Nitrogen and carbon regulation of glutamine synthetase and glutamate synthase in Corynebacterium glutamicum ATCC 13032.

The effect of nitrogen and carbon status on the regulation of glutamine synthetase (GS) and glutamate synthase (GOGAT) were investigated in Corynebacterium glutamicum 13032. Under carbon-sufficient, nitrogen-limiting conditions, GS and GOGAT activities were five- and seven-fold higher, respectively, and transcription of the corresponding genes (glnA and gltBD) was similarly induced. GS activity was also induced in complete medium with added glucose, while GOGAT activity was unaffected. Under carbon-limiting, nitrogen-limiting conditions, the level of GS induction was reduced approximately three-fold, whereas GOGAT activity did not respond. Disruption of the hkm gene, encoding a putative histidine kinase upstream of gltBD, reduced the levels of GOGAT activity two-fold under both nitrogen-rich and nitrogen-limiting conditions. Promoter studies using a hkm-chloramphenicol acetylase fusion plasmid revealed that transcription of hkm is moderately induced (ca. 1.5-fold) by nitrogen starvation, indicating that the Hkm protein may play a role in signal transduction of the nutritional status of the growth medium.

Characterisation of a transposon-induced pleiotropic mutant of Clostridium acetobutylicum P262.

Transposon-induced metronidazole resistance was used as a selection system for the isolation of Clostridium acetobutylicum P262 mutants with altered electron transport pathways. The metronidazole resistant transconjugant of interest, mutant 3R, displayed resistance to DNA damaging agents, UV and bleomycin, and harboured a single transposon insertion within a structural gene, designated sum(susceptibility to metronidazole). The sum gene encoded a 334 amino-acid protein, with 36% identity and 57-58% similarity at the amino acid level to two archaebacterial protein sequences which appear to represent a class of uncharacterised reductase enzymes. Physiological studies of mutant 3R revealed a number of pleiotropic characteristics which included enhanced autolysin activity, increased motility, impaired clostridial cell formation, and resistance to the toxic tripeptide analogue, bialaphos. The introduction of the sum gene in multiple copies on a plasmid vector into the related strain Clostridium beijerinckii NCIMB 8052, resulted in inhibition of cell division, motility and autolysin activity. The sum gene appears to be a member of a new subgroup of activases with reducing activity, which may control a regulon affecting different stationary phase processes such as clostridial differentiation and sporulation in C. acetobutylicum P262. The metronidazole resistant phenotype of the sum mutant can be attributed to an increased capacity for DNA repair.

Transposon mutagenesis of Clostridium acetobutylicum P262: isolation and characterization of solvent deficient and metronidazole resistant mutants.

An efficient transposon mutagenesis system using conjugative transposons Tn916 and Tn925::Tn917 was established for Clostridium acetobutylicum P262, an industrial strain which has proved difficult to manipulate genetically. Transposon insertions occurred at several different locations to produce a variety of mutants. An oligosporogenous mutant deficient in acetone and butanol production, and two sporulation-deficient and metronidazole resistant mutants were characterized with respect to differentiation and solvent production. Tn925::Tn917 inserted near a string of adenosine residues and transposon insertion was often multiple.