Streptomycin-resistant and streptomycin-dependent mutants of the extreme thermophile Thermus thermophilus

Gregory, Steven T.; Cate, J.H.D.; Dahĺberg, Albert E.

doi:10.1006/jmbi.2001.4676

Cited by 55 publications

(79 citation statements)

References 38 publications

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“…Consistent with the presence of an RsmG ortholog is the recent experimental demonstration of the presence of m 7 G527 in T. thermophilus HB8 16S rRNA (Guymon et al 2006). To confirm the identity of TTHA1971 as rsmG, we identified three mutant alleles of rsmG, which had arisen in previous selections for streptomycin resistance (Gregory et al 2001Carr et al 2005;), but could not be shown to carry mutations in either rpsL, encoding ribosomal protein S12, or in rrsB, encoding 16S rRNA.…”

Section: Identification Of T Thermophilus Rsmg Isolation Of Spontansupporting

confidence: 63%

Structural and functional studies of the Thermus thermophilus 16S rRNA methyltransferase RsmG

Gregory¹,

DeMi̇rci̇²,

Belardinelli³

et al. 2009

RNA

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The RsmG methyltransferase is responsible for N 7 methylation of G527 of 16S rRNA in bacteria. Here, we report the identification of the Thermus thermophilus rsmG gene, the isolation of rsmG mutants, and the solution of RsmG X-ray crystal structures at up to 1.5 Å resolution. Like their counterparts in other species, T. thermophilus rsmG mutants are weakly resistant to the aminoglycoside antibiotic streptomycin. Growth competition experiments indicate a physiological cost to loss of RsmG activity, consistent with the conservation of the modification site in the decoding region of the ribosome. In contrast to Escherichia coli RsmG, which has been reported to recognize only intact 30S subunits, T. thermophilus RsmG shows no in vitro methylation activity against native 30S subunits, only low activity with 30S subunits at low magnesium concentration, and maximum activity with deproteinized 16S rRNA. Cofactor-bound crystal structures of RsmG reveal a positively charged surface area remote from the active site that binds an adenosine monophosphate molecule. We conclude that an early assembly intermediate is the most likely candidate for the biological substrate of RsmG.

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Section: Identification Of T Thermophilus Rsmg Isolation Of Spontansupporting

confidence: 63%

Structural and functional studies of the Thermus thermophilus 16S rRNA methyltransferase RsmG

Gregory¹,

DeMi̇rci̇²,

Belardinelli³

et al. 2009

RNA

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“…Several single nucleotide mutations to the rpsL gene, which encodes the ribosomal protein S12, have been characterized as conferring Sm resistance in different bacteria (Funatsu and Wittmann, 1972; Timms et al ., 1992; Gregory et al ., 2001). This array of modifications make the antibiotic marker a robust tool for genomic mutagenesis in E. coli (Jiang et al ., 2013) and P. syringae (Swingle et al ., 2010).…”

Section: Resultsmentioning

confidence: 99%

A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities

Ricaurte

Martínez‐García

Nyerges

et al. 2017

Microbial Biotechnology

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SummaryBacterial recombineering typically relies on genomic incorporation of synthetic oligonucleotides as mediated by Escherichia coli λ phage recombinase β – an occurrence largely limited to enterobacterial strains. While a handful of similar recombinases have been documented, recombineering efficiencies usually fall short of expectations for practical use. In this work, we aimed to find an efficient Recβ homologue demonstrating activity in model soil bacterium Pseudomonas putida EM42. To this end, a genus‐wide protein survey was conducted to identify putative recombinase candidates for study. Selected novel proteins were assayed in a standardized test to reveal their ability to introduce the K43T substitution into the rpsL gene of P. putida. An ERF superfamily protein, here termed Rec2, exhibited activity eightfold greater than that of the previous leading recombinase. To bolster these results, we demonstrated Rec2 ability to enter a range of mutations into the pyrF gene of P. putida at similar frequencies. Our results not only confirm the utility of Rec2 as a Recβ functional analogue within the P. putida model system, but also set a complete workflow for deploying recombineering in other bacterial strains/species. Implications range from genome editing of P. putida for metabolic engineering to extended applications within other Pseudomonads – and beyond.

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“…The P90L mutation on its own has been identified previously as a streptomycin-or paromomycin-dependence mutation in a number of organisms including E. coli (Kurland et al 1996) and T. thermophilus (Gregory et al 2001;Carr et al 2005). Such ''ancillary'' mutations in combination with P90L have been observed previously in E. coli (Timms and Bridges 1993) but their phenotypes have never been examined separately from the primary drug-dependence mutation.…”

Section: Resultsmentioning

confidence: 99%

“…Here we describe streptomycin-resistance mutations in ribosomal protein S12 at sites distant from the streptomycinbinding site and positions of other previously identified resistance mutations (Carter et al 2000;Gregory et al 2001;Sharma et al 2007). One of these substitutions, H76R, is located in a QEH triplet consisting of residues Q74, E75, and H76, which has been interpreted in low-resolution cryo-EM maps as a protrusion contacting the aminoacyltRNA acceptor helix at or near residue U69 (Stark et al 2002;Valle et al 2003).…”

Section: Introductionmentioning

confidence: 97%

A signal relay between ribosomal protein S12 and elongation factor EF-Tu during decoding of mRNA

Gregory¹,

Carr²,

Dahĺberg³

2008

RNA

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Codon recognition by aminoacyl-tRNA on the ribosome triggers a process leading to GTP hydrolysis by elongation factor Tu (EF-Tu) and release of aminoacyl-tRNA into the A site of the ribosome. The nature of this signal is largely unknown. Here, we present genetic evidence that a specific set of direct interactions between ribosomal protein S12 and aminoacyl-tRNA, together with contacts between S12 and 16S rRNA, provide a pathway for the signaling of codon recognition to EF-Tu. Three novel amino acid substitutions, H76R, R37C, and K53E in Thermus thermophilus ribosomal protein S12, confer resistance to streptomycin. The streptomycin-resistance phenotypes of H76R, R37C, and K53E are all abolished by the mutation A375T in EF-Tu. A375T confers resistance to kirromycin, an antibiotic freezing EF-Tu in a GTPase activated state. H76 contacts aminoacyl-tRNA in ternary complex with EF-Tu and GTP, while R37 and K53 are involved in the conformational transition of the 30S subunit occurring upon codon recognition. We propose that codon recognition and domain closure of the 30S subunit are signaled through aminoacyl-tRNA to EF-Tu via these S12 residues.

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Streptomycin-resistant and streptomycin-dependent mutants of the extreme thermophile Thermus thermophilus

Cited by 55 publications

References 38 publications

Structural and functional studies of the Thermus thermophilus 16S rRNA methyltransferase RsmG

Structural and functional studies of the Thermus thermophilus 16S rRNA methyltransferase RsmG

A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities

A signal relay between ribosomal protein S12 and elongation factor EF-Tu during decoding of mRNA

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