This work is composed of two separate projects that contribute to the field’s understanding of Neisseria gonorrhoeae genetics and physiology. First, I showed that N. gonorrhoeae mounts a substantial transcriptional program in response to hydrogen peroxide (HP), a prominent reactive oxygen species (ROS) encountered during infection. We measured which strain FA1090 genes show differential transcript abundance in response to sublethal amounts of HP to differentiate HP-responsive signaling from widespread cellular death and dysregulation. RNA sequencing (RNA- Seq) revealed that 150 genes were significantly upregulated and 143 genes downregulated following HP exposure. We annotated HP-responsive operons and all transcriptional start sites (TSSs) and identified which TSSs responded to HP treatment. We compared the HP responses with other previously-reported genes and found only partial overlapping of other regulatory networks, indicating that the response to HP involves multiple biological functions. Using a representative subset of responsive genes, we validated the RNA-Seq results by RT-qPCR and found that the HP transcriptome was similar to that of sublethal organic peroxide. None of the genes in the representative subset, however, responded to sublethal levels of HOCl or O2-, other prominent ROS encountered during infection. The magnitude of the transcriptional response to sublethal HP, comprising 293 genes, is comparable to other gene regulatory networks known to significantly affect N. gonorrhoeae biology, such as networks affected by iron and oxygen availability. The magnitude of the transcriptional response to sublethal HP, as well as the specificity of this response for sublethal HP, support the idea that N. gonorrhoeae may use variations in HP levels as a signal for different stages of infection. In addition, we developed a new method for measuring pilin antigenic variation (Av) using droplet digital polymerase chain reaction technology (ddPCR), wherein all ddPCR reagents are partitioned into emulsified oil droplets, each containing a single DNA template. Two fluorescent Taqman probes were designed to detect a portion of the pilS3- copy 1 sequence (S3C1) when it recombines into the pilE locus, or to detect a conserved sequence present in all pilE variants. This assay utilizes a N. gonorrhoeae strain containing IPTG-inducible RecA recombinase required for pilin Av. In addition, it utilizes a starting pilE gene that does not contain the S3C1 sequences and strains grown with or without IPTG. The ddPCR assay measures the pilin Av frequencies with a level of detection limit of 0.50% S3C1 variants/pilE. S3C1 pilin variants were not detected without IPTG in the growth medium, but occurred at a frequency of 1% in N. gonorrhoeae grown for 22 hours with IPTG induction. The frequency of pilin antigenic variation (Av) was reduced to 0.5% and to below the level of detection in recX and recQ mutants, respectively, which were previously shown to be partially deficient in pilin Av. We used the ddPCR assay to determine whether colony density affects Av frequencies as suggested by pilus phase variation phenotypes. N. gonorrhoeae grown on solid medium with confluent colonies exhibited one third lower Av frequencies than N. gonorrhoeae grown on solid medium with colonies that were spaced farther apart than 1,580 colonies per cm2. These data show colony density affects antigenic variation frequency. ddPCR is an accurate, efficient way to measure Av frequency in N. gonorrhoeae without next-generation sequencing, and shows colony density affects Av frequency.

Creator

• Quillin, Sarah Jane

DOI

• https://doi.org/10.21985/n2-c10h-1n52

Subject

• Microbiology

Language

• en

Alternate Identifier

• http://dissertations.umi.com/northwestern:14977
• etdadmin_upload_716337

Keyword

• Bacterial genetics
• Microbiology
• Neisseria gonorrhoeae
• Bacteriology

Date created

• 2018-01-01

Resource type

• Dissertation

Rights statement

• In Copyright