Spectinomycin Resistance in rpsE Mutants is Recessive in Streptomyces roseosporus

Abstract

Eight spontaneous mutants of Streptomyces roseosporus resistant to spectinomycin (SpcR) were mapped to distinct transversions or deletions in the rpsE gene. Most of the mutations were strongly recessive to the wild type SpcS allele. This suggests that some SpcR alleles of rpsE may be useful in a spectinomycin based counterselection system. Keywords spectinomycin, rpsE, ribosomal protein S5, Streptomyces reseosporus, resistance Spectinomycin is a bacteriostatic aminocyclitol antibiotic that inhibits protein synthesis. It affects ribosome function by binding to the 30S subunit and inhibiting translocation of peptidyl-tRNA from the A-site to the P-site [1,2]. Translocation involves local pivoting of the head of the ribosome relative to the body, and is sterically hindered by the presence of the antibiotic [3]. Amino acid substitutions in ribosomal protein S5 [4], the product of the rpsE gene, is associated with resistance to spectinomycin (SpcR). This protein is located on the 30S subunit and interacts with 16S rRNA and other proteins. Crystallographic studies of the Bacillus stearothermophilus S5 protein showed that it contains an N-terminal dsRNA binding region that interacts with 16S rRNA helix H3 [4,5].Early experiments on ribosome morphogenesis in E. coli hinted that sensitivity to spectinomycin might be a dominant phenotype. Studies demonstrating assembly of ribosomes from a pool of subunits showed an excess of SpcS strains when isogenic merodiploids were constructed by introducing an SpcS allele episomally into a SpcR background [6]. Other merodiploids constructed between SpcR and SpcS strains were unstably SpcS, and segregated SpcR derivatives more frequently than expected from spontaneous mutation [7]. It is possible that SpcS alleles could be dominant in a heterozygote through incorporation of sensitive subunits into spcR ribosomes [6,8]. In this study, we isolated a collection of spontaneous SpcR rpsE mutants of S. roseosporus, complemented them with a cloned rpsE gene and showed that SpcS is dominant over SpcR. Cloning of the S. roseosporus rpsE geneThe approach used for cloning the rpsE gene took advantage of the synteny observed in the chromosomes of model actinomycetes [9,10], in this case, the local order of genes around rpsE (Fig. 1A). Primers P949 and P950 (5ЈCGSGARGCSGGVCTSAAGTTC3Ј and 5ЈCTTSAG-CTTSGGVAGVCGCATGTG3Ј) targeting rplR and rplO were used to amplify (92°C 15 seconds, 52°C 15 seconds, 72°C 45 seconds, 30 cycles) an intervening region from S. roseosporus UA343 (NRRL 15998). The sequenced PCR product (deposited as Genbank AY772011) was found to include rpsE and rpmD, indicating that the rplR · rpsE · rpmD · rplO continguity was also conserved in S. roseosporus (Fig. 1B). The rpsE gene sequence was GC rich (69.3%) and the 200 residue deduced S5 peptide was

Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor

“…subtilis ( Supplementary Figure S16A ). The ribosomal protein S5 (encoded by rpsE ) from Neisseria gonorrhoeae ( 51 , 52 ) and Streptomyces filamentosus ( 53 ) were aligned with RpsE of B . subtilis to assign potential targets for conferring spectinomycin resistance (V21, A22, K26, and R30), and ribosomal protein S12 (encoded by rpsL ) from Borrelia burgdorferi ( 54 ), Mycobacterium tuberculosis ( 55 ), and Streptomyces coelicolor ( 56 ) were aligned with RpsL of B .…”

Translational protein RpsE as an alternative target for novel nucleoside analogues to treat MDR Enterobacter cloacae ATCC 13047: network analysis and molecular dynamics study

“…The ‘RpsE-inhibitor’ docked complexes were generated by considering Ile30 as the active site for being a crucial residue in Spectinomycin (standard antibiotic) binding (Bollen et al 1969 ; He et al 2005 ; Dharuman et al 2021 ). Lead_1 and Lead_2 were bound to RpsE with BEs of − 5.06 ± 0.06and − 4.48 ± 0.30 kcal/mol with average inhibition constant (IC) of 0.20 and 0.56 mM respectively.…”

Spontaneous and induced mutations to rifampicin, streptomycin and spectinomycin resistances in actinomycetes: mutagenic mechanisms and applications for strain improvement

“…Regarding the SpcR mutations, only a small number have been sequenced from a single Streptomyces species, and all contained transversions at GC sites or in-frame deletions in the rpsE gene. 25 As in-frame RifR deletions also occurred in the rpoB gene, both markers are suitable for analyzing deletion mutagenesis. It would be useful to expand the numbers of SpcR mutations in several actinomycetes to determine the full spectrum of available base-pair substitution pathways.…”

“…In a recent study, spontaneous mutations to SpcR in the daptomycinproducing Streptomyces roseosporus were sequenced, and all were mapped to the rpsE gene. 25 Each of the nine mutations caused aminoacid changes in a restricted region (positions 43-52) in loop 2 of the S5 ribosomal protein. The targeted peptide region (VAKVVKGGRR) is rich in basic and aliphatic amino acids.…”