The MisR regulon in Neisseria gonorrhoeae strain FA19
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ABSTRACT: Whole transcriptome analysis of N. gonorrhoeae FA19 and isogenic NGEG_00293 (misR) mutant using RNA-Seq (note that NGO0177 is the misR ORF designation in mapping strain FA1090)
Project description:Whole transcriptome analysis of N. gonorrhoeae FA19 and isogenic NGEG_00293 (misR) mutant using RNA-Seq (note that NGO0177 is the misR ORF designation in mapping strain FA1090) Examination of total transcriptomes in N. gonorrhoeae FA19 WT and an FA19 misR::kan isogenic mutant to determine the regulatory impact of the MisR response regulator on cells grown under laboratory conditions. Illumina HiSeq-2000 next generation sequencing was used to sequence the transcriptomes of each strain. Reads were mapped against the N. gonorrhoeae FA1090 genome (NCBI accession number NC_002946) because at the time this experiment was run the FA19 genome was incomplete.
Project description:In Neisseria gonorrhoeae, Fur (ferric uptake regulator) protein regulates iron homeostasis gene expression through binding to conserved sequences in promoters of iron-responsive genes. We have expanded the gonococcal Fur regulon using a custom microarray to monitor iron-responsive gene expression throughout the growth curve combined with a genome-wide in silico analysis to predict Fur boxes (FB), and in vivo FuRTA assays to detect genes able to bind Fur. Keywords: time course: (1hr ,2hr, 3hr, 4hr)
Project description:Neisseria gonorrhoeae, the etiologic agent of gonorrhea, is frequently asymptomatic in women, often leading to chronic infections. One factor contributing to this may be biofilm formation. N. gonorrhoeae can form biofilms over glass and plastic surfaces. There is also evidence that biofilm formation may occur during natural cervical infection. To further study the mechanism of this biofilm formation, transcriptional profiles of N. gonorrhoeae biofilm were compared to planktonic profiles. Biofilm RNA was extracted from N. gonorrhoeae 1291 grown for 48 hours in continuous flow chambers over glass. Planktonic RNA was extracted from the biofilm runoff. When biofilm was compared to planktonic growth, 3.8 % of the genome was differentially regulated. Genes highly up-regulated in biofilm included aniA, norB, and ccp, which play critical roles in anaerobic metabolism and oxidative stress tolerance. Down-regulated genes included the nuo gene cluster (NADH dehydrogenase) and the cytochrome bcI complex, which are involved in aerobic respiration and are thought to contribute to endogenous oxidative stress. Furthermore, we determined that aniA, ccp, and norB insertional mutants are attenuated for biofilm formation over glass and transformed human cervical epithelial cells (THCEC). This data suggests that biofilm formation could minimize oxidative stress during cervical infection and allow N. gonorrhoeae to maintain a nitric oxide steady state that may be anti-inflammatory.
Project description:The overall goals and objectives of this study are to investigate the transcriptomics of Neisseria gonorrhoeae using RNA-seq. This work will look at gene expression, start points of transcription, transcriptional termination, and differences between these in different conditions and between strains and growing cultures over time.
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.
Project description:Transcriptional profiling of N. gonorrhoeae comparing wild type cells to cells with inactivated by chloramphenicol cassette (cm) dam replacing gene (drg) or wild type cells comparing to cells with inserted dam gene. The Goal was to study the role of drg or dam presence in overall expression profile.
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.
Project description:Hfq is an RNA chaperone, which functions as a pleiotropic regulator for RNA metabolism in bacteria. To characterize the role of Hfq in pathogenicity of Neisseria gonorrhoeae we generated a N. gonorrhoeae hfq mutant, MS11hfq.Transcriptional analysis using a custom-made N. gonorrhoeae microarray revealed that 369 open reading frames were differentially regulated in MS11hfq compared to the wild-type (wt) strain (202 were upregulated, 167 were downregulated).