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

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.

Keywords: 6S; dRNA-seq; lactic acid bacteria; small regulatory RNA; transcriptome.

Figure 1.

TSS mining, 5′-UTR distribution and promoter analysis. (A) Different types of transcription start sites identified in the L. lactis MG1363 genome from mapped reads of the TEX-treated RNA-seq data set (gray arrows: annotated ORFs, blue: Reads from the + strand, green: Reads from the - minus strand, red blocks: Positions of putative regulatory RNAs predicted by SIPHT. (B) Length distribution of 5′-UTRs. 5′-UTRs up to a length of 100 nt are plotted in stepwise increments of 10 nt in gray, those larger than 100 nt are shown with increments of 25 nt (separated by the dotted line). Color code is given in the inset. The RBS consensus sequence and the consensus sequence in the first 7 nt of the 111 leaderless mRNAs were determined by MEME. (C) Top: Analysis using MEME of motifs in the 50 nt upstream of all 1819 TSSs predicted by TSSer. Curved dotted line: periodic AT stretches. Bottom: Reconstruction of the L. lactis promoter consensus using MAST. In both: −35 and −10 sequences are indicated.

Figure 2.

Experimental validation of novel RNAs. Detection of 9 sRNAs and 3 asRNAs by Northern hybridization in total RNA isolated from L. lactis MG1363 grown in GM17 under various conditions (Ex: exponential phase, St: stationary phase, pH: 10 min acid (pH 4.5) stress, Sa: 10 min salt (2.5% w/v extra NaCl) stress, St*: 10 min starvation in PBS). The positions of the sRNAs and asRNAs are indicated with asterisks (*). As a control, all blots were probed with an oligonucleotide targeting 5S RNA. Visualization of the relevant chromosomal locus and the expression levels of the genes (as derived from the TEX-treated RNA-seq dataset) are given below each blot, // signifies that not the entire gene is shown. Probes used to identify the various RNAs are given in Table S13.

Figure 3.

Analysis of the L. lactis non-coding 6S RNA. (A) Genomic region of the 6S (LLMGnc_004) gene of L. lactis MG1363. Open reading frames are depicted as grey arrows, 6S is shown in blue. Solid black arrows: promoters. The nucleotide sequence of the 6S promoter (P_6S) is given in capitals, including a predicted cre-site (small letter type) upstream of the −35 box. (B) Structure of 6S RNA predicted by Mfold. (C) Detection of 6S RNA 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 served as an RNA concentration control. In addition, one lane of the 8% polyacrylamide gel was stained after electrophoresis with ethidium bromide to visualize the relative amounts of 5S and 6S RNAs. O.N.: overnight culture, OD 2h 2.0: cells taken from a culture maintained at OD₆₀₀ = 2.0 for 2 hours. Primers used for these experiments are given in Table S13. (D) Volcano plot showing the differentially (p-value <0.01 and ≥2 -fold) expressed genes upon overexpression of 6S RNA in comparison with the control, using a short (10-min) pulse of nisin addition to a culture of L. lactis SVDM2001 in GM17 and at an OD₆₀₀ = 0.45. Indicated in yellow: differentially expressed genes, gray circles: measure of expression level.

Figure 4.

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 (P_{LLMGnc_147}) is given, including a predicted cre-site that overlaps the −35 box. (B) Structure of LLMGnc_147 using Mfold. (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) P_{LLMGnc_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

Figure 5.

LLMGnc_147 is involved in the utilization of galactose. (A) Volcano plot of genes that are differentially expressed (p-value <0.001 and ≥2 -fold change) after pulse-expression of LLMGnc_147 via a 10-min addition of nisin to a culture at an OD₆₀₀ = 0.45. For clarification of symbols, see the legend to Fig. 3. (B) Nisin-induced overexpression for 20 min of LLMGnc_147 (red triangles) in L. lactis SVDM2002 in comparison with the empty vector control (blue squares), after which the strain was re-inoculated 1:20 in fresh M17 medium containing 1% galactose. (C) Identical experimental set-up as described in (B) for growth after re-inoculation in M17 with 1% (w/v) of the indicated carbon sources. The experiments were repeated twice and the lines represent averages of 4 microtiter plate measurements.