Characterization of M. tuberculosis SerB2, an Essential HAD-Family Phosphatase,Reveals Novel Properties

M. tuberculosis harbors an essential phosphoserine phosphatase (MtSerB2, Rv3042c) that contains two small- molecule binding ACT-domains (Pfam 01842) at the N-terminus followed by the phosphoserine phosphatase (PSP) domain. We found that exogenously added MtSerB2 elicits microtubule rearrangements in THP-1 cells. Mutational analysis demonstrates that phosphatase activity is co-related to the elicited rearrangements, while addition of the ACT-domains alone elicits no rearrangements. The enzyme is dimeric, exhibits divalent metal- ion dependency, and is more specific for l- phosphoserine unlike other classical PSPases. Binding of a variety of amino acids to the ACT-domains influences MtSerB2 activity by either acting as activators/inhibitors/have no effects. Additionally, reduced activity of the PSP domain can be enhanced by equimolar addition of the ACT domains. Further, we identified that G18 and G108 of the respective ACT-domains are necessary for ligand-binding and their mutations to G18A and G108A abolish the binding of ligands like l- serine. A specific transition to higher order oligomers is observed upon the addition of l- serine at ∼0.8 molar ratio as supported by Isothermal calorimetry and Size exclusion chromatography experiments. Mutational analysis shows that the transition is dependent on binding of l- serine to the ACT-domains. Furthermore, the higher-order oligomeric form of MtSerB2 is inactive, suggesting that its formation is a mechanism for feedback control of enzyme activity. Inhibition studies involving over eight inhibitors, MtSerB2, and the PSP domain respectively, suggests that targeting the ACT-domains can be an effective strategy for the development of inhibitors.     

Isolation and Identification of Natural Endophytic Rhizobia from Rice (Oryza sativa L.) Through rDNA PCR-RFLP and Sequence Analysis

Three novel endophytic rhizobial strains (RRE3, RRE5, and RRE6) were isolated from naturally growing surface sterilized rice roots. These isolates had the ability to nodulate common bean (Phaseolus vulgaris). Polymerase chain reaction–restriction fragment length polymorphism and sequencing of 16S rDNA of these isolates revealed that RRE3 and RRE5 are phylogenetically very close to Burkholderia cepacia complex, whereas RRE6 has affinity with Rhizobium leguminosarum bv. phaseoli. Plant infection test using gusA reporter gene tagged construct of these isolates indicated that bacterial cells can go inside and colonize the rice root interiors. A significant increase in biomass and grain yield was also recorded in greenhouse-grown rice plants inoculated with these isolates.     

Pyrroloquinoline-quinone synthesized in Escherichia coli by pyrroloquinoline-quinone synthase of Deinococcus radiodurans plays a role beyond mineral phosphate solubilization

Deinococcus radiodurans, an extremely radioresistant bacterium, synthesizes coenzyme pyrroloquinoline-quinone (PQQ) but exhibits a negative phenotype for mineral phosphate solubilization. Gene for the putative PQQ synthesizing protein was PCR amplified and cloned from Deinococcus, sequenced, and expressed in Escherichia coli, under an inducible E. coli promoter. The transgenic E. coli expressed PQQ synthase protein of 42kDa and complemented the mineral phosphate solubilization phenotype of E. coli, suggesting the synthesis of an active protein. The cells expressing high levels of this protein showed increased protection against photodynamically produced reactive oxygen species. The effect could be attributed to the upregulation of antioxidant enzymes such as catalase and superoxide dismutase by PQQ in transgenic E. coli through an unknown mechanism. The study elucidates a hitherto unknown possible function of PQQ in bacteria.     

Antiproliferative and Apoptotic Effects of pH-Responsive Veratric Acid-Loaded Polydopamine Nanoparticles in Human Triple Negative Breast Cancer Cells

Veratric acid (VA) is plant-derived phenolic acid known for its therapeutic potential, but its anticancer effect on highly invasive triple-negative breast cancer (TNBC) is yet to be evaluated. Polydopamine nanoparticles (nPDAs) were chosen as the drug carrier to overcome VA's hydrophobic nature and ensure a sustained release of VA. We prepared pH-sensitive nano-formulations of VA-loaded nPDAs and subjected them to physicochemical characterization and in vitro drug release studies, followed by cell viability and apoptotic assays on TNBC cells (MDA-MB-231 cells). The SEM and zeta analysis revealed spherical nPDAs were uniform size distribution and good colloidal stability. In vitro drug release from VA-nPDAs was sustained, prolonged and pH-sensitive, which could benefit tumor cell targeting. MTT and cell viability assays showed that VA-nPDAs (IC50=17.6 μM) are more antiproliferative towards MDA-MB-231 cells than free VA (IC50=437.89 μM). The induction of early and late apoptosis by VA-nPDAs in the cancer cells was identified using annexin V and dead cell assay. Thus, the pH response and sustained release of VA from nPDAs showed the potential to enter the cell, inhibit cell proliferation, and induce apoptosis in human breast cancer cells, indicating the anticancer potential of VA.     

Patterns of variation in cultivated common bean (Phaseolus vulgaris,Fabaceae)

More than 18,000 accessions of common bean (Thaseolus vulgaris, Fabaceae) from the Centro Internacional de Agricultura Tropical (CIAT) germplasm bank were examined at two locations in Colombia. A large variation in cultivated dry bean was found among accessions from primary centers of domestication in Middle and South America. For some bean types, such as medium- and large-seeded white, variation was greater among germplasm from western Asia (Turkey) and Europe (Portugal, Spain, Greece, France, Italy, and Bulgaria). Based on growth habit, on seed, pod, and leaf characteristics, and on ecological regions of adaptation, dry-bean germplasm was divided into a total of six gene pools from Middle American and four gene pools from South American centers of domestication. Most of the variation in the snap or stringless bean appears to be of relatively recent origin; it was greatest among cultivars from China, Europe, and the United States. These could be grouped into two additional gene pools. A strategy for breeding and transfer of genes across gene pools is also discussed.     

A novel serralysin metalloprotease from Deinococcus radiodurans

A hypothetical protein (DR2310) from the radiation resistant organism Deinococcus radiodurans harbors highly conserved Zn(+2)-binding (HEXXH) domain and Met-turn (SVMSY), characteristic of the serralysin family of secreted metalloproteases from Gram negative bacteria. Deletion mutagenesis of DR2310 confirmed that the ORF is expressed in Deinococcus radiodurans as a secreted protease of 85 kDa. Biochemical analysis revealed DR2310 to be a Ca(+2) and Zn(+2)-requiring metalloprotease. Unique features such as a long N-terminus, replacement of the highly conserved C-terminal glycine rich Ca(+2)-binding repeats with a single N-terminal aspartate rich eukaryotic thrombospondin type-3 Ca(+2)-binding repeat and absence of C-terminal secretion signals make it a novel member of serralysin family. This is the first report of a functional serralysin family metalloprotease from a Gram positive organism.