BACHELOR’S THESIS Nanjing Agricultural University · 2023
MICROBIOLOGY & OXIDATIVE STRESS
Exploring DinF’s role in bacterial stress responses
An initial investigation of DinF-mediated oxidative stress resistance in Klebsiella pneumoniae
English summary of my undergraduate thesis, originally written in Chinese.
My bachelor’s thesis investigated the contribution of DinF to oxidative stress resistance in Klebsiella pneumoniae. The project followed a candidate from a genetic screen through insertional inactivation, laboratory stress-response comparisons, and a competitive intestinal colonization model.
01 / BACKGROUND
Why investigate a DNA damage-associated protein?
Bacteria encounter reactive oxygen species (ROS), including hydrogen peroxide, which can damage DNA, proteins, and other cellular components. Understanding how bacteria tolerate oxidative stress helps connect laboratory stress responses with the challenges they encounter in a host.
Why DinF? My screening work identified dinF as a candidate associated with an altered oxidative stress response. Previous research in Escherichia coli had linked DinF to protection against oxidative stress and bile salts. This provided a reason to investigate DinF in K. pneumoniae, rather than assuming that its function was identical across bacterial species.
Why examine the SOS response? The SOS system coordinates bacterial responses to DNA damage, with LexA acting as a key regulator. The reported association of dinF with this system motivated an exploratory lexA comparison. A shared stress phenotype, however, would not on its own establish that DinF acts through a particular SOS-dependent mechanism.
The research gap: did disruption of dinF affect resistance to different oxidative stresses in K. pneumoniae, and would any laboratory phenotype correspond to a measurable difference in intestinal colonization?
Background: undergraduate thesis Introduction. Related primary study: Rodríguez-Beltrán et al., 2012 (E. coli DinF and oxidative stress). This earlier study provides context, not evidence of the mechanism in my K. pneumoniae experiments.
02 / RESEARCH QUESTIONS
From a screening hit to a testable phenotype
- Stress response: does insertional inactivation of dinF alter survival after hydrogen peroxide or cumene hydroperoxide (CHP) exposure, and is this accompanied by a baseline growth defect?
- Possible regulatory context: what does a separate lexA insertion-inactivation comparison reveal about oxidative stress sensitivity, and how does its growth phenotype limit interpretation?
- Host context: is a difference between wild-type and dinF-disrupted bacteria detectable in the tested competitive mouse intestinal colonization model?
Gene disruption: the strains described here were insertion-inactivated. The Δ notation in the original figures should not be read as evidence of a precise, clean gene deletion.
03 / EXPERIMENTAL APPROACH
Connecting the questions to experimental comparisons
Candidate screening
I screened a transposon mutant library for an altered oxidative stress response and used insertion-site analysis to identify dinF as a candidate.
Functional comparisons
I studied insertion-inactivated strains and compared growth and responses to different oxidative stresses. A separate lexA comparison explored the possible SOS connection.
Physiological context
I compared the laboratory findings with competitive colonization results in an adult mouse intestinal model, and interpreted the data using GraphPad Prism.
Methods at a glance
The thesis combined transposon-library screening, arbitrary PCR, sequencing and insertion-site analysis, genetic validation, growth-curve comparisons, stress-response measurements, and competitive colonization analysis.
The project illustrates a research progression from an initial screen to a candidate-gene study and a comparison with an animal model.
Source: thesis Chapters 2–3; defence slides 5–17. This overview summarizes the research rather than reproducing experimental protocols.
04 / RESULTS
What the experiments showed
- 01
The dinF phenotype depended on the stress tested
Disruption of dinF increased sensitivity to hydrogen peroxide. The thesis reported no clear change in sensitivity to the organic peroxide CHP under the tested conditions, and no evident baseline growth defect in the dinF comparison.
View dataHide data ＋Hydrogen peroxide responseThe disrupted strain showed lower survival in several comparisons. The effect varied across the tested conditions.Thesis Fig. 2-11, printed p. 22 (PDF p. 27). Original labels and bilingual caption retained. Student’s t-test as reported in the thesis. Supporting comparisons in the thesis reported no evident baseline growth defect and no clear change in CHP sensitivity. The figure above specifically shows the hydrogen peroxide experiment.
- 02
The SOS connection remained preliminary
The lexA-disrupted strain showed a strong stress-response phenotype alongside a substantial growth defect. This was consistent with a possible SOS connection, but did not establish how DinF acts or isolate a specific causal pathway.
View dataHide data ＋Exploratory LexA comparisonThe disrupted strain showed pronounced hydrogen peroxide sensitivity. The accompanying growth defect prevents attributing this phenotype solely to a specific oxidative stress pathway.Thesis, printed p. 26 (PDF p. 31). The text refers to Fig. 2-14, while the original caption is labelled Fig. 2-12; original wording and labels are retained. Student’s t-test as reported in the thesis. - 03
The mouse study did not mirror the laboratory phenotype
No significant difference in intestinal colonization was detected between wild-type and dinF-disrupted bacteria in the model studied. The in vitro finding therefore did not translate into a demonstrated colonization disadvantage in this experiment.
View dataHide data ＋Intestinal colonizationThe displayed comparisons did not show a statistically significant difference between the tested strains.Thesis Fig. 3-1, printed p. 28 (PDF p. 33). Mann–Whitney test as described in the text. The original English caption contains a threshold typo; the Chinese caption specifies P < 0.05.
05 / INTERPRETATION
A laboratory phenotype is one part of the story
This project taught me to connect screening results with follow-up evidence, distinguish a proposed mechanism from a demonstrated one, and interpret a finding within the limits of its experimental model.
Limitations & next questions
- The study was an initial investigation, and the specific mechanism connecting DinF to oxidative stress remained unresolved.
- The growth defect in the lexA comparison complicates attribution of its phenotype to a specific stress-response mechanism.
- A non-significant colonization result does not demonstrate that DinF has no role in every host environment.
- The observed difference between laboratory and animal results should not be treated as evidence for a clinical intervention.
METHODS I WORKED WITH
Based on my undergraduate thesis, dated 9 May 2023, and its accompanying defence presentation. The thesis title is summarized in natural English; original figures retain their source wording.
MOMENTS FROM MY UNDERGRADUATE YEARS