Lysine catabolism in Streptomyces spp. is primarily through cadaverine: beta-lactam producers also make alpha-aminoadipate

Madduri, Krishna M.; Stuttard, C.; Vining, L. C.

doi:10.1128/jb.171.1.299-302.1989

Cited by 55 publications

(41 citation statements)

References 17 publications

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“…Previously, we showed that lysine r-aminotransferase (LAT) activity and a-aminoadipate synthesis occur only in 3-lactam-producing actinomycetes (12). Here we report that LAT is governed by DNA located within the cluster of ,B-lactam biosynthesis genes in S. clavuligerus.…”

Section: Streptomyces Clavuligerus Is a Gram-positive Mycelialmentioning

confidence: 84%

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Cloning and location of a gene governing lysine epsilon-aminotransferase, an enzyme initiating beta-lactam biosynthesis in Streptomyces spp

1991

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In actinomycetes that produce P-lactam antibiotics of the cephem type, lysine r-aminotransferase is the initial enzyme in the conversion of lysine to a-aminoadipic acid. We used a two-stage process ("chromosome walking") to screen a lambda library of Streptomyces clavuligerus genomic DNA for fragments that expressed lysine r-aminotransferase activity in S. lividans. Restriction analysis of the cloned DNA confirmed the location of the putative lat gene within the cluster of , -lactam biosynthesis genes, roughly midway between pcbC, the structural gene for isopenicillin N synthetase, and the putative cefE gene encoding deacetoxycephalosporin C synthetase.Streptomyces clavuligerus is a gram-positive mycelial procaryote that produces a number of P-lactam-containing metabolites. Among these are antibiotics of the penicillin and cephalosporin types which are synthesized through a series of reactions involving condensation of ot-aminoadipate, cysteine, and valine to the tripeptide 8-(L-a-aminoadipyl)-L-cysteinyl-D-valine (ACV), cyclization of ACV to isopenicillin N (IPN), and modification of this initial penicillin to a variety of end products (reviewed in reference 2).In recent years, some P-lactam biosynthesis genes have been cloned from several organisms (1,15). Kovacevic et al. (9) provided evidence of gene clustering when they used pcbC DNA encoding IPN synthetase from S. lipmanii as a probe to isolate cefE encoding deacetoxycephalosporin synthetase (DAOCS) from a genomic library of S. clavuligerus and to locate adjacent genes. Also, a DNA fragment containing both cefD (isopenicillin N epimerase) and cefE was cloned from S. clavuligerus and shown to direct the synthesis of a polygenic mRNA of about 10 kb (8), indicating that 1-lactam genes are organized in at least one operon.Previously, we showed that lysine r-aminotransferase (LAT) activity and a-aminoadipate synthesis occur only in 3-lactam-producing actinomycetes (12). Here we report that LAT is governed by DNA located within the cluster of ,B-lactam biosynthesis genes in S. clavuligerus. MATERIALS AND METHODSOrganisms and growth conditions. The Streptomyces and Escherichia coli strains used in this study are listed in Table 1. Streptomyces spp. were grown at 30°C on either MYM agar (17) or R5 agar (3) modified by omitting sucrose and substituting maltose for glucose; mycelium was propagated in either tryptic soya broth (Difco Laboratories) or YEME medium (3). Streptomyces protoplasts were prepared and transformed by standard procedures (3); for protoplast regeneration R5 medium was used. Strains of E. coli were grown in Luria broth (LB) or on LB gelled with 1.5% agar; E. coli DH5a was transformed by the CaCl2 method (13).

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Section: Streptomyces Clavuligerus Is a Gram-positive Mycelialmentioning

confidence: 84%

“…Labeling with [32P]dCTP was by random primer extension, using a kit from Boehringer Mannheim, Dorval, Quebec. LAT activities were assayed as described previously (12).…”

Section: Methodsmentioning

confidence: 99%

Cloning and location of a gene governing lysine epsilon-aminotransferase, an enzyme initiating beta-lactam biosynthesis in Streptomyces spp

1991

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“…In actinomycetes, LAT is found only in the P-lactamproducing species and is not needed for sustaining growth on L-lysine (21). The gram-negative bacterium Flavobacterium sp.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, Madduri et al (21) reported that in these actinomycetes, L-lysine-e-aminotransferase (LAT) is specific to P-lactam (cephamycin C) producers and provides the precursor for antibiotic synthesis; the pathway via cadaverine is obligatory for lysine catabolism (Fig. 1).…”

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confidence: 99%

Localization of the lysine epsilon-aminotransferase (lat) and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (pcbAB) genes from Streptomyces clavuligerus and production of lysine epsilon-aminotransferase activity in Escherichia coli

et al. 1991

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Lysine e-aminotransferase (LAT) in the ,I-lactam-producing actinomycetes is considered to be the first step in the antibiotic biosynthetic pathway. putative N and C termini of S. clavuligerus pcbAB suggests that the coding region occupies 12 kbp and codes for a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the I8-lactam biosynthetic cluster between pcbC and cefE (-25 kbp) is nearly complete.Many naturally occurring P-lactam antibiotics (isopenicillin N, cephalosporin C, and cephamycins) contain a side chain derived from a-aminoadipic acid (a-AAA). When the producer is a filamentous fungus (Penicillium chrysogenum, Aspergillus nidulans, Cephalosporium acremonium), this amino acid is formed as an obligate intermediate in the synthesis of lysine. However, in procaroytes, lysine is synthesized via the diaminopimelic acid pathway without the production of a-AAA (for reviews, see references 23 and 36). Whitney et al. (37) established that in the P-lactamproducing Streptomyces spp., ot-AAA is derived from the breakdown of lysine. Although the catabolism of lysine in bacteria is diverse, conversion of lysine to a-AAA by removal of the E-amino group was tentatively linked in Streptomyces lipmanii to an aminotransferase (16) which was later conclusively shown to be present in Nocardia (previously Streptomyces) lactamdurans (15). Recently, Madduri et al. (21) reported that in these actinomycetes, L-lysine-e-aminotransferase (LAT) is specific to P-lactam (cephamycin C) producers and provides the precursor for antibiotic synthesis; the pathway via cadaverine is obligatory for lysine catabolism (Fig. 1). Moreover, a gene governing LAT production was found exclusively in Streptomyces spp. producing P-lactams (22) MATERIALS AND METHODS Bacterial strains, growth conditions, DNA manipulations, and DNA sequence determination. E. coli strains were grown in TY broth (18) supplemented with either 5 ,ug of tetracycline per ml or 80 jig of ampicillin per ml under standard conditions, except as noted.Restriction endonuclease digestion, ligation, and determination of DNA restriction fragments by agarose or acrylamide gel electrophoresis followed established procedures (29). DNA fragments were subcloned into pUC or M13 vectors for further analysis. DNA sequencing was performed by the dideoxy method with TAQ-TRACK (Promega Biotec) and followed the supplier's recommendations. Fragments were sequenced either directly from the plasmid or after subcloning into M13 bacteriophage vectors, using forward and reverse sequencing primers for pUC and M13 templates, as well as several custom DNA oligonucleotide primers. Custom DNA oligonucleotide primers were synthesized by using an Applied Biosystems DNA Synthesizer (model 380A) according to the manufacturer's instructions.Computing. DNA sequences were analyzed with the GCG

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“…In streptomycetes, where aspartate is a precursor not only of the related protein amino acids but sometimes also of secondary metabolites, there is evidence that regulatory mechanisms acting on core reactions in the aspartate pathway control the flow of metabolic intermediates to such products as the p-lactam antibiotics, which are derived from lysine via L-a-aminoadipic acid (Whitney e t al., 1972;Madduri et al, 1989; Vining e t a/., 1990). I3oth Ask and Asd have been partially purified from StreptornJyces clavuligerus (Mendelovitz & Aharonowitz, 1982 Sambrook et al (1989).…”

Section: Introductionmentioning

confidence: 99%

Streptomyces akiyoshiensis differs from other Gram-positive bacteria in the organization of a core biosynthetic pathway gene for aspartate family amino acids

Vining

1996

Microbiology

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A partial Sau3Al digest of genomic DNA from Streptomyces akiyoshiensis was cloned in a Streptomyces-Escherichia coli shuttle vector, and the recombinant plasmids were used to transform E. coli CGSC 6212, which carries a mutation in the gene for aspartate semialdehyde dehydrogenase (Asd). One of 39000 transformants tested grew on LB medium lacking diaminopimelate. A 17 kb plasmid (pJV21) isolated from this strain conferred prototrophy when used to transform E. coli CGSC 6212. The gene responsible was located on a 2.2 kb DNA fragment by subcloning. Nucleotide sequencing and codon preference analysis of the subcloned insert and of the 3.3 kb insert in the Asd--complementing plasmid pJV36 located three complete and two incomplete open reading frames (ORFs). One of these (ORF3), encoding a polypeptide of 338 amino acids (M, 354848 was identified as the gene for Asd by comparing i t s sequence with database sequences of asd from other bacteria. The inability of pJV30, in which a segment of ORF3 had been deleted, to transform E. coli CGSC 6212 to prototrophy supported this assignment. Southern hybridization indicated that the sequenced region of the cloned DNA fragment represented a continuous segment of the 5. akiyoshiensis chromosome. The deduced amino acid sequences of the ORFs adjacent to asd showed no similarity to sequences for aspartate kinase (Ask); also, transformation with plasmids containing asd and adjacent regions from the 5. akiyoshiensis chromosome did not complement the ask mutant E. coli CGSC 5074. It is concluded that asd and ask in 5. akiyoshiensis are not present in an operon, and thus are organized differently from these genes in the Gram-positive bacteria previously examined.

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Lysine catabolism in Streptomyces spp. is primarily through cadaverine: beta-lactam producers also make alpha-aminoadipate

Cited by 55 publications

References 17 publications

Cloning and location of a gene governing lysine epsilon-aminotransferase, an enzyme initiating beta-lactam biosynthesis in Streptomyces spp

Cloning and location of a gene governing lysine epsilon-aminotransferase, an enzyme initiating beta-lactam biosynthesis in Streptomyces spp

Localization of the lysine epsilon-aminotransferase (lat) and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (pcbAB) genes from Streptomyces clavuligerus and production of lysine epsilon-aminotransferase activity in Escherichia coli

Streptomyces akiyoshiensis differs from other Gram-positive bacteria in the organization of a core biosynthetic pathway gene for aspartate family amino acids

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