In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 20 ( 2012-05-15)
Abstract:
This study represents a comprehensive resource for scientists working on gene regulation and Salmonella infection biology. We anticipate that these data will allow scientists to rapidly identify promoters that show important patterns of expression and facilitate discoveries that relate to gene regulation. The findings can be visualized at www.imib-wuerzburg.de/research/salmonella . The combination of chromatin immunoprecipitation of RNA polymerase and the primary σ 70 sigma factor with global identification of transcriptional start sites is unique for a bacterial pathogen and is likely to be a valuable approach in many microbial systems. Small, noncoding RNAs have recently been found to control major aspects of bacterial gene regulation. Unfortunately, only a fraction of this small regulatory RNA (sRNA) complement had been identified in S . Typhimurium ( 4 ). Our approach has revealed the suite of 140 small, noncoding RNAs that are expressed by Salmonella during infection-relevant growth. To identify small, regulatory RNAs with confidence, the analysis of RNA sequencing (RNA-seq) data was combined with the analysis of sequences of RNA bound to the Hfq chaperone protein. In general, a chaperone protein assists RNA in binding to other relevant molecules. To analyze these data, we used an approach that we developed in 2008 ( 4 ) and identified 60 newly identified Salmonella sRNAs, half of which were confirmed by Northern blotting, an independent method of detecting RNA. One of these sRNAs, IsrJ, has already been shown to play a role in virulence ( 5 ). We look forward to the identification of more sRNAs that control the ability of Salmonella to cause disease. In this study we identified the transcriptional start sites that drive expression of the virulence genes present in Salmonella pathogenicity island 1 that play a role in the invasion of epithelial cells. In addition to the expected primary transcriptional start sites that facilitate expression of the key operons, we also report evidence for several internal transcriptional start sites that allow expression of individual virulence genes. Fig. P1 shows the transcriptional map of the main regulatory region of Salmonella pathogenicity island 2 (SPI2) and reveals a secondary transcriptional start site for the ssrAB genes that encode the two-component master regulator of SPI2. We also identified an antisense transcript within ssrA that could have regulatory significance. Specifically, the location and nature of S . Typhimurium gene promoters was not known. Transcription in bacteria is initiated at promoter regions of DNA, where the core RNA polymerase associates with a protein called a sigma factor to drive transcription and produce mRNA, the crucial first step for the activity of a gene. Here, we used a combination of RNA sequencing techniques and chromatin immunoprecipitation to identify the first nucleotide of transcripts and to locate the transcription machinery on the chromosome of S . Typhimurium. Sequence analyses of 〉 800 promoters revealed a sequence motif for binding of RNA polymerase with Sigma70 and defined a consensus S . Typhimurium gene promoter. The publication of the annotated genome of S . Typhimurium in 2001, and advances in genomics and global mutagenesis, led to the functional characterization of more virulence proteins than for other bacterial pathogens ( 1 ). Gene expression analyses have revealed the classes of genes that react to environmental stressors, both in vitro and during infection ( 2 , 3 ). Our knowledge of the bacterial genes that are critical for survival, adaptation, and disease has helped to make S . Typhimurium the best-understood bacterial infection model in biology. However, the availability of basic transcriptional information for S . Typhimurium has lagged behind that for other bacteria. A few bacterial species have evolved the ability to thrive inside mammalian cells by producing so-called effector proteins that hijack the cellular machinery of the host. Foremost among these intracellular pathogens is Salmonella enterica serovar Typhimurium ( S . Typhimurium), which produces ∼40 effector proteins with a broad range of functions. These proteins are encoded by virulence genes located on chromosomal regions called pathogenicity islands. The expression of these virulence genes must be tightly regulated and choreographed to ensure that the right gene is transcribed at the right time during infection. However, our understanding of this important aspect of gene regulation remains limited.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1201061109
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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