Roles of RpoN in the resistance of Campylobacter jejuni under various stress conditions

<p>Abstract</p> <p>Background</p> <p><it>Campylobacter jejuni </it>is a leading foodborne pathogen worldwide. Despite the fastidious nature of <it>C. jejuni </it>growth, increasing numbers of human campylobacteriosis suggest that <it>C. jejuni </it>may possess unique mechanisms to survive under various stress conditions. <it>C. jejuni </it>possesses only three sigma factors (FliA, RpoD, and RpoN) and lacks stress-defense sigma factors. Since FliA and RpoD are dedicated to flagella synthesis and housekeeping, respectively, in this study, we investigated the role of RpoN in <it>C. jejuni</it>'s defense against various stresses.</p> <p>Results</p> <p>Survivability of an <it>rpoN </it>mutant was compared with the wild-type <it>C. jejuni </it>under various stress conditions. While the growth of the <it>rpoN </it>mutant was as comparably as that of the wild type in shaking cultures, the <it>rpoN </it>mutant exhibited significant survival defects when cultured statically. The <it>rpoN </it>mutant was more sensitive to osmotic stress (0.8% NaCl) with abnormally-elongated cell morphology. Compared to the wile type, the <it>rpoN </it>mutant was more susceptible to acid stress (pH 5) and more resistant to hydrogen peroxide. However, the <it>rpoN </it>mutation had little effect on the resistance of <it>C. jejuni </it>to alkaline pH, heat, cold and antimicrobials.</p> <p>Conclusions</p> <p>The results demonstrate that RpoN plays an important role in <it>C. jejuni</it>'s defense against various stresses which this bacterial pathogen may encounter during transmission to and infection of humans.</p

Transducer-Like Protein in Campylobacter jejuni With a Role in Mediating Chemotaxis to Iron and Phosphate

Chemotaxis-mediated motility enables Campylobacter jejuni to navigate through complex environmental gradients and colonize diverse niches. C. jejuni is known to possess several methyl accepting chemotaxis proteins (MCPs), also called transducer-like proteins (Tlps). While the role of some of the Tlps in chemotaxis has been identified, their regulation and role in virulence is still not very clear. Here, we investigated the contribution of Tlp2 to C. jejuni chemotaxis, stress survival and colonization of the chicken gastrointestinal tract. The Δtlp2 deletion mutant showed decreased chemotaxis toward aspartate, pyruvate, inorganic phosphate (Pi), and iron (FeSO4). Transcriptional analysis of tlp2 with a promoter fusion reporter assay revealed that the tlp2 promoter (Ptlp2) was induced by Pi and iron, both in the ferrous (Fe2+) and ferric form (Fe3+). RT-PCR analysis using overlapping primers indicated that the phoX gene, located immediately downstream of tlp2, is co-transcribed with tlp2. A transcription start site was identified at 53 bp upstream of the tlp2 start codon. The Δtlp2 mutant showed decreased colonization of the chicken gastrointestinal tract. Collectively, our findings revealed that the tlp2 plays a role in C. jejuni pathogenesis and colonization in the chicken host and its expression is regulated by iron

ATAD5 restricts R-loop formation through PCNA unloading and RNA helicase maintenance at the replication fork

R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflict (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or ATAD5-interacting RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors

Regulation of Oxidative Stress Response by CosR, an Essential Response Regulator in Campylobacter jejuni

CosR (Campylobacter oxidative stress regulator; Cj0355c) is an OmpR-type response regulator essential for the viability of Campylobacter jejuni, a leading foodborne pathogen causing human gastroenteritis worldwide. Despite importance, the function of CosR remains completely unknown mainly because of cell death caused by its knockout mutation. To overcome this technical limitation, in this study, antisense technology was used to investigate the regulatory function of CosR by modulating the level of CosR expression. Two-dimensional gel electrophoresis (2DGE) was performed to identify the CosR regulon either by suppressing CosR expression with antisense peptide nucleic acid (PNA) or by overexpressing CosR in C. jejuni. According to the results of 2DGE, CosR regulated 32 proteins involved in various cellular processes. Notably, CosR negatively regulated a few key proteins of the oxidative stress response of C. jejuni, such as SodB, Dps, Rrc and LuxS, whereas CosR positively controlled AhpC. Electrophoretic mobility shift assay showed that CosR directly bound to the promoter region of the oxidative stress genes. DNase I footprinting assays identified 21-bp CosR binding sequences in the sodB and ahpC promoters, suggesting CosR specifically recognizes and binds to the regulated genes. Interestingly, the level of CosR protein was significantly reduced by paraquat (a superoxide generator) but not by hydrogen peroxide. Consistent with the overall negative regulation of oxidative stress defense proteins by CosR, the CosR knockdown by antisense rendered C. jejuni more resistant to oxidative stress compared to the wild type. Overall, this study reveals the important role played by the essential response regulator CosR in the oxidative stress defense of C. jejuni

Transcriptional Regulation of the CmeABC Multidrug Efflux Pump and the KatA Catalase by CosR in Campylobacter jejuni

CosR is an essential response regulator in Campylobacter jejuni, a major food-borne pathogen causing enteritis worldwide. A transcriptomic analysis performed in this study discovered 93 genes whose transcriptional levels were changed >2-fold due to the repression of CosR expression by antisense peptide nucleic acid. The identified CosR-regulated genes are involved in various cellular functions, such as energy production, protein synthesis and folding, flagellum biogenesis, and lipid metabolism. Interestingly, 17 of the 93 CosR-regulated genes (18.3%) are predicted essential genes, indicating that CosR may participate in the regulation of vital biological processes in C. jejuni. In particular, CosR knockdown increased the transcriptional levels of cmeA, cmeB, and cmeC genes, whose protein product (CmeABC) is an important determinant conferring multidrug resistance in Campylobacter. Negative regulation of cmeABC by CosR was verified by quantitative real-time PCR (qRT-PCR) and PcmeABC::lacZ assay. The results of electrophoretic mobility shift assays (EMSAs) and DNase I footprinting assays demonstrated that CosR directly binds to the cmeABC promoter. Another notable finding is that CosR regulates the transcription of katA, the sole catalase gene in C. jejuni. Further characterization with qRT-PCR, the catalase enzyme assay, EMSA, and DNase I footprinting assays successfully demonstrated that CosR affects the katA transcription and the catalase activity by direct interactions with the katA promoter. The findings in this study clearly demonstrated that CosR regulates resistance mechanisms in C. jejuni by controlling the expression of genes involved in oxidative stress defense and extrusion of toxic compounds out of the cell.This article is published as Hwang, Sunyoung, Qijing Zhang, Sangryeol Ryu, and Byeonghwa Jeon. "Transcriptional regulation of the CmeABC multidrug efflux pump and the KatA catalase by CosR in Campylobacter jejuni." Journal of bacteriology 194, no. 24 (2012): 6883-6891. doi: 10.1128/JB.01636-12. Posted with permission.</p

Three-dimensional solar steam generation device with additional non-photothermal evaporation

The evaporation sites of a solar desalination device were expanded from conventional 2D to a new type of 3D by leaving the side area of the porous water transporter exposed to the air. The 3D solar desalination device permits not only photothermal distillation by the photoabsorbers at the top under sunlight illumination, but also additional non-photothermal evaporation on the side of the water transporter that works even at night by exploiting environmental heat. For the first time, we developed a unique configuration of water transport exposed to the environment with a great contribution to an active site increase and confirmed the significant impact of the active site increase on the solar desalination performance by systematic and strong pieces of evidence. Due to the effective utilization of enormous evaporation sites on the top and side surfaces in the 3D configuration, the device exhibited a significant steam generation rate of similar to 0.74 g/h under 1 sun illumination, which is similar to 1.5 times higher than the maximum value achieved with photothermal evaporation only. Our study suggests an innovative change which incorporates additional non-photothermal evaporation in the solar desalination device can be a straightforward and efficient way to address clean water deficiencies worldwide in the future

The assessment of memes as digital multimodal composition in L2 classrooms

Digital multimodal writing has become predominant in our students’ lives in and outside the L2 classrooms. While many L2 educators integrate various multimodal projects into their curriculum, internet memes and their assessment have not been explored in depth in L2 settings, although memes have potential to be valuable multimodal writing tasks. The purpose of this study is to better understand how memes can be incorporated and assessed in L2 classrooms. Twenty-seven student-created memes in a low-intermediate Korean as a Foreign Language course at a large university were collected and analyzed. The findings indicated that the student-created memes successfully addressed the multimodal aspect of the meme genre, universal and specific cultural references, and language aspects specific to the genre of memes. Our analyses of the memes suggest three key components to assess this multimodal writing project in addition to its overall task/functions: 1) multimodal aspects that include understanding the interplay between multiple modes for an effective message in a given context; 2) cultural aspects, demonstrating cultural knowledge and its application, recognizing the semiotic importance of multimodal expression in the target community; and 3) language aspects as an effective communication medium, demonstrating genre knowledge of the specific task and language accuracy.24 month embargo; available online: 13 August 2022This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]