Cellular response to oxidative stress is a crucial mechanism that promotes the survival of Pseudomonas aeruginosa during infection. However, the translational regulation of oxidative stress response remains largely unknown. Here, we reveal a tRNA modification-mediated translational response to H2O2 in P. aeruginosa. We demonstrated that the P. aeruginosa trmB gene encodes a tRNA guanine (46)-N7-methyltransferase that catalyzes the formation of m7G46 in the tRNA variable loop. Twenty-three tRNA substrates of TrmB with a guanosine residue at position 46 were identified, including 11 novel tRNA substrates. We showed that loss of trmB had a strong negative effect on the translation of Phe- and Asp-enriched mRNAs. The trmB-mediated m7G modification modulated the expression of the catalase genes katA and katB, which are enriched with Phe/Asp codons at the translational level. In response to H2O2 exposure, the level of m7G modification increased, consistent with the increased translation efficiency of Phe- and Asp-enriched mRNAs. Inactivation of trmB led to decreased KatA and KatB protein abundance and decreased catalase activity, resulting in H2O2-sensitive phenotype. Taken together, our observations reveal a novel role of m7G46 tRNA modification in oxidative stress response through translational regulation of Phe- and Asp-enriched genes, such as katA and katB.

Yeasts are generally recognized as contaminants in the production of white-brined cheeses, such as Feta and Feta-type cheeses. The most predominant yeasts species are Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces marxianus, Kluyveromyces lactis, Rhodotorula mucilaginosa, and Trichosporon spp. Although their spoilage potential varies at both species and strain levels, yeasts will, in case of excessive growth, present a microbiological hazard, effecting cheese quality. To evaluate the hazard and trace routes of contamination, the exact taxonomic classification of yeasts is required. Today, identification of dairy yeasts is mainly based on DNA sequencing, various genotyping techniques, and, to some extent, advanced phenotypic identification technologies. Even though these technologies are state of the art at the scientific level, they are only hardly implemented at the industrial level. Quality defects, caused by yeasts in white-brined cheese, are mainly linked to enzymatic activities and metabolism of fermentable carbohydrates, leading to production of metabolites (CO 2 , fatty acids, volatile compounds, amino acids, sulfur compounds, etc.) and resulting in off-flavors, texture softening, discoloration, and swelling of cheese packages. The proliferation of spoilage yeast depends on maturation and storage conditions at each specific dairy, product characteristics, nutrients availability, and interactions with the co-existing microorganisms. To prevent and control yeast contamination, different strategies based on the principles of HACCP and Good Manufacturing Practice (GMP) have been introduced in white-brined cheese production. These strategies include milk pasteurization, refrigeration, hygienic sanitation, air filtration, as well as aseptic and modified atmosphere packaging. Though a lot of research has been dedicated to yeasts in dairy products, the role of yeast contaminants, specifically in white-brined cheeses, is still insufficiently understood. This review aims to summarize the current knowledge on the identification of contaminant yeasts in white-brined cheeses, their occurrence and spoilage potential related to different varieties of white-brined cheeses, their interactions with other microorganisms, as well as guidelines used by dairies to prevent cheese contamination.

This study investigated the spoilage potential of yeast strains Kluyveromyces marxianus (Km1, Km2 and Km3), Pichia kudriavzevii Pk1 and Torulaspora delbrueckii Td1 grown in skyr in cold storage. Yeast strains were isolated from skyr and identified by sequencing of the 26S rRNA gene. K. marxianus yeasts were grown in skyr to high numbers, generating large amounts of volatile organic compounds (VOC) associated with off-flavours, among them were alcohols (3-methyl-1-butanol, 2-methyl-1-propanol and 1-hexanol), esters (ethyl acetate and 3-methylbutyl acetate) and aldehydes (hexanal, methylbutanal and methylpropanal). Growth of P. kudriavzevii Pk1 led to moderate increases in several alcohols and esters (mostly, 3-methyl-1-butanol and ethyl acetate), whereas only minor shifts in VOCs were associated with T. delbrueckii Td2. The levels of the key aroma compounds, diacetyl and acetoin, were significantly decreased by all K. marxianus strains and P. kudriavzevii Pk1. In contrast to the other yeast species, K. marxianus was able to utilize lactose, producing ethanol and carbon dioxide. Based on the overall results, K. marxianus was characterised by the highest spoilage potential. The study revealed the differences between the yeast species in fermentative and spoilage activities, and clarified the role of yeast metabolites for off-flavour formation and quality defects in skyr during cold storage.

No abstract

Pseudomonas aeruginosa gidA, which encodes a putative tRNA-modifying enzyme, is associated with a variety of virulence phenotypes. Here, we demonstrated that P. aeruginosa gidA is responsible for the modifications of uridine in tRNAs in vivo. Loss of gidA was found to have no impact on the mRNA levels of katA and katB, but it decreased KatA and KatB protein levels, resulting in decreased total catalase activity and a hydrogen peroxide-sensitive phenotype. Furthermore, gidA was found to affect flagella-mediated motility and biofilm formation; and it was required for the full virulence of P. aeruginosa in both Caenorhabditis elegans and macrophage models. Together, these observations reveal the posttranscriptional impact of gidA on the oxidative stress response, highlight the complexity of catalase gene expression regulation, and further support the involvement of gidA in the virulence of P. aeruginosa.