Regulation of Acetate Kinase Isozymes and Its Importance for MixedAcid Fermentation in Lactococcus lactis
Puri, P. ; Goel, A. ; Bochynska, A. ; Poolman, B. - \ 2014
Journal of Bacteriology 196 (2014)7. - ISSN 0021-9193 - p. 1386 - 1393.
streptococcus-lactis - escherichia-coli - methanosarcina-thermophila - lysine acetylation - membrane-proteins - product formation - light-scattering - in-vivo - metabolism - phosphate
Acetate kinase (ACK) converts acetyl phosphate to acetate along with the generation of ATP in the pathway for mixed-acid fermentation in Lactococcus lactis. The reverse reaction yields acetyl phosphate for assimilation purposes. Remarkably, L. lactis has two ACK isozymes, and the corresponding genes are present in an operon. We purified both enzymes (AckA1 and AckA2) from L. lactis MG1363 and determined their oligomeric state, specific activities, and allosteric regulation. Both proteins form homodimeric complexes, as shown by size exclusion chromatography and static light-scattering measurements. The turnover number of AckA1 is about an order of magnitude higher than that of AckA2 for the reaction in either direction. The K-m values for acetyl phosphate, ATP, and ADP are similar for both enzymes. However, AckA2 has a higher affinity for acetate than does AckA1, suggesting an important role under acetate-limiting conditions despite the lower activity. Fructose-1,6-bisphosphate, glyceraldehyde- 3-phosphate, and phospho-enol-pyruvate inhibit the activities of AckA1 and AckA2 to different extents. The allosteric regulation of AckA1 and AckA2 and the pool sizes of the glycolytic intermediates are consistent with a switch from homolactic to mixed-acid fermentation upon slowing of the growth rate.
Quantitative physiology of Lactococcus lactis at extreme low-growth rates
Ercan, O. ; Smid, E.J. ; Kleerebezem, M. - \ 2013
Environmental Microbiology 15 (2013)8. - ISSN 1462-2912 - p. 2319 - 2332.
maintenance energy - subsp lactis - streptococcus-cremoris - continuous-culture - product formation - stationary-phase - acid bacteria - carbohydrate starvation - enterococcus-faecalis - stress resistance
This paper describes the metabolic adaptation of Lactococcus lactis during the transition from a growing to a non-growing state using retentostat cultivation. Under retentostat cultivation, the specific growth rate decreased from 0.025 h-1 to 0.0001 h-1 in 42 days, while doubling time increased to more than 260 days. Viability of the overall culture was maintained above 90% but included approximately 20% damaged cells, which had lost their colony forming capacity on solid media. Although culture biomass and viability had reached a steady-state after 14 days of retentostat cultivation, the morphology of the cells changed from coccus-to-rod shape at later stages of retentostat cultivation, by which the cell’s surface to volume ratio was estimated to increase 2.4-fold. Furthermore, the metabolic patterns switched between homolactic and mixed-acid fermentation during the retentostat cultivation. Retentostat cultivation enabled the calculation of accurate substrate- and energy-related maintenance coefficients and biomass yields under nongrowing conditions, which were in good agreement with those calculated by extrapolation from chemostat cultivations at high dilution rates. In this study, we illustrate how retentostat cultivation allows decoupling of growth and non-growth associated processes in L. lactis, enabling the analysis of quantitative physiological responses of this bacterium to near zero-specific growth rates.