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Background: Clostridium autoethanogenum is an acetogenic bacterium that autotrophically converts carbon monoxide (CO) and carbon dioxide (CO2) gases into bioproducts and fuels via the Wood–Ljungdahl pathway (WLP). To facilitate overall carbon capture efficiency, the reaction stoichiometry requires supplementation of hydrogen at an increased ratio of H2:CO to maximize CO2 utilization; however, the molecular details and thus the ability to understand the mechanism of this supplementation are largely unknown. Results: In order to elucidate the microbial physiology and fermentation where at least 75% of the carbon in ethanol comes from CO2, we established controlled chemostats that facilitated a novel and high (11:1) H2:CO uptake ratio. We compared and contrasted proteomic and metabolomics profiles to replicate continuous stirred tank reactors (CSTRs) at the same growth rate from a lower (5:1) H2:CO condition where ~ 50% of the carbon in ethanol is derived from CO2. Our hypothesis was that major changes would be observed in the hydrogenases and/or redox-related proteins and the WLP to compensate for the elevated hydrogen feed gas. Our analyses did reveal protein abundance differences between the two conditions largely related to reduction–oxidation (redox) pathways and cofactor biosynthesis, but the changes were more minor than we would have expected. While the Wood–Ljungdahl pathway proteins remained consistent across the conditions, other post-translational regulatory processes, such as lysine-acetylation, were observed and appeared to be more important for fine-tuning this carbon metabolism pathway. Metabolomic analyses showed that the increase in H2:CO ratio drives the organism to higher carbon dioxide utilization resulting in lower carbon storages and accumulated fatty acid metabolite levels. Conclusions: This research delves into the intricate dynamics of carbon fixation in C. autoethanogenum, examining the influence of highly elevated H2:CO ratios on metabolic processes and product outcomes. The study underscores the significance of optimizing gas feed composition for enhanced industrial efficiency, shedding light on potential mechanisms, such as post-translational modifications (PTMs), to fine-tune enzymatic activities and improve desired product yields. [ABSTRACT FROM AUTHOR]

Metaproteomics is an increasingly popular methodology that provides information regarding the metabolic functions of specific microbial taxa and has potential for contributing to ocean ecology and biogeochemical studies. A blinded multi-laboratory intercomparison was conducted to assess comparability and reproducibility of taxonomic and functional results and their sensitivity to methodological variables. Euphotic zone samples from the Bermuda Atlantic Time-Series Study in the North Atlantic Ocean collected by in situ pumps and the AUV Clio were distributed with a paired metagenome, and one-dimensional liquid chromatographic data dependent acquisition mass spectrometry analyses was stipulated. Analysis of mass spectra from seven laboratories through a common informatic pipeline identified a shared set of 1056 proteins from 1395 shared peptides constituents. Quantitative analyses showed good reproducibility: pairwise regressions of spectral counts between laboratories yielded R2 values ranging from 0.43 to 0.83, and a Sørensen similarity analysis of the top 1,000 proteins revealed 70–80 % similarity between laboratory groups. Taxonomic and functional assignments showed good coherence between technical replicates and different laboratories. An informatic intercomparison study, involving 10 laboratories using 8 software packages successfully identified thousands of peptides within the complex metaproteomic datasets, demonstrating the utility of these software tools for ocean metaproteomic research. Future efforts could examine reproducibility in deeper metaproteomes, examine accuracy in targeted absolute quantitation analyses, and develop standards for data output formats to improve data interoperability. Together, these results demonstrate the reproducibility of metaproteomic analyses and their suitability for microbial oceanography research including integration into global scale ocean surveys and ocean biogeochemical models. [ABSTRACT FROM AUTHOR]

Metaproteomics is an increasingly popular methodology that provides information regarding the metabolic functions of specific microbial taxa and has potential for contributing to ocean ecology and biogeochemical studies. A blinded multi-laboratory intercomparison was conducted to assess comparability and reproducibility of taxonomic and functional results and their sensitivity to methodological variables. Euphotic zone samples from the Bermuda Atlantic Time-Series Study in the North Atlantic Ocean collected by in situ pumps and the AUV Clio were distributed with a paired metagenome, and one-dimensional liquid chromatographic data dependent acquisition mass spectrometry analyses was stipulated. Analysis of mass spectra from seven laboratories through a common informatic pipeline identified a shared set of 1056 proteins from 1395 shared peptides constituents. Quantitative analyses showed good reproducibility: pairwise regressions of spectral counts between laboratories yielded R² values ranging from 0.43 to 0.83, and a Sørensen similarity analysis of the top 1,000 proteins revealed 70-80% similarity between laboratory groups. Taxonomic and functional assignments showed good coherence between technical replicates and different laboratories. An informatic intercomparison study, involving 10 laboratories using 8 software packages successfully identified thousands of peptides within the complex metaproteomic datasets, demonstrating the utility of these software tools for ocean metaproteomic research. Future efforts could examine reproducibility in deeper metaproteomes, examine accuracy in targeted absolute quantitation analyses, and develop standards for data output formats to improve data interoperability. Together, these results demonstrate the reproducibility of metaproteomic analyses and their suitability for microbial oceanography research including integration into global scale ocean surveys and ocean biogeochemical models. [ABSTRACT FROM AUTHOR]

Abstract Background High-resolution tandem mass spectra can now be readily acquired with hybrid instruments, such as LTQ-Orbitrap and LTQ-FT, in high-throughput shotgun proteomics workflows. The improved spectral quality enables more accurate de novo sequencing for identification of post-translational modifications and amino acid polymorphisms. Results In this study, a new de novo sequencing algorithm, called Vonode, has been developed specifically for analysis of such high-resolution tandem mass spectra. To fully exploit the high mass accuracy of these spectra, a unique scoring system is proposed to evaluate sequence tags based primarily on mass accuracy information of fragment ions. Consensus sequence tags were inferred for 11,422 spectra with an average peptide length of 5.5 residues from a total of 40,297 input spectra acquired in a 24-hour proteomics measurement of Rhodopseudomonas palustris. The accuracy of inferred consensus sequence tags was 84%. According to our comparison, the performance of Vonode was shown to be superior to the PepNovo v2.0 algorithm, in terms of the number of de novo sequenced spectra and the sequencing accuracy. Conclusions Here, we improved de novo sequencing performance by developing a new algorithm specifically for high-resolution tandem mass spectral data. The Vonode algorithm is freely available for download at http://compbio.ornl.gov/Vonode.

The Salicaceae family is of growing interest in the study of dioecy in plants because the sex determination region (SDR) has been shown to be highly dynamic, with differing locations and heterogametic systems between species. Without the ability to transform and regenerate Salix in tissue culture, previous studies investigating the mechanisms regulating sex in the genus Salix have been limited to genome resequencing and differential gene expression, which are mostly descriptive in nature, and functional validation of candidate sex determination genes has not yet been conducted. Here, we used Arabidopsis to functionally characterize a suite of previously identified candidate genes involved in sex determination and sex dimorphism in the bioenergy shrub willow Salix purpurea. Six candidate master regulator genes for sex determination were heterologously expressed in Arabidopsis, followed by floral proteome analysis. In addition, 11 transcription factors with predicted roles in mediating sex dimorphism downstream of the SDR were tested using DAP‐Seq in both male and female S. purpurea DNA. The results of this study provide further evidence to support models for the roles of ARR17 and GATA15 as master regulator genes of sex determination in S. purpurea, contributing to a regulatory system that is notably different from that of its sister genus Populus. Evidence was also obtained for the roles of two transcription factors, an AP2/ERF family gene and a homeodomain‐like transcription factor, in downstream regulation of sex dimorphism. [ABSTRACT FROM AUTHOR]

Mutualistic association can improve a plant's health and productivity. G-type lectin receptor-like kinase (PtLecRLK1) is a susceptibility factor in Populus trichocarpa that permits root colonization by a beneficial fungus, Laccaria bicolor. Engineering PtLecRLK1 also permits L. bicolor root colonization in non-host plants similar to Populus trichocarpa. The intracellular signaling reprogramed by PtLecRLK1 upon recognition of L. bicolor to allow for the development and maintenance of symbiosis is yet to be determined. In this study, phosphoproteomics was utilized to identify phosphorylation-based relevant signaling pathways associated with PtLecRLK1 recognition of L. bicolor in transgenic switchgrass roots. Our finding shows that PtLecRLK1 in transgenic plants modifies the chitin-triggered plant defense and MAPK signaling along with a significant adjustment in phytohormone signaling, ROS balance, endocytosis, cytoskeleton movement, and proteasomal degradation in order to facilitate the establishment and maintenance of L. bicolor colonization. Moreover, protein–protein interaction data implicate a cGMP-dependent protein kinase as a potential substrate of PtLecRLK1. [ABSTRACT FROM AUTHOR]