Isolation and characterization of bacterial phosphorous metabolism genes from complex microbial communities

Phosphorous (P) is an essential nutrient, playing a central role in the life of a bacterial cell. It is involved in cellular metabolic pathways, cell signaling and is a component of many of the cell’s macromolecules. Since a majority of the biosphere’s microorganisms have not yet been cultured, much more can be learned about the biochemical and genetic mechanisms that govern bacterial P metabolism. The function-driven approach to metagenomics was applied to study P metabolism in the bacterial communities present in pulp and municipal wastewater treatment plant activated sludge and soil, leading to the isolation and identification of three new phosphatases, genes involved in P transport, regulation of P related functions and additional genes which may be important for the bacterial cell’s adaptation to the above communities. The identification of two new nonspecific acid phosphatases (NSAPs) phoNACX6.13 and phoNBCX4.10 and an alkaline phosphatase, phoAACX6.71, belonging to the nucleotide pyrophosphatase phosphodiesterase (NPP) family is reported here. The genes for the three phosphatases were cloned, sequenced, and analysed for upstream regulatory sequences in addition to biochemical characterization of their protein products. PhoB-binding sites were found upstream to phoAACX6.71 and NSAP phoNACX6.13, suggesting these genes are governed by the mechanisms of the previously described “pho” regulon. The two NSAPs have pH optima in the acidic neutral range while the alkaline phosphatase has an optimal pH at 9.5. The three phosphatases appear to be distantly related to known bacterial phosphatase enzymes. Phylogenetic analysis shows the newly identified NSAPs appear on a separate clade from known bacterial NSAPs. Key amino acid residues involved in the catalytic site of these NSAPs were identified in PhoNACX6.13 and PhoNBCX4.10.In PhoAACX6.71, key amino acid residues involved in catalysis and metal cofactor coordination were identified. The roles of these residues were confirmed based on the predicted molecular structure of these proteins. The structures indicate the three proteins are globular with folding patterns suitable for catalytic residues to bind and cleave the P substrate. This is the first report of functional characterization of phosphatases from uncultured bacteria. In addition to exploring the hydrolysis of phosphate esters, the transport and metabolism of other P compounds was also investigated. By phenotypic complementation of phosphonate growth deficient mutants of the legume symbiont, Sinorhizobium meliloti and large scale sequencing of selected metagenomic clones, 92 ORFs were isolated. As expected, about 25% of these ORFs are P transport proteins and P related regulators. Genes involved in other regulatory functions made up about 12% of the total while genes related to Nitrogen metabolism and assimilation account for about 8% of the newly identified ORFs. About 30% of the ORFs encoded general cellular functions or hypothetical proteins of unknown function. The results of this investigation demonstrate the effectiveness of functional metagenomics in studying genetic diversity of bacteria inhabiting complex microbial communities and in identifying new proteins of interest

Cytogenetic Prognostication Within Medulloblastoma Subgroups

PURPOSE: Medulloblastoma comprises four distinct molecular subgroups: WNT, SHH, Group 3, and Group 4. Current medulloblastoma protocols stratify patients based on clinical features: patient age, metastatic stage, extent of resection, and histologic variant. Stark prognostic and genetic differences among the four subgroups suggest that subgroup-specific molecular biomarkers could improve patient prognostication. PATIENTS AND METHODS: Molecular biomarkers were identified from a discovery set of 673 medulloblastomas from 43 cities around the world. Combined risk stratification models were designed based on clinical and cytogenetic biomarkers identified by multivariable Cox proportional hazards analyses. Identified biomarkers were tested using fluorescent in situ hybridization (FISH) on a nonoverlapping medulloblastoma tissue microarray (n = 453), with subsequent validation of the risk stratification models. RESULTS: Subgroup information improves the predictive accuracy of a multivariable survival model compared with clinical biomarkers alone. Most previously published cytogenetic biomarkers are only prognostic within a single medulloblastoma subgroup. Profiling six FISH biomarkers (GLI2, MYC, chromosome 11 [chr11], chr14, 17p, and 17q) on formalin-fixed paraffin-embedded tissues, we can reliably and reproducibly identify very low-risk and very high-risk patients within SHH, Group 3, and Group 4 medulloblastomas. CONCLUSION: Combining subgroup and cytogenetic biomarkers with established clinical biomarkers substantially improves patient prognostication, even in the context of heterogeneous clinical therapies. The prognostic significance of most molecular biomarkers is restricted to a specific subgroup. We have identified a small panel of cytogenetic biomarkers that reliably identifies very high-risk and very low-risk groups of patients, making it an excellent tool for selecting patients for therapy intensification and therapy de-escalation in future clinical trials

Regulation of Expression of Scaffoldin-Related Genes in Clostridium thermocellum

Clostridium thermocellum produces an extracellular multienzyme complex, termed the cellulosome, that allows efficient solubilization of crystalline cellulose. The complex is organized around a large noncatalytic protein subunit, termed CipA or scaffoldin, and is found either free in the supernatant or cell bound. The binding of the complex to the cell is mediated by three cell surface anchoring proteins, OlpB, Orf2p, and SdbA, that interact with the CipA scaffoldin. The transcriptional level of the olpB, orf2, sdbA, and cipA genes was determined quantitatively by RNase protection assays in batch and continuous cultures, under carbon and nitrogen limitation. The mRNA level of olpB, orf2, and cipA varied with growth rate, reaching 40 to 60 transcripts per cell under carbon limitation at a low growth rate of 0.04 h(−1) and 2 to 10 transcripts per cell at a growth rate of 0.35 h(−1) in batch culture. The mRNA level of sdbA was about three transcripts per cell and was not influenced by growth rate. Primer extension analysis revealed two major transcriptional start sites, at −81 and −50 bp, upstream of the translational start site of the cipA gene. The potential promoters exhibited homology to the known sigma factors σ(A) and σ(L) (σ(54)) of Bacillus subtilis. Transcription from the σ(L)-like promoter was found under all growth conditions, whereas transcription from the σ(A)-like promoter was significant only under carbon limitation. The overall expression level obtained in the primer extension analysis was in good agreement with the results of the RNase-protection assays