Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany1
Author for correspondence: Josef Altenbuchner. Tel: +49 711 685 7591. Fax: +49 711 685 6973. e-mail: Josef.Altenbuchner{at}po.uni-stuttgart.de
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ABSTRACT |
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Keywords: Streptomyces, -glucosidase, ABC transporter, repressor, amplification
Abbreviations: ABC, ATP-binding cassette; AUD, amplifiable unit of DNA; LDR, left direct repeat; LIP, left imperfect palindrome; MDR, middle direct repeat; PBS, potential binding sequence; p-NPG, p-nitrophenyl -D-glucopyranoside; RDR, right direct repeat; RIP, right imperfect palindrome
The GenBank accession number for the sequence reported in this paper is U22894.
a Present address: Biozentrum/Physiologische Chemie I, Am Hubland, 97074 Würzburg, Germany.
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INTRODUCTION |
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AUD1 is composed of three 1 kb and two 4·7 kb repeats alternately arranged in direct orientation (Fig. 1; Altenbuchner & Cullum, 1985
). The 4·7 kb repeats are identical and encode putative proteins having some similarities to chitinases (Volff et al., 1996
). They serve as substrate for the recA-dependent recombination event leading to amplification. When they are replaced by direct repeats generated from Escherichia coli DNA the modified AUD1 structure is still able to amplify (Volff et al., 1996
). The left and middle 1 kb repeats are identical (LDR and MDR, 1009 bp in length) and differ from the right 1 kb repeat (RDR, 1012 bp in length) at 63 positions (6·2% divergence; Piendl et al., 1994
). The 1 kb repeats encode DNA-binding proteins displaying similarities to repressors of E. coli inducible operons, such as LacI, CytR and GalR. The RDR-encoded protein binds upstream and downstream of the right 1 kb repeat and upstream of the middle 1 kb repeat (Volff et al., 1996
). The two identical upstream binding sites named LIP (for left imperfect palindrome) are 36 bp long and are located 8 nt upstream of the start codon of MDR and RDR (Volff et al., 1996
). The downstream binding site is located in a 147 nt fragment flanking RDR on the right side. This fragment contains another imperfect palindromic sequence, RIP (for right imperfect palindrome). RIP is 32 bp long and is found 90 nt downstream from RDR. However, RIP is not a binding site for the 1-kb-repeat-encoded proteins (Volff et al., 1996
). A 34 nt sequence with similarities to LIP, called PBS (for potential binding sequence), is also present in the 147 nt fragment 32 nt downstream of RDR. The presence of both up- and downstream binding sites as well as a functional protein encoded by the RDR is important for efficient amplification of AUD1 (Volff et al., 1996
).
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METHODS |
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E. coli JM109 (Yanisch-Perron et al., 1984) was used as host in cloning experiments. All E. coli cultures, including those for selective expression, were done in dYT medium or on dYT agar plates (Sambrook et al., 1989 ).
phage were propagated on E. coli NM538 (Frischauf et al., 1983
) in dYT liquid medium or dYT soft agar supplemented with 10 mM Mg2+ and 0·2% maltose. For E. coli transformation, competent cells were prepared as described by Chung et al. (1989)
and transformants were selected with ampicillin (100 mg l-1).
DNA manipulations and sequencing.
S. lividans and E. coli plasmid preparations were done using the alkaline lysis procedure of Kieser (1984) . Genomic DNA was isolated according to Hopwood et al. (1985)
. Southern blot experiments and DIG labelling of DNA were done as described by Volff et al. (1996)
. Construction of a genomic library of S. lividans TK64 in
EMBL4, plaque hybridization with [
-32P]dCTP-labelled DNA and isolation of
DNA were done as described by Eichenseer & Altenbuchner (1994)
.
Sequencing reactions were performed on double-stranded DNA using the Autoread Sequencing kit and an automated laser fluorescent ALF Sequencer (Pharmacia Biotech). Nucleotide sequences were analysed using programs of the GCG Wisconsin package (Devereux et al., 1984 ). For the codon preference program (Gribskov et al., 1984
), a codon usage table from eight Streptomyces genes was generated as described by Pfeifer et al. (1992)
. Database searches were run with the programs BLASTN, BLASTX and TBLASTN (Altschul et al., 1990
) on the BLAST e-mail server from the National Center for Biotechnology Information, Bethesda, MD, USA.
Gel redardation assays.
LDR was amplified from plasmid pMT680 as described for RDR (Volff et al., 1996 ) using the primers S485 (5'-GCG CTA GTC TTG CGC ATA TGA CGC GAC GAC TTG C-3') and S1014 (5'-AAA AGG ATC CCG CAA GGA ACT TGC GGT-3'). The restriction sites for BamHI and NdeI in the primer sequences allowed insertion of the PCR fragment into the L-rhamnose-inducible vector pJOE2702 (Volff et al., 1996
). The resulting plasmid, pJOE2935, and plasmid pJOE2797 (RDR in pJOE2702) constructed earlier (Volff et al., 1996
), were transferred into E. coli JM109. Expression of the genes was induced by growing the strains at 30 °C in dYT medium to an OD600 of 0·5, followed by addition of 0·2% rhamnose and incubation for 3 h before preparation of crude extracts. Gel mobility shifts were done in 5% polyacrylamide gels using the LIP-containing HindIII fragment of plasmid pJOE2201 (Volff et al., 1996
) or the PBS-containing HindIII fragment of pJOE2864-2. The plasmid pJOE2864-2 contains the two complementary oligonucleotides S973 (5'-GATCCATTTACGAAATCAGCGCGAGATATTGCGCCACTTGCATG-3') and S974 (5'-CAAGTGGGCGCAATATCTCGCGCTGATTTCGTAAATG-3'), inserted between the SphI and BamHI site of pIC20H. The two HindIII fragments of pJOE2201 and pJOE2864-2 were labelled by filling in the ends with [
-32P]dATP and Klenow polymerase as described by Volff et al. (1996)
. Sugars (sucrose, maltose, C2C8 maltooligosaccharides and trehalose) were included in the binding assay at a final concentration of 1 mM.
Selective expression of aglA.
aglA was PCR-amplified in the presence of 10% DMSO (30 cycles, annealing temperature 58 °C) from plasmid pJOE683. Primers (S1069, 5'-AAA ACA TAT GGC AGC CCC CAA CTC GCA CC-3'; and S1070, 5'-AAA AAG ATC TGG CGC GCA GCC ACA CCG TGT-3') were designed to introduce a NdeI site overlapping the ATG start codon of aglA and a BglII site directly flanking its last C-terminal codon. For selective expression in E. coli, aglA was inserted as an NdeIBglII fragment into pJOE2775 (Krebsfänger et al., 1998 ) under the control of the rhaB promoter. The six histidine codons present in the vector were fused to aglA. The correct sequence of the amplified aglA gene was verified by DNA sequencing of the resulting plasmid, pJOE2951. E. coli JM109 cells transformed with pJOE2951 were induced with 0·2% rhamnose for 3 h at 30 °C.
Determination of -glucosidase activities with p-nitrophenyl
-D-glucopyranoside (p-NPG).
Enzyme activity was measured spectrophotometrically by monitoring the release of p-nitrophenol from p-NPG. Cells of S. lividans and E. coli were grown in 50 ml medium, washed twice and resuspended in enzyme buffer (20 mM Tris, 100 mM NaCl, 0·02% NaN3, pH 8·0). Crude extracts (20 µl, prepared either by sonication or by French press) or purified enzyme were added to 600 µl 0·1 M sodium phosphate pH 7·0 and 400 µl of a 13 mM pNPG solution in the same buffer and incubated at 25 °C until a yellow colour appeared. The reaction was stopped by addition of 500 µl 0·4 M Na2BO3 pH 9·8 and the absorbance at 405 nm was measured. For Km determination, the final p-NPG concentration was varied from 0·1 to 10 mM. Protein concentration was determined using the method of Bradford (1976) . One unit of
-glucosidase activity was defined as the amount of enzyme hydrolysing 1 µmol p-NPG under these conditions. The temperature stability of AglA was determined by incubation of the enzyme for 15 min at temperatures between 25 °C and 60 °C and cooling on ice for several minutes before adding it to the reaction mixture. The pH optimum of AglA was determined by using 600 µl 0·66 M potassium/sodium phosphate buffer pH 5·09·0 or 0·05 M Tris/HCl pH 7·09·0 as reaction buffer.
Gene disruption experiments.
To disrupt the chromosomal aglA gene in S. lividans TK64, the tsr gene conferring resistance to thiostrepton (TsR, Thompson et al., 1980 ) was inserted into the BamHI site of aglA contained in the 3·8 kb insert of pJOE683 (this work). This insert ligated with tsr was then cloned into the E. coliStreptomyces shuttle vector pJOE1082. This temperature-sensitive vector carries the minimal replicon and kanamycin resistance gene of Streptomyces plasmid pGM11 (Muth et al., 1989
) ligated to the E. coli plasmid pUC19 (Vieira & Messing, 1982
). The resulting plasmid, pJni5, was used to disrupt aglA. To inactivate the 1 kb repeat(s), the tsr gene was inserted into the PstI site of the right 1 kb repeat RDR (Piendl et al., 1994
). The resulting disrupted repeat was cloned into pJOE1082 and the resulting plasmid was called pJOE2369. Protoplasts of S. lividans were transformed with pJni5 and pJOE2369. After selection of the transformants with thiostrepton (50 µg ml-1) at 30 °C, spores were separated from mycelium by filtration (Hopwood et al., 1985
) and plated on HT medium at 40 °C to inhibit the replication of the temperature-sensitive vector pJOE1082. After a further plating of spores on HT medium plus thiostrepton at 40 °C, single colonies were tested for their growth on HT medium plus kanamycin (15 µg ml-1). Thiostrepton-resistant and kanamycin-sensitive colonies were retained and further cultivated. The success of the gene disruption and the loss of vector pJOE1082 were confirmed by Southern blot hybridization using pJni5, pJOE2369, the tsr gene and pJOE1082 as probes (data not shown).
Construction of plasmids pJOE3072-9, pJOE3073-1 and pJOE3076-2.
These three plasmids are derivatives of the E. coliS. lividans shuttle vector pJOE3069, which is similar to pEI16 described previously (Volff et al., 1996 ). The plasmid pJOE3069 contains the SCP2 origin of replication on a SalIXhoI fragment and the SCP2 stability region on a BamHISacI fragment, both inserted into pUC13. Plasmid pJOE3076-2 contains the tsr gene inserted into pJOE3069 as a HindIII fragment from plasmid pJOE803 (Altenbuchner & Eichenseer, 1991
). The agl genes, together with the C-terminal end of RDR, were obtained from
559-1 as an EcoRIPstI fragment and inserted into pJOE803 (pJOE3063-2). The agl genes were isolated again from pJOE3063-2 together with the tsr gene as a HindIIIPstI fragment and combined with a HindIIIPstI fragment from pJOE2434 (Volff et al., 1996
) containing the N-terminal end of RDR together with 73 bp upstream sequence. This HindIII fragment was inserted into pJOE3069 to give pJOE3072-9. For pJOE3073-1, the PstI site in the RDR was destroyed by Klenow polymerase. Nucleotide sequencing of the deleted region showed that instead of a 4 bp deletion, actually 11 bp were missing.
Substrate specificity of the -glucosidase.
To 30 µl of reaction buffer (100 mM sodium phosphate pH 7·0) 10 µl substrate (500 mM stock solution in 100 mM sodium phosphate) and 10 µl enzyme (usually 2·4 µg purified enzyme, but when sucrose was used as substrate, the enzyme solution was tenfold diluted) were added and incubated for 60 min at 25 °C. To determine the Km for sucrose, the final concentration of sucrose in the reaction mixture was varied from 10 mM up to 900 mM. The reaction was stopped by heating to 90 °C for 2 min. Granutest 250 (E. Merk; 1 ml) was added and after 45 min at 25 °C the absorbance at 340 nm was determined on a Kontron spectrophotometer. The test is based on reduction of NAD+ by glucose dehydrogenase oxidizing the glucose which is generated by the enzyme reaction to gluconolactone. The amounts of glucose were obtained from a calibration curve generated with defined glucose concentrations. In addition, the products of enzyme reactions were determined and quantified by HPLC using a Hamilton RCX-10 column (250x4·1 mm) and an electrochemical detection unit, CoulochemII (ESA), with the analytical cell 5040. The mobile phase was 0·1 M NaOH and the flow rate was 0·75 ml min-1. For tri- and tetrasaccharides and phosphorylated disaccharides, the mobile phase was 0·22 M NaOH/0·02 M sodium acetate.
Purification of AglA.
E. coli JM109(pJOE2951) was grown in 50 ml dYT at 37 °C to an OD600 of 0·5, the culture shifted to 30 °C, L-rhamnose added to a final concentration of 0·1% and incubation continued for a further 3 h. The cells were harvested by centrifugation (Sorvall SS34, 10 min, 6000 r.p.m., 4 °C), washed in enzyme buffer (20 mM Tris, 100 mM NaCl, 0·02% NaN3, pH 8·0) and resuspended in 3 ml enzyme buffer. The cells were disrupted using a French press (two cycles at approx. 800 bar), the crude extract clarified by centrifugation (Sorvall SS34, 30 min, 20000 r.p.m., 4 °C) and the supernatant (1 ml) applied to a metal affinity spin column (Talon, Clontech Laboratories) equilibrated with enzyme buffer. After centrifugation at 600 r.p.m. for 2 min in a Heraeus Megafuge with a swingout rotor, the column was washed twice with 1 ml enzyme buffer and the enzyme eluted with 2x1 ml enzyme buffer supplemented with 50 mM imidazole. Finally, the two fractions were combined and dialysed twice against enzyme buffer.
Gel filtration of AglA.
The native molecular mass of AglA was determined by gel filtration on a Superdex 75, PC 3.2/30 column (Smart system, Pharmacia), calibrated with ribonuclease A (13700 Da), chymotrypsin (25000 Da), ovalbumin (43000 Da) and bovine serum albumin (67000 Da). The column was equilibrated with enzyme buffer and the enzyme eluted with this buffer at a flow rate of 0·04 ml min-1. The elution profile was monitored at 280 nm and the presence of active enzyme in the fractions by enzyme assays with p-NPG as substrate.
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RESULTS |
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The similarities indicated that the ORFs may form an operon for uptake and degradation of an -glycosidic sugar and were named tentatively aglE, aglF, aglG, aglA and aglX (Fig. 1
and Table 1
). The first ORF, aglE, begins 151 bp downstream of the stop codon of RDR (right 1 kb repeat of AUD1) and encodes a protein of 471 amino acids. The deduced protein shows similarities to periplasmic sugar-binding proteins. The N-terminal amino acid sequence is in accordance with consensus sequences of signal peptides of secreted proteins with a hydrophilic region containing six arginine residues followed by a hydrophobic region of 14 aa and a signal petide cleavage site, LAAC, which is recognized by signal peptidase II (van Heijne, 1989
). This indicates that AglE is a lipoprotein. The next two predicted gene products, AglF and AglG, have sizes of 231 and 291 aa, respectively, and show similarity to the MalF-MalG proteins, integral membrane components of the E. coli maltose transport system (Boos & Lucht, 1996
). AglF and AglG are hydrophobic proteins according to a KyteDoolittle plot as expected (data not shown). AglA (534 aa) is an
-glucosidase, as indicated by high aa sequence identity with a maltooligosaccharide hydrolase (61% identity, Table 1
) and the E. coli trehalose-6-phosphate hydrolase (38% identity), and later proved by enzyme assays. The aglX-encoded protein of 547 aa shows significant similarities to a putative mycobacterial protein of unknown function.
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Cmls Arg- mutants of S. lividans are highly unstable and therefore the possibility that there were additional rearrangements in strain Jni27 leading to increased expression of the agl genes could not be completely excluded. A further experiment was performed to definitively corroborate the role of the RDR-encoded protein as repressor of the agl genes. All five agl genes together with RDR and a thiostrepton resistance gene were inserted into a low-copy SCP2 plasmid derivative to give plasmid pJOE3072-9. A second plasmid was generated by deleting the PstI site in pJOE3072-9, which introduced a frameshift mutation in RDR (pJOE3073-1), and for a control, the tsr gene alone was inserted into the SCP2 vector (pJOE3076-2). All three plasmids were introduced into S. lividans WP, a Cmls Arg- mutant of S. lividans which has AUD1 and the agl genes deleted. After growth in modified HT medium without dextrin, the -glucosidase activity was determined. As can be seen from Fig. 4
, there was a slight increase in enzyme activity in S. lividans WP with pJOE3072-9 compared to the vector control and a 10-fold increase when RDR was inactivated, which confirms the repressor function of the RDR-encoded protein.
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DISCUSSION |
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No function could be attributed to the fifth gene, aglX, found in S. lividans. It is not known whether aglX belongs to the putative agl operon and if there are even more agl genes downstream since no transcription terminator sequence was found behind aglA and aglX.
The 1-kb-repeat-encoded protein binds 8 nt upstream of its start codon (for MDR and RDR) at a sequence called LIP which would allow autoregulation of its own synthesis. It also binds in the region between RDR and aglE at a sequence called PBS which shows significant similarity to LIP. A nucleotide sequence fitting the consensus sequence of a Streptomyces vegetative promoter was identified within the 151 bp intercistronic region (Fig. 6). PBS overlaps the -35 sequence of this putative promoter sequence and therefore the binding of the RDR-encoded protein to PBS would inhibit binding of the RNA polymerase to this promoter. When the agl genes are transcribed by this promoter the mRNA would start at the beginning of RIP. The function of RIP remains unclear. It could be the target of a global regulatory system. As a part of the leader region of the agl mRNA it could also stabilize the mRNA by a stem-and-loop secondary structure as described by Deng et al. (1990)
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According to its amino acid sequence, AglA clearly belongs to the family 13 of glycoside hydrolases. Members of this family retain the configuration at the anomeric centre of the substrate during hydrolysis and the catalytic domain of the enzymes shows a characteristic (ß/)8 barrel structure. Secreted and intracellular enzymes with various substrate specificities, such as
-glucosidases,
-amylases, pullulanases, cyclodextrinases or trehalose-6-phosphate hydrolase, belong to this family (Henrissat, 1991
; Henrissat & Bairoch, 1993
; Warren, 1996
). For the
-glucosidase MalL of Bacillus subtilis, a member of this family, sucrose was also the preferred substrate (Schönert et al., 1998
). But the specific activity of MalL was about 10-fold higher and the Km for sucrose 10-fold lower than for AglA. Despite the preference for sucrose, the expression of the corresponding gene was induced by maltose and not by sucrose and the biological function of MalL seems to be in the breakdown of internal storage polysaccharides (Schönert et al., 1998
).
The function of AglA and of the other Agl proteins remains unclear since S. lividans does not grow on sucrose. Colonies of S. lividans grew equally well on minimal medium agar plates with sucrose or without any carbon source. Also, addition of sucrose to complete medium did not induce expression of aglA. The most obvious explanation would be that we have not identified the natural substrate of the agl genes. The very high Km of AglA for sucrose (100 mM) might point in this direction.
Another reason might be that AUD1 and the agl genes, which are located in the unstable region of the S. lividans chromosome, were accidentally fused. This would mean that the inducer is different from the substrate of AglA. A further possibility would be that there was originally only one regulatory gene for the agl genes. This regulatory gene was duplicated twice during the evolution of the strain and by mutagenesis evolved a different function, for example to protect the chromosome in unstable mutants from further deletions by promoting amplification of the AUD1 sequence. When strain Jni27 with the inactivated regulatory gene was plated on agar plates with minimal medium and sucrose as carbon source there was no improvement in growth compared to TK64, AJ100 or Jni26, which makes the latter two possibilities unlikely. Alternatively, the agl genes may have lost their function by mutation in a similar manner to pseudogenes in higher eukaryotes.
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ACKNOWLEDGEMENTS |
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Received 25 October 1999;
revised 31 December 1999;
accepted 14 January 2000.