Maximillian T. Follettie

Nucleotide sequence and fine structural analysis of the Corynebacterium glutamicum hom-thrB operon.

The complete nucleotide sequence of the Corynebacterium glutamicum hom‐thrB operon has been determined and the structural genes and promoter region mapped. A polypeptide of Mr 46136 is encoded by hom and a polypeptide of Mr, 32618 is encoded by thrB. Both predicted protein sequences show amino acid sequence homology to their counterparts in Escherichia coli and Bacillus subtilis. The promoter region has been mapped by S1‐nuclease and deletion analysis. Located between −88, RNA start site and −219 (smallest deletion clone with complete activity) are sequence elements similar to those found in E. coli and B. subtills promoters. Although there are no obvious attenuator‐like structures in the 5′‐untranslated region, there is a dyad‐symmetry element, which may act as an operator.

Organization and regulation of the Corynebacterium glutamicum hom-thrB and thrC loci

The genes encoding the three terminal enzymes in the threonine biosynthetic pathway, homoserine dehydrogenasa (hom), homoserine kinase (thrB) and threonine synthase (thrC) have been isolated from Corynebacterium glutamicum. The C. glutamicum hom and thrB genes were subcloned on a 3.6 kb Sal l‐generated chromosomal fragment. The C. glutamicum thrC gene was shown not to be linked to the hom‐thrB locus. L‐methionine represses the cloned homoserine dehydrogenase and homoserine kinase similar to that of the chromosomally encoded hom and thrB gene products. Northern hybridization analysis demonstrates that this repression is mediated at the level of transcription and that hom‐thrB represents an operon in C. glutamicum.

Regulation of phospho(enol)-pyruvate-and oxaloacetate-converting enzymes in Corynebacterium glutamicum

The presence and properties of the enzymes involved in the synthesis and conversion of phospho(enol)pyruvate (PEP) and oxaloacetate (OAA), the precursors for aspartate-derived amino acids, were investigated in three different Corynebacterium strains. This study revealed the presence of both PEP carboxykinase 0.29 μmol·min−1·mg−1 of protein [units (U)·mg−1] and PEP synthetase (0.13 U·mg−1) in C. 2 glutamicum as well as pyruvate kinase (1.4 U·mg−1) and PEP carboxylase (0.16 U·mg−1). With the exception of PEP carboxykinase these activities were also present in glucose-grown C. flavum and C. lactofermentum. Pyruvate carboxylase activity was not detected in all three species cultivated on glucose or lactate. At least five enzyme activities that utilize OAA as a substrate were detected in crude extracts of C. glutamicum: citrate synthase (2 U·mg−1), malate dehydrogenase (2.5 U·mg−1), glutamate: OAA transaminase (1 U·mg−1), OAA-decarboxylating activity (0.89 U·mg−1) and the previously mentioned PEP carboxykinase (0.29 U·mg−1). The partially purified OAA-decarboxylase activity of C. glutamicum was completely dependent on the presence of inosine diphosphate and Mn2+, had a Michaelis constant (Km) of 2.0mm for OAA and was inhibited by ADP and coenzyme A (CoA). Examination of the kinetic properties showed that adenine nucleotides and CoA derivatives have reciprocal but reinforcing effects on the enzymes catalyzing the interconversion of pyruvate, PEP and OAA in C. glutamicum. A model for the regulation of the carbon flow based on these findings is presented.

Effect of different levels of aspartokinase of the lysine production by Corynebacterium lactofermentum

A 2.9-kb SacI fragment containing the ask-asd operon, encoding aspartokinase and aspartate-semialdehyde dehydrogenase, was cloned from an aminoethylcysteine-resistant, lysine-producing Corynebacterium lactofermentum strain. Enzymatic analysis showed that the aspartokinase (ASK) activity was completely resistant to inhibition by mixtures of lysine and threonine. Comparison of the deduced amino acid sequence of the β submit of the ask gene showed three amino acid residue changes with ask genes encoding wild-type, feedback-sensitive enzymes. Three C. lactofermentum strains, one being aspartokinase-negative, one carrying two ask genes on the chromosome and one having a sixfold higher specific ASK activity than the parental strain, were constructed by transconjugation and electroporation, and used to analyse the role of ASK in the lysine production by C. lactofermentum. The results indicate that, in this study, feed-back-resistant ASK is necessary for high-level lysine production, but dispensable for lysine and diaminopimelate synthesis required for cell growth.

Genetic engineering of coryneform bacteria

Abstract The coryneforms are a diverse group of bacteria which includes animal and plant pathogens as well as non-pathogenic bacteria. Although they are of significant economic and health importance, their genetics is poorly understood. The development of genetic engineering techniques for coryneforms and initial gene cloning studies are discussed.