Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Determination of the transcription start sites of heterologous promoters in Actinoplanes sp. SE50/110 by 5'-end specific transcriptome sequencing

ABSTRACT: The promoter structure influences binding and clearance of RNA polymerase and therefore substantially influences expression of a gene. A promoter usually consists of a -10 and a -35-region, an extended -10-motif and A+T-rich upstream promoter elements. Most of these elements are optional, whereas the -10-region is essential (Albersmeier et al. 2017). Knowledge about the transcription start sites (TSS) of genes allows genome-wide localization and determination of the promoter regions. In our group, a special protocol for the amplification of primary transcripts was developed, including the capture of primary transcripts, rewriting them into cDNA (complementary DNA) and amplification in the further course of the protocol (Pfeifer-Sancar et al. 2013). Here, TSS were manually determined with special regard to the heterologous promoters. For each construct, at least one and up to three different TSS were found, leading to the identification of one or several -10-core-hexamers. These were located mostly 6 to 7 nucleotides upstream of each TSS, which corresponds to the average distance of 6.4 nt described for Actinoplanes sp. SE50/110 by Schwientek et al. (2014).

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Actinoplanes sp. SE50/110

SUBMITTER: Schaffert, Lena

PROVIDER: E-MTAB-8433 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Evaluation of vector systems and promoters for overexpression of the acarbose biosynthesis gene acbC in Actinoplanes sp. SE50/110.

Schaffert Lena L März Camilla C Burkhardt Lisa L Droste Julian J Brandt David D Busche Tobias T Rosen Winfried W Schneiker-Bekel Susanne S Persicke Marcus M Pühler Alfred A Kalinowski Jörn J

Microbial cell factories 20190628 1

<h4>Background</h4>Actinoplanes sp. SE50/110 is a natural producer of acarbose. It has been extensively studied in the last decades, which has led to the comprehensive analysis of the whole genome, transcriptome and proteome. First genetic and microbial techniques have been successfully established allowing targeted genome editing by CRISPR/Cas9 and conjugal transfer. Still, a suitable system for the overexpression of singular genes does not exist for Actinoplanes sp. SE50/110. Here, we discuss, ...[more]

PMID: 31253141

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Project description:Background: Lactococcus garvieae is a bacterial pathogen that affects different animal species and human. Despite the widespread distribution and emerging clinical significance of L. garvieae in both veterinary and human medicine, there is almost a complete lack of knowledge about the genetic content of this microorganism. In the present study the genomic content of L. garvieae CECT 4531 was analyzed by bioinformatic tools and microarray-based comparative genomic hybridizations (CGH) experiments, using Lactococcus lactis subsp. lactis IL1403 and Streptococcus pneumoniae TIGR4 as reference microorganisms. Results: The combination and integration of in silico analyses and in vitro (CGH) experiments performed between the reference microorganisms allowed establishing an inter-species hybridization framework with a detection threshold based on the sequence similarity ?70%. With this threshold value, 267 genes were identified as having an analogue in L. garvieae, most of which (n = 258) have been documented for the first time in this pathogen. Most of these genes are related to ribosomal, sugar metabolism or energy conversion systems. Some identified genes could be involved in the pathogenesis of L. garvieae infections. Conclusions: In this study a comparative analysis based on microarray interspecies hybridization and the use of bioinformatic tools were used for the first time to study the genetic content of L. garvieae CECT 4531. Towards this approach, we identified 267 potentially present genes in L. garvieae CECT 4531, some of which could be involved in the pathogenesis of L. garvieae infections, such as als or mycA. These results provide the first insight into the genome content of L. garvieae. Array-based comparative genome hybridization (CGH): The L. lactis subsp. lactis IL1403 and S. pneumoniae TIGR4 microarrays used for the CGH analysis were purchased from Eurogentec (Serain, Belgium). The L. lactis microarray contains 4608 spots: 2126 duplicated ORFs, 32 negative controls and 324 empty spots. The S. pneumoniae microarray contains 4608 spots: 2087 duplicated ORFs, 224 negative controls and 210 empty spots. The CGH experiments were performed by means of competitive hybridizations using DNA of L. lactis subsp. lactis IL1403 or S. pneumoniae TIGR4, depending on the array as positive controls. DNAs to be hybridized on the same array were labelled with Cy3-dUTP and Cy5-dUTP, respectively. For each microarray hybridization reaction, aliquots (1–2 µg) of labelled genomic DNAs of reference (labelled with Cy3) and test (labelled with Cy5) strains, were mixed in 45 µl EGT hybridization solution (Eurogentec, Serain, Belgium) and denatured at 65ºC for 2 min. The hybridization mixture was then loaded onto a microarray slide, covered with a coverslip and incubated at 38ºC overnight. Following hybridization, the slides were washed in 2 X SSC, 0.5% SDS for 5 min followed by 5 min in 1 X SSC, 0.25% SDS. Finally, slides were rinsed in 0.2 X SSC and dried by centrifugation. The results presented herein represent a compilation of sixteen separate CGH experiments: L. lactis subsp. lactis IL1403 arrays (reference microorganism) were hybridized with S. pneumoniae TIGR4 (test microorganism) (n=2); S. pneumoniae TIGR4 arrays (reference microorganism) were hybridized with L. lactis subsp. lactis IL1403 (test microorganism) (n=2); L. lactis subsp. lactis IL1403 arrays (reference microorganism) were hybridized with L.garvieae CECT 4531 (test microorganism) (n=8); S. pneumoniae TIGR4 arrays (reference microorganism) were hybridized with L.garvieae CECT 4531 (test microorganism) (n=4). Data acquisition and analysis: The microarray was scanned after hybridization using a Scanarray HT microarray scanner (Perkin-Elmer). The signal intensity of the two fluors was determined using ImaGene software (BioDiscovery, El Segundo, CA, USA). Microarray data were analysed using ImaGene software, Microsoft Excel and an in-house designed and built Microsoft Access database [30]. Gene calling was based on a signal-to-noise ratio (SNR) > 3 for each spot. After CGH experiments, a gene was considered to show a positive result when it was present in at least three of the four CGH assays. In the case of the L. garvieae CECT 4531 hybridizations with L. lactis subsp. lactis IL1403 arrays, it was necessary to perform a greater number of assays (n=8) due to the poor quality of one of the array batches used. Thus, the criteria chosen to determine a positive result in this case was when the gene was present in at least five of the eight CGH assays.

Dataset Information

Determination of the transcription start sites of heterologous promoters in Actinoplanes sp. SE50/110 by 5'-end specific transcriptome sequencing

Publications

Evaluation of vector systems and promoters for overexpression of the acarbose biosynthesis gene acbC in Actinoplanes sp. SE50/110.

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