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Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism.

van der Meulen SB, de Jong A, Kok J - RNA Biol (2016)

Bottom Line: L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding.Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs.Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species.

View Article: PubMed Central - PubMed

Affiliation: a Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute , University of Groningen , Groningen , The Netherlands.

ABSTRACT
RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species.

No MeSH data available.


Related in: MedlinePlus

The sRNA LLMGnc_147 is involved in carbon metabolism. (A) Genomic region of LLMGnc_147. Open reading frames are depicted as gray arrows, LLMGnc_147 in shown in blue. Solid black arrows: promoters. The nucleotide sequence of the LLMGnc_147 promoter (PLLMGnc_147) is given, including a predicted cre-site that overlaps the −35 box. (B) Structure of LLMGnc_147 using Mfold.83 (C) Detection of LLMGnc_147 by Northern hybridization in samples of L. lactis grown in GM17 until the indicated ODs at 600 nm, or until OD600 = 0.6 in M17 with 1% of the indicated sugars. 5S RNA serves as a loading control. Labeled primers used for identification of the RNAs are given in Table S13. (D) PLLMGnc_147::gfp activity in L. lactis MG1363 (wt) and SVDM2003 cells grown in M17 containing 1% (w/v) of the indicated carbon source. Fluorescence and optical density were measured 5 hours after re-inoculation from an overnight culture growing in GM17. The experiment was repeated 3 times and error bars are indicated
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f0004: The sRNA LLMGnc_147 is involved in carbon metabolism. (A) Genomic region of LLMGnc_147. Open reading frames are depicted as gray arrows, LLMGnc_147 in shown in blue. Solid black arrows: promoters. The nucleotide sequence of the LLMGnc_147 promoter (PLLMGnc_147) is given, including a predicted cre-site that overlaps the −35 box. (B) Structure of LLMGnc_147 using Mfold.83 (C) Detection of LLMGnc_147 by Northern hybridization in samples of L. lactis grown in GM17 until the indicated ODs at 600 nm, or until OD600 = 0.6 in M17 with 1% of the indicated sugars. 5S RNA serves as a loading control. Labeled primers used for identification of the RNAs are given in Table S13. (D) PLLMGnc_147::gfp activity in L. lactis MG1363 (wt) and SVDM2003 cells grown in M17 containing 1% (w/v) of the indicated carbon source. Fluorescence and optical density were measured 5 hours after re-inoculation from an overnight culture growing in GM17. The experiment was repeated 3 times and error bars are indicated

Mentions: As a further demonstration of the validity of the reported sRNAs and to initiate the functional analysis of these putative regulator molecules in L. lactis, we characterized one of them in more detail. LLMGnc_147 is a 102-nt-long sRNA of which the gene is located between llmg_1960 and the transcriptional activator gene tenA (Fig. 4B). Its promoter carries a possible cre site overlapping the −35 box, suggesting that LLMGnc_147 is under control of CcpA (Fig. 4A) and related to carbon utilization. Northern analysis shows that LLMGnc_147 is highly expressed in cells growing on cellobiose (Fig. 4C). A transcriptional fusion of the promoter of LLMGnc_147 to gfp confirmed that it was most active in the presence of cellobiose and to a lesser extent with galactose (Fig. 4D).Figure 4.


Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism.

van der Meulen SB, de Jong A, Kok J - RNA Biol (2016)

The sRNA LLMGnc_147 is involved in carbon metabolism. (A) Genomic region of LLMGnc_147. Open reading frames are depicted as gray arrows, LLMGnc_147 in shown in blue. Solid black arrows: promoters. The nucleotide sequence of the LLMGnc_147 promoter (PLLMGnc_147) is given, including a predicted cre-site that overlaps the −35 box. (B) Structure of LLMGnc_147 using Mfold.83 (C) Detection of LLMGnc_147 by Northern hybridization in samples of L. lactis grown in GM17 until the indicated ODs at 600 nm, or until OD600 = 0.6 in M17 with 1% of the indicated sugars. 5S RNA serves as a loading control. Labeled primers used for identification of the RNAs are given in Table S13. (D) PLLMGnc_147::gfp activity in L. lactis MG1363 (wt) and SVDM2003 cells grown in M17 containing 1% (w/v) of the indicated carbon source. Fluorescence and optical density were measured 5 hours after re-inoculation from an overnight culture growing in GM17. The experiment was repeated 3 times and error bars are indicated
© Copyright Policy - open-access
Related In: Results  -  Collection

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f0004: The sRNA LLMGnc_147 is involved in carbon metabolism. (A) Genomic region of LLMGnc_147. Open reading frames are depicted as gray arrows, LLMGnc_147 in shown in blue. Solid black arrows: promoters. The nucleotide sequence of the LLMGnc_147 promoter (PLLMGnc_147) is given, including a predicted cre-site that overlaps the −35 box. (B) Structure of LLMGnc_147 using Mfold.83 (C) Detection of LLMGnc_147 by Northern hybridization in samples of L. lactis grown in GM17 until the indicated ODs at 600 nm, or until OD600 = 0.6 in M17 with 1% of the indicated sugars. 5S RNA serves as a loading control. Labeled primers used for identification of the RNAs are given in Table S13. (D) PLLMGnc_147::gfp activity in L. lactis MG1363 (wt) and SVDM2003 cells grown in M17 containing 1% (w/v) of the indicated carbon source. Fluorescence and optical density were measured 5 hours after re-inoculation from an overnight culture growing in GM17. The experiment was repeated 3 times and error bars are indicated
Mentions: As a further demonstration of the validity of the reported sRNAs and to initiate the functional analysis of these putative regulator molecules in L. lactis, we characterized one of them in more detail. LLMGnc_147 is a 102-nt-long sRNA of which the gene is located between llmg_1960 and the transcriptional activator gene tenA (Fig. 4B). Its promoter carries a possible cre site overlapping the −35 box, suggesting that LLMGnc_147 is under control of CcpA (Fig. 4A) and related to carbon utilization. Northern analysis shows that LLMGnc_147 is highly expressed in cells growing on cellobiose (Fig. 4C). A transcriptional fusion of the promoter of LLMGnc_147 to gfp confirmed that it was most active in the presence of cellobiose and to a lesser extent with galactose (Fig. 4D).Figure 4.

Bottom Line: L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding.Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs.Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species.

View Article: PubMed Central - PubMed

Affiliation: a Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute , University of Groningen , Groningen , The Netherlands.

ABSTRACT
RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species.

No MeSH data available.


Related in: MedlinePlus