Abstract
Bacillus subtilis strains produce a wide range of antibiotics, including ribosomal and nonribosomal peptide antibiotics, as well as bacilysocin and neotrehalosadiamine. Mutations in B. subtilis strain 168 that conferred resistance to drugs such as streptomycin and rifampin resulted in overproduction of the dipeptide antibiotic bacilysin. Cumulative drug resistance mutations, such as mutations in the mthA and rpsL genes, which confer low- and high-level resistance, respectively, to streptomycin, and mutations in rpoB, which confer resistance to rifampin, resulted in cells that overproduced bacilysin. Transcriptional analysis demonstrated that the enhanced transcription of biosynthesis genes was responsible for the overproduction of bacilysin. This approach was effective also in activating the cryptic genes of Bacillus amyloliquefaciens, leading to actual production of antibiotic(s).
TEXT
Bacillus subtilis strains have been reported to produce three ribosomal antibiotics (TasA, subtilosin, and sublancin), four nonribosomal antibiotics (bacitracin, bacilysin, plipastatin, and surfactin), the novel phospholipid antibiotic bacilysocin, and an amino-sugar antibiotic (neotrehalosadiamine [NTD]) (1, 2). The primary structures of bacitracin, surfactin, plipastatin, and bacilysocin are shown in Fig. 1. The dipeptide bacilysin is one of the simplest peptide antibiotics produced by B. subtilis, with an l-alanine residue at its N terminus and an unusual amino acid, l-anticapsin, at its C terminus (Fig. 1). Although the biosynthesis of bacilysin has been studied extensively (3, 4), little is known about its regulation. Our laboratory has focused on strain improvement for antibiotic overexpression and has developed a new method (i.e., ribosome engineering) to activate or to enhance antibiotic production in bacteria. Current methods of antibiotic production, ranging from classic random approaches to metabolic engineering, are either costly or labor-intensive. In contrast, ribosome engineering is characterized by simplicity, consisting of the isolation of spontaneously developing drug-resistant mutants. Therefore, this method does not require induced mutagenesis and provides a rational approach to enhance bacterial capabilities with industrial applications (5–7).
FIG 1.
Structures of antibiotics produced by B. subtilis strains.
The mechanisms underlying the generation of these mutants have been studied extensively in Streptomyces and Bacillus. The streptomycin-resistant (Smr) rpsL-mutant ribosomes, carrying an amino acid substitution in the ribosomal protein S12 that confers a high level of resistance to streptomycin, are more stable than wild-type ribosomes, indicating that increased stability could enhance protein synthesis in the late growth phase (8). We later found that increased expression of the translation factor ribosome recycling factor also contributes to the enhanced protein synthesis observed in the late growth phase with the rpsL K88E mutant (9). This finding suggested that both the greater stability of the 70S ribosomes and the elevated levels of ribosome recycling factor resulting from the K88E mutation are responsible for enhanced protein synthesis in the late growth phase, with the latter being responsible for antibiotic overproduction by the K88E mutant (9). In contrast, certain rifampin-resistant (Rifr) rpoB mutations, which yield RNA polymerases (RNAPs) with mutant β-subunits, activate antibiotic production by increasing the affinities of mutant RNAPs for promoters of certain genes, eventually enhancing the transcription of antibiotic biosynthesis genes (10, 11). Recently, we identified a novel Smr mutation that confers low-level streptomycin resistance in B. subtilis. This mutation, located in the mthA gene, affects S-adenosylhomocysteine/methylthioadenosine nucleosidase, an enzyme involved in S-adenosylmethionine (SAM)-recycling pathways. Bacteria carrying the mthA mutation had elevated intracellular SAM levels, apparently due to the arrest of SAM-recycling pathways (12). These increases in SAM levels were directly responsible for bacilysin overproduction by the mthA mutants. Using B. subtilis and Bacillus amyloliquefaciens, we report here that cumulative drug resistance mutations markedly activate antibiotic production by these organisms. This approach involves less time, cost, and labor than other methods and may be widely applicable to bacterial strain improvement.
Cells were spread on solid LB medium (10 g tryptone, 5 g yeast extract, and 5 g NaCl per liter) containing 100 μg/ml streptomycin. Colonies that grew within 3 days after plating were defined as spontaneous Smr mutants. Strain KJ04 (mthA1) was obtained by transformation with congression, using B. subtilis strain YO-005 (hisC101) as the recipient (Table 1). All B. subtilis strains were grown at 30°C, with vigorous shaking, in LB medium or NG medium (10 g nutrient broth, 10 g glucose, 2 g NaCl, 5 mg CuSO4·5H2O, 7.5 mg FeSO4·7H2O, 3.6 mg MnSO4·5H2O, 15 mg CaCl2·2H2O, and 9 mg ZnSO4·7H2O per liter), a medium developed for antibiotic production by B. subtilis (13). Antibiotic production was determined by paper disk-agar diffusion assays, using half-strength Mueller-Hinton agar (Difco) plates inoculated with Staphylococcus aureus 209P as the test organism (14). To assess the production of neotrehalosadiamine (NTD), strains were grown in S7N medium at 37°C (10) and assayed as described above, except for the inclusion of 1 mM glucosamine in the Mueller-Hinton assay plates to negate the effects of any bacilysin. Spontaneous Rifr or Smr mutants of B. amyloliquefaciens were obtained by spreading spores on LB agar containing 1 μg/ml rifampin or 500 μg/ml streptomycin, respectively, followed by 3 days of incubation at 37°C. In the assay of antibiotic(s) produced by B. amyloliquefaciens, agar disks (diameter of 8 mm) were cut from the plates and put on test plates inoculated with S. aureus or Penicillium chrysogenum, followed by incubation for the indicated time. For real-time quantitative reverse transcription-PCR (qRT-PCR) analysis, total RNA was prepared as described previously (15); contaminating DNA was removed by incubation of 2 μg total RNA with 2 U of DNase I (Invitrogen) for 15 min at 25°C. RNAs were reverse transcribed using a high-capacity RNA-to-cDNA kit (ABI), according to the manufacturer's instructions, with the reaction being terminated by incubation for 5 min at 95°C. Samples were analyzed using an Applied Biosystems 7300 real-time qRT-PCR system (ABI) and Thunder Bird SYBER qPCR mix (Toyobo, Osaka, Japan). Amplification of the 16S rRNA gene was used as an internal control. The primers used are shown in Table S1 in the supplemental material.
TABLE 1.
Strains of B. subtilis and B. amyloliquefaciens used in this study
| Strain | Relevant genotype or description | MIC (μg/ml)a |
Source or reference | |
|---|---|---|---|---|
| Sm | Rif | |||
| B. subtilis | ||||
| 168 | Wild type (trpC2) | 10 | 0.3 | Standard strain |
| KJ04 | mthA1 (low-level Sm resistance) | 50 | 0.3 | 12 |
| 60009 | rpsL (K56I) (high-level Sm resistance) | >10,000 | 0.3 | 13 |
| KO-263 | rpsL (K56N) (high-level Sm resistance) | >10,000 | 0.3 | 13 |
| KO-267 | rpsL (K56R) (high-level Sm resistance) | >10,000 | 0.3 | 13 |
| KO-1236 | rpoB (Q469E) (Rif resistance) | 20 | >200 | This study |
| KO-1237 | rpoB (R485S) (Rif resistance) | 20 | >200 | This study |
| KO-1238 | rpoB (S487L) (Rif resistance) | 20 | 20 | This study |
| KO-1239 | rpoB (R485H) (Rif resistance) | 30 | >200 | This study |
| KO-1240 | rpoB (H482Y) (Rif resistance) | 10 | >200 | This study |
| KO-1241 | rpoB (H482P) (Rif resistance) | 10 | >200 | This study |
| KO-1242 | rpoB (H482R) (Rif resistance) | 10 | >200 | This study |
| KO-1243 | rpoB (L467P) (Rif resistance) | 20 | 10 | This study |
| KO-1244 | rpoB (S468P) (Rif resistance) | 20 | >200 | This study |
| KO-1245 | mthA1 rpoB (R485H) (KO-1239→KJ04)b | 50 | 200 | This study |
| KO-1246 | mthA1 rpoB (L467P) (KO-1243→KJ04)b | 50 | 5 | This study |
| KO-1250 | mthA1 rpoB (L467P) rpsL (K56R) (KO-267→KO-1246)b | >10,000 | 5 | This study |
| B. amyloliquefaciens | ||||
| NBRC 15535 | Wild type | 10 | 0.02 | From NITEc |
| KO-1265 | rpoB (H482Y) | 10 | >200 | This study |
| KO-1267 | rpoB (H482Y) rpsL (K56R) | >10,000 | >200 | This study |
Determined after incubation for 24 h at 37°C on LB plates. Sm, streptomycin; Rif, rifampin.
Transformation of the right strain by DNA from the left strain.
NITE, National Institute of Technology and Evaluation.
To investigate the effects of mutations in rpsL (encoding the ribosomal protein S12) or rpoB (encoding the RNAP β–subunit) on bacilysin production, several spontaneous Smr and Rifr mutants were isolated. When spores of wild-type strain 168 were spread and incubated on LB agar containing various concentrations of streptomycin or rifampin, Smr or Rifr mutants developed after 2 to 3 days (Table 1). DNA sequencing, using the primers as shown in Table S1 in the supplemental material, showed that most of the mutants had mutations in the rpsL or rpoB gene. Strikingly, the introduction of certain rpsL (e.g., K56R) and rpoB (e.g., R485H and L467P) mutations effectively increased bacilysin production (Table 2). These mutant strains grew as well as the parental strain 168 (data not shown). In contrast, the rpoB S487L mutation, which effectively activated NTD production (10), abolished bacilysin production entirely (Table 2).
TABLE 2.
Locations of mutations in B. subtilis mthA, rpsL, and rpoB genes and resulting amino acid changes in MthA, RpsL, and RpoB
| Strain | Bacilysin production (μg/ml)a | Position of mutation or substitution (change observed) |
Frequency (no. with mutation/no. sequenced)b | |||||
|---|---|---|---|---|---|---|---|---|
| mthA | MthA | rpsL | RpsL | rpoB | RpoB | |||
| 168 | 2.8 ± 0.3 | —c | — | — | — | — | — | — |
| KJ04 | 6.0 ± 0.4 | mthA1 | Frameshift | — | — | — | — | — |
| 60009 | 3.1 | — | — | 167 (A→T) | 56 (Lys→Ile) | — | — | — |
| KO-263 | 2.9 | — | — | 168 (A→T) | 56 (Lys→Asn) | — | — | — |
| KO-267 | 5.8 ± 0.7 | — | — | 167 (A→G) | 56 (Lys→Arg) | — | — | — |
| KO-1236 | 2.3 | — | — | — | — | 1405 (C→G) | 469 (Gln→Glu) | 7/62 |
| KO-1237 | 2.7 | — | — | — | — | 1453 (C→A) | 485 (Arg→Ser) | 3/62 |
| KO-1238 | 0 | — | — | — | — | 1460 (C→T) | 487 (Ser→Leu) | 5/62 |
| KO-1239 | 4.9 ± 0.4 | — | — | — | — | 1454 (G→A) | 485 (Arg→His) | 3/62 |
| KO-1240 | 0 | — | — | — | — | 1444 (C→T) | 482 (His→Tyr) | 10/62 |
| KO-1241 | 2.5 | — | — | — | — | 1445 (A→C) | 482 (His→Pro) | 2/62 |
| KO-1242 | 2.4 | — | — | — | — | 1445 (A→G) | 482 (His→Arg) | 5/62 |
| KO-1243 | 4.9 ± 0.7 | — | — | — | — | 1400 (T→C) | 467 (Leu→Pro) | 1/62 |
| KO-1244 | 2.5 | — | — | — | — | 1402 (T→C) | 468 (Ser→Pro) | 1/62 |
| KO-1245 | 11.0 ± 0.6 | mthA1 | Frameshift | — | — | 1454 (G→A) | 485 (Arg→His) | — |
| KO-1246 | 10.6 ± 0.1 | mthA1 | Frameshift | — | — | 1400 (T→C) | 467 (Leu→Pro) | — |
| KO-1250 | 16.6 ± 0.9 | mthA1 | Frameshift | 167 (A→G) | 56 (Lys→Arg) | 1400 (T→C) | 467 (Leu→Pro) | — |
The strains were grown in NG medium at 30°C for 24 h. Antibiotic production was determined by the paper disk-agar diffusion method, as described in the text. Experiments were conducted in duplicate or triplicate, and the data are expressed as means ± standard deviations.
Frequency of mutants with the same mutation among the rifampin-resistant mutants subjected to DNA sequencing.
—, not applicable.
To assess the effects of cumulative drug resistance mutations on bacilysin production, we introduced the rpoB L467P and rpsL K56R mutations, in that order, into strain KJ04, which contains an mthA mutation, by transformation using PCR-amplified mutation-encoding DNA fragments (Table 1). Mutations in the transformants were confirmed by DNA sequencing. As expected, the double and triple mutants produced larger amounts of bacilysin than did the starting strain KJ04 (Fig. 2A and B and Table 2), demonstrating that the rpoB L467P and rpsL K56R mutations are directly responsible for the observed increases in antibiotic production. The triple mutant KO-1250 grew as well as the wild-type strain 168, sporulated well, and produced a 5-fold greater amount of bacilysin than did the wild-type strain (Fig. 2A). Bacilysin has been reported to interfere with glucosamine synthesis (16). The antibiotic activity detected was due to bacilysin, as the addition of 1 mM glucosamine to the assay plates completely abolished antibiotic activity (Fig. 2B).
FIG 2.
Growth and antibiotic production of parental (strain 168 or NBRC 15535) and mutant strains of B. subtilis or B. amyloliquefaciens. (A) B. subtilis strains were grown in NG medium at 30°C. Bacilysin production was determined by the paper disk-agar diffusion method, as described in the text. Growth (closed symbols) and bacilysin production (open symbols) of strains 168 and KO-1250 are shown. OD650, optical density at 650 nm. (B) B. subtilis strains 168 (wild type), KJ04 (mthA), KO-1246 (mthA rpoB), and KO-1250 (mthA rpoB rpsL) were grown at 30°C for 24 h, as described for panel A, and bacilysin production was determined by the paper disk-agar diffusion method. In the lower panel (but not the upper panel), 1 mM glucosamine was included in the Mueller-Hinton assay medium. (C) B. subtilis strains were grown in S7N medium (containing 0.5% nutrient broth) at 37°C for 36 h, and NTD production was determined by the paper disk-agar diffusion method, with 1 mM glucosamine included in the bioassay plates to negate the effects of any bacilysin that might be produced. (D) B. amyloliquefaciens strains were grown at 25°C for 3 days on Landy agar plates (28) containing 0.1% yeast extract (upper) or for 1 day on plates containing 1% yeast extract (lower). Agar disks (diameter, 8 mm) were cut from the plates and put onto test plates inoculated with S. aureus (upper) or P. chrysogenum (lower), followed by incubation at 37°C for 24 h (upper) or at 25°C for 48 h (lower).
B. subtilis 168 can produce another antibiotic, namely, neotrehalosadiamine (NTD) (Fig. 1), although the genes responsible for its biosynthesis are dormant under ordinary culture conditions (10). However, introduction of the mthA mutation, conferring low-level resistance to streptomycin, activated the production of NTD (12). Therefore, we compared the abilities of strain KJ04 (mthA) and the double (mthA rpoB) and triple (mthA rpoB rpsL) mutants to produce NTD. Strains were grown at 37°C for 36 h in S7N medium and developed for NTD production (10); NTD production was assayed by the paper disk-agar diffusion method, using Mueller-Hinton assay plates containing 1 mM glucosamine to negate any effects of bacilysin. Unlike bacilysin production, introduction of the rpoB L467P and rpsL K56R mutations into strain KJ04 did not further enhance NTD production (Fig. 2C), suggesting that the mechanisms underlying the activation of antibiotic production differ for NTD and bacilysin.
The biosynthesis of bacilysin is controlled by a polycistronic operon (ywfBCDEFG) and a monocistronic operon (ywfH) (1), both of which are regarded as structural genes for bacilysin biosynthesis. Using real-time quantitative PCR, we analyzed the level of transcription of the ywfB gene, which may encode the enzyme that synthesizes the anticapsin moiety (1). Although expression of ywfB was detected in wild-type strain 168, its expression was transient (Fig. 3A). In contrast, the rpsL K56R and rpoB L467P mutants displayed higher levels of expression and prolonged expression, respectively, which accounted for the increased production of bacilysin by these mutants. Strikingly, enhancement of ywfB expression was much more pronounced for the double mutant KO-1246 (mthA rpoB) and the triple mutant KO-1250 (mthA rpoB rspL) (Fig. 3B), in accordance with a burst of bacilysin production (Fig. 2B). Importantly, the double and triple mutants showed both higher levels of and more sustained ywfB expression.
FIG 3.
Transcriptional analysis of the ywfB gene, involved in bacilysin biosynthesis. (A) The B. subtilis wild-type strain 168 and the single mutant strains KO-267 (rpsL) and KO-1243 (rpoB) were grown as described for Fig. 2A, and the levels of expression of the ywfB gene were determined by real-time quantitative PCR. (B) The double (mthA rpoB) and triple (mthA rpoB rpsL) mutant strains were grown and the levels of expression of the ywfB gene were determined as described for panel A. (C) The wild-type strain 168 was grown in NG medium or 3× NG medium, and the levels of expression of the ywfB gene were determined as described for panel A. The data are expressed as means ± standard deviations from three or more samples.
The results presented above indicate that the double and triple mutants, i.e., KO-1246 and KO-1250, acquired greater ability to initiate antibiotic production. Therefore, these mutants are likely capable of producing greater amounts of bacilysin when cultured in denser media, although initiation of secondary metabolism is usually abolished in wild-type strains under such nutrient-rich culture conditions. To assess this possibility, the triple mutant KO-1250 and the wild-type strain 168 were grown in media containing various nutrient concentrations (1-, 1.5-, 2-, or 3-fold concentrations) or in NG media supplemented with various amounts of soybean powder and glucose. After cultivation for 24 h, the amounts of bacilysin produced were determined by the paper disk-agar diffusion method. We found that bacilysin production by the triple mutant KO-1250 was enhanced, showing an 8.8-fold higher titer than that of the wild-type strain, by cultivation in dense medium (2× NG medium), which allow a greater supply of substrates for bacilysin biosynthesis, whereas the wild-type strain 168 no longer produced detectable amounts of bacilysin under similar culture conditions (Table 3), possibly due to a lack of sufficient initiation for secondary metabolism (i.e., reduced expression of the genes for biosynthesis) (Fig. 3C).
TABLE 3.
Bacilysin production by B. subtilis triple mutant KO-1250 in various media
| Medium | Bacilysin production (μg/ml)a |
|
|---|---|---|
| KO-1250 | Strain 168 (wild type) | |
| NG medium | 15.2 ± 1.1 | 2.4 ± 0.1 |
| 1.5× NG medium | 19.0 ± 1.7 | 2.1 ± 0.3 |
| 2× NG medium | 21.1 ± 0.5 | 0 |
| 3× NG medium | 20.0 ± 0.3 | 0 |
| NG medium plus 0.5% soybean powder and 0.5% glucose | 16.3 ± 0.6 | 0 |
| NG medium plus 1% soybean powder and 1% glucose | 17.7 ± 0.4 | 0 |
| NG medium plus 2% soybean powder and 2% glucose | 20.0 ± 2.1 | 0 |
The triple mutant KO-1250 (mthA rpoB rpsL) was grown in various media at 30°C for 24 h. Antibiotic production was determined by the paper disk-agar diffusion method, as described in the text. Experiments were performed in triplicate or more, and the data are expressed as means ± standard deviations.
Like streptomycetes and fungi, members of the genus Bacillus are characterized by the presence of many genes coding for biosynthesis of secondary metabolites. A typical example is the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42, the genome of which reveals an unexpected potential to produce secondary metabolites; more than 8.5% of the genome is devoted to the synthesis of antibiotics and siderophores, including polyketides and nonribosomal peptides (e.g., bacillibactin, macrolactin, difficidin, bacillaene, surfactin, fengycin, bacillomycin D, and bacilysin) (17). The majority of the genes coding for antibiotics, however, are silent or weakly expressed and thus are called “cryptic” genes. Therefore, working with B. amyloliquefaciens NBRC 15535 (the type strain of this species), we studied whether cumulative drug resistance mutations were effective in activating the cryptic genes of this organism. The wild-type strain 15535 did not produce any detectable amounts of antibiotics in various media when assayed using S. aureus as the test organism. We found, however, that introduction of certain rpoB and rpsL mutations, in that order, into strain 15535 by selecting for resistance to rifampin or streptomycin (Table 1) enabled cells to produce an antibiotic effective against S. aureus, reflecting the existence of abundant cryptic genes in this organism (Fig. 2D, upper). The rpoB H482Y mutation (but not the rpsL K56R mutation) was effective also for activation of production of another antibiotic, characterized by Penicillium chrysogenum growth inhibition (Fig. 2D, lower). It is notable that the rpoB H482Y mutation (His482→Tyr) corresponds to the rpoB H437Y mutation (His437→Tyr) of Streptomyces coelicolor A3(2), which was most often effective in activating the cryptic pathways of actinomycetes (18), although the H482Y mutation was not effective at all in enhancing bacilysin production in B. subtilis (Table 1). These results show widespread applicability of this approach in activating and enhancing antibiotic production in Bacillus spp.
Cryptic gene activation and strain improvement are both important in applied microbiological research, especially for production of clinically important antibiotics and those important in veterinary medicine and agriculture. We previously reported that antibiotic production by S. coelicolor A3(2) was activated 180-fold by cumulative (i.e., octuple) drug resistance mutations, including mutations of str, gen, rif, par, gnt, fus, tsp, and lin (representing resistance to streptomycin, gentamicin, rifampin, paromomycin, Geneticin, fusidic acid, thiostrepton, and lincomycin, respectively) (19). Among prokaryotes, B. subtilis provides a feasible system for the study of various biological functions, with the presence of a transformation system and the availability of genomic information from the completed genome project. In the present study, we demonstrated that cumulative drug resistance mutation technology can also be used in eubacteria such as Bacillus spp., although the observed effects were not as dramatic as shown in S. coelicolor A3(2). It should be emphasized that the improved strains (double and triple mutants) displayed not only higher levels of expression but also more sustained expression of the biosynthesis genes (Fig. 3B), suggesting a fundamental notion underlying strain improvement. A similar notion comes from the study of enzyme production by Paenibacillus agaridevorans, given that certain drug-resistant mutants with cycloisomaltooligosaccharide glucanotransferase overproduction showed more widespread expression of the gene encoding the enzyme throughout the growth phase (Y. Tanaka, K. Funane, T. Hosaka, K. Murakami, K. Kasahara, T. Inaoka, Y. Hiraga, and K. Ochi, unpublished results). The presence in actinomycetes and certain bacteria of many cryptic secondary metabolite gene clusters (20–23) is of interest for the activation and/or exploitation of biosynthetic pathways for useful metabolites (18, 24–27). Such cryptic biosynthetic pathways have the potential to generate novel bioactive compounds, which may have clinical importance. The key issue for the success of this approach is to find ways to induce and to enhance the expression of cryptic or poorly expressed pathways, providing material sufficient for structure elucidation and biological testing. Our approaches may be helpful in solving early-stage discovery problems by (i) inducing some level of expression of cryptic biosynthesis gene clusters (waking the sleeping genes) and (ii) rapidly increasing product yields to obtain enough material for chemical and biological characterization (early-stage yield enhancement).
Supplementary Material
ACKNOWLEDGMENTS
This work was supported by grants to K.O. from the National Agriculture and Food Research Organization (Program for Promotion of Basic and Applied Research for Innovations in Bio-oriented Industry) and the MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2014 to 2016 (grant S1413002).
We thank G. Ozcengiz for giving us useful advice and Yu Morimoto and Yasuko Tanaka for providing experimental assistance.
Footnotes
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01932-15.
REFERENCES
- 1.Inaoka T, Takahashi K, Ohnishi-Kameyama M, Yoshida M, Ochi K. 2003. Guanine nucleotides guanosine 5′-diphosphate 3′-diphosphate and GTP co-operatively regulate the production of an antibiotic bacilysin in Bacillus subtilis. J Biol Chem 278:2169–2176. doi: 10.1074/jbc.M208722200. [DOI] [PubMed] [Google Scholar]
- 2.Tamehiro N, Okamoto-Hosoya Y, Okamoto S, Ubukata M, Hamada M, Naganawa H, Ochi K. 2002. Bacilysocin, a novel phospholipid antibiotic produced by Bacillus subtilis 168. Antimicrob Agents Chemother 46:315–320. doi: 10.1128/AAC.46.2.315-320.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Hilton MD, Alaeddinoglu NG, Demain AL. 1988. Bacillus subtilis mutant deficient in the ability to produce the dipeptide antibiotic bacilysin: isolation and mapping of the mutation. J Bacteriol 170:1018–1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Koroglu TE, Ogulur I, Mutlu S, Yazgan-Karatas A, Ozcengiz G. 2011. Global regulatory systems operating in bacilysin biosynthesis in Bacillus subtilis. J Mol Microbiol Biotechnol 20:144–155. doi: 10.1159/000328639. [DOI] [PubMed] [Google Scholar]
- 5.Ochi K, Okamoto S, Tozawa Y, Inaoka T, Hosaka T, Xu J, Kurosawa K. 2004. Ribosome engineering and secondary metabolite production. Adv Appl Microbiol 56:155–184. doi: 10.1016/S0065-2164(04)56005-7. [DOI] [PubMed] [Google Scholar]
- 6.Ochi K. 2007. From microbial differentiation to ribosome engineering. Biosci Biotechnol Biochem 71:1373–1386. doi: 10.1271/bbb.70007. [DOI] [PubMed] [Google Scholar]
- 7.Ochi K, Hosaka T. 2013. New strategies for drug discovery: activation of silent or weakly expressed microbial gene clusters. Appl Microbiol Biotechnol 97:87–98. doi: 10.1007/s00253-012-4551-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Okamoto-Hosoya Y, Hosaka T, Ochi K. 2003. An aberrant protein synthesis activity is linked with antibiotic overproduction in rpsL mutants of Streptomyces coelicolor A3(2). Microbiology 149:3299–3309. doi: 10.1099/mic.0.26490-0. [DOI] [PubMed] [Google Scholar]
- 9.Hosaka T, Xu J, Ochi K. 2006. Increased expression of ribosome recycling factor is responsible for the enhanced protein synthesis during the late growth phase in an antibiotic-overproducing Streptomyces coelicolor ribosomal rpsL mutant. Mol Microbiol 61:883–897. doi: 10.1111/j.1365-2958.2006.05285.x. [DOI] [PubMed] [Google Scholar]
- 10.Inaoka T, Takahashi K, Yada H, Yoshida M, Ochi K. 2004. RNA polymerase mutation activates the production of a dormant antibiotic 3,3′-neotrehalosadiamine via an autoinduction mechanism in Bacillus subtilis. J Biol Chem 279:3885–3892. [DOI] [PubMed] [Google Scholar]
- 11.Hosaka T, Ohnishi-Kameyama M, Muramatsu H, Murakami K, Tsurumi Y, Kodani S, Yoshida M, Fujie A, Ochi K. 2009. Antibacterial discovery in actinomycetes strains with mutations in RNA polymerase or ribosomal protein S12. Nat Biotechnol 27:462–464. doi: 10.1038/nbt.1538. [DOI] [PubMed] [Google Scholar]
- 12.Tojo S, Kim J-Y, Tanaka Y, Inaoka T, Hiraga Y, Ochi K. 2014. The mthA mutation conferring low-level resistance to streptomycin enhances antibiotic production in Bacillus subtilis by increasing the S-adenosylmethionine pool size. J Bacteriol 196:1514–1524. doi: 10.1128/JB.01441-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Hosoya Y, Okamoto S, Muramatsu H, Ochi K. 1998. Acquisition of certain streptomycin-resistant (str) mutations enhances antibiotic production in bacteria. Antimicrob Agents Chemother 42:2041–2047. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Ochi K, Ohsawa S. 1984. Initiation of antibiotic production by the stringent response of Bacillus subtilis Marburg. J Gen Microbiol 130:2473–2482. [DOI] [PubMed] [Google Scholar]
- 15.Inaoka T, Satomura T, Fujita Y, Ochi K. 2009. Novel gene regulation mediated by overproduction of secondary metabolite neotrehalosadiamine in Bacillus subtilis. FEMS Microbiol Lett 291:151–156. doi: 10.1111/j.1574-6968.2008.01450.x. [DOI] [PubMed] [Google Scholar]
- 16.Kenig M, Abraham EP. 1976. Antimicrobial activities and antagonists of bacilysin and anticapsin. J Gen Microbiol 94:37–45. doi: 10.1099/00221287-94-1-37. [DOI] [PubMed] [Google Scholar]
- 17.Chen XH, Koumoutsi A, Schols R, Eisenreich A, Scheneider K, Heinemeyer I, Morgenstern B, Voss B, Hess WR, Reva O, Junge H, Voigt B, Jungblut R, Vater J, Sussmuth R, Liesegang H, Strittmatter A, Gottschalk G, Borriss R. 2007. Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42. Nat Biotechnol 25:1007–1014. doi: 10.1038/nbt1325. [DOI] [PubMed] [Google Scholar]
- 18.Tanaka Y, Kasahara K, Hirose Y, Murakami K, Kugimiya R, Ochi K. 2013. Activation and products of the cryptic secondary metabolite biosynthetic gene clusters by rifampin resistance (rpoB) mutations in actinomycetes. J Bacteriol 195:2959–2970. doi: 10.1128/JB.00147-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Wang G, Hosaka T, Ochi K. 2008. Dramatic activation of antibiotic production in Streptomyces coelicolor by cumulative drug resistance mutations. Appl Environ Microbiol 74:2834–2840. doi: 10.1128/AEM.02800-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Bentley SD, Chater KF, Cerdeno-Tarraga AM, Challis GL, Thomson NR, James KD, Harris DE, Quail MA, Kieser H, Harper D, Bateman A, Brown S, Chandra G, Chen CW, Collins M, Cronin A, Fraser A, Goble A, Hidalgo J, Hornsby T, Howarth S, Huang CH, Kieser T, Larke L, Murphy L, Oliver K, O'Neil S, Rabbinowitsch E, Rajandream MA, Rutherford K, Rutter S, Seeger K, Saunders D, Sharp S, Squares R, Squares S, Taylor K, Warren T, Wietzorrek A, Woodward J, Barrell BG, Parkhill J, Hopwood DA. 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417:141–147. doi: 10.1038/417141a. [DOI] [PubMed] [Google Scholar]
- 21.Ikeda H, Ishikawa J, Hanamoto A, Shinose M, Kikuchi H, Shiba T, Sakaki Y, Hattori M, Omura S. 2003. Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat Biotechnol 21:526–531. doi: 10.1038/nbt820. [DOI] [PubMed] [Google Scholar]
- 22.Ohnishi Y, Ishikawa J, Hara H, Suzuki H, Ikenoya M, Ikeda H, Yamashita A, Hattori M, Horinouchi S. 2008. Genome sequence of the streptomycin-producing microorganism Streptomyces griseus IFO 13350. J Bacteriol 190:4050–4060. doi: 10.1128/JB.00204-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Oliynyk M, Samborskyy M, Lester JB, Mironenko T, Scott N, Dickens S, Haydock SF, Leadlay PF. 2007. Complete genome sequence of the erythromycin-producing bacterium Saccharopolyspora erythraea NRRL23338. Nat Biotechnol 25:447–453. doi: 10.1038/nbt1297. [DOI] [PubMed] [Google Scholar]
- 24.Hopwood DA. June 2008. The tip of the iceberg. Small Things Considered. http://schaechter.asmblog.org/schaechter/2008/06/the-tip-of-the.html.
- 25.Zhu H, Sandiford SK, van Wezel GP. 2014. Triggers and cues that activate antibiotic production by actinomycetes. J Ind Microbiol Biotechnol 41:371–386. doi: 10.1007/s10295-013-1309-z. [DOI] [PubMed] [Google Scholar]
- 26.Ochi K, Tanaka Y, Tojo S. 2014. Activating the expression of bacterial cryptic gene by rpoB mutations in RNA polymerase or by rare earth elements. J Ind Microbiol Biotechnol 41:403–414. doi: 10.1007/s10295-013-1349-4. [DOI] [PubMed] [Google Scholar]
- 27.Gomez-Escribano JP, Bibb MJ. 2014. Heterologous expression of natural product biosynthetic gene clusters in Streptomyces coelicolor: from genome mining to manipulation of biosynthetic pathways. J Ind Microbiol Biotechnol 41:425–431. doi: 10.1007/s10295-013-1348-5. [DOI] [PubMed] [Google Scholar]
- 28.Gordillo MA, Navarro AR, Benitez LA, Plaza MIT, Maldonado MC. 2009. Preliminary study and improve the production of metabolites with antifungal activity by a Bacillus sp strain IBA 33. Microbiol Insights 2:15–24. [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.