Project description:Amino acid assimilation and metabolism are crucial for bacterial growth and survival and this is particularly obvious for lactic acid bacteria (LAB) that are generally auxotroph for various amino acids. However, amino acid assimilation is poorly characterized and a complete description of the response during amino acid starvation is still lacking in LAB. In this context, the global response of the LAB model Lactococcus lactis was characterized during isoleucine starvation in batch culture. The stress was imposed by isoleucine natural consumption in an initially rich chemically defined medium. Dynamic analyses were performed both using transcriptomic and proteomic approaches. The response was found to occur gradually and could be divided into three major parts that were firstly deduced from transcriptomic analysis and generally corroborated by proteomic results: (i) a global repression of biogenic processes (transcription, translation, and carbon metabolism and transport), (ii) a specific response related to the limiting nutrient (numerous pathways belonging to carbon or nitrogen metabolism and leading to isoleucine supply were activated) and (iii) an additional response connected to oxidative stress (induction of aerobic metabolism, electron transport, thioredoxin metabolism and pyruvate dehydrogenase). The involvement of various regulatory mechanisms such as growth rate regulation, stringent response, CodY, GlnR, and CcpA regulations, was discussed on the basis of transcriptomic data comparisons. Above the full description of L. lactis isoleucine starvation response, this work additionally provided a complex but realistic outlook of the regulation network involved in isoleucine starvation. Such integrated and comparative approach will allow, by its implementation to other regulations and environmental conditions, the whole regulatory network of L. lactis or any other microorganism to be deciphered. Batch cultivation of Lactococcus lactis IL1403 were carried out on a chemically defined medium and under controlled conditions (30 °C, pH 6.6, nitrogen atmosphere). Cell samples were harvested at steady state. Total RNA was extracted from these samples and radiolabelled cDNA were prepared and hybridized on nylon arrays. 1948 amplicons specific of Lactococcus lactis IL1403 genes were spotted twice on the array. Samples corresponding to various growth rates were analyzed simultaneously and 3 independent repetitions were performed.

Project description:The development of transcriptomic tools has allowed exhaustive description of stress responses. These responses always superimpose a general response associated to growth rate decrease and a specific one corresponding to the stress. The exclusive growth rate response can be achieved through chemostat cultivation, enabling all parameters to remain constant except the growth rate. We analysed metabolic and transcriptomic responses of Lactococcus lactis in continuous cultures at different growth rates ranging from 0.09 to 0.47 h-1. Growth rate was conditioned by isoleucine supply. Although the metabolism was constant and homolactic, a widespread transcriptomic response involving 30 % of the genome was observed. The expression of genes encoding physiological functions associated with biogenesis increased with growth rate (transcription, translation, fatty acid and phospholipids metabolism). Many phages, prophages and transposons related genes were down regulated by growth rate suggesting genome plasticity to be involved in the adaptation to slow growth. The growth rate response was compared to carbon and amino-acid starvation transcriptomic responses, revealing constant and significant involvement of growth rate regulations in these two stressful conditions (overlap 26%). Although stringent response mechanism is considered as the one governing growth deceleration in bacteria, the rigorous comparison of the two transcriptomic responses clearly indicated the mechanisms are distinct. Moreover it was established that genes positively regulated by growth rate are preferentially located in the vicinity of replication origin while those negatively regulated are mainly encountered at the opposite. This result demonstrates the often neglected relationship between genes expression and their location on chromosome. Keywords: growth rate impact, continuous cultures Continuous cultivation of Lactococcus lactis IL1403 were carried out on a chemically defined medium and under controlled conditions (30 Â°C, pH 6.6, nitrogen atmosphere). Cell samples were harvested at steady state. Total RNA was extracted from these samples and radiolabelled cDNA were prepared and hybridized on nylon arrays. 1948 amplicons specific of Lactococcus lactis IL1403 genes were spotted twice on the array. Samples corresponding to various growth rates were analyzed simultaneously and 3 independent repetitions were performed.

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Lactococcus lactis

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