Institute of Genetics, National Yang-Ming University, Shih-Pai, Taipei 112, Taiwan1
Department of Chemistry, Chung-Yuan Christian University, Chung-Li, Taiwan2
Author for correspondence: Carton W. Chen. Tel: +886 2 2826 7040. Fax: +886 2 2826 4930. e-mail: cwchen{at}ym.edu.tw
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ABSTRACT |
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Keywords: Streptomyces, chromosome, linear plasmid, telomere, helicase
Abbreviations: TIR, terminal inverted repeat; TP, terminal protein
The GenBank accession number for the sequence determined in this work is AF194023.
a Present address: Cell and Developmental Biology, Graduate Group Complex, University of California, Davis, CA 95616, USA.
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INTRODUCTION |
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An origin of replication (oriC) is located in approximately the centre of the chromosomes (Calcutt & Schmidt, 1992 ; Zakrzewska-Czerwinska & Schrempf, 1992
). Here, bidirectional replication is initiated and proceeds to the telomeres (Musialowski et al., 1994
). Such internal initiation of replication has also been observed in the linear plasmids of Streptomyces (Chang & Cohen, 1994
; Shiffman & Cohen, 1992
). In the linear plasmid pSLA2, replication leaves single-stranded gaps of about 280 nt at the 3' ends (Chang & Cohen, 1994
). For the Streptomyces chromosomes, the sizes of the intermediate single-stranded gaps at the 3' end are not known. Various models have been proposed for the patching of these gaps involving the TP either as a primer for replication or as a nicking enzyme for processing of the reaction intermediates (Chen, 1996
; Qin & Cohen, 1998
).
The terminal DNA of several chromosomes and linear plasmids of Streptomyces have been isolated and sequenced for about 200 bp (Huang et al., 1998 ). Most of these terminal regions share conserved sequences for the first 166168 nt, beyond which the sequences diverge. The terminal homology is constituted mainly of seven palindromes, with potential to form very stable and complex secondary structures at the 3' ends of the linear replicons.
Linear plasmids are abundant in Streptomyces (Hinnebusch & Tilly, 1993 ; Kinashi & Shimaji, 1987
; Kinashi et al., 1987
; Netolitzky et al., 1995
; Sakaguchi, 1990
). In S. lividans, two copies of SLP2 per chromosome are present. Of the 30 kb TIR of the S. lividans chromosome, the first 15·4 kb is shared by one (right) end of SLP2 (Chen et al., 1993
; Lin et al., 1993
). A 5·3 kb transposon, Tn4811 (Chen et al., 1992
), is present in all three homologous sequences (Fig. 1
). Sequence analysis of Tn4811 revealed three potential coding sequences for transposition functions (including a transposase) and two for accessory proteins (an oxidoreductase and its regulator).
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METHODS |
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Reagents and buffers.
Restriction enzymes and T4 DNA ligase were purchased from Boehringer Mannheim and New England Biolabs. Lysozyme, kanamycin and ampicillin were from Sigma. Thiostrepton was a gift from Dr. S. J. Lucania (Squibb Institute for Medical Research, Princeton, NJ, USA).
DNA sequence determination and analyses.
Restriction fragments isolated from the 3B11 cosmid (Lin & Chen, 1997 ) were cloned in the pBluescript II KS(-) vector (Stratagene). Sequential deletions from each end were obtained by exonuclease III digestion using the Erase-a-Base system (Promega). Dye primer sequencing reactions were cycled on a 9700 thermocycler, using Tag FS polymerase and a Big Dye terminator kit (Perkin Elmer). The sequencing reactions were electrophoresed on an ABI 377 sequencer (Applied Biosystems). To overcome ambiguities due to secondary structures formed by the terminal palindromes, supplementary manual sequencing was performed using a Sequenase kit (US Biochemical) with three modifications. Firstly, chain elongation time was extended from 2 to 8 min to obtain longer sequences. Secondly, a terminal deoxynucleotide transferase reaction was performed after chain termination according to Fawcett & Bartlett (1990)
to remove band ambiguities caused by abnormal termination or pausing of polymerase. Thirdly, in addition to 7 M urea, 10% (v/v) formamide was added to the polyacrylamide gel to eliminate distorted separations of the bands (Huang et al., 1998
).
Nucleotide and amino acid sequence analyses.
General sequence analysis was performed using the GCG package (version 10) and GeneWorks (IntelliGenetics; release 2·4). Database searches for homology to individual polypeptide sequences were performed on the NCBI BLAST server (http://www.ncbi.nlm.nih.gov/BLAST/) using basic BLAST (BLASTN, BLASTP and BLASTX), PSI-BLAST (Altschul et al., 1997 ) and PHI-BLAST (Zhang et al., 1998
). Codon bias preference analysis was performed with MacFRAME (version 1·3; K. Kendall, Tulane University, LA, USA). Searches for protein pattern/motif matches were performed on the ProDom database (http://protein.toulouse.inra.fr/prodom.html). The energy-optimized secondary structure was constructed using M. Zukers DNA Fold Server (http://www.ibc.wustl.edu/~zuker/dna/form1.cg) based on a folding temperature of 30 °C in 1 M NaCl. Some manual adjustments were introduced to incorporate appropriate purinepurine sheared pairings in the secondary structures (Chou et al., 1997
).
Recombinant DNA procedures.
General recombinant DNA procedures were based on those described by Sambrook et al. (1989) . Genetic manipulations of Streptomyces cultures were according to Hopwood et al. (1985)
. For hybridization, DNA probes were labelled with digoxigenin with random primers or by nick translation with a DIG DNA labelling and detection kit (Boehringer Mannheim). DNA fragments separated by agarose gel electrophoresis were transferred to Hybond-N+ membrane (Amersham) according to Southern (1975)
, and hybridized with digoxigenin-labelled DNA probes as specified by the supplier. For knockout of ORF1 by targeted recombination, the 3·6 kb BglII fragment containing the truncated ORF1::tsr sequence was isolated from pLUS695 DNA and used to transform S. lividans ZX7(SLP2) according to Oh & Chater (1997)
.
PFGE.
This was performed at 13 °C in a Rotaphor unit (Biometra) in 0·5x TBE buffer with a reorientation angle of 120 °. Preparation and restriction digestion of intact genomic DNA of Streptomyces in agar plugs and PFGE of the restricted DNA were according to Kieser et al. (1992). Other conditions are specified in the figure legends.
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RESULTS AND DISCUSSION |
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Overlapping fragments spanning the relevant regions on pLUS450 and 3B11 were subcloned in pBluescript vectors and their sequences determined in both directions. The final assembled sequence linked the telomere to the Tn4811 sequences previously characterized. The 10141 nt from the end of the chromosome to (not including) Tn4811 was subjected to subsequent analysis.
Strand bias in the terminal sequence
The overall G+C content of the 10·1 kb was 67·9 mol%. There was a slightly skewed G vs C distribution in the two strands. The forward strand contained more Gs (35·5 mol%) than Cs (32·4 mol%). This skew was most striking in the last 1 kb, just preceding Tn4811. In this stretch of sequence, the strand bias was almost two to one for G vs C (about one to one for A vs T). On close inspection, many of the biased distributions were in the form of GG vs CC, and, more strikingly, GGCGG vs CCGCC. Of all 95 GGCGG pentanucleotides in the total 10·1 kb, 69 (73%) were in the forward strand and 26 in the reverse strand. Whereas the GGCGG pentanucleotides were evenly distributed throughout the reverse strand, they were concentrated in the last 1 kb of the forward strand (Fig. 2a). In this stretch of DNA, the ratio of GGCGG occurrences on the two strands was 16:1. No such strand bias was detected in the downstream 5·3 kb Tn4811 sequence. Whether or how this striking sequence organization plays a role in the structure or replication of the telomeres can only be speculated on at present.
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The structure we proposed (Fig. 3) contained eight hairpins and one long stem. As in the previous model of Huang et al. (1998)
, the first four palindromes assumed a Y-shaped (rabbit ears) secondary structure with the long duplex stem formed by palindromes I and IV, and two hairpin structures formed by palindromes II and III. Each of the remaining six palindromes (VX) formed a hairpin structure. The nucleotide sequences of all the hairpin loops were subsets of the palindrome CGCGCGCAGCGCG (loop sequence underlined; Fig. 3
, shaded), which interestingly is also present at a 3' hairpin of autonomous parvovirus genomes (Chou et al., 1997
). The GCA loop in this type of hairpins has been proposed by Chou et al. (1997)
based on 13C-NMR studies (Zhu et al., 1995b
) to be closed by the sheared G:A pairing, and is resistant to single-strand specific nucleases (Chou et al., 1997
).
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Potential coding sequence
The 10·1 kb terminal sequence was subjected to codon preference analysis in all six reading frames to find potential protein-encoding genes. Only one open reading frame (ORF1; nt 16414283) exhibited significant preference for codons with a G or C in the third position (Fig. 2b), typical for Streptomyces protein coding sequences (Bibb et al., 1984
), and contained translational initiation triplets GTG or ATG. The GTG at nt 1641 was preceded by potential ribosomal binding sequences (1622AAAGGA1627 and 1628AAGG1631) with extensive complementarity to the 3' end sequence of the Streptomyces 16S rRNA (Baylis & Bibb, 1988
).
ORF1 encodes a protein product of 881 aa that is enriched in charged amino acids, particularly Arg (82) Glu (69) and Asp (47). The pI of the protein product is 6·2. BLAST searches found strong homology of this protein sequence to five hypothetical protein products predicted from the genome sequences of Mycobacterium and Helicobacter pylori. The two with highest homology were a hypothetical 1606 aa protein RvD1-Rv2024c in Mycobacterium bovis BCG (Gordon et al., 1999 ) and its putative deletion variant (minus 515 aa) in Mycobacterium tuberculosis H37Rv (Cole et al., 1998
). More sensitive PSI-BLAST (Altschul et al., 1997
) and PHI-BLAST (Zhang et al., 1998
), which searched for relevant sequence similarities using position-specific scoring matrices, found homology between the N-terminal 500 aa of ORF1 and a group of nucleic-acid-binding proteins, mostly the DEAD and DEAH families of helicases. A DEAH motif was present at ORF1 aa 174177, which was part of the signature of a subfamily of ATP-dependent DNA and RNA helicases ([GSAH]-x-[LIVMF](3)-D-E-[ALIV]-H-[NECR]). Like other proteins classified in this group, the sequence of the ORF1 product also contained an ATP/GTP-binding motif A(P-loop; Gorbalenya & Koonin, 1993
) at aa 3845.
Search against a protein domain database (http://protein.toulouse.inra.fr/prodom.html) found a match in aa 33268 of the ORF1 product to domain PD035493, which was contained in five proteins (Fig. 4a) a putative helicase C of H. pylori 26695 (Tomb et al., 1997
), a hypothetical 57·3 kDa protein in M. tuberculosis H37RV (Cole et al., 1998
) and the HsdR subunit of the EcoKI, EcoAI and EcoE type I restriction-modification enzyme systems (Murray et al., 1993
). All these five proteins also contained the ATP/GTP-binding motif and three had the DEAH motif.
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Deletions of the ORF1 sequence from the chromosome and plasmid
The likelihood that the palindromes in the terminal sequence of the S. lividans chromosome might form very extensive stable secondary structures during replication suggested the active participation of a helicase in the process(es). The protein produced by the terminally located ORF1, with its helicase motifs, might play this biochemical role. If this were true, ORF1 could be essential for the replication of the linear chromosomes (and probably the linear plasmid SLP2). To test this possibility, attempts were made to delete the ORF1 sequences on the chromosome and/or SLP2 by targeted recombination. The 3·6 kb BglII fragment from pLUS448 containing the ORF1 sequence (nt 17105340) was subcloned in an E. coli vector, and the 1·2 kb NotIEcoNI internal segment (nt 26613854) was replaced by the thiostrepton resistance gene, tsr, plus downstream multiple cloning sites (including HindIII and PstI; Fig. 1a). The resulting 3·6 kb BglII fragment containing the truncated ORF1::tsr sequence was cleaved from plasmid pLUS695 and used to transform S. lividans ZX7(SLP2).
Eighteen thiostrepton-resistant transformants were isolated, and their genomic DNA was analysed by EcoRV digestion and hybridization using the 1·1 kb tsr probe (Fig. 1a). One transformant did not contain the tsr sequence (not shown) and was excluded from further analysis. All the remaining transformants contained the tsr sequence and the SLP2 plasmid (data not shown).
To locate and analyse the integration sites of the truncated ORF1::tsr sequence, genomic DNA of these transformants was digested with HindIII and hybridized with three different probe preparations (Fig. 1a): (i) end probe (the 2·5 kb PstI terminal fragment on the chromosome and the right end of SLP2 plasmid); (ii) ORF1 probe (the 1·2 kb NotIEcoNI fragment of ORF1), which would reveal the presence of the resident ORF1 sequence but not the integrated truncated copy; and (iii) a mixture of two probes specific to the two arms of the chromosome (probes 511 and 436).
HindIII sites were located at 180, 75 and 47 kb from the left and the right telomere of the S. lividans chromosome, and the right telomere of SLP2, respectively (Fig. 1a). Three fragments of these sizes could be identified in ZX7(SLP2) by hybridization to the end probe and ORF1 probe (Fig. 5a
, b
). Homologous replacement of the ORF1 sequence by the truncated ORF1::tsr sequence at one arm of the replicons would result in hybridization of the end probe to a new 3·8 kb terminal HindIII fragment, accompanied by the loss of hybridization of the end probe to one of the three hybridizing HindIII fragments (180, 75 and 47 kb) of ZX7(SLP2). On the other hand, if replacement occurred at all the ORF1 sequences, the end probe would hybridize only to the new 3·8 kb fragment (Fig. 1b
).
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Type II, representing four transformants, also contained the unexpected 7·4 kb hybridizing fragment (Fig. 5a, b
), but lacked hybridization to the three fragments in ZX7(SLP2). The absence of the three hybridizing fragments of ZX7(SLP2) suggested that the aberrant integration had occurred on both ends of the chromosome as well as SLP2 (Fig. 1c
). Alternatively, the lack of hybridization of the end probe to the 180 and 75 kb fragments could reflect deletions of the chromosomal ends. However, hybridization using the 511 and 436 probes (Fig. 5c
) revealed the expected chromosomal fragments, whose sizes (176 and 71 kb; Fig. 1c
), were electrophoretically indistinguishable from those of ZX7(SLP2) (180 and 75 kb). Thus, if deletions had occurred on the ends of the chromosome, they would have to be just larger than the end probe sequence (2·5 kb) and yet small enough to cause no detectable changes in electrophoretic mobilities.
In these observed aberrant integrations of truncated ORF1::tsr, one crossover appeared to be between homologous sequences, whilst the other was at an unexpected position (Fig. 1a). The aberrant integration occurred rather frequently: 5 out of 17 total transformants examined (29%). Using the same transformation procedure (with linearized DNA), Oh & Chater (1997)
had also observed a similar kind of aberrant integration of a disrupted whiB gene fragment into the S. coelicolor A3(2) chromosome at a frequency of about 20%. It was not clear whether this was an artefact specific for the transformation of Streptomyces using linearized DNA, or specific for the particular target sequences.
ORF1 is not essential for replication of linear replicons
Type III, representing the majority (12) of the transformants, showed hybridization of the end probe to a 3·8 kb fragment only (Fig. 5a). The ORF1 probe did not hybridize to any DNA fragments (Fig. 5b
). These results indicated that all the ORF1 sequences in ZX7(SLP2) had been either replaced by the truncated ORF1::tsr or deleted. Growth and sporulation characteristics of these type III transformants in two solid media (R2 and SP medium 2) and two liquid media (TSB and YEME) were indistinguishable from ZX7(SLP2) despite the lack of functional ORF1. Furthermore, they exhibited normal SLP2-type pocks during conjugation with plasmidless S. lividans TK21 or S. coelicolor M145 (H.-H. Lee, unpublished results), indicating that the conjugal transfer function of SLP2 was not affected (Hopwood et al., 1983
).
To address the question regarding the requirement of ORF1 for the replication of linear chromosomes, it was necessary to determine whether the type III chromosomes had remained linear or had become circularized. Circularized chromosomes would supposedly be free from the need to replicate the telomeres and thus any enzymes specific for the task. The absence of the three hybridizing fragments of ZX7(SLP2) could mean (i) all three termini (both ends of the chromosome and the right end of SLP2) had suffered the homologous replacement by ORF1::tsr and these replicons remained linear, or (ii) that only one or two ends had suffered the homologous replacement, whilst the remaining one(s) had undergone deletions. In the latter case, the deletions might lead to circularization of the chromosomes (Lin et al., 1993 ; Redenbach et al., 1993
).
To distinguish between the two alternative possibilities, genomic DNA from the twelve type III transformants was digested with HpaI and hybridized to the end probe. HpaI cuts the two arms of the chromosome at 200 and 340 kb from the ends (Fig. 1a). Homologous replacement of the ORF1 sequence on the chromosome would not introduce extra HpaI sites, and therefore would essentially not change the sizes of these two hybridizing fragments (Fig. 1b
). The same would be expected for SLP2, i.e. the end probe would hybridize to a 19 kb HpaI fragment of SLP2 with or without homologous replacement. In the type III transformants, the end probe hybridized to three such fragments (Fig. 5d
).
On the other hand, if circularization of the chromosome had occurred without completely deleting the end probe sequences, the end probe would hybridize to a single chromosomal fragment of at least 500 kb, the fusion product of the two terminal HpaI fragments. No such fragments were seen in the type III transformants. We therefore concluded that the chromosomes in these transformants had remained linear, and thus that ORF1 was not essential for the maintenance of the linear chromosome or the SLP2 plasmid in S. lividans.
Despite the evidence against the absolute requirement of ORF1 in the maintenance of linear chromosomes in S. lividans, it is possible that the putative helicase encoded by ORF1 plays a non-essential accessory role in a process such as transcription through the secondary structures. It should be noted that, in the S. lividans chromosome, ORF1 is located in the 30 kb TIR, the overall lengths and sequences of which vary widely among different species (Huang et al., 1998 ). Under relatively low stringent conditions, the ORF1 probe revealed weak hybridization signals in the genomic DNA of the closely related S. coelicolor and Streptomyces parvulus (and two other species to lesser degrees) among eight species examined (Fig. 6
). The chromosomal DNA of S. lividans exhibited no other hybridizing fragments other than the expected 6·8 and 1·0 kb BamHI fragments (prediction from the sequence and Lin, 1995
). This ruled out the possibility that an ORF1 paralogue elsewhere in the chromosome might suppress the ORF1 defect. On the other hand, it was highly unlikely that all the twelve type III ORF1 null mutants contained such conjectural suppressor mutations.
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ACKNOWLEDGEMENTS |
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Received 19 October 1999;
revised 19 December 1999;
accepted 5 January 2000.