The EMBO Journal vol. 1 1 no.2 pp.785 - 792, 1992
Cloning of the complete biosynthetic gene cluster for an aminonucleoside antibiotic, puromycin, and its regulated expression in heterologous hosts Rosa ALacalle, Jose A.Tercero and Antonio Jimenez1 Centro de Biologia Molecular (CSIC and UAM). Universidad Aut6noma, Canto Blanco, 28049 Madrid, Spain 'Corresponding author
Communicated by J.Davies
Puromycin, produced by Streptomyces alboniger, is a member of the large group of aminonucleoside antibiotics. The genes pac and dmpM, encoding a puromycin N-acetyl transferase and an O-demethyl puromycin O-methyltransferase, respectively, are tightly linked in the DNA of S.alboniger. The entire set of genes encoding the puromycin biosynthesis pathway was cloned by screening a gene library from S.alboniger, raised in the low copy number cosmid pKC505, with a DNA fragment containing pac and dmpM. Puromycin was identified by biochemical and physicochemical methods, including 'H NMR, in the producing transformants. This pathway was located in a single DNA fragment of 15 kb which included the resistance, structural and regulatory genes and was expressed when introduced into two heterologous hosts Streptomyces lividans and Streptomyces griseofuscus. In addition to pac and dmpM, two other genes have been identified in the pur cluster: pacHY, which determines an N-acetylpuromycin hydrolase and prgl, whose deduced amino acid sequence is significantly similar to that of degT, a Bacillus stearothermophilus pleiotropic regulatory gene. Key words: antibiotic genes/cosmid vectors/puromycin biosynthesis/regulation/Streptomyces
Introduction Nucleoside antibiotics constitute one of the largest group of microbial secondary metabolites (for a review, see Isono, 1988) and possess a wide range of biological activity. They act as agents against bacteria, viruses, insects, protozoans, mycelial fungi and yeasts, helminths, plants and tumour cells. In addition, immunosuppressing and immunostimulating activities have been reported. Therefore, nucleoside antibiotics are extremely useful in research and can be used as specific inhibitors of biochemical reactions since most metabolic pathways may be affected by one or more of these antibiotics, (as would be expected given the fundamental role played by nucleosides and nucleotides in the metabolism of living organisms and their viruses). Moreover, they provide examples of important therapeutic and phytopathological compounds, especially as antiviral (ribavarin or AraA), plant antifungal agents (mildiomycin) and herbicides (polyoxins). An important drawback in the therapeutic use of these compounds derives from their high toxicity. This problem (©) Oxford University Press
may be solved in some cases by structural modifications resulting in derivatives with lower toxicity. Another approach is expected to result from the application of recombinant DNA techniques. The pioneering work of Hopwood et al. (1985a) demonstrated the production of novel ('hybrid') antibiotics by gene transfer between Streptomyces strains producing different members of the same chemical class of compounds. These studies required the availability of the cloned genes, which, in principle, may be obtained by complementation of antibiotic non-producing mutants. Cloned genes, encoding specific enzymic activity from a well characterized cloned antibiotic biochemical pathway, can be used as probes to clone the corresponding genes from organisms producing chemically related antibiotics. The best example derives from the cloning of the whole set of genes for actinorhodin production (Malpartida and Hopwood, 1984) and the characterization of the DNA region complementing the actI mutation of S. coelicolor. This sequence contained part of the polyketide synthase (PKS) from S.coelicolor (Malpartida and Hopwood, 1986). By using act! as a probe (for a review, see Hopwood and Sherman, 1990), strongly hybridizing DNA sequences were found in most polyketide-producing streptomycetes, including the anthracyclins (adriamycin), macrolides (tylosine), milbemycin, polyethers (nonactin) as well as the isochromanequinone group (granaticin) of which actinorhodin is a member (Malpartida et al., 1987). Subsequently, the actI DNA region was employed to clone genes encoding the PKSs from the milbemycin-, tetracenomycin- and granaticin-producing Streptomyces (Malpartida et al., 1987; Bibb et al., 1989; Sherman et al., 1989) and to identify the corresponding genes from the oxytetracyclin-producing S. rimosus. Additionally, this strategy may permit the identification of cryptic genes for the biosynthesis of a particular class of antibiotics, as was shown by detecting the genes for nonactin biosynthesis in S.parvulus, a (normally) nonactin non-producing strain (Malpartida et al., 1987). In view of these findings, it is clear that recombinant DNA studies can provide fundamental data on secondary metabolite biosynthesis, which could be used to improve the biological activity of certain antibiotics. Puromycin (PUR) is an aminonucleoside antibiotic produced by S. alboniger. It specifically inhibits peptidyl transfer on both 70S and 80S ribosomes and has been widely used as a basic tool for studying protein synthesis (for reviews, see Vaizquez, 1979 and Cundliffe, 1981). The puromycin biochemical pathway is not well understood, although some data are available (Figure 1). It is known that adenosine is a direct precursor for the 3'-amino-3'-desoxyadenosine moiety of puromycin (Suhadolnik, 1981; Goodchild, 1982). In addition, two enzymes of this pathway, an O-demethyl puromycin O-methyltransferase (DMPM) and a puromycin N-acetyltransferase (PAC) have been characterized (Rao et al., 1969; Sankaran and Pogell, 1975; Vara et al., 1985b). Biochemical studies suggest that PAC
785
R.A.Lacalle, J.A.Tercero and A.Jimenez
inactivates the intermediate N6,N6,O-tridemethyl-puromycin by acetylation, and that DMPM methylates N-acetyl-Odemethyl-puromycin (AcODMP) to form N-acetylpuromycin (AcPUR) the last intermediate of the pathway (Vara et al., 1985b). Recently, an esterase activity which hydrolyses AcPUR to release PUR has been found in culture filtrates from S. alboniger (unpublished data). The expression of PAC and DMPM is strictly, and coordinately, regulated in S.alboniger (Sankaran and Pogell, 1975; Vara et al., 1985b). The genes pac (encoding PAC) and dmpM (encoding DMPM) have been cloned and sequenced and are transcribed on the same mRNA (Lacalle, 1990; Lacalle et al., 1989, 1991). In antibiotic-producing organisms, it has been found that the genes for self-resistance are often located within the biosynthetic gene clusters (for a review, see Seno and Baltz, 1989). Therefore, the finding that PAC and DMPM are members of the same operon suggests that other, and possibly all the genes for the puromycin biosynthesis pathway could be linked. This report describes the cloning of the genes of the puromycin biosynthesis pathway in a discrete and single DNA segment and its regulated expression in heterologous hosts. To our knowledge this is the first report of cloning the gene cluster required for the complete biosynthesis of a nucleoside antibiotic.
S.griseofuscus were then transformed with DNA from 15 of the isolated plasmids and culture filtrates from the resulting transformants were examined for the presence of material(s) acting as PAC substrate. Table I records the results of the six most relevant plasmids. It was subsequently proved that this material is indeed puromycin, and will be described as such hereafter. Therefore, puromycin is produced, in addition to others, by Streptomyces transformants containing either pPB4.34, 5.13, 11.37 or 11.40 (Figure 2A and Table I). In general, S. lividans and S. griseofuscus transformants produced about 10- to 20- and 50-fold less puromycin respectively, than did S.alboniger (Table I). Plasmid DNA could not be detected in the puromycinproducing and non-producing Streptomyces transformants by either agarose gel electrophoresis or CsCl gradients (unpublished data). A similar situation was found previously with other recombinant plasmids derived from pKC505 (Rao et al., 1987). However, their existence was proved by transforming E. coli with total DNA from the relevant Streptomyces clones and examining the restriction maps of the resulting plasmids, which were identical to those of the original E. coli isolates. In addition, Southern blot hybridization indicated that the structure of the plasmid inserts corresponded to that present in the S. alboniger DNA (unpublished data). S. lividans and S. griseofuscus were then retransformed with total DNA from the puromycinproducing clones. About 95 % of the resulting transformants
Results Cloning of the pur cluster From a S.alboniger gene library, constructed in the low copy number cosmid pKC505, 20 000 Escheria coli clones were screened with a 32P-labelled probe, which contained both pac and dmpM genes. A total of 27 positive clones was isolated. The restriction map of the recombinant plasmids from all these clones were determined, and that of the six most relevant is presented in Figure 2A. Both S. lividans and H\ N /H
produced puromycin. A comparison of the restriction maps of puromycinproducing and non-producing plasmids indicated that puromycin biosynthesis (pur cluster) was encoded by a single DNA fragment of 15 kb (Figure 2A). A detailed study of the restriction map of pPB5. 13 showed the presence of a 18 kb SpeI-XbaI DNA fragment which should contain the whole pur cluster (Figure 2). This sequence was subcloned
HsNN H
HN/H
H>N/H
N
N
PAC
HOH2C
~~HOH2C~~ Acetyl CoA
HOH
A HOH2C- < \ / NH OH
HS-CoA
CO
I
H,C, N' NCH3 -N
H3C .NCH3 N'
~~N-ACETYL
0
FHYDROLASE
HOH2Ce NH OH
Acetate
H2N- HC-CH2
OCH3
HOH2
H3C- N' CH3
H% ICH3 N
0Nz -4 DMPM
N"
SAH
H
HOH2C
NH OH
H3C-CO-HN-HC-CH,
f
OCH3
SAM
-=
HOH2C-.>
H3C-CO-HN-HHH -CH2
NH OH
H3C-CO-HN-HC-CH2QOH
PUROMYCIN Fig. 1. Schematic view of the puromycin biosynthesis pathway in S.alboniger. Based on data from Pattabiraman and Pogell (1969) and Vara et al.
(1985b).
786
Puromycin biosynthesis gene cluster
from pPB5. 13, via 'Bluescript' plasmid and E.coli, in cosmid pJAR4. The resulting construct (pCXS; Figure 2A) directed the synthesis of puromycin in S. lividans and S.griseofiuscus, as shown by PAC (Table I), TLC and HPLC assays (see below), in similar amounts to those produced by pPB4.34. Table 1. Puromycin biosynthesis by several Streptomvces
Plasmid
Puromycin production (in itg/ml) S. griseofuscus S. lividans 0.0 0.0 0.0 15
None pKC505 pPB2.49 pPB4.34 pPB4.6 pPB5.13 pPBl 1.37 pPBl 1.40 pCXS
S. alboniger
0.0 0.0 0.0 3 0.0 2 4 2 3
0.0 13 10 10 8
150 -
-
Streptomyces transformants containing the indicated constructs and S.alboniger were grown in S medium. Culture filtrates were then collected at 40 h (S.alboniger) and 50 h (clones) of fermentation and extracted with chloroform. PUR was quantified in these extracts by the PAC assay. It should be noted that the indicated data do not accurately represent the maximal levels of PUR production due to the sharp peak of the PUR biosynthesis levels during the growth curve.
Identification of puromycin Chloroform extracts of culture filtrates from S. alboniger and the other Streptomyces clones were examined by the TLC systems indicated in Materials and methods. In all clones giving a PAC-positive reaction, a spot moving with an Rf identical to that of puromycin was seen. In addition, samples derived from S.alboniger and the Streptomyces clones contained traces of a compound migrating as AcPUR. Neither were detected in either S. lividans (pKC5O5), S.griseofiuscus (pKC5O5), S. lividans (pJAR4), S.griseofiiscus (pJAR4) or transformants whose culture filtrates gave a PAC-negative reaction. When the chloroform extracts were used as substrates for a PAC assay in the presence of [3H]acetyl-coenzyme A and the reaction products were examined by TLC, a radioactive spot migrating as AcPUR was detected only in PAC-positive samples (unpublished data). In addition, the chloroform extracts were also analysed by HPLC, and the resulting profiles from presumed puromycin-containing samples had a component with retention time identical to that of puromycin. As an example, Figure 3 presents the profiles of samples from S. lividans (pPB4.34) and (pKC5O5) and S.alboniger. These results suggest that puromycin was produced by the relevant Streptomyces strains. The antimicrobial activity of the relevant culture filtrates was that expected for puromycin. Finally, to confirm that puromycin biosynthesis takes place
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dmnpM
dnMA
gene;
pac
.....
pac
pacHY
gene; C] pg gene; E
pcgeYe
regon
1_
Fig. 2. (A) Restriction maps of Salboniger DNA inserts on Streptomvces cosmids pKC505 and pJAR4. The single XbaI and Spel restriction sites of pPB5.13 are present also in pPB4.6. However, pPB4.6 contains some extra, unmapped, XbaI restriction sites in the left hand region. The presence of XbaI and SpeI sites was not examined in the other plasmids. + and indicate that PAC activity was detected or undetected in chloroform extracts from the relevant culture filtrates, respectively. (B) Organization of the identified puromycin biosynthetic genes. -
787
R.A.Lacalle, J.A.Tercero and A.Jim6nez
in S. lividans (pPB4.34), puromycin was purified from culture filtrates of this clone as indicated in Materials and methods. 1H NMR spectra of the final product were indistinguishable from that of authentic puromycin (unpublished data). We conclude, therefore, that the biochemical pathway for the biosynthesis of puromycin is contained in pPB4.34 and therefore in a 15 kb DNA fragment defined by the restriction maps of several puromycin-producing plasmids (Figure 2). Regulation of puromycin biosynthesis In S.alboniger, the expression of both PAC and DMPM is strictly growth regulated. It commences at mid- to lateexponential growth and reaches a maximum at the entrance of the stationary phase, to decay drastically during this stage to the growth curve (Rao et al., 1969; Vara et al., 1985b). These findings suggest that the genetic system of the biochemical pathway for puromycin production from S. alboniger includes functions which regulate the expression of the biosynthetic genes. To examine if the pur cluster
S. lividans pKC505
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alboniger ~~~~~~~~~~~~~~S.
CD
0 CD
S. fividans pPB4.34
0
5
10o
15
20
25
30
35
40
50
45
RETENTION TIME (minutes)
Fig. 3. HPLC of chloroform extracts from culture filtrates of several Streptomyces. Samples were processed as indicated under Materials and methods.
included such regulatory gene(s), the biosynthesis of PAC, DMPM and puromycin was followed during the growth curve in liquid cultures of both S. lividans (pPB4.34) and S.alboniger. The results of this experiment (Figure 4) indicated that puromycin biosynthesis and the expression of both PAC and DMPM activities are regulated in a similar way in both S.alboniger and the transformant S. lividans (pPB4.34). Interestingly, whereas the specific activities of PAC and DMPM in the Streptomyces clones are similar or higher than in S.alboniger, puromycin production is 10-fold lower than in the parental strain (Figure 4). Nucleotide sequence analysis has shown the presence of one potential regulatory gene (prgl) of 1287 nt in the cloned pur cluster (Figure 2B). The amino-terminal of its deduced amino acid sequence (PRG1) presents significant similarity to the corresponding regions of the gene products from degT, a pleiotropic regulatory gene of Bacillus stearothermophilus (Takagi et al., 1990), and the eryCl and orfl0.4 genes of the erythromycin biosynthesis pathway from Saccharopolyspora erythraea (Dhillon et al., 1989) and the rjb cluster of Salmonella typhimurium (Jiang et al., 1991), respectively. In contrast, whereas the carboxy-terminus of DEGT, ERYC1 and ORF 10.4 are homologous, they differ totally from that of PRG1 (Figures 5 and 6). The central region (amino acids 198 -220) of PRG1 has a helix-turn-helix structure, deduced from amino acid sequence similarities with putative DNA-binding domains of DEGT and X Cro-like DNA binding domains (Figure 7). By applying the Dodd -Egan method for predicting these structures, the score (0.0) of PRG1 (Figure 7) is too low for it to be considered to be involved in DNA binding. However, other putative helix-turn-helix domains from proteins which bind to DNA, such as LEXA and SV40 large T antigen, give very low or negative scores from the Dodd-Egan matrix (Figure 7; Dodd and Egan, 1990). In addition, the amino acids at positions 394-407 at the carboxy end of PRG1 (DWPEDVALIEAYAE) could form an amphipathic a-helix structure formed of negatively charged residues (D or E) separated by either two or four hydrophobic amino acids. This structure, present in DEGT (Takagi et al., 1990), is considered to be a consensus region for gene activation present in transcription activator proteins (Ptashne, 1988). Taken together, these considerations suggest that prgl is a regulatory gene in the pur cluster. -
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Fig. 4. Expression of PAC and DMPM and biosynthesis of puromycin during cell growth. S.alboniger (A) and S.lividans (pPB5.13) (B) were grown in S medium at 30°C. At the indicated times, samples were taken to determine optical density and then filtered. Cell extracts were used to assay PAC and DMPM activities. Chloroform extracts from the filtrates were used to quantify puromycin by the PAC assay. Similar results were obtained by examining the antibacterial activity of these extracts. 0: optical density 660 nm; O: PAC activity; 0 DMPM activity; A: puromycin production.
788
Puromycin biosynthesis
al.
(1979). These plasmids
most probably linear plasmids which would be positions shown in Figure 8.
Genomic location of the pur cluster
et
Streptomyces, the gene clusters for antibiotic biosynthesis pathways are usually located on the chromosome. A well known exception is the methylenomycin cluster, which is found on the giant linear plasmid SCPI from S. coelicolor
molecules and not small circular
In
(Chater
S.alboniger,
1985;
Bruton,
and
Sankaran and
Kinashi
et
Pogell (1973)
al.,
1987).
expected
to
migrate
those
to
are
Indeed, such circular elements have
gel electrophoresis (unpublished data).
agarose
In
by curing determining
gene cluster
been detected in
not
of total DNA from S.
alboniger
deduced
studies with ethidium bromide that the genes
Discussion
production were not plasmid encoded. Other studies provided evidence suggesting the existence, in S. alboniger, of an extrachromosomal element implicated in the formation of aerial mycelium (Redshaw et al., 1976, 1979). The availability of the pur cluster and the CHEF technique for large plasmid identification has allowed the re-examination of these questions. Total DNA from S.alboniger was submitted to CHEF electrophoresis, transferred to a nylon membrane and hybridized to the 18 kb Spel Xbal fragment containing the pur cluster. The results suggested that this cluster is present in the chromosomal DNA of S. alboniger (Figure 8), in agreement with the findings from Sankaran and Pogeil (1 973). However, the possibility of the pur cluster being located in a giant circular plasmid cannot yet be completely eliminated. Two fragments migrating at positions 1000 and 2000 kb can be clearly seen in Figure 8. It is not yet known whether PUR
these represent monomeric and dimeric forms of the
plasmid
two
or
different elements,
Complementation of antibiotic non-producing mutants has successfully used to clone the whole biosynthesis gene clusters for several antibiotics, including actinorhodin (Malpartida and Hopwood, 1984), tetracenomycin (Motamedi and Hutchinson, 1987) undecylprodigiosin (Feitelson et al., 1985) and oxytetracycline (Binnie et al., 1989). Because puromycin non-producing mutants from S. alboniger were not available, we used a DNA fragment containing the biosynthetic genes pac and dmpM as probe to search for the puromycin gene cluster in a genomic library raised in cosmid pKC5O5 (Rao et al., 1987). Of these two genes, pac was known to determine puromycin resistance (Vara et al., 1985a) and dmpM an O-demethylpuromycin O-methyltransferase (Vara et al., 1988). Consequently, the complete set of genes from S. alboniger for puromycin production have been cloned and then located in a single DNA fragment of 15 kb which was expressed in two heterologous hosts, S. lividans and S.griseofuscus. This been
same
the different
although
discrete
intensities of the signals in lines 2 and 3 suggest the former possibility. One of these plasmids may encode the aerial mycelium function(s) suggested by the work of Redshaw
0
100
300
200
0
300
sequence
0
must
carry
200
100
400
31300
31100
EL
/2(?00
/
21!00
/
00
100
0
0
ERYCl
DEGT
0
the
400
300
/
4100
structural,
the
regulatory elements of the pathway, regulation of puromycin production and the the
and
because the
200
100
DNA
resistance
100
200
300
0
l90
290
ORFi 0.4
390,
400
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200
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-
ERYCl Fig. 5. COMPARE/DOTPLOT analysis of the deduced amino was used at a stringency of 16 (Devereux et al., 1984).
ERYCl
ORFi 0.4
acid sequences of
PRGI,
DEGT,
ERYCI1
and
ORFIO.4.
A window size of 30 amino
acids
789
R.A.Lacalle, J.A.Tercero and A.Jimenez PRG1 DEGT ERYCl
ORF10 .4 PRG1 DEGT
51
GQ
35 35 61
11-
PRG1
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.
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103 85
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PRG1
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V P GAPESDDPALVI E tTI LEVRLYGHPADLAm
rFIX EL
4VAERYG
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GTFGD
EDR
Q
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hIADRHGI
LVED
QAV
RG
- - - -
a Lr-%,PL,r-lnIll rv LAMWO-111 f -J l'R IF'.v WV F
TLIECL
M
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"Iolpi
M.,
g6TTTj4 EKtVIFI HKPK KKI DF~~
CYCAPGC
Fig. 6. Alignment of the deduced amino acid sequences of PRG1. DEGT, ERYCI and ORF10.4. Alignment was derived from the BESTFIT and PILEUP programs (Devereux et al., 1984). Bold letters represent identical residues. These residues and conservative replacements are boxed.
expression of the genes pac and dmpM in the relevant S. lividans transformant were similar to those of the producing organism. The demonstration of puromycin production in clones from two independent Streptomyces species eliminates the possibility of either activation or complementation of cryptic genes in the hosts. The gene pac, which inactivates puromycin by Nacetylation is the presumptive resistance determinant in S.alboniger (Vara et al., 1985a), although it also appears to be a key enzyme in directing the puromycin biosynthesis pathway through the correct inactivated intermediates (Vara et al., 1985b). This gene is located close to the end of the pur cluster, but within the pur biosynthetic genes, similar to other resistance determinants identified within biosynthetic genes in antibiotic-producing Streptomyces (Seno and Baltz, 1989). Whether pac is the only resistance determinant is not known, although existing evidence is in favour of this supposition (Vara et al., 1985a). Nonetheless, S.alboniger failed to incorporate [3H]puromycin from the culture medium whereas its ribosomes are fully sensitive to the antibiotic (unpublished data) and the presence of a transmembrane-bound protein involved in establishing a puromycin permeability barrier cannot be eliminated. Such is the case for both S. rimosus and S. coelicolor, the oxytetracycline and methylenomycin producers, respectively, where genes for resistance apparently encode specific membrane proteins whose function is secretion of these antibiotics (Neal and Chater, 1987; Butler et al., 1989). A similar protein is likely to be required for the export of N-acetylpuromycin in S.alboniger. This was inferred from the detection of traces of N-acetylpuromycin and a hydrolase activity, which converts the latter intermediate into puromycin in the culture medium of the producing strain. This enzyme has also been detected in culture filtrates of all puromycin-producing clones reported in this work (unpublished data), allowing the relevant gene (pacHY) to be located in the pur cluster (Figure 2B). Sequencing work in progress has detected a gene (prgl ), located upstream of pac, whose deduced amino acid sequence suggests an involvement in gene regulation. Thus, its amino terminus is highly homologous to that of degT, a pleiotropic regulatory gene from B.stearothermophilus. The degTgene, similarly to the regulatory degSU, degQ and degR genes from Bacillus subtilis (Msadek et al., 1990) confers pleiotropic effects on this bacterium, including altered control of sporulation, filamentous cells and enhancement of extracellular enzyme production. It was suggested 790
*
*
*
Score
*
PRG1
SL
Q A N K A V Y A G E G G I L V T D D A L
DEGT
AA FG LS LN IR
Q Q Q Q Q
X x
cro cI
434 CI P22 C2
A T E A A
I K S E A
G A K Y N G T A K D L G V A D K M G L A Q K V G L G K M V G
K V M T V
C Y G T S
V G E L G T A A Q S A I N K A I Q S G V G A L F Q Q S I E Q L E N V A I S Q W Q
0
T H N N R
.0
-3. 1 4.0 4.6 5.6 6.1
Fig. 7. Possible helix-turn-helix domain in the PRG1 protein. Amino acid sequence similarities were found with some DNA-binding domains (Dodd and Egan, 1990; Tagaki et al., 1990). The asterisk indicates the conserved amino acids. The indicated scores correspond to predictions of DNA-binding motifs by using the weight matrix of Dodd and Egan (1990).
that DEGT functions as a sensor protein for environmental stimuli and also as a regulator to activate or repress transcription of target genes (Takagi et al., 1990). If this holds true for DEGT, then given the sequence similarities, we suggest that PRG1 is a transcriptional regulator of the pur cluster. The similarities in sequence of DEGT and PRG1 with ERYC 1 (an enzymatic/regulatory protein of the erythromycin biosynthesis pathway; Dhillon et al., 1989) and ORF10.4 (a putative protein of the rbf cluster of S. typhimurium; Jiang et al., 1991) may imply a regulatory function to the genes eryC1 and orfl0.4. As proposed for degT, the prgl gene (and by extension eryC1 and orfl0.4 in their particular genetic systems) might be the effector gene for transcriptional regulation of a two-component system of a signal transductional pathway (Stock et al., 1990; Msadeck et al., 1990) to mediate gene expression from the pur cluster. Puromycin biosynthesis in the heterologous transformants was 10- to 50-fold lower than in S.alboniger. A similar result was found in cloning the pathway genes for oxytetracycline (Binnie et al., 1989) and cephamycin C (Chen et al., 1988). Several factors may account for these results, including biomass production (S.alboniger cultures reached a 9-fold higher cell density than S. lividans clones) and plasmid copy number. In addition, the supply of primary metabolites in the Streptomyces clones may limit the production of puromycin. This is consistent with the finding that production of S.griseofuscus is 5-fold lower than in S. lividans. The biosynthesis of PAC and DMPM does not seem to be limiting, because their rates of expression in the S. lividans transformants are higher than in S. alboniger. Characterization of all the genes involved in puromycin biosynthesis will be useful in detecting homologous genes from other actinomycetes, to identify biosynthetic clusters for other -
-
Puromycin biosynthesis gene cluster
MgSO4) was employed to study PUR production. In this case, mycelia Qz
12 1
%
cr
3 4
1
1
2 1
4
3 1
1
1
__.
2200,1600 1125-
1020 945
850 800
-
-
-
770 -700
-
630580 460
-
Fig. 8. Genomic locations of the pur cluster in S.alboniger. Total DNA from S.alboniger was developed by CHEF. The gel was blotted on a nylon membrane, which was subsequently probed with a 18 kb Xbal-SpeI DNA fragment containing the pur cluster. Left panel is a photograph of the gel stained with ethidium bromide. Right panel is the X-ray film of the Southern blot. DNA on lanes 2 and 3 was obtained from independent S.alboniger cultures. Numbers to the left indicate the size (in kb) of the Saccharoinvces cerevisiae chromosomal DNA markers. Numbers to the right denote the size (in kb) of plasmid DNAs. which are identified by arrows.
nucleoside antibiotics and, subsequently, production of novel 'hybrid' antibiotics.
Materials and methods Strains, vectors, media and DNA methodology S.alboniger ATCC 12461. the puromycin producer (Porter et e/l.. 1952). S. /ividans 66 (1326) (Hopwood t cil. 1985b), S. griseofiiscus (Larson and Hershberger. 1984) and E.coli strains DH5 (Hanahan, 1985). HBIOI (Bolivar and Backman. 1979) and AM6 (Vara et al.. 1985a) are described in the indicated references. Strcptomnvces-E.coli shuttle cosmid pKC505 (Rao et Cl/.. 1987) was provided by Dr R.H.Baltz (Eli Lilly. Indianapolis. USA). Streptotns'ces-E.coli cosmid pJAR4 is similar to pKC505 with hygromycin B resistance instead of apramycin resistance (unpublished data). StreptomYces plasmids were pFV8 and pFV I (Vara eti/l.. 1985a). E. ('li plassmids were 'Bluescript' SK and pVN7. 1 (a pUC 19 derivatives containing the genes pac1 and tdnmpM from S.al/boniger: Vara et al.. 1988). Plasmid DNA from E. coli and Strcptomoi\ves was prepared as described by Hopwood ct (1985b). Unless otherw\ise specified. Streptomyccs were grown in YEME liquid medium supplemented with 34% sucrose and 5 mM MgSO4 (Hopwvood et (i/.. 1985b). Medium S (2 % starch, 0.4% Bacto-peptone. 0.4 % yeast extract, 0.4% K.HPO4, 0.2% KH2PO4, 0.02% CoCI, and 2 mM a/.
were dispersed by introducing a stainless steel spring in baffled flasks. To prepare protoplasts from Streptomyces, YEME medium was supplemented as above and with either 0.5% or 1% glycine for S.lividans and S. griseofiiscus, respectively. Solid medium for S. lividans and its transformants was R5 (Hopwood et al., 1985b) and that for S.alboniger and S.griseofiJscus was S medium solidified with 2% agar. S.lividans and S.griseofiscus protoplasts were transformed as described by Hopwood et al. (1985b) and Larson and Hershberger (1984), respectively. When required. antibiotics were added at the following concentrations: 10 and 25 yg/ml thiostreptone for liquid and solid media, respectively; 25 and 100 itg/ml apramycin for Streptomyces and E.coli, respectively; 200 and 50 yg/ml hygromycin B for Streptomnvces, and E.coli, respectively, and 100 ,ug/ml ampicillin. Antimicrobial activity of chloroform extracts of culture filtrates was determined by the paper disc method using a variety of bacteria as testing organisms. Transformation of E.coli and Streptoinvces was performed according to Hopwood et al. (1985b). A S.alboniger gene library was prepared in cosmid pKC505 as described by Rao et al. (1987). Essentially, total DNA from S.albonziger was partially digested with Sau3A and the resulting 20-40 kb DNA fragments were collected from sucrose gradients and then dephosphorylated. To prepare cosmid arms, pKC505 was cut with HpaI, dephosphorylated and then digested with BamnHI. These arms were then ligated to the S.alboniger 20-40 kb DNA fragments. Packaging of this ligation mixture was performed by using the X inz vitro packaging kit from Amersham as described by the supplier. Packaged recombinant cosmids were transfected into E. coli HB101. A total of 150 000 apramycin resistant clones were obtained, at a frequency of 1.7 x 105 colonies per DNA tg. Southern blot hybridizations were carried out as described by Maniatis et al. (1982). Labelling of probes with [Q-32P]dCTP was achieved by the 'random primers' method of Feinberg and Vogelstein (1983). Colony hybridization was accomplished as reported by Grunstein and Hogness (1975). Pulsed field gel electrophoresis was performed by contour clamped homogeneous electric fields (CHEF), using an LKB-2015 Pulsaphor equipment (Chu et al., 1986). Total DNA from S.alboniger (Kinashi et al., 1987), and the Saccharomvces cerevisiae control DNA (Bio-Rad) were separated on a 15 cm square, 0.8% agarose gel at 14°C in 0.5 x TBE buffer (1 x TBE: 0.089 M Tris-borate, 0.089 M boric acid, 0.002 M EDTA, pH 8.0). The electrophoresis conditions were: pulse time of 120 s for 87 h, constant voltage at 150 V throughout. After electrophoresis, the gel was processed as described by Kinashi et al. (1987).
Enzyme assays PAC and DMPM activities were determined radiochemically by using either [3H]acetyl-coenzyme A or [35S]adenosyl-methionine. respectively, as cosubstrates, as described by Vara et al. (1985b, 1988). As a source of PAC activity, log phase cells from a S. lividans transformant containing plasmid pFV8 (Vara et al., 1985b) were broken by sonication on ice, centrifuged in the cold for 15 min at 15 000 r.p.m. in a microfuge and the resulting supernatant was then collected and kept at -70°C until use (Vara et al.. 1985b). To quantify PAC and DMPM activities from Streptomynces transformants. cell extracts were prepared similarly from mycelia collected at different times during cell growth in S medium. One unit of PAC activity corresponds to 1 tmol of AcPUR synthesized per min per mg of protein. One unit of DMPM activity corresponds to 1 nmol of AcODMP synthesized per min per mg of protein. Protein concentration was estimated by the method of Bradford (1976). Puromycin assays In general, PUR or PUR-like material was extracted from culture filtrates at pH 11.0 by three successive chloroform extractions in a rotary shaker (Vara et al., 1985b). The combined fractions were then evaporated to dryness and dissolved in water. PUR or PUR-like material was then identified and/or quantified by the PAC enzymic assay. This system allows an accurate estimation of puromycin at concentrations as low as 0.5 /g/ml. In addition, it is extremely specific for the puromycin structural backbone (Vara et al., 1985b). Physicochemical identification of AcPUR, AcODMP, ODMP (all known PUR precursors) and PUR was unambiguously carried out by either TLC or HPLC. The TLC systems were: I[ethyl acetate-methanol (3:1)] and II [acetonitrile-0. 1 M ammonium acetate (5:95)], both on silica gel 60 F,54 (Kodak) and III [acetonitrile -0.1 M ammonium acetate (20:80)] and IV [butanol acetic acid-water (12:3:5)] both on cellulose F,54 (Merck, Darmstadt). The Rf values of PUR, AcPUR, ODMP and AcODMP were, rcspectively, 0.55, 0.82 0.5and 0.80.21 0.3, 0.27 and 0.49: 0.41, 0.62, 0.49 and 0.85 and 0.78, 1, 0.69 and 0.96 for systems l, II, III and IV, respectively. These compounds were detected by exposing to UV light. If [ 3H]acetyl derivatives were studied, chromatograms were cut into 0.5 cm
791
R.A.Lacalle, J.A.Tercero and A.Jimenez pieces, which were then quantified in a liquid scintillation spectrometer (Vara et al., 1985b). HPLC was performed on spectra Physics SP8700 equipment and a Spectra Physics SP8440 254/280 nm detector. Chromatography was performed through a reversed phase of C18 column (5 pm, 250 mm x 4 mm. Lichrosorbe Merck, Darmstadt). Solvents used were A (0.1 M ammonium acetate, pH 6.0) and B (0.2% trifluoroacetic acid in acetonitrile). Gradient conditions were 80-60% A in 30 min; 60-40% A in 15 min; 80-20% A in 10 min, at a flow rate of 0.5 ml per min. Detection was by UV absorption at 254 nm. Under these conditions the retention times of ODMP, AcODMP, PUR and AcPUR were 13.3, 17.4, 23 and 27 min, respectively. Puromycin purification Filtrates from a 5 1 culture (60 h) of S.lividans (pPB4.34) were collected and extracted twice with 1 I chloroform each. Extracts were combined and concentrated up to 0.3 ml by vacuum. This sample was developed by preparative TLC on five plates (2 mm thick) by using system I (see above). Bands migrating as PUR were removed, combined and then extracted with 30 ml of chloroform:methanol (1:9). The extract was taken to dryness under vacuum and then dissolved in 0.1 ml water. Samples (25 Al) of this solution were subjected to HPLC as described above. Fractions eluting with a PUR retention time were collected and then combined. These steps were repeated three times for each sample. Finally, the relevant fractions were combined and lyophilized. The final product (1.3 mg) was dissolved in D2O pH 3.0 to perform 'H NMR spectroscopy. 1H NMR studies High resolution 'H NMR spectra at 360 MHz were obtained on a Bruker WM-360 spectrometer at a probe temperature of 20°C on D20. Conditions were: 8 As pulse width (900 flip angle), 4000 Hz spectral width, 16 K data points, 3 s recycle time and 256 scans. Intensity of the water signal was reduced when necessary with the help of a 0.5 s presaturation pulse.
Acknowledaements We are indebted to Dr R.H.Baltz (Eli Lilly) for the gifts of cosmid pKC505 and S.griseoftuscus, to Prof. M.Rico for his help with 'H NMR analysis, to Prof. Julian E.Davis for critically correcting the manuscript and to Mrs Asunci6n Martin for expert technical assistance. This research was supported by grants from the CAICYT (No. 613/615), Smith Kline and French and by an institutional grant from Fundacion Ram6n Areces to the Centro de Biologia Molecular.
References Bibb,M.H., Bir6,S., Motamedi,H., Collins,J.F. and Hutchinson,C.R. (1989) EMBO J., 8, 2727-2736. Binnie,C., Warren,M. and Butler,M.J. (1989) J. Bacteriol, 171, 887 - 895. Bolivar,F. and Backman,K. (1979) Methods Enzvmol., 68, 245-267. Bradford,M.M. (1976) Anal. Biochem., 72, 248-254. Butler,M.J., Friend,E.J., Hunter,I.S., Kaczmarek,F.S., Sugden,D.A. and Warren,M. (1989) Mol. Gen. Genet., 215, 231-238. Chater,K.F. and Bruton,C.J. (1985) EMBO J., 4, 1893-1897. Chen,C.W., Lin,H., Kuo,C.L., Tsai,H. and Tsai,J.F. (1988) BiolTechnology, 6, 1222-1224. Chu,G., Vollrath,D. and Davis,R.W. (1986) Science, 234, 1582-1585. Cundliffe,E. (1981) In Gale,E.F. Cundliffe,E., Reynolds,P.E., Richmond,M.H. and Waring,M.J. (eds), 7he Molecular Basis ofAntibiotic Action. John Wiley and Sons Ltd, New York, pp. 402-547. Devereux,J., Haeberli,P. and Smithies,O. (1984) Nucleic Acids Res., 12, 387 -395. Dhillon,N., Hale,R.S., Cortes,J. and Leadlay,P.F. (1989) Mol. Microbiol., 3, 1405-1414. Dodd,I.B. and Egan,J.B. (1990) Nucleic Acids Res., 18, 5019-5026. Feinberg,A.P. and Vogelstein,B. (1983) Anal. Biochem., 132, 6-13. Feitelson,J.S., Malpartida,F. and Hopwood,D.A. (1985) J. Gen. Microbiol., 131, 2431-2441. Goodchild,J. (1982) In Sammes,P. (ed.), Topics in Antibiotic Chemistry. Ellis Horwood Publishers, Chichester. Vol. VI, pp. 99-227. Grunstein,M. and Hogness,D. (1975) Proc. Natl. Acad. Sci. USA, 72, 3961 -3965. Hanahan,D. (1985) In Glover,D.M. (ed.), DNA Cloning: A Practical Approach. IRL Press, Oxford, pp. 109-136. Hopwood,D.A., Malpartida.F., Kieser,H.M., Ikeda,H., Duncan,J., Fujii,I., Rudd,B.A.M., Floss,H.G. and Omura,S. (1985a) Nature, 314, 642-644.
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Hopwood,D.A., Bibb,M.J., Chater,K.F., Kieser,T., Bruton,C.J. Kieser,H.M., Lydiate,D.J., Smith,C.P. and Ward,J.M,. (1985b) Genetic Manipulation of Streptomyces: A Laboratory Manual. The Johns Innes Foundation, Norwich, UK. Hopwood,D.A. and Sherman,D.H. (1990) Annu. Rev. Genet., 24, 37-66. Isono,K.J. (1988) J. Antibiotics, 41, 171 1-1739. Jiang,X.-M., Neal,B., Santiago,F., Lee,S.J., Romana,L.K. and Reeves,P.R. (1991) Mol. Microbiol., 5, 695-713. Kinashi,H., Shimaji,M. and Sakai,A. (1987) Nature, 328, 454-456. Lacalle,R.A., Pulido,D., Vara,J., Zalacain,M. and Jimenez,A. (1989) Gene, 79, 375-380. Lacalle,R.A. (1990) Aislamiento del cluster pur detenninante de la bios(tntesis de puromicina en Streptomyces alboniger. PhD Thesis, Facultad de Ciencias, Universidad Aut6noma de Madrid, Madrid, Spain. Lacalle,R.A., Ruiz,D. and Jimenez,A. (1991) Gene, 109, 55-61. Larson,J.L. and Hershberger,C.L. (1984) J. Bacteriol., 157, 314-317. Malpartida,F. and Hopwood,D.A. (1984) Nature, 309, 462-464. Malpartida,F. and Hopwood,D.A. (1986) Mol. Gen. Genet., 205, 66-73. Malpartida,F., Hallam,S., Kieser,H.M., Motamedi,H., Hutchinson,C.R., Butler,M.J., Sugden,D.A., Warren,M., McKillop,C., Baily,C.R., Humphreys,G.O. and Hopwood,D.A. (1987) Nature, 325, 818-821. Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Motamedi,H. and Hutchinson,R. (1987) Proc. Natl. Acad. Sci. USA, 84, 4445-4449. Msadek,T., Kunst,F., Henner,D., Klier,A., Rapoport,G. and Dedonder,.R. (1990) J. Bacteriol., 172, 824-834. Neal,R.J. and Chater,K.F. (1987) Gene, 58, 229-241. Pattabiraman,T.N. and Pogell,B.M. (1969) Biochim. Biophys. Acta, 182, 245 -247. Porter,J.N., Hewitt,R.I., Hesseltine,C.W., Krupka,C.W., Lowery,J.A., WallaceW.S., Bohonos,N. and Williams,J.H. (1952), Antibiot. Chemother., 2, 409-415. Ptashne,M. (1988) Nature, 335, 683-689. Rao,M.M., Rebello,P.F.and Pogell,B.M. (1969) J. Biol. Chem., 244, 112-118. Rao,R.N., Richardson,M.A. and Kuhstoss,S. (1987) Methods Enzymnol., 153, 166-198. Redshaw,P.A., McCann,P.A., Sankaran,L. and Pogell,B.M. (1976) J. Bacteriol., 125, 698-705. Redshaw,P.A., McCann,P.A., Pentella,M.A. and Pogell,B.M. (1979) J. Bacteriol., 137, 891-899. Sankaran,L. and Pogell,B.M. (1973) Nature, 245, 257-260. Sankaran,L. and Pogell,B.M. (1975) Antimicrob. Agents Chemother., 8, 721 -732. Seno,E.T. and Baltz,R.H. (1989) In Shapiro,S. (ed.), Regulationl of Secondary Metabolism in Actinomycetes. CRC, Boca Raton, FL, pp. 1-48. Sherman,D.H., Malpartida,F., Bibb,M.J., Kieser,H.M., Bibb,M.J. and Hopwood,D.A. (1989) EMBO J., 8, 2717-2725. Stock,J.B., Stock,A.M. and Mottonen,J.M. (1990) Nature, 344, 395-400. Suhadolnik,R.J. (1981) In Corcoran,J.W. (ed.), Antibiotics. Springer-Verlag, Berlin, Vol. IV, pp. 353-370. Takagi,M., Takada,H. and ImanakaT. (1990) J. Bacteriol, 172, 411-418. Vara,J., Malpartida,F., Hopwood,D.A. and Jimenez,A. (1985a) Gene, 33, 197-206. Vara,J., Perez-Gonzalez,J.A. and Jimenez,A. (1985b) Biochemistry, 24, 8074-8081. Vara,J.A., Pulido,D., Lacalle,R.A. and Jimenez,A. (1988) Gene, 69, 135-140. Vazquez,D. (1979) Inhibitors of Protein Biosynthesis. Springer Verlag, Berlin. Received on October 1, 1991; revised oni October 29, 1991
Cloning of the complete biosynthetic gene cluster for an aminonucleoside antibiotic, puromycin, and its regulated expression in heterologous hosts Rosa ALacalle, Jose A.Tercero and Antonio Jimenez1 Centro de Biologia Molecular (CSIC and UAM). Universidad Aut6noma, Canto Blanco, 28049 Madrid, Spain 'Corresponding author
Communicated by J.Davies
Puromycin, produced by Streptomyces alboniger, is a member of the large group of aminonucleoside antibiotics. The genes pac and dmpM, encoding a puromycin N-acetyl transferase and an O-demethyl puromycin O-methyltransferase, respectively, are tightly linked in the DNA of S.alboniger. The entire set of genes encoding the puromycin biosynthesis pathway was cloned by screening a gene library from S.alboniger, raised in the low copy number cosmid pKC505, with a DNA fragment containing pac and dmpM. Puromycin was identified by biochemical and physicochemical methods, including 'H NMR, in the producing transformants. This pathway was located in a single DNA fragment of 15 kb which included the resistance, structural and regulatory genes and was expressed when introduced into two heterologous hosts Streptomyces lividans and Streptomyces griseofuscus. In addition to pac and dmpM, two other genes have been identified in the pur cluster: pacHY, which determines an N-acetylpuromycin hydrolase and prgl, whose deduced amino acid sequence is significantly similar to that of degT, a Bacillus stearothermophilus pleiotropic regulatory gene. Key words: antibiotic genes/cosmid vectors/puromycin biosynthesis/regulation/Streptomyces
Introduction Nucleoside antibiotics constitute one of the largest group of microbial secondary metabolites (for a review, see Isono, 1988) and possess a wide range of biological activity. They act as agents against bacteria, viruses, insects, protozoans, mycelial fungi and yeasts, helminths, plants and tumour cells. In addition, immunosuppressing and immunostimulating activities have been reported. Therefore, nucleoside antibiotics are extremely useful in research and can be used as specific inhibitors of biochemical reactions since most metabolic pathways may be affected by one or more of these antibiotics, (as would be expected given the fundamental role played by nucleosides and nucleotides in the metabolism of living organisms and their viruses). Moreover, they provide examples of important therapeutic and phytopathological compounds, especially as antiviral (ribavarin or AraA), plant antifungal agents (mildiomycin) and herbicides (polyoxins). An important drawback in the therapeutic use of these compounds derives from their high toxicity. This problem (©) Oxford University Press
may be solved in some cases by structural modifications resulting in derivatives with lower toxicity. Another approach is expected to result from the application of recombinant DNA techniques. The pioneering work of Hopwood et al. (1985a) demonstrated the production of novel ('hybrid') antibiotics by gene transfer between Streptomyces strains producing different members of the same chemical class of compounds. These studies required the availability of the cloned genes, which, in principle, may be obtained by complementation of antibiotic non-producing mutants. Cloned genes, encoding specific enzymic activity from a well characterized cloned antibiotic biochemical pathway, can be used as probes to clone the corresponding genes from organisms producing chemically related antibiotics. The best example derives from the cloning of the whole set of genes for actinorhodin production (Malpartida and Hopwood, 1984) and the characterization of the DNA region complementing the actI mutation of S. coelicolor. This sequence contained part of the polyketide synthase (PKS) from S.coelicolor (Malpartida and Hopwood, 1986). By using act! as a probe (for a review, see Hopwood and Sherman, 1990), strongly hybridizing DNA sequences were found in most polyketide-producing streptomycetes, including the anthracyclins (adriamycin), macrolides (tylosine), milbemycin, polyethers (nonactin) as well as the isochromanequinone group (granaticin) of which actinorhodin is a member (Malpartida et al., 1987). Subsequently, the actI DNA region was employed to clone genes encoding the PKSs from the milbemycin-, tetracenomycin- and granaticin-producing Streptomyces (Malpartida et al., 1987; Bibb et al., 1989; Sherman et al., 1989) and to identify the corresponding genes from the oxytetracyclin-producing S. rimosus. Additionally, this strategy may permit the identification of cryptic genes for the biosynthesis of a particular class of antibiotics, as was shown by detecting the genes for nonactin biosynthesis in S.parvulus, a (normally) nonactin non-producing strain (Malpartida et al., 1987). In view of these findings, it is clear that recombinant DNA studies can provide fundamental data on secondary metabolite biosynthesis, which could be used to improve the biological activity of certain antibiotics. Puromycin (PUR) is an aminonucleoside antibiotic produced by S. alboniger. It specifically inhibits peptidyl transfer on both 70S and 80S ribosomes and has been widely used as a basic tool for studying protein synthesis (for reviews, see Vaizquez, 1979 and Cundliffe, 1981). The puromycin biochemical pathway is not well understood, although some data are available (Figure 1). It is known that adenosine is a direct precursor for the 3'-amino-3'-desoxyadenosine moiety of puromycin (Suhadolnik, 1981; Goodchild, 1982). In addition, two enzymes of this pathway, an O-demethyl puromycin O-methyltransferase (DMPM) and a puromycin N-acetyltransferase (PAC) have been characterized (Rao et al., 1969; Sankaran and Pogell, 1975; Vara et al., 1985b). Biochemical studies suggest that PAC
785
R.A.Lacalle, J.A.Tercero and A.Jimenez
inactivates the intermediate N6,N6,O-tridemethyl-puromycin by acetylation, and that DMPM methylates N-acetyl-Odemethyl-puromycin (AcODMP) to form N-acetylpuromycin (AcPUR) the last intermediate of the pathway (Vara et al., 1985b). Recently, an esterase activity which hydrolyses AcPUR to release PUR has been found in culture filtrates from S. alboniger (unpublished data). The expression of PAC and DMPM is strictly, and coordinately, regulated in S.alboniger (Sankaran and Pogell, 1975; Vara et al., 1985b). The genes pac (encoding PAC) and dmpM (encoding DMPM) have been cloned and sequenced and are transcribed on the same mRNA (Lacalle, 1990; Lacalle et al., 1989, 1991). In antibiotic-producing organisms, it has been found that the genes for self-resistance are often located within the biosynthetic gene clusters (for a review, see Seno and Baltz, 1989). Therefore, the finding that PAC and DMPM are members of the same operon suggests that other, and possibly all the genes for the puromycin biosynthesis pathway could be linked. This report describes the cloning of the genes of the puromycin biosynthesis pathway in a discrete and single DNA segment and its regulated expression in heterologous hosts. To our knowledge this is the first report of cloning the gene cluster required for the complete biosynthesis of a nucleoside antibiotic.
S.griseofuscus were then transformed with DNA from 15 of the isolated plasmids and culture filtrates from the resulting transformants were examined for the presence of material(s) acting as PAC substrate. Table I records the results of the six most relevant plasmids. It was subsequently proved that this material is indeed puromycin, and will be described as such hereafter. Therefore, puromycin is produced, in addition to others, by Streptomyces transformants containing either pPB4.34, 5.13, 11.37 or 11.40 (Figure 2A and Table I). In general, S. lividans and S. griseofuscus transformants produced about 10- to 20- and 50-fold less puromycin respectively, than did S.alboniger (Table I). Plasmid DNA could not be detected in the puromycinproducing and non-producing Streptomyces transformants by either agarose gel electrophoresis or CsCl gradients (unpublished data). A similar situation was found previously with other recombinant plasmids derived from pKC505 (Rao et al., 1987). However, their existence was proved by transforming E. coli with total DNA from the relevant Streptomyces clones and examining the restriction maps of the resulting plasmids, which were identical to those of the original E. coli isolates. In addition, Southern blot hybridization indicated that the structure of the plasmid inserts corresponded to that present in the S. alboniger DNA (unpublished data). S. lividans and S. griseofuscus were then retransformed with total DNA from the puromycinproducing clones. About 95 % of the resulting transformants
Results Cloning of the pur cluster From a S.alboniger gene library, constructed in the low copy number cosmid pKC505, 20 000 Escheria coli clones were screened with a 32P-labelled probe, which contained both pac and dmpM genes. A total of 27 positive clones was isolated. The restriction map of the recombinant plasmids from all these clones were determined, and that of the six most relevant is presented in Figure 2A. Both S. lividans and H\ N /H
produced puromycin. A comparison of the restriction maps of puromycinproducing and non-producing plasmids indicated that puromycin biosynthesis (pur cluster) was encoded by a single DNA fragment of 15 kb (Figure 2A). A detailed study of the restriction map of pPB5. 13 showed the presence of a 18 kb SpeI-XbaI DNA fragment which should contain the whole pur cluster (Figure 2). This sequence was subcloned
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(1985b).
786
Puromycin biosynthesis gene cluster
from pPB5. 13, via 'Bluescript' plasmid and E.coli, in cosmid pJAR4. The resulting construct (pCXS; Figure 2A) directed the synthesis of puromycin in S. lividans and S.griseofiuscus, as shown by PAC (Table I), TLC and HPLC assays (see below), in similar amounts to those produced by pPB4.34. Table 1. Puromycin biosynthesis by several Streptomvces
Plasmid
Puromycin production (in itg/ml) S. griseofuscus S. lividans 0.0 0.0 0.0 15
None pKC505 pPB2.49 pPB4.34 pPB4.6 pPB5.13 pPBl 1.37 pPBl 1.40 pCXS
S. alboniger
0.0 0.0 0.0 3 0.0 2 4 2 3
0.0 13 10 10 8
150 -
-
Streptomyces transformants containing the indicated constructs and S.alboniger were grown in S medium. Culture filtrates were then collected at 40 h (S.alboniger) and 50 h (clones) of fermentation and extracted with chloroform. PUR was quantified in these extracts by the PAC assay. It should be noted that the indicated data do not accurately represent the maximal levels of PUR production due to the sharp peak of the PUR biosynthesis levels during the growth curve.
Identification of puromycin Chloroform extracts of culture filtrates from S. alboniger and the other Streptomyces clones were examined by the TLC systems indicated in Materials and methods. In all clones giving a PAC-positive reaction, a spot moving with an Rf identical to that of puromycin was seen. In addition, samples derived from S.alboniger and the Streptomyces clones contained traces of a compound migrating as AcPUR. Neither were detected in either S. lividans (pKC5O5), S.griseofiuscus (pKC5O5), S. lividans (pJAR4), S.griseofiiscus (pJAR4) or transformants whose culture filtrates gave a PAC-negative reaction. When the chloroform extracts were used as substrates for a PAC assay in the presence of [3H]acetyl-coenzyme A and the reaction products were examined by TLC, a radioactive spot migrating as AcPUR was detected only in PAC-positive samples (unpublished data). In addition, the chloroform extracts were also analysed by HPLC, and the resulting profiles from presumed puromycin-containing samples had a component with retention time identical to that of puromycin. As an example, Figure 3 presents the profiles of samples from S. lividans (pPB4.34) and (pKC5O5) and S.alboniger. These results suggest that puromycin was produced by the relevant Streptomyces strains. The antimicrobial activity of the relevant culture filtrates was that expected for puromycin. Finally, to confirm that puromycin biosynthesis takes place
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Fig. 2. (A) Restriction maps of Salboniger DNA inserts on Streptomvces cosmids pKC505 and pJAR4. The single XbaI and Spel restriction sites of pPB5.13 are present also in pPB4.6. However, pPB4.6 contains some extra, unmapped, XbaI restriction sites in the left hand region. The presence of XbaI and SpeI sites was not examined in the other plasmids. + and indicate that PAC activity was detected or undetected in chloroform extracts from the relevant culture filtrates, respectively. (B) Organization of the identified puromycin biosynthetic genes. -
787
R.A.Lacalle, J.A.Tercero and A.Jim6nez
in S. lividans (pPB4.34), puromycin was purified from culture filtrates of this clone as indicated in Materials and methods. 1H NMR spectra of the final product were indistinguishable from that of authentic puromycin (unpublished data). We conclude, therefore, that the biochemical pathway for the biosynthesis of puromycin is contained in pPB4.34 and therefore in a 15 kb DNA fragment defined by the restriction maps of several puromycin-producing plasmids (Figure 2). Regulation of puromycin biosynthesis In S.alboniger, the expression of both PAC and DMPM is strictly growth regulated. It commences at mid- to lateexponential growth and reaches a maximum at the entrance of the stationary phase, to decay drastically during this stage to the growth curve (Rao et al., 1969; Vara et al., 1985b). These findings suggest that the genetic system of the biochemical pathway for puromycin production from S. alboniger includes functions which regulate the expression of the biosynthetic genes. To examine if the pur cluster
S. lividans pKC505
Ec
ltLO
cm
w 0 z
alboniger ~~~~~~~~~~~~~~S.
CD
0 CD
S. fividans pPB4.34
0
5
10o
15
20
25
30
35
40
50
45
RETENTION TIME (minutes)
Fig. 3. HPLC of chloroform extracts from culture filtrates of several Streptomyces. Samples were processed as indicated under Materials and methods.
included such regulatory gene(s), the biosynthesis of PAC, DMPM and puromycin was followed during the growth curve in liquid cultures of both S. lividans (pPB4.34) and S.alboniger. The results of this experiment (Figure 4) indicated that puromycin biosynthesis and the expression of both PAC and DMPM activities are regulated in a similar way in both S.alboniger and the transformant S. lividans (pPB4.34). Interestingly, whereas the specific activities of PAC and DMPM in the Streptomyces clones are similar or higher than in S.alboniger, puromycin production is 10-fold lower than in the parental strain (Figure 4). Nucleotide sequence analysis has shown the presence of one potential regulatory gene (prgl) of 1287 nt in the cloned pur cluster (Figure 2B). The amino-terminal of its deduced amino acid sequence (PRG1) presents significant similarity to the corresponding regions of the gene products from degT, a pleiotropic regulatory gene of Bacillus stearothermophilus (Takagi et al., 1990), and the eryCl and orfl0.4 genes of the erythromycin biosynthesis pathway from Saccharopolyspora erythraea (Dhillon et al., 1989) and the rjb cluster of Salmonella typhimurium (Jiang et al., 1991), respectively. In contrast, whereas the carboxy-terminus of DEGT, ERYC1 and ORF 10.4 are homologous, they differ totally from that of PRG1 (Figures 5 and 6). The central region (amino acids 198 -220) of PRG1 has a helix-turn-helix structure, deduced from amino acid sequence similarities with putative DNA-binding domains of DEGT and X Cro-like DNA binding domains (Figure 7). By applying the Dodd -Egan method for predicting these structures, the score (0.0) of PRG1 (Figure 7) is too low for it to be considered to be involved in DNA binding. However, other putative helix-turn-helix domains from proteins which bind to DNA, such as LEXA and SV40 large T antigen, give very low or negative scores from the Dodd-Egan matrix (Figure 7; Dodd and Egan, 1990). In addition, the amino acids at positions 394-407 at the carboxy end of PRG1 (DWPEDVALIEAYAE) could form an amphipathic a-helix structure formed of negatively charged residues (D or E) separated by either two or four hydrophobic amino acids. This structure, present in DEGT (Takagi et al., 1990), is considered to be a consensus region for gene activation present in transcription activator proteins (Ptashne, 1988). Taken together, these considerations suggest that prgl is a regulatory gene in the pur cluster. -
B
A 70
10
aT) -60
.5
0.
_50 -a
E
_40 Z C)
0
a.I 0
-30 02 _20 X
C)
-
TIME (HOURS)
E
-6
z 0
1
z
E
w
a -J
E
0
0.
0
4 0
0,1. 0
_2
-10
w
0.
U)
8
-
E
~3
CO) z w 0
a.
CO
-o
_ O
TIME (HOURS)
Fig. 4. Expression of PAC and DMPM and biosynthesis of puromycin during cell growth. S.alboniger (A) and S.lividans (pPB5.13) (B) were grown in S medium at 30°C. At the indicated times, samples were taken to determine optical density and then filtered. Cell extracts were used to assay PAC and DMPM activities. Chloroform extracts from the filtrates were used to quantify puromycin by the PAC assay. Similar results were obtained by examining the antibacterial activity of these extracts. 0: optical density 660 nm; O: PAC activity; 0 DMPM activity; A: puromycin production.
788
Puromycin biosynthesis
al.
(1979). These plasmids
most probably linear plasmids which would be positions shown in Figure 8.
Genomic location of the pur cluster
et
Streptomyces, the gene clusters for antibiotic biosynthesis pathways are usually located on the chromosome. A well known exception is the methylenomycin cluster, which is found on the giant linear plasmid SCPI from S. coelicolor
molecules and not small circular
In
(Chater
S.alboniger,
1985;
Bruton,
and
Sankaran and
Kinashi
et
Pogell (1973)
al.,
1987).
expected
to
migrate
those
to
are
Indeed, such circular elements have
gel electrophoresis (unpublished data).
agarose
In
by curing determining
gene cluster
been detected in
not
of total DNA from S.
alboniger
deduced
studies with ethidium bromide that the genes
Discussion
production were not plasmid encoded. Other studies provided evidence suggesting the existence, in S. alboniger, of an extrachromosomal element implicated in the formation of aerial mycelium (Redshaw et al., 1976, 1979). The availability of the pur cluster and the CHEF technique for large plasmid identification has allowed the re-examination of these questions. Total DNA from S.alboniger was submitted to CHEF electrophoresis, transferred to a nylon membrane and hybridized to the 18 kb Spel Xbal fragment containing the pur cluster. The results suggested that this cluster is present in the chromosomal DNA of S. alboniger (Figure 8), in agreement with the findings from Sankaran and Pogeil (1 973). However, the possibility of the pur cluster being located in a giant circular plasmid cannot yet be completely eliminated. Two fragments migrating at positions 1000 and 2000 kb can be clearly seen in Figure 8. It is not yet known whether PUR
these represent monomeric and dimeric forms of the
plasmid
two
or
different elements,
Complementation of antibiotic non-producing mutants has successfully used to clone the whole biosynthesis gene clusters for several antibiotics, including actinorhodin (Malpartida and Hopwood, 1984), tetracenomycin (Motamedi and Hutchinson, 1987) undecylprodigiosin (Feitelson et al., 1985) and oxytetracycline (Binnie et al., 1989). Because puromycin non-producing mutants from S. alboniger were not available, we used a DNA fragment containing the biosynthetic genes pac and dmpM as probe to search for the puromycin gene cluster in a genomic library raised in cosmid pKC5O5 (Rao et al., 1987). Of these two genes, pac was known to determine puromycin resistance (Vara et al., 1985a) and dmpM an O-demethylpuromycin O-methyltransferase (Vara et al., 1988). Consequently, the complete set of genes from S. alboniger for puromycin production have been cloned and then located in a single DNA fragment of 15 kb which was expressed in two heterologous hosts, S. lividans and S.griseofuscus. This been
same
the different
although
discrete
intensities of the signals in lines 2 and 3 suggest the former possibility. One of these plasmids may encode the aerial mycelium function(s) suggested by the work of Redshaw
0
100
300
200
0
300
sequence
0
must
carry
200
100
400
31300
31100
EL
/2(?00
/
21!00
/
00
100
0
0
ERYCl
DEGT
0
the
400
300
/
4100
structural,
the
regulatory elements of the pathway, regulation of puromycin production and the the
and
because the
200
100
DNA
resistance
100
200
300
0
l90
290
ORFi 0.4
390,
400
0
100
200
300
.400
300 3/300
WL
20
)o
V2(200
0
Ur-
200
0
/~~~~~~~~ 300
Ic
~~~~~~~~100
10,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 0~~~~~ 0
-
ERYCl Fig. 5. COMPARE/DOTPLOT analysis of the deduced amino was used at a stringency of 16 (Devereux et al., 1984).
ERYCl
ORFi 0.4
acid sequences of
PRGI,
DEGT,
ERYCI1
and
ORFIO.4.
A window size of 30 amino
acids
789
R.A.Lacalle, J.A.Tercero and A.Jimenez PRG1 DEGT ERYCl
ORF10 .4 PRG1 DEGT
51
GQ
35 35 61
11-
PRG1
163 145 144 ORF10.4 179
DEGT ERYCl
222 205 204 ORF10 .4 238
DEGT ERYCl
.
AEFAAY
JA-TSG g eV___ GVGDEVI T T GSS
LAL
KPGDEVI
SPKLGE
103 85
ERYCl 85 ORF10 .4 119
PRG1
&-TI-------NgLGTFs.61-.,bkIC.-' E.. ,x
V P GAPESDDPALVI E tTI LEVRLYGHPADLAm
rFIX EL
4VAERYG
F
GTFGD
EDR
Q
GGILVD
FIAHGI IEDAIQ(Y¢KGYTYSFFNLGAGGMII Ufl
S-F-- ft-Ek .jp..b 46givK!t..ULIYPII' I --
hIADRHGI
LVED
QAV
RG
- - - -
a Lr-%,PL,r-lnIll rv LAMWO-111 f -J l'R IF'.v WV F
TLIECL
M
-I
"Iolpi
M.,
g6TTTj4 EKtVIFI HKPK KKI DF~~
CYCAPGC
Fig. 6. Alignment of the deduced amino acid sequences of PRG1. DEGT, ERYCI and ORF10.4. Alignment was derived from the BESTFIT and PILEUP programs (Devereux et al., 1984). Bold letters represent identical residues. These residues and conservative replacements are boxed.
expression of the genes pac and dmpM in the relevant S. lividans transformant were similar to those of the producing organism. The demonstration of puromycin production in clones from two independent Streptomyces species eliminates the possibility of either activation or complementation of cryptic genes in the hosts. The gene pac, which inactivates puromycin by Nacetylation is the presumptive resistance determinant in S.alboniger (Vara et al., 1985a), although it also appears to be a key enzyme in directing the puromycin biosynthesis pathway through the correct inactivated intermediates (Vara et al., 1985b). This gene is located close to the end of the pur cluster, but within the pur biosynthetic genes, similar to other resistance determinants identified within biosynthetic genes in antibiotic-producing Streptomyces (Seno and Baltz, 1989). Whether pac is the only resistance determinant is not known, although existing evidence is in favour of this supposition (Vara et al., 1985a). Nonetheless, S.alboniger failed to incorporate [3H]puromycin from the culture medium whereas its ribosomes are fully sensitive to the antibiotic (unpublished data) and the presence of a transmembrane-bound protein involved in establishing a puromycin permeability barrier cannot be eliminated. Such is the case for both S. rimosus and S. coelicolor, the oxytetracycline and methylenomycin producers, respectively, where genes for resistance apparently encode specific membrane proteins whose function is secretion of these antibiotics (Neal and Chater, 1987; Butler et al., 1989). A similar protein is likely to be required for the export of N-acetylpuromycin in S.alboniger. This was inferred from the detection of traces of N-acetylpuromycin and a hydrolase activity, which converts the latter intermediate into puromycin in the culture medium of the producing strain. This enzyme has also been detected in culture filtrates of all puromycin-producing clones reported in this work (unpublished data), allowing the relevant gene (pacHY) to be located in the pur cluster (Figure 2B). Sequencing work in progress has detected a gene (prgl ), located upstream of pac, whose deduced amino acid sequence suggests an involvement in gene regulation. Thus, its amino terminus is highly homologous to that of degT, a pleiotropic regulatory gene from B.stearothermophilus. The degTgene, similarly to the regulatory degSU, degQ and degR genes from Bacillus subtilis (Msadek et al., 1990) confers pleiotropic effects on this bacterium, including altered control of sporulation, filamentous cells and enhancement of extracellular enzyme production. It was suggested 790
*
*
*
Score
*
PRG1
SL
Q A N K A V Y A G E G G I L V T D D A L
DEGT
AA FG LS LN IR
Q Q Q Q Q
X x
cro cI
434 CI P22 C2
A T E A A
I K S E A
G A K Y N G T A K D L G V A D K M G L A Q K V G L G K M V G
K V M T V
C Y G T S
V G E L G T A A Q S A I N K A I Q S G V G A L F Q Q S I E Q L E N V A I S Q W Q
0
T H N N R
.0
-3. 1 4.0 4.6 5.6 6.1
Fig. 7. Possible helix-turn-helix domain in the PRG1 protein. Amino acid sequence similarities were found with some DNA-binding domains (Dodd and Egan, 1990; Tagaki et al., 1990). The asterisk indicates the conserved amino acids. The indicated scores correspond to predictions of DNA-binding motifs by using the weight matrix of Dodd and Egan (1990).
that DEGT functions as a sensor protein for environmental stimuli and also as a regulator to activate or repress transcription of target genes (Takagi et al., 1990). If this holds true for DEGT, then given the sequence similarities, we suggest that PRG1 is a transcriptional regulator of the pur cluster. The similarities in sequence of DEGT and PRG1 with ERYC 1 (an enzymatic/regulatory protein of the erythromycin biosynthesis pathway; Dhillon et al., 1989) and ORF10.4 (a putative protein of the rbf cluster of S. typhimurium; Jiang et al., 1991) may imply a regulatory function to the genes eryC1 and orfl0.4. As proposed for degT, the prgl gene (and by extension eryC1 and orfl0.4 in their particular genetic systems) might be the effector gene for transcriptional regulation of a two-component system of a signal transductional pathway (Stock et al., 1990; Msadeck et al., 1990) to mediate gene expression from the pur cluster. Puromycin biosynthesis in the heterologous transformants was 10- to 50-fold lower than in S.alboniger. A similar result was found in cloning the pathway genes for oxytetracycline (Binnie et al., 1989) and cephamycin C (Chen et al., 1988). Several factors may account for these results, including biomass production (S.alboniger cultures reached a 9-fold higher cell density than S. lividans clones) and plasmid copy number. In addition, the supply of primary metabolites in the Streptomyces clones may limit the production of puromycin. This is consistent with the finding that production of S.griseofuscus is 5-fold lower than in S. lividans. The biosynthesis of PAC and DMPM does not seem to be limiting, because their rates of expression in the S. lividans transformants are higher than in S. alboniger. Characterization of all the genes involved in puromycin biosynthesis will be useful in detecting homologous genes from other actinomycetes, to identify biosynthetic clusters for other -
-
Puromycin biosynthesis gene cluster
MgSO4) was employed to study PUR production. In this case, mycelia Qz
12 1
%
cr
3 4
1
1
2 1
4
3 1
1
1
__.
2200,1600 1125-
1020 945
850 800
-
-
-
770 -700
-
630580 460
-
Fig. 8. Genomic locations of the pur cluster in S.alboniger. Total DNA from S.alboniger was developed by CHEF. The gel was blotted on a nylon membrane, which was subsequently probed with a 18 kb Xbal-SpeI DNA fragment containing the pur cluster. Left panel is a photograph of the gel stained with ethidium bromide. Right panel is the X-ray film of the Southern blot. DNA on lanes 2 and 3 was obtained from independent S.alboniger cultures. Numbers to the left indicate the size (in kb) of the Saccharoinvces cerevisiae chromosomal DNA markers. Numbers to the right denote the size (in kb) of plasmid DNAs. which are identified by arrows.
nucleoside antibiotics and, subsequently, production of novel 'hybrid' antibiotics.
Materials and methods Strains, vectors, media and DNA methodology S.alboniger ATCC 12461. the puromycin producer (Porter et e/l.. 1952). S. /ividans 66 (1326) (Hopwood t cil. 1985b), S. griseofiiscus (Larson and Hershberger. 1984) and E.coli strains DH5 (Hanahan, 1985). HBIOI (Bolivar and Backman. 1979) and AM6 (Vara et al.. 1985a) are described in the indicated references. Strcptomnvces-E.coli shuttle cosmid pKC505 (Rao et Cl/.. 1987) was provided by Dr R.H.Baltz (Eli Lilly. Indianapolis. USA). Streptotns'ces-E.coli cosmid pJAR4 is similar to pKC505 with hygromycin B resistance instead of apramycin resistance (unpublished data). StreptomYces plasmids were pFV8 and pFV I (Vara eti/l.. 1985a). E. ('li plassmids were 'Bluescript' SK and pVN7. 1 (a pUC 19 derivatives containing the genes pac1 and tdnmpM from S.al/boniger: Vara et al.. 1988). Plasmid DNA from E. coli and Strcptomoi\ves was prepared as described by Hopwood ct (1985b). Unless otherw\ise specified. Streptomyccs were grown in YEME liquid medium supplemented with 34% sucrose and 5 mM MgSO4 (Hopwvood et (i/.. 1985b). Medium S (2 % starch, 0.4% Bacto-peptone. 0.4 % yeast extract, 0.4% K.HPO4, 0.2% KH2PO4, 0.02% CoCI, and 2 mM a/.
were dispersed by introducing a stainless steel spring in baffled flasks. To prepare protoplasts from Streptomyces, YEME medium was supplemented as above and with either 0.5% or 1% glycine for S.lividans and S. griseofiiscus, respectively. Solid medium for S. lividans and its transformants was R5 (Hopwood et al., 1985b) and that for S.alboniger and S.griseofiJscus was S medium solidified with 2% agar. S.lividans and S.griseofiscus protoplasts were transformed as described by Hopwood et al. (1985b) and Larson and Hershberger (1984), respectively. When required. antibiotics were added at the following concentrations: 10 and 25 yg/ml thiostreptone for liquid and solid media, respectively; 25 and 100 itg/ml apramycin for Streptomyces and E.coli, respectively; 200 and 50 yg/ml hygromycin B for Streptomnvces, and E.coli, respectively, and 100 ,ug/ml ampicillin. Antimicrobial activity of chloroform extracts of culture filtrates was determined by the paper disc method using a variety of bacteria as testing organisms. Transformation of E.coli and Streptoinvces was performed according to Hopwood et al. (1985b). A S.alboniger gene library was prepared in cosmid pKC505 as described by Rao et al. (1987). Essentially, total DNA from S.albonziger was partially digested with Sau3A and the resulting 20-40 kb DNA fragments were collected from sucrose gradients and then dephosphorylated. To prepare cosmid arms, pKC505 was cut with HpaI, dephosphorylated and then digested with BamnHI. These arms were then ligated to the S.alboniger 20-40 kb DNA fragments. Packaging of this ligation mixture was performed by using the X inz vitro packaging kit from Amersham as described by the supplier. Packaged recombinant cosmids were transfected into E. coli HB101. A total of 150 000 apramycin resistant clones were obtained, at a frequency of 1.7 x 105 colonies per DNA tg. Southern blot hybridizations were carried out as described by Maniatis et al. (1982). Labelling of probes with [Q-32P]dCTP was achieved by the 'random primers' method of Feinberg and Vogelstein (1983). Colony hybridization was accomplished as reported by Grunstein and Hogness (1975). Pulsed field gel electrophoresis was performed by contour clamped homogeneous electric fields (CHEF), using an LKB-2015 Pulsaphor equipment (Chu et al., 1986). Total DNA from S.alboniger (Kinashi et al., 1987), and the Saccharomvces cerevisiae control DNA (Bio-Rad) were separated on a 15 cm square, 0.8% agarose gel at 14°C in 0.5 x TBE buffer (1 x TBE: 0.089 M Tris-borate, 0.089 M boric acid, 0.002 M EDTA, pH 8.0). The electrophoresis conditions were: pulse time of 120 s for 87 h, constant voltage at 150 V throughout. After electrophoresis, the gel was processed as described by Kinashi et al. (1987).
Enzyme assays PAC and DMPM activities were determined radiochemically by using either [3H]acetyl-coenzyme A or [35S]adenosyl-methionine. respectively, as cosubstrates, as described by Vara et al. (1985b, 1988). As a source of PAC activity, log phase cells from a S. lividans transformant containing plasmid pFV8 (Vara et al., 1985b) were broken by sonication on ice, centrifuged in the cold for 15 min at 15 000 r.p.m. in a microfuge and the resulting supernatant was then collected and kept at -70°C until use (Vara et al.. 1985b). To quantify PAC and DMPM activities from Streptomynces transformants. cell extracts were prepared similarly from mycelia collected at different times during cell growth in S medium. One unit of PAC activity corresponds to 1 tmol of AcPUR synthesized per min per mg of protein. One unit of DMPM activity corresponds to 1 nmol of AcODMP synthesized per min per mg of protein. Protein concentration was estimated by the method of Bradford (1976). Puromycin assays In general, PUR or PUR-like material was extracted from culture filtrates at pH 11.0 by three successive chloroform extractions in a rotary shaker (Vara et al., 1985b). The combined fractions were then evaporated to dryness and dissolved in water. PUR or PUR-like material was then identified and/or quantified by the PAC enzymic assay. This system allows an accurate estimation of puromycin at concentrations as low as 0.5 /g/ml. In addition, it is extremely specific for the puromycin structural backbone (Vara et al., 1985b). Physicochemical identification of AcPUR, AcODMP, ODMP (all known PUR precursors) and PUR was unambiguously carried out by either TLC or HPLC. The TLC systems were: I[ethyl acetate-methanol (3:1)] and II [acetonitrile-0. 1 M ammonium acetate (5:95)], both on silica gel 60 F,54 (Kodak) and III [acetonitrile -0.1 M ammonium acetate (20:80)] and IV [butanol acetic acid-water (12:3:5)] both on cellulose F,54 (Merck, Darmstadt). The Rf values of PUR, AcPUR, ODMP and AcODMP were, rcspectively, 0.55, 0.82 0.5and 0.80.21 0.3, 0.27 and 0.49: 0.41, 0.62, 0.49 and 0.85 and 0.78, 1, 0.69 and 0.96 for systems l, II, III and IV, respectively. These compounds were detected by exposing to UV light. If [ 3H]acetyl derivatives were studied, chromatograms were cut into 0.5 cm
791
R.A.Lacalle, J.A.Tercero and A.Jimenez pieces, which were then quantified in a liquid scintillation spectrometer (Vara et al., 1985b). HPLC was performed on spectra Physics SP8700 equipment and a Spectra Physics SP8440 254/280 nm detector. Chromatography was performed through a reversed phase of C18 column (5 pm, 250 mm x 4 mm. Lichrosorbe Merck, Darmstadt). Solvents used were A (0.1 M ammonium acetate, pH 6.0) and B (0.2% trifluoroacetic acid in acetonitrile). Gradient conditions were 80-60% A in 30 min; 60-40% A in 15 min; 80-20% A in 10 min, at a flow rate of 0.5 ml per min. Detection was by UV absorption at 254 nm. Under these conditions the retention times of ODMP, AcODMP, PUR and AcPUR were 13.3, 17.4, 23 and 27 min, respectively. Puromycin purification Filtrates from a 5 1 culture (60 h) of S.lividans (pPB4.34) were collected and extracted twice with 1 I chloroform each. Extracts were combined and concentrated up to 0.3 ml by vacuum. This sample was developed by preparative TLC on five plates (2 mm thick) by using system I (see above). Bands migrating as PUR were removed, combined and then extracted with 30 ml of chloroform:methanol (1:9). The extract was taken to dryness under vacuum and then dissolved in 0.1 ml water. Samples (25 Al) of this solution were subjected to HPLC as described above. Fractions eluting with a PUR retention time were collected and then combined. These steps were repeated three times for each sample. Finally, the relevant fractions were combined and lyophilized. The final product (1.3 mg) was dissolved in D2O pH 3.0 to perform 'H NMR spectroscopy. 1H NMR studies High resolution 'H NMR spectra at 360 MHz were obtained on a Bruker WM-360 spectrometer at a probe temperature of 20°C on D20. Conditions were: 8 As pulse width (900 flip angle), 4000 Hz spectral width, 16 K data points, 3 s recycle time and 256 scans. Intensity of the water signal was reduced when necessary with the help of a 0.5 s presaturation pulse.
Acknowledaements We are indebted to Dr R.H.Baltz (Eli Lilly) for the gifts of cosmid pKC505 and S.griseoftuscus, to Prof. M.Rico for his help with 'H NMR analysis, to Prof. Julian E.Davis for critically correcting the manuscript and to Mrs Asunci6n Martin for expert technical assistance. This research was supported by grants from the CAICYT (No. 613/615), Smith Kline and French and by an institutional grant from Fundacion Ram6n Areces to the Centro de Biologia Molecular.
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