Effects of Interventions to Prevent Work-Related Asthma, Allergy, and Other Hypersensitivity Reactions in Norwegian Salmon Industry Workers (SHInE): Protocol for a Pragmatic Allocated Intervention Trial and Related Substudies.

BACKGROUND: Workers in the salmon processing industry have an increased risk of developing respiratory diseases and other hypersensitivity responses due to occupational exposure to bioaerosols containing fish proteins and microorganisms, and related allergens. Little is known about effective measures to reduce bioaerosol exposure and about the extent of skin complaints among workers. In addition, while identification of risk factors is a core activity in disease prevention strategies, there is increasing interest in health-promoting factors, which is an understudied area in the salmon processing industry. OBJECTIVE: The overall aim of this ongoing study is to generate knowledge that can be used in tailored prevention of development or chronification of respiratory diseases, skin reactions, protein contact dermatitis, and allergy among salmon processing workers. The main objective is to identify effective methods to reduce bioaerosol exposure. Further objectives are to identify and characterize clinically relevant exposure agents, identify determinants of exposure, measure prevalence of work-related symptoms and disease, and identify health-promoting factors of the psychosocial work environment. METHODS: Data are collected during field studies in 9 salmon processing plants along the Norwegian coastline. Data collection comprises exposure measurements, health examinations, and questionnaires. A wide range of laboratory analyses will be used for further analysis and characterization of exposure agents. Suitable statistical analysis will be applied to the various outcomes of this comprehensive study. RESULTS: Data collection started in September 2021 and was anticipated to be completed by March 2023, but was delayed due to the COVID-19 pandemic. Baseline data from all 9 plants included 673 participants for the health examinations and a total of 869 personal exposure measurements. A total of 740 workers answered the study's main questionnaire on demographics, job characteristics, lifestyle, health, and health-promoting factors. Follow-up data collection is not completed yet. CONCLUSIONS: This study will contribute to filling knowledge gaps concerning salmon workers' work environment. This includes effective workplace measures for bioaerosol exposure reduction, increased knowledge on hypersensitivity, allergy, respiratory and dermal health, as well as health-promoting workplace factors. Together this will give a basis for improving the work environment, preventing occupational health-related diseases, and developing occupational exposure limits, which in turn will benefit employees, employers, occupational health services, researchers, clinicians, decision makers, and other stakeholders. TRIAL REGISTRATION: ClinicalTrials.gov NCT05039229; https://www.clinicaltrials.gov/study/NCT05039229. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/48790.

Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes.

BACKGROUND: Heterologous production of cold-adapted proteins currently represents one of the greatest bottlenecks in the ongoing bioprospecting efforts to find new enzymes from low-temperature environments, such as, the polar oceans that represent essentially untapped resources in this respect. In mesophilic expression hosts such as Escherichia coli, cold-adapted enzymes often form inactive aggregates. Therefore it is necessary to develop new low-temperature expression systems, including identification of new host organisms and complementary genetic tools. Psychrophilic bacteria, including Pseudoalteromonas haloplanktis, Shewanella and Rhodococcus erythropolis have all been explored as candidates for such applications. However to date none of these have found widespread use as efficient expression systems, or are commercially available. In the present work we explored the use of the sub-Arctic bacterium Aliivibrio wodanis as a potential host for heterologous expression of cold-active enzymes. RESULTS: We tested 12 bacterial strains, as well as available vectors, promoters and reporter systems. We used RNA-sequencing to determine the most highly expressed genes and their intrinsic promoters in A. wodanis. In addition we examined a novel 5'-fusion to stimulate protein production and solubility. Finally we tested production of a set of "difficult-to-produce" enzymes originating from various bacteria and one Archaea. Our results show that cold-adapted enzymes can be produced in soluble and active form, even in cases when protein production failed in E. coli due to the formation of inclusion bodies. Moreover, we identified a 60-bp/20-aa fragment from the 5'-end of the AW0309160_00174 gene that stimulates expression of Green Fluorescent Protein and improves production of cold-active enzymes when used as a 5'-fusion. A 25-aa peptide from the same protein enhanced secretion of a 25-aa-sfGFP fusion. CONCLUSIONS: Our results indicate the use of A. wodanis and associated genetic tools for low-temperature protein production and indicate that A. wodanis represents an interesting platform for further development of a protein production system that can promote further cold-enzyme discoveries.

DNA binding with a minimal scaffold: structure-function analysis of Lig E DNA ligases.

DNA ligases join breaks in the phosphodiester backbone of DNA by catalysing the formation of bonds between opposing 5'P and 3'OH ends in an adenylation-dependent manner. Catalysis is accompanied by reorientation of two core domains to provide access to the active site for cofactor utilization and enable substrate binding and product release. The general paradigm is that DNA ligases engage their DNA substrate through complete encirclement of the duplex, completed by inter-domain kissing contacts via loops or additional domains. The recent structure of a minimal Lig E-type DNA ligase, however, implies it must use a different mechanism, as it lacks any domains or loops appending the catalytic core which could complete encirclement. In the present study, we have used a structure-guided mutagenesis approach to investigate the role of conserved regions in the Lig E proteins with respect to DNA binding. We report the structure of a Lig-E type DNA ligase bound to the nicked DNA-adenylate reaction intermediate, confirming that complete encirclement is unnecessary for substrate engagement. Biochemical and biophysical measurements of point mutants to residues implicated in binding highlight the importance of basic residues in the OB domain, and inter-domain contacts to the linker.

Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4.

Cytosine-specific DNA methyltransferases are important enzymes in most living organisms. In prokaryotes, most DNA methyltransferases are members of the type II restriction-modification system where they methylate host DNA, thereby protecting it from digestion by the accompanying restriction endonucleases. DNA methyltransferases can also act as solitary enzymes having important roles in controlling gene expression, DNA replication, cell cycle and DNA post-replicative mismatch repair. They have potential applications in biotechnology, such as in labeling of biopolymers, DNA mapping or epigenetic analysis, as well as for general DNA-protein interaction studies. The parI gene from the psychrophilic bacterium Psychrobacter arcticus 273-4 encodes a cytosine-specific DNA methyltransferase. In this work, recombinant ParI was expressed and purified in fusion to either an N-terminal hexahistidine affinity tag, or a maltose binding protein following the hexahistidine affinity tag, for solubility improvement. After removal of the fusion partners, recombinant ParI was found to be monomeric by size exclusion chromatography, with its molecular mass estimated to be 54 kDa. The apparent melting temperature of the protein was 53 °C with no detectable secondary structures above 65 °C. Both recombinant and native ParI showed methyltransferase activity in vivo. In addition, MBP- and His-tagged ParI also demonstrated in vitro activity. Although the overall structure of ParI exhibits high thermal stability, the loss of in vitro activity upon removal of solubility tags or purification from the cellular milieu indicates that the catalytically active form is more labile. Horizontal gene transfer may explain the acquisition of a protein-encoding gene that does not display common cold-adapted features.

Properties and distribution of a metallo-ß-lactamase (ALI-1) from the fish pathogen Aliivibrio salmonicida LFI1238.

OBJECTIVES: To characterize the chromosome-encoded metallo-ß-lactamase (MBL) from the psychrophilic, marine fish-pathogenic bacterium Aliivibrio salmonicida LFI1238 and check for the presence of the gene in other Aliivibrio isolates both connected to the fish-farming industry and from the environment. METHODS: The MBL gene was cloned and intracellularly expressed in Escherichia coli. Kinetic parameters, NaCl dependence, pH optimum and temperature optimum were determined using purified enzyme. The VIM-2 enzyme from a Pseudomonas aeruginosa hospital isolate was used as a counterpart in comparative analysis. PCRs with degenerate MBL primers were used to screen different A. salmonicida isolates for the presence of the gene. RESULTS: A. salmonicida MBL (ALI-1) is an Ambler class B ß-lactamase sharing 39% and 29% amino acid identity with IMP-1 and VIM-2, respectively. ALI-1 hydrolysed all ß-lactam antibiotics tested, except for the monobactam aztreonam and the penicillin piperacillin. A profound increase in activity was observed when adding NaCl to the assay mixture (60% active without addition of NaCl, increasing to 100% at 0.5 M NaCl). The increase was less noticeable for VIM-2 (100% active at 0.2 M NaCl). ALI-1 appears to be ubiquitous in nature as it is found in Aliivibrio isolates not affected by human activity. CONCLUSIONS: This work provides more data for the ever-expanding MBL group of enzymes. These periplasmic enzymes are activated by addition of NaCl, and the marine enzyme is highly salt tolerant and cold active. The observed enzyme properties very likely reflect the conditions that the enzymes face in situ.