Effect of glutamate synthase (GOGAT) activity on Bacillus subtilis spore properties.
The role of glutamate as osmoprotector was investigated through the study of a mutation in its biosynthetic pathway. A glt::Tn917-lacZ-cat insertion mutant (N1) conferring glutamate auxotrophy and enhanced beta-galactosidase expression on high-salt media was selected. Co-transformation experiments and PCR analysis allowed locating the insertion into the gltB gene corresponding to the small unit of the glutamate synthase (GOGAT). The N1 mutant strain presented a glutamate requirement for growth and a tenfold decrease in GOGAT activity. Transcriptional activity of GOGAT, measured as beta-galactosidase from the transposon fusion, correlated with enzymatic activity; expression was enhanced at the stationary phase and in high-ionic-strength media. However, osmotolerance of cultures of N1 mutant were as wild-type (wt), at least in semi-rich medium. In contrast, sporulation was slightly reduced (75% of wt), and spores were less resistant to UV, heat, and osmolarity, properties linked to the content of small, acid-soluble proteins (SASP). The content of these proteins was, in fact, reduced, in particular the SASP-gamma type. The peptidoglycan-cortex, however, was not impaired since spores maintained lysozyme resistance. Addition of glutamate during sporulation partially rescued spore resistance, but germination and outgrowth remained impaired. Deficiencies in germination and outgrowth were also observed with spores from a gltA mutant strain. Taken together, these results pointed to the importance of GOGAT activity during sporulation, in particular for the synthesis SASPs.
Ruzal
Sandra M
SM
Facultad de Ciencias Exactas y Naturales, Departamento de Quimica Biologica, Ciudad Universitaria, Pabellon II Piso 4, (1428) Buenos Aires, Argentina. sandra@qb.fcen.uba.ar
Sanchez-Rivas
Carmen
C
D019677:Artificial Gene Fusion; D001412:Bacillus subtilis / Q000201:enzymology / Q000502:physiology*; D001426:Bacterial Proteins / Q000096:biosynthesis; D004251:DNA Transposable Elements; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005970:Glutamate Synthase / Q000235:genetics* / Q000378:metabolism*; D018698:Glutamic Acid / Q000096:biosynthesis; D009113:Muramidase / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009997:Osmotic Pressure; D010457:Peptidoglycan / Q000096:biosynthesis; D013171:Spores, Bacterial / Q000254:growth & development*; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D010457:Peptidoglycan; C030069:spore-specific proteins, Bacillus; D018698:Glutamic Acid; D005970:Glutamate Synthase; D009113:Muramidase; D001616:beta-Galactosidase