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Role of mRNA stability during bacterial adaptation

ABSTRACT: Bacterial adaptation involves extensive cellular reorganization. In particular, growth rate adjustments are associated with substantial modifications of gene expression and mRNA abundance. In this work we aimed to assess the role of mRNA degradation during such variations. A genome-wide transcriptomic-based method was used to determine mRNA half-lives. The model bacterium Lactococcus lactis was used and five growth rates were studied in continuous cultures under isoleucine-limitation and in batch cultures during the adaptation to the isoleucine starvation. During continuous isoleucine-limited growth, the mRNAs of different genes had different half-lives. The stability of most of the transcripts was not constant, and increased as the growth rate decreased. This half-life diversity was analyzed to investigate determinants of mRNA stability. The concentration, length, codon adaptation index and secondary structures of mRNAs were found to contribute to the regulation of mRNA stability in these conditions. However, the growth rate was, by far, the most influential determinant. The respective influences of mRNA degradation and transcription on the regulation of intra-cellular transcript concentration were estimated. The role of degradation on mRNA homeostasis was clearly evidenced: for more than 90 % of the mRNAs studied during continuous isoleucine-limited growth of L. lactis, degradation was antagonistic to transcription. Although both transcription and degradation had, opposite effects,, the mRNA changes in response to growth rate were driven by transcription. Interestingly, degradation control increased during the dynamic adaptation of bacteria as the growth rate reduced due to progressive isoleucine starvation in batch cultures. This work shows that mRNA decay differs between gene transcripts and according to the growth rate. It demonstrates that mRNA degradation is an important regulatory process involved in bacterial adaptation. However, its impact on the regulation of mRNA levels is smaller than that of transcription in the conditions studied.

ORGANISM(S): Lactococcus lactis

PROVIDER: GSE43875 | GEO | 2014/06/16

SECONDARY ACCESSION(S): PRJNA188582

REPOSITORIES: GEO

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Project description:Bacterial adaptation involves extensive cellular reorganization. In particular, growth rate adjustments are associated with substantial modifications of gene expression and mRNA abundance. In this work we aimed to assess the role of mRNA degradation during such variations. A genome-wide transcriptomic-based method was used to determine mRNA half-lives. The model bacterium Lactococcus lactis was used and five growth rates were studied in continuous cultures under isoleucine-limitation and in batch cultures during the adaptation to the isoleucine starvation. During continuous isoleucine-limited growth, the mRNAs of different genes had different half-lives. The stability of most of the transcripts was not constant, and increased as the growth rate decreased. This half-life diversity was analyzed to investigate determinants of mRNA stability. The concentration, length, codon adaptation index and secondary structures of mRNAs were found to contribute to the regulation of mRNA stability in these conditions. However, the growth rate was, by far, the most influential determinant. The respective influences of mRNA degradation and transcription on the regulation of intra-cellular transcript concentration were estimated. The role of degradation on mRNA homeostasis was clearly evidenced: for more than 90 % of the mRNAs studied during continuous isoleucine-limited growth of L. lactis, degradation was antagonistic to transcription. Although both transcription and degradation had, opposite effects,, the mRNA changes in response to growth rate were driven by transcription. Interestingly, degradation control increased during the dynamic adaptation of bacteria as the growth rate reduced due to progressive isoleucine starvation in batch cultures. This work shows that mRNA decay differs between gene transcripts and according to the growth rate. It demonstrates that mRNA degradation is an important regulatory process involved in bacterial adaptation. However, its impact on the regulation of mRNA levels is smaller than that of transcription in the conditions studied. In the study presented here mRNA stabilities were analyzed at 5 growth rates. For each growth rate mRNA levels were measured in a time course experiment following rifampicin addition. At least 12 time points per growth rate are available, including 3 replicates of the zero.

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.

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:Degradation of mRNA is one of the key processes that control the steady-state level of gene expression. However, the rate of mRNA decay for the majority of genes is not known. We successfully obtained the rate of mRNA decay for 19 977 non-redundant genes by microarray analysis of RNA samples obtained from mouse embryonic stem (ES) cells. Median estimated half-life was 7.1 h and only <100 genes, including Prdm1, Myc, Gadd45 g, Foxa2, Hes5 and Trib1, showed half-life less than 1 h. In general, mRNA species with short half-life were enriched among genes with regulatory functions (transcription factors), whereas mRNA species with long half-life were enriched among genes related to metabolism and structure (extracellular matrix, cytoskeleton). The stability of mRNAs correlated more significantly with the structural features of genes than the function of genes: mRNA stability showed the most significant positive correlation with the number of exon junctions per open reading frame length, and negative correlation with the presence of PUF-binding motifs and AU-rich elements in 30 -untranslated region (UTR) and CpG di-nucleotides in the 50 -UTR. The mRNA decay rates presented in this report are the largest data set for mammals and the first for ES cells. Keywords: growth condition design,reference design,replicate design,RNA stability design,time series design We used two mouse ESC lines: MC1 derived from mouse strain 129S6/SvEvTac and MC2-B6 derived from strain C57BL/6J. To remove contaminating feeder cells, ES cells were first cultured for two passages on gelatin-coated culture dish in complete ES medium. Differentiated ES cells were obtained from MC1 cells by LIF withdrawal (complete medium without LIF, below referenced as LIF-) or by retinoic acid (RA) treatment. To measure mRNA stability, transcription was blocked by adding actinomycin D to the medium. RNA was hybridized in duplicate for each cell type and time point. All hybridizations were carried out by combining a Cy3-CTP-labeled experimental target and a Cy5-CTP-labeled Universal Mouse Reference target.

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Role of mRNA stability during bacterial adaptation

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