The dapA and dapB genes, encoding, respectively, dihydrodipicolinate synthase and dihydrodipicolinate reductase, the two first enzymes of the lysine branch of the aspartic amino acid family, were cloned from the DNA of the amino acid-producing bacterium Brevibacterium lactofermentum. The two genes were clustered in a 3.5-kb Sau3AI-BamHI fragment but were separated by an open reading frame of 750 nucleotides. The protein encoded by this open reading frame had little similarity to any protein in the data banks, and its function remains unknown. The three genes were translated in Escherichia coli, giving the corresponding polypeptides.The lysine biosynthetic pathway in corynebacteria is still poorly understood despite the use of Corynebacterium glutamicum and Brevibacterium lactofermentum for the industrial production of lysine. This pathway shares the first two steps with the threonine and methionine biosynthetic pathways, which involve the activation of aspartic acid to aspartyl phosphate and the reduction of the activated amino acid to aspartyl semialdehyde. In the lysine branch, the aspartyl semialdehyde is converted to dihydrodipicolinate by condensation with pyruvate. This first "branching" reaction, which involves the condensation of a C4 with a C3 fragment to form the seven-carbon dihydrodipicolinate, is carried out by dihydrodipicolinate synthase, encoded in Escherichia coli by the dapA gene. The condensation step is followed by a cyclization reaction which results in the formation of 2,3-dihydrodipicolinate and may involve an enzyme-bound C7 linear intermediate (Fig. 1). Whether this reaction requires more than one protein is uncertain. The dihydrodipicolinate formed is later converted to Al-piperideine-2,6-dicarboxylate by dihydrodipicolinate reductase, which in E. coli is encoded by the dapB gene. In the last steps of the pathway the A'-piperideine-2,6-dicarboxylate is converted to D,L-diaminopimelic acid by at least two alternative pathways (6, 20). In addition to the succinylase and acetylase variants common to other procaryotes, a shunt pathway in which A'-piperideine-2,6-dicarboxylate is converted in a single step to diaminopimelate by the enzyme D-diaminopimelate dehydrogenase has been described in corynebacteria (11). Finally, diaminopimelic acid is decarboxylated to form lysine by diaminopimelate decarboxylase (lysA gene).Only preliminary information is available on the nature of the dapA and dapB genes of C. glutamicum (1,5), and no information has been published on that in B. lactofermen-* Corresponding author. tum. We describe here that the dapA and dapB genes of B.lactofermentum are clustered together with a third open reading frame (ORF) located between them. The three ORFs are translated into polypeptides.Cloning the dapB gene and deletion mapping. Total DNA of B. lactofermentum ATCC 13869 (Table 1) was isolated as described previously (7) and partially digested with Sau3AI or totally digested with HindIII in different experiments. DNA fragments of 4 to 10 kb were ligated with pUC13, and th...

An rplK SummaryThe transcriptional and translational control of the biosynthesis of the b-lactamase inhibitor clavulanic acid is a subject of great scientific and industrial interest. To study the role of the ribosomal protein L11 on control of clavulanic acid gene transcription, the DNA region aspC-tRNA trp -secE-rplK-rplA-rplJ-rplL of Streptomyces clavuligerus was cloned and characterized. An S. clavuligerus rplK DPALG mutant, with an internal 12 nucleotides in-frame deletion in the rplK gene, encoding the L11 (RplK) ribosomal protein lacking amino acids 29 PALG 32 , was constructed by gene replacement. This deletion alters the L11 N-terminal domain that interacts with the RelA and class I releasing factors-mediated translational termination. The mutant grew well, showed threefold higher resistance to thiostrepton, did not form spores and lacked diffusible brown pigments, as compared with the wild-type strain. The wild-type phenotype was recovered by complementation with the native rplK gene. S. clavuligerus rplK DPALG produced reduced levels of clavulanic acid (15-26% as compared with the wild type) and cephamycin C (40-50%) in cultures grown in defined SA and complex TSB media. The decreased yields resulted from an impaired transcription of the regulatory genes ccaR and claR and the cefD and ceaS2 genes for cephamycin and clavulanic acid biosynthesis respectively. Expression of ceaS2 encoding carboxyethylarginine synthase (CEAS), the precursor-committing enzyme for clavulanic acid biosynthesis, was particularly affected in this mutant. In the wild-type strain polyphosphorylated nucleotides peaked at 36-48 h of growth in SA cultures whereas expression of the cephamycin and clavulanic acid genes occurred 12-24 h earlier than the increase in ppGpp indicating that there is no strict correlation between the peak of ppGpp and the onset of transcription of the clavulanic acid and cephamycin C biosynthesis. The drastic effect of the rplK DPALG mutation on the onset of expression of the ceaS2 and the regulatory ccaR and claR genes and the lack of correlation with ppGpp levels suggest that the onset of transcription of these genes is modulated by the conformational alteration of the N-terminal region of L11 probably by interaction with the nascent peptide releasing factors and with RelA.

The rplK gene of Corynebacterium glutamicum ATCC13032 comprises 438 nucleotides and encodes a protein of 145 amino acids with a molecular mass of 153 kDa. The amino acid sequence revealed extensive similarities to the large ribosomal subunit protein L11 from several Gram-positive and Gram-negative bacteria. The C. glutamicum rplK gene is located downstream of secE, representing part of the protein export apparatus, and of nusG, encoding a transcription antiterminator protein. The rplK gene is followed by an ORF homologous to rplA encoding the 50S ribosomal protein L1. Northern analysis revealed that transcription of the rplK-rplA cluster resulted in two different transcripts of 15 and 06 kb. The 15 kb transcript corresponds to the entire rplK-rplA cluster and the short transcript originates from the rplK gene. A C. glutamicum rplK mutant strain carrying a 12 bp in-frame deletion within rplK, which resulted in the loss of the tetrapeptide Pro-Ala-Leu-Gly in the L11 protein, was constructed. The mutant failed to accumulate (p)ppGpp in response to amino acid starvation and exhibited an increased tolerance to the antibiotic thiostrepton. Evidently, the C. glutamicum rplK gene is required for (p)ppGpp accumulation upon nutritional starvation.

The Brevibacterium lactofermentum argS gene, which encodes an arginyl-tRNA synthetase, was identified in the upstream region of the lysA gene. The cloned gene was sequenced; it encodes a 550-amino-acid protein with an Mr of 59,797. The deduced amino acid sequence showed 28% identical and 49% similar residues when compared with the sequence of the Escherichia coli arginyl-tRNA synthetase. The B. lactofermentum enzyme showed the highly conserved motifs of class I aminoacyl-tRNA synthetases. Expression of the argS gene in B. lactofermentum and E. coli resulted in an increase in aminoacyl-tRNA synthetase activity, correlated with the presence in sodium dodecyl sulfate-polyacrylamide gels of a clear protein band that corresponds to this enzyme. One single transcript of about 3,000 nucleotides and corresponding to the B. lactofermentum argS-lysA operon was identified. The transcription of these genes is repressed by lysine and induced by arginine, showing an interesting pattern of biosynthetic interlock between the pathways of both amino acids in corynebacteria.

Two genes, hom (encoding homoserine dehydrogenase) and thrB (encoding homoserine kinase), of the threonine biosynthetic pathway are clustered in the chromosome of Brevibacterium lactofermentum in the order 5' hom-thrB 3', separated by only 10 bp. The Brevibacterium thrB gene is expressed in Escherichia coli, in Brevibacterium lactofermentum, and in Corynebacterium gluamicum and complements auxotrophs of all three organisms deficient in homoserine kinase, whereas the Brevibacterium hom gene did not complement two different E. coil auxotrophs lacking homoserine dehydrogenase. However, complementation was obtained when the homoserine dehydrogenase was expressed as a fusion protein in E. coil. Northern (RNA) analysis showed that the hom-thrB cluster is transcribed, giving two diferent transcripts of 2.5 and 1.1 kb. The 2.5-kb transcript corresponds to the entire cluster hom-thrB (i.e., they form a bicistronic operon), and the short transcript (1.1 kb) originates from the thrB gene. The promoter in front of hom and the hom-internal promoter in front of thrB were subcloned in promoter-probe vectors of E. col and corynebacteria. The thrB promoter is efficiently recognized both in E. coil and corynebacteria, whereas the hom promoter is functional in corynebacteria but not in E. coil. The transcription start points of both promoters have been identified by primer extension and S1 mapping analysis. The thrB promoter was located in an 87-bp fragment that overlaps with the end of the hom gene. A functional transcriptional terminator located downstream from the cluster was subcloned in terminator-probe vectors.Threonine is synthesized from aspartic acid in five enzymatic reactions. The initial two reactions which convert aspartic acid to aspartate-13-semialdehyde are common to the lysine pathway. Conversion of aspartate-p-semialdehyde into homoserine (catalyzed by homoserine dehydrogenase [HD]) is common for threonine and methionine biosynthesis. Homoserine is converted into threonine by the action of two other enzymes, homoserine kinase (HK) and threonine synthase (TS).In Escherichia coli, the genes encoding four of the five enzyme activities involved in threonine biosynthesis are clustered together in the thrABC operon. The thrA gene encodes the bifunctional enzyme aspartokinase-HD (AKI-HDI), whereas thrB and thrC encode HK and TS, respectively (7,20). In corynebacteria there is one monofunctional HD (17), whereas in E. coli there are two isoenzymes (HDI and HDII, respectively) that form part of bifunctional polypeptides AKI-HDI and AKII-HDII.We previously cloned and sequenced the Brevibacterium lactofermentum hom gene encoding the monofunctional HD (26) and the thrB gene encoding HK (24,25) and showed that both are clustered. The thrC gene encoding TS was found to be at a separate position in the chromosome (21). An arrangement of the three genes identical to that in B. lactofermentum was found in Corynebacterium glutamicum (12,13,32). These two microorganisms are closely related nonpathogenic corynebacteria (7a). The nuc...