of ribosomes with an excess of Alexa488 dye. 70 S ribo- somes (0.5 mM) (continuous line) were incubated for 15 minutes at 30 8C with 50-fold excess Alexa488-maleimide (dotted line) and subjected to analytical gel filtration on a Superdex S-200 at a flow rate of 0.5 ml/minute.

Article Snippet: Purification of 70 S ribosomes from Thermus thermophilus T. thermophilus strain HB27 (ATCC BAA-163) was grown at 70 8C under strong aeration in a medium containing 4 g Peptone-140, 2 g yeast extract and 1 g of NaCl per liter supplemented with Castenholz salts (pH 7.5).44 Cells were harvested and stored at 280 8C until use.

Techniques: In Vitro, Binding Assay, Labeling, Western Blot, Incubation, SDS Page

Journal: Journal of Bacteriology

Article Title: Mlc of Thermus thermophilus : a Glucose-Specific Regulator for a Glucose/Mannose ABC Transporter in the Absence of the Phosphotransferase System

doi: 10.1128/jb.00715-06

Figure Lengend Snippet: FIG. 4. Mlc glucose/mannose transport operon and TMSP ABC transport operon organization in T. thermophilus. The TTC numbers refer to the gene numbering in the genome sequencing of T. thermophilus HB27 (10). We propose the names GlcE, GlcF, and GlcG for the glucose/ mannose-binding protein and the two membrane components of the glucose/mannose ABC transporter.

Article Snippet: Strains and plasmids used in this study Strain or plasmid Known genotypea Source orreference Strains E. coli SF120 ptr32::cm degP4::kan ompT 2 JM-G2 ptsG-lacZ mlc::Tn10 22 T. thermophilus DSM7029 Wild-type strain (HB27) DSMZ CL3 mlc::kan This study HB27 malK1::kan malK1::kan 28 JN1 malF::bleo Jutta Nesper CL4 mlc::kan malF::bleo This study Plasmids pFC4 pGDR11 lacIq; N-terminally His6-tagged MlcTth (gene TTC0329); Amp r This study pEM1 pCS19 lacIq; wild-type MlcTth (gene TTC0329); Amp r This study pCL2 pQE30 (QIAGEN) lacIq; N-terminally His6-tagged Mlc916Tth::Kan; Amp r Kanr This study pTTC0328 pQE30 (QIAGEN) lacIq; N-terminally His6-tagged glucose-binding protein (gene TTC0328) without its signal sequence This study pCL10 pQE30(QIAGEN) lacIq; N-terminal His6-tagged TMSP transporter binding protein (gene TTC1627) without its signal sequence This study pCS19 pQE60 (QIAGEN) lacIq; Ampr 29 pGDR11 pQE31 (QIAGEN) lacIq; N-terminal His6 tag; Amp r 20 pMK18 pUC18 derivative with a thermostable resistance to kanamycin 6 pREP4 pACYC derivative containing the p15A replicon; Kanr QIAGEN on M arch 7, 2015 by M A H ID O L U N IV F A C O F M E D http://jb.asm .org/ D ow nloaded from ampicillin/ml and 25 g of kanamycin/ml.

Techniques: Sequencing, Binding Assay, Membrane

Journal: Journal of Bacteriology

Article Title: Mlc of Thermus thermophilus : a Glucose-Specific Regulator for a Glucose/Mannose ABC Transporter in the Absence of the Phosphotransferase System

doi: 10.1128/jb.00715-06

Figure Lengend Snippet: FIG. 7. Glucose transport kinetics of T. thermophilus. (A) Wild type (HB27); (B) malF::bleo mutant (JN1); (C) mlc::kan mutant (CL3). Transport by JN1 represents the activity of the glucose/man- nose ABC transporter, and transport by CL3 represents the activity of the TMSP ABC transporter. The curves can be fitted according to the Michaelis-Menten equation and yield a Km of 0.15 M for glucose uptake via the glucose/mannose transporter and a Km of 1.4 M for the TMSP transporter. A double mutant harboring both malF::bleo and mlc::kan exhibits neither glucose nor maltose transport activity. TABLE 2. Transport of glucose and maltose in T. thermophilus

Article Snippet: Strains and plasmids used in this study Strain or plasmid Known genotypea Source orreference Strains E. coli SF120 ptr32::cm degP4::kan ompT 2 JM-G2 ptsG-lacZ mlc::Tn10 22 T. thermophilus DSM7029 Wild-type strain (HB27) DSMZ CL3 mlc::kan This study HB27 malK1::kan malK1::kan 28 JN1 malF::bleo Jutta Nesper CL4 mlc::kan malF::bleo This study Plasmids pFC4 pGDR11 lacIq; N-terminally His6-tagged MlcTth (gene TTC0329); Amp r This study pEM1 pCS19 lacIq; wild-type MlcTth (gene TTC0329); Amp r This study pCL2 pQE30 (QIAGEN) lacIq; N-terminally His6-tagged Mlc916Tth::Kan; Amp r Kanr This study pTTC0328 pQE30 (QIAGEN) lacIq; N-terminally His6-tagged glucose-binding protein (gene TTC0328) without its signal sequence This study pCL10 pQE30(QIAGEN) lacIq; N-terminal His6-tagged TMSP transporter binding protein (gene TTC1627) without its signal sequence This study pCS19 pQE60 (QIAGEN) lacIq; Ampr 29 pGDR11 pQE31 (QIAGEN) lacIq; N-terminal His6 tag; Amp r 20 pMK18 pUC18 derivative with a thermostable resistance to kanamycin 6 pREP4 pACYC derivative containing the p15A replicon; Kanr QIAGEN on M arch 7, 2015 by M A H ID O L U N IV F A C O F M E D http://jb.asm .org/ D ow nloaded from ampicillin/ml and 25 g of kanamycin/ml.

Techniques: Mutagenesis, Activity Assay

Journal: Antibiotics

Article Title: Tiamulin-Resistant Mutants of the Thermophilic Bacterium Thermus thermophilus

doi: 10.3390/antibiotics9060313

Figure Lengend Snippet: Spontaneous tiamulin-resistant mutants identified in this study.

Article Snippet: All mutants were derived from the T. thermophilus strains HB27 (ATCC BAA-163) [ ] or IB-21 [ ].

Techniques: Mutagenesis, Selection

Journal: Antibiotics

Article Title: Tiamulin-Resistant Mutants of the Thermophilic Bacterium Thermus thermophilus

doi: 10.3390/antibiotics9060313

Figure Lengend Snippet: Sites of mutations and inferred mechanisms of resistance. ( A ) Sequence alignment of ribosomal protein uL3 encompassing the sites of mutations identified in this study. Uses organisms for whom tiamulin-resistance mutations in uL3 have been found. Tth, Thermus thermophilus (Accession number Q72I04); Dra, Deinococcus radiodurans (Accession number Q9RXK2); Eco, Escherichia coli (Accession number P60438); Sau, Staphylococcus aureus (Accession number P60449); Bhy, Brachyspira hyodysenteriae (Accession number A0A2K9JGD3) ( B ) Interaction of ribosomal protein uL3 and 23S rRNA at the tiamulin binding site, based on the crystal structures of ribosomes from T. thermophilus (PDB entry 4y4p.cif; 36) and D. radiodurans (PDB entry 1xbp.cif; 8). Structures were aligned and images rendered using PyMOL (Schrodinger). R149 is within hydrogen bonding distance of the 2’ hydroxyl of U2504 and the phosphate oxygen of U2506. ( C ) Interactions between tiamulin and 23S rRNA in the D. radiodurans (PDB entry 1xbp.cif; 8) 50S subunit structure. Sites of mutation conferring resistance are colored red, sites of mutations not conferring resistance are colored blue.

Article Snippet: All mutants were derived from the T. thermophilus strains HB27 (ATCC BAA-163) [ ] or IB-21 [ ].

Techniques: Sequencing, Binding Assay, Mutagenesis

Journal: Antibiotics

Article Title: Tiamulin-Resistant Mutants of the Thermophilic Bacterium Thermus thermophilus

doi: 10.3390/antibiotics9060313

Figure Lengend Snippet: Cross-resistance phenotypes of tiamulin-resistant mutants.

Article Snippet: All mutants were derived from the T. thermophilus strains HB27 (ATCC BAA-163) [ ] or IB-21 [ ].

Techniques: Inhibition