Abstract
The stability of mRNA was investigated for the first time at the genomic scale during carbon starvation adaptation of Lactococcus lactis IL1403. In exponential phase, mRNA half-lives were correlated positively to open reading frame length. A polypurine sequence, AGGAG, was identified as a putative 5'-stabilizer and inverted repeated sequences as a 3'-destabilizer. These original findings suggested that multiple pathways of mRNA degradation should coexist: internal cleavage, endonuclease cleavage initiated at the 5'-end, and exonuclease attack at the 3'-end. During carbon starvation adaptation, mRNA stability globally increased, but specific mechanisms allowing a wide range of stabilization factors between genes and differential kinetic evolution were involved. A formal method allowing the quantification of the relative influences of transcription and degradation on the mRNA pool control was developed and applied in L. lactis. Gene expression was mostly controlled by altered transcription prior to carbon source exhaustion, while the influence of mRNA stability increased during the starvation phase. This study highlighted that stability modulation in response to adverse growth conditions can govern gene regulation to the same extent as transcription in bacteria.
Highlights
Tococcus lactis during acidic conditions was published (9), but it was restricted to a particular functional category
Stability Determinants in Exponential Growth—mRNA stability was examined with DNA arrays during the exponential growth of L. lactis on glucose CDM medium, and 817 different mRNA half-lives were quantified simultaneously. mRNA half-lives of genes adhE, ccpA, and tpi were confirmed with Northern blot analysis and previous data obtained with fluorescent labeling (17)
MRNA Degradation Mechanisms—This study provided the first characterization of whole genome mRNA decay in L. lactis and more generally in a lactic acid bacterium
Summary
Organism and Growth Conditions—The bacterium used throughout this study was L. lactis ssp. lactis IL1403, whose genome was entirely sequenced (13). Determination of mRNA Half-lives—For mRNA half-life quantification, three growth conditions (exponential, deceleration, and carbon starvation phases) were studied in independent but physiologically identical cultures. After centrifugation to eliminate cell debris and phenol (4 °C, 25 min, 13,000 rpm), half of the aqueous phase containing RNA was extracted with a RNeasy midi kit (Qiagen), including the DNase I treatment described in the manufacturer’s instructions. The three growth conditions (exponential, deceleration, and carbon starvation phases) were previously characterized from the transcriptional viewpoint (12), but values were recalculated to obtain mRNA concentrations rather than abundances. Raw data were standardized by the all spots’ mean intensity of the exponential phase and corrected by total RNA concentration in each growth condition (11.7 Ϯ 1.3, 7.6 Ϯ 0.8, and 8.2 Ϯ 1.4 g (100 g dried cellsϪ1) in exponential, deceleration, and starvation phases respectively)
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