Approaches to Identify Novel Non‐messenger RNAs in Bacteria and to Investigate their Biological Functions: Functional Analysis of Identified Non‐mRNAs

Wagner, E. Gerhart H.; Vogel, Jörg

doi:10.1002/9783527619504.ch37

Cited by 8 publications

(6 citation statements)

References 94 publications

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“…RACE. 5Ј RACE was carried out as described previously (24). Reverse transcription of the 5Ј-UTR of inlJ mRNA was performed with the inlJ-ext primer, while the inlJ-ext2 primer (5Ј-CTCTATAATCATTTATTATACGCCGTTTTA TTTAC-3Ј) was used for PCR amplification.…”

Section: Methodsmentioning

confidence: 99%

The Listeria monocytogenes Virulence Factor InlJ Is Specifically Expressed In Vivo and Behaves as an Adhesin

et al. 2008

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The food-borne pathogen Listeria monocytogenes is adapted to a diversity of environments, such as soil, food, body fluids, and the cytosol of eukaryotic cells. The transition between saprophytic and pathogenic life is mediated through complex regulatory pathways that modulate the expression of virulence factors. Here we examined the expression of inlJ, a recently identified gene encoding a protein of the LPXTG-internalin family and involved in pathogenesis. We show that inlJ expression is controlled neither by the major listerial regulator of virulence genes, PrfA, nor by AxyR, a putative AraC regulator encoded by a gene adjacent to inlJ and divergently transcribed. The InlJ protein is not produced by bacteria grown in vitro in brain heart infusion medium or replicating in the cytosol of tissue-cultured cells. In contrast, it is efficiently produced and localized at the surface of bacteria present in the liver and blood of infected animals. Strikingly, the expression of inlJ by a heterologous promoter in L. monocytogenes or L. innocua promotes bacterial adherence to human cells in vitro. Taken together, these results strongly suggest that InlJ acts as a novel L. monocytogenes sortase-anchored adhesin specifically expressed during infection in vivo.

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Section: Methodsmentioning

confidence: 99%

The Listeria monocytogenes Virulence Factor InlJ Is Specifically Expressed In Vivo and Behaves as an Adhesin

et al. 2008

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“…RNA was extracted using an RNeasy kit (QIAGEN) as recommended by the manufacturer, with the modification that 100-m glass beads and bead beating were used to enhance cell lysis. Rapid amplification of 5Ј cDNA ends (5Ј-RACE) identification of transcription start sites was performed as described previously (14). PCR products were sequenced and analyzed using the M. tuberculosis H37Rv genome sequence as a reference (8).…”

Section: Methodsmentioning

confidence: 99%

Identification of Mycobacterial σ Factor Binding Sites by Chromatin Immunoprecipitation Assays

et al. 2007

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Mycobacterium tuberculosis and Mycobacterium bovis are responsible for infections that cause a substantial amount of death, suffering, and loss around the world. Still, relatively little is known about the mechanisms of gene expression in these bacteria. Here, we used genome-wide location assays to identify direct target genes for mycobacterial factors. Chromatin immunoprecipitation assays were performed with M. bovis BCG for Myc-tagged proteins expressed using an anhydrotetracycline-inducible promoter, and enriched DNA fragments were hybridized to a microarray representing intergenic regions from the M. tuberculosis H37Rv genome. Several putative target genes were validated by quantitative PCR. The corresponding transcriptional start sites were identified for F , C , and K , and consensus promoter sequences are proposed. Our conclusions were supported by the results of in vitro transcription assays. We also examined the role of each holoenzyme in the expression of factor genes. Our results revealed that many factors are expressed from autoregulated promoters.Several pathogenic mycobacteria cause infections that lead to tuberculosis or related diseases in a variety of organisms. For example, Mycobacterium tuberculosis infects nearly onethird of the human population (10), while Mycobacterium bovis infections in cattle are responsible for important agricultural losses (12). Advanced knowledge of the biology of these bacteria could significantly contribute to prevention and treatment of the associated diseases. A key step toward this end occurred with the publication of the complete M. tuberculosis and M. bovis genome sequences (8, 12). Several genes have also been interrupted in M. tuberculosis, leading to different outcomes for virulence (for reviews, see references 7 and 38). More recently, in many studies workers have reported gene expression profiles for various growth conditions and during different stages of infection in mice and human macrophages (21, 37, 41), providing new insights into the strategies used by the pathogens to adapt to various environments. Nevertheless, much remains to be discovered about the molecular mechanisms controlling the genetic programs in pathogenic mycobacteria.Thirteen factor genes were predicted based on the genome sequence of M. tuberculosis, and almost identical M. bovis counterparts were also predicted (25,35). Some of these genes may orchestrate critical responses in the pathogenesis of tuberculosis and therefore may be attractive targets for development of new drugs and vaccines. Indeed, in mouse models infections with sigC, sigD, sigE, sigF, sigh, and sigL mutant strains were all attenuated compared to infections with the wild-type strains (5,9,13,16,20,23,24,26,27,32,33,40). Still, the conditions leading to the activities of most factors remain elusive (23,35). Differential gene expression profiles were nonetheless obtained for the factor mutant strains, and consensus promoter sequences have been proposed (5,9,13,16,20,26,27,32,33,40). However, direct and indirect effec...

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“…Based on the TSS information, potential sigma factor binding sites were determined by searching for the consensus sites in the upstream 20 nt. Due to the lack of prediction programs for finding the consensus binding sites for M. smegmatis, the known consensus sites were identified manually (Gerhart et al 2008). A putative SigA consensus was found for IGR-2, IGR-3, AS-5, and AS-11 (Supplemental Fig.…”

Section: Identification Of Transcription Start Sites (Tss) By 59 Racementioning

confidence: 99%

Identification of small RNAs in Mycobacterium smegmatis using heterologous Hfq

Qin

et al. 2012

RNA

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Gene regulation by small RNAs (sRNAs) has been extensively studied in various bacteria. However, the presence and roles of sRNAs in mycobacteria remain largely unclear. Immunoprecipitation of RNA chaperone Hfq to enrich for sRNAs is one of the effective methods to isolate sRNAs. However, the lack of an identified mycobacterial hfq restricts the feasibility of this approach. We developed a novel method that takes advantage of the conserved inherent sRNAs-binding capability of heterologous Hfq from Escherichia coli to enrich sRNAs from Mycobacterium smegmatis, a model organism for studying Mycobacterium tuberculosis. We validated 12 trans-encoded and 12 cis-encoded novel sRNAs in M. smegmatis. Many of these sRNAs are differentially expressed at exponential phase compared with stationary phase, suggesting that sRNAs are involved in the growth of mycobacteria. Intriguingly, five of the cis-encoded novel sRNAs target known transposases. Phylogenetic conservation analysis shows that these sRNAs are pathogenicity dependent. We believe that our findings will serve as an important reference for future analysis of sRNAs regulation in mycobacteria and will contribute significantly to the development of sRNAs prediction programs. Moreover, this novel method of using heterologous Hfq for sRNAs enrichment can be of general use for the discovery of bacterial sRNAs in which no endogenous Hfq is identified.

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Approaches to Identify Novel Non‐messenger RNAs in Bacteria and to Investigate their Biological Functions: Functional Analysis of Identified Non‐mRNAs

Cited by 8 publications

References 94 publications

The Listeria monocytogenes Virulence Factor InlJ Is Specifically Expressed In Vivo and Behaves as an Adhesin

The Listeria monocytogenes Virulence Factor InlJ Is Specifically Expressed In Vivo and Behaves as an Adhesin

Identification of Mycobacterial σ Factor Binding Sites by Chromatin Immunoprecipitation Assays

Identification of small RNAs in Mycobacterium smegmatis using heterologous Hfq

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