FEMS Microbiology Letters 115 (1994) 119-124 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier

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FEMSLE 05756

Characterization of yeast DNA sequences of directing transcription in Streptomyces and Escherichia coli

capable

Miguel A. Alvarez, Rosaura Rodicio, M. Cruz Martin, Luis A. Diaz 1 and M. Rosario Rodicio *

Departamento de Biolog[a Funcional, Universidad de Oviedo, 33006 Oviedo, Spain

(Received 3 September 1993; revision received 15 October 1993; accepted 22 October 1993)

Abstract: Random genomic DNA fragments from Saccharomyces cerevisiae were tested for their ability to activate transcription of a promoterless aminoglycoside phosphotransferase-encoding gene in Streptomyces. About 10% of the insertions led to kanamycin resistance when selected at low concentration (5 /zg ml- t ) . The nucleotide sequences of five insertions that allowed growth at different concentrations of the antibiotic were determined. Three of them contained - 10 and - 3 5 consensus sequences for the major class of eubacterial promoters. In two others, a - 10 sequence could be identified, but a - 35 element was absent at the appropriate distance. All of the five inserts were also transcriptionally active in Escherichia coli and therefore probably belong to the major class of eubacterial promoters. Three of the characterized insertions found to match known yeast sequences did not derive from promoter regions. We conclude that sequences that function as eubacterial promoters occur at random in the yeast genome.

Key words: Streptomyces; Saccharomyces cerevisiae; Promoter; Promoter-probe vector; Transcription

Introduction

Functional expression of Saccharomyces (Sa.) cerevisiae genes in Escherichia coli has been achieved frequently [1]. Occasionally, expression of the eukaryotic gene has relied on prokaryotic promoter elements present in the adjacent vector

* Corresponding author. Tel.: (085) 103652; Fax: (085) 103534. i Present address: FYSE, Departamento de Investigaci6n Mi-

crobiol6gica, Paseo del Deleite sn., 18300 Aranjuez, Madrid, Spain.

DNA. However, in a number of cases, the 5'-non- coding sequences of the yeast gene were shown to contain promoter elements functional in E. coli, coexisting with the elements controlling expres- sion in Saccharomyces [2-5]. In addition, cross- recognition of the 5'-noncoding region of the Sa. cerevisiae GALIO gene by yeast RNA polymerase II, yeast mitochondrial RNA polymerase and E. coli RNA polymerase has been reported [6].

Apart from expression of yeast genes in E. coli, functional expression of the TRP1 gene of Sa. cerevisiae can be achieved in Gram-positive eubacteria of the genus Streptomyces (M.A. A1-

SSDI 0 3 7 8 - 1 0 9 7 ( 9 3 ) E 0 4 4 8 - L

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varez, unpublished results). It has been shown that only a minority of Streptomyces promoters can function in E. coli. They are similar to the major class of eubacterial promoters, recognized by cr70-1ike factors. The consensus sequences proposed for the - 3 5 and - 10 regions of these promoters (TTGACPu and TAGPuPuT, respec- tively; [7]), differ slightly from those accepted for E. coli promoters (TTGACA and TATAAT; [8]). However, most of the characterized Streptomyces promoters are not active in E. coli and their sequences do not conform to the hexameric ele- ments described above [7].

As transcriptional initiation signals in prokary- otes and eukaryotes are different, the existence of yeast sequences that show promoter activity in eubacteria may be fortuitous. Alternatively, recognition sequences for an ancestral ~r factor may have been conserved through evolution, as suggested by the similarity that exists between the - 1 0 region of typical eubacterial promoters and the TATA-Iike elements that are recognized by eukaryotic transcription initiation factors (includ- ing TFIID from Sa. cerevisiae; [9]). In the present work we have tested these hypotheses by investi- gating DNA sequences able to direct transcrip- tion in Streptomyces, that exist in the genome of Sa. cerevisiae. The number of genes already se- quenced from Sa. cerevisiae (a project to deter- mine the complete genomic nucleotide sequence of this organism is in progress; [10]) facilitated the identification of the origin of randomly cloned DNA fragments.

Materials and Methods

Microorganisms and culture conditions The following strains were used in this work:

Streptomyces (St.) lividans 1326 [11], Sa. cerevisiae RMI-1 c (MATa, leul, trpl, MALI-lC; [12] and E. coli JM103 [13]. Standard media and culture con- ditions for Streptomyces were as in [11] and for E. coli as described [13].

DNA isolation, manipulation and characterization Plasmids plJ486 [14] and pBR322 [13], as well

as the M13 derivatives mpl8 and mpl9 [15] were

used. Genomic DNA from Saccharomyces was isolated according to [12]. Techniques for trans- formation and DNA manipulation were as previ- ously described [11,13]. Computer-aided data-base searching was carried out using the University of Wisconsin Genetics Computer Group programs package.

Results and Discussion

Transcriptional fusions Random DNA segments of Sa. cerevisiae were

tested for their ability to activate transcription of the promoterless aphH gene, present in the Streptomyces vector pIJ486. Expression of the gene confers resistance to kanamycin and other aminoglycoside antibiotics. A transcriptional ter- minator, from E. coli phage fd, is located up- stream of the aphH coding region to prevent transcriptional read-through from vector promot- ers. A polylinker sequence, downstream of this terminator, facilitates insertion and recovery of DNA fragments. To generate a yeast genomic library in pIJ486, 4 /~g of DNA obtained from Sa. cerevisiae were digested with Sau3A1 (8 units, 6 min, 37°C) and ligated to 1 ~g of vector DNA, digested with BamHI and dephosphorylated by treatment with calf intestinal alkaline phos- phatase. After transformation of St. lividans pro- toplasts, about 50 000 thiostrepton-resistant colonies were obtained (the tsr gene, conferring resistance to thiostrepton is the selectable marker present in pIJ486). To determine the frequency and size of insertions obtained, plasmids were isolated from 23 independent transformants and digested with BglII, sites for which are present at both ends of the polylinker of pIJ486, flanking the unique BamHI site used for cloning. Analysis by electrophoresis on 1.5% agarose gels showed that 14 out of 23, i.e. 60.8% of the transformants, contained insertions ranging in size from approx. 100-1000 bp.

Once the presence of insertions was estab- lished, serial dilutions of a spore suspension de- rived from the transformants were plated in par- allel onto MMT solid medium with and without 5 /~g m1-1 of kanamycin. 6.5% of the colonies

turned out to be resistant to the antibiotic. Plas- mid DNA was extracted from 25 resistant clones, digested with BgllI, and analyzed by elec- trophoresis on agarose gels. Twenty-three of these plasmids contained DNA insertions (the remain- ing two may have inserts too small to be detected).

The experiments described above strongly sug- gest the existence of Sa. cerevisiae sequences which are transcriptionally active in Streptomyces. These sequences, designated as YSP (yeast DNA fragments functioning as Streptomyces pro- moters), are relatively abundant. Thus, about ]0% of the colonies with recombinant plasmids (calculated considering that the frequency of in- serts in the genomic library was 60.8%, see above) were resistant to at least 5/zg ml-1 of kanamycin. However, this value is lower than that obtained by Kieser et al. [16] testing a library of BgllI fragments of St. lividans DNA for their ability to express the aphlI gene of plJ486 in the homolo- gous host. There, a frequency of about 78% of promoter-active recombinants was reported.

Quantitative analysis of the promoter strengths The level of kanamycin resistance conferred by

plJ486 clones to individual transformants can be used as an indicator of the relative strengths of the isolated promoters [14]. To assess this, serial dilutions of a spore suspension derived from the original transformants (see above) were plated onto MMT medium containing different concen- trations of the antibiotic (5-500 ~g ml-1). As shown in Fig. 1, most of the insertions with pro- moter activity allow growth at relatively low con- centrations (5, 10 and 20/xg ml-1), whereas only about 12% conferred resistance to 50/~g ml- l or more.

Characterization of YSP fragments In order to investigate the nature of the YSP

sequences, five different recombinant plasmids, isolated from kanamycin-resistant clones, were further characterized. The levels of resistance conferred to St. lividans by insertions YSP1 to YSP5, present in these plasmids, are shown in Table 1. To elucidate whether these sequences are also functional in E. coli, pBR322 was ligated to plJ486 derivatives containing YSP1 to YSP4,

50-

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O t -

| 20-

10-

5 10 2O 50 100 250 500

pg/ml kanlunvein

Fig. 1. Kanamycin resistance levels conferred to St. lividans by plJ486 recombinant derivatives containing Sau3AI fragments of Sa. cerevisiae D N A (transcriptional fusions). Percentages given correspond to St. lividans clones which grew on medium with the indicated concentration of the antibiotic but not on that with the next higher concentration. The total number of

resistant clones was set at 100%.

after digestion of the plasmids with ClaI and EcoRV (both are unique restriction sites present in pBR322 as well as in plJ486, where they are located within the tsr gene). Since YSP5 contains an internal ClaI site, EcoRV digestion was used to insert pBR322 into plJ486-YSP5. The resulting

Table 1

Promoter activity of YSP sequences in Streptornyces and E. coli, assessed by the level of kanamycin (Km) resistance con- ferred to the host

Insertion Size (bp) Km R (/xgm1-1)

Streptomyces E. coli

YSP1 290 250 100 YSP2 390 100 100 YSP3 619 50 250 YSP4 132 20 5 YSP5 137 20 20

For Streptomyces, serial dilutions of spores containing recom- binant clones were plated and the concentration at which colonies grew in the same number and size as on control plates lacking the antibiotic are presented. For E. coli, colonies were picked onto media with and without kanamycin.

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YSP-I 1 GATCCGCTTT CTTGTTTGCA TGGTCCGTCC AAGAAGGTGA GATTTGAACA

51 GCAAAAGCAG CAACAGCAAC ATCAGCAACT TCATAATGAC TTCAATACAG

101 ATTTCAACCT GAAAAGCCCG TCCAGTAAAA AAATGGGGGT AGAACAGCTG

151 ATCCTTTAAA ACTACAACAG CTCTTCTTAG AATGCTCGTT AAAATCGAAA

201 TTTGCTACGC TTAGACCTCA AATTTAT&GT GAGCCCACTA GGACAAAGCC

251 TCAATTAGTC AGTGAGAACC AGAATTATGA GATGTGGATC

YSP-2 1 GATCTTCAAA GTGGAGACGA

51 AGAGCCTATA AGAGAGCCAC

101 ACTGGCATCC ATGCATGTGT

151 TTTACACGCT TTCCATTAAC

201 TTTTTCACTT TATCTTGTCC

251 TGgCGCACGT GACCAGCGAC

301 AGCTAAAATA CATACATACA

351 CCCCTCTCAA TTTTAAGTAC

TGCTGGACAC AGGAATAGTG AGAAACTTAT

AGACATGCTA AAACCGTGGT TATTACAGAG

TGGTAGTCCT AGGTAGGAGC GTAATGTGTC

AATATTCCTT TTTTTctTTT TTATTATTTT

TTTTTAGCTT TACTCTACAC TATTTTGCAT

AAACTATGTA AGcGTGAAAG GTTGTCTACA

TATACAATGC CTATTTTAGA ACGAAGTTGG

TCCTGATC

YSP-3 1 GATCCAGCCT GTTCAAGTCG AGTTCTGCTG TGGGAGATAT TAAGTCGACC

51 TTTTCACCAC CAATAACAAA CAACTTTCCA CTTTTATATT GGTGGCTCAA

101 AGCGTTGTTG AAAGCCATGG AATAAACCTT GGATGGTAGC TCTGTAGTAT

151 AGTCGTTTGG TGCAGTTCTT GCCAATGCCC TACCACCATT ATGTCTTGTT

201 GGAGAGTTAG CATCCCCGAC TCTTGCCCTA CCAGAACCCT TTTGGCATCA

251 ATTTACGTCT GGAAAACCCG TTTTCACTTC TACCGGGAGG ATTTGAGGCG

301 CCTACTCTTC TATTATCGTT TTCATAAACT ACGGCTCTCC ATAGTATATC

351 ACGCCTTAAT GGAGCAGCTA CAGTTGATGT GGGGACCGGC ACAAATGTCA

401 ATGGTTCTAA TGATGGAAAG GAGCGAACGG TAACTAAGGC GTATTTGGGA

451 GGAATGGCTG CGTTTGGCAG AGGGTTTAAC GTGGATTCTG CATGTGCGGT

501 GGCAGTGGTA GCTTGGTAGC GTATAGATTG TAAAGTCTAT ZGCATAAAAT

551 GGTTGTAATG TTAGTATACG GATTCATCTT CAGAAAATCG ACAACCAAAT

601 GGAAAAAGAG CGAGGGACGA TC

YSP-4 1 GATCTGATAG AGAATGTCCC AAATGTCACT CTCGGGAaAA TGTGTTTTTT

51 CAATCACAAC AAAGAAGAAA GGATACTTCG ATGGTTTTAT TCTTTGTGTG

101 CTTATCTTGC TCACACATAT TTACTTCAGA TC

YSP-5 1 GATCGATTTG CACGTCAGAA CCGCTACGAG CCTCCACCAG AGTTTCCTCT

51 GGCTTCACCC TATTCAGGCA TAGTTCACCA TCTTTCGGGT CCCAACAGCT

i01 ATC~TCTTAC TCAAATCCAT CCGAAGACAT CAGGATC

Fig. 2. Nucleotide sequences of yeast D N A insertions in pH486. Putative - 1 0 and - 3 5 elements are shown bold and underl ined ( - 10) or bold and overlined ( - 35). Only elements located within about 100 nucleotides ups t ream of the translation start codon of the aphll gene are designated, as most of the Streptomyces promoter elements reported have been located within this distance [7].

Lower case letters designate nucleotides that differ from the published sequences, presumably due to strain polymorphisms.

constructs, in which the expression un i t ' o f yeast D N A sequences and the aphH gene remained intact, were transformed into E. coli. The kanamycin resistance level of the different trans- formants was estimated on 2 x YT medium. Table 1 shows that all five YSP segments are also tran- scriptionally active in E. coli. Although the re- sults in physiologically different hosts cannot be directly compared, in general, sequences confer- ring relatively high resistance levels in Strepto- myces also behave as strong promoters in E. coli (YSP1-YSP3) and vice versa (YSP4 and YSP5).

In order to find out about the genomic origin of YSP1-YSP5 and to identify possible promoter elements, their nucleotide sequences were deter- mined. Therefore, the five YSP insertions were subcloned into both M13mpl8 and M13mpl9 as Hind l I I -EcoRI fragments. Analysis of the result- ing sequences (Fig. 2) revealed the presence in YSP1, YSP3 and YSP4 of appropriately spaced hexanucleotides, showing similarity with the - 10 and - 3 5 consensus regions for the major class of eubacterial promoters [8]. In the case of YSP2 and YSP5, several possible - 1 0 consensus-like sequences can be identified but no - 3 5 consen- sus-like region is present at an appropriate dis- tance.

The D N A sequences obtained from the YSP fragments were used in a search of the GeneBank and EMBL databases. Three out of the five se- quences tested were found to match known yeast sequences. Thus, YSP4 derives from the gene encoding the RPB9 subunit of yeast R N A poly- merase II [17] and YSP5 contains part of the gene encoding the yeast 25S rRNA [18]. There- fore, the elements showing weak promoter activi- ties in both Streptomyces and E. coli, belong to intragenic regions in yeast. YSP2 originates from Sa. cerevisiae chromosome V, where it spans the region between two open reading frames, SYGP- ORF13 and SYGP-ORF14 (unpublished data; in- formation obtained from the sequence databases). Considering the direction of aphH gene tran- scription, the element functions in Streptomyces in opposite orientation to the two putative yeast genes. Sequences corresponding to YSP1 and YSP4 have not been entered to the databases as yet.

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The results presented above demonstrate the existence of yeast D N A sequences capable of acting as promoters in Streptomyces. Five out of five sequences that have been characterized were also functional in E. coli. All seem to belong to the major class of eubacterial promoters. Rela- tively G + C-rich Streptomyces specific promot- ers, if present in yeast DNA, are probably very rare. Moreover, the results obtained support the idea that sequences with promoter activity in eubacteria appear randomly in the yeast genome. Thus, at least three of the characterized YSP insertions did not originate from 5'-noncoding regions. In addition, allowing for degeneration in the consensus - 1 0 and - 3 5 sequences as ob- served in bacterial promoters, the high A + T content of the yeast genome (60%) predicts a random appearance of appropriately spaced A + T rich bacterial promoter elements with a fre- quency in the range found in this work.

Acknowledgements

We would like to thank Dr. Pelayo Casares (Depar tamento de Biologla Funcional, Area de Gen6tica, Universidad de Oviedo) and Dr. Jiirgen Heinisch (Institut fiir Mikrobiologie, Universit~it Diisseldorf, FRG) for helpful comments on the manuscript. The work was supported by Grant CAICYT PBT87-0025-C03-03 from the Programa Movilizador de Biotecnologla (Spain).

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