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The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages.

Mraheil MA, Billion A, Mohamed W, Mukherjee K, Kuenne C, Pischimarov J, Krawitz C, Retey J, Hartsch T, Chakraborty T, Hain T - Nucleic Acids Res. (2011)

Bottom Line: Currently extensive information exists on the sRNAs of Listeria monocytogenes expressed during growth in extracellular environments.A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly.Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Justus-Liebig-University, Frankfurter Strasse 107, 35392 Giessen, Germany.

ABSTRACT
Small non-coding RNAs (sRNAs) are widespread effectors of post-transcriptional gene regulation in bacteria. Currently extensive information exists on the sRNAs of Listeria monocytogenes expressed during growth in extracellular environments. We used deep sequencing of cDNAs obtained from fractioned RNA (<500 nt) isolated from extracellularly growing bacteria and from L. monocytogenes infected macrophages to catalog the sRNA repertoire during intracellular bacterial growth. Here, we report on the discovery of 150 putative regulatory RNAs of which 71 have not been previously described. A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly. We validated highly expressed sRNAs by northern blotting and demonstrated by the construction and characterization of isogenic mutants of rli31, rli33-1 and rli50* for intracellular expressed sRNA candidates, that their expression is required for efficient growth of bacteria in macrophages. All three mutants were attenuated when assessed for growth in mouse and insect models of infection. Comparative genomic analysis revealed the presence of lineage specific sRNA candidates and the absence of sRNA loci in genomes of naturally occurring infection-attenuated bacteria, with additional loss in non-pathogenic listerial genomes. Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.

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Discovery of the intracellular sRNome of L. monocytogenes using RNA-Seq. (A) Extracellular and intracellular transcriptional landscape of L. monocytogenes is represented using GenomeViz (46). Circles display following information from outside to inside: (1) COG categories; (2) rRNAs and tRNAs (blue), a prophage-like locus (light brown) and the virulence gene cluster (red); (3) intracellular regulatory RNAs (outer circle) and extracellular regulatory RNAs (inner circle); (4) intracellular asRNAs (outer circle) and extracellular asRNAs (inner circle); (5) intracellular cis-regulatory RNAs including riboswitches (outer circle) and extracellular cis-regulatory RNAs including riboswitches (inner circle); (6) regulation of intracellular sRNAs; (7) regulation of intracellular asRNAs and (8) intracellular cis-regulatory RNAs including riboswitches; (B and C) Distribution of mapped sequence reads used for extracellular and intracellular transcriptome analysis; (D) Comparative analysis of sRNA transcriptome data using ‘cumulative’ values which can be summarized since sRNA candidates would not be counted multiple times (see Supplementary Figure S1 for a ‘non-cumulative’ version). Comparison of our RNA-seq results, whole genome tiling array from Toledo-Arana and coworkers (31), RNA-seq data of L. monocytogenes 10403S (32) and in silico regulatory RNA predictions (1).
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Figure 1: Discovery of the intracellular sRNome of L. monocytogenes using RNA-Seq. (A) Extracellular and intracellular transcriptional landscape of L. monocytogenes is represented using GenomeViz (46). Circles display following information from outside to inside: (1) COG categories; (2) rRNAs and tRNAs (blue), a prophage-like locus (light brown) and the virulence gene cluster (red); (3) intracellular regulatory RNAs (outer circle) and extracellular regulatory RNAs (inner circle); (4) intracellular asRNAs (outer circle) and extracellular asRNAs (inner circle); (5) intracellular cis-regulatory RNAs including riboswitches (outer circle) and extracellular cis-regulatory RNAs including riboswitches (inner circle); (6) regulation of intracellular sRNAs; (7) regulation of intracellular asRNAs and (8) intracellular cis-regulatory RNAs including riboswitches; (B and C) Distribution of mapped sequence reads used for extracellular and intracellular transcriptome analysis; (D) Comparative analysis of sRNA transcriptome data using ‘cumulative’ values which can be summarized since sRNA candidates would not be counted multiple times (see Supplementary Figure S1 for a ‘non-cumulative’ version). Comparison of our RNA-seq results, whole genome tiling array from Toledo-Arana and coworkers (31), RNA-seq data of L. monocytogenes 10403S (32) and in silico regulatory RNA predictions (1).

Mentions: Sequencing reads were mapped to the genome of L. monocytogenes using BLASTN with an e-value of 0.001 and default word size with rewards for a nucleotide match that had been set to two. Additionally nucleotide identity was required to be >60% combined with coverage of 80% between query and subject sequence. Reads that did not fulfil these requirements were removed from the dataset. After clipping, additional linker removal, quality control and mapping against the genome of L. monocytogenes EGD-e, 114 459 unique reads with at least 80% sequence identity with 80% coverage and a minimum length of 21 nt remained for detailed analysis. We observed that ∼49% of the IC reads perfectly match the genome with 100% sequence identity compared with only 28% of the reads from the EC cDNA library. The ‘intergenome’ of L. monocytogenes, i.e. the non-coding sequence between annotated ORFs, comprises ∼10% (∼300 000 bp) of the entire genome. In the intergenome fraction we observed expression of nearly one-third under intracellular condition which additionally shows the importance of the intergenome (Figure 1A). Approximately 60% of all sequence reads mapped to annotated rRNA and tRNA genes (Figure 1B and C).Figure 1.


The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages.

Mraheil MA, Billion A, Mohamed W, Mukherjee K, Kuenne C, Pischimarov J, Krawitz C, Retey J, Hartsch T, Chakraborty T, Hain T - Nucleic Acids Res. (2011)

Discovery of the intracellular sRNome of L. monocytogenes using RNA-Seq. (A) Extracellular and intracellular transcriptional landscape of L. monocytogenes is represented using GenomeViz (46). Circles display following information from outside to inside: (1) COG categories; (2) rRNAs and tRNAs (blue), a prophage-like locus (light brown) and the virulence gene cluster (red); (3) intracellular regulatory RNAs (outer circle) and extracellular regulatory RNAs (inner circle); (4) intracellular asRNAs (outer circle) and extracellular asRNAs (inner circle); (5) intracellular cis-regulatory RNAs including riboswitches (outer circle) and extracellular cis-regulatory RNAs including riboswitches (inner circle); (6) regulation of intracellular sRNAs; (7) regulation of intracellular asRNAs and (8) intracellular cis-regulatory RNAs including riboswitches; (B and C) Distribution of mapped sequence reads used for extracellular and intracellular transcriptome analysis; (D) Comparative analysis of sRNA transcriptome data using ‘cumulative’ values which can be summarized since sRNA candidates would not be counted multiple times (see Supplementary Figure S1 for a ‘non-cumulative’ version). Comparison of our RNA-seq results, whole genome tiling array from Toledo-Arana and coworkers (31), RNA-seq data of L. monocytogenes 10403S (32) and in silico regulatory RNA predictions (1).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105390&req=5

Figure 1: Discovery of the intracellular sRNome of L. monocytogenes using RNA-Seq. (A) Extracellular and intracellular transcriptional landscape of L. monocytogenes is represented using GenomeViz (46). Circles display following information from outside to inside: (1) COG categories; (2) rRNAs and tRNAs (blue), a prophage-like locus (light brown) and the virulence gene cluster (red); (3) intracellular regulatory RNAs (outer circle) and extracellular regulatory RNAs (inner circle); (4) intracellular asRNAs (outer circle) and extracellular asRNAs (inner circle); (5) intracellular cis-regulatory RNAs including riboswitches (outer circle) and extracellular cis-regulatory RNAs including riboswitches (inner circle); (6) regulation of intracellular sRNAs; (7) regulation of intracellular asRNAs and (8) intracellular cis-regulatory RNAs including riboswitches; (B and C) Distribution of mapped sequence reads used for extracellular and intracellular transcriptome analysis; (D) Comparative analysis of sRNA transcriptome data using ‘cumulative’ values which can be summarized since sRNA candidates would not be counted multiple times (see Supplementary Figure S1 for a ‘non-cumulative’ version). Comparison of our RNA-seq results, whole genome tiling array from Toledo-Arana and coworkers (31), RNA-seq data of L. monocytogenes 10403S (32) and in silico regulatory RNA predictions (1).
Mentions: Sequencing reads were mapped to the genome of L. monocytogenes using BLASTN with an e-value of 0.001 and default word size with rewards for a nucleotide match that had been set to two. Additionally nucleotide identity was required to be >60% combined with coverage of 80% between query and subject sequence. Reads that did not fulfil these requirements were removed from the dataset. After clipping, additional linker removal, quality control and mapping against the genome of L. monocytogenes EGD-e, 114 459 unique reads with at least 80% sequence identity with 80% coverage and a minimum length of 21 nt remained for detailed analysis. We observed that ∼49% of the IC reads perfectly match the genome with 100% sequence identity compared with only 28% of the reads from the EC cDNA library. The ‘intergenome’ of L. monocytogenes, i.e. the non-coding sequence between annotated ORFs, comprises ∼10% (∼300 000 bp) of the entire genome. In the intergenome fraction we observed expression of nearly one-third under intracellular condition which additionally shows the importance of the intergenome (Figure 1A). Approximately 60% of all sequence reads mapped to annotated rRNA and tRNA genes (Figure 1B and C).Figure 1.

Bottom Line: Currently extensive information exists on the sRNAs of Listeria monocytogenes expressed during growth in extracellular environments.A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly.Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Justus-Liebig-University, Frankfurter Strasse 107, 35392 Giessen, Germany.

ABSTRACT
Small non-coding RNAs (sRNAs) are widespread effectors of post-transcriptional gene regulation in bacteria. Currently extensive information exists on the sRNAs of Listeria monocytogenes expressed during growth in extracellular environments. We used deep sequencing of cDNAs obtained from fractioned RNA (<500 nt) isolated from extracellularly growing bacteria and from L. monocytogenes infected macrophages to catalog the sRNA repertoire during intracellular bacterial growth. Here, we report on the discovery of 150 putative regulatory RNAs of which 71 have not been previously described. A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly. We validated highly expressed sRNAs by northern blotting and demonstrated by the construction and characterization of isogenic mutants of rli31, rli33-1 and rli50* for intracellular expressed sRNA candidates, that their expression is required for efficient growth of bacteria in macrophages. All three mutants were attenuated when assessed for growth in mouse and insect models of infection. Comparative genomic analysis revealed the presence of lineage specific sRNA candidates and the absence of sRNA loci in genomes of naturally occurring infection-attenuated bacteria, with additional loss in non-pathogenic listerial genomes. Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.

Show MeSH
Related in: MedlinePlus