Area de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain1
Author for correspondence: Paloma Liras. Tel: +34 987 291504. Fax: +34 987 291506. e-mail: degplp{at}unileon.es
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ABSTRACT |
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Keywords: aspartic semialdehyde dehydrogenase, beta-lactams
Abbreviations: -AAA,
-aminoadipic acid; Asd, aspartate semialdehyde dehydrogenase; Ask, aspartokinase; DAP, diaminopimelate
The GenBank accession number for the sequence reported in this paper is AJ298904.
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INTRODUCTION |
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The second step of the lysine pathway has been paid more attention. Genes for Asd (asd) have been isolated from several organisms, including enterobacteria (Haziza et al., 1982 ; Galán et al., 1990
), Pseudomonas aeruginosa (Hoang et al., 1997
), and the Gram-positive bacteria Streptococcus mutans (Jagusztyn-Krynicka et al., 1982
), Corynebacterium glutamicum (Kalinowski et al., 1990
), Mycobacterium smegmatis (Cirillo et al., 1994
), Streptomyces akiyoshiensis (Le et al., 1996
) and A. mediterranei (Zhang et al., 1999
, 2000
).
In mycobacteria, corynebacteria, Amycolatopsis and bacilli the ask and asd genes are clustered in an operon (Kalinowski et al., 1990 ; Chen et al., 1993
; Cirillo et al., 1994
; Zhang et al., 1999
), although that is not the case in Streptomyces akiyoshiensis (Le et al., 1996
).
In the ß-lactam-producing actinomycetes Streptomyces clavuligerus and Nocardia lactamdurans (reclassified recently as Amycolatopsis lactamdurans; Barreiro et al., 2000 ) L-lysine is converted additionally to piperidine-6-carboxylate (P6C) by the enzyme lysine-6-aminotransferase (Madduri et al., 1991
; Coque et al., 1991
) and then oxidized to
-aminoadipic acid (
-AAA) by the P6C dehydrogenase (Fuente et al., 1997
; Pérez-Llarena et al., 1998
).
-AAA is a direct precursor of cephamycin C, cephalosporin C and other ß-lactam antibiotics and its availability is normally a limiting step for ß-lactam biosynthesis (Malmberg et al., 1993
, 1995
; Rius et al., 1996
; Martín, 1998
). It was therefore of great interest to characterize the first steps of the lysine pathway in A. lactamdurans and the regulation of the metabolic flux leading to lysine and cephamycin C, particularly the subunit composition of Ask and the feedback regulation of this enzyme. We report in this article the characterization of the genes ask and asd and the nature of the encoded enzymes that catalyse the first steps of lysine biosynthesis in this micro-organism.
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METHODS |
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Southern hybridization analysis and DNA sequencing.
DNA fragments used as probes were eluted from agarose gels and purified with QIAEX II (Qiagen). These probes were labelled by nick translation with [-32P]dCTP (11·3x1013 Bq mmol-1; Amersham) using the nick translation kit from Promega. Labelled probes were purified and concentrated with Wizard columns (Promega). DNA was transferred to Hybond-NX membranes (Amersham) and hybridized in 50% (w/v) formamide at 42 °C as indicated by Sambrook et al. (1989)
. The nucleotide sequence was obtained by the dideoxy chain-termination method (Sanger et al., 1977
) using Sequenase (USB) in the sequencing reaction in an automatic ABI PRISM Sequencer 310 (Perkin Elmer).
Transcriptional analysis.
Total RNA was isolated from cultures of A. lactamdurans in complex NYG medium using the RNeasy Kit (Qiagen). After separation in denaturing agarose gels the RNA was transferred to nylon membranes. Northern hybridization was performed by standard methods (Sambrook et al., 1989 ). The RNA molecular mass I kit from Boehringer was used as size standards.
Ask assays.
Ask activity was assayed by measuring the amount of aspartyl-ß-hydroxamate formed (Stadman et al., 1961 ). One unit was defined as the activity required to form 1 nmol aspartyl-ß-hydroxamate min-1. Proteins were precipitated from the crude extract by adding 5 vols saturated ammonium sulfate solution, collected by centrifugation and resuspended in 1 ml Tris/HCl (40 mM, pH 8·0). The assay mixture contained in a 1 ml volume: 40 mM Tris/HCl (pH 8·0), 20 mM MgCl2, 400 mM hydroxylamine, 25 mM ATP, 200 mM L-aspartic acid and protein extract. The reaction mixture was incubated for 60 min at 30 °C and 1·5 ml ferric chloride solution (10%, w/v, FeCl3; 3·3%, w/v, trichloroacetic acid; 5·83%, v/v, HCl) was added. After centrifugation at 10000 r.p.m. for 5 min, the A515 was measured in the supernatant. Background activity was measured in the absence of aspartate or ATP.
SDS-PAGE.
SDS-PAGE of the cell extracts was performed in 12 or 15% (w/v) polyacrylamide gel as described by Laemmli (1970) and the proteins were stained with Coomassie blue.
Antibodies and immunoblotting of Ask.
The and ß Ask subunits were resolved by SDS-PAGE of crude extracts (20 µg per lane) of A. lactamdurans, Streptomyces clavuligerus and Streptomyces coelicolor. The proteins were transferred to an Immobilon-P membrane using a Mini-Trans Electrophoretic Cell (Bio-Rad). Rabbit antibodies against a consensus sequence of the C-terminal region common to several Ak
and Akß of Streptomyces Ask sequences (aa 405421 in the A. lactamdurans sequence) were synthesized by Neosystems (Strasbourg, France) and provided by the Institute of Biotechnology (INBIOTEC, León, Spain). Positive immunoreactive bands were visualized using the anti-rabbit IG alkaline phosphatase conjugated system (Sigma).
In vitro mutagenesis of the ask gene.
Mutagenesis was carried out using the QuickChange site-directed mutagenesis kit (Stratagene). The serine at position 301 was mutated by PCR using oligonucleotides S1 (5'-GCAGAACGTTTACAACACCTCGTCGG-3') and S2 (5'-CCGACGAGGTGTTGTAAACGTTCTGC-3'). To mutate the glycine at position 345, oligonucleotides G1 (5'-GACGACCACGTCGACAAGGTCTCC-3') and G2 (5'-GGGAGACCTTGTCGACGTGGTCGT-3') were used. The nucleotide giving rise to the mutation is underlined in each oligonucleotide.
Sequence analysis.
Computer-assisted nucleotide sequence analyses were performed utilizing the DNAstar package (Madison, WI, USA). Deduced protein sequences were compared using the SWISS-PROT and Gene Bank databases by the FASTA and BLITZ search programs. Protein sequence comparisons were analysed by the CLUSTAL V program (Higgins et al., 1992 ).
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RESULTS |
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The complete nucleotide sequence of a 2·7 kb EcoRIHindIII fragment located in the central region of pF2 was obtained and the ORFs in the nucleotide sequence were identified by using the Gribskov algorithm (Gribskov et al., 1984 ) with data on codon usage for A. lactamdurans (Coque et al., 1993
). Two complete ORFs were present in this insert (Fig. 1
). ORF1 of 1266 nt (nt 651331), starting with the initiation codon GTG (nt 6567), was preceded by a putative RBS, GAGGAGG, located 8 bp upstream. This ORF1 ends at the TAG codon (nt 13281330) and has a G+C content of 67·1 mol%.
A second ORF (ORF2), 1064 nt long with a G+C content of 69·1 mol%, is located downstream and in the same orientation as the previous one but in a different reading frame (Fig. 1). ORF1 and ORF2 are separated by only 2 nt. The proximity of the ORF1 and ORF2 genes suggests that they form a functional operon structure. Downstream of ORF1/ORF2 there is a 10 bp inverted repeated region (nt 24192439) that may form a stemloop structure in the transcript with a calculated free energy of -17 kcal mol-1. This inverted repeat is followed by a stretch of four U residues that supports its role as a transcriptional terminator.
ORF1 and ORF2 encode Ask and Asd of A. lactamdurans
Computer-aided analysis of ORF1 showed that it encodes a protein of 421 aa with a deduced Mr of 44108 and significant sequence similarity to the -subunit of Ask (Ask-
), ranging from 91% for A. mediterranei to 7072% for Mycobacterium or Corynebacterium species, 42% with the AskI and AskII proteins of Bacillus subtilis and 20% with the AskIII and the Ask domains of Ask-HdhI and II of E. coli. Comparisons of the whole amino acid sequence of the A. lactamdurans Ask protein revealed considerable sequence conservation between species. Five regions (ae) are highly conserved in all Ask sequences, revealing regions essential for structural and functional enzyme requirements. This conservation is especially high in region c (aa residues 247265 in Fig. 2
) of A. lactamdurans Ask. Computer analysis showed a putative RBS, GAGGAG internal to the ask gene. It precedes a putative polypeptide, ß, starting at the GTG codon present in nt 812 (Fig. 2b
), suggesting an organization similar to that proposed in corynebacteria in which the lysCß gene is in the same frame as lysC
and overlaps with its 3' end (Follettie et al., 1993
; Kalinowski et al., 1990
). ORF1-ß, starting at the valine residue indicated in Fig. 2(a)
(GTG in Fig. 2b
) would encode a polypeptide of 172 aa and an Mr of 18145.
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Functional analysis and transcription of the ask and asd genes
A point of interest is whether the ask and asd genes form part of a transcriptional unit or are expressed as independent transcripts from two separate promoters. To confirm the linkage of asd and ask in A. lactamdurans, different DNA fragments containing ask and/or asd genes were subcloned in both orientations (Fig. 1) in relation to the lacZ promoter of plasmid pBSKSII. Complementation to prototrophy of E. coli asd mutants was only observed when the plasmids contained the asd gene downstream and in the same orientation as the lacZ promoter (Fig. 1
). The need of an external promoter for expression of the A. lactamdurans asd gene suggests that this gene either lacks an adjacent promoter (being expressed from the upstream ask promoter) or that the Amycolatopsis asd promoter is not recognized by the E. coli RNA polymerase.
Northern hybridization experiments were carried out to elucidate how many transcripts were formed from the ask-asd genes. A single RNA transcript of about 2·6 kb (Fig. 3) hybridized with both probes B and C (including ask
and askß-asd, respectively), indicating that both genes are transcribed as a single transcriptional unit under the culture conditions used. Therefore these data indicate that the A. lactamdurans ask and asd genes are transcribed from a promoter located upstream of the transcriptional start site of ask, although we cannot exclude the possibility that they might be transcribed separately under other culture conditions.
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Ask activity of A. lactamdurans is feedback-regulated by the concerted action of lysine and threonine
Ask is feedback-regulated in most micro-organisms (Eikmanns et al., 1993 ; Malumbres & Martín, 1996
). To determine if there is concerted regulation of the A. lactamdurans Ask by threonine and lysine, enzyme activity was measured in ammonium sulphate (70% saturation) precipitates of extracts of A. lactamdurans LC411 and E. coli transformants containing the A. lactamdurans ask gene in the presence or absence of L-lysine (5 mM), L-threonine (5 mM), and L-lysine and L-threonine (5 mM each).
The results (Table 3) showed that addition of L-lysine resulted in an increase of activity of 16% but addition of L-threonine barely affected the activity (94%). However, the addition of both L-lysine and L-threonine to the assay resulted in an activity of 0·52 U (mg protein)-1, corresponding to 76% of the activity in the control unsupplemented assay. These results suggest that in A. lactamdurans there is a concerted inhibition by lysine and threonine, similar to that of C. glutamicum.
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Separate mutations of Ser301 to Tyr and of Gly345 to Asp were obtained with the QuickChange kit of Stratagene using modified oligonucleotides S1 and S2, or G1 and G2, and plasmid F2 as DNA template. The presence of the mutations was confirmed by sequencing the modified DNA fragments. Concerted regulation by lysine and threonine of the mutated Asks was tested in E. coli transformants (Table 3) in the presence of 5 mM lysine, threonine or both, since we were unable to obtain ask gene replacement mutants in A. lactamdurans. As shown in Table 3
the control strain E. coli DH5
(pUC18) shows a weak inhibitory regulation by lysine and threonine which results in a 30% decrease in activity. In E. coli(pF2) the decrease in activity is lower and corresponds to 23% feedback inhibition. However, in the transformants carrying mutant Ask proteins the enzyme activity is higher than in the transformants containing wild-type A. lactamdurans Ask and, what is more relevant, this activity is not reduced (mutation Ser301Tyr) or even slightly increased (mutation Gly345Asp) by addition of lysine and threonine. These results are consistent with the idea that feedback inhibition by lysine and threonine of A. lactamdurans Ask is exerted at a regulatory centre that involves Ser301 and Gly345, both of which occur in the
and ß subunits.
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DISCUSSION |
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The ask and asd genes are closely linked in A. lactamdurans (separated by only 2 nt). The RBS for the asd gene is located within the 5' region of the ask gene, an organization typical of coordinated translation. Linkage of the ask and asd genes occurs in corynebacteria (Kalinowski et al., 1990 ), mycobacteria (Cirillo et al., 1994
) and in Gram-negative bacteria, but not in Streptomyces akiyoshiensis, the producer strain of 5-hydroxy-4-oxonorvaline (Le et al., 1996
). This different organization of the ask-asd genes in various actinomycetes opens the question of whether the organization in the genus Streptomyces is different from that in Amycolatopsis, but the recent sequencing of the Streptomyces coelicolor genome indicates that at least in this species (ALO79348.1, locus SC66T3.26 and SC8E4A.10) the organization is similar to that found in A. lactamdurans.
Both ask and asd genes are transcribed as a single bicistronic mRNA of 2·6 kDa, expressed from a single promoter upstream of ask. The promoter may also be regulated at the transcriptional level, facilitating coordinate expression.
Nucleotide sequence analysis suggests that the ask gene is translated into two overlapping polypeptides since there is an internal GAGGAG ribosome-binding site within the ask gene at nucleotide positions 10551060. This hypothesis was supported by Western analysis using antibodies raised against the C-terminal region of Ask. Two polypeptides of the expected sizes (48 and 17 kDa), if translation occurs from the initial ATG and also from the internal GTG at position 812 of the gene (amino acid residue 269), were clearly observed in Western analysis. Since the antibodies were raised against the amino acid sequence in the C-terminal region of Ask it is concluded that both polypeptides contain the same amino acid sequence. This mechanism of formation of both polypeptides from a single gene has also been reported for the ask gene of corynebacteria. As described in this article the same mechanism is apparently at work in other Streptomyces species such as Streptomyces clavuligerus and Streptomyces coelicolor.
We have shown that the A. lactamdurans Ask is feedback-regulated by the concerted action of lysine and threonine both in A. lactamdurans extracts and when the enzyme was expressed in E. coli. This result differs from those of Zhang et al. (2000) who reported inhibition of the A. mediterranei Ask by lysine alone. The amino acid concentrations used in our work (5 mM) are higher than those used by Zhang et al. (2000)
who used a purified recombinant enzyme, but these differences are unlikely to be responsible for the patterns of inhibition observed. Rather, it suggests that different regulatory mechanisms may occur in different actinomycetes. In that regard, Ask of Streptomyces clavuligerus, a ß-lactam producer, has also been reported to be feedback-regulated by lysine plus threonine (Mendelovitz & Aharonowitz, 1982
).
Two amino acids of Ask, S301 and G345, were shown to be involved in the regulatory site involved in the concerted feedback inhibition of lysine and threonine. Our directed mutagenesis studies support previous observations of the role of these two amino acids in feedback regulation of Ask of C. glutamicum and C. flavum (Kalinowski et al., 1991 ). Indeed, mutation of either of these amino acids results in isolation of S-aminoethylcysteine (a lysine analogue) resistant mutants of these corynebacteria.
Ask activity of the parental A. lactamdurans strain is much lower than that observed in E. coli. This may indicate that the flux of intermediates entering the aspartic acid family pathway is somehow limited in the actinomycete, which is certainly a slow-growing bacterium compared to E. coli. The low Ask activity of A. lactamdurans may also explain the low level of cephamycin biosynthesis in this actinomycete.
Amplification of the ask and asd operon and deregulation of Ask in A. lactamdurans is being used for the improvement of cephamycin production in this actinomycete (P. Liras & J. F. Martín, unpublished). Despite the extreme difficulty in transforming A. lactamdurans (Kumar et al., 1994 ) initial experiments indicate that the ask-asd genes can be amplified in autonomously replicating plasmids and efforts to integrate additional copies of the ask-asd cluster into the chromosome in A. lactamdurans are now in progress.
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ACKNOWLEDGEMENTS |
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Received 1 November 2000;
revised 2 February 2001;
accepted 20 February 2001.