Pathway analysis for intracellular Porphyromonas gingivalis using a strain ATCC 33277 specific database

<p>Abstract</p> <p>Background</p> <p><it>Porphyromonas gingivalis </it>is a Gram-negative intracellular pathogen associated with periodontal disease. We have previously reported on whole-cell quantitative proteomic analyses to investigate the differential expression of virulence factors as the organism transitions from an extracellular to intracellular lifestyle. The original results with the invasive strain <it>P. gingivalis </it>ATCC 33277 were obtained using the genome sequence available at the time, strain W83 [GenBank: <ext-link ext-link-id="AE015924" ext-link-type="gen">AE015924</ext-link>]. We present here a re-processed dataset using the recently published genome annotation specific for strain ATCC 33277 [GenBank: <ext-link ext-link-id="AP009380" ext-link-type="gen">AP009380</ext-link>] and an analysis of differential abundance based on metabolic pathways rather than individual proteins.</p> <p>Results</p> <p>Qualitative detection was observed for 1266 proteins using the strain ATCC 33277 annotation for 18 hour internalized <it>P. gingivalis </it>within human gingival epithelial cells and controls exposed to gingival cell culture medium, an improvement of 7% over the W83 annotation. Internalized cells showed increased abundance of proteins in the energy pathway from asparagine/aspartate amino acids to ATP. The pathway producing one short chain fatty acid, propionate, showed increased abundance, while that of another, butyrate, trended towards decreased abundance. The translational machinery, including ribosomal proteins and tRNA synthetases, showed a significant increase in protein relative abundance, as did proteins responsible for transcription.</p> <p>Conclusion</p> <p>Use of the ATCC 33277 specific genome annotation resulted in improved proteome coverage with respect to the number of proteins observed both qualitatively in terms of protein identifications and quantitatively in terms of the number of calculated abundance ratios. Pathway analysis showed a significant increase in overall protein synthetic and transcriptional machinery in the absence of significant growth. These results suggest that the interior of host cells provides a more energy rich environment compared to the extracellular milieu. Shifts in the production of cytotoxic fatty acids by intracellular <it>P. gingivalis </it>may play a role in virulence. Moreover, despite extensive genomic re-arrangements between strains W83 and 33277, there is sufficient sequence similarity at the peptide level for proteomic abundance trends to be largely accurate when using the heterologous strain annotated genome as the reference for database searching.</p

Quantitative proteomics of nutrient limitation in the hydrogenotrophic methanogen Methanococcus maripaludis

<p>Abstract</p> <p>Background</p> <p>Methanogenic Archaea play key metabolic roles in anaerobic ecosystems, where they use H₂and other substrates to produce methane. <it>Methanococcus maripaludis </it>is a model for studies of the global response to nutrient limitations.</p> <p>Results</p> <p>We used high-coverage quantitative proteomics to determine the response of <it>M. maripaludis </it>to growth-limiting levels of H₂, nitrogen, and phosphate. Six to ten percent of the proteome changed significantly with each nutrient limitation. H₂limitation increased the abundance of a wide variety of proteins involved in methanogenesis. However, one protein involved in methanogenesis decreased: a low-affinity [Fe] hydrogenase, which may dominate over a higher-affinity mechanism when H₂is abundant. Nitrogen limitation increased known nitrogen assimilation proteins. In addition, the increased abundance of molybdate transport proteins suggested they function for nitrogen fixation. An apparent regulon governed by the euryarchaeal nitrogen regulator NrpR is discussed. Phosphate limitation increased the abundance of three different sets of proteins, suggesting that all three function in phosphate transport.</p> <p>Conclusion</p> <p>The global proteomic response of <it>M. maripaludis </it>to each nutrient limitation suggests a wider response than previously appreciated. The results give new insight into the function of several proteins, as well as providing information that should contribute to the formulation of a regulatory network model.</p

Functional responses of methanogenic archaea to syntrophic growth.

Methanococcus maripaludis grown syntrophically with Desulfovibrio vulgaris was compared with M. maripaludis monocultures grown under hydrogen limitation using transcriptional, proteomic and metabolite analyses. These measurements indicate a decrease in transcript abundance for energy-consuming biosynthetic functions in syntrophically grown M. maripaludis, with an increase in transcript abundance for genes involved in the energy-generating central pathway for methanogenesis. Compared with growth in monoculture under hydrogen limitation, the response of paralogous genes, such as those coding for hydrogenases, often diverged, with transcripts of one variant increasing in relative abundance, whereas the other was little changed or significantly decreased in abundance. A common theme was an apparent increase in transcripts for functions using H(2) directly as reductant, versus those using the reduced deazaflavin (coenzyme F(420)). The greater importance of direct reduction by H(2) was supported by improved syntrophic growth of a deletion mutant in an F(420)-dependent dehydrogenase of M. maripaludis. These data suggest that paralogous genes enable the methanogen to adapt to changing substrate availability, sustaining it under environmental conditions that are often near the thermodynamic threshold for growth. Additionally, the discovery of interspecies alanine transfer adds another metabolic dimension to this environmentally relevant mutualism

Roles of Coenzyme F420-Reducing Hydrogenases and Hydrogen- and F420-Dependent Methylenetetrahydromethanopterin Dehydrogenases in Reduction of F420 and Production of Hydrogen during Methanogenesis▿

Reduced coenzyme F420 (F420H2) is an essential intermediate in methanogenesis from CO2. During methanogenesis from H2 and CO2, F420H2 is provided by the action of F420-reducing hydrogenases. However, an alternative pathway has been proposed, where H2-dependent methylenetetrahydromethanopterin dehydrogenase (Hmd) and F420H2-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd) together reduce F420 with H2. Here we report the construction of mutants of Methanococcus maripaludis that are defective in each putative pathway. Their analysis demonstrates that either pathway supports growth on H2 and CO2. Furthermore, we show that during growth on formate instead of H2, where F420H2 is a direct product of formate oxidation, H2 production occurs. H2 presumably arises from the oxidation of F420H2, and the analysis of the mutants during growth on formate suggests that this too can occur by either pathway. We designate the alternative pathway for the interconversion of H2 and F420H2 the Hmd-Mtd cycle

The Alternative Sigma Factor RpoN Is Required for hrp Activity in Pseudomonas syringae pv. Maculicola and Acts at the Level of hrpL Transcription

�-Glucuronidase (uidA) reporter gene fusions were constructed for the hrpZ, hrpL, and hrpS genes from the phytopathogen Pseudomonas syringae pv. maculicola strain ES4326. These reporters, as well as an avrRpt2-uidA fusion, were used to measure transcriptional activity in ES4326 and a ES4326 rpoN mutant. rpoN was required for the expression of avrRpt2, hrpZ, and hrpL in vitro in minimal media and in vivo when infiltrated into Arabidopsis thaliana leaves. In contrast, the expression of hrpS was essentially the same in wild-type and rpoN mutant strains. Constitutive expression of hrpL in an rpoN mutant restored hrpZ transcription to wild-type levels, restored the hypersensitive response when infiltrated into tobacco (Nicotiana tobacum), and partially restored the elicitation of virulence-related symptoms but not growth when infiltrated into Arabidopsis leaves. These data indicate that rpoN-mediated control of hrp gene expression acts at the level of hrpL and that in planta growth of P. syringae is not required for the elicitation of disease symptoms

Public interest in ∆8-Tetrahydrocannabinol (delta-8-THC) increased in US states that restricted ∆9-Tetrahydrocannabinol (delta-9-THC) use

BackgroundThere is an expanding unregulated market for a psychotropic compound called ∆8-Tetrahydrocannabinol (delta-8-THC) that is being derived from hemp, but there are no empirical estimates of public interest in this compound.MethodsTo measure public interest, we obtained yearly Google query fractions (QFs) that mentioned delta-8-THC (i.e., "delta 8," "delta-8," or "Δ8") for the past decade (from January 2011 through August 2021) for every country and territory in the world and every state in the United States (US) from Google Trends. We also obtained the same trends for the last complete month of data for all US states (July 2021) to compare across cannabis use policies. We summarized QFs across years, countries, US states and cannabis policies in US states using linear regression, means and ratios. We estimated raw search counts for the US using comscore.com.ResultsThe global rate of delta-8-THC searches was stable between 2011 and 2019 before increasing by 257.0% from 2019 to 2020 and 705.0% from 2020 to 2021. In 2021, the rate of delta-8-THC searches in the US was at least 10 times higher than the rates in other countries or territories. In absolute terms, there were 22.3 million delta-8-THC searches in the US in the first 8 months of 2021 alone. Increases in delta-8-THC searches from 2020 to 2021 occurred in all 50 US states and the District of Columbia (Mean 854.2%; range&nbsp;=&nbsp;256.4% - 2831.2%) but continued to vary substantially between states in 2021. In July 2021, the legal status of delta-9-THC use across US states explained 49.0% of the variance in delta-8-THC QFs between US states (R2&nbsp;=&nbsp;0.490; p &lt; 0.001) and was inversely associated, where delta-8-THC QFs were higher in jurisdictions with stricter cannabis use policies.ConclusionPublic interest in delta-8-THC increased rapidly in 2020 and 2021 and was particularly high in US states that restricted delta-9-THC use. Jurisdictions should clarify whether delta-8-THC can be sold as a hemp product