Structure of the ask-asd operon and formation of aspartokinase subunits in the cephamycin producer ‘Amycolatopsis lactamdurans

Victor Hernándo-Rico1, Juan F. Martín1, Irene Santamarta1 and Paloma Liras1

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The first two genes of the lysine pathway are closely linked forming a transcriptional operon in the cephamycin producer Amycolatopsis lactamdurans’. The asd gene, encoding the enzyme aspartic semialdehyde dehydrogenase, has been cloned by complementation of Escherichia coli asd mutants. It encodes a protein of 355 aa with a deduced Mr of 37109. The ask gene encoding the aspartokinase (Ask) is located upstream of the asd gene as shown by determination of Ask activity conferred to E. coli transformants. asd and ask are separated by 2 nt and are transcribed in a bicistronic 2·6 kb mRNA. As occurs in corynebacteria, the presence of a ribosome-binding site within the ask sequence suggests that this ORF encodes two overlapping proteins, Ask{alpha} of 421 aa and Mr 44108, and Askß of 172 aa and Mr 18145. The formation of both subunits of Ask from a single gene (ask) was confirmed by using antibodies against the C-terminal end of Ask which is identical in both subunits. Ask activity of ‘A. lactamdurans is regulated by the concerted action of lysine plus threonine and this inhibition is abolished in E. coli transformants containing Ser301 to Tyr, or Gly345 to Asp mutations of the ‘A. lactamdurans ask gene.

Keywords: aspartic semialdehyde dehydrogenase, beta-lactams

Abbreviations: {alpha}-AAA, {alpha}-aminoadipic acid; Asd, aspartate semialdehyde dehydrogenase; Ask, aspartokinase; DAP, diaminopimelate

The GenBank accession number for the sequence reported in this paper is AJ298904.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
L-Lysine in bacteria is formed from aspartic acid in a pathway that leads to the formation of L,D-diaminopimelate (meso-DAP), a building block for peptidoglycan biosynthesis. meso-DAP is decarboxylated in the last step of the pathway to form L-lysine. Aspartokinase (Ask) and aspartate semialdehyde dehydrogenase (Asd) catalyse the two initial reactions in the pathway leading to the formation of aspartate semialdehyde, the branching point to lysine and homoserine that is then converted into methionine and threonine (Eikmanns et al., 1993 ). Interestingly the lysine biosynthetic gene organization and the regulation of the lysine pathway are distinct in different organisms (Malumbres & Martín, 1996 ). In E. coli there are three Ask isoenzymes which are regulated specifically by lysine, methionine or threonine (Thèze et al., 1974 ). There are three Asks in bacilli but they are regulated by diaminopimelic acid, lysine, or lysine and threonine (Zhang et al., 1990 ). A single Ask has been described in Pseudomonas, Brevibacterium and Corynebacterium (Shiio & Mijayama, 1969 ; Cohen et al., 1969 ; Cremer et al., 1988 ). In corynebacteria Ask is a complex of {alpha} and ß subunits that are formed from the same gene (ask) (Follettie et al., 1993 ). Lysine is the precursor of a variety of secondary metabolites produced by filamentous actinomycetes (Lanzini & Lorenzetti, 1993 ; Martín et al., 2000 ). In actinomycetes there are reports of a concerted feedback inhibition by lysine and threonine on the Ask of Streptomyces clavuligerus (Mendelovitz & Aharonowitz, 1982 ) and recently the purified recombinant Ask of Amycolatopsis mediterranei was found to be inhibited by lysine (Zhang et al., 2000 ).

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 {alpha}-aminoadipic acid ({alpha}-AAA) by the P6C dehydrogenase (Fuente et al., 1997 ; Pérez-Llarena et al., 1998 ). {alpha}-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.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, media, culture conditions and plasmids.
The bacterial strains and plasmids used for this study are listed in Table 1. ‘Amycolatopsis lactamdurans LC 411 was grown in complex NYG medium (Ginther, 1979 ) at 28 °C. Escherichia coli was grown in LB (Sambrook et al., 1989 ) or in Vogel–Bonner minimal medium containing (g l-1): citric acid, 2; glucose, 2; MgSO4 . 7H2O, 0·2; KH2PO4, 10; NaH(NH4)PO4 . 4H2O, 3·5; pH 7·2. A mixture of L,L-, D,D- and meso-DAP was added when required. E. coli competent cells were prepared as described by Chung et al. (1989) and transformed by standard procedures (Sambrook et al., 1989 ). E. coli transformants were selected in presence of ampicillin (100 µl ml-1).


View this table:
[in this window]
[in a new window]
 
Table 1. Strains and plasmids used in this study

 
DNA isolation and manipulation.
Total DNA of ‘A. lactamdurans’ was obtained by using the Kirby method (Kirby et al., 1967 ) as modified by Hopwood et al. (1985) and using sodium tri-isopropylnaphthalene sulphonate instead of SDS. Plasmid DNA from E. coli was prepared by the alkaline lysis procedure (Birnboim & Doly, 1979 ), whereas rapid screenings of plasmid DNA were carried out by the boiling method (Holmes & Quigley, 1981 ). Two gene libraries of ‘A. lactamdurans’ total DNA were prepared: one in plasmid pUC13 using 4–10 kb Sau3A DNA fragments and a second one in pUC18 using HindIII fully digested DNA. The recombinant plasmids obtained were used to transform E. coli asd strains CGSC 5080 and CGSC 5081.

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 [{alpha}-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 {alpha} 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{alpha} and Akß of Streptomyces Ask sequences (aa 405–421 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 ).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning of asd from ‘A. lactamdurans
Plasmid libraries of ‘A. lactamdurans’ total DNA were used to transform E. coli asd mutant strains CGSC 5080 and CGSC 5081. Recombinant plasmids pB2 and pF2 that complemented both mutants to prototrophy were isolated. Restriction analysis of these plasmids showed that they overlap in a 2·2 kb region (Fig. 1). To confirm that both inserts were adjacent in the ‘A. lactamdurans’ genome, total DNA of ‘A. lactamdurans’ was separately digested with BamI, HindIII, NcoI, NotI, PstI and XhoI and hybridized with a [{alpha}-32P]-labelled EcoRI–PvuII fragment of 1·3 kb from pB2 (Fig. 1, probe A). Bands of hybridization corresponding to the expected sizes if both inserts belong to the same DNA region were found in each digested DNA (not shown).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Restriction map of the ‘A. lactamdurans’ DNA fragments complementing the asd mutation of E. coli CGSC5080 and E. coli CGSC5081. The plasmid constructs used for complementation are shown in the lower part of the figure. The arrow indicates the location and orientation of the lacZ promoter.

 
Complementation analysis and nucleotide sequence of the cloned ‘A. lactamdurans’ DNA fragment
The ‘A. lactamdurans’ DNA insert cloned in pF2 or pB2 was digested with several restriction endonucleases. The fragments obtained, separated by gel electrophoresis, were subcloned in both orientations in the SphI and SmaI sites of pUC119 and pBSII-SK(+) respectively, to give plasmids pF22, pF25, pF26, pF221 and pB21. Complementation analysis showed that a 1·5 kb DNA insert common to plasmid pF25 and pB21 was sufficient to complement E. coli asd mutants. Ordered sets of DNA fragments were generated from pF221 and pF25 inserts and sequenced.

The complete nucleotide sequence of a 2·7 kb EcoRI–HindIII 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 65–1331), starting with the initiation codon GTG (nt 65–67), was preceded by a putative RBS, GAGGAGG, located 8 bp upstream. This ORF1 ends at the TAG codon (nt 1328–1330) 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 2419–2439) that may form a stem–loop 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 {alpha}-subunit of Ask (Ask-{alpha}), ranging from 91% for A. mediterranei to 70–72% 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 (a–e) 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 247–265 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{alpha} 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.



View larger version (80K):
[in this window]
[in a new window]
 
Fig. 2. (a) Comparison of the amino acid sequence of Ask of ‘A. lactamdurans’, A. mediterranei (accession no. Q9RQ25), Mycobacterium smegmatis (P41403), C. glutamicum (P26512) and Bacillus stearothermophilus (P53553). The horizontal arrow indicates the valine residue that starts the Askß protein. Several regions conserved in different Asks (a to e) are indicated by bars. (b) Comparison of a portion of the nucleotide sequence upstream of the askß gene from ‘A. lactamdurans’, A. mediterranei, Mycobacterium tuberculosis, Mycobacterium smegmatis, C. glutamicum, Methanobacterium jannaschii and Thermus flavus. Potential ribosome-binding sites are underlined and initiation codons are boxed in the respective Askß proteins. The two vertical arrows indicate amino acids changed in the in vitro mutagenesis studies.

 
ORF2 encodes a protein of 355 aa with a deduced Mr of 37109. Comparison of the protein encoded by ORF2 with the SWISS-PROT database showed significant homology with Asd of different organisms. At the amino acid level the identity was about 35% with the homologous proteins of Streptomyces, Mycobacterium and Corynebacterium species as well as with the Asd of A. mediterranei (36·5% similarity). This percentage of similarity is significantly lower than that found for Ask and indicates that the amino acid sequence of ‘A. lactamdurans’ Asd is quite different from that of other Gram-positive bacteria. The motif GAGKEG (aa 162–167) appears to correspond to the NADH-binding site (GXGXXG) of the dehydrogenase.

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. lactamduransasd 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{alpha} 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.



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 3. Total RNA from ‘A. lactamdurans’ was blotted onto nylon membranes and hybridized with a SacI 0·9 kb DNA probe complementary to the 5' end of the ask (ask{alpha}) gene (probe B) and with a SacI 1·3 kb DNA probe containing the 3' region of the ask (askß) gene and the whole asd gene (probe C).

 
Ask activity of the ‘A. lactamduransask gene
To confirm that the cloned ask gene encodes a functional Ask, this activity was measured in E. coli DH5{alpha} carrying plasmids with the different inserts shown in Fig. 1. As a positive control the activity of E. coli(pULM301), a transformant carrying the ask-asd cluster from ‘Corynebacterium lactofermentum (formerly ‘Brevibacterium lactofermentum’) that is well expressed in E. coli (Malumbres, 1993 ), was also measured. When the ask gene from ‘A. lactamdurans’ was expressed in the multicopy vector pF2 in E. coli, the Ask activity in the transformant increased to 5·7 mU (mg protein)-1, 8·1-fold in relation to the E. coli(pUC18) control strain (Table 2), confirming that the insert contains a functional ask gene. The activity was still higher in the positive control E. coli(pULM301) containing the ask-asd cluster from ‘Corynebacterium lactofermentum’ (18·8-fold higher than the control strain), in agreement with the observation that many corynebacteria promoters are known to be well expressed in E. coli.


View this table:
[in this window]
[in a new window]
 
Table 2. Ask activity in Escherichia coli DH5{alpha} transformants

 
Two immunoreactive proteins are formed from the ask gene
Since Ask of corynebacteria is composed of two subunits, {alpha} and ß formed from a single ask gene, inmunoblot studies were performed with extracts of ‘A. lactamdurans using antibodies against the 20-aa C-terminal region of Ask. Extracts of Streptomyces coelicolor and Streptomyces clavuligerus were also included for comparative purposes. Results showed that two immunoreactive bands of about 48 and 17 kDa (Fig. 4) corresponding to the deduced size of the {alpha} and ß subunits of Ask were detected in ‘A. lactamdurans and in the two Streptomyces strains tested. In addition a diffuse immunoreactive band of 34–45 kDa was found in all preparations of ‘A. lactamdurans’, but not in Streptomyces clavuligerus or Streptomyces coelicolor that may correspond to a processed or partially degraded Ask. This band was only detected with anti-Ask antibodies, but not with antibodies against other Streptomyces proteins.



View larger version (58K):
[in this window]
[in a new window]
 
Fig. 4. Immunodetection of the Ask{alpha} and Askß proteins (indicated by arrows) using anti-Ask antibodies in cell-free extracts (20 µg per lane) from Streptomyces coelicolor (lane 1), ‘A. lactamdurans’ (2) and Streptomyces clavuligerus (3).

 
These results indicate that, indeed, two forms ({alpha} and ß) of Ask with the same amino acid sequence as detected by immunoblotting studies are formed from the ask gene and that this phenomenon also appears to be a general feature in Streptomyces species.

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. lactamduransask 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.


View this table:
[in this window]
[in a new window]
 
Table 3. Concerted inhibition by lysine and threonine of ‘A. lactamdurans’ Ask and of wild-type and mutant Asks of ‘A. lactamdurans’ expressed in E. coli

 
Involvement of Ask Ser301 and Gly345 in the regulatory site for concerted inhibition
In corynebacteria the concerted inhibition by threonine and lysine is exerted at a regulatory site that involves Ser301 of Ask (Kalinowski et al., 1991 ). A mutant resistant to the lysine analogue aminoethylcysteine (AEC) showed a Ser301 to Tyr301 change. Similarly, in Corynebacterium flavum N13, Gly345 of Ask has been reported to be involved in the concerted inhibition of Ask by lysine and threonine (Follettie et al., 1993 ). Both Ser301 and Gly345 are present in the amino acid sequence of A. lactamdurans’ Ask. Therefore, we decided to confirm the presence of a concerted feedback regulation of Ask in ‘A. lactamdurans’ by in vitro mutagenesis of these two amino acid residues.

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{alpha}(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 {alpha} and ß subunits.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The {alpha}-AAA component of the ß-lactam antibiotic intermediate {delta}-(L-{alpha}-aminoadipyl-L-cysteinyl-D-valine) (ACV) (Martín, 2000 ) derives from the lysine pathway in several cephamycin-producing actinomycetes (Martín, 1998 ). In bacteria L-lysine derives from the aspartic acid family biosynthetic pathway that starts with the activation of aspartic acid by Ask and the subsequent reduction to aspartic semialdehyde catalysed by Asd. In corynebacteria and other Gram-positive bacteria the lysine pathway is feedback-regulated by the concerted action of lysine and threonine (Eikmanns et al., 1993 ; Malumbres & Martín, 1996 ).

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 1055–1060. 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.


   ACKNOWLEDGEMENTS
 
This research was supported by the Spanish Ministry of Science and Education (CICYT, BIO 97-0289-CO2-O2). I.S. and V.H. received fellowships from the University of León and from Grant BIO 97-0289, respectively. We are grateful to Marcos Malumbres for kindly providing the ‘A. lactamdurans’ genomic libraries and to INBIOTEC for providing antibodies against Ask.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Barreiro, C., Pisabarro, A. & Martín, J. F. (2000). Characterization of the ribosomal rrnD operon of the cephamycin producer ‘Nocardia lactamdurans’ shows that this actinomycete belongs to the genus Amycolatopsis. Syst Appl Microbiol 23, 15-24.[Medline]

Birnboim, H. C. & Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7, 1513-1523.[Abstract]

Chen, N. Y., Jiang, S. Q., Klein, D. A. & Paulus, H. (1993). Organization and nucleotide sequence of the Bacillus subtilis diaminopimelate operon, a cluster of genes encoding the first three enzymes of diaminopimelate synthesis and dipicolinate synthase. J Biol Chem 268, 9448-9465.[Abstract/Free Full Text]

Chung, C. T., Niemela, S. L. & Miller, R. H. (1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci USA 86, 2172-2175.[Abstract]

Cirillo, J. D., Weisbrod, T. R., Pascopella, L., Bloom, B. R. & Jacobs, W. R.Jr (1994). Isolation and characterization of the aspartokinase and aspartate semialdehyde dehydrogenase operon from mycobacteria. Mol Microbiol 11, 629-639.[Medline]

Cohen, G. N., Stainer, R. Y. & LeBras, G. (1969). Regulation of the biosynthesis amino acids of the aspartate family in coliform bacteria and Pseudomonas. J Bacteriol 99, 791-801.[Medline]

Coque, J. J. R., Liras, P., Laíz, L. & Martín, J. F. (1991). A gene encoding lysine 6-aminotransferase, which forms the ß-lactam precursor {alpha}-aminoadipic acid, is located in the cluster of cephamycin biosynthetic genes in Nocardia lactamdurans. J Bacteriol 173, 6258-6264.[Medline]

Coque, J. J. R., Malumbres, M., Martín, J. F. & Liras, P. (1993). Analysis of the codon usage of the cephamycin C producer A. lactamdurans. FEMS Microbiol Lett 110, 91-96.

Cremer, J., Creptow, C., Eggeling, L. & Sahm, H. (1988). Regulation of enzymes of lysine biosynthesis in Corynebacterium glutamicum. J Gen Microbiol 134, 3221-3229.[Medline]

Eikmanns, B. J., Eggeling, L. & Sahm, H. (1993). Molecular aspects of lysine, threonine, and isoleucine biosynthesis in Corynebacterium glutamicum. Antonie Leeuwenhoek 64, 145-163.[Medline]

Follettie, M. T., Peoples, O. P., Agoropoulou, C. & Sinskey, A. J. (1993). Gene structure and expression of the Corynebacterium flavum N13 ask-asd operon. J Bacteriol 175, 4096-4103.[Abstract]

de la Fuente, J. L., Rumbero, A., Martín, J. F. & Liras, P. (1997). {Delta}-1-Piperideine-6-carboxylate dehydrogenase, a new enzyme that forms {alpha}-aminoadipate in Streptomyces clavuligerus and other cephamycin C-producing actinomycetes. Biochem J 327, 59-64.[Medline]

Galán, J. E., Nakayama, K. & Curtiss, R. I. (1990). Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strains. Gene 94, 29-35.[Medline]

Ginther, C. L. (1979). Sporulation and the production of serine proteases and cephamycin C by Streptomyces lactamdurans. Antimicrob Agents Chemother 15, 522-526.[Medline]

Gribskov, M., Devereux, J. & Burgess, R. R. (1984). The codon preference plot: graphic analysis of protein coding sequences and prediction of gene expression. Nucleic Acids Res 12, 539-549.[Abstract]

Haziza, C., Strategier, P. & Patte, J. C. (1982). Nucleotide sequence of the asd gene of Escherichia coli: absence of a typical attenuation signal. EMBO J 1, 379-384.[Medline]

Higgins, D. G., Bleasby, A. J. & Fuchs, R. (1992). CLUSTAL V: improved software for multiple sequencing alignment. Comput Appl Biosci 8, 189-191.[Abstract]

Hoang, T. T., Williams, S., Schweizer, H. P. & Lam, J. S. (1997). Molecular genetic analysis of the region containing the essential Pseudomonas aeruginosa asd gene encoding aspartate-ß-semialdehyde dehydrogenase. Microbiology 143, 899-907.[Abstract]

Holmes, D. S. & Quigley, M. (1981). A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114, 193-197.[Medline]

Hopwood, D. A., Bibb, M. J., Chater, K. F. & 7 other authors (1985). Genetic Manipulation of Streptomyces: a Laboratory Manual. Norwich: John Innes Foundation.

Jagusztyn-Krynicka, E. K., Smorawinska, M. & Curtiss, R.III (1982). Expression of Streptococcus mutans aspartate-semialdehyde dehydrogenase gene cloned into plasmid pBR322. J Gen Microbiol 128, 1135-1145.[Medline]

Kalinowski, J., Bachmann, B., Thierbach, G. & Pühler, A. (1990). Aspartokinase genes lysC{alpha} and lysCß overlap and are adjacent to the aspartate ß-semialdehyde dehydrogenase gene asd in Corynebacterium glutamicum. Mol Gen Genet 224, 317-324.[Medline]

Kalinowski, J., Cremer, B., Bachmann, B., Eggeling, L., Sahm, H. & Puhler, A. (1991). Genetic and biochemical analysis of the aspartokinase from Corynebacterium glutamicum. Mol Microbiol 5, 1197-1204.[Medline]

Kirby, K. S., Fox-Carter, E. & Guest, M. (1967). Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from bacteria. Biochem J 104, 258-262.[Medline]

Kumar, C. V., Coque, J. J. R. & Martín, J. F. (1994). Efficient transformation of the cephamycin C producer Nocardia lactamdurans and development of shuttle and promoter-probe cloning vectors. Appl Environ Microbiol 60, 4086-4093.[Abstract]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]

Lanzini, G. & Lorenzetti, R. (1993). Biotechnology of Antibiotics and Other Bioactive Microbial Metabolites. New York: Plenum.

Le, Y., He, J. & Vining, L. C. (1996). Streptomyces akiyoshiensis differs from other Gram-positive bacteria in the organization of a core biosynthetic pathway gene for aspartate family amino acids. Microbiology 142, 791-798.[Abstract]

Madduri, K., Stuttard, C. & Vining, L. C. (1991). Cloning and location of a gene governing lysine {epsilon}-aminotransferase, an enzyme initiating ß-lactam biosynthesis in Streptomyces spp. J Bacteriol 173, 985-988.[Medline]

Malmberg, L. H., Hu, W. S. & Sherman, D. H. (1993). Precursor flux control through targeted chromosomal insertion of the lysine epsilon-aminotransferase (lat) gene in cephamycin C biosynthesis. J Bacteriol 175, 6916-6924.[Abstract]

Malmberg, L. H., Hu, W.-S. & Sherman, D. H. (1995). Effects of enhanced lysine epsilon-aminotransferase activity on cephamycin biosynthesis in Streptomyces clavuligerus. Appl Microbiol Biotechnol 44, 198-205.[Medline]

Malumbres, M. (1993). Clonación y caracterización molecular de los genes biosintéticos de treonina (thrC) y lisina de Brevibacterium lactofermentum. PhD thesis, University of León, Spain.

Malumbres, M. & Martín, J. F. (1996). Molecular control mechanisms of lysine and threonine biosynthesis in amino acid-producing corynebacteria: Redirecting carbon flow. FEMS Microbiol Lett 143, 103-114.[Medline]

Martín, J. F. (1998). New aspects of genes and enzymes for ß-lactam antibiotic biosynthesis. Appl Microbiol Biotechnol 50, 1-15.[Medline]

Martín, J. F. (2000). {alpha}-Aminoadipyl-cysteinyl-valine synthetases in ß-lactam producing organisms. From Abraham’s discoveries to novel concepts of non-ribosomal peptide synthesis. J Antibiot 53, 1008-1021.[Medline]

Martín, J. F., Gutiérrez, S. & Aparicio, J. F. (2000). Secondary metabolites. In Encyclopedia of Microbiology , pp. 213-237. Edited by J. Lederberg. San Diego:Academic Press.

Mendelovitz, S. & Aharonowitz, Y. (1982). Regulation of cephamycin C synthesis, aspartokinase, dihydrodipicolinic acid synthetase, and homoserine dehydrogenase by aspartic acid family amino acids in Streptomyces clavuligerus. Antimicrob Agents Chemother 21, 74-84.[Medline]

Pérez-Llarena, F. J., Rodríguez-García, A., Enguita, F. J., Martín, J. F. & Liras, P. (1998). The pcd gene encoding piperideine-6-carboxylate dehydrogenase involved in biosynthesis of {alpha}-aminoadipic acid is located in the cephamycin cluster of Streptomyces clavuligerus. J Bacteriol 180, 4753-4756.[Abstract/Free Full Text]

Rius, N., Maeda, K. & Demain, A. L. (1996). Induction of L-lysine {epsilon}-aminotransferase by L-lysine in Streptomyces clavuligerus, producer of cephalosporins. FEMS Microbiol Lett 144, 207-211.[Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[Abstract]

Shiio, I. & Mijayama, R. (1969). Concerted inhibition and its reversal by end products of aspartate kinase in Brevibacterium flavum. J Biochem 65, 849-859.[Medline]

Stadman, E. R., Cohen, G. W., Lebras, G. & Robichon-Szulmajster, M. (1961). Feedback inhibition and repression of aspartokinase activity in E. coli and Saccharomyces cerevisiae. J Biol Chem 236, 2033-2038.

Thèze, J., Margarita, D., Cohen, G. N., Borne, F. & Patte, J. C. (1974). Mapping of the structural genes of three aspartokinases and of the two homoserine dehydrogenases of Escherichia coli K12. J Bacteriol 117, 133-143.[Medline]

Zhang, J. J., Hu, F. M., Chen, N. Y. & Paulus, H. (1990). Comparison of the three aspartokinase isozymes in Bacillus subtilis Marburg and 168. J Bacteriol 172, 701-708.[Medline]

Zhang, W., Jiang, W., Zhao, G., Yang, Y. & Chiao, J. (1999). Sequence analysis and expression of the aspartokinase and aspartate semialdehyde dehydrogenase operon from rifamycin SV-producing Amycolatopsis mediterranei. Gene 237, 413-419.[Medline]

Zhang, W.-W., Jiang, W.-H., Zhao, G.-P., Yang, Y.-L. & Chiao, J.-S. (2000). Expression in Escherichia coli, purification and kinetic analysis of the aspartokinase and aspartate semialdehyde dehydrogenase from the rifamycin SV-producing Amycolatopsis mediterranei U32. Appl Microbiol Biotechnol 44, 52-58.

Received 1 November 2000; revised 2 February 2001; accepted 20 February 2001.