Pascal Loubiere

Respiration Capacity of the Fermenting Bacterium Lactococcus lactis and Its Positive Effects on Growth and Survival

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.

Kinetic analysis of red pigment and citrinin production by Monascus ruber as a function of organic acid accumulation.

In submerged cultures performed in synthetic medium containing glucose and glutamate, the filamentous fungus Monascus ruber produced a red pigment and a mycotoxin, citrinin. In oxygen-limiting conditions, the production of these two metabolites was growth-associated, as was the production of primary metabolites. In oxygen-excess conditions, the profile of citrinin production was typical of a secondary metabolite, since it was produced mostly during the stationary phase. In contrast, the production of the pigment decreased rapidly throughout the culture, showing a profile characteristic of an inhibitory mechanism. The organic acids produced during the culture, L-malate and succinate, were shown to be slightly inhibitory against pigment production, while citrinin production was unaffected. However, this inhibition could not account for the observed profile of pigment production in batch cultures. Other dicarboxylic acids such as fumarate or tartrate showed a similar effect to that provoked by malate and succinate as regards pigment production. It was concluded that the decrease in red pigment production during the culture was due to the inhibitory effect of an unknown product whose accumulation was favored in aerobic conditions.

Dynamic response of catabolic pathways to autoacidification in Lactococcus lactis : transcript profiling and stability in relation to metabolic and energetic constraints

Summary The dynamic response of the central metabolic pathways to autoacidification (accumulation of organic acid fermentation products) in Lactococcus lactis was investigated in a global manner by integrating molecular data (cellular transcript concentrations, mRNA turnover) within physiological investigations of metabolic and energetic parameters. The decrease in pH associated with the accumulation of organic acids modified the physiological state of the cell considerably. Cytoplasmic acidification led to inhibition of enzyme activities and, consequently, to a diminished catabolic flux through glycolysis and a decreased rate of biochemical energy synthesis. This decrease in energy production together with the increased energy expenditure to counter cytoplasmic acidification led to energetic limitations for biomass synthesis. In these conditions, the specific growth rate decreased progressively, and growth ultimately stopped, although a diminished catabolic flux was maintained in the absence of growth. The cellular response to this phenomenon was to maintain significant levels of mRNA of catabolic genes, involving both continued transcription of the genes and also, in certain cases, an increase in transcript stability. Thus, translation was maintained, and intracellular concentration of certain enzymes increased, partially compensating for the inhibition of activity provoked by the diminished pH. When catabolic activity ceased after prolonged exposure to stress‐induced stationary phase, endogenous RNA catabolism was observed.

Staphylococcus aureus Virulence Expression Is Impaired by Lactococcus lactis in Mixed Cultures

ABSTRACT Staphylococcus aureus is responsible for numerous food poisonings due to the production of enterotoxins by strains contaminating foodstuffs, especially dairy products. Several parameters, including interaction with antagonistic flora such as Lactococcus lactis, a lactic acid bacterium widely used in the dairy industry, can modulate S. aureus proliferation and virulence expression. We developed a dedicated S. aureus microarray to investigate the effect of L. lactis on staphylococcal gene expression in mixed cultures. This microarray was used to establish the transcriptomic profile of S. aureus in mixed cultures with L. lactis in a chemically defined medium held at a constant pH (6.6). Under these conditions, L. lactis hardly affected S. aureus growth. The expression of most genes involved in the cellular machinery, carbohydrate and nitrogen metabolism, and stress responses was only slightly modulated: a short time lag in mixed compared to pure cultures was observed. Interestingly, the induction of several virulence factors and regulators, including the agr locus, sarA, and some enterotoxins, was strongly affected. This work clearly underlines the complexity of L. lactis antagonistic potential for S. aureus and yields promising leads for investigations into nonantibiotic biocontrol of this major pathogen.

Transcriptome Analysis of the Progressive Adaptation of Lactococcus lactis to Carbon Starvation

Adaptation of Lactococcus lactis towards progressive carbon starvation is mediated by three different types of transcriptomic responses: (i) global responses, i.e., general decreases of functions linked to bacterial growth and lack of induction of the general stress response; (ii) specific responses functionally related to glucose exhaustion, i.e., underexpression of central metabolism genes, induction of alternative sugar transport and metabolism, and induction of the arginine deiminase pathway; and (iii) other responses never described previously during carbon starvation.

Metabolic and Transcriptomic Adaptation of Lactococcus lactis subsp. lactis Biovar diacetylactis in Response to Autoacidification and Temperature Downshift in Skim Milk

ABSTRACT For the first time, a combined genome-wide transcriptome and metabolic analysis was performed with a dairy Lactococcus lactis subsp. lactis biovar diacetylactis strain under dynamic conditions similar to the conditions encountered during the cheese-making process. A culture was grown in skim milk in an anaerobic environment without pH regulation and with a controlled temperature downshift. Fermentation kinetics, as well as central metabolism enzyme activities, were determined throughout the culture. Based on the enzymatic analysis, a type of glycolytic control was postulated, which was shared by most of the enzymes during the growth phase; in particular, the phosphofructokinase and some enzymes of the phosphoglycerate pathway during the postacidification phase were implicated. These conclusions were reinforced by whole-genome transcriptomic data. First, limited enzyme activities relative to the carbon flux were measured for most of the glycolytic enzymes; second, transcripts and enzyme activities exhibited similar changes during the culture; and third, genes involved in alternative metabolic pathways derived from some glycolytic metabolites were induced just upstream of the postulated glycolytic bottlenecks, as a consequence of accumulation of these metabolites. Other transcriptional responses to autoacidification and a decrease in temperature were induced at the end of the growth phase and were partially maintained during the stationary phase. If specific responses to acid and cold stresses were identified, this exhaustive analysis also enabled induction of unexpected pathways to be shown.

Influence of end‐products inhibition and nutrient limitations on the growth of Lactococcus lactis subsp. lactis

Lactococcus lactis was grown in a simple synthetic medium with glucose as substrate, enabling the precise quantification of each nutrients contribution to growth. As expected, for the growth of lactic acid bacteria, the growth rate decreased progressively during the cultivation after a short period of exponential growth. End‐products of fermentation, predominantly lactate and in minor amounts formate, acetate and ethanol, accumulated within the medium. Growth of the bacterium in fresh media supplemented with these end‐products showed that the concentrations attained in the fermentor had no significant influence on the growth rate. As regards nutrients, vitamins and magnesium were never limiting during the culture. On the other hand, amino acid concentrations decreased, some of them being totally consumed and exhausted from the medium before growth ceased. However, growth in reconstituted media constructed with the amino acid concentrations remaining at different times of cultivation showed that amino acid depletion could not account for the observed growth decrease. Batch culture supernatant fluid was used as cultivation medium. Growth rates observed in supernatant cultures supplemented with various nutrients, compared to non‐supplemented supernatant, showed that no addition improved growth. Finally, it was concluded that in the experimental conditions used in this study, growth inhibition was predominantly due to phenomena other than lactate inhibition and nutritional limitations, and hence associated with unidentified compounds produced in the fermentation.

Anaerobic sugar catabolism in Lactococcus lactis: genetic regulation and enzyme control over pathway flux.

Abstract. Lactic acid bacteria and particularly Lactococcus lactis are widely used for the production of lactic acid in fermented foods. Control of the catabolic rate in L. lactis, i.e., the rate of lactic acid production, appears to be determinant for dairy product quality. While the mechanisms involved in control have not been totally elucidated, they seem to depend upon the strain and the growth conditions. Furthermore, it remains unclear whether the catabolic rate is controlled at the level of transcription, translation or enzyme activity. The recent sequencing of the L. lactis genome has brought novel insights to physiologic studies of the bacteria. This review discusses both genetic information and metabolic studies concerning anaerobic sugar catabolism in L. lactis.

Transcriptome Analysis of Lactococcus lactis in Coculture with Saccharomyces cerevisiae

ABSTRACT The study of microbial interactions in mixed cultures remains an important conceptual and methodological challenge for which transcriptome analysis could prove to be the essential method for improving our understanding. However, the use of whole-genome DNA chips is often restricted to the pure culture of the species for which the chips were designed. In this study, massive cross-hybridization was observed between the foreign cDNA and the specific Lactococcus lactis DNA chip. A very simple method is proposed to considerably decrease this nonspecific hybridization, consisting of adding the microbial partners DNA. A correlation was established between the resulting cross-hybridization and the phylogenetic distance between the microbial partners. The response of L. lactis to the presence of Saccharomyces cerevisiae was analyzed during the exponential growth phase in fermentors under defined growth conditions. Although no differences between growth kinetics were observed for the pure and the mixed cultures of L. lactis, the mRNA levels of 158 genes were significantly modified. More particularly, a strong reorientation of pyrimidine metabolism was observed when L. lactis was grown in mixed cultures. These changes in transcript abundance were demonstrated to be regulated by the ethanol produced by the yeast and were confirmed by an independent method (quantitative reverse transcription-PCR).

Transcriptomic response of Lactococcus lactis in mixed culture with Staphylococcus aureus.

ABSTRACT The mechanisms of interaction between Lactococcus lactis and the food pathogen Staphylococcus aureus are of crucial importance, as one major role of lactic acid bacteria (LAB) in fermented foods is to inhibit undesirable and pathogenic flora. It was never questioned if the presence of a pathogen can actively modify the gene expression patterns of LAB in a shared environment. In this study, transcriptome and biochemical analyses were combined to assess the dynamic response of L. lactis in a mixed culture with S. aureus. The presence of S. aureus hardly affected the growth of L. lactis but dramatically modified its gene expression profile. The main effect was related to earlier carbon limitation and a concomitantly lower growth rate in the mixed culture due to the consumption of glucose by both species. More specific responses involved diverse cellular functions. Genes associated with amino acid metabolism, ion transport, oxygen response, menaquinone metabolism, and cell surface and phage expression were differentially expressed in the mixed culture. This study led to new insights into possible mechanisms of interaction between L. lactis and S. aureus. Moreover, new and unexpected effects of L. lactis on the virulence of S. aureus were discovered, as described elsewhere (S. Even, C. Charlier, S. Nouaille, N. L. Ben Zakour, M. Cretenet, F. J. Cousin, M. Gautier, M. Cocaign-Bousquet, P. Loubière, and Y. Le Loir, Appl. Environ. Microbiol. 75:4459-4472, 2009).