NORMAL GENOMIC

• Weitnauer G et al.
• An ATP-binding cassette transporter and two rRNA methyltransferases areinvolved in resistance to avilamycin in the producer organism Streptomycesviridochromogenes Tu57.
• Antimicrob Agents Chemother. 2001; 45: 690-5
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• Three different resistance factors from the avilamycin biosynthetic genecluster of Streptomyces viridochromogenes Tu57, which confer avilamycinresistance when expressed in Streptomyces lividans TK66, were isolated.Analysis of the deduced amino acid sequences showed that AviABC1 issimilar to a large family of ATP-binding transporter proteins and thatAviABC2 resembles hydrophobic transmembrane proteins known to act jointlywith the ATP-binding proteins. The deduced amino acid sequence of aviRbshowed similarity to those of other rRNA methyltransferases, and AviRa didnot resemble any protein in the databases. Independent expression in S.lividans TK66 of aviABC1 plus aviABC2, aviRa, or aviRb conferred differentlevels of resistance to avilamycin: 5, 10, or 250 microg/ml, respectively.When either aviRa plus aviRb or aviRa plus aviRb plus aviABC1 plus aviABC2was coexpressed in S. lividans TK66, avilamycin resistance levels reachedmore than 250 microg/ml. Avilamycin A inhibited poly(U)-directedpolyphenylalanine synthesis in an in vitro system using ribosomes of S.lividans TK66(pUWL201) (GWO), S. lividans TK66(pUWL201-Ra) (GWRa), or S.lividans TK66(pUWL201-Rb) (GWRb), whereas ribosomes of S. lividans TK66containing pUWL201-Ra+Rb (GWRaRb) were highly resistant. aviRa and aviRbwere expressed in Escherichia coli, and both enzymes were purified asfusion proteins to near homogeneity. Both enzymes showed rRNAmethyltransferase activity using a mixture of 16S and 23S rRNAs from E.coli as the substrate. Coincubation experiments revealed that the enzymesmethylate different positions of rRNA.

• Cheng X, Roberts RJ
• AdoMet-dependent methylation, DNA methyltransferases and base flipping.
• Nucleic Acids Res. 2001; 29: 3784-95
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• Twenty AdoMet-dependent methyltransferases (MTases) have beencharacterized structurally by X-ray crystallography and NMR. These includeseven DNA MTases, five RNA MTases, four protein MTases and four smallmolecule MTases acting on the carbon, oxygen or nitrogen atoms of theirsubstrates. The MTases share a common core structure of a mixedseven-stranded beta-sheet (6 downward arrow 7 upward arrow 5 downwardarrow 4 downward arrow 1 downward arrow 2 downward arrow 3 downward arrow)referred to as an 'AdoMet-dependent MTase fold', with the exception of aprotein arginine MTase which contains a compact consensus fold lacking theantiparallel hairpin strands (6 downward arrow 7 upward arrow). Theconsensus fold is useful to identify hypothetical MTases during structuralproteomics efforts on unannotated proteins. The same core structure worksfor very different classes of MTase including those that act on substratesdiffering in size from small molecules (catechol or glycine) tomacromolecules (DNA, RNA and protein). DNA MTases use a 'base flipping'mechanism to deliver a specific base within a DNA molecule into atypically concave catalytic pocket. Base flipping involves rotation ofbackbone bonds in double-stranded DNA to expose an out-of-stacknucleotide, which can then be a substrate for an enzyme-catalyzed chemicalreaction. The phenomenon is fully established for DNA MTases and for DNAbase excision repair enzymes, and is likely to prove general for enzymesthat require access to unpaired, mismatched or damaged nucleotides withinbase-paired regions in DNA and RNA. Several newly discovered MTasefamilies in eukaryotes (DNA 5mC MTases and protein arginine and lysineMTases) offer new challenges in the MTase field.

• Tait-Kamradt A, Davies T, Cronan M, Jacobs MR, Appelbaum PC, Sutcliffe J
• Mutations in 23S rRNA and ribosomal protein L4 account for resistance inpneumococcal strains selected in vitro by macrolide passage.
• Antimicrob Agents Chemother. 2000; 44: 2118-25
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• The mechanisms responsible for macrolide resistance in Streptococcuspneumoniae mutants, selected from susceptible strains by serial passage inazithromycin, were investigated. These mutants were resistant to 14- and15-membered macrolides, but resistance could not be explained by anyclinically relevant resistance determinant [mef(A), erm(A), erm(B),erm(C), erm(TR), msr(A), mph(A), mph(B), mph(C), ere(A), ere(B)]. Aninvestigation into the sequences of 23S rRNAs in the mutant and parentalstrains revealed individual changes of C2611A, C2611G, A2058G, and A2059G(Escherichia coli numbering) in four mutants. Mutations at these residuesin domain V of 23S rRNA have been noted to confer erythromycin resistancein other species. Not all four 23S rRNA alleles have to contain themutation to confer resistance. Some of the mutations also confercoresistance to streptogramin B (C2611A, C2611G, and A2058G), 16-memberedmacrolides (all changes), and clindamycin (A2058G and A2059G).Interestingly, none of these mutations confer high-level resistance totelithromycin (HMR-3647). Further, two of the mutants which had no changesin their 23S rRNA sequences had changes in a highly conserved stretch ofamino acids ((63)KPWRQKGTGRAR(74)) in ribosomal protein L4. One mutantcontained a single amino acid change (G69C), while the other mutant had a6-base insert, resulting in two amino acids (S and Q) being insertedbetween amino acids Q67 and K68. To our knowledge, this is the firstdescription of mutations in 23S rRNA genes or ribosomal proteins inmacrolide-resistant S. pneumoniae strains.

• Lampson BC, Parisi JT
• Naturally occurring Staphylococcus epidermidis plasmid expressingconstitutive macrolide-lincosamide-streptogramin B resistance contains adeleted attenuator.
• J Bacteriol. 1986; 166: 479-83
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• A naturally occurring constitutive macrolide-lincosamide-streptogramin B(MLS) resistance plasmid, pNE131, from Staphylococcus epidermidis waschosen to study the molecular basis of constitutive expression.Restriction and functional maps of pNE131 are presented along with thenucleotide sequence of ermM, the gene which mediates constitutive MLSresistance. Sharing 98% sequence homology within the 870-base-pairSau3A-TaqI fragment, ermM appears to be almost identical to ermC, theinducible MLS resistance determinant from S. aureus (pE194). The two genesshare nearly identical sequences, except in the 5' promoter region ofermM. Constitutive expression of ermM is due to the deletion of 107 basepairs relative to ermC; the deletion removes critical sequences forattenuation, resulting in constitutive methylase expression.