Investigation of the adaptation of Lactococcus lactis to isoleucine starvation integrating dynamic transcriptome and proteome information

Abstract

Background: Amino acid assimilation is crucial for bacteria and this is particularly true for Lactic Acid Bacteria (LAB) that are generally auxotroph for amino acids. The global response of the LAB model Lactococcus lactis ssp. lactis was characterized during progressive isoleucine starvation in batch culture using a chemically defined medium in which isoleucine concentration was fixed so as to become the sole limiting nutriment. Dynamic analyses were performed using transcriptomic and proteomic approaches and the results were analysed conjointly with fermentation kinetic data.

Results: The response was first deduced from transcriptomic analysis and corroborated by proteomic results. It occurred progressively and could be divided into three major mechanisms: (i) a global down-regulation of processes linked to bacterial growth and catabolism (transcription, translation, carbon metabolism and transport, pyrimidine and fatty acid metabolism), (ii) a specific positive response related to the limiting nutrient (activation of pathways of carbon or nitrogen metabolism and leading to isoleucine supply) and (iii) an unexpected oxidative stress response (positive regulation of aerobic metabolism, electron transport, thioredoxin metabolism and pyruvate dehydrogenase). The involvement of various regulatory mechanisms during this adaptation was analysed on the basis of transcriptomic data comparisons. The global regulator CodY seemed specifically dedicated to the regulation of isoleucine supply. Other regulations were massively related to growth rate and stringent response.

Conclusion: This integrative biology approach provided an overview of the metabolic pathways involved during isoleucine starvation and their regulations. It has extended significantly the physiological understanding of the metabolism of L. lactis ssp. lactis. The approach can be generalised to other conditions and will contribute significantly to the identification of the biological processes involved in complex regulatory networks of micro-organisms.

Figure 1

Kinetic profile of L. lactis IL1403 during isoleucine starvation (average of at least four independent repetitions) A. Concentrations of biomass (♦), glucose (■), lactate (▲) and isoleucine (black line). B. Specific rates of growth (♦), glucose consumption (■) and lactate production (▲). The numbered arrows indicate the sampling for both proteome and transcriptome analysis. (1): exponential phase corresponding to the reference; (2): 20 minutes of starvation; (3): 1.34 h of starvation; (4): 3 h of starvation.

Figure 2

Macroarray and qRT-PCR comparison in L. lactis IL1403. Log-log scatter plot of expression ratios between exponential phase and 1.34 h of isoleucine starvation obtained by macroarray (y-axis) and qRT-PCR (x-axis) analyses for 9 genes (adhE, busAB, clpE, glpF1, ilvD, optC, ptnAB, rplM and rpoB).

Figure 3

Coordinated expression of genes and proteins involved in amino acids metabolism during isoleucine starvation in L. lactis IL1403. One arrow represents one metabolic reaction and dashed line arrows correspond to more than one reaction. Protein names, if they are known, are indicated beside the arrows. Brackets stand for genes that are absent from the macro-array. Thick black arrows indicate up-regulation or, if slashed, down-regulation at the transcriptional level. White arrows stand for over-expression of the corresponding protein. Framed amino acids are those that can be synthetised by L. lactis IL1403 while the other ones correspond to natural auxotrophies of the strain.

Figure 4

Double hierarchical clustering to identify gene pattern similarity between various experiments. Each row correspond to a single gene, the strongest is expression compared to the reference, the darker the color and vice- versa. The dendrogramme on top of the figure shows how similar are the compared conditions. Ile = isoleucine starvation response (this study), aer = aeration stimulon [38], µ = growth rate effect [17], SR = stringent response [17].