A variety of microorganisms have the ability to use phosphonic acids as sole sources of phosphorus. in the formation of inorganic phosphate and pyruvic acid (Fig. 1(13) discovered both known and novel pathways for 2-AEPn catabolism by expression of genes encoded in a marine metagenomic library in 1021. can use a number of phosphonates via the C-P lyase pathway and the corresponding gene cluster has been identified (15, 16). The genes in are induced under phosphate-limiting growth conditions. However, when C-P lyase was genetically inactivated, retained the ability to grow on 2-AEPn as a sole phosphorus source. Therefore, it was suggested that encoded genes for both a C-P lyase and phosphonatase pathway (16), as had previously been shown for (17). Later, the complete genome sequence of was determined, revealing a chromosome (3.65 Mbp) and two megaplasmids, pSymA (1.35 Mbp) and pSymB (1.68 Mbp) (15, 18C20). Surprisingly, genes for a phosphonatase pathway were absent. Instead, the genetic complement suggests that catabolizes 2-AEPn via a novel pathway involving (i) conversion of 2-AEPn to PnAA, (ii) oxidation of PnAA to PnA, and (iii) hydrolysis of PnA to acetate and inorganic phosphate by a metal-dependent phosphonoacetate hydrolase similar to Zanosar the enzyme described above. Here we report the genetic and biochemical characterization of this novel pathway. EXPERIMENTAL PROCEDURES Materials Chemical reagents used in this study were obtained from Sigma-Aldrich (St. Louis, MO) or Thermo Fisher Scientific (Pittsburgh, PA) Zanosar and were used without further purification. Media components were purchased from Thermo Fisher Scientific or VWR (West Chester, PA). Bacterial Strains, Plasmids, and Culture Conditions The strains and plasmids used in this study are listed in supplemental Table S1. strains were grown at 37 C unless indicated otherwise. strains were grown at 30 C. Luria-Bertani (LB) liquid or solid media were used for most purposes with the addition of appropriate antibiotics at the following concentrations: 200 g/ml neomycin, 100 g/ml ampicillin, 50 g/ml kanamycin. SOC media used for transformation of DH5 pir and BL21 (DE3) cells with plasmid DNA was prepared as previously described (21). To test for utilization of various phosphorus sources, strains were first grown to saturation at 30 C in 0.2% (w/v) glucose-MOPS medium (22) containing phosphate (50 m), biotin (100 ng/ml) and l-methionine (5 g/ml). Subcultures were then inoculated into 0.2% (w/v) glucose-MOPS medium containing biotin and l-methionine and the desired phosphorus source at 500 m. Growth was measured by monitoring optical density at 410 nm using a Bausch & Lomb Spectronic 21 spectrophotometer. DNA Isolation and Manipulation All cloning procedures were done using established cloning methods (23). Restriction endonucleases and T4 DNA ligase were purchased from Invitrogen (Carlsbad, CA). Plasmid DNA was isolated using the Qiagen (Valencia, CA) Miniprep kit. The Qiagen QIAquick kit was used for the purification of DNA fragments from enzymatic reactions and agarose gels. PCR amplifications of DNA fragments were done using high-fidelity KOD polymerase (Novagen, EMD Chemicals, Inc., Gibbstown, NJ). FailSafe PCR 2J premix Zanosar buffer purchased from Epicentre (Madison, WI) was used in all of the PCR amplifications. Oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA). The recombinant plasmids were Rabbit polyclonal to ZW10.ZW10 is the human homolog of the Drosophila melanogaster Zw10 protein and is involved inproper chromosome segregation and kinetochore function during cell division. An essentialcomponent of the mitotic checkpoint, ZW10 binds to centromeres during prophase and anaphaseand to kinetochrore microtubules during metaphase, thereby preventing the cell from prematurelyexiting mitosis. ZW10 localization varies throughout the cell cycle, beginning in the cytoplasmduring interphase, then moving to the kinetochore and spindle midzone during metaphase and lateanaphase, respectively. A widely expressed protein, ZW10 is also involved in membrane traffickingbetween the golgi and the endoplasmic reticulum (ER) via interaction with the SNARE complex.Both overexpression and silencing of ZW10 disrupts the ER-golgi transport system, as well as themorphology of the ER-golgi intermediate compartment. This suggests that ZW10 plays a criticalrole in proper inter-compartmental protein transport confirmed by DNA sequencing in the W. M. Keck Middle for Practical and Comparative Genomics in the College or university of Illinois, Urbana-Champaign. 1021 (WM5130) crude DNA was made by scraping an individual colony into 100 l of sterile drinking water accompanied by incubation at 100 C for 5 min. The cell particles was eliminated by centrifugation at 14,000 for 5 min. NMR Spectroscopy and Mass Spectrometry (MS) Instrumentation All NMR tests had been performed in the Varian Oxford Middle for Quality in NMR lab at the College or university of Illinois, Urbana-Champaign. The current presence of phosphorus-containing substances was recognized using 1H-decoupled 31P NMR spectroscopy. All the spectra had been gathered in H2O supplemented.