SUMMARY In addition to RNA polymerases I, II, and III, the essential RNA polymerases present in all eukaryotes, plants have two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V, that play nonredundant roles in siRNA-directed DNA methylation and gene silencing. We show that Arabidopsis Pol IV and Pol V are composed of subunits that are paralogous or identical to the 12 subunits of Pol II. Four subunits of Pol IV are distinct from their Pol II paralogs, six subunits of Pol V are distinct from their Pol II paralogs, and four subunits differ between Pol IV and Pol V. Importantly, the subunit differences occur in key positions relative to the template entry and RNA exit paths. Our findings support the hypothesis that Pol IV and Pol V are Pol II-like enzymes that evolved specialized roles in the production of noncoding transcripts for RNA silencing and genome defense.

The northwestern Sargasso Sea undergoes annual cycles of productivity with increased production in spring corresponding to periods of upwelling, and oligotrophy in summer and autumn, when the water column becomes highly stratified. The biological productivity of this region is reduced during stratified periods as a result of low concentrations of phosphorus and nitrogen in the euphotic zone. To better understand the mechanisms of microbial survival in this oligotrophic environment, we used capillary liquid chromatography (LC)-tandem mass spectrometry to detect microbial proteins in surface samples collected in September 2005. A total of 2215 peptides that mapped to 236 SAR11 proteins, 1911 peptides that mapped to 402 Prochlorococcus proteins and 2407 peptides that mapped to 404 Synechococcus proteins were detected. Mass spectra from SAR11 periplasmic substrate-binding proteins accounted for a disproportionately large fraction of the peptides detected, consistent with observations that these extremely small cells devote a large proportion of their volume to periplasm. Abundances were highest for periplasmic substrate-binding proteins for phosphate, amino acids, phosphonate, sugars and spermidine. Proteins implicated in the prevention of oxidative damage and protein refolding were also abundant. Our findings support the view that competition for multiple nutrients in oligotrophic systems is extreme, but nutrient flux is sufficient to sustain microbial community activity.

Summary In Arabidopsis, RNA-dependent DNA methylation and transcriptional silencing involves three nuclear RNA polymerases that are biochemically undefined: the presumptive DNA-dependent RNA polymerases, Pol IV and Pol V and the putative RNA-dependent RNA polymerase, RDR2. Here, we demonstrate their RNA polymerase activities in vitro. Unlike Pol II, Pols IV and V require an RNA primer, are insensitive to alpha-amanitin and differ in their ability to displace the non-template DNA strand during transcription. Biogenesis of 24 nt small interfering RNAs (siRNAs), which guide cytosine methylation to corresponding sequences, requires both Pol IV and RDR2, which physically associate in vivo. Whereas Pol IV does not require RDR2 for activity, RDR2 is non-functional in the absence of associated Pol IV. These results suggest that the physical and mechanistic coupling of Pol IV and RDR2 results in the channeled synthesis of double-stranded precursors for 24 nt siRNA biogenesis.

Strategies for removal of high abundance proteins have been increasingly utilized in proteomic studies of serum/ plasma and other body fluids to enhance the detection of low abundance proteins and achieve broader proteome coverage; however, both the reproducibility and specificity of the high abundance protein depletion process still represent common concerns. Here we report a detailed evaluation of immunoaffinity subtraction performed applying the ProteomeLab IgY-12 system that is commonly used in human serum/plasma proteome characterization in combination with high resolution LC-MS/MS. Plasma samples were repeatedly processed using this approach, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. The removal of target proteins by the immunoaffinity subtraction system and the overall process was highly reproducible. Non-target proteins, including one spiked protein standard (rabbit glyceraldehyde-3-phosphate dehydrogenase), were also observed to bind to the column at different levels but also in a reproducible manner. The results suggest that multiprotein immunoaffinity subtraction systems can be readily integrated into quantitative strategies to enhance detection of low abundance proteins in biomarker discovery studies.

Marine oxygen minimum zones (OMZs) are intrinsic water column features arising from respiratory oxygen demand during organic matter degradation in stratified waters. Currently OMZs are expanding due to global climate change with resulting feedback on marine ecosystem function. Here we use metaproteomics to chart spatial and temporal patterns of gene expression along defined redox gradients in a seasonally stratified fjord to better understand microbial community responses to OMZ expansion. The expression of metabolic pathway components for nitrification, anaerobic ammonium oxidation (anammox), denitrification, and inorganic carbon fixation were differentially expressed across the redoxcline and covaried with distribution patterns of ubiquitous OMZ microbes including Thaumarchaeota, Nitrospina, Nitrospira, Planctomycetes, and SUP05/ARCTIC96BD-19 Gammaproteobacteria. Nitrification and inorganic carbon fixation pathways affiliated with Thaumarchaeota dominated dysoxic waters, and denitrification, sulfur oxidation, and inorganic carbon fixation pathways affiliated with the SUP05 group of nitrate-reducing sulfur oxidizers dominated suboxic and anoxic waters. Nitrifier nitrite oxidation and anammox pathways affiliated with Nirospina, Nitrospira, and Planctomycetes, respectively, also exhibited redox partitioning between dysoxic and suboxic waters. The numerical abundance of SUP05 proteins mediating inorganic carbon fixation under anoxic conditions suggests that SUP05 will become increasingly important in global ocean carbon and nutrient cycling as OMZs expand.M arine oxygen (O 2 ) minimum zones (OMZs) are widespread and naturally occurring water column features that arise when respiratory O 2 demand during decomposition of organic matter exceeds O 2 availability in stratified waters. Operationally defined by dissolved O 2 concentrations <20 μM, OMZs promote the use of alternative terminal electron acceptors (TEAs) in microbial energy metabolism that results in climate active gas production including carbon dioxide (CO 2 ), nitrous oxide (N 2 O), and methane (CH 4 ) (1). Currently, OMZs constitute ∼7% of the ocean volume (1, 2). However, global warming promotes conditions for OMZ expansion and intensification, e.g., reduced O 2 solubility and increased stratification, with resulting feedback on the climate system (3, 4).Within OMZs, the use of nitrate (NO 3 − ) and nitrite (NO 2 − ) as TEAs in dissimilatory nitrate reduction (denitrification) and anaerobic ammonium oxidation (anammox) results in fixed nitrogen loss in the form of N 2 O and dinitrogen gas (N 2 ), respectively (5, 6). Because OMZs account for up to 50% of oceanic N 2 production, they have the potential to limit primary production in overlying surface waters (7,8). A recent model suggests that nitrogen fixation in proximity to OMZ waters can balance nitrogen loss processes (9), and several studies along redoxclines in the Eastern Tropical South Pacific and Baltic Sea have measured nitrogen fixation rates that support a close spatial coupling between nitro...

Summary Unlike nuclear multisubunit RNA polymerases I, II and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA Polymerases IV and V are non-essential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their twelve subunits, respectively, and differ from one another in three subunits, proteomic analyses show that maize Pols IV and V differ from Pol II in six subunits, but differ from each other only in their largest subunits. Use of alternative catalytic second-subunits, which are non-redundant for development and paramutation, yields at least two subtypes of Pol IV, and three subtypes of Pol V in maize. Pol IV/V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.

Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium) is a facultative intracellular pathogen that causes ϳ8,000 reported cases of acute gastroenteritis and diarrhea each year in the United States. Although many successful physiological, biochemical, and genetic approaches have been taken to determine the key virulence determinants encoded by this organism, the sheer number of uncharacterized reading frames observed within the S. enterica genome suggests that many more virulence factors remain to be discovered. We used a liquid chromatography-mass spectrometry-based "bottom-up" proteomic approach to generate a more complete picture of the gene products that S. typhimurium synthesizes under typical laboratory conditions as well as in culture media that are known to induce expression of virulence genes. When grown to logarithmic phase in rich medium, S. typhimurium is known to express many genes that are required for invasion of epithelial cells. Conversely stationary phase cultures of S. typhimurium express genes that are needed for both systemic infection and growth within infected macrophages. Lastly bacteria grown in an acidic, magnesium-depleted minimal medium (MgM) designed to mimic the phagocytic vacuole have been shown to up-regulate virulence gene expression. Initial comparisons of protein abundances from bacteria grown under each of these conditions indicated that the majority of proteins do not change significantly. However, we observed subsets of proteins whose expression was largely restricted to one of the three culture conditions. For example, cells grown in MgM had a higher abundance of Mg 2؉ transport proteins than found in other growth conditions. A second more virulent S. typhimurium strain (14028) was also cultured under these same growth conditions, and the results were directly compared with those obtained for strain LT2. This comparison offered a unique opportunity to contrast protein populations in these closely related bacteria. Among a number of proteins displaying a higher abundance in strain 14028 were the products of the pdu operon, which encodes enzymes required for propanediol utilization. These pdu operon proteins were validated in culture and during macrophage infection. Our work provides further support for earlier observations that suggest pdu gene expression contributes to S. typhimurium pathogenesis.

Lignocellulosic biofuels are promising as sustainable alternative fuels, but lignin inhibits access of enzymes to cellulose, and by-products of lignin degradation can be toxic to cells. The fast growth, high efficiency and specificity of enzymes employed in the anaerobic litter deconstruction carried out by tropical soil bacteria make these organisms useful templates for improving biofuel production. The facultative anaerobe Enterobacter lignolyticus SCF1 was initially cultivated from Cloud Forest soils in the Luquillo Experimental Forest in Puerto Rico, based on anaerobic growth on lignin as sole carbon source. The source of the isolate was tropical forest soils that decompose litter rapidly with low and fluctuating redox potentials, where bacteria using oxygen-independent enzymes likely play an important role in decomposition. We have used transcriptomics and proteomics to examine the observed increased growth of SCF1 grown on media amended with lignin compared to unamended growth. Proteomics suggested accelerated xylose uptake and metabolism under lignin-amended growth, with up-regulation of proteins involved in lignin degradation via the 4-hydroxyphenylacetate degradation pathway, catalase/peroxidase enzymes, and the glutathione biosynthesis and glutathione S-transferase (GST) proteins. We also observed increased production of NADH-quinone oxidoreductase, other electron transport chain proteins, and ATP synthase and ATP-binding cassette (ABC) transporters. This suggested the use of lignin as terminal electron acceptor. We detected significant lignin degradation over time by absorbance, and also used metabolomics to demonstrate moderately significant decreased xylose concentrations as well as increased metabolic products acetate and formate in stationary phase in lignin-amended compared to unamended growth conditions. Our data show the advantages of a multi-omics approach toward providing insights as to how lignin may be used in nature by microorganisms coping with poor carbon availability.