Characterization of Three Lactic Acid Bacteria and Their Isogenic ldh Deletion Mutants Shows Optimization for Y _ATP (Cell Mass Produced per Mole of ATP) at Their Physiological pHs

Abstract

Several lactic acid bacteria use homolactic acid fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three homolactic acid bacteria Lactococcus lactis, Enterococcus faecalis, and Streptococcus pyogenes. Of note, deletion of the ldh genes hardly affected the growth rate in chemically defined medium under microaerophilic conditions. However, the growth rate was affected in rich medium. Furthermore, deletion of ldh affected the ability for utilization of various substrates as a carbon source. A switch to mixed acid fermentation was observed during glucose-limited continuous growth and was dependent on the growth rate for S. pyogenes and on the pH for E. faecalis. In S. pyogenes and L. lactis, a change in pH resulted in a clear change in Y ATP (cell mass produced per mole of ATP). The pH that showed the highest Y ATP corresponded to the pH of the natural habitat of the organisms.Comparative analyses, as demonstrated by comparative genomics and bioinformatics, are extremely powerful for (i) transfer of information from (experimentally) well-studied organisms to other organisms and (ii) when coupled to functional and phenotypic information, insight into the relative importance of components to the observed differences and similarities. The central principle is that important aspects of the functional differences between organisms derive not only from the differences in genetic components (which underlies comparative genomics) but also from the interactions between their components. Although this type of analysis is much discussed, only a very few studies focus on cross-species comparisons.Here we study three relatively simple and highly related lactic acid bacteria (LAB) which nevertheless exhibit stark and important differences in their functional relationship with humans: these organisms are homofermentative lactic acid bacteria, namely, Lactococcus lactis, the major microorganism used in the dairy industry (21); Enterococcus faecalis, a major LAB in the human intestinal microbiota and a (fecal) contaminant in food and water as well as a contributor to food fermentation (16); and Streptococcus pyogenes, an important human pathogen (9, 15). These organisms have a similar primary metabolism but persist in completely different environments (milk, feces, skin/mucous membranes/blood).L. lactis is by far the best-studied lactic acid bacterium (3,13,14,23,24,30), and a kinetic model for its complete glycolysis, including some branching pathways, has been developed (2, 10). For the other two lactic acid bacteria, less information is readily available.Generally, observations made with L. lactis are quickly translated to other LAB. With this approach we are able to separate organism-specific observations from observations that are general for LAB.Here three LAB and their isogenic ldh deletion strains were characterized with respect to growth rates, catabolic flux distribution, ATP demand, and their ability to utilize different carbon sources. Our da...

Redox Balance via Lactate Dehydrogenase Is Important for Multiple Stress Resistance and Virulence in Enterococcus faecalis

“…This demonstrates that the loss of the characteristic metabolic reaction for lactic acid bacteria does not have an obvious impact on fitness under normal growth conditions in E. faecalis. Similar results have been obtained for Lactococcus lactis (8) and Streptococcus pyogenes (7), where little or no difference in the growth rate between the parent and ⌬ldh mutants has been observed. The growth yields were also comparable in E. faecalis when the strains grew under aerobic conditions, but final cell densities were even higher than those for the wild type when the ⌬ldh-1 mutant and the ⌬ldh-1 ⌬ldh-2 double mutant strain grew under anaerobic conditions (2).…”

“…Deficiency in either LDH-1, LDH-2, or both activities causes a significant increase in the levels of mixed acid fermentation end products in strain V583 (2,3,6). All LDH mutants showed growth rates similar to that of the parental strain (2,6,7). This demonstrates that the loss of the characteristic metabolic reaction for lactic acid bacteria does not have an obvious impact on fitness under normal growth conditions in E. faecalis.…”

Loss of Antibiotic Tolerance in Sod-Deficient Mutants Is Dependent on the Energy Source and Arginine Catabolism in Enterococci

“…We then tried the hexose gluconate as the energy source, but neither the JH2-2 strain nor the OG1RF strain grew significantly on this substrate. A similar result has been published for E. faecalis strain V583 (27). In the next step, we tested the pentose ribose, which is metabolized through the pentose phosphate pathway (Fig.…”

Engineering a Cyanobacterial Cell Factory for Production of Lactic Acid

“…Based in part on the earlier work carried out by our research group in this area (6,7,11) and on the catalytic properties of the respective enzymes, we selected a lactate dehydrogenase (ldh) gene from Escherichia coli (ldhA, encoding a D-LDH, EC 1.1.1.28 [41]), from Bacillus subtilis (ldh, encoding an L-LDH, EC 1.1.1.27 [32]), and from Lactococcus lactis (ldh, encoding an L-LDH, EC 1.1.1.27 [13,14]). The three genes were cloned using the "BioBrick" cloning strategy (19) and subsequently placed in (separate) integration vectors to achieve stable chromosomal incorporation of these genes within the neutral docking site slr0168 in Synechocystis sp.…”