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The pilus retraction protein PilT has a global influence on the transcriptome of Neisseria gonorrhoeae

ABSTRACT: The ATPase protein PilT mediates retraction of type IV pili. We performed microarrays comparing the transcription profile of the Neisseria gonorrhoeae wild-type strain MS11 and its isogenic pilT mutant. 63 open reading frames were found to be differentially regulated in the pilT mutant. Most interestingly, a loss of function mutation in pilT leads to an upregulation of pilE, which encodes the pilus subunit protein.

ORGANISM(S): Neisseria gonorrhoeae

PROVIDER: GSE12258 | GEO | 2008/09/01

SECONDARY ACCESSION(S): PRJNA113637

REPOSITORIES: GEO

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The pilus retraction protein PilT has a global influence on the transcriptome of Neisseria gonorrhoeae

Project description:The ATPase protein PilT mediates retraction of type IV pili. We performed microarrays comparing the transcription profile of the Neisseria gonorrhoeae wild-type strain MS11 and its isogenic pilT mutant. 63 open reading frames were found to be differentially regulated in the pilT mutant. Most interestingly, a loss of function mutation in pilT leads to an upregulation of pilE, which encodes the pilus subunit protein. Ngo arrays were generated as custom 8x15 K microarrays (Agilent Technologies) covering all open reading frames (ORFs) from N. gonorrhoeae strain FA1090 (accession number AE004969) supplemented with all ORFs from the gonoccoccal genetic island of strain MS11 (accession number AY803022) by 6 specific 60-mer oligonucleotides per gene on average. Microarray analysis was performed as dual-color hybridization with dye-reversal color-swaps as technical replicates for Cy-dye specific effect compensation and three independent biological replicates. Microarray features were extracted with the FE 9.5.3.1 software from Agilent Technologies using the GE2-v4_95_Feb07 protocol with default settings. Data were loaded into Resolver (Rosetta Biosoftware) using the MAGE-ML loader and analyzed for differential gene expression. Ratio profiles were combined into ratio experiments and regulated genes were identified with a threshold of 1.75 fold-change and anti-correlation of the color-swapped dye reversals, rendering the analysis highly stringent and robust with P values <0.0001 of the results.

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Project description:Purpose: The goal of this study was the identification of target genes of the Neisseria gonorrhoeae sRNAs NgncR_237, NgncR_162 and NgncR_163. Methods: RNA was isolated from N. gonorrhoeae using the miRNeasy Micro Kit (Qiagen). Enrichment of mRNA was done using the Universal Ribodepletion Kit followed by Next Ultra Directional Library Preparation Kit for Illumina (NEB). The cDNA was sequenced on Illumina HiSeq 3000 platform with 100 bp paired end reads. Adapters from Fastq sequences were removed using cutadapt version 1.2.1. Only reads exceeding a mean base quality 5 within all sliding windows of 5 bp were mapped to the genome of strain N. gonorrhoeae MS11 (reference genome ASM15685v2). Annotation of non-coding RNAs was according to Remmele et al.(2014). Read mapping was conducted using Bowtie2 v2.1.0. DeSEQ2 version 1.6.2 was used to identify differentially regulated transcripts. Results: N. gonorrhoeae genes were identified which were upregulated or downregulated upon induced expression of sRNAs NgncR_237, NgncR_162 and NgncR_163. Conclusions: sRNA NgncR_237, NgncR_162 and NgncR_163 are involved in the regulation of genes involved in type IV pilus biogenesis, energy metabolism and transport processes.

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