Campylobacteriosis is the primary cause of bacterial diarrhea and sickens approximately 1 in 10 people annually (WHO, 2020). Campylobacteriosis generally lasts for 3–6 d, and symptoms incited by Campylobacter spp. include diarrhea, fever, headache, abdominal pain, nausea, and vomiting (WHO, 2020). Complications from Campylobacter infection can be serious and even fatal, with most fatalities occurring in the very young, elderly, and immunocompromised (WHO, 2020). Various post-infection complications are possible including arthritis, irritable bowel syndrome, and Guillain-Barré syndrome (CDC, 2024b); the latter causes muscle weakness that can progress into paralysis (CDC, 2024a). Recovery from Guillain-Barré syndrome can take months or even years with some cases leading to permanent paralysis (CDC, 2024a), and Campylobacter infections are one of the leading causes of the syndrome (Poropatich et al., 2010; CDC, 2024a).

One of the primary causal agents of campylobacteriosis is Campylobacter jejuni (WHO, 2020), which is a gram-negative, motile bacteria (Perez-Perez and Blaser, 1996). The bacteria is frequently cultured in vitro at 42°C, the body temperature of chickens, or 37°C, the body temperature of humans, but can survive a range of temperatures (Aroori et al., 2013; Khan et al., 2013). C. jejuni is microaerophilic and requires an O₂ concentration of 3–10% for growth; however, some strains were reported to be aerotolerant (Oh et al., 2015a), which allows the bacterium to survive meat processing procedures. Strains of Campylobacter that are capable of surviving for more than 12 h of exposure to atmospheric oxygen levels during incubation with agitation are classified as aerotolerant, and those that survive more than 24 h are considered hyper-aerotolerant (Oh et al., 2015a). Aerotolerant Campylobacter spp. are highly prevalent in strains isolated from raw meat, with some studies reporting 71.4–86.6% of isolates to be aerotolerant (Oh et al., 2015a; Kim et al., 2019). Clinical isolates also exhibit aerotolerance (Oh et al., 2018), and this trait increases the likelihood of human infections. The prevalence of aerotolerant isolates in raw meat and human patients highlights the importance of studying how microaerophilic Campylobacter spp. develop aerotolerance and survive in atmospheric oxygen.

Despite this significance, the genetic mechanisms underlying aerotolerance in C. jejuni are not well understood. Genes known to be involved in aerotolerance include katA, ahpC, and sodB, which encode catalase, alkyl hydroperoxide reductase, and superoxide dismutase, respectively (Vercellone et al., 1990; Purdy and Park, 1994; Baillon et al., 1999). Numerous studies have demonstrated relationships between these three genes and resistance to O₂ exposure or oxidative stress (Kikuchi and Suzuki, 1984; Grant and Park, 1995; Purdy et al., 1999; van Vliet et al., 1999; Lee et al., 2005; Handley et al., 2015; Oh et al., 2015a, 2015b, 2019; Rodrigues et al., 2016; Jeon et al., 2022). Other Campylobacter genes have been noted as potential contributors to aerotolerance, but most are not well-characterized and many have unknown functions (Stahl and Stintzi, 2011). Certain strategies have been implicated in Campylobacter’s aerotolerance as well such as biofilm formation (Joshua et al., 2006; Asakura et al., 2007; Reuter et al., 2010; Turonova et al., 2015; Feng et al., 2016; Oh et al., 2016; Kassem et al., 2017; Efimochkina et al., 2019; Zhong et al., 2020; Mouftah et al., 2021; Kanaan, 2024), the viable-but-nonculturable (VBNC) state (Moran and Upton, 1987a; Lee et al., 2005; Zhong et al., 2020), and polymicrobial interactions with other bacteria and even amoeba (Axelsson-Olsson et al., 2007; Wongjaroen et al., 2008; Hilbert et al., 2010; Bui et al., 2012; Karki et al., 2021; Anis et al., 2022; Šimunović et al., 2022). Delaporte et al. (2024) have summarized previous research on potential genetic mechanisms and other strategies which may contribute to aerotolerance (Delaporte et al., 2024).

In this study, we use RNA-Seq to compare gene expression in strains of C. jejuni in response to O₂ exposure. Gene expression during microaerobic conditions (Time 0) were compared to aerobic conditions (Times 0.5, 6, 12, and 24 h) in the aerosensitive T1-21 and aerotolerant WP2202 strains of C. jejuni. The data were analyzed to detect differences in gene expression in response to microaerobic growth and O₂ exposure and provide valuable information on how aerotolerant and aerosensitive C. jejuni strains differ in their response to oxygen exposure.

In this study, we compare differences in gene expression in the aerosensitive T1-21 and aerotolerant WP2202 strains of C. jejuni during microaerobic conditions (time 0) and aerobic conditions (times 0.5, 6, 12, and 24 h). The goal of comparing gene expression in the aerotolerant and aerosensitive strains was to identify potential genetic mechanisms that allow the aerotolerant strain to survive the same level of O₂ exposure that is lethal to the aerosensitive strain. Additionally, we expected that gene expression in the two strains would change in aerobic conditions as the bacterial cells attempt to survive O₂ exposure, which is why the comparison of gene expression in aerobic and microaerobic conditions is important. Identifying the genetic mechanisms underlying aerotolerance in C. jejuni and its response to oxidative stress is vital for understanding the ability of this bacterium to survive O₂ exposure during meat processing and for developing possible strategies to reduce the contamination of our food supply with this pathogen.

Clear differences in gene regulation were found in the two strains of C. jejuni. Interestingly, the aerotolerant WP2202 differentially regulated a greater percentage of genes under aerobic conditions than the aerosensitive T1-21 strain at 0.5, 6, and 12 h but not at 24 h. It is possible that the T1-21 cells begin to die at 24 h due to prolonged exposure to oxidative stress, and the uptick in gene expression at this timepoint may be a final attempt at survival. Although prolonged exposure to oxidative stress at 24 h would likely be lethal to the aerosensitive T1-21 strain, the continued changes in gene expression suggest that some of the bacterial cells were surviving. At 24 h, the T1-21 strain may be entering the viable-but-nonculturable (VBNC) state, which is a strategy used by C. jejuni to cope with oxidative stress (Moran and Upton, 1987a; Lee et al., 2005; Zhong et al., 2020). One study indicates cj1254 as potentially vital for the VBNC state (Ohno et al., 2024), and a close homolog to the gene (locus tag LBCKBIAG_01152) was found to be upregulated (fold change of 2.7) at 24 h in the aerosensitive strain in our study, however its regulation was nonsignificant at the other timepoints (Supplementary Table S3). In contrast, gene expression in the aerotolerant WP2202 strain began to increase earlier than in the aerosensitive T1-21, with an initial spike in regulation starting at 0.5 h that continued to increase up to 12 h. The rapid change in gene expression exhibited by WP2202 in response to aerobic stress likely improves its ability to survive O₂ exposure for longer durations than the aerosensitive T1-21 strain. Additionally, WP2202 exhibited widespread downregulation of genes in its plasmid, possibly as a strategy to conserve resources under oxidative stress (Supplementary Table S6).

The number of genes significantly regulated in each COG category was recorded for the purpose of noting general trends in regulation that could indicate what pathways are differentially regulated in response to aerobic conditions. The majority of genes were assigned to COG category S, which includes all genes of unknown function; this data reveals that much of the C. jejuni genome remains uncharacterized. The category with the second largest number of genes differentially regulated was COG category J (translation, ribosomal structure, and biogenesis). In the aerotolerant WP2202, a large number of genes in COG category J were downregulated at all timepoints, possibly because the strain was conserving resources during aerobic stress. Unlike the WP2202 strain, the T1-21 strain upregulated several category J genes at 24 h, perhaps as part of a last-minute survival attempt. The other COG categories represented in the RNA-Seq data contained fewer DEGs than those in category J. In the aerosensitive T1-21, the upregulation of genes in COG category C (energy production and conversion) may have been to produce the additional energy needed to fuel the oxidative stress response. In the aerotolerant WP2202 strain, category C genes were primarily downregulated at 0.5 and 6 h, followed by upregulation at 12 and 24 h. If the aerotolerant strain was coping well with oxidative stress, upregulation of category C genes may have been unnecessary until prolonged oxidative stress occurred. The aerotolerant WP2202 upregulated COG category E (amino acid transport and metabolism) genes at 6 h, followed by downregulation at 12 and 24 h. The early upregulation of category E genes in aerotolerant WP2202 could be part of the strain’s stress response, whereas downregulation at 12 h and 24 h may reflect a need to save resources after prolonged stress. Both strains apparently benefit from upregulating genes from COG category F (nucleotide transport and metabolism) at the later timepoints, suggesting that nucleotide transport and metabolism is important after prolonged stress. Genes in COG category H (coenzyme transport and metabolism) were upregulated in the aerotolerant WP2202 at 6 h, perhaps as part of the oxidative stress response. Differential regulation of category H genes in the aerosensitive T1-21 was not obvious until 12 h. Based on these differences in the two strains, it is plausible that the early upregulation of category H genes in the aerotolerant strain is important for O₂ tolerance. Coenzymes are known to have roles in the oxidative stress response in bacteria (Gout, 2019), which suggests that this category of genes might help the organism survive O₂ exposure.

The katA, ahpC, and sodB genes have been well-studied for their roles in the oxidative stress response in Campylobacter (Vercellone et al., 1990; Purdy and Park, 1994; Baillon et al., 1999). Our data show that these three genes were differentially regulated in both the aerosensitive T1-21 and the aerotolerant WP2202. Expression of katA gene, which encodes a catalase that helps protect bacteria from hydrogen peroxide (Verhoeff-Bakkenes et al., 2008), was highly upregulated (fold change > 60) in both strains at 12 h and expression was over two-fold higher in the WP2202 as compared to T1-21. Furthermore, katA expression was higher in the aerotolerant WP2202 strain versus T1-21 at 6 and 24 h, which likely contributes to the higher aerobic fitness of the aerotolerant strain. With respect to katA, our results agree with other studies showing increased levels of catalase activity in Campylobacter spp. during oxidative stress, including studies conducted with aerotolerant strains (Vercellone et al., 1990; Rodrigues et al., 2016; Oh et al., 2019).

The ahpC gene encodes an alkyl hydroperoxide reductase that protects DNA from oxidative stress (Jacobson et al., 1989). Similar to katA, an uptick in ahpC expression was observed at 12 h for both WP2202 and T1-21, with a slightly higher magnitude (11-fold change) in the aerotolerant strain. This data provides further evidence that the oxidative stress response is particularly important at the 12 h timepoint in both strains. Our data show a similar level of ahpC upregulation in both T1-21 and WP2202, which differs from another study where this gene was more highly regulated in an aerotolerant strain of C. jejuni as compared to an aerosensitive reference strain (Rodrigues et al., 2016).

Superoxide dismutase, which is encoded by sodB, is involved in the detoxification of reactive oxygen species (ROS) (Fridovich, 1983; Purdy and Park, 1994). Mild upregulation of sodB was observed at all timepoints in the aerotolerant WP2202 strain, suggesting a role in the oxidative stress response. In the aerosensitive strain T1-21, upregulation of sodB was observed only at the 12-h timepoint and was downregulated at 24 h, with the other timepoints nonsignificant. Our data for sodB agrees with prior studies that indicate its role in aerotolerance (Kikuchi and Suzuki, 1984; Lee et al., 2005; Rodrigues et al., 2016; Oh et al., 2019).

Numerous other genes were noted in the data based on previous studies showing a relationship with aerotolerance or the oxidative stress response. Three genes, acnB, tpx, and trxB, were upregulated at statistically significant timepoints in both strains, which agrees with studies showing a potential role for these genes in the oxidative stress response (Atack et al., 2008; Flint et al., 2014; Rodrigues et al., 2016). Another gene, czcD, was not found in the RNA-Seq data for the aerosensitive T1-21 strain but was upregulated at all statistically significant timepoints in the aerotolerant WP2202. A prior study suggested a role for czcD in resistance to H₂O₂ (Stoakes et al., 2024), so it remains possible that czcD contributes to aerotolerance in WP2202. CzcD is also involved in zinc export, providing protection against toxic zinc levels potentially imposed by the host as an antimicrobial strategy (Barnawi et al., 2020). Although fusA and mreB were previously shown to be induced by paraquat, which causes oxidative stress (Garénaux et al., 2008), these two genes were downregulated in our RNA-Seq data, indicating they may not have roles in T1-21 or WP2202 aerotolerance. A previous study indicated that a msrA mutant exhibited decreased resistance to oxidative stress (Atack and Kelly, 2008). In the aerotolerant WP2202, msrA was downregulated at 12 and 24 h; however, msrA expression at other timepoints in WP2202 and all timepoints in T1-21 were statistically nonsignificant. The waaF/rfaF gene is known to be involved in capsule formation, and a waaF mutant had decreased aerotolerance (Kim et al., 2023). Although waaF was not present in the aerosensitive T1-21 strain, it was downregulated in the aerotolerant WP2202 strain. In S. aureus, yabJ2 was reported to have a potential role in capsule formation (Meier et al., 2007). In the RNA-Seq data for both T1-21 and WP2202, yabJ2 was upregulated at all statistically significant timepoints with an uptick in expression at 12 h. In a previous study, dps was upregulated in C. jejuni under aerobic conditions (Rodrigues et al., 2016) and may function to protect the species from oxidative stress by sequestering iron (Ishikawa et al., 2003). In WP2202, dps was upregulated at all timepoints, which suggests a role in aerotolerance. The flagellin gene flaA was shown to be induced during exposure of C. jejuni to paraquat (Garénaux et al., 2008). In our RNA-Seq data, flaA was induced in both T1-21 and WP2202 at 12 and 24 h, suggesting this gene may function after prolonged exposure to oxidative stress. In C. jejuni, flgI, pseB, and flgH function in motility, and mutations in these genes render the bacterium more susceptible to oxidants (Flint et al., 2014). Oddly, our data showed that flgI was downregulated at all significant timepoints in both strains except 12 h where it was slightly upregulated. Similarly, pseB was slightly upregulated at the 12 h timepoint in T1-21 but was downregulated at all other significant timepoints. Most of the RNA-Seq data for pseB expression in strain WP2202 was not significant. Furthermore, flgH showed a similar pattern of initial downregulation followed by upregulation at the 12 and 24 h timepoints in both strains. It remains plausible that C. jejuni is upregulating motility genes in an attempt to escape oxidative stress. When the quorum-sensing gene luxS was mutated in C. jejuni, the bacterium showed increased sensitivity to certain oxidants (He et al., 2008). Our data for both strains showed that luxS was mildly upregulated at 6 h and downregulated at 24 h, suggesting that it could have an early function in the oxidative stress response.

Our RNA-Seq data revealed additional genes that might have dual roles in responding to both heat and oxidative stress. The clpB, dnaK, grpE, and hrcA gene products function as proteins or regulators of the heat shock response (Thies et al., 1999a, 1999b; Andersen et al., 2005; Ayllón et al., 2017), and groS and groL are known to be upregulated during heat stress in E. coli (Chueca et al., 2017). Our data for both T1-21 and WP2202 showed that clpB, dnaK, groL, groS, and grpE were upregulated at 6, 12 and 24 h. The expression pattern in the aerosensitive T1-21 strain was similar for hrcA except upregulation occurred later at 12 and 24 h. In both C. jejuni strains, these genes exhibited an uptick in upregulation at 12 h, which supports the dual function of these genes in response to both the heat and oxidative stress.

Iron is generally viewed as a causal agent in oxidative stress due to its role in the formation of ROS (Touati, 2000). Interestingly, the addition of iron to growth media was shown to increase aerotolerance in Campylobacter (Bowdre et al., 1976; Bolton and Coates, 1983; Moran and Upton, 1987b; Tangwatcharin et al., 2007) and several genes involved in iron acquisition function in aerotolerance, including dps, cft, and tonB2 (Wai et al., 1996; Ishikawa et al., 2003; Flint et al., 2014). In our RNA-Seq data, tpd (encoding P19), hugZ (Cj1613c), and hmuU were highly upregulated in the aerosensitive T1-21 strain but only slightly induced in the aerotolerant WP2202. These genes have presumptive roles in iron acquisition and regulation (Ridley et al., 2006; Chan et al., 2010; Woo et al., 2012). Iron uptake in some prokaryotes is associated with cirA2 (Uljanovas et al., 2023), which was highly upregulated in T1-21 but only slightly induced in the aerotolerant WP2202 strain. Since iron also functions in the formation of ROS, induction in the aerosensitive T1-21 strain render this strain more vulnerable to O₂ exposure. Another gene involved in iron regulation is dps, which was mentioned earlier for its role in the oxidative stress response. Interestingly, dps differs from other iron-related genes in that it is upregulated at all timepoints in the aerotolerant WP2202 but is initially downregulated in the aerosensitive T1-21 strain. Since the Dps protein binds and sequesters iron, the aerotolerant strain may be recruiting this protein as a defense against iron-induced oxidative stress (Ishikawa et al., 2003).

A total of 17 ribosomal genes were downregulated in the T1-21 and WP2202 strains. Fifteen of these genes were more highly downregulated in the aerotolerant WP2202 strain than the aerosensitive T1-21. Ribosome biogenesis is quite costly to the bacterial cell (Zegarra et al., 2023), so expression of these genes may be downregulated when the bacterium is under stress as a means of conserving resources. Based on the differences in regulation of these genes in the two strains, the aerotolerant WP2202 is more effective at downregulating the expression of these genes, which could assist with its survival when it encounters O₂.

Several genes were identified with contrasting regulation in the two C. jejuni strains. For example, cheW, nrfA, tolQ2, and yknY were upregulated in the aerosensitive T1-21 but downregulated in the aerotolerant WP2202 strain. Previous studies demonstrated that cheW mutants of C. jejuni more readily form biofilms as compared to wild-type strains (Tram et al., 2020). Furthermore, hyper-aerotolerant strains of C. jejuni were better at biofilm formation than other strains (Mouftah et al., 2021), suggesting that the aerotolerant WP2202 strain may downregulate cheW as a way of increasing its biofilm formation and enhancing tolerance to oxidative stress (Joshua et al., 2006; Asakura et al., 2007; Reuter et al., 2010; Turonova et al., 2015). It is unclear why the aerosensitive T1-21 strain induces cheW rather than downregulating it; however, cheW also has a role in chemotaxis (Reuter et al., 2020), and its upregulation may be part of an escape strategy for C. jejuni. The nrfA gene encodes a nitrite reductase that is involved in the nitrate respiration pathway, which allows nitrate to be used as an alternate electron acceptor when O₂ is limiting (Pittman et al., 2007). The aerotolerant strain may downregulate this pathway under aerobic conditions when O₂ is plentiful; however, it is unclear why nrfA was upregulated when O₂ levels were high. Several genes in the aerotolerant strain (tolQ2, yknY) may be downregulated to more effectively manage resources under oxidative stress; however, it remains unclear why these genes were upregulated in the aerosensitive T1-21. In contrast to the previous genes discussed, dcuA was upregulated in the aerotolerant WP2202 strain but downregulated in the aerosensitive T1-21. In E. coli, dcuA encodes a membrane protein involved in transporting C4-dicarboxylates under anaerobic conditions (Six et al., 1994). Although C. jejuni does not grow well anaerobically, a prior study demonstrated that dcuA was upregulated in microaerobic conditions (Woodall et al., 2005). The aerosensitive T1-21 might downregulate dcuA in aerobic conditions during its transition to utilizing the aerobic C4-dicarboxylate transport system. It remains unclear why the aerotolerant WP2202 upregulated dcuA in aerobic conditions.

We hypothesized that the changes in gene expression associated with the oxidative stress response may occur rapidly after O₂ exposure, and special note was taken of genes with large fold changes (magnitude > 10) at the 0.5 and 6 h timepoints. The genes with large spikes in gene expression at 0.5 or 6 h in the aerosensitive T1-21 included btuB1, btuB2, cirA2, btuF, tpd, hmuU, and yknY. The genes cirA2, tpd, and hmuU were discussed above for their roles in iron transport and metabolism, whereas yknY was mentioned due to its contrasting patterns of regulation in the two strains. The btuB1, btuB2, and btuF genes are involved in the acquisition of vitamin B12 in other bacterial species (Degnan et al., 2014; Poudel et al., 2023). Since B12 is critical for the functionality of many enzymes (Chimento et al., 2003), the high upregulation (fold changes ranging from 12 to 125) of these genes in the aerosensitive T1-21 strain may be a reaction to aerobic stress. Interestingly, WP2202 displayed only mild upregulation of btuB1 and btuB2 (fold changes < 10) and the data for btuF was not significant, indicating a less important role for these genes in the aerotolerant strain. Two unknown genes were also observed with high upregulation in the aerosensitive strain, including GenBank locus tags ASB61_RS03560 and a region overlapping locus tags ASB61_RS07715, ASB61_RS07720, and ASB61_RS07725. The locus ASB61_RS03560 was described in GenBank as “energy transducer TonB.” In Campylobacter spp., TonB proteins function in energy transduction from the inner to outer membrane, provide energy for the acquisition of ferric enterobactin (Zeng et al., 2013), and may also function in the oxidative stress response (Flint et al., 2014). It is tempting to speculate that locus ASB61_RS03560 encodes a gene that functions in iron acquisition in response to oxidative stress. The ASB61_RS07715, ASB61_RS07720, and ASB61_RS07725 loci are described in GenBank as a Fe-S protein, an ABC transporter permease, and a FtsX-like permease family protein. FtsX functions in the remodeling of bacterial cell walls during cell division (Mavrici et al., 2014), and it is possible that the aerosensitive T1-21 strain could upregulate this unknown gene in an attempt to reinforce the integrity of its cell wall during oxidative stress. Another unknown gene upregulated in T1-21 mapped to a region overlapping chuA and chuB, which are involved in heme uptake in Campylobacter (Ridley et al., 2006); this provides additional evidence that the aerosensitive strain upregulate iron acquisition genes during oxidative stress. Finally, an unknown gene on the pcjDM plasmid of the aerosensitive strain (locus tag ASB61_RS08465) was observed to be highly downregulated at 6 h. According to GenBank, this gene likely encodes a phage holin family protein, suggesting evidence of past phage infection. Since holin proteins are generally detrimental to bacteria (Young, 1992), it is reasonable to assume that this gene would be downregulated under most circumstances. The increase in downregulation of the gene under oxidative stress conditions as compared to microaerobic conditions might be evidence of the bacteria protecting itself from additional damage during stress.

Several genes that exhibited spikes in up- or down-regulation at the 0.5 or 6 h timepoints were present in the aerotolerant WP2202 strain as well, including clpB, grpE, hrcA, katA, and kdgR, and unknown genes with GenBank locus tags A0W69_06090, A0W69_06100, A0W69_08085, a region overlapping locus tags A0W69_08115 and A0W69_08120, A0W69_01600, and a region overlapping locus tags A0W69_08545 and A0W69_08550. Several of these genes were discussed above for their roles in heat shock (clpB, hrcA, grpE) or oxidative stress (katA). A spike in downregulation was observed at the 6 h timepoint for kdgR in the aerotolerant WP2202, but the data for kdgR expression in T1-21 was not significant. In other bacterial species, kdgR functions as a repressor in the metabolism of sugars (Pujic et al., 1998). Although Campylobacter was thought to lack carbohydrate metabolism, there is now evidence that it may have limited metabolism of carbohydrates (Garber et al., 2020); therefore, the downregulation of kdgR may be an attempt to use carbohydrates as an additional energy source during stress. The unknown gene with locus tag A0W69_01600 is highly downregulated at 6 h (fold change −141) and is described in GenBank as a “chemotaxis protein.” As discussed above, the chemotaxis gene cheW was somewhat downregulated in the aerotolerant WP2202. Since cheW mutants are better at biofilm formation (Tram et al., 2020), this gene may be downregulated for the purpose of biofilm formation and protection from oxidative stress. Two unknown genes, A0W69_06090 and A0W69_06100, have large amounts of downregulation (fold changes 11 and 12) at 0.5 h, however, both encode hypothetical proteins. An unknown gene (A0W69_08085) described as encoding a “fumarate reductase” was upregulated at 0.5 and 6 h. Oddly, fumarate reductases are thought to contribute to oxidative stress in bacteria via the formation of ROS (Messner and Imlay, 2002; Meehan and Malamy, 2012). Additionally, genes encoding fumarate reductases were upregulated when O₂ was limiting in Campylobacter and are thought to function in anaerobic rather than aerobic respiration (Guccione et al., 2010). Further investigation is required to clarify the role of this enzyme during oxidative stress. Another unknown gene (overlapping locus tags A0W69_08115 and A0W69_08120) was described as a putative C4-dicarboxylate ABC transporter and was highly upregulated at 6 and 12 h. Similarly, the C4-dicarboxylate transporter gene dcuA was upregulated in the aerotolerant WP2202, suggesting that the aerotolerant strain may upregulate these transporters as a response to oxidative stress.

Several genes displayed spikes in up- or down-regulation at both the 12 and 24 h timepoints. In the aerosensitive strain, these included acnB and ahpC (possible oxidative stress genes), clpB and hugZ (iron acquisition or iron transport genes), and tolQ2 and nrfA. Other genes with spikes in regulation at 12 or 24 h in the aerosensitive T1-21 strain include flaC, mdtC, pfs, purL, secD, trpB, ttca, and seven unknown genes on plasmid pcjDM (locus tags ASB61_RS08740, ASB61_RS08640, ASB61_RS08525, ASB61_RS08545, ASB61_RS08600, ASB61_RS08440, and ASB61_RS08430). In the aerotolerant strain, genes with spikes in regulation at 12 or 24 h included atpC, dedA, ligA2, mmpA, modA, pth, purH, tal, tktA, torC, and torZ. Many ribosomal genes were sharply downregulated at 12 or 24 h in one or both strains as well.

This study revealed several promising avenues for future research. Notably, the aerotolerant strain differentially regulated a greater number of genes at all timepoints than the aerosensitive strain, suggesting that it has a broader range of strategies for responding to oxidative stress. The spike in gene regulation commonly observed at 12 h in both strains is particularly intriguing and suggests that a potential shift in gene expression may occur at this time in response to prolonged oxidative stress. Further study of genes involved in iron acquisition that are upregulated in the aerosensitive T1-21 strain under aerobic conditions could reveal whether the phenomenon increases the incidence of iron-induced oxidative stress. Furthermore, the upregulation of heat shock genes in both strains was compelling, and further study of the connections between the oxidative and heat stress responses would be valuable. Additionally, the potential tendency of the aerotolerant strain to downregulate resource-intensive tasks warrants further study. One limitation of this study is the inclusion of only two strains of C. jejuni, which may not demonstrate the full range of genetic diversity in this species. Examining the gene expression of additional strains will be important to verify the patterns of expression observed. Moreover, transcriptomics can only tell us about changes in gene expression without fully illustrating the functional effects of each gene. Further evaluation in the form of knockout mutations or complementation studies will be required to verify the contribution of each gene to aerotolerance. This study builds on previous lines of research by providing additional data for the role of previously-characterized oxidative stress genes in the response of C. jejuni to aerobic conditions.