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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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Comparative overview of known and putative regulatory RNAs of L. monocytogenes EGD-e. EGD-e was compared with 3 L. monocytogenes serotypes (3 × 1/2a, 1 × 4b and 1 × 4a) and three non-pathogenic Listeria species (L. innocua, L. welshimeri and L. seeligeri). To determine the distribution of regulatory RNA inside the genus a BLAST analysis was conducted using sRNAdb (unpublished software). Candidates were considered present inside a strain in the case of a sequence identity of 60% and a coverage of 80%. Since the surrounding locus is often important for the function of the regulatory RNA, information about the conservation of adjacent genes was included using the same cutoff. Possible cases for direction, presence and absence of each regulatory RNA and its flanking genes was color-coded below. A white square indicates the absence of the regulatory RNA. As a reference for this analysis the relevant loci of L. monocytogenes EGD-e were chosen. The small black arrow depicted in the legend indicates the regulatory RNA, while larger arrows in black and white symbolize the left and right flanking gene, respectively. A large gray arrow denotes a gene overlapping the regulatory RNA in sense or antisense direction. The arrow direction is not representative for the strand but for the relation to the locus in the reference genome of L. monocytogenes EGD-e. (E) indicates extracellular and (I) intracellular expression of the regulatory RNA.
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Figure 6: Comparative overview of known and putative regulatory RNAs of L. monocytogenes EGD-e. EGD-e was compared with 3 L. monocytogenes serotypes (3 × 1/2a, 1 × 4b and 1 × 4a) and three non-pathogenic Listeria species (L. innocua, L. welshimeri and L. seeligeri). To determine the distribution of regulatory RNA inside the genus a BLAST analysis was conducted using sRNAdb (unpublished software). Candidates were considered present inside a strain in the case of a sequence identity of 60% and a coverage of 80%. Since the surrounding locus is often important for the function of the regulatory RNA, information about the conservation of adjacent genes was included using the same cutoff. Possible cases for direction, presence and absence of each regulatory RNA and its flanking genes was color-coded below. A white square indicates the absence of the regulatory RNA. As a reference for this analysis the relevant loci of L. monocytogenes EGD-e were chosen. The small black arrow depicted in the legend indicates the regulatory RNA, while larger arrows in black and white symbolize the left and right flanking gene, respectively. A large gray arrow denotes a gene overlapping the regulatory RNA in sense or antisense direction. The arrow direction is not representative for the strand but for the relation to the locus in the reference genome of L. monocytogenes EGD-e. (E) indicates extracellular and (I) intracellular expression of the regulatory RNA.

Mentions: Finally, we compared our RNA sequencing identified regulatory RNA to the genomes of four human pathogenic strains of L. monocytogenes (36,53,54), an attenuated L. monocytogenes 4a strain (HCC23) (56) and the three apathogenic species of L. innocua, L. welshimeri and L. seeligeri (36,43,44) to investigate the regulation of virulence. Here we detected that the number of regulatory RNAs among the L. monocytogenes serotypes 4b and 1/2a belonging to Lineage I and II were highly similar compared with L. monocytogenes 1/2a EGD-e as reference (II > I > III), but decreased rapidly, when listerial species were more distantly related (Figure 6).Figure 6.


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)

Comparative overview of known and putative regulatory RNAs of L. monocytogenes EGD-e. EGD-e was compared with 3 L. monocytogenes serotypes (3 × 1/2a, 1 × 4b and 1 × 4a) and three non-pathogenic Listeria species (L. innocua, L. welshimeri and L. seeligeri). To determine the distribution of regulatory RNA inside the genus a BLAST analysis was conducted using sRNAdb (unpublished software). Candidates were considered present inside a strain in the case of a sequence identity of 60% and a coverage of 80%. Since the surrounding locus is often important for the function of the regulatory RNA, information about the conservation of adjacent genes was included using the same cutoff. Possible cases for direction, presence and absence of each regulatory RNA and its flanking genes was color-coded below. A white square indicates the absence of the regulatory RNA. As a reference for this analysis the relevant loci of L. monocytogenes EGD-e were chosen. The small black arrow depicted in the legend indicates the regulatory RNA, while larger arrows in black and white symbolize the left and right flanking gene, respectively. A large gray arrow denotes a gene overlapping the regulatory RNA in sense or antisense direction. The arrow direction is not representative for the strand but for the relation to the locus in the reference genome of L. monocytogenes EGD-e. (E) indicates extracellular and (I) intracellular expression of the regulatory RNA.
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Related In: Results  -  Collection

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Figure 6: Comparative overview of known and putative regulatory RNAs of L. monocytogenes EGD-e. EGD-e was compared with 3 L. monocytogenes serotypes (3 × 1/2a, 1 × 4b and 1 × 4a) and three non-pathogenic Listeria species (L. innocua, L. welshimeri and L. seeligeri). To determine the distribution of regulatory RNA inside the genus a BLAST analysis was conducted using sRNAdb (unpublished software). Candidates were considered present inside a strain in the case of a sequence identity of 60% and a coverage of 80%. Since the surrounding locus is often important for the function of the regulatory RNA, information about the conservation of adjacent genes was included using the same cutoff. Possible cases for direction, presence and absence of each regulatory RNA and its flanking genes was color-coded below. A white square indicates the absence of the regulatory RNA. As a reference for this analysis the relevant loci of L. monocytogenes EGD-e were chosen. The small black arrow depicted in the legend indicates the regulatory RNA, while larger arrows in black and white symbolize the left and right flanking gene, respectively. A large gray arrow denotes a gene overlapping the regulatory RNA in sense or antisense direction. The arrow direction is not representative for the strand but for the relation to the locus in the reference genome of L. monocytogenes EGD-e. (E) indicates extracellular and (I) intracellular expression of the regulatory RNA.
Mentions: Finally, we compared our RNA sequencing identified regulatory RNA to the genomes of four human pathogenic strains of L. monocytogenes (36,53,54), an attenuated L. monocytogenes 4a strain (HCC23) (56) and the three apathogenic species of L. innocua, L. welshimeri and L. seeligeri (36,43,44) to investigate the regulation of virulence. Here we detected that the number of regulatory RNAs among the L. monocytogenes serotypes 4b and 1/2a belonging to Lineage I and II were highly similar compared with L. monocytogenes 1/2a EGD-e as reference (II > I > III), but decreased rapidly, when listerial species were more distantly related (Figure 6).Figure 6.

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