Influence of oxygen on NADH recycling and oxidative stress resistance systems in Lactobacillus panis PM1

Abstract

Lactobacillus panis strain PM1 is an obligatory heterofermentative and aerotolerant microorganism that also produces 1,3-propanediol from glycerol. This study investigated the metabolic responses of L. panis PM1 to oxidative stress under aerobic conditions. Growth under aerobic culture triggered an early entrance of L. panis PM1 into the stationary phase along with marked changes in end-product profiles. A ten-fold higher concentration of hydrogen peroxide was accumulated during aerobic culture compared to microaerobic culture. This H2O2 level was sufficient for the complete inhibition of L. panis PM1 cell growth, along with a significant reduction in end-products typically found during anaerobic growth. In silico analysis revealed that L. panis possessed two genes for NADH oxidase and NADH peroxidase, but their expression levels were not significantly affected by the presence of oxygen. Specific activities for these two enzymes were observed in crude extracts from L. panis PM1. Enzyme assays demonstrated that the majority of the H2O2 in the culture media was the product of NADH: H2O2 oxidase which was constitutively-active under both aerobic and microaerobic conditions; whereas, NADH peroxidase was positively-activated by the presence of oxygen and had a long induction time in contrast to NADH oxidase. These observations indicated that a coupled NADH oxidase - NADH peroxidase system was the main oxidative stress resistance mechanism in L. panis PM1, and was regulated by oxygen availability. Under aerobic conditions, NADH is mainly reoxidized by the NADH oxidase - peroxidase system rather than through the production of ethanol (or 1,3-propanediol or succinic acid production if glycerol or citric acid is available). This system helped L. panis PM1 directly use oxygen in its energy metabolism by producing extra ATP in contrast to homofermentative lactobacilli.

Figure 1

Effect of oxygen and time on growth and end-product formation in L. panis PM1. Growth response (OD₆₀₀) and end-product formation of L. panis PM1 cultivated in mMRS under aerobic (a) and microaerobic (b) conditions. Samples for cell density and HPLC analysis were withdrawn from the cultures after 24, 48, 72 and 96 hours.

Figure 2

Effect of oxygen and time on the accumulation of hydrogen peroxide and growth by L. panis. Growth response (OD₆₀₀) and H₂O₂ production by L. panis PM1 cultured in mMRS under aerobic (circles) and microaerobic (triangles) conditions after 24, 48, and 72 hours (a). The inhibitory concentrations of H₂O₂ were determined from L. panis PM1 cultures grown in MRS broth containing H₂O₂ at concentration of 0, 30, 60, 120, 240, and 480 μM for 2 days under microaerobic conditions. Optical density was measured at 600 nm with a spectrophotometer (b).

Figure 3

Effect of oxygen availability on accumulation of hydrogen peroxide in L. panis PM1 culture media. H₂O₂ production by L. panis PM1 cultured in mMRS in 15 ml conical tubes with 3 (circles), 6 (squares), and 9 (triangles) ml mMRS, respectively, under aerobic conditions. H₂O₂ concentration was measured at 6, 12, and 24 hours.

Figure 4

Effect of external electron acceptors on end-product formation by L. panis PM1. End-product formation by L. panis PM1 cultured in mMRS containing either 24 mM citric acid (a) or 160 mM glycerol (b) as external electron acceptor under aerobic conditions. Samples for HPLC analysis were withdrawn from the cultures after 24 and 48 hours.