Lactobacillus brevis L62 evolution during prolonged stationary phase of growth

Cilj ovog rada bio je proučiti evoluciju bakterije mliječne kiseline Lactobacillus brevis L62 tijekom produljene stacionarne faza rasta. Tijekom šaržnog uzgoja u produljenoj stacionarnoj fazi rasta stanice su bile izložene nedostatku nutrijenata i niskoj pH vrijednosti. Adaptivne promjene koje uzrokuju nastanak mutanata s prednošću rasta u navedenim uvjetima stresa istražene su na razini genoma, proteoma i metaboloma. Istraživanja genoma pokazala su da tijekom produljene stacionarne faze rasta dolazi do genetičkih preraspodjela kromosoma u 50% 75 dana starih bakterijskih klonova te do gubitka najmanjeg plazmida kod 33% klonova. Istraživanja proteoma su ukazala na promjene koje imaju ključnu ulogu u adaptaciji stanica tijekom produljene stacionarne faze rasta. Navedene promjene se mogu podijeliti na četiri osnovne skupine: I) metabolizam ugljikohidrata, II) oksidoredukcijske reakcije u kojima sudjeluju koenzimi NAD(P)H, III) katabolizam alternativnih izvora energije i IV) odgovor stanica na stres. Istraživanjima metaboloma ispitan je utjecaj navedenih promjena u proteomu na metabolizam glukoze i metabolizam aminokiselina. Rezultati su pokazali da 75 dana stari klonovi proizvode manje mliječne kiseline u usporedbi sa stanicama divljeg tipa te da mutanti L. brevis L62 tijekom produljene stacionarne faze rasta metaboliziraju glicin, histidin i arginin kao alternativne izvore nutrijenata.The aim of this study was to investigate the evolution of lactic acid bacteria Lactobacillus brevis L62 during prolonged stationary phase of growth. During growth in batch system in prolonged stationary phase, the cells were stressed with starvation and low pH. Genomic, proteomic and metabolomic analyses were used to investigate adaptive changes responsible for mutant occuranse with growth advantage and survival in stressful conditions. Genome analyses pointed out genetic rearrangements in 50% of 75 day old clones and plasmid reduction in 33% 75 day old clones. Proteome investigation point to changes responsible for adaptation during growth in prolonged stationary phase of growth. The changes can be grouped in four main groups: i) carbohydrate metabolism, ii) energy metabolism, iii) alternative energy source catabolism and iv) response to stress. The effect of proteome changes to glucose metabolism and aminoacid metabolism was examined by matabolomic analyses. Results showed that 75 day old clones produce less lactic acid in comparison to parent cells and that L. brevis L62 started to metabolize glycine, histidine and arginine during long term incubation as alternative source of nutrients