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Identification of Bacterial Populations in Dairy Wastewaters by Use of 16S rRNA Gene Sequences and Other Genetic Markers. Jeffery A. McGarvey, 2004.Hydraulic flush waste removal systems coupled to solid/liquid separators and circulated treatment lagoons are commonly utilized to manage the large amounts of animal waste produced on high-intensity dairy farms . Although these systems are common, little is known about the microbial populations that inhabit them or how they change as they traverse the system . Using culture-based and non-culture-based methods, we characterized the microbial community structure of manure, water from the separator pit, and water from the circulated treatment lagoon from a large dairy in the San Joaquin Valley of California . Our results show that both total bacterial numbers and bacterial diversity are highest in manure, followed by the separator pit water and the lagoon water . The most prevalent phylum in all locations was the Firmicutes (low-G+C, gram-positive bacteria) . The most commonly occurring operational taxonomic unit (OTU) had a 16S rRNA gene (rDNA) sequence 96 to 99% similar to that of Clostridium lituseburense and represented approximately 6% of the manure derived sequences, 14% of the separator pit-derived sequences and 20% of the lagoon-derived sequences . Also highly prevalent was an OTU with a 16S rDNA sequence 97 to 100% similar to that of Eubacterium tenue, comprising approximately 3% of the manure-derived sequences, 6% of the separator pit-derived sequences and 9% of the lagoon-derived sequences . Taken together, these sequences represent approximately one-third of the total organisms in the lagoon waters, suggesting that they are well adapted to this environment . The RNA Polymerase  Subunit from Sinorhizobium meliloti Can Assemble with RNA Polymerase Subunits from Escherichia coli and Function in Basal and Activated Transcription both In Vivo and In Vitro.Melicent C. Peck, 2002.Sinorhizobium meliloti, a gram-negative soil bacterium, forms a nitrogen-fixing symbiotic relationship with members of the legume family . To facilitate our studies of transcription in S . meliloti, we cloned and characterized the gene for the subunit of RNA polymerase (RNAP) . S . meliloti rpoA encodes a 336-amino-acid, 37-kDa protein . Sequence analysis of the region surrounding rpoA identified six open reading frames that are found in the conserved gene order secY (SecY)-adk (Adk)-rpsM (S13)-rpsK (S11)-rpoA ()-rplQ (L17) found in the
-proteobacteria . In vivo, S . meliloti rpoA expressed in Escherichia coli complemented a temperature sensitive mutation in E . coli rpoA, demonstrating that S . meliloti
supports RNAP assembly, sequence-specific DNA binding, and interaction with transcriptional activators in the context of E . coli. In vitro, we reconstituted RNAP holoenzyme from S . meliloti
and E . coli ß, ß', and
 subunits . Similar to E . coli RNAP, the hybrid RNAP supported transcription from an E . coli core promoter and responded to both upstream (UP) element- and Fis-dependent transcription activation . We obtained similar results using purified RNAP from S . meliloti . Our results demonstrate that S . meliloti
functions are conserved in heterologous host E . coli even though the two
subunits are only 51% identical . The ability to utilize E . coli as a heterologous system in which to study the regulation of S . meliloti genes could provide an important tool for our understanding and manipulation of these processes .
A Novel Adenylate Binding Site Confers Phosphopantetheine Adenylyltransferase Interactions with Coenzyme A. Tina Izard, 2003.Phosphopantetheine adenylyltransferase (PPAT) regulates the key penultimate step in the essential coenzyme A (CoA) biosynthetic pathway . PPAT catalyzes the reversible transfer of an adenylyl group from Mg2+:ATP to 4'-phosphopantetheine to form 3'-dephospho-CoA (dPCoA) and pyrophosphate . The high-resolution crystal structure of PPAT complexed with CoA has been determined . Remarkably, CoA and the product dPCoA bind to the active site in distinct ways . Although the phosphate moiety within the phosphopantetheine arm overlaps, the pantetheine arm binds to the same pocket in two distinct conformations, and the adenylyl moieties of these two ligands have distinct binding sites . Moreover, the PPAT:CoA crystal structure confirms the asymmetry of binding to the two trimers within the hexameric enzyme . Specifically, the pantetheine arm of CoA bound to one protomer within the asymmetric unit displays the dPCoA-like conformation with the adenylyl moiety disordered, whereas CoA binds the twofold-related protomer in an ordered and unique fashion . Production of Native-Type Streptoverticillium mobaraense Transglutaminase in Corynebacterium glutamicum. Masayo Date, 2003.We previously observed secretion of active-form transglutaminase in Corynebacterium glutamicum by coexpressing the subtilisin-like protease SAM-P45 from Streptomyces albogriseolus to process the prodomain . However, the N-terminal amino acid sequence of the transglutaminase differed from that of the native Streptoverticillium mobaraense enzyme . In the present work we have used site-directed mutagenesis to generate an optimal SAM-P45 cleavage site in the C-terminal region of the prodomain . As a result, native-type transglutaminase was secreted . Peptide Surface Display and Secretion Using Two LPXTG-Containing Surface Proteins from Lactobacillus fermentum BR11. Mark S. Turner, 2003.A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from Lactobacillus fermentum BR11 has been identified by using an antiserum raised against whole L . fermentum BR11 cells . The first protein, Rlp, is similar to the Rib surface protein from Streptococcus agalactiae, while the other protein, Mlp, is similar to the mucus binding protein Mub from Lactobacillus reuteri . It was shown that multiple copies of mlp exist in the genome of L . fermentum BR11 . Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in L . fermentum . The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His6) . The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall . Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion) . The mlp promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His6 and CFTR peptides or His6 peptide, respectively . Therefore, these newly described proteins from L . fermentum BR11 have potential as protein production and targeting vectors .
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