JOURNAL OF BACTERIOLOGY, Jan. 1992, p.

367-376

0021-9193/92/020367-10$02.00/0 Copyright 1992, American Society for Microbiology

Vol. 174, No. 2

Construction and Application of Plasmid- and Transposon-Based Promoter-Probe Vectors for Streptomyces spp. That Employ a Vibrio harveyi Luciferase Reporter Cassette
CHARLES D. SOHASKEY, HANA IM, AND ALAN T. SCHAUER* Department of Microbiology, University of Texas, Austin, Texas 78712-1095 Received 12 July 1991/Accepted 13 November 1991

Several versatile promoter-probe vectors have been constructed for Streptomyces strains which utilize the production of blue-green light as a measure of transcription activity. Three plasmid vectors (two high and one low copy number) and two transposons are described. The multicopy plasmids pRS1106 and pRS1108 contain a transcription terminator and multiple-cloning polylinker upstream of promoterless luciferase (lux) and neomycin resistance reporter genes. Plasmid pHI90 is similar in structure to the pRS vectors except that its single copy number is an advantage for regulation studies or situations in which overexpression is otherwise toxic to the cell. The two transposons carry a promoterless lux cassette cloned such that transposition into a target DNA and fusion to the target's transcription unit occur simultaneously. TnS351 was created by inserting the luciferase genes near the right end of the viomycin resistance transposon Tn4563. TnS353 carries the luciferase genes near the right end of a neomycin resistance transposon derived from Tn4556. The size of TnS353 was minimized by deleting nonessential transposon sequences, making this element small enough to be cloned into 4)C31 bacteriophages for efficient transposon delivery to target cells of Streptomyces strains. The two Tnlux transposons have been used to generate Streptomyces coelicolor morphological mutants and to monitor transcription from chromosomal promoters during development.
The ability to create gene fusions has proved to be extremely useful in the isolation and characterization of transcriptional signals (4). Promoter-probe plasmids utilizing a variety of reporter genes have been described for Streptomyces spp. (1, 12, 18, 21, 36, 44). Many of these vectors have simplified the analysis of gene regulation in this filamentous bacterium. In this report, we describe five new gene fusion vectors (three plasmids and two transposons) that employ a luciferase (lux) gene cassette from Vibrio harveyi as the reporter of transcription. Experience in other microbial systems has demonstrated the value of gene fusion transposons for analyzing transcription at the insertion locus (23, 27, 34). They are ideal for scanning the chromosome for transcripts that respond to developmental changes or to externally applied signals. Transposons designed to fuse luciferase genes to target transcription units have been described for other bacteria (2, 11). In Streptomyces spp., a transposon (TnS099) capable of fusing the colorimetric xylE gene to target promoters has recently been constructed (15). The luciferase transposons described here, TnS351 and TnS353, are derivatives of Tn4556, a well-characterized Streptomyces transposon (6-8, 41). Tn4556-vph (Tn4563) has been used both to localize genes on cloned DNA (9) and to generate developmental mutants of Streptomyces coelicolor (39). Originally isolated from S. fradiae, Tn4556 is a member of the Tn3 family. The entire DNA sequence has been determined and, by comparison with Tn3, the putative transposase and resolvase genes have been identified (41). Because of transposition immunity, Tn4556 derivatives exist in only one copy per replicon (8, 30). TnS351 and TnS353 have been used to generate a collection of insertions into the S. coelicolor chromosome. Different insertions emit light over a wide range of different intensities.

MATERIALS AND METHODS Bacterial strains, transformation, and culture methods. All of the Streptomyces strains used in this work were derived from S. coelicolor A3(2) or S. lividans 66 and are shown in Table 1. Most in vitro DNA manipulations were transformed into S. lividans TK24 prior to being moved into S. coelicolor. Biological assay of promoter activity for plasmid fusions was carried out in S. coelicolor J1501. Transposition experiments were performed by using S. coelicolor 2612 as the host for donor plasmids. Escherichia coli plasmids generated during TnS353 construction were propagated in CQ21 (ara leu lacP1 purE gal his argG rpsL xyl mtl ilv) (31) or TB1 [hsdR A(lac-proAB) ara rspL lacZAM15] (40). Standard Streptomyces media and methods have been published elsewhere (17). R4 (39), R5, and HT (16) were used for phenotypic characterization and some antibiotic resistance tests. Spores of TK24 strains were prepared from R5 medium, while R4 medium was used for spore preparations of S. coelicolor strains. Viomycin was added to a final concentration of 50 ,ug/ml. Florimycin (purified viomycin) was a generous gift of V. Lanzov, Institute of Nuclear Physics, Leningrad, USSR. Thiostrepton, a generous gift of S. J. Lucania (Squibb), was used at a concentration of 50 jig/ml in plates and 20 jig/ml in liquid medium. The final concentration of neomycin in plates was 10 jxg/ml. Carbenicillin was kindly provided by I. Molineux. Construction of pRS1106 and pRS1108. pRS1105, a Streptomyces promoter-probe plasmid that carries two transcriptional reporter cassettes in tandem, was constructed previously (36). One cassette is a promoterless neomycin resistance gene, and the other is the luxAB luciferase cassette from V. harveyi. Although there are a variety of
367

Corresponding author.

368

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TABLE 1. Streptomyces strains

J. BACTERIOL.

Strain

Genotype

Source or reference

J1501 2612 TK24 UT1000 UT1001

S. coelicolor A3(2) hisAl uraAl strAl pglAl

K. Chater

ScP1S. coelicolor A3(2) argAl proAl cysD18 SCP1+ (NF) SCP+ S. lividans 66 str6 TB1/pMDK2

K. Chater J. M. Weber This work This work This work This work Laboratory collection; 35 This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work

UT1002
UT1003 UT1004 UT1005 UT1006 UT1007 UT1008 UT1009 UTA901 UTA1010 UTA1011 UTJ90 UTJ1105 UTJ1106 UTJ1108 UTJ960 UTJ961 UTJ962

TB1/pMDK3 CQ21/pMDK4 CQ21/pMLS3 CQ21/pAL1002

CQ21/pDD10 CQ21/pMDK7 CQ21/pCJO9 CQ21/pCJO10 CQ21/pMAP11 2612/pAL901 TK24/pMAP12 2612/pMAP12 J1501/pHI90 J1501/pRS1105 J1501/pRS1106 J1501/pRS1108 J1501/pS960 J1501/pS961 J1501/pS962

restriction enzyme sites located in the pRS1105 polylinker region, most of these are not useful because they are not unique. Therefore, we have modified the multiple cloning site by substituting portions of the polylinkers from (i) the E. coli vector pBLUESCRIPT SK+ (Stratagene, Inc., La Jolla, Calif.; hereafter designated pSK), creating pRS1106, and (ii) pSL1180, yielding pRS1108.

Plasmids pRS1106 and pRS1108 are diagrammed in Fig. 2 along with their parent molecules. In step 1, the polylinker of pRS1105 was expanded by the addition of a fragment from the multiple cloning polylinker of pSK. The pSK polylinker was excised by a SacI and KpnI; single-stranded ends were made blunt with T4 DNA polymerase and ligated to pRS1105 (pRS1105 had been linearized with XbaI and made blunt ended by treatment with the Klenow fragment of E. coli DNA polymerase I). The resulting plasmid, pRS1106, has a polylinker with unique sites for restriction enzymes HindIIl, BstXI, and BgIII along with two sites each for XbaI and BamHI. pRS1108 was created from pRS1106 to maximize the number of unique restriction sites in the polylinker. A portion of the pRS1106 polylinker was replaced by sequences taken from the E. coli vector pSL1180 (Fig. 2, step 2). The pRS1108 polylinker contains unique sites for BamHI, HindIII, DraI, Eco 47-3 AflhI, ApaLI, AvrII, Stul, and XcaI. Sites for BglII and XbaI are not unique but cut only in the pRS1108 polylinker. Expression of the sapA promoter from pRS1106 or pRS1108 was indistinguishable from the pattern reported for expression from pRS1105 (14, 19). Construction of pHI90. Construction of the single-copy lux fusion plasmid pHI90 is shown in Fig. 3. The multiplecloning polylinker and luciferase genes were transferred from pRS1108 to the SCP2* vector pIJ698. The fragment was excised from pRS1108 by SmaI digestion, HindIII linkers were added, and the resulting fragment was cloned into HindIII-cut pIJ698, yielding pHI90. In theory, the lux cassette could ligate with pIJ698 in either orientation, but only the orientation shown in Fig. 3 was obtained. Attempts to reorient the cassette, by HindIII digestion and religation of pHI90, have not been successful, suggesting that it may not be possible to obtain a stable plasmid that carries the lux cassette in the orientation opposite that shown. Construction of TnS351. TnS351 (Fig. 1) is a derivative of Tn4563 (7) which carries the lux cassette near the right-hand inverted repeat of the element. Tn4S63, carried on plasmid
Miul pal Nsil SnaBI EcoRI Sspi Asel Stul Sspl Aap7l8
pi

Tn5351
11112 bp
Bgll

Scil Chol EcoRV

Noti Sphl Pstl II SphiI Ncol

-I

LIL-l

.IsH

Sall bal

IR

tnpA

-npA

tnp

IR

tnpR

vph
MIul Hpal Nsil BspMII Eco47-3

luxB luxA

SnaBI EcoRI

Sspl
Aset

Tn5353

8062 bp

EcoRV Asull Ncol

SapI

BspHi
HindlIl
~ ~ ~ ~

.Sphl

M eal
-

luxB luxA tnpR neo tnpA FIG. 1. Luciferase gene fusion transposons. TnS351 was derived from Tn4563, and TnS353 was constructed from Tn4556. See text and Fig. 4 for construction details. Genes and symbols are as described for Fig. 2 except that tnpA encodes transposase and tnpR is the resolvase gene. IR denotes the 38-bp inverted repeat sequences at the ends of the elements. Restriction sites shown are unique on each element, except SspI
(two sites).

No* 4.m

IR

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FIG. 2. Construction of pRS1106 and pRS1108. See text for detailed discussion of construction. luxA and luxB encode the a and P subunits, respectively, of V. harveyi luciferase. The 5' portion of the luxE gene is included in the clones, but in addition to being truncated, it carries a small, internal deletion (33). neo is the TnS neomycin phosphotransferase gene, encoding resistance to neomycin or kanamycin. tsr is the thiostrepton resistance ribosomal methylase from S. azureus (43). Symbols: shaded arrows, transcription directions; hashed box, a DNA fragment taken from coliphage fd which carries a strong transcription terminator (13); asterisks, restriction enzyme sites within the polylinker that are unique on that vector. pSK is shorthand for pBLUESCRIPT SK+ (Stratagene). MCS (multiple cloning site) denotes the superpolylinker of pSL1180 that carries 42 unique restriction enzyme sites (3). pRS1105 has been described previously (36).

pUC1172 (7), was cut with BstXI and made blunt ended by treatment with T4 DNA polymerase. The luciferase genes were removed from pAL1002 (Fig. 4B) (35) as a BamHIBglII fragment, treated with the Klenow fragment of E. coli

DNA polymerase I to blunt the ends, and ligated into pUC1172 at the BstXI site. The resulting plasmid, carrying TnS351, was named pAL901. Ligation products carrying the lux genes in the opposite orientation were also obtained;

BamHI a Hindlil

FIG. 3. Construction of pHI90. See text for details. Symbols and genes are as described for Fig. 2. hyg is the hygromycin phosphotransferase from S. hygroscopicus (28). pIJ698 has been described previously (25).

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pMDK4

IuxB

pMDK7
EcoRV

FIG. 4. Construction of Tn5353. See text for details. Symbols and genes are as described for Fig. 2. Wide, filled boxes on plasmids represent transposon sequences. pIJ648 and pIJ702 have been described elsewhere (22, 25).

cells containing these plasmids produced significantly more light than did pAL901-containing cells, apparently because of transcription by an endogenous transposon promoter (results not shown). Construction of Tn5353. Figures 4A and B document construction of four modules that were required to build TnS353. The cloning strategy minimized the amount of putative nonessential spacer DNA and permitted modules to

be assembled by ligation at unique restriction sites. Tn4556 was transferred to the BamHI site of pUC7 from the BamHI site of pMDK2 (Fig. 4A, step 1). The ligation was treated with HindIII to counterselect against re-formation of pMDK2. The new plasmid, pUC7: :Tn4556, was named pMDK3. An internal BglII Tn4556 fragment was also prepared from pMDK2 (Fig. 4A, step 2). The parent replicon (pMDK2) was inactivated by NruI digestion. The V. harveyi

372

SOHASKEY ET AL.

J. BACTERIOL.

luxAB cassette was transferred from pAL1002 (35) as an SalI-EcoRV fragment to EcoRV and Sall-digested pSK, forming pMLS3 (Fig. 4B, step 3). ApaLI and BamHI digestion of pAL1002 were included to disable the parent replicon and thus prevent its religation. The TnS aminoglycoside phosphotransferase gene (encoding neomycin or kanamycin resistance) was obtained from pIJ648 (25) as a HindIII-SmaI
fragment which was ligated into pSK that had also been digested with HindlIl and SmaI, creating pDD10 (step 4). Assembly of the component parts into the final Tn53S3 transposon is shown in Fig. 4C. First, the lux genes were linked to the Tn4556 internal fragment (step 5). pMDK4 was opened by digestion with KpnI and SmaI. EcoRI was also included to inactivate the insert: it cleaved the small KpnISmaI fragment released from pMDK4 to prevent its recloning during the subsequent ligation. The luciferase genes were obtained from pMLS3 as a KpnI-SmaI fragment. The pMLS3 replicon was eliminated by both ApaLI digestion and agarose gel purification of the lux fragment (ApaLI also eliminated comigration of the two similar-sized fragments on the gel). The resulting plasmid was named pMDK7. Second, the neomycin resistance gene was added (step 6). The gene was removed from pDD10 by SmaI and HincIl digestion, and it was purified away from the pDD10 replicon by elution from agarose. pMDK7 was cut with BglII and SmaI; the single-stranded DNA ends of the BglII site were removed with mung bean nuclease. Ligation yielded the kanamycin resistance plasmid pCJO9. Third, the lux-neo cassette was moved into Tn4556 (step 7). pMDK3 was linearized with EcoRV and KpnI; Sacl was included to prevent subsequent religation of the small piece of transposon DNA that was released. The cassette was removed from pCJO9 by EcoRV and KpnI digestion; the pCJO9 replicon was inactivated by digestion with ApaLI. The ligated plasmid, carrying the transposon, luciferase and neomycin resistance genes, and a BglII-KpnI deletion, was named pCJO10. Fourth, the new transposon's size was minimized by an internal deletion of putative nonessential transposon sequences (step 8). pCJO10 was cleaved with BstXI and KpnI. Ends were then made blunt with mung bean nuclease, and the product was religated. The resulting plasmid, carrying Tn5353, was named pMAP11. Finally, TnS353 was transferred from the pMAP11 E. coli replicon to pIJ702 for delivery to Streptomyces strains by transformation (step 9). TnS353 was removed from pMAP11 with BamHI, gel purified, and ligated into the BglII site of pIJ702, resulting in pMAP12. Visualization and quantitation of light production. For the experiment shown in Fig. 6, the strength of light production from colonies was measured by punching out a uniformly sized portion of a lawn of bacteria from a plate. This was typically accomplished by using the broad end of a standard yellow 200-,ul Pipetman tip. The excess portion of the yellow tip was then removed with a razor blade, and the colony plug (still in the pipet tip) was placed face down in the bottom of a glass scintillation vial. The vial contained 2 ,lI of n-decanal (Sigma) on a thin paper wick in order to provide substrate to the luciferase enzyme as a vapor. The vial was sealed, and photon output was measured in a Turner Designs model 20e luminometer (30-s delay followed by 45-s integration). For the experiment shown in Fig. 7, measurements were made as described above except that slants were used instead of plates. At each time point, the cap of the slant to be assayed was temporarily replaced by a cap which had (i) a clear, plastic window in its center and (ii) a paper wick on its inside surface that contained 2 ,ul of n-decanal. The slant was then placed into the luminometer such that the photo-

multiplier tube was facing the top surface of the colony in the slant culture. Petri plates with colonies carrying luciferase fusions were photographed in the dark with a photon-counting camera system (Hamamatsu Photonics VIM; Photonic Microscopy, Oak Brook, Ill.). Ten microliters of n-decanal was applied to a circular paper ring (100 mm in diameter, 4 mm wide) that was attached to the side of a glass petri plate lid. The plastic petri dish lid was replaced by a glass lid for observation of colonies. Transposition. Transposition from plasmid to chromosome was performed as described previously (39). Plasmids carrying the transposons were transformed into S. coelicolor 2612 with selection for thiostrepton resistance. Individual transformants were picked, amplified in liquid (with thiostrepton), and plated on drug-free plates (R5, R4, or HT plates). Spores were then collected, and dilutions were plated onto R5 medium containing viomycin (for TnS351) or HT neomycin (for Tn5353). The relative plasmid-curing frequency was calculated for each experiment by transferring some of these colonies to R5 medium containing thiostrepton; typically, the donor plasmid was detected in <1% of viomycin- or neomycin-resistant colonies.

RESULTS

Construction of new luciferase fusion vectors for Streptomyces strains. A group of new promoter-probe vectors was designed to overcome limitations to the use of pRS1105 (36). These limitations included (i) too few unique restriction sites for promoter fragment cloning and (ii) nonstringent control of plasmid copy number. In addition, development of a delivery system for Tn4556 mutagenesis of the S. coelicolor chromosome (39) inspired construction of Tnlux transposons. See Materials and Methods for details of vector constructions. Plasmids pRS1106 and pRS1108 (Fig. 2) were derived from pRS1105. pRS1106 has been used to analyze, in Streptomyces strains, promoters that have been manipulated in the E. coli pBLUESCRIPT plasmids (19). The homology of this polylinker to pBLUESCRIPT plasmids makes pRS1106 a versatile vehicle for DNA sequencing or site-directed mutagenesis of promoter fragments when small size and single strandedness are helpful (e.g., sequencing; pSK plasmids carry a filamentous phage packaging site). The polylinker of pRS1108 has the largest number of unique cloning sites because of inclusion of much of the pSL1180 superpolylinker. pHI90 (Fig. 3) is a low-copy-number (one to two copies per cell) promoter-probe plasmid carrying the pRS1108 multiple-cloning polylinker. Two transposable elements that carry the promoterless lux cassette were constructed (Fig. 1). While these elements can be used to integrate promoter-lux fusions that have been constructed in vitro (42), they were designed to create random transcriptional fusions throughout the Streptomyces genome or into DNA cloned on Streptomyces plasmids or phages. TnS351 was derived by a simple modification of Tn4563 (see Materials and Methods). To allow packaging and delivery by 4C31 phage, a smaller Tnlux transposon (TnS353) was generated by deleting Tn4556 sequences that were thought to be nonessential for transposition. In addition, a more useful drug selection marker (neo from TnS) was added to bypass the requirement for viomycin, which is not commercially available. DNA sequences upstream of the lux genes in both Tnlux transposons contain stop codons in all

VOL. 174, 1992

STREPTOMYCES LUCIFERASE VECTORS

373

5
4

3
'

~0
10I
100
10o1

1o-2
1 0-3

FIG. 5. Visualization of chromosomal promoter activity in Tn5353 luciferase fusions. (A) Transposition selection plate photographed in incident room light. Transposition was performed as described in Materials and Methods. The colonies shown are at an early stage of aerial hypha production. (B) Graphic representation of light produced by the colonies on the same plate. The height of the peaks is directly proportional to the photon flux from each colony.

10

three possible reading frames, preventing formation of translational fusions. Transposition of Tnlux to the S. coelcolor chromosome. The rationale behind insertion into and deletion of transposon DNA described above was based almost entirely on sequence analysis (41). Therefore, it was necessary to show that transposition functions in TnS351 or TnS353 had not been destroyed during the construction of these transposons. Both TnS351 and TnS353 were tested for transposition from plasmid pIJ702 to the chromosome of S. coelicolor. After transformation into strain 2612, the donor plasmids were cured from cells (39) and viomycin-resistant (in the case of TnS351) or neomycin-resistant (for TnS353) colonies were selected. Resulting colonies were tested for sensitivity to thiostrepton, the phenotype associated with loss of the donor plasmid. Southern blotting confirmed that both TnS351 and TnS353 were competent for transposition and, like their parent elements, inserted randomly and only once per chromosome (data not shown). Despite their single copy number, fusions to transcription units on the chromosome of S. coelicolor are easily detected.

Time (days) FIG. 6. Quantitation of light emitted from TnS351-containing mycelia during a developmental time course. Time zero is the time of inoculation. Symbols: circles, 2612/pAL901; squares, 2612:: TnS351-CS70; diamonds, 2612. Shading of symbols denotes morphological state: open, only substrate hyphae present; half-filled, aerial hyphae present; filled, sporulation evident. Cells were grown on solid media and analyzed as described in Materials and Methods. Measurements were integrated for 45 s (after a 30-s delay). Media used were R2YE (2612 and 2612::TnS351) and R2YE plus thiostrepton (2612/pAL901).

Figure 5 is a photograph taken with a low-light camera showing luminescence from colonies carrying insertions of TnS353. The wide range of different light intensities is consistent with transposon insertion into a variety of transcription units. Similar results were obtained with TnSS51 (data not shown). A quantitative time course experiment on one TnS351 insertion (CS70) is shown in Fig. 6. Luciferase expression from this isolate was bimodal, with peaks during substrate mycelium development and during sporulation. The signal remained well above background levels (until sporulation was complete) but was lower than the emission from cells carrying the multicopy transposon donor plasmid pAL901. Isolation and characterization of Tn5353-induced morphological mutants. A collection of bld and whi sporulation mutants was obtained after TnS353 mutagenesis of S. coeli-

374

SOHASKEY ET AL.
TABLE 2. Phenotypic characteristics of bld::TnS353 morphological insertion mutants
Character
Wild type

J. BACTERIOL.

Morphological classa
I II III IV

Colony morphology

Sporulating

Soft fragmenting; sculptured surface

Hard, nonfragmenting; smooth surface

+ Antibiotic productionb Morphology on different carbon sources + Glucose + + Galactose + + Maltose + Mannose + + Mannitol + + Arabinose 3 3d No. of mutants isolated a (5, 29). Morphological classes have been defined previously
b

Soft, fragmenting; smooth surface +

Hard, fragmenting
+C

+ + +
-

+ +

12

+ + 0

Reflects appearance of the pigmented antibiotic actinorhodin or undecylprodigiosin. Production of antibiotics occurs when aerial mycelium is produced on certain carbon sources. d Three TnS353 insertions which did not exhibit any morphological defects were used as controls in this experiment.
c

color. Southern analysis showed that, with one possible exception, the transposon had inserted at a different chromosomal location in each mutant tested (data not shown). The bld mutants have been assigned to classes (Table 2) based on antibiotic production and phenotypic suppression of the sporulation defect by growth on alternative carbon
sources.

Figure 7 shows the profile of light production over time for several bld insertion mutants. Figure 7A illustrates the wide variation in light output from the different insertion mutants. The brightest mutant, bld-161, was visible to the naked eye. At the other extreme, bld-137 produced light at near-background levels, suggesting that it inserted in the antisense orientation for lux expression with respect to the target transcription unit. As shown in Fig. 7B, the same data plotted as percent of maxima show considerable variation in the temporal kinetics of luciferase expression over the course of colonial development.

DISCUSSION
The new luciferase vectors described here greatly expand the repertoire of possible experimental approaches to Streptomyces transcription analysis. Although there are other gene fusion vectors available for Streptomyces strains, many have serious drawbacks. For example, lacZ, the reporter gene of choice in most other systems, cannot be used in most Streptomyces species. This is due both to multiple, endogenous galactosidase activities (10) and to the instability of the gene itself when present on plasmids (26). Neomycin resistance reporter plasmids are a second example (44). Although new promoters, or up-mutations in cloned promoters, can be directly selected with these vectors, the selection does not work for late, stationary-phase genes such as those that are involved in spore development or antibiotic production. Observation of stationary-phase transcription is also a problem with vectors employing the colorimetric xyiE reporter gene (21) because spore and antibiotic pigments mask the yellow pigment produced by the xylE gene product. In addition, xylE fusions can be difficult to assay when present in single copy on the chromosome. The bacterial lux cassette as the reporter of transcription offers a solution to these problems and also provides additional advantages (37): (i) extremely low levels of light can be

accurately measured, and light can be quantified linearly over many orders of magnitude; (ii) there is no significant endogenous background activity; (iii) transcription can be monitored noninvasively over time in vivo because the repeated application of the substrate, n-decanal, is generally nontoxic; (iv) the assays are simple and inexpensive (except when extremely dim sources must be photographed); (v) stationary-phase gene expression can be monitored; and most important, (vi) gene expression can be spatially localized within a colony because light does not accumulate at its source. In addition, the luxAB cassette carries restriction sites for the enzymes AseI and SspI. These two enzymes recognize A:T palindromes and are thus valuable for physical mapping of insertions by pulse-field electrophoresis because they cut so rarely in Streptomyces DNA (73% G+C). We modified the polylinker region of the broad-host-range luciferase promoter-probe plasmid pRS1105 (36) in order to facilitate ongoing studies of the promoter for sapA, a late gene that encodes a spore-associated protein in S. coelicolor (14). The new vectors, pRS1106 and pRS1108, contain additional unique promoter cloning sites, greatly simplifying the steps required to remove a cloned promoter. The new sites are especially important when a unique site is destroyed during the cloning of a promoter fragment with incompatible restriction sites at its ends. Like their predecessor pRS1105, the plasmid vectors described here can be used to monitor promoter activity over time or space. Several regulated promoters that have been studied in detail exhibit normal patterns of regulation in the pRS plasmids, despite their high copy number (14, 20, 32, 36, 38). However, we have observed that the copy number of these plasmids can vary considerably depending on growth conditions. In addition, the pRS vectors undergo deletions at a relatively high frequency; they have been observed to break down to small derivatives, especially during protoplast regeneration or vector DNA preparation when large (>100 ml) cultures are grown (19). Most gene fusion vectors that have been described previously are based on a multicopy plasmid replicon. While this may be beneficial for analysis of weak promoters or in DNA manipulations, it also may distort observations of a promoter's regulation, for example by titrating a DNA-binding protein. Concerns about alterations in promoter regulation

VOL. 174, 1992

STREPTOMYCES LUCIFERASE VECTORS

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A
-c
0)

50 40
30

p.

L.

a,
L.

c
co I--

C:
0 -9.0

20
10
* ..-...... 0

(L

di--EL,fX

Time (days)

B
I I I I t
I I I I

100

5bld-139

bld- I 10-

80

60
40

INI

E
x

20
0
100

7\!j*
-

<

bld-161

bld- 137

80
0 0.-c 0~

60
40

20
| oT | |
| | I

~~~~~~

0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 8

Time (days)
FIG. 7. Quantitation of light emitted from bld::Tn5353 insertion mutants during a developmental time course. (A) Absolute value of light expression in time after inoculation. Symbols: circles, bld-161; squares, bld-139; upward triangles, bld-110; downward triangles, bld-137. (B) Data of panel A plotted as percent of maxima to accentuate temporal profiles. Colonies were confluent HT slant cultures and were analyzed as described in Materials and Methods. Measurements were integrated as described in the legend to Fig. 6.

bility of pIJ702 and SCP1 (39), it was relatively easy to isolate and identify transposition events. Thus, transposition of a Tn4556 derivative is possible with only tnpA and tnpR. The other open reading frames (41) are not essential for transposition. The addition of the neo gene from Tn5 has made TnS353 more practical than other Tn4556 derivatives. Unlike viomycin, neomycin is commercially available, and neo provides resistance in both E. coli and Streptomyces spp. The neo cassette also has its own promoter and does not rely on externally supplied transcription (as does the vph gene of TnS351). In addition, both Tnlux elements carry restriction enzyme sites for Asel and SspI. These enzymes have been used to correlate the physical and genetic maps of the S. coelicolor genome (24). Thus, Tnlux insertions can be quickly located on the chromosome without the need for conjugation or Southern analysis. The Tnlux transposons do not interfere with normal colony development. Earlier work with pRS1105 suggested that high-level expression of the lux genes in S. coelicolor might yield a bld phenotype. This is not the case with Tnlux; colonies that express manyfold more light than the bald pRS1105 clones sporulate normally. The Tn5351 insertion in Fig. 1 is an example of such a bright colony; its luminescence can be seen with the naked eye. Apparently, the growth inhibition caused by pRS1105 is not attributable to lux expression. It was expected that a transposon which inserts randomly into the chromosome would fuse the lux genes to a variety of transcription units and therefore a variety of different lux expression patterns would result. Some of the insertions would be in the antisense orientation, facing away from promoters, yielding little or no expression. The observed phenotypes met the expectations. Expression from different insertions can vary over several orders of magnitude, from background (e.g., dark colonies in Fig. 5) to colonies which glow brightly enough to be seen with the unaided eye. While most randomly isolated insertions appear to produce light in proportion to the vegetative growth rate, unusual temporal control patterns are sometimes observed, such as the double peak shown in Fig. 6. Developmental regulation of Tnlux insertions is much more apparent in Fig. 7. These colonies were selected because they all fail to differentiate. Figure 7B clearly demonstrates that there are distinct temporal patterns of transcription among bld::Tn5353 insertions. Peak times of expression range from 12 h to 3.5 days. As has been done for other microbial systems, it may be possible to sort mutants into classes based on time of expression of the reporter. In addition, insertion mutations can easily be moved into other morphological mutant backgrounds. Such investigations might provide a foundation for modeling the interdependent network of developmental gene expression in S. coelicolor.
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