N sensitive and is only considered active under anaerobic conditions [36]. The presence of genes predicted to encode PFL or genes that resemble the PDH-genes of other organisms does not preclude that a species still depends on Met-Enkephalin site pyruvate oxidase under aerobic conditions for the production of C2 and C1 components, analogous to what we concluded for L. johnsonii. Clear data to support this hypothesis are lacking, although CO2 dependency of L. plantarum was also reported to cause a characteristic 
growth stagnation under aerobic conditions [19]. In addition, another study showed that a pyruvate-oxidase deficient mutant of L. plantarum is hampered in its acetate production capacity [37,38], supporting the role of this enzyme in aerobic acetate supply in lactobacilli that have a broader genetic arsenal. The effect of deletion of pox in L. johnsonii confirms the role of POX in the generation of both C1 and C2 sources (CO2 and acetate) required for growth. However, a byproduct of pyruvate oxidation by POX is hydrogen peroxide, of which the accumu-lation induces oxidative stress that leads to premature growth arrest under aerobic conditions [24]. This brings us to the intriguing conclusion that oxygen appears to both benefit and harm L. johnsonii. Under aerobic conditions, clearly, a lower biomass yield is reached (Supplemental material, Figure S1) on the one hand, presumably as a consequence of hydrogen peroxide production. On the other, our data also establish clearly that oxygen can increase the metabolic capacity of the strain, relieving some of its fastidious growth requirements. These opposing consequences of oxygen presence suggest that a micro-aerobic environment may be optimal for growth of L. johnsonii NCC 533. Here we have refined 
the metabolic requirements of L. johnsonii NCC 533 and pinpointed the pivotal role of the pox gene in the requirement for C1 and C2 sources. These findings can provide novel clues for the optimization of growth conditions of these commercially relevant microbes, and may in more general terms facilitate a more efficient regime for the production of probiotics belonging to this group of lactobacilli.Supporting 23727046 InformationFigure S1 Effect of catalase on aerobic growth. Figure 1: Growth of L. johnsonii NCC 533 in MRS medium supplemented with 0.5 mg/ml catalase (open symbols) and regular MRS medium (closed symbols) in either static tubes with limited headspace (round symbols) or in shake flasks (square symbols). Depicted are the averages of duplicate experiments 6 standard error of the mean. (TIF) Figure S2 Superimposed image of baclight-stained microcolonies. Composite picture in which images of colonies after 7 hours of growth in environments that vary in oxygen and CO2 content are grouped. Images were thresholded, colors were assigned artificially and superimposed as MK-8931 chemical information described in Materials Methods. (TIF) Table 15755315 S1 Presence of genes for pyruvate dehydrogenase or pyruvate formate lyase in Lactobacilli. Overview of pyruvate dehydrogenase and pyruvate formate lyase encoding gene prevalence in lactobacilli (Table A) and in species belonging to the Lactobacillus acidophilus group (Table 1B). If no gene was found, a BLAST search was performed using the protein sequence of the homologue in L. plantarum WCFS1. Shown are the query coverage and the e-value. (DOCX)AcknowledgmentsWe would like to thank the Molecular Cytology group at SILS, University of Amsterdam for letting us use the BX fluorescence microscope and in part.N sensitive and is only considered active under anaerobic conditions [36]. The presence of genes predicted to encode PFL or genes that resemble the PDH-genes of other organisms does not preclude that a species still depends on pyruvate oxidase under aerobic conditions for the production of C2 and C1 components, analogous to what we concluded for L. johnsonii. Clear data to support this hypothesis are lacking, although CO2 dependency of L. plantarum was also reported to cause a characteristic growth stagnation under aerobic conditions [19]. In addition, another study showed that a pyruvate-oxidase deficient mutant of L. plantarum is hampered in its acetate production capacity [37,38], supporting the role of this enzyme in aerobic acetate supply in lactobacilli that have a broader genetic arsenal. The effect of deletion of pox in L. johnsonii confirms the role of POX in the generation of both C1 and C2 sources (CO2 and acetate) required for growth. However, a byproduct of pyruvate oxidation by POX is hydrogen peroxide, of which the accumu-lation induces oxidative stress that leads to premature growth arrest under aerobic conditions [24]. This brings us to the intriguing conclusion that oxygen appears to both benefit and harm L. johnsonii. Under aerobic conditions, clearly, a lower biomass yield is reached (Supplemental material, Figure S1) on the one hand, presumably as a consequence of hydrogen peroxide production. On the other, our data also establish clearly that oxygen can increase the metabolic capacity of the strain, relieving some of its fastidious growth requirements. These opposing consequences of oxygen presence suggest that a micro-aerobic environment may be optimal for growth of L. johnsonii NCC 533. Here we have refined the metabolic requirements of L. johnsonii NCC 533 and pinpointed the pivotal role of the pox gene in the requirement for C1 and C2 sources. These findings can provide novel clues for the optimization of growth conditions of these commercially relevant microbes, and may in more general terms facilitate a more efficient regime for the production of probiotics belonging to this group of lactobacilli.Supporting 23727046 InformationFigure S1 Effect of catalase on aerobic growth. Figure 1: Growth of L. johnsonii NCC 533 in MRS medium supplemented with 0.5 mg/ml catalase (open symbols) and regular MRS medium (closed symbols) in either static tubes with limited headspace (round symbols) or in shake flasks (square symbols). Depicted are the averages of duplicate experiments 6 standard error of the mean. (TIF) Figure S2 Superimposed image of baclight-stained microcolonies. Composite picture in which images of colonies after 7 hours of growth in environments that vary in oxygen and CO2 content are grouped. Images were thresholded, colors were assigned artificially and superimposed as described in Materials Methods. (TIF) Table 15755315 S1 Presence of genes for pyruvate dehydrogenase or pyruvate formate lyase in Lactobacilli. Overview of pyruvate dehydrogenase and pyruvate formate lyase encoding gene prevalence in lactobacilli (Table A) and in species belonging to the Lactobacillus acidophilus group (Table 1B). If no gene was found, a BLAST search was performed using the protein sequence of the homologue in L. plantarum WCFS1. Shown are the query coverage and the e-value. (DOCX)AcknowledgmentsWe would like to thank the Molecular Cytology group at SILS, University of Amsterdam for letting us use the BX fluorescence microscope and in part.