An immunodiffusion activity stain for yeast histidinol dehydrogenase.

A procedure is described for detecting enzymatically active yeast histidinol dehydrogenase-antibody complexes in immunodiffusion agar. The method employs a coupled dye system, consisting of a tetrazolium salt and a phenazine methosulfate intermediate, that produces an insoluble formazan and stains active precipitin lines red. The specificity of the reaction is indicated by its dependence on substrate and by its dependence on an intact HIS4C region, based on observations with mutant forms of the yeast HIS4 multifunctional protein.     

The product of the his4 gene cluster in Saccharomyces cerevisiae. A trifunctional polypeptide.

The his4 region of yeast encodes the information for the third (phosphoribosyl-AMP cyclohydrolase), second (phosphoribosyl-ATP pyrophosphohydrolase), and tenth (histidinol dehydrogenase) steps in the histidine biosynthetic pathway. These three activities co-purify with a single protein which has a subunit molecular weight of 95,000 (95,000 protein), as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Extracts of yeast strains which carry nonsense or deletion mutations in various portions of the his4 region, purified in parallel by affinity chromatography on AMP-agarose columns, were examined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis slabs. All such mutant extracts examined were found to lack the 95,000 protein found in a strain carrying a wild type his4 allele. The presence of a protease inhibitor, phenylmethylsulfonyl fluoride, during the purification of the trifunctional enzyme prevented the degradation of the 95,000 protein to polypeptides of lower molecular weight. Monospecific antibody prepared against the 95,000 protein removed all three of the activities specified by his4 from solution; active 95,000 protein was recovered in the resuspended immunoprecipitates. All this evidence shows that the product of the his4 region is a trifunctional, 95,000-dalton protein. Preliminary evidence from two-dimensional gel electrophoresis, NH2-terminal analysis, and gel filtration column chromatography indicates that the native trifunctional enzyme is a dimer of identical 95,000-dalton subunits.     

Cutting edge: myeloid differentiation factor 88 deficiency improves resistance against sepsis caused by polymicrobial infection

Toll-like receptors (TLRs) are important for the activation of innate immune cells upon encounter of microbial pathogens. The present study investigated the potential roles of TLR2, TLR4, and the signaling protein myeloid differentiation factor 88 (MyD88) in polymicrobial septic peritonitis. Whereas both TLR2 and TLR4 were dispensable for host defense against septic peritonitis, MyD88-deficient mice were protected in this infection model. Recruitment of neutrophils to the septic focus and bacterial clearance were normal in MyD88-deficient mice. In contrast, the systemic inflammatory response was strongly attenuated in the absence of MyD88. Surprisingly, MyD88 deficiency did not alter cytokine and chemokine production in spleen, but markedly reduced the inflammatory response in liver and lung. Production of monocyte chemoattractant protein-1 and macrophage-inflammatory protein-1alpha was entirely independent of MyD88. These results imply a central role of MyD88 for the systemic immune pathology of polymicrobial sepsis and show that cytokine production in spleen and induction of certain chemokines are MyD88 independent.     

Capturing single cell genomes of active polysaccharide degraders: an unexpected contribution of Verrucomicrobia

Microbial hydrolysis of polysaccharides is critical to ecosystem functioning and is of great interest in diverse biotechnological applications, such as biofuel production and bioremediation. Here we demonstrate the use of a new, efficient approach to recover genomes of active polysaccharide degraders from natural, complex microbial assemblages, using a combination of fluorescently labeled substrates, fluorescence-activated cell sorting, and single cell genomics. We employed this approach to analyze freshwater and coastal bacterioplankton for degraders of laminarin and xylan, two of the most abundant storage and structural polysaccharides in nature. Our results suggest that a few phylotypes of Verrucomicrobia make a considerable contribution to polysaccharide degradation, although they constituted only a minor fraction of the total microbial community. Genomic sequencing of five cells, representing the most predominant, polysaccharide-active Verrucomicrobia phylotype, revealed significant enrichment in genes encoding a wide spectrum of glycoside hydrolases, sulfatases, peptidases, carbohydrate lyases and esterases, confirming that these organisms were well equipped for the hydrolysis of diverse polysaccharides. Remarkably, this enrichment was on average higher than in the sequenced representatives of Bacteroidetes, which are frequently regarded as highly efficient biopolymer degraders. These findings shed light on the ecological roles of uncultured Verrucomicrobia and suggest specific taxa as promising bioprospecting targets. The employed method offers a powerful tool to rapidly identify and recover discrete genomes of active players in polysaccharide degradation, without the need for cultivation.     

Flow-Cytometric Cell Sorting and Subsequent Molecular Analyses for Culture-Independent Identification of Bacterioplankton Involved in Dimethylsulfoniopropionate Transformations

Marine bacterioplankton transform dimethylsulfoniopropionate (DMSP) into the biogeochemically important and climatically active gas dimethylsulfide. In order to identify specific bacterial taxa mediating DMSP processing in a natural marine ecosystem, we amended water samples from a southeastern U.S. salt marsh with 20 μM DMSP and tracked community shifts with flow cytometry (FCM) coupled to 16S rRNA gene analyses. In two out of four seasons studied, DMSP amendments induced the formation of distinct bacterioplankton populations with elevated nucleic acid (NA) content within 24 h, indicative of cells actively utilizing DMSP. The 16S rRNA genes of the cells with and without elevated NA content were analyzed following cell sorting and PCR amplification with sequencing and terminal restriction fragment length polymorphism approaches. Compared to cells in the control FCM populations, bacteria with elevated NA content in the presence of DMSP were relatively enriched in taxa related to Loktanella, Oceanicola, and Sulfitobacter (Roseobacter lineage, α-Proteobacteria); Caulobacter (α-Proteobacteria); and Brachymonas and Xenophilus (β-Proteobacteria) in the May-02 sample and to Ketogulonicigenium (Roseobacter lineage, α-Proteobacteria) and novel γ-Proteobacteria in the Sept-02 sample. Our study suggests that diverse bacterioplankton participate in the metabolism of DMSP in coastal marine systems and that their relative importance varies temporally.     

Single-cell genomics shedding light on marine Thaumarchaeota diversification

Previous studies based on analysis of amoA, 16S ribosomal RNA or accA gene sequences have established that marine Thaumarchaeota fall into two phylogenetically distinct groups corresponding to shallow- and deep-water clades, but it is not clear how water depth interacts with other environmental factors, including light, temperature and location, to affect this pattern of diversification. Earlier studies focused on single-gene distributions were not able to link phylogenetic structure to other aspects of functional adaptation. Here, we analyzed the genome content of 46 uncultivated single Thaumarchaeota cells sampled from epi- and mesopelagic waters of subtropical, temperate and polar oceans. Phylogenomic analysis showed that populations diverged by depth, as expected, and that mesopelagic populations from different locations were well mixed. Functional analysis showed that some traits, including putative DNA photolyase and catalase genes that may be related to adaptive mechanisms to reduce light-induced damage, were found exclusively in members of the epipelagic clade. Our analysis of partial genomes has thus confirmed the depth differentiation of Thaumarchaeota populations observed previously, consistent with the distribution of putative mechanisms to reduce light-induced damage in shallow- and deep-water populations.     

In vivo behavior of copper-64-labeled methanephosphonate tetraaza macrocyclic ligands

Copper-64 ( T(1/2)=12.7 h; beta(+): 0.653 MeV, 17.4%; beta(-): 0.578 MeV, 39%) is produced in a biomedical cyclotron and has applications in both imaging and therapy. Macrocyclic chelators are widely used as bifunctional chelators to bind copper radionuclides to antibodies and peptides owing to their relatively high kinetic stability. In this paper, we evaluated three tetraaza macrocyclic ligands with two, three, and four pendant methanephosphonate functional groups. DO2P [1,4,7,10-tetraazacyclododecane-1,7-di(methanephosphonic acid)], DO3P [1,4,7,10-tetraazacyclododecane-1,4,7-tri(methanephosphonic acid)], and DOTP [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methanephosphonic acid)] were all radiolabeled with (64)Cu in high radiochemical yields. Copper-64-labeled DO2P and DOTP were highly stable in rat serum out to 24 h, while (64)Cu-DO3P remained 73% intact, with the remainder possibly forming a (64)Cu(.)2DO3P dimer by 24 h. The biodistribution experiments were performed in normal Sprague-Dawley rats. Of the three complexes, (64)Cu-DO2P demonstrated the most optimal clearance through the blood and liver. Copper-64-DO3P and (64)Cu-DOTP exhibited higher liver uptake and longer retention of liver activity, possibly because of the large negative charge of the complexes under physiological conditions. All three (64)Cu-labeled complexes showed high accumulation in bone, likely due to the binding of the methanephosphonate groups to hydroxyapatite. These results suggest that this series of methanephosphonate macrocyclic ligands may be useful as potential bone-imaging agents. The thermodynamic stability constants of the Cu(II) complexes with these three ligands were determined, and were found to be significantly higher than those of their acetate analogues. The Cu(II)-DO2P complex exhibited the highest stability constant among divalent transition metal ion DO2P complexes. Metabolism studies of (64)Cu-DO2P in rat liver suggest that the DO2P ligand may be used as a bifunctional chelator for copper radionuclides in radiodiagnostic or radiotherapeutic studies.     

Single-cell enabled comparative genomics of a deep ocean SAR11 bathytype

Bacterioplankton of the SAR11 clade are the most abundant microorganisms in marine systems, usually representing 25% or more of the total bacterial cells in seawater worldwide. SAR11 is divided into subclades with distinct spatiotemporal distributions (ecotypes), some of which appear to be specific to deep water. Here we examine the genomic basis for deep ocean distribution of one SAR11 bathytype (depth-specific ecotype), subclade Ic. Four single-cell Ic genomes, with estimated completeness of 55%–86%, were isolated from 770 m at station ALOHA and compared with eight SAR11 surface genomes and metagenomic datasets. Subclade Ic genomes dominated metagenomic fragment recruitment below the euphotic zone. They had similar COG distributions, high local synteny and shared a large number (69%) of orthologous clusters with SAR11 surface genomes, yet were distinct at the 16S rRNA gene and amino-acid level, and formed a separate, monophyletic group in phylogenetic trees. Subclade Ic genomes were enriched in genes associated with membrane/cell wall/envelope biosynthesis and showed evidence of unique phage defenses. The majority of subclade Ic-specfic genes were hypothetical, and some were highly abundant in deep ocean metagenomic data, potentially masking mechanisms for niche differentiation. However, the evidence suggests these organisms have a similar metabolism to their surface counterparts, and that subclade Ic adaptations to the deep ocean do not involve large variations in gene content, but rather more subtle differences previously observed deep ocean genomic data, like preferential amino-acid substitutions, larger coding regions among SAR11 clade orthologs, larger intergenic regions and larger estimated average genome size.