Cloning and sequences of two macrolide-resistance-encoding genes from mycinamicin-producing Micromonospora griseorubida

doi:10.1016/0378-1119(94)90125-2

Gene

Volume 141, Issue 1, 8 April 1994, Pages 39-46

https://doi.org/10.1016/0378-1119(94)90125-2 Get rights and content

Abstract

Two macrolide-resistance determinants were cloned from a mycinamicin (Mm)-producing Micromonospora griseorubida strain in Streptomyces lividans and Streptomyces parvulus. One of the cloned genes, designated myrA, was cloned as a gene which conferred strong resistance to Mm and tyiosin (Ty), but not to erythromycin (Er) or josamycin (Jm) on S. lividans. Another gene, named myrB, was cloned as an Er^Rencoding gene which conferred MLS resistance (to macrolide, lincosamide and streptogramine B antibiotics) on S. parvulus. Both myrA and myrB were sequenced and the corresponding ORFs were determined. The deduced amino acid (aa) sequence of myrA showed no similarity to proteins in the available databases, suggesting that an unknown mechanism of macrolide resistance is exerted by the MyrA protein. The deduced aa sequence of myrB exhibited high similarity to 23S rRNA methyltransferases (MTases), such as ErmE and CarB, from a variety of microorganisms.

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Cited by (21)

Genetic characterization, mechanisms and dissemination risk of antibiotic resistance of multidrug-resistant Rothia nasimurium
2021, Infection, Genetics and Evolution
Citation Excerpt :
Three different types of efflux pumps were detected: Type I efflux pumps are from the major facilitator superfamily (MFS) and included tetZ and cmx, with resistance to tetracyclines and chloramphenicols (Tauch et al. 2000; Tauch et al. 1998); Type II is from ATP-binding cassette transporter families (ABC) including pstB, with resistance to fluoroquinolones (Lu et al. 2014); Type III efflux pumps are from small multidrug resistance families (SMR) including qacEΔ1, with resistance to disinfectants (Shafaati et al., 2016). MBLs, mysA, and rpoB variants provide resistance to antibiotic beta-Lactams (Sacha et al. 2008), macrolides (Inouye et al. 1994), and rifamycins (Ergeshov et al. 2017). Additionally, the presence of four resistance-related protein sequences, including the Fe-S cluster assembly ATPase sufC, phage shock protein (pspB), cysteine synthase (Cys) and phosphomannomutase (PMM), were consistent with previous reports (Zuo, 2011).
Rothia nasimurium is part of the commensal flora of humans and other animals and has recently received increasing attention for its multidrug-resistance (MDR) and pathogenicity. Currently, no systematic reports characterize the genetics, mechanisms, and dissemination risks of antibiotic resistance in MDR R. nasimurium. Here, we present the first report outlining a MDR strain of R. nasimurium, E1706032a, isolated from ducks exhibiting clinical sickness. Phylogenetic analysis indicates that E1706032a mostly likely originated in the commensal bacteria of Amazona aestiva in Florida. E1706032a is resistant to beta-lactams, aminoglycosides, macrolides, sulfonamides, fluoroquinolones, rifamycins, tetracyclines, lincosamides and chloramphenicol. Genetic sequences related to drug resistance were detected, including resistance genes (aac(6′)-Ib, ant(3″)-Ia, sul1, dfrA7, erm(X)), efflux pumps (tetZ, qacEΔ1, cmx, phosphate ABC transporter ATP-binding protein), and resistance-related proteins (hydrolase of the metallo-beta-lactamase (MBLs), mycinamicin resistance protein (myrA), DNA-directed RNA polymerase subunit beta (rpoB) variants, etc). E1706032a carries an IS481-like element, IS5564 and IS6-like elements, and IS6100 along with several novel transposases of the IS3 family. E1706032a also carries the class 1 integron gene IntI1, which is downstream adjacent to the gene cassettes aac(6′)-Ib, tetZ, dfrA27, ant(3″)-Ia, qacEΔ1, sul1, cmx and upstream adjacent to gene tnpA of IS6100. Genetic analysis suggests that E1706032a carries wide antibiotic resistance and dissemination potential through movable elements and thus has the potential to cause difficult-to-treat infections in animals and humans. The dissemination of E1706032a from parrots in Florida to ducks in eastern China indicates a cross-regional public health infection risk that should be evaluated for risk of global spreading.
The tylosin-resistance methyltransferase RlmA<sup>II</sup> (TlrB) modifies the N-1 position of 23 S rRNA nucleotide G748
2004, Journal of Molecular Biology
The methyltransferase RlmA^II (TlrB) confers resistance to the macrolide antibiotic tylosin in the drug-producing strain Streptomyces fradiae. The resistance conferred by RlmA^II is highly specific for tylosin, and no resistance is conferred to other macrolide drugs, or to lincosamide and streptogramin B (MLS_B) drugs that bind to the same region on the bacterial ribosome. In this study, the methylation site of RlmA^II is identified unambiguously by liquid chromatography/electrospray ionization mass spectrometry as the N-1 position of 23 S rRNA nucleotide G748. This position is contacted by the mycinose sugar moiety of tylosin, which is absent from the other drugs. The selective resistance to tylosin conferred by m¹G748 illustrates how differences in drug structure facilitate the drug fit at the MLS_B-binding site. This observation is of relevance for the rational design of novel antimicrobials targeting the MLS_B site, especially if the antimicrobials are to be used against pathogens possessing m¹G748.
Mutational analysis defines the roles of conserved amino acid residues in the predicted catalytic pocket of the rRNA:m<sup>6</sup>A methyltransferase ErmC′
2003, Journal of Molecular Biology
Methyltransferases (MTases) from the Erm family catalyze S-adenosyl-l-methionine-dependent modification of a specific adenine residue in bacterial 23 S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide and streptogramin B antibiotics. Despite the available structural data and functional analyses on the level of the RNA substrate, still very little is known about the mechanism of rRNA:adenine-N⁶ methylation. Only predictions regarding various aspects of this reaction have been made based on the analysis of the crystal structures of methyltransferase ErmC′ (without the RNA) and their comparison with the crystallographic and biochemical data for better studied DNA:m⁶A MTases. To validate the structure-based predictions of presumably essential residues in the catalytic pocket of ErmC′, we carried out the site-directed mutagenesis and studied the function of the mutants in vitro and in vivo. Our results indicate that the active site of rRNA:m⁶A MTases is much more tolerant to amino acid substitutions than the active site of DNA:m⁶A MTases. Only the Y104 residue implicated in stabilization of the target base was found to be indispensable. Remarkably, the N101 residue from the “catalytic” motif IV and two conserved residues that form the floor (F163) and one of the walls (N11) of the base-binding site are not essential for catalysis in ErmC′. This somewhat surprising result is discussed in the light of the available structural data and in the phylogenetic context of the Erm family.
Organization of the biosynthetic gene cluster for the polyketide macrolide mycinamicin in Micromonospora griseorubida
2003, FEMS Microbiology Letters
Mycinamicin, composed of a branched lactone and two sugars, desosamine and mycinose, at the C-5 and C-21 positions, is a 16-membered macrolide antibiotic produced by Micromonospora griseorubida A11725, which shows strong antimicrobial activity against Gram-positive bacteria. The nucleotide sequence (62 kb) of the mycinamicin biosynthetic gene cluster, in which there were 22 open reading frames (ORFs), was completely determined. All of the products from the 22 ORFs are responsible for the biosynthesis of mycinamicin II and self-protection against the compounds synthesized. Central to the cluster is a polyketide synthase locus (mycA), which encodes a seven-module system comprised of five multifunctional proteins. Immediately downstream of mycA, there is a set of genes for desosamine biosynthesis (mydA–G and mycB). Moreover, mydH, whose product is responsible for the biosynthesis of mycinose, lies between mydA and B. On the other hand, eight ORFs were detected upstream of the mycinamicin PKS gene. The myrB, mycG, and mycF genes had already been characterized by Inouye et al. The other five ORFs (mycCI, mycCII, mydI, mycE, and mycD) lie between mycA1 and mycF, and these five genes and mycF are responsible for the biosynthesis of mycinose. In the PKS gene, four regions of KS and AT domains in modules 1, 4, 5, and 6 indicated that it does not show the high GC content typical for Streptomyces genes, nor the unusual frame plot patterns for Streptomyces genes. Methylmalonyl-CoA was used as substrate in the functional units of those four modules. The relationship between the substrate and the unusual frame plot pattern of the KS and AT domains was observed in the other PKS genes, and it is suggested that the KS-AT original region was horizontally transferred into the PKS genes on the chromosomal DNA of several actinomycetes strains.
Mode of Action and Resistance Mechanisms of Antimicrobial Macrolides
2003, Macrolide Antibiotics: Chemistry, Biology, and Practice: Second Edition
This chapter reviews the molecular-biological mode of action of macrolide antibiotics and the biochemical and genetic mechanisms of resistance to MLS antibiotics. It discusses the mode of action and mechanisms of drug resistance in clinically isolated bacteria. The chapter also briefly describes recent major developments in macrolide antibiotics. Macrolide antibiotics essentially consist of a large lactone ring to which one or more sugars are linked. They include macrolide, lincosamide, and type B streptogramin antibiotics. It is noted that MLS antibiotics inhibit protein synthesis by binding to closely related sites on the 50S subunit of the 70S ribosome of bacteria despite being structurally different from each other. MLS antibiotics exhibit narrow spectrum of activity against bacteria that include gram-positive cocci and bacilli and against gram-negative cocci. Macrolide antibiotics are often used as a safe remedy against infection by one of these bacteria, because they do not exhibit severe adverse effects. Gram-negative bacilli, such as Pseudomonasaeruginosa and Escherichia coli, occasionally cause opportunistic infection which is usually intrinsically resistant to MLS antibiotics.
TetZ, a new tetracycline resistance determinant discovered in gram-positive bacteria, shows high homology to gram-negative regulated efflux systems
2000, Plasmid
The complete nucleotide sequence of the tetracycline resistance plasmid pAG1 from the gram-positive soil bacterium Corynebacterium glutamicum 22243 (formerly Corynebacterium melassecola 22243) was determined. The R-plasmid has a size of 19,751 bp and contains at least 18 complete open reading frames. The resistance determinant of pAG1 revealed homology to gram-negative tetracycline efflux and repressor systems of Tet classes A through J. The highest levels of amino acid sequence similarity were observed to the transmembrane tetracycline efflux protein TetA(A) and to the tetracycline repressor TetR(A) of transposon Tn1721 with 64 and 56% similarity, respectively. This is the first time a repressor-regulated tet gene has been found in gram-positive bacteria. A new class of tetracycline resistance and repressor proteins, termed TetA(Z) and TetR(Z), is proposed.

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Abstract

References (28)

Developments in Streptomyces cloning

The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences

Gene

Cloning and nucleotide sequence of a carbomycin-resistance gene from Streptomyces thermo-tolerans

Gene

Cloning and characterization of two genes from Streptomyces lividans that confer inducible resistance to lincomycin and macrolide antibiotics

Gene

The Streptomyces plasmid SCP2∗: its functional analysis and development into useful cloning vectors

Gene

A new shuttle cosmid vector, pKC505, for streptomycetes: its use in the cloning of three different spiramycin-resistance genes from a Streptomyces ambofaciens library

Gene

Homology between protein controlling Streptomyces fradiae tylosin resistance and ATP-binding transport

Gene

N-Methyl transferase of Streptomyces erythraeus that confers resistance to the macrolide-lincosamide-streptogramin B antibiotics: amino acid sequence and its homology to cognate R-factor enzymes from pathogenic bacilli and cocci

Gene

Cloning of tlrD, a fourth resistance gene, from the tyiosin producer. Streptomyces fradiae

Gene

A cluster of tylosin biosynthetic genes is interrupted by a structurally unstable segment containing four repeated sequences

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae

Mol. Gen. Genet.

How antibiotic-producing organisms avoid suicide

Annu. Rev. Microbiol.

Molecular cloning and expression of carbomycin biosynthetic and resistance genes from Streptomyces thermotolerans

Involvement of cell impermeability in resistance to macrolides in some producer streptomycetes

J. Antibiot.

Cited by (21)

Genetic characterization, mechanisms and dissemination risk of antibiotic resistance of multidrug-resistant Rothia nasimurium

The tylosin-resistance methyltransferase RlmA<sup>II</sup> (TlrB) modifies the N-1 position of 23 S rRNA nucleotide G748

Mutational analysis defines the roles of conserved amino acid residues in the predicted catalytic pocket of the rRNA:m<sup>6</sup>A methyltransferase ErmC′

Organization of the biosynthetic gene cluster for the polyketide macrolide mycinamicin in Micromonospora griseorubida

Mode of Action and Resistance Mechanisms of Antimicrobial Macrolides

TetZ, a new tetracycline resistance determinant discovered in gram-positive bacteria, shows high homology to gram-negative regulated efflux systems