A Genomic Screen Revealing the Importance of Vesicular Trafficking Pathways in Genome Maintenance and Protection against Genotoxic Stress in Diploid Saccharomyces cerevisiae Cells

The ability to survive stressful conditions is important for every living cell. Certain stresses not only affect the current well-being of cells but may also have far-reaching consequences. Uncurbed oxidative stress can cause DNA damage and decrease cell survival and/or increase mutation rates, and certain substances that generate oxidative damage in the cell mainly act on DNA. Radiomimetic zeocin causes oxidative damage in DNA, predominantly by inducing single- or double-strand breaks. Such lesions can lead to chromosomal rearrangements, especially in diploid cells, in which the two sets of chromosomes facilitate excessive and deleterious recombination. In a global screen for zeocin-oversensitive mutants, we selected 133 genes whose deletion reduces the survival of zeocin-treated diploid Saccharomyces cerevisiae cells. The screen revealed numerous genes associated with stress responses, DNA repair genes, cell cycle progression genes, and chromatin remodeling genes. Notably, the screen also demonstrated the involvement of the vesicular trafficking system in cellular protection against DNA damage. The analyses indicated the importance of vesicular system integrity in various pathways of cellular protection from zeocin-dependent damage, including detoxification and a direct or transitional role in genome maintenance processes that remains unclear. The data showed that deleting genes involved in vesicular trafficking may lead to Rad52 focus accumulation and changes in total DNA content or even cell ploidy alterations, and such deletions may preclude proper DNA repair after zeocin treatment. We postulate that functional vesicular transport is crucial for sustaining an integral genome. We believe that the identification of numerous new genes implicated in genome restoration after genotoxic oxidative stress combined with the detected link between vesicular trafficking and genome integrity will reveal novel molecular processes involved in genome stability in diploid cells.     

In-line monitoring of protein concentration with MIR spectroscopy during UFDF

Rapid increase of product titers in upstream processes has presented challenges for downstream processing, where purification costs increase linearly with the increase of the product yield. Hence, innovative solutions are becoming increasingly popular. Process Analytical Technology (PAT) tools, such as spectroscopic techniques, are on the rise due to their capacity to provide real-time, precise analytics. This ensures consistent product quality and increased process understanding, as well as process control. Mid-infrared spectroscopy (MIR) has emerged as a highly promising technique within recent years, owing to its ability to monitor several critical process parameters at the same time and unchallenging spectral analysis and data interpretation. For in-line monitoring, Attenuated Total Reflectance—Fourier Transform Infrared Spectroscopy (ATR-FTIR) is a method of choice, as it enables reliable measurements in a liquid environment, even though water absorption bands are present in the region of interest. Here, we present MIR spectroscopy as a monitoring tool of critical process parameters in ultrafiltration/diafiltration (UFDF). MIR spectrometer was integrated in the UFDF process in an in-line fashion through a single-use flow cell containing a single bounce silicon ATR crystal. The results indicate that the one-point calibration algorithm applied to the MIR spectra, predicts highly accurate protein concentrations, as compared with validated offline analytical methods.     

Drought stress and pathogen infection alter feeding behavior of a phytopathogen vector

The impact of drought stress on tripartite plant-pathogen-vector interactions constitutes a complex and largely understudied field of plant-insect interaction. A number of studies explored these topics using aphid vectors of plant pathogens, but few have considered the interactions between drought-stressed plants and pathogen-transmitting psyllids. The potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), is one of the key pests of solanaceous crops in the USA that causes direct injury as well as indirect injury through transmission of a bacterial pathogen, Candidatus Liberibacter solanacearum (Lso), the causal agent of zebra chip. Previous studies explored the impact of Lso infection and drought stress on B. cockerelli development and reproductive rate separately, but no research to date has evaluated whether drought stress and Lso infection alter feeding behavior of the insects. We explored this using the electrical penetration graph (EPG) technique and monitored feeding behavior of Lso-infected and uninfected potato psyllids on well-watered and drought-stressed tomato (Solanum lycopersicum L., Solanaceae). We found that drought stress had a significant effect on feeding behavior associated with salivation into the phloem and phloem ingestion, both linked to Lso transmission. Furthermore, infected potato psyllids in particular produced a higher number of events associated with these feeding behaviors and remained in these phases longer in well-watered plants than in plants that were under drought stress. We also reported a new and previously undescribed waveform H of unknown biological function that was produced by the psyllids. This is the first study that considered the impact of bacterial infection and concomitant drought stress on feeding behavior of an insect quantified using EPG.     

The antimutagenic effect of a truncated ε subunit of DNA polymerase III inEscherichia coli cells irradiated with UV light

It has previously been suggested that inhibition of the proofreading 3′-5′ exonuclease activity of DNA polymerase may play an important role in generation of UV-induced mutations inEscherichia coli. Our previous work showing that overproduction of ε, the proofreading subunit of DNA polymerase III, counteracts the SOS mutagenic response ofE. coli seemed to be consistent with this hypothesis. To explore further the nature of the antimutagenic effect of ε we constructed plasmid pMK17, which encodes only two of the three highly conserved segments of ε — Exol and ExoII; the third segment, ExoIII, which is essential for 3′–5′ exonuclease activity, is deleted. We show that at 40°C, over-production of the truncated e subunit significantly delays production of M13 phage, suggesting that the protein retains its capacity to bind to DNA. On the other hand, the presence of pMK17 in atrpE65 strain growing at 40°C causes a 10-fold decrease in the frequency of UV-induced Trp⁺ mutations. This antimutagenic effect of the truncated s is effectively relieved by excess UmuD,C proteins. We also show that the presence of plasmid pIP21, which contains thednaQ49 allele encoding an ε subunit that is defective in proofreading activity, almost completely prevents generation of UV-induced mutations in thetrpE65 strain. We propose that the DNA binding ability of free ε, rather than its 3′–5′ exonuclease activity, affects processing of premutagenic UV-induced lesions, possibly by interfering with the interaction between the UmuC-UmuD′-RecA complex and Pol III holoenzyme. This interaction is probably a necessary condition for translesion synthesis.