General Materials Science

Candidatus Saccharibacteria is a well-described candidate phylum that has not been successfully isolated. Nevertheless, its presence was suggested by 16S rRNA gene sequencing data, and it is frequently detected in natural environments and activated sludge. Because pure culture representatives of Candidatus Saccharibacteria are lacking, the specificity of primers for the determination of their abundance and diversity should be carefully evaluated. In this study, eight Candidatus Saccharibacteria-specific primers were selected from previous studies and evaluated for their coverage against a public database, annealing temperature of the combined primer sets, as well as their utilization to determine the detection frequencies and phylogenetic diversity by cloning analysis, and in quantification by quantitative polymerase chain reaction (PCR). Among the eight primers, four primers (TM7314F, TM7580F, TM7-910R, and TM7-1177R) showed high coverage. Cloning analysis showed that four primer sets (TM7314F and TM7-910R, TM7314F and TM7-1177R, TM7580F and TM7-910R, and TM7580F and TM7-1177R) yielded high detection frequencies for Candidatus Saccharibacteria in activated sludge from a wastewater treatment plant in Higashihiroshima City, Japan. Quantitative PCR results indicated that the primer set containing TM7314F and TM7-910R was superior for the specific detection of Candidatus Saccharibacteria in activated sludge.

Candidatus Liberibacter asiaticus is a fastidious prokaryotic α-proteobacterium that has not been cultured as yet. It is the causative agent of Citrus Huanglongbing (HLB) is a disease that infects citrus plants causing huge losses to the citrus industry. The plants employ a variety of defense mechanisms to combat the pathogen attack which mainly involves the formation of reactive oxygen species (ROS) mainly hydrogen peroxide, peroxynitrite and organic hydroperoxides. The microbe on the other hand has antioxidant proteins that counter the reactive oxygen species. Peroxiredoxin (Prx) which is known to play a crucial role in peroxide detoxification is a super family of one of such antioxidant proteins. In this work, we have determined the crystal structure of Bacterioferritin Comigratory Protein (BCP) that belongs to 1-Cys Prx enzyme having peroxidatic/sulfenic acid cysteine (C-47) but lacks the resolving cysteine. Furthermore, we are investigating structural aspects to gain insight into the binding of different peroxide substrates at the active site. Site directed mutagenesis has been done to introduce the non-conserved resolving cysteine to study its effect on peroxidase activity in both wild type and mutant form. Biochemically, both BCP 1-cys and BCP 2-cys have shown peroxidase activity. DNA protection activity against oxidative damage and cell-line based assays has implicated its protective role towards the hypoxic condition of cells. Hence, the protein becomes a lucrative target for ligands which might be potential antimicrobials.