The marine viromes of four oceanic regions

Viruses are the most common biological entities in the marine environment. There has not been a global survey of these viruses, and consequently, it is not known what types of viruses are in Earth's oceans or how they are distributed. Metagenomic analyses of 184 viral assemblages collected over a decade and representing 68 sites in four major oceanic regions showed that most of the viral sequences were not similar to those in the current databases. There was a distinct “marine-ness” quality to the viral assemblages. Global diversity was very high, presumably several hundred thousand of species, and regional richness varied on a North-South latitudinal gradient. The marine regions had different assemblages of viruses. Cyanophages and a newly discovered clade of single-stranded DNA phages dominated the Sargasso Sea sample, whereas prophage-like sequences were most common in the Arctic. However most viral species were found to be widespread. With a majority of shared species between oceanic regions, most of the differences between viral assemblages seemed to be explained by variation in the occurrence of the most common viral species and not by exclusion of different viral genomes. These results support the idea that viruses are widely dispersed and that local environmental conditions enrich for certain viral types through selective pressure.     

Modulation of the ComA-dependent quorum response in Bacillus subtilis by multiple Rap proteins and Phr peptides

In Bacillus subtilis, extracellular peptide signaling regulates several biological processes. Secreted Phr signaling peptides are imported into the cell and act intracellularly to antagonize the activity of regulators known as Rap proteins. B. subtilis encodes several Rap proteins and Phr peptides, and the processes regulated by many of these Rap proteins and Phr peptides are unknown. We used DNA microarrays to characterize the roles that several rap-phr signaling modules play in regulating gene expression. We found that rapK-phrK regulates the expression of a number of genes activated by the response regulator ComA. ComA activates expression of genes involved in competence development and the production of several secreted products. Two Phr peptides, PhrC and PhrF, were previously known to stimulate the activity of ComA. We assayed the roles that PhrC, PhrF, and PhrK play in regulating gene expression and found that these three peptides stimulate ComA-dependent gene expression to different levels and are all required for full expression of genes activated by ComA. The involvement of multiple Rap proteins and Phr peptides allows multiple physiological cues to be integrated into a regulatory network that modulates the timing and magnitude of the ComA response.     

Expanding the subproteome of the inner mitochondria using protein separation technologies: one- and two-dimensional liquid chromatography and two-dimensional gel electrophoresis

Currently no single proteomics technology has sufficient analytical power to allow for the detection of an entire proteome of an organelle, cell, or tissue. One approach that can be used to expand proteome coverage is the use of multiple separation technologies especially if there is minimal overlap in the proteins observed by the different methods. Using the inner mitochondrial membrane subproteome as a model proteome, we compared for the first time the ability of three protein separation methods (two-dimensional liquid chromatography using the ProteomeLab PF 2D Protein Fractionation System from Beckman Coulter, one-dimensional reversed phase high performance liquid chromatography, and two-dimensional gel electrophoresis) to determine the relative overlap in protein separation for these technologies. Data from these different methods indicated that a strikingly low number of proteins overlapped with less than 24% of proteins common between any two technologies and only 7% common among all three methods. Utilizing the three technologies allowed the creation of a composite database totaling 348 non-redundant proteins. 82% of these proteins had not been observed previously in proteomics studies of this subproteome, whereas 44% had not been identified in proteomics studies of intact mitochondria. Each protein separation method was found to successfully resolve a unique subset of proteins with the liquid chromatography methods being more suited for the analysis of transmembrane domain proteins and novel protein discovery. We also demonstrated that both the one- and two-dimensional LC allowed for the separation of the alpha-subunit of F1F0 ATP synthase that differed due to a change in pI or hydrophobicity.     

Mapping of Neutralizing Epitopes on Renibacterium salmoninarum p57 by Use of Transposon Mutagenesis and Synthetic Peptides

Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish.     

Polarized trafficking of E-cadherin is regulated by Rac1 and Cdc42 in Madin-Darby canine kidney cells

E-cadherin is a major cell-cell adhesion protein of epithelia that is trafficked to the basolateral cell surface in a polarized fashion. The exact post-Golgi route and regulation of E-cadherin transport have not been fully described. The Rho GTPases Cdc42 and Rac1 have been implicated in many cell functions, including the exocytic trafficking of other proteins in polarized epithelial cells. These Rho family proteins are also associated with the cadherin-catenin complexes at the cell surface. We have used functional mutants of Rac1 and Cdc42 and inactivating toxins to demonstrate specific roles for both Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Dominant-negative mutants of Cdc42 and Rac1 accumulate E-cadherin at a distinct post-Golgi step. This accumulation occurs before p120^ctn interacts with E-cadherin, because p120^ctn localization was not affected by the Cdc42 or Rac1 mutants. Moreover, the GTPase mutants had no effect on the trafficking of a targeting mutant of E-cadherin, consistent with the selective involvement of Cdc42 and Rac1 in basolateral trafficking. These results provide a new example of Rho GTPase regulation of basolateral trafficking and demonstrate novel roles for Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Rho family GTPases; catenin; polarity; sorting; actin     

Delineation of xenobiotic substrate sites in rat glutathione S-transferase M1-1

Glutathione S-transferases catalyze the conjugation of glutathione with endogenous and exogenous xenobiotics. Hu and Colman (1995) proposed that there are two distinct substrate sites in rat GST M1-1, a 1-chloro-2,4-dintrobenzene (CDNB) substrate site located in the vicinity of tyrosine-115, and a monobromobimane (mBBr) substrate site. To determine whether the mBBr substrate site is distinguishable from the CDNB substrate site, we tested S-(hydroxyethyl)bimane, a nonreactive derivative of mBBr, for its ability to compete kinetically with the substrates. We find that S-(hydroxyethyl)bimane is a competitive inhibitor (K(I) = 0.36 microM) when mBBr is used as substrate, but not when CDNB is used as substrate, demonstrating that these two sites are distinct. Using site-directed mutagenesis, we have localized the mBBr substrate site to an area midway through alpha-helix 4 (residues 90-114) and have identified residues that are important in the enzymatic reaction. Substitution of alanine at positions along alpha-helix 4 reveals that mutations at positions 103, 104, and 109 exhibit a greater perturbation of the enzymatic reaction with mBBr than with CDNB as substrate. Various other substitutions at positions 103 and 104 reveal that a hydrophobic residue is necessary at each of these positions to maintain optimal affinity of the enzyme for mBBr and preserve the secondary structure of the enzyme. Substitutions at position 109 indicate that this residue is important in the enzyme's affinity for mBBr but has a minimal effect on Vmax. These results demonstrate that the promiscuity of rat GST M1-1 is in part due to at least two distinct substrate sites.