Homozygous triplicate mutations in three 16S rRNA genes responsible for high-level aminoglycoside resistance in Nocardia farcinica clinical isolates from a Canada-wide bovine mastitis epizootic.

Nocardia farcinica strains showing high-level resistance to amikacin were isolated from clinical cases in a Canada-wide bovine mastitis epizootic. Shotgun cloning of the resistance genes in the amikacin-resistant mastitis isolate N. farcinica IFM 10580 (W6220 [Centers for Disease Control and Prevention]) using a multicopy vector system revealed that the 16S rRNA gene with an A-to-G single-point mutation at position 1408 (in Escherichia coli numbering) conferred "moderate" cross-resistance to amikacin and other aminoglycosides to an originally susceptible N. farcinica strain IFM 10152. Subsequent DNA sequence analyses revealed that, in contrast to the susceptible strain, all three chromosomal 16S rRNA genes of IFM 10580, the epizootic clinical strain, contained the same A1408G point mutations. Mutant colonies showing high-level aminoglycoside resistance were obtained when the susceptible strain N. farcinica IFM 10152 was transformed with a multicopy plasmid carrying the A1408G mutant 16S rRNA gene and was cultured in the presence of aminoglycosides for 3 to 5 days. Of these transformants, at least two of the three chromosomal 16S rRNA genes contained A1408G mutations. A triple mutant was easily obtained from a strain carrying the two chromosomal A1408G mutant genes and one wild-type gene, even in the absence of the plasmid. The triple mutant showed the highest level of resistance to aminoglycosides, even in the absence of the plasmid carrying the mutant 16S rRNA gene. These results suggest that the homozygous mutations in the three 16S rRNA genes are responsible for the high-level aminoglycoside resistance found in N. farcinica isolates of the bovine mastitis epizootic.

An ABC transporter involved in the control of streptomycin production in Streptomyces griseus.

We screened for a gene that inhibits streptomycin production in Streptomyces griseus when it is introduced on a high-copy-number plasmid pIJ702, and obtained a plasmid pKM545. The introduction of pKM545 abolished streptomycin production on all media tested including YMP-sugar and Nutrient broth. S1 protection analysis demonstrated that the introduction of this plasmid downregulated the transcriptional activity of the promoter preceding strR, the pathway-specific transcriptional regulator for streptomycin biosynthesis. The 2.8-kb BamHI fragment cloned onto pKM545 contained two coding sequences SGR_5442 and 5443. These coding sequences and the two downstream ones (SGR_5444 and 5445) constituted a possible operon structure designated to be rspABCD (regulation of streptomycin production). RspB and RspC exhibited a marked similarity with an ATP-binding domain and a membrane-associating domain of an ABC-2 type transporter, respectively, suggesting that the Rsp proteins comprise a membrane exporter. The gene cluster consisting of the rsp operon and the upstream divergent small coding sequence (SGR_5441) was widely distributed to Streptomyces genome. An rspB mutant of S. griseus produced 3-fold streptomycin of the parental strain in YMP liquid medium. The evidence implies that the Rsp translocator is involved in the export of a substance that specifies the expression level of streptomycin biosynthesis genes in S. griseus.