Metabolic and Transcriptional Analysis of Acid Stress in Lactococcus lactis, with a Focus on the Kinetics of Lactic Acid Pools

• Type of Material: Article
• Authors: Ana Lúcia Carvalho, Universidade Nova de Lisboa; David L. Turner, Universidade Nova de Lisboa; Luís L. Fonseca, Universidade Nova de Lisboa; Ana Solopova, University of Groningen; Teresa Catarino, Universidade Nova de Lisboa; Oscar P. Kuipers, University of Groningen; Eberhard Otto Voit, Emory University; Ana Rute Neves, Universidade Nova de Lisboa; Helena Santos, Universidade Nova de Lisboa
• Language: English
• Date: 2013-07-03
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: © 2013 Carvalho et al
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.pone.0068470
• Title of Journal or Parent Work: PLoS ONE
• ISSN: 1932-6203
• Volume: 8
• Issue: 7
• Start Page: e68470
• End Page: e68470
• Grant/Funding Information: AL Carvalho and LL Fonseca were supported by fellowships (SFRH/BD/30419/2006 and SFRH//BPD/26902/2006) from Fundação para a Ciência e a Tecnologia (FCT). The NMR spectrometers are part of the National NMR Network (REDE/1517/RMN/2005), supported by "Programa Operacional Ciência e Inovação (POCTI) 2010” and FCT.; This work was funded in part by the U.S. National Science Foundation Project MCB-0946595, Principal Investigator EO Voit, and by FCT Project PTDC/EBB-EBI/113727/2009 (to AR Neves).; The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
• Keywords: CREMORIS; SUBSP LACTIS; ESCHERICHIA-COLI; STREPTOCOCCUS-LACTIS; TRANSPORT; IN-VIVO; LOW PH; SYSTEM; NUCLEAR-MAGNETIC-RESONANCE; Science & Technology; PROTONMOTIVE FORCE; Science & Technology - Other Topics; Multidisciplinary Sciences
• Research Categories: Biology, Genetics; Biology, Microbiology

Abstract

The effect of pH on the glucose metabolism of non-growing cells of L. lactis MG1363 was studied by in vivo NMR in the range 4.8 to 6.5. Immediate pH effects on glucose transporters and/or enzyme activities were distinguished from transcriptional/translational effects by using cells grown at the optimal pH of 6.5 or pre-adjusted to low pH by growth at 5.1. In cells grown at pH 5.1, glucose metabolism proceeds at a rate 35% higher than in non-adjusted cells at the same pH. Besides the upregulation of stress-related genes (such as dnaK and groEL), cells adjusted to low pH overexpressed H + -ATPase subunits as well as glycolytic genes. At sub-optimal pHs, the total intracellular pool of lactic acid reached approximately 500 mM in cells grown at optimal pH and about 700 mM in cells grown at pH 5.1. These high levels, together with good pH homeostasis (internal pH always above 6), imply intracellular accumulation of the ionized form of lactic acid (lactate anion), and the concomitant export of the equivalent protons. The average number, n, of protons exported with each lactate anion was determined directly from the kinetics of accumulation of intra- and extracellular lactic acid as monitored online by 13 C-NMR. In cells non-adjusted to low pH, n varies between 2 and 1 during glucose consumption, suggesting an inhibitory effect of intracellular lactate on proton export. We confirmed that extracellular lactate did not affect the lactate: proton stoichiometry. In adjusted cells, n was lower and varied less, indicating a different mix of lactic acid exporters less affected by the high level of intracellular lactate. A qualitative model for pH effects and acid stress adaptation is proposed on the basis of these results.