ABSTRACT: 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. Overall design: The starting baseline for this study is Neisseria gonorrhoeae strain NCCP11945, minimally passaged since it was genome sequenced, grown at 37C in 5% CO2. All other experiments would be based off of this, both in terms of number of samples and replicates.
INSTRUMENT(S): Ion Torrent PGM (Neisseria gonorrhoeae NCCP11945)
Project description:Microarray comparative genome hybridization (mCGH) data was collected from one Neisseria cinerea, two Neisseria lactamica, two Neisseria gonorrhoeae, and 48 Neisseria meningitidis isolates. For N. meningitidis, these isolates are from diverse clonal complexes, invasive and carriage strains, and all major serogroups. The microarray platform represented N. meningitidis strains MC58, Z2491, and FAM18 and N. gonorrhoeae FA1090.
Project description:Deep sequencing of cDNA from Neisseria gonorrhoeae bacteria and human neutrophils, alone and after coincubation. Overall design: Total RNA was isolated from Neisseria gonorrhoeae strains FA1090/H041 and adherent primary human neutrophils, alone and after co-culture over time. Ribosomal RNA was depleted and the remaining RNA was reverse-transcribed into a cDNA library for Illumina sequencing. Reads were mapped to the FA1090, H041 and/or human genomes.
Project description:We report for the first time movement of Correia Repeat Enclosed Elements, through inversion of the element at its chromosomal location. Analysis of Ion Torrent generated genome sequence data from Neisseria gonorrhoeae strain NCCP11945 passaged for 8 weeks in the laboratory under standard conditions and stress conditions revealed a total of 37 inversions: 24 were exclusively seen in the stressed sample; 7 in the control sample; and the remaining 3 were seen in both samples. These inversions have the capability to alter gene expression in N. gonorrhoeae through the previously determined activities of the sequence features of these elements. In addition, the locations of predicted non-coding RNAs were investigated to identify potential associations with CREE. Associations varied between strains, as did the number of each element identified. The analysis indicates a role for CREE in disrupting ancestral regulatory networks, including non-coding RNAs. RNA-Seq was used to examine expression changes related to Correia repeats in the strain
Project description:To better understand the role of Neisseria gonorrhoeae CpxRA in controlling virulence determinants, here we defined genes potentially regulated by CpxRA by using RNA-Seq. We identified approximately 139 genes differentially expressed between cpxA (Cpx system active) and cpxR (Cpx system inactive) mutants. A large number of the differentially expressed genes encode envelope-localized proteins. Overall design: RNA of Neisseria gonorrhoeae wildtype, cpxA and cpxR mutants were collected at late log phase of growth, in quadruplicate.
Project description:In this study wild-type, fur mutant, and complemented fur mutant strains of the human pathogen Neisseria gonorrhoeae F62 were grown under high (100 uM iron) or low (100 uM desferal) iron conditions to identify genes whose expression was regulated by iron and/or Fur Overall design: Examination of each of three strains under two conditions by RNA-seq
Project description:We assessed the small RNA transcriptome of Neisseria gonorrhoeae strain MS11 in two genetic backgrounds; using wild type cells as well as cells carrying a rppH insertional mutation. It was found that the presence of the RppH enzyme affected both the quantity and length of small RNAs in various chromosomal locations. However, in comparing the two transcriptomes, we found that not all small RNAs were differentially expressed, suggesting that RppH targets only a subset of transcripts. Analysis of the small RNA transcriptome of N. gonorrhoeae in a wild type and ΔrppH mutant background.
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. Overall design: Six biofilm flow chambers were inoculated with N. gonorrhoeae strain 1291 wildype. Flow chambers were inoculated with approximately 1 ml of a cell suspension (in biofilm media) from an overnight plate that was suspended to an OD600 of 0.3. Two flow cells each were supplied by a single reservoir of media and the runoff from these two flow cells was passed through a sterile glass wool filter for the removal of detached biofilm flocs, and collected in a sterile waste flask and cultured to assess culture purity. At this time, the waste flask was replaced with a second sterile flask containing 10 ml of RNAlater® (Qiagen Corporation, Valencia, CA) and 100 µl of 10% sodium azide. For the final 24 hours of biofilm growth, the filtered planktonic effluent was collected in this solution to preserve the RNA and prevent transcriptional changes from occurring. Planktonic RNA was extracted from the collected effluent of the two flow cells, while each biofilm RNA was extracted directly from these two flow cells. RNA purity was assessed on an Agilent 2100 Bioanalyzer (Quantum Analytics, Foster City, CA), and only samples with RNA integrity numbers of 7.5 or greater were used for hybridization to microarrays. All six samples had RNA integrity numbers of 7.5 or greater. Therefore, we elected to hybridize the two samples with the highest integrity numbers.
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) Overall design: The effect of iron on global gene expression was determined over a 4-h time period by comparing the RNA profile of the organism grown in iron-deplete conditions to growth in iron-replete conditions.