Development of Antibiotic-Overproducing Strains by Site-Directed Mutagenesis of the rpsL Gene in Streptomyces lividans

Certain rpsL (which encodes the ribosomal protein S12) mutationsthat confer resistance to streptomycin markedly activate theproduction of antibiotics in Streptomyces spp. These rpsL mutationsare known to be located in the two conserved regions withinthe S12 protein. To understand the roles of these two regionsin the activation of silent genes, we used site-directed mutagenesisto generate eight novel mutations in addition to an alreadyknown (K88E) mutation that is capable of activating antibioticproduction in Streptomyces lividans. Of these mutants, two (L90Kand R94G) activated antibiotic production much more than theK88E mutant. Neither the L90K nor the R94G mutation conferredan increase in the level of resistance to streptomycin and paromomycin.Our results demonstrate the efficacy of the site-directed mutagenesistechnique for strain improvement.

INTRODUCTION

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Introduction

It was previously reported that certain mutations that conferresistance to streptomycin or paromomycin can activate antibioticproduction (actinorhodin and undecylprodigiosin) in Streptomycescoelicolor A3(2) and Streptomyces lividans 66 (13, 15). Thesemutations are located in the rpsL gene, which encodes the ribosomalprotein S12, and can effectively activate antibiotic productioneven in the genetic background of relA and relC (11, 15), themutations of which are known to severely inhibit productionof antibiotics in wild-type cells due to a failure to produceppGpp (2, 11). The introduction of the mutation (str) causingstreptomycin resistance is also effective in enhancing antibioticproduction in other bacteria, including members of the generaBacillus and Pseudomonas (7). Recently, the str mutation wasalso shown to confer tolerance of organic chemicals to Pseudomonasputida (6). The level of antibiotic production depended on boththe type and position of amino acid substitution in the protein.The replacement of Lys-88 by Glu was the most effective in increasingactinorhodin production of S. lividans and S. coelicolor A3(2),while a replacement of Lys-43 by Asn had no effect on antibioticproduction (15). The rpsL mutations that were found in S. coelicolorA3(2) and S. lividans that confer resistance to streptomycinare K43N, K43R, K43T, K88E, K88R, and P91S (see references 5,8, 13, and 15). Of these, only two (K88E and P91S) prominentlyincreased antibiotic production. It thus appears that certainmutations around the K88E region may distinctively affect antibioticproduction. Since most of those mutations are not likely toconfer resistance to streptomycin, it would be difficult orimpossible to select for such mutations by resistance to thedrug. Therefore, we used site-directed mutagenesis to generaterpsL mutations that may have an effect on antibiotic production.

Plasmids were constructed and amplified in Escherichia colistrain DH5 ${alpha}$ . The strain was grown at 37°C in Luria-Bertanimedium. S. lividans TK21 and its derivatives were cultured at30°C on YEME, R2YE, TSB(10), GYM, or R4 medium (15). Forselection of transformants, the media were supplemented with50 µg of ampicillin or thiostrepton per ml. The plasmidpUC18 was purchased from Takara Shuzo. The single-copy-numberplasmid pV1, an E. coli-Streptomyces shuttle plasmid, was constructedby Kawamoto et al. (9). General techniques for plasmid isolationand transformation with Streptomyces and E. coli have been previouslydescribed by Hopwood et al. (10) and Sambrook et al. (14), respectively.PCR amplification was carried out by using PE480 and PE9700(PE Biosystems), and DNA sequencing was performed by using theDNA sequencer ABI310 (PE Biosystems).

Primers were designed by using the data obtained from the S.coelicolor genome sequence (1). Total DNA was isolated fromS. lividans TK21, and a 684-bp DNA fragment containing a putativepromoter region (300 bp) and a coding region for the rpsL genewas amplified with pS12BmN (5'-CGGGATCCCGTACTTCGTCCGCCACGACACGGC)and pS12BmC (5'-CCGCGGATCCCGCTTACTTCTCCTTCTTGGCGCCG) primers.The amplified fragment was inserted into the BamHI site of pUC18to obtain the recombinant plasmid pU-TK21, which carries therpsL gene in the same direction as the lacZ gene. pU-TK21 wasused as a template for the following PCR mutagenesis experiments:the primers used for mutagenesis are listed in Table 1. PCRexperiments were performed as illustrated in Fig. 1. After thesecond PCR, the products digested with EcoRI and HindIII wereligated into pUC18 to generate pUCmut1 through -mut9. The plasmidsthus obtained were sequenced, and nine clones that had the desiredmutation without a PCR error were selected. The wild-type andmutant rpsL genes were excised by digesting with BamHI and wereligated with the pV1 vector to generate plasmids, i.e., pVWT,pVK43R, pVR86V, pVV87K, pVK88G, pVD89R, pVL90K, pVG92D, pVR94G,and pVK88E. These plasmids were then used to transform S. lividansTK21. The transformants were selected with thiostrepton (50µg/ml).

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TABLE 1. Primers used for site-directed mutagenesis^a

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FIG. 1. Schematic representation of the overlapping PCR strategy for introducing mutations into rpsL. pMut represents a primer containing a mutation site. In the first-step PCR, two fragments were amplified. After the overlap extension, the reconstituted mutant genes were further amplified in the second-step PCR by using primers M13-Fw and Rv.

Construction of mutation plasmid.

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Construction of mutation...

Two regions in protein S12 are highly conserved among eukaryoticand prokaryotic microorganisms; these are illustrated as regionI (TPKKPNS) and region II (RVKDLPGVR) in Fig. 2. Recent X-raycrystallographic analysis of the ribosome from Thermus thermophilusdemonstrated that each region consists of two independent loopstructures (12, 17). rpsL mutations known to confer resistanceto streptomycin in E. coli and Streptomyces spp. are all situatedin either of these two regions. Interestingly, in Streptomycesspp., mutations that activate antibiotic production (K88E andP91S) are found in region II, justifying our choice of thisregion for the present study. Since the basic amino acid residuesin region II play a role in the interaction with the rRNA phosphatebackbone (3), we replaced the basic amino acids (Arg and Lys)with neutral amino acids and vice versa. Eventually, we constructedthe following mutants: KO-471 (K43R), KO-472 (R86L), KO-473(V87K), KO-474 (K88G), KO-475 (D89R), KO-476 (L90K), KO-477(G92D), and KO-478 (R94G) (Fig. 2). The K88E mutation was alsoconstructed as a control. The mutation K43R confers streptomycinresistance in Bacillus subtilis and markedly enhanced the productionof an unidentified antibiotic in this organism (7). The G92Dmutation displays a streptomycin-dependent phenotype with E.coli (16). A mutation at the position Arg-94 was also knownto give rise to a streptomycin-dependent phenotype for E. coliwhen this residue was deleted (16). We changed Arg-94 to thesmallest amino acid glycine instead of deleting it (Fig. 2).

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FIG. 2. Mutations that were constructed by site-directed mutagenesis. a.a., amino acids.

Antibiotic production by transformants.

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Antibiotic production by...

The transformants with a single-copy-number plasmid thus constructed(Table 2) were inoculated on GYM agar medium and R4 agar mediumsupplemented with 0.5% Casamino Acids (R4C). All transformantsand the parental strain grew on GYM agar medium as shown forseveral representative transformants, except that transformantsKO-476 (L90K) and KO-478 (R94G) displayed a somewhat retardedaerial mycelium formation (Fig. 3A). Importantly, transformantsL90K and R94G produced extensive amounts of the red antibioticundecylprodigiosin, but the transformant K88E produced onlya slight amount of this antibiotic under the culture conditionsused. The transformants KO-468 (vector control) and KO-469 (transformedwith the wild-type rpsL gene) produced virtually no antibiotic.These results were confirmed by using the R4C medium. The transformantKO-470 (K88E) produced a considerable amount of undecylprodigosinunder this set of culture conditions. Strikingly, productionof the antibiotic was more pronounced in the transformants KO-476(L90K) and KO-478 (R94G) (Fig. 3B). The amounts of undecylprodigiosinproduced were 2.9-fold (KO-476) and 1.9-fold (KO-478) greaterthan those of the transformant KO-470 (K88E), as determinedquantitatively by the method described by Kieser et al. (10).None of the other mutations tested (K43R, R86L, V87K, K88G,D89R, and G92D) were effective in activating antibiotic production.Although R4C medium is a medium suitable for production of bothactinorhodin and undecylprodigiosin (15), we could not detectactinorhodin production in any transformants. This is probablybecause the S. lividans strains created here are hemizygous,carrying a wild type as well as a mutant copy of the rpsL gene(see below).

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TABLE 2. Strains used in this study and their level of resistance to streptomycin and paromomycin

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FIG. 3. Effect of a single copy of the mutant rpsL gene on antibiotic production. Wild-type S. lividans TK21 cells were transformed with the plasmid pV1, which expressed the mutant rpsL or wild-type rpsL. The strain KO-468 represents the vector control. The strains were grown for 4 days at 30°C on GYM (A) or R4C (B) medium. Redness represents the antibiotic undecylprodigiosin.

The ability of these transformants to exhibit resistance tostreptomycin and paromomycin was further tested. The experimentalsystem chosen in this study to evaluate the new rpsL mutationsinvolved expressing the mutant S12 proteins from their nativepromoters on a single-copy-number plasmid. This means that theS. lividans strains created carry both wild-type and mutant-typerpsL genes. Wild-type copies of the rpsL gene are generallyconsidered to encode dominant streptomycin sensitivity, andin fact we detected no streptomycin resistance when K88E transformantwas incubated for 24 h on a medium containing streptomycin.However, the K88E transformant exhibited a considerable resistance(up to 40 µg/ml [Table 2]) when incubated for a longertime (48 h or more), although the resistance level is lowerthan that for the previously described K88E mutants that areresistant to 100 µg of streptomycin per ml (5, 8, 11,15). Based on this fact, we evaluated the transformants withrespect to the level of resistance to streptomycin. Althoughthe transformants KO-470 (K88E), KO-471 (K43R), and KO-474 (K88G)were resistant to streptomycin, the parental strain and othertransformants, including KO-476 and KO-478, showed entirelyno resistance to streptomycin (1.5 µg/ml) (Table 2). Itis notable that even the transformant KO-477, which has a G92Dmutant-type rpsL gene that confers a streptomycin-dependentphenotype on E. coli (16), showed neither resistance to, nordependence on, streptomycin. None of the transformants was resistantto paromomycin. Similar results were obtained when the resistancelevel was determined by using R4 agar medium (data not shown).Thus, unlike K88E, the mutations L90K and R94G did not conferresistance to either streptomycin or paromomycin.

Region I has been reported to interact directly with the spacebetween the 16S rRNA 530 loop and the 1492-1493 strand of thedecoding site (17). Most of the mutations in this region, foundpreviously in E. coli and other bacteria, can increase translationalaccuracy. These mutations could have the effect of wideningthe space between the tRNA-mRNA complex and the 30S A site (17).On the other hand, only a limited number of data is availableconcerning the role of region II, although certain mutationsfound in this region can be responsible for the increased translationalaccuracy (16). The mutation G92D, which confers a hyperaccuracyphenotype to E. coli, did not activate antibiotic production(Fig. 3), implying that the observed activation of antibioticproduction in Streptomyces spp. is not correlated with the translationaccuracy. Recently, a study in our laboratory with an in vitrotranslation assay system raised the possibility that an increasedrigidity of 70S ribosome particles caused by specific rpsL mutationsmay result in antibiotic production activation (T. Hosaka andK. Ochi, unpublished results). Since neither the L90K nor R94Gmutation could confer resistance to streptomycin or paromomycin,it is impossible to find these mutations among the resistantisolates. In conclusion, the site-directed mutagenesis techniquecould be effective for improving the ribosomal protein S12 byfunctional modulation. Although we have no explanation for thefailure to activate actinorhodin production, it is possiblethat the activation of actinorhodin biosynthetic genes requiresmore forceful metabolic modulation than that of undecylprodigiosinbiosynthetic genes, as could be achieved by the presence ofonly the mutant-type rpsL gene.

ACKNOWLEDGMENTS

This work was supported by a grant from the Organized ResearchCombination System (ORCS) of the Ministry of Education, Culture,Sports, Science and Technology, Japan.

FOOTNOTES

* Corresponding author. Mailing address: National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. Phone: 81-29-838-8125. Fax: 81-29-838-7996. E-mail: kochi{at}affrc.go.jp.

REFERENCES

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