SummaryStreptomycin has been an important drug for the treatment of tuberculosis since its discovery in 1944. But numerous strains of Mycobacterium tuberculosis, the bacterial pathogen that causes tuberculosis, are now streptomycin resistant. Although such resistance is often mediated by mutations within rrs, a 16S rRNA gene or rpsL, which encodes the ribosomal protein S12, these mutations are found in a limited proportion of clinically isolated streptomycinresistant M. tuberculosis strains. Here we have succeeded in identifying a mutation that confers lowlevel streptomycin resistance to bacteria, including M. tuberculosis. We found that mutations within the gene gidB confer low-level streptomycin resistance and are an important cause of resistance found in 33% of resistant M. tuberculosis isolates. We further clarified that the gidB gene encodes a conserved 7-methylguanosine (m and, once emerged, result in vigorous emergence of high-level streptomycin-resistant mutants at a frequency more than 2000 times greater than that seen in wild-type strains. Further studies on the precise function of GidB may provide a basis for developing strategies to suppress pathogenic bacteria, including M. tuberculosis.

Certain str mutations that confer high-or low-level streptomycin resistance result in the overproduction of antibiotics by Streptomyces spp. The str mutations that confer the high-level resistance occur within rpsL, which encodes the ribosomal protein S12, while those that cause low-level resistance are not as well known. We have used comparative genome sequencing to determine that low-level resistance is caused by mutations of rsmG, which encodes an S-adenosylmethionine (SAM)-dependent 16S rRNA methyltransferase containing a SAM binding motif. Deletion of rsmG from wild-type Streptomyces coelicolor resulted in the acquisition of streptomycin resistance and the overproduction of the antibiotic actinorhodin. Introduction of wild-type rsmG into the deletion mutant completely abrogated the effects of the rsmG deletion, confirming that rsmG mutation underlies the observed phenotype. Consistent with earlier work using a spontaneous rsmG mutant, the strain carrying ⌬rsmG exhibited increased SAM synthetase activity, which mediated the overproduction of antibiotic. Moreover, high-performance liquid chromatography analysis showed that the ⌬rsmG mutant lacked a 7-methylguanosine modification in the 16S rRNA (possibly at position G518, which corresponds to G527 of Escherichia coli). Like certain rpsL mutants, the ⌬rsmG mutant exhibited enhanced protein synthetic activity during the late growth phase. Unlike rpsL mutants, however, the ⌬rsmG mutant showed neither greater stability of the 70S ribosomal complex nor increased expression of ribosome recycling factor, suggesting that the mechanism underlying increased protein synthesis differs in the rsmG and the rpsL mutants. Finally, spontaneous rsmG mutations arose at a 1,000-fold-higher frequency than rpsL mutations. These findings provide new insight into the role of rRNA modification in activating secondary metabolism in Streptomyces.One of the most intriguing challenges in biology is the elucidation of the mechanisms whereby cells switch from primary to secondary metabolism in response to extracellular nutritional conditions. Among prokaryotes, Streptomyces spp. provide a tractable experimental system for studying such mechanisms due to their display of a wide range of adaptations to extreme nutrient limitations, including the production and secretion of antibiotics and enzymes and the formation of aerial mycelium and spores (4, 9). Streptomyces coelicolor A3(2) is the best-characterized strain in this genus and has been used to study mechanisms regulating antibiotic production (7). This strain produces several chemically diverse antibiotics, including the red-pigmented tripyrrole undecylprodigiosin (Red) and the deep blue-pigmented polyketide actinorhodin (Act). We previously reported that the introduction of certain str mutations that confer streptomycin (Sm) resistance enhances Act production in both S. coelicolor A3(2) and S. lividans (12,35). Moreover, an impaired ability to produce Act, resulting from certain developmental mutations (relA, relC, and brgA) in S. coeli...

The genes required for ␥-polyglutamic acid (PGA) production were cloned from Bacillus subtilis IFO16449, a strain isolated from fermented soybeans. There were four open reading frames in the cloned 4.2-kb DNA fragment, and they were almost identical to those in the ywsC and ywtABC genes of B. subtlis 168. Northern blot analysis showed that the four genes constitute an operon. Three genes, ywsC, ywtA, and ywtB, were disrupted to determine which gene plays a central role in PGA biosynthesis. No PGA was produced in ⌬ywsC and ⌬ywtA strains, indicating that both of these genes are essential for PGA production. To clarify the function of the YwsC protein, histidine-tagged YwsC (YwsC-His) was produced in the ⌬ywsC strain and purified from the lysozyme-treated lysate of the transformant by Ni-nitrilotriacetic acid affinity chromatography. Western blot analysis revealed that the YwsC-His protein consists of two subunits, the 44-kDa and 33-kDa proteins, which are encoded by in-phase overlapping in the ywsC gene.14 C-labeled PGA was synthesized by the purified proteins from L- Some Bacillus strains produce ␥-polyglutamic acid (PGA), an amino acid polymer that consists of only D-glutamic acid or D-and L-glutamic acid polymerized through ␥-glutamyl bonds, as a capsular or an extracellular viscous material (6). PGA was first discovered as a component of the capsule of Bacillus anthracis (19) and Bacillus mesentericus (18) and was isolated from the culture medium of Bacillus subtilis (7). Since then, a number of bacteria producing PGA, including B. subtilis (8,17,21,22), Bacillus licheniformis (9, 42), and Bacillus megaterium (13, 41), have been reported. PGA is a main constituent of the sticky material in natto, a Japanese traditional food made from soybeans that have been steamed and then fermented by B. subtilis (14).Concerning PGA biosynthesis, Makino et al. reported cloning of three genes, capBCA, responsible for capsular PGA biosynthesis from B. anthracis, and the gene products occurred together as membrane-associated proteins in the Escherichia coli transformant (26,27,43). The complete genome sequence of B. subtilis 168, in which ywsC and ywtAB were found to be highly homologous to the capBCA genes of B. anthracis, has been made available in databases (31). Recently, pgsBCA genes for PGA biosynthesis were also cloned from B. subtilis IFO3336, and their sequences were found to be the same as those of the ywsC and ywtAB genes of B. subtilis 168 (2). These three genes seem to be involved in PGA production; however, little is known about the function of each gene product in PGA biosynthesis.In this paper, we describe the cloning and gene disruption of the ywsC and ywtAB genes, which are responsible for PGA production in B. subtilis IFO16449, a strain isolated from natto, and we also describe the characterization of the YwsC 44-kDa and 33-kDa proteins, which catalyze the biosynthesis of PGA from L-glutamate, a crucial enzyme in PGA production. MATERIALS AND METHODSBacterial strains, plasmids, and media. Bacillus subtilis IF...

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