High-Throughput Screening for Streptomyces Antibiotic Biosynthesis Activators

Abstract

A genomic cosmid library of Streptomyces clavuligerus was constructed and transferred efficiently by conjugation to Streptomyces lividans, and 12 distinct groups of overlapping cosmid clones that activated the silent actinorhodin biosynthesis gene cluster were identified. This generally applicable high-throughput screening procedure greatly facilitates the identification of antibiotic biosynthesis activators.

TEXT

The expression of secondary metabolic gene clusters in streptomycetes is controlled by regulatory genes, many of which are activators (2, 16, 24). Increasing the copy number of such activators often increases antibiotic production or activates silent gene clusters. This property of activators has been used for the cloning of abaA, afsQ1/Q2, afsR, and relA/spoT from Streptomyces coelicolor (6, 9, 10, 18) and afsR2 from Streptomyces lividans (25). Testing many clones for increased antibiotic biosynthesis is a major effort. We chose to generate a Streptomyces clavuligerus ATCC 27064 (NRRL3585) genomic library of 2,400 cosmids (average insert size of 33 kb, 10 genome equivalents) in Escherichia coli and then transfer the clones directly to S. lividans to test their ability to induce the near-silent actinorhodin (Act; blue type II aromatic polyketide) and calcium-dependent antibiotic (CDA; a colorless nonribosomal peptide). Here we describe a high-throughput procedure for transferring genomic cosmids individually from E. coli cultures grown in 96-well plates to S. lividans. Mass transfer from 96-well plates was used before, but the initial clones needed to be transferred to a suitable conjugation donor by transformation (17). We introduced the conjugative helper plasmid pUZ8002 into E. coli XL1-Blue MR (Stratagene) to produce strain XLUZ. pUZ8002 mobilizes oriT-plasmids efficiently from E. coli to streptomycetes (7). XLUZ was then used as the cloning host to construct the S. clavuligerus genomic library. We used pJTU2554, which is a 9.5-kb integrative (attP ϕC31) cos oriT vector, as the cloning vector (14). As a result, the clones of the genomic library could be mobilized into S. lividans directly, without an additional DNA transformation step (see below).

The E. coli strains containing the S. clavuligerus cosmids were grown in 100 μl LB broth for 6 h at 37°C in 96-well plates. About 10⁵ heat-shocked spores of S. lividans 1326 (13) were added to each well and mixed. The 96-well plates were centrifuged, the liquid supernatant was removed, and about 3 μl from each pellet was transferred to a petri dish (diameter, 15 cm) with MS agar (8) using a 96-pin needle block. The plates were incubated at 30°C for 16 to 20 h and then flooded with 3 ml water containing antibiotics (apramycin to select for exconjugants and nalidixic acid to select against the E. coli donor). After 7 days of incubation at 30°C, apramycin-resistant S. lividans exconjugants were picked onto fresh MS plates containing the same antibiotics. From the 2,400 cosmid clones, 2,310 (96%) produced apramycin-resistant exconjugants. In total, we found 105 exconjugants (4%) that produced bluish pigment, indicating that the near-silent actinorhodin biosynthesis gene cluster of S. lividans had been activated by the introduced cosmid clones. All 105 cosmids were reintroduced individually into S. lividans by conjugation, and it was confirmed that antibiotic production was indeed caused by the presence of the cosmids and not by spontaneous mutations of the host.

Seventy-one antibiotic-activating cosmids were analyzed by terminal sequencing of the inserts and aligned with the S. clavuligerus genome sequence (20, 23). Forty-six of these cosmids overlapped, forming 12 contigs, and the remaining 25 were unique. Ten of the shared regions of these contigs encoded putative transcriptional regulators, some of which have well-characterized homologues in other streptomycetes, e.g., the two-component regulatory system PhoR-PhoP in contig 5 and the OsaB homologue (SCLAV_4638) and the ClgR homologue (SCLAV_4644) in contig 9 (Table 1) (1, 4, 22). In addition, all 14 cosmids of contig 1 contained the two genes SCLAV_3382 and SCLAV_3383, which encode putative proteins resembling the well-known antibiotic biosynthesis activators AfsR and AfsS. The SCLAV_3382 product was similar to AfsR of S. coelicolor (9) (69% amino acid [aa] identity) and to AfsR-p from Streptomyces peucetius (21) (74% aa identity). SCLAV_3383 encodes a 143-aa hypothetical protein that resembled the 63-aa AfsS of S. coelicolor (19) (46% identity in a 52-aa overlap). We therefore designated SCLAV_3382 and SCLAV_3383 afsR_cla and afsS_cla, respectively.

Table 1.

Genes present in the overlapping regions of 12 contigs consisting of two or more cosmids that activated actinorhodin production in S. lividans

Contig	No. of cosmids	No. of bp of shared DNA	Shared genes (no. of genes in overlap)	Putative transcriptional regulator(s) encoded by the overlap DNA
1	14	5,531	SCLAV_3382-SCLAV_3383 (2)	AfsRScla
2	6	15,689	SCLAV_5591-SCLAV_5602 (12)	SCLAV_5602, a putative SRCAP-like transcriptional activatora
3	4	25,786	SCLAV_0066-SCLAV_0084 (19)	SCLAV_0070, XRE family; SCLAV_0082, TetR family
4	4	10,563	SCLAV_2781-SCLAV_2787 (7)	No regulator
5	3	16,679	SCLAV_3221-SCLAV_3233 (13)	SCLAV_3221, PhoR (80% identity in a 415-aa overlap with PhoR of S. coelicolor [22]); SCLAV_3222, PhoP (84% identity in a 223-aa overlap with PhoP of S. coelicolor [22]); SCLAV_3224, CarD family
6	3	16,335	SCLAV_p1044-SCLAV_p1053 (10)	No regulator
7	2	16,911	SCLAV_0411-SCLAV_0427 (17)	SCLAV_0412, AfsR-like; SCLAV_0415, TetR family; SCLAV_0426, TetR family; SCLAV_0425, TetR family
8	2	28,726	SCLAV_2357-SCLAV_2375 (19)	SCLAV_2362, sigma 24 homologue
9	2	26,644	SCLAV_4638-SCLAV_4654 (17)	SCLAV_4638, OsaB (92% identity in a 222-aa overlap with OsaB of S. coelicolor [4]); SCLAV_4644, ClgR (90% identity in a 129-aa overlap with ClgR of S. lividans [1]); SCLAV_4650, AraC family
10	2	24,818	SCLAV_p0481-SCLAV_p0502 (22)	SCLAV_p0483; SCLAV_p0499
11	2	30,540	SCLAV_p0593-SCLAV_p0621 (29)	SCLAV_p0607; SCLAV_p0610, SARP familyb; SCLAV_p0620, TetR family
12	2	15,342	SCLAV_p1434-SCLAV_p1451 (18)	SCLAV_p1438, two-component system sensor; SCLAV_p1439, two-component system response regulator

^aSRCAP, Snf2-related cAMP response-element binding protein (CREB)-binding protein (CBP) activator protein (12).

^bSARP, Streptomyces antibiotic regulatory protein.

To verify that afsRS_cla can promote actinorhodin production, a 4,423-bp BamHI-PvuII fragment, probably containing the native promoter and the coding sequences of afsRS_cla, was excised from cosmid 1G6 and cloned into the integrative vector pSET152 (3) to give pHL851. pHL851 was introduced by conjugation into S. lividans 1326 and produced exconjugants that were indistinguishable from S. lividans::1G6 with regard to actinorhodin production, while the wild-type S. lividans and the vector control S. lividans::pSET152 did not produce actinorhodin (Fig. 1a). This proved that the actinorhodin overproduction in S. lividans was caused by the heterologous expression of afsRS_cla.

Fig 1.

Actinorhodin and CDA production in S. lividans containing afsRS_cla expressed from the native promoter. (a) Actinorhodin (dark blue, showing as dark gray) production by S. lividans strains containing pHL851 or cosmid 1G6. The S. lividans wild-type and S. lividans::pSET152 controls did not produce actinorhodin. The MS agar plates were incubated for 6 days at 28°C and photographed. (b) Bioassay of CDA production. Agar plugs of S. lividans and S. lividans::pHL851 cultures (indicated at the top) were placed onto LB agar with or without Ca²⁺ (8 mM) that was spread with Bacillus mycoides. The plates were incubated for 12 h at 37°C. A 15-mm-diameter inhibition zone was observed with S. lividans::pHL851 on LB agar, and this zone increased to 23 mm on LB with Ca²⁺, a result which is characteristic for CDA. No inhibition zones were observed with the S. lividans wild type or the vector control. S. liv::pHL851, S. lividans containing pHL851 carrying afsRS_cla; S. liv::1G6, S. lividans containing the entire cosmid 1G6; S. liv::pSET152, S. lividans containing pSET152 (vector control); S. liv wt, S. lividans 1326 (wild type).

Calcium-dependent antibiotic (CDA) production was measured according to the procedure in reference 13. S. lividans and S. lividans::pHL851 were grown on nutrient agar (Oxoid), and agar plugs were placed onto LB agar with or without Ca²⁺ that had been preinoculated with Bacillus mycoides. After 12 h at 37°C, inhibition zones were observed with S. lividans::pHL851. The addition of Ca²⁺ increased the zone of inhibition, a result which is characteristic for CDA (Fig. 1b).

We also transferred pHL851 into S. clavuligerus. The strain was cultivated in shake flasks, and clavulanic acid production was analyzed according to the procedure in reference 11. Compared to the vector control, clavulanic acid production was increased 5-fold in S. clavuligerus::pHL851 (Fig. 2a). We were also surprised that S. clavuligerus::pHL851 growing on starch casein agar (15) was slightly yellow compared to the wild-type strain. The high-performance liquid chromatography (HPLC) analysis, UV-visible light (UV-Vis) spectrum (peaks at 246, 300, and 386 nm), and mass spectrum (Fig. 2b) were identical to those of a holomycin standard sample. Holomycin is a dithiolopyrrolone antibacterial and anticancer antibiotic produced by a special mutant of S. clavuligerus which was defective in late steps of clavulanic acid biosynthesis, but only minute quantities are synthesized by the wild-type strain (5). This indicated that increasing the copy number of afsRS_cla in S. clavuligerus also activated the normally silent holomycin biosynthetic gene cluster.

Fig 2.

An additional copy of afsRS_cla increased clavulanic acid production and induced holomycin production in S. clavuligerus. (a) Clavulanic acid production by S. clavuligerus::pHL851, S. clavuligerus::pSET152, and the wild-type strain, grown in shake flask cultures. Values are the means from three repeat cultures using the same clone, and the error bars show the standard deviations. (b) HPLC analysis (390-nm trace) of holomycin production (★) by S. clavuligerus::pHL851, cultivated for 4 days at 30°C on starch casein agar. The mass peaks typical for holomycin are shown in the inset box. S. cla::pHL851, S. clavuligerus::pHL851; S. cla::pSET152, vector control; wt, S. clavuligerus wild type.

These experiments have shown that a Streptomyces genomic cosmid library can be established in E. coli XLUZ containing a conjugative helper plasmid and that the resulting cosmid clones can be transferred to S. lividans with high efficiency from 96-well microtiter plates using a multiple inoculator. We have also shown that multiple groups of overlapping S. clavuligerus cosmid clones induced antibiotic production in S. lividans. Subcloning of the previously known regulatory genes afsRS_cla confirmed that the observed increase in antibiotic production was indeed caused by these two global antibiotic biosynthesis regulators.