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Delicate variances in the translational machinery affect how efficiently different organisms approach protein synthesis. Determining the scale of this effect, however, requires knowledge on the differences of mistranslation levels. Here, we used a dual-luciferase reporter assay cloned into a broad host range plasmid to reveal the translational fidelity profiles of Pseudomonas putida, Pseudomonas aeruginosa and Escherichia coli . We observed that these profiles are surprisingly different, whereas species more prone to translational frameshifting are not necessarily more prone to stop codon readthrough. As tRNA modifications are among the factors that have been implicated to affect translation accuracy, we also show that translational fidelity is context-specifically influenced by pseudouridines in the anticodon stem-loop of tRNA, but the effect is not uniform between species.

Pseudouridines are known to be important for optimal translation. In this study we demonstrate an unexpected link between pseudouridylation of tRNA and mutation frequency in Pseudomonas species. We observed that the lack of pseudouridylation activity of pseudouridine synthases TruA or RluA elevates the mutation frequency in Pseudomonas putida 3 to 5-fold. The absence of TruA but not RluA elevates mutation frequency also in Pseudomonas aeruginosa . Based on the results of genetic studies and analysis of proteome data, the mutagenic effect of the pseudouridylation deficiency cannot be ascribed to the involvement of error-prone DNA polymerases or malfunctioning of DNA repair pathways. In addition, although the deficiency in TruA-dependent pseudouridylation made P. putida cells more sensitive to antimicrobial compounds that may cause oxidative stress and DNA damage, cultivation of bacteria in the presence of reactive oxygen species (ROS)-scavenging compounds did not eliminate the mutator phenotype. Thus, the elevated mutation frequency in the absence of tRNA pseudouridylation could be the result of a more specific response or, alternatively, of a cumulative effect of several small effects disturbing distinct cellular functions, which remain undetected when studied independently. This work suggests that pseudouridines link the translation machinery to mutation frequency.

Nucleoid-associated proteins (NAPs) such as IHF, HU, Fis, and H-NS alter the topology of bound DNA and may thereby affect accessibility of DNA to repair and recombination processes. To examine this possibility, we investigated the effect of IHF on the frequency of homologous recombination (HR) and point mutations in soil bacterium Pseudomonas putida by using plasmidial and chromosomal assays. We observed positive effect of IHF on the frequency of HR, whereas this effect varied depending both on the chromosomal location of the HR target and the type of plasmid used in the assay. The occurrence of point mutations in plasmid was also facilitated by IHF, whereas in the chromosome the positive effect of IHF appeared only at certain DNA sequences and/or chromosomal positions. We did not observe any significant effects of IHF on the spectrum of mutations. However, despite of the presence or absence of IHF, different mutational hot spots appeared both in plasmid and in chromosome. Additionally, the frequency of frameshift mutations in the chromosome was also strongly affected by the location of the mutational target sequence. Taking together, our results indicate that IHF facilitates the occurrence of genetic changes in P. putida, whereas the location of the target sequence affects both the IHF-dependent and IHF-independent mechanisms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Toxoplasma gondii is a zoonotic parasite of major veterinary and public health importance. The parasite is common in many host species in Estonia, however, information about the seroprevalence in the local sheep population is lacking. We estimated T. gondii seroprevalence and evaluated a limited selection of possible risk factors for T. gondii seropositivity in sheep raised in Estonia. Sera from 1599 sheep from 122 flocks, collected during 2012-2013, were diluted to 1:40 and screened for the presence of anti- T. gondii immunoglobulin G antibodies using a commercial direct agglutination test. The cutoff titer for seropositivity was 40. A total of 667 of the 1599 sheep were T. gondii seropositive, yielding an apparent seroprevalence estimate of 41.71% (95% confidence interval 39.31-44.14). At least one seropositive sheep was detected on the majority (88.53%) of the farms. T. gondii seroprevalence was significantly lower in lambs (age <12 months) than in adults (age ≥12 months) (11.11% and 42.16%, respectively). The seroprevalence increased steeply with age in the first years of life. A multivariable random-effects logistic regression model identified being an adult (odds ratio [OR] 9.55) and being from a farm located on the islands (OR 2.51) as risk factors for T. gondii seropositivity, while being of Texel breed appeared as a protective factor (OR 0.34) when compared with the Estonian Blackhead breed. Exposure to T. gondii was widespread among the investigated sheep. Undercooked meat and unpasteurized milk from sheep from Estonia should be considered possible sources of T. gondii infections to other hosts, including humans.

Background: Toxoplasma gondii is an important foodborne zoonotic parasite. Meat of infected animals is presumed to constitute a major source of human infection and may be a driver of geographical variation in the prevalence of anti- T. gondii antibodies in humans, which is substantial in the Nordic-Baltic region in northern Europe. However, data on seroprevalence of T. gondii in different animal species used for human consumption are scattered., Methods: We conducted a systematic review of seroprevalence studies and meta-analysis to estimate the seroprevalence of T. gondii in five animal species that are raised or hunted for human consumption in the Nordic-Baltic region: domestic pigs ( Sus scrofa domesticus ), sheep ( Ovis aries ), cattle ( Bos taurus ), wild boars ( Sus scrofa ), and moose ( Alces alces ). We searched for studies that were conducted between January 1990 and June 2018, and reported in articles, theses, conference abstracts and proceedings, and manuscripts. Subgroup analyses were performed to identify variables influencing the seroprevalence., Findings: From a total of 271 studies identified in the systematic review, 32 were included in the meta-analysis. These comprised of 13 studies on domestic pigs, six on sheep, three on cattle, six on wild boars, and four on moose. The estimated pooled seroprevalence of T. gondii was 6% in domestic pigs (CI 95% : 3-10%), 23% in sheep (CI 95% : 12-36%), 7% in cattle (CI 95% : 1-21%), 33% in wild boars (CI 95% : 26-41%), and 16% in moose (CI 95% : 10-23%). High heterogeneity was observed in the seroprevalence data within each species. In all host species except wild boars, the pooled seroprevalence estimates were significantly higher in animals >1 year of age than in younger animals. Not all studies provided information on animal age, sensitivity and specificity of the serological method employed, and the cut-off values used for defining an animal seropositive., Conclusions: A substantial proportion of animals raised or hunted for human consumption in the region had tested positive for T. gondii . This indicates widespread exposure to T. gondii among animals raised or hunted for human consumption in the region. Large variations were observed in the seroprevalence estimates between the studies in the region; however, studies were too few to identify spatial patterns at country-level.

In Estonia, northeastern Europe, antibodies against Toxoplasma gondii are common in many host species, including wildlife, domestic animals, and humans. Our nationwide study aimed to estimate T. gondii seroprevalence and its geographical distribution, and to evaluate plausible risk factors for seropositivity in free-ranging moose (Alces alces) hunted for human consumption in 2015. We screened sera or plasma from 463 moose for presence of anti-T. gondii immunoglobulin G antibodies with a commercial direct agglutination test, using a cut-off titer 40 for seropositivity. Altogether 111 moose tested seropositive, yielding a seroprevalence estimate of 23.97%. Seropositive moose were detected in all the 13 counties where the samples originated from. Based on a multivariable logistic regression model, 'being female', 'being adult', and 'being from the northern part of the country' were significant risk factors, with odds ratios of 2.91, 3.07, and 3.11, respectively, and there was interaction between the variables 'being female' and 'being from the northern part of the country'. A substantial proportion of the moose investigated had been exposed to T. gondii. Presence of the parasite in edible tissues of the moose was not shown in the present study, but moose hunted in Estonia should be considered a potential T. gondii infection source to other hosts, including humans. Seropositivity indicates previous exposure, and because the seroprevalence was higher in adults than in calves and because moose are herbivores, the exposure was likely exposure to and ingestion of T. gondii oocysts. The results can thus be interpreted to indicate that the environment in Estonia was widely contaminated with T. gondii oocysts, in particular in the northern part of the country. Investigation of samples from a free-ranging herbivorous host that is hunted in large numbers appears useful in revealing environmental distribution patterns of T. gondii., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Toxoplasma gondii is a widespread occurring parasite infecting warm-blooded animals, including pigs and humans. The aims of this study were to estimate the prevalence of anti-T. gondii antibodies and to evaluate risk factors for T. gondii seropositivity in breeding pigs raised in Estonia. Sera from 382 pigs were tested with a commercial direct agglutination test, using a cut-off titer of 40 for seropositivity, for the presence of anti-T. gondii immunoglobulin G antibodies., Results: Twenty-two (5.8%) of the 382 pigs tested seropositive for T. gondii, and 6 of the 14 herds had at least one seropositive pig. The proportion of seropositive pigs within the herds ranged between 0 and 43%. Gender appeared as a significant factor, with sows having 5.6 times higher odds to be seropositive to T. gondii than boars. Seroprevalence did not increase with age., Conclusions: Anti-T. gondii antibodies were present in a substantial proportion of breeding pig herds in Estonia. On the other hand, the presence of herds without seropositive pigs illustrates that porcine T. gondii infections can be avoided even in a country where the parasite is endemic and common in several other host species.

Fasciolosis, an infectious disease caused by the liver fluke Fasciola hepatica, affects grazing cattle world-wide. Liver fluke F. hepatica is prevalent and well-documented in cattle in many European countries, but for the Baltic countries such information is limited. This study investigated the seroprevalence and distribution of F. hepatica in cattle in Estonia. A total of 2461 individual serum samples from 218 farms distributed throughout all 15 Estonian counties, collected between February 2012 and March 2013, were tested for specific anti-F. hepatica antibodies using an in-house enzyme-linked immunosorbent assay (ELISA). In total, 144 individual animals tested seropositive, yielding an animal-level seroprevalence of 5.9% (95% CI 5.0-6.9). The herd-level seroprevalence was 28.4% (95% CI 22.8-34.7) and the herds with at least one seropositive animal were located in 13 of the 15 counties. Of the 62 F. hepatica-positive herds, 14 (6.4%) had an in-herd seroprevalence higher than 25%. With respect to production type, the herd-level seroprevalence was 20.2%, 35.6%, and 36.4% in dairy, mixed, and beef herds, respectively. Animals from the two large islands had higher odds of testing F. hepatica-seropositive than animals from the mainland. Animals from mixed and beef herds had higher odds of testing F. hepatica-seropositive than animals from dairy herds. Mixed and beef herds, and herds with more than 100 cattle, had higher odds of having at least one seropositive animal. This study provided the first serological evidence of the presence and distribution of F. hepatica in cattle herds in Estonia., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Toxoplasma gondii is a zoonotic protozoan parasite that thrives in Estonia. In this nationwide cross-sectional study, we tested sera from 3991 cattle, collected from 228 farms in 2012-2013, for anti-T. gondii immunoglobulin G antibodies using a commercial direct agglutination test. Titer of 100 was set as cut-off: samples that tested positive at the dilution 1:100 were defined as positive. The apparent animal-level seroprevalence was 18.62%. At least one seropositive animal was found on 68.86% of the farms, and seropositive cattle were detected in all counties. The seroprevalence appeared to increase with age until five years (60-71 months) of age, but had no obvious pattern in the older animals. Animals of the local Estonian Red breed had higher odds to test seropositive than did animals of the Estonian Holstein breed. Whether the farm focused on dairy or beef cattle was not associated with an animal testing T. gondii seropositive nor with finding at least one T. gondii seropositive animal on the farm. The odds of finding at least one T. gondii seropositive animal on the farm were higher if the herd size was above median (105 in dairy and mixed dairy farms; 35 in beef and mixed beef farms). The results indicate that T. gondii is endemic within the agricultural setting in Estonia and present on the majority of cattle farms., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Formation of microcolonies (papillae) permits easy visual screening of mutational events occurring in single colonies of bacteria. In this study, we have established a novel papillation assay employable in a wide range of pseudomonads including Pseudomonas aeruginosa and Pseudomonas putida for monitoring mutation frequency in distinct colonies. With the aid of this assay, we conducted a genome-wide search for the factors affecting mutation frequency in P. putida. Screening ∼27,000 transposon mutants for increased mutation frequency allowed us to identify 34 repeatedly targeted genes. In addition to genes involved in DNA replication and repair, we identified genes participating in metabolism and transport of secondary metabolites, cell motility, and cell wall synthesis. The highest effect on mutant frequency was observed when truA (tRNA pseudouridine synthase), mpl (UDP-N-acetylmuramate-alanine ligase) or gacS (multi-sensor hybrid histidine kinase) were inactivated. Inactivation of truA elevated the mutant frequency only in growing cells, while the deficiency of gacS affected mainly stationary-phase mutagenesis. Thus, our results demonstrate the feasibility of the assay for isolating mutants with elevated mutagenesis in growing as well as stationary-phase bacteria., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Bacterial type II toxin-antitoxin systems consist of a potentially poisonous toxin and an antitoxin that inactivates the toxic protein by binding to it. Most of the toxins regulate stress survival, but their activation depends on the stability of the antitoxin that has to be degraded in order for the toxin to be able to attack its cellular targets. The degradation of antitoxins is usually rapid and carried out by ATP-dependent protease Lon or Clp, which is activated under stress conditions. The graTA system of Pseudomonas putida encodes the toxin GraT, which can affect the growth rate and stress tolerance of bacteria but is under most conditions inactivated by the unusually stable antitoxin GraA. Here, we aimed to describe the stability features of the antitoxin GraA by analyzing its degradation rate in total cell lysates of P. putida. We show that the degradation rate of GraA depends on the growth phase of bacteria being fastest in the transition from exponential to stationary phase. In accordance with this, higher ATP levels were shown to stabilize GraA. Differently from other antitoxins, the main cellular proteases Lon and Clp are not involved in GraA stability. Instead, GraA seems to be degraded through a unique pathway involving an endoprotease that cleaves the antitoxin into two unequal parts. We also identified the global transcriptional regulator MexT as a factor for destabilization of GraA, which indicates that the degradation of GraA may be induced by conditions similar to those that activate MexT., Importance: Toxin-antitoxin (TA) modules are widespread in bacterial chromosomes and have important roles in stress tolerance. As activation of a type II toxin is triggered by proteolytic degradation of the antitoxin, knowledge about the regulation of the antitoxin stability is critical for understanding the activation of a particular TA module. Here, we report on the unusual degradation pathway of the antitoxin GraA of the recently characterized GraTA system. While GraA is uncommonly stable in the exponential and late-stationary phases, its degradation increases in the transition phase. The degradation pathway of GraA involves neither Lon nor Clp, which usually targets antitoxins, but rather an unknown endoprotease and the global regulator MexT, suggesting a new type of regulation of antitoxin stability., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)