Bacteriophage origin of some minimal ATP-dependent DNA ligases: a new structure from Burkholderia pseudomallei with striking similarity to Chlorella virus ligase

DNA ligases, the enzymes responsible for joining breaks in the phosphodiester backbone of DNA during replication and repair, vary considerably in size and structure. The smallest members of this enzyme class carry out their functions with pared-down protein scaffolds comprising only the core catalytic domains. Here we use sequence similarity network analysis of minimal DNA ligases from all biological super kingdoms, to investigate their evolutionary origins, with a particular focus on bacterial variants. This revealed that bacterial Lig C sequences cluster more closely with Eukaryote and Archaeal ligases, while bacterial Lig E sequences cluster most closely with viral sequences. Further refinement of the latter group delineates a cohesive cluster of canonical Lig E sequences that possess a leader peptide, an exclusively bacteriophage group of T7 DNA ligase homologs and a group with high similarity to the Chlorella virus DNA ligase which includes both bacterial and viral enzymes. The structure and function of the bacterially-encoded Chlorella virus homologs were further investigated by recombinantly producing and characterizing, the ATP-dependent DNA ligase from Burkholderia pseudomallei as well as determining its crystal structure in complex with DNA. This revealed that the enzyme has similar activity characteristics to other ATP-dependent DNA ligases, and significant structural similarity to the eukaryotic virus Chlorella virus including the positioning and DNA contacts of the binding latch region. Analysis of the genomic context of the B. pseudomallei ATP-dependent DNA ligase indicates it is part of a lysogenic bacteriophage present in the B. pseudomallei chromosome representing one likely entry point for the horizontal acquisition of ATP-dependent DNA ligases by bacteria

DNA repair during bacterial competence? A potential role for the periplasmic ligase, Lig E

The rapid rise in antibiotic resistance among pathogenic bacteria calls for better understanding of drivers of resistance and pathways by which it occurs. Identification of such mechanisms could identify novel pathways that can be targeted for therapeutic interventions. In particular, disruption of the DNA repair system, which includes ligases that seal breaks in DNA, would greatly discourage bacterial growth. Recently, an enigmatic ATP-dependent DNA ligase, Lig E, has been discovered in many species of antibiotic-resistant Gram-negative bacteria. This enzyme is not only minimal and compact, but also contains an N (amino)-terminal signal peptide that indicates a likely periplasmic location, as opposed to the cytoplasm, the location of genomic DNA. It is thus hypothesised that Lig E has a potential role in bacterial competence and aids in repairing breaks in damaged DNA obtained from the environment, a role which may enhance the acquisition of antibiotic resistance genes under DNA damaging conditions. In recent years, there has been growing public concern about the human pathogen Neisseria gonorrhoeae, which causes the sexually transmitted disease, gonorrhoea. Previously easily treatable, this disease is now spreading at alarming rates due to the bacterium’s rapid acquisition of antibiotic resistance genes, attributable to its ability to take up pieces of DNA without regulation. It is in relation to this natural competence that Lig E is hypothesised to function, not only by accelerating the uptake process, but also by increasing the uptake efficiency. Thus, the aim of this thesis was to identify the cellular location and biological function of Lig E in N. gonorrhoeae (Ngo-Lig) by generating in vivo mutants, the effects of which were characterised both in vivo (growth experiment) and in vitro (ligation assays). Steps were also made towards developing an in vivo assay to test the role of Ngo-Lig in DNA uptake by optimisation of uptake reporter constructs. Results obtained over the course of this thesis showed that disruption of Lig E in N. gonorrhoeae leads to a decrease in the rates of gonococcal growth. From this, the hypothesis of the role of Lig E evolved to also consider its potential role in biofilm formation, which is important for the bacterium’s attachment and infection. Although further research into the biological role of Lig E are necessary, the results collected demonstrated potentially novel pathways involving Lig E that may be targeted by future drug developments to tackle this emerging threat in our community

Design of novel nozzles for higher interlayer strength of 3D printed cement paste

In this study, novel nozzles for cement paste 3D printing are designed and optimized for higher interlayer strength via experiment and volume-of-fluid (VOF) based simulation, in terms of various outlet shapes and two nozzle components namely the interface shaper and the side trowel. These nozzles are evaluated experimentally and theoretically based on their performances in the specimen interlayer strength, interfacial shear stress, and cross-sectional geometry. It is concluded that the “Cir3” and the “Kidney” outlet shapes achieve the best performance with the paste water-cement (w/c) ratio ranging from 0.21 to 0.23, subject to the nozzle stand-off distance of 12 mm and printing speed of 60 mm/s. In addition, the interface shaper and the side trowel are able to further enhance the interlayer strength significantly by up to 2 times, through optimization of the interfacial geometry and minimization of the interlayer notch of 3D printed cement paste. It is also confirmed that the optimal nozzle varies with the w/c ratio of cement paste due to different notch depths that are generated, such that nozzle optimization is required along with material development for cement paste 3D printing

Immunogenic fusion proteins induce neutralizing SARS-CoV-2 antibodies in the serum and milk of sheep

Antigen-specific polyclonal immunoglobulins derived from the serum, colostrum, or milk of immunized ruminant animals have potential as scalable therapeutics for the control of viral diseases including COVID-19. Here we show that the immunization of sheep with fusions of the SARS-CoV-2 receptor binding domain (RBD) to ovine IgG2a Fc domains promotes significantly higher levels of antigen-specific antibodies compared to native RBD or full-length spike antigens. This antibody population contained elevated levels of neutralizing antibodies that suppressed binding between the RBD and hACE2 receptors in vitro. A second immune-stimulating fusion candidate, Granulocyte-macrophage colony-stimulating factor (GM-CSF), induced high neutralizing responses in select animals but narrowly missed achieving significance. We further demonstrated that the antibodies induced by these fusion antigens were transferred into colostrum/milk and possessed cross-neutralizing activity against diverse SARS-CoV-2 variants. Our findings highlight a new pathway for recombinant antigen design in ruminant animals with applications in immune milk production and animal health