The physiology of Lactobacillus plantarum at extremely low growth rates, through cultivation in retentostats, is much closer to carbon-limited growth than to stationary phase, as evidenced from transcriptomics data, metabolic fluxes, and biomass composition and viability.Using a genome-scale metabolic model and constraint-based computational analyses, amino-acid fluxes—in particular, the rather paradoxical excretion of Asp, Arg, Met, and Ala—could be rationalized as a means to allow extensive metabolism of other amino acids, that is, that of branched-chain and aromatic amino acids.Catabolic products from aromatic amino acids are known to have putative plant-hormone action. The metabolism of amino acids, as well as transcription data, strongly suggested a plant environment-like response in slow-growing L. plantarum, which was confirmed by significant effects of fermented medium on plant root formation.
Natural ecosystems are usually characterized by extremely low and fluctuating nutrient availability. Hence, microorganisms in these environments live a ‘feast-and-famine' existence, with famine the most habitual state. As a result, extremely slow or no growth is the most common state of bacteria, and maintenance processes dominate their life.
In the present study, Lactobacillus plantarum was used as a model microorganism to investigate the physiology of slow growth. Besides fermented foods, this microorganism can be observed in a variety of environmental niches, including plants and lakes, in which nutrient supply is limited. To mimic these conditions, L. plantarum was grown in a glucose-limited chemostat with complete biomass retention (retentostat). During cultivation, biomass progressively accumulated, resulting in steadily decreasing specific substrate availability. Less energy was thus available for growth, and the specific growth rate decreased accordingly, with a final calculated doubling time greater than one year. Detailed measurements of metabolic fluxes were used as constraints in a genome-scale metabolic model to precisely calculate the amount of energy used for net biomass synthesis and for maintenance purposes: at the lowest growth rate investigated (μ=0.0002 h−1), maintenance accounted for 94% of all energy expenses.
Genome-scale metabolic analysis was used in combination with transcriptomics to study the adaptation of L. plantarum to extremely slow growth under limited carbon and energy supply. Importantly, slow growth as investigated here was fundamentally different from the widely studied carbon starvation-induced stationary phase: non-growing cells in retentostat conditions were glucose limited rather than starved, and the transition from a growing to a non-growing state under retentostat conditions was progressive, in contrast with the abrupt transition in batch cultures. These differences were reflected in various aspects of the cell physiology.
The metabolic behavior was remarkably stable during adaptation to slow growth. Although carbon catabolite repression was clearly relieved, as indicated by the upregulation of genes for the utilization of alternative carbohydrates, the metabolism remained largely based on the conversion of glucose to lactate.
Stress resistance mechanisms were also not massively induced. In particular, analysis of the biomass composition—which remained similar to fast-growing cells even under virtually non-growing conditions—and of the gene expression profile, failed to reveal clear stringent or general stress responses, which are generally triggered in glucose-starved cells. The observation that genes involved in growth-associated processes were not downregulated suggested that active synthesis of biomass components (RNA, proteins, and membranes) was required to account for the observed stable biomass and that turnover of macromolecules was high in slow-growing cells. Biomass viability or morphology was also not affected, compared with faster growth conditions. The only typical stress response was the induction of an SOS response—in particular, the upregulation of the two error-prone DNA polymerases—suggesting an increased potential for genetic diversity under adverse conditions. Although diversity was not apparent under the conditions studied here, such mechanisms of increased rates of mutagenesis are likely to have an important role in the adaptation of L. plantarum to slow growth.
A surprising response of L. plantarum during adaptation to slow growth was the production of several amino acids (Arg, Asp, Met, and Ala). A priori, this metabolic behavior seemed inefficient in a context of energy limitation. However, reduced cost analysis using the genome-scale metabolic model indicated that it had a positive effect on energy generation. In-depth analysis of metabolic flux distributions showed that biosynthesis of these amino acids was connected to the catabolism of branched-chain and aromatic amino acids (BCAAs and AAAs), under conditions of limited ammonium efflux. At a fixed ammonium efflux—fixed at the measured value—flux balance analysis indicated that BCAAs and AAAs were expensive to metabolize, because the regeneration of 2-ketoglutarate through glutamate dehydrogenase was limited by ammonium dissipation. Therefore, alternative pathways had to be active to supply the necessary pool of 2-ketoglutarate. At low growth rates, amino-acid production (Arg, Asp, Ala, and Met) accounted for most of the 2-ketoglutarate regeneration. Although it came at the expense of ATP, this metabolic alternative to glutamate dehydrogenase was less energy costly than other solutions such as purine biosynthesis. This is thus an excellent example in which precise, quantitative modeling results in new insights in physiology that intuition would never have achieved. It also shows that flux balance analysis can be used to accurately predict energetically inefficient metabolism, provided the appropriate fluxes are constrained (here, ammonium efflux).
The observation that BCAAs and AAAs were catabolized at the expense of energy was intriguing. However, several end products of these catabolic pathways can serve as signaling molecules for interactions with other organisms. In particular, precursors of plant hormones were predicted as possible end products in the model simulations. Accordingly, the production of compounds interfering with plant root development was demonstrated in slow-growing L. plantarum. The metabolic analysis thus suggested that slow-growing L. plantarum produced plant hormones—or precursors thereof—as a strategy to divert the plant metabolism towards its own interest. In support of this view, transcriptome analysis indicated the upregulation of genes involved in the catabolism of β-glucosides—typical sugars from plant cell wall—as well as a very high induction of six gene clusters encoding cell-surface protein complexes predicted to have a role in the utilization of plant polysaccharides (csc clusters). In such a plant context, limited ammonium production would also make sense, because of the well-documented toxicity of ammonium for plants: production of amino acids could represent an alternative to ammonium excretion while keeping both parties satisfied.
In conclusion, the physiology of L. plantarum at extremely low growth rates, as studied by genome-scale metabolic modeling and transcriptomics, is fundamentally different from that of starvation-induced stationary phase cells. Excitingly, these conditions seem to trigger responses that favor interactions with the environment, more specifically with plants. The reported observations were made in the absence of any plant-derived material, suggesting that this response might constitute a hardwired behavior.
Situations of extremely low substrate availability, resulting in slow growth, are common in natural environments. To mimic these conditions, Lactobacillus plantarum was grown in a carbon-limited retentostat with complete biomass retention. The physiology of extremely slow-growing L. plantarum—as studied by genome-scale modeling and transcriptomics—was fundamentally different from that of stationary-phase cells. Stress resistance mechanisms were not massively induced during transition to extremely slow growth. The energy-generating metabolism was remarkably stable and remained largely based on the conversion of glucose to lactate. The combination of metabolic and transcriptomic analyses revealed behaviors involved in interactions with the environment, more particularly with plants: production of plant hormones or precursors thereof, and preparedness for the utilization of plant-derived substrates. Accordingly, the production of compounds interfering with plant root development was demonstrated in slow-growing L. plantarum. Thus, conditions of slow growth and limited substrate availability seem to trigger a plant environment-like response, even in the absence of plant-derived material, suggesting that this might constitute an intrinsic behavior in L. plantarum.