Respiration capacity of the fermenting bacterium Lactococcus lactis and its positive effects on growth and survival

Abstract

Oxygen is a major determinant of both survival and mortality of aerobic organisms. For the facultative anaerobe Lactococcus lactis, oxygen has negative effects on both growth and survival. We show here that oxygen can be beneficial to L. lactis if heme is present during aerated growth. The growth period is extended and long-term survival is markedly improved compared to results obtained under the usual fermentation conditions. We considered that improved growth and survival could be due to the capacity of L. lactis to undergo respiration. To test this idea, we confirmed that the metabolic behavior of lactococci in the presence of oxygen and hemin is consistent with respiration and is most pronounced late in growth. We then used a genetic approach to show the following. (i) The cydA gene, encoding cytochrome d oxidase, is required for respiration and plays a direct role in oxygen utilization. cydA expression is induced late in growth under respiration conditions. (ii) The hemZ gene, encoding ferrochelatase, which converts protoporphyrin IX to heme, is needed for respiration if the precursor, rather than the final heme product, is present in the medium. Surprisingly, survival improved by respiration is observed in a superoxide dismutase-deficient strain, a result which emphasizes the physiological differences between fermenting and respiring lactococci. These studies confirm respiratory metabolism in L. lactis and suggest that this organism may be better adapted to respiration than to traditional fermentative metabolism.

FIG. 1

Altered growth and pH of L. lactis grown under Ox/H conditions. An overnight 30°C MG1363 culture in M17-glu was used to inoculate media (at a 1/1,000 dilution) for growth and pH measurements under four conditions at 30°C: Ox/H, No Ox/H, Ox/No H, and No Ox/No H. As growth curves and final pHs for the No Ox/H, Ox/No H, and No Ox/No H conditions were similar, only data for the No Ox/No H (open squares; solid line for growth, broken line for pH) and Ox/H (closed circles; solid line for growth, broken line for pH) conditions are shown. Growth of all cultures was saturated at 24 h. Experiments were performed six times and yielded comparable results. Results of a representative experiment are shown.

FIG. 2

Improved long-term survival of L. lactis after growth under Ox/H conditions. MG1363 cells were grown under four conditions at 30°C as described in the legend to Fig. 1: Ox/H (closed circles), No Ox/H (closed squares), Ox/No H (open circles), and No Ox/No H (open squares). After 24 h, cultures were transferred to 4°C. Cell viability was determined by plating dilutions on solid M17-glu at the indicated times. Experiments were performed four times and yielded comparable results. Results of a representative experiment are shown.

FIG. 3

Metabolic alterations in Ox/H cultures are greatest in late exponential phase. L. lactis MG1363 was grown aerobically in M17-glu in the presence or absence of hemin or anaerobically in the absence of hemin. Samples were taken at various intervals to monitor growth, glucose consumption, and lactate, acetate, acetoin, and diacetyl accumulation (see Materials and Methods). Determinations were performed at least twice, with a maximum deviation between measurements of 10%.

FIG. 4

Effects of cydA and sodA mutations on L. lactis long-term survival under respiration conditions. MG1363 and cydA and sodA mutants were grown at 30°C as described in the legend to Fig. 2 under Ox/H (closed circles), Ox/No H (open circles; for MG1363 and sodA strains), and No Ox/No H (open squares) conditions. After 24 h, cultures were transferred to 4°C. Cell viability was determined by plating dilutions on solid M17-glu at the indicated times. Experiments were performed at least twice; the results of a representative experiment are shown.

FIG. 5

Expression of cydA is induced under Ox/H conditions in late-exponential-phase cells. L. lactis strain MG1363 was grown under aeration and respiration conditions (see Materials and Methods), and samples were removed for total RNA extraction at different OD₆₀₀s during growth (times, from left to right, correspond to approximately 3, 4, 6, and 8 h after the start of growth). Northern blot analysis was performed using an internal cydA fragment as a probe. Membranes were subsequently dehybridized and rehybridized with a 16S rDNA probe to verify that the amounts deposited in the wells were identical. Northern blotting was performed three times and yielded results similar to those shown here.

FIG. 6

Heme-dependent oxygen consumption by L. lactis is abolished in the mutant cydA strain. Aerated cultures of MG1363 and the cydA mutant, grown with or without hemin (H), were harvested after overnight growth. Oxygen consumption was evaluated with cells resuspended in saline buffer containing 1% glucose (see Materials and Methods). Oxygen remaining in the medium was measured using an Oxymeter. Ox/H and Ox/No H conditions are represented by closed and open symbols, respectively. Duplicate samples showed less than a 10% difference in measurements. Experiments were performed twice and yielded comparable results.