SummaryPhylogenetic analysis of independent gains of C4 photosynthesis in Suaedoideae shows differences compared with other C4 lineages in amino acids of phosphoenolpyruvate carboxylase under positive selection and their position relative to functional residues.

The use of plant materials to generate renewable biofuels and other high-value chemicals is the sustainable and preferable option, but will require considerable improvements to increase the rate and efficiency of lignocellulose depolymerization. This review highlights novel and emerging technologies that are being developed and deployed to characterize the process of lignocellulose degradation. The review will also illustrate how microbial communities deconstruct and metabolize lignocellulose by identifying the necessary genes and enzyme activities along with the reaction products. These technologies include multi-omic measurements, cell sorting and isolation, nuclear magnetic resonance spectroscopy (NMR), activity-based protein profiling, and direct measurement of enzyme activity. The recalcitrant nature of lignocellulose necessitates the need to characterize the methods microbes employ to deconstruct lignocellulose to inform new strategies on how to greatly improve biofuel conversion processes. New technologies are yielding important insights into microbial functions and strategies employed to degrade lignocellulose, providing a mechanistic blueprint in order to advance biofuel production.

BackgroundIn the model single-cell C4 plant Bienertia sinuspersici, chloroplast- and nuclear-encoded photosynthetic enzymes, characteristically confined to either bundle sheath or mesophyll cells in Kranz-type C4 leaves, all occur together within individual leaf chlorenchyma cells. Intracellular separation of dimorphic chloroplasts and key enzymes within central and peripheral compartments allow for C4 carbon fixation analogous to NAD-malic enzyme (NAD-ME) Kranz type species. Several methods were used to investigate dimorphic chloroplast differentiation in B. sinuspersici.ResultsConfocal analysis revealed that Rubisco-containing chloroplasts in the central compartment chloroplasts (CCC) contained more photosystem II proteins than the peripheral compartment chloroplasts (PCC) which contain pyruvate,Pi dikinase (PPDK), a pattern analogous to the cell type-specific chloroplasts of many Kranz type NAD-ME species. Transient expression analysis using GFP fusion constructs containing various lengths of a B. sinuspersici Rubisco small subunit (RbcS) gene and the transit peptide of PPDK revealed that their import was not specific to either chloroplast type. Immunolocalization showed the rbcL-specific mRNA binding protein RLSB to be selectively localized to the CCC in B. sinuspersici, and to Rubisco-containing BS chloroplasts in the closely related Kranz species Suaeda taxifolia. Comparative fluorescence analyses were made using redox-sensitive and insensitive GFP forms, as well comparative staining using the peroxidase indicator 3,3-diaminobenzidine (DAB), which demonstrated differences in stromal redox potential, with the CCC having a more negative potential than the PCC.ConclusionsBoth CCC RLSB localization and the differential chloroplast redox state are suggested to have a role in post-transcriptional rbcL expression.

HighlightEnzyme kinetic measurements and positive selection analysis show that C4 species in Suaedoideae have PEPC and Rubisco kinetics similar to other C4 species despite different amino acid convergence.

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Understanding the factors that regulate microbe function and microbial community assembly, function, and fitness is a grand challenge. A critical factor and an important enzyme cofactor and regulator of gene expression is cobalamin (vitamin B). Our knowledge of the roles of vitamin B is limited because technologies that enable characterization of microbial metabolism and gene regulation with minimal impact on cell physiology are needed. To meet this need we show that a synthetic probe mimic of B supports growth of B auxotrophic bacteria and archaea. We demonstrate that a B activity-based probe (B-ABP) is actively transported into cells, and converted to adenosyl-B-ABP akin to native B Identification of the proteins that bind the B-ABP in, a sp. and, demonstrate the specificity for known and novel B protein targets. The B-ABP also regulates the B dependent RNA riboswitch and the transcription factor EutR. Our results demonstrate a new approach to gain knowledge about the role of B in microbe functions. Our approach provides a powerful, non-disruptive tool to analyze B interactions in living cells, and can be used to discover the role of B in diverse microbial systems. We demonstrate that a cobalamin chemical probe can be used to investigate roles of vitamin B in microbial growth and regulation, by supporting growth of B auxotrophic bacteria and archaea, enabling biological activity with three different cell macromolecules (RNA, DNA, and proteins), and facilitating functional proteomics to characterize B-protein interactions. The B-ABP is both transcriptionally and translationally able to regulate gene expression analogous to natural vitamin B The application of the B-ABP at biologically relevant concentrations facilitates a unique way to measure B microbial dynamics and identify new B protein targets in bacteria and archaea. We demonstrate that the B-ABP can be used to identify protein interactions across diverse microbes, from to microbes isolated from naturally occurring phototrophic biofilms, to the salt-tolerant archaea .

The gut microbiome is a complex microbial community with important impacts on human health. One of the major groups within the gut microbiome, the Bacteroidetes , rely on their ability to capture vitamin B 12 and related molecules for fitness in the gut.

To study the developmental transition of chloroplasts from C(3) to C(4) photosynthesis in the terrestrial single-cell C(4) species Bienertia sinuspersici, a regeneration protocol was developed. Stem explant material developed callus either with or without red nodular structures (RNS) when cultured on Murashige-Skoog (MS) salts and vitamins, supplemented with 5 mM phosphate, plus 1 mg L(-1) dichloropenoxy-acetic acid (2,4-D), and 87 mM sucrose (Stage 1 media). Only calli having RNS were able to regenerate plantlets. MS media plus phosphate was used throughout regeneration, with the Stage 2 media containing 2 mg L(-1) 6-benzylaminopurine, 43 mM sucrose and 1.5% soluble starch. Stage 3 media had no hormones or organic sources of carbon, and cultures were grown under ambient (~400 ppm) versus CO(2) enrichment (1.2% CO(2)). When calli without RNS were cultured under Stage 3 conditions with 1.2% CO(2), there was an increase in growth, protein content, and photosystem II yield, while structural and biochemical analyses indicated the cells in the calli had C(3) type photosynthesis. CO(2) enrichment during growth of RNS during Stage 3 had a large effect on regeneration success, increasing efficiency of shoot and root development, size of plantlets, leaf soluble protein, and chlorophyll concentration. Anatomical analysis of plantlets, which developed under 1.2% CO(2), showed leaves developed C(4) type chlorenchyma cells, including expression of key C(4) biochemical enzymes. Increasing salinity in the media, from 0 to 200 mM NaCl, increased tissue osmolality, average plantlet area and regeneration success, but did not affect protein or chlorophyll content.