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

Abstract

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.

Homologous expression of aspartokinase (ask) gene inStreptomyces clavuligerusand itshom-deleted mutant: Effects on cephamycin C production

“…5 The ask (encoding the aspartokinase; EC 2.7.2.4) and asd (aspartate semialdehyde dehydrogenase; EC 1.2.1.11) genes are clustered in an operon in mycobacteria, corynebacteria and bacilli [6][7][8][9] as well as in Amycolatopsis 10 and S. clavuligerus, 11 but not in Streptomyces akiyoshiensis. 12 The regulation of the aspartate pathway involves the multiplicity of the aspartokinase, first enzyme of the pathway, in certain bacteria like Escherichia coli and bacilli. 13,14 In other organisms, including actinomycetes, only one aspartokinase has been described.…”

Structure of the ask-asd operon and formation of aspartokinase subunits in the cephamycin producer ‘Amycolatopsis lactamdurans’ The GenBank accession number for the sequence reported in this paper is AJ298904.

“…The RBS for the asd gene is located within the 5h 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 '.…”

“…n et al, 1990), Pseudomonas aeruginosa (Hoang et al, 1997), and the Gram-positive bacteria Streptococcus mutans (Jagusztyn-Krynicka et IP: 34.214.212.150 On: Fri, 11 May 2018 11:26:02 V. HERNA ! NDO- RICO and OTHERS 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(Zhang et al, , 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).…”

“…NDO- RICO and OTHERS 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(Zhang et al, , 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 β-lactam-producing actinomycetes Streptomyces clavuligerus and ' Nocardia lactamdurans ' (reclassified recently as ' Amycolatopsis lactamdurans ' ; Barreiro et al, 2000) -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 α-aminoadipic acid (α-AAA) by the P6C dehydrogenase (Fuente et al, 1997 ;Pe!…”