Vascularization of the Selaginella rhizophore: anatomical fingerprints of polar auxin transport with implications for the deep fossil record

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138403/1/nph14478_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138403/2/nph14478-sup-0005-Legends.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138403/3/nph14478.pd

Genomic and microarray analysis of aromatics degradation in Geobacter metallireducens and comparison to a Geobacter isolate from a contaminated field site

<p>Abstract</p> <p>Background</p> <p>Groundwater and subsurface environments contaminated with aromatic compounds can be remediated <it>in situ </it>by <it>Geobacter </it>species that couple oxidation of these compounds to reduction of Fe(III)-oxides. <it>Geobacter metallireducens </it>metabolizes many aromatic compounds, but the enzymes involved are not well known.</p> <p>Results</p> <p>The complete <it>G. metallireducens </it>genome contained a 300 kb island predicted to encode enzymes for the degradation of phenol, <it>p</it>-cresol, 4-hydroxybenzaldehyde, 4-hydroxybenzoate, benzyl alcohol, benzaldehyde, and benzoate. Toluene degradation genes were encoded in a separate region. None of these genes was found in closely related species that cannot degrade aromatic compounds. Abundant transposons and phage-like genes in the island suggest mobility, but nucleotide composition and lack of synteny with other species do not suggest a recent transfer. The inferred degradation pathways are similar to those in species that anaerobically oxidize aromatic compounds with nitrate as an electron acceptor. In these pathways the aromatic compounds are converted to benzoyl-CoA and then to 3-hydroxypimelyl-CoA. However, in <it>G. metallireducens </it>there were no genes for the energetically-expensive dearomatizing enzyme. Whole-genome changes in transcript levels were identified in cells oxidizing benzoate. These supported the predicted pathway, identified induced fatty-acid oxidation genes, and identified an apparent shift in the TCA cycle to a putative ATP-yielding succinyl-CoA synthase. Paralogs to several genes in the pathway were also induced, as were several putative molybdo-proteins. Comparison of the aromatics degradation pathway genes to the genome of an isolate from a contaminated field site showed very similar content, and suggested this strain degrades many of the same compounds. This strain also lacked a classical dearomatizing enzyme, but contained two copies of an eight-gene cluster encoding redox proteins that was 30-fold induced during benzoate oxidation.</p> <p>Conclusion</p> <p><it>G. metallireducens </it>appears to convert aromatic compounds to benzoyl-CoA, then to acetyl-CoA via fatty acid oxidation, and then to carbon dioxide via the TCA cycle. The enzyme responsible for dearomatizing the aromatic ring may be novel, and energetic investments at this step may be offset by a change in succinate metabolism. Analysis of a field isolate suggests that the pathways inferred for <it>G. metallireducens </it>may be applicable to modeling <it>in situ </it>bioremediation.</p

Geobacter Strains Expressing Poorly Conductive Pili Reveal Constraints on Direct Interspecies Electron Transfer Mechanisms

Cytochrome-to-cytochrome electron transfer and electron transfer along conduits of multiple extracellular magnetite grains are often proposed as strategies for direct interspecies electron transfer (DIET) that do not require electrically conductive pili (e-pili). However, physical evidence for these proposed DIET mechanisms has been lacking. To investigate these possibilities further, we constructed Geobacter metallireducens strain Aro-5, in which the wild-type pilin gene was replaced with the aro-5 pilin gene that was previously shown to yield poorly conductive pili in Geobacter sulfurreducens strain Aro-5. G. metallireducens strain Aro-5 did not reduce Fe(III) oxide and produced only low current densities, phenotypes consistent with expression of poorly conductive pili. Like G. sulfurreducens strain Aro-5, G. metallireducensstrain Aro-5 displayed abundant outer surface cytochromes. Cocultures initiated with wild-type G. metallireducens as the electron-donating strain and G. sulfurreducens strain Aro-5 as the electron-accepting strain grew via DIET. However, G. metallireducens Aro-5/G. sulfurreducenswild-type cocultures did not. Cocultures initiated with the Aro-5 strains of both species grew only when amended with granular activated carbon (GAC), a conductive material known to be a conduit for DIET. Magnetite could not substitute for GAC. The inability of the two Aro-5 strains to adapt for DIET in the absence of GAC suggests that there are physical constraints on establishing DIET solely through cytochrome-to-cytochrome electron transfer or along chains of magnetite. The finding that DIET is possible with electron-accepting partners that lack highly conductive pili greatly expands the range of potential electron-accepting partners that might participate in DIET. IMPORTANCE DIET is thought to be an important mechanism for interspecies electron exchange in natural anaerobic soils and sediments in which methane is either produced or consumed, as well as in some photosynthetic mats and anaerobic digesters converting organic wastes to methane. Understanding the potential mechanisms for DIET will not only aid in modeling carbon and electron flow in these geochemically significant environments but will also be helpful for interpreting meta-omic data from as-yet-uncultured microbes in DIET-based communities and for designing strategies to promote DIET in anaerobic digesters. The results demonstrate the need to develop a better understanding of the diversity of types of e-pili in the microbial world to identify potential electron-donating partners for DIET. Novel methods for recovering as-yet-uncultivated microorganisms capable of DIET in culture will be needed to further evaluate whether DIET is possible without e-pili in the absence of conductive materials such as GAC

Expressing the Geobacter metallireducens PilA in Geobacter sulfurreducens Yields Pili with Exceptional Conductivity

ABSTRACT The electrically conductive pili (e-pili) of Geobacter sulfurreducens serve as a model for a novel strategy for long-range extracellular electron transfer. e-pili are also a new class of bioelectronic materials. However, the only other Geobacter pili previously studied, which were from G. uraniireducens , were poorly conductive. In order to obtain more information on the range of pili conductivities in Geobacter species, the pili of G. metallireducens were investigated. Heterologously expressing the PilA gene of G. metallireducens in G. sulfurreducens yielded a G. sulfurreducens strain, designated strain MP, that produced abundant pili. Strain MP exhibited phenotypes consistent with the presence of e-pili, such as high rates of Fe(III) oxide reduction and high current densities on graphite anodes. Individual pili prepared at physiologically relevant pH 7 had conductivities of 277 ± 18.9 S/cm (mean ± standard deviation), which is 5,000-fold higher than the conductivity of G. sulfurreducens pili at pH 7 and nearly 1 million-fold higher than the conductivity of G. uraniireducens pili at the same pH. A potential explanation for the higher conductivity of the G. metallireducens pili is their greater density of aromatic amino acids, which are known to be important components in electron transport along the length of the pilus. The G. metallireducens pili represent the most highly conductive pili found to date and suggest strategies for designing synthetic pili with even higher conductivities. IMPORTANCE e-pili are a remarkable electrically conductive material that can be sustainably produced without harsh chemical processes from renewable feedstocks and that contain no toxic components in the final product. Thus, e-pili offer an unprecedented potential for developing novel materials, electronic devices, and sensors for diverse applications with a new “green” technology. Increasing e-pili conductivity will even further expand their potential applications. A proven strategy is to design synthetic e-pili that contain tryptophan, an aromatic amino acid not found in previously studied e-pili. The studies reported here demonstrate that a productive alternative approach is to search more broadly in the microbial world. Surprisingly, even though G. metallireducens and G. sulfurreducens are closely related, the conductivities of their e-pili differ by more than 3 orders of magnitude. The ability to produce e-pili with high conductivity without generating a genetically modified product enhances the attractiveness of this novel electronic material

A hypothetico-deductive approach to assessing the social function of chemical signalling in a non-territorial solitary carnivore

The function of chemical signalling in non-territorial solitary carnivores is still relatively unclear. Studies on territorial solitary and social carnivores have highlighted odour capability and utility, however the social function of chemical signalling in wild carnivore populations operating dominance hierarchy social systems has received little attention. We monitored scent marking and investigatory behaviour of wild brown bears Ursus arctos, to test multiple hypotheses relating to the social function of chemical signalling. Camera traps were stationed facing bear ‘marking trees’ to document behaviour by different age sex classes in different seasons. We found evidence to support the hypothesis that adult males utilise chemical signalling to communicate dominance to other males throughout the non-denning period. Adult females did not appear to utilise marking trees to advertise oestrous state during the breeding season. The function of marking by subadult bears is somewhat unclear, but may be related to the behaviour of adult males. Subadults investigated trees more often than they scent marked during the breeding season, which could be a result of an increased risk from adult males. Females with young showed an increase in marking and investigation of trees outside of the breeding season. We propose the hypothesis that females engage their dependent young with marking trees from a young age, at a relatively ‘safe’ time of year. Memory, experience, and learning at a young age, may all contribute towards odour capabilities in adult bears

Phylogenetic Signal Variation in the Genomes of Medicago (Fabaceae)

Genome-scale data offer the opportunity to clarify phylogenetic relationships that are difficult to resolve with few loci, but they can also identify genomic regions with evolutionary history distinct from that of the species history. We collected whole-genome sequence data from 29 taxa in the legume genus Medicago, then aligned these sequences to the Medicago truncatula reference genome to confidently identify 87 596 variable homologous sites. We used this data set to estimate phylogenetic relationships among Medicago species, to investigate the number of sites needed to provide robust phylogenetic estimates and to identify specific genomic regions supporting topologies in conflict with the genome-wide phylogeny. Our full genomic data set resolves relationships within the genus that were previously intractable. Subsampling the data reveals considerable variation in phylogenetic signal and power in smaller subsets of the data. Even when sampling 5000 sites, no random sample of the data supports a topology identical to that of the genome-wide phylogeny. Phylogenetic relationships estimated from 500-site sliding windows revealed genome regions supporting several alternative species relationships among recently diverged taxa, consistent with the expected effects of deep coalescence or introgression in the recent history of Medicago. [Medicago; phylogenomics; whole-genome resequencing.

Exploring structural variation and gene family architecture with De Novo assemblies of 15 Medicago genomes

Abstract Background Previous studies exploring sequence variation in the model legume, Medicago truncatula, relied on mapping short reads to a single reference. However, read-mapping approaches are inadequate to examine large, diverse gene families or to probe variation in repeat-rich or highly divergent genome regions. De novo sequencing and assembly of M. truncatula genomes enables near-comprehensive discovery of structural variants (SVs), analysis of rapidly evolving gene families, and ultimately, construction of a pan-genome. Results Genome-wide synteny based on 15 de novo M. truncatula assemblies effectively detected different types of SVs indicating that as much as 22% of the genome is involved in large structural changes, altogether affecting 28% of gene models. A total of 63 million base pairs (Mbp) of novel sequence was discovered, expanding the reference genome space for Medicago by 16%. Pan-genome analysis revealed that 42% (180 Mbp) of genomic sequences is missing in one or more accession, while examination of de novo annotated genes identified 67% (50,700) of all ortholog groups as dispensable – estimates comparable to recent studies in rice, maize and soybean. Rapidly evolving gene families typically associated with biotic interactions and stress response were found to be enriched in the accession-specific gene pool. The nucleotide-binding site leucine-rich repeat (NBS-LRR) family, in particular, harbors the highest level of nucleotide diversity, large effect single nucleotide change, protein diversity, and presence/absence variation. However, the leucine-rich repeat (LRR) and heat shock gene families are disproportionately affected by large effect single nucleotide changes and even higher levels of copy number variation. Conclusions Analysis of multiple M. truncatula genomes illustrates the value of de novo assemblies to discover and describe structural variation, something that is often under-estimated when using read-mapping approaches. Comparisons among the de novo assemblies also indicate that different large gene families differ in the architecture of their structural variation

Anode Biofilm Transcriptomics Reveals Outer Surface Components Essential for High Density Current Production in Geobacter sulfurreducens Fuel Cells

The mechanisms by which Geobacter sulfurreducens transfers electrons through relatively thick (>50 µm) biofilms to electrodes acting as a sole electron acceptor were investigated. Biofilms of Geobacter sulfurreducens were grown either in flow-through systems with graphite anodes as the electron acceptor or on the same graphite surface, but with fumarate as the sole electron acceptor. Fumarate-grown biofilms were not immediately capable of significant current production, suggesting substantial physiological differences from current-producing biofilms. Microarray analysis revealed 13 genes in current-harvesting biofilms that had significantly higher transcript levels. The greatest increases were for pilA, the gene immediately downstream of pilA, and the genes for two outer c-type membrane cytochromes, OmcB and OmcZ. Down-regulated genes included the genes for the outer-membrane c-type cytochromes, OmcS and OmcT. Results of quantitative RT-PCR of gene transcript levels during biofilm growth were consistent with microarray results. OmcZ and the outer-surface c-type cytochrome, OmcE, were more abundant and OmcS was less abundant in current-harvesting cells. Strains in which pilA, the gene immediately downstream from pilA, omcB, omcS, omcE, or omcZ was deleted demonstrated that only deletion of pilA or omcZ severely inhibited current production and biofilm formation in current-harvesting mode. In contrast, these gene deletions had no impact on biofilm formation on graphite surfaces when fumarate served as the electron acceptor. These results suggest that biofilms grown harvesting current are specifically poised for electron transfer to electrodes and that, in addition to pili, OmcZ is a key component in electron transfer through differentiated G. sulfurreducens biofilms to electrodes

Construction of a Geobacter Strain With Exceptional Growth on Cathodes

Insoluble extracellular electron donors are important sources of energy for anaerobic respiration in biogeochemical cycling and in diverse practical applications. The previous lack of a genetically tractable model microorganism that could be grown to high densities under anaerobic conditions in pure culture with an insoluble extracellular electron donor has stymied efforts to better understand this form of respiration. We report here on the design of a strain of Geobacter sulfurreducens, designated strain ACL, which grows as thick (ca. 35 μm) confluent biofilms on graphite cathodes poised at -500 mV (versus Ag/AgCl) with fumarate as the electron acceptor. Sustained maximum current consumption rates were &gt;0.8 A/m2, which is &gt;10-fold higher than the current consumption of the wild-type strain. The improved function on the cathode was achieved by introducing genes for an ATP-dependent citrate lyase, completing the complement of enzymes needed for a reverse TCA cycle for the synthesis of biosynthetic precursors from carbon dioxide. Strain ACL provides an important model organism for elucidating the mechanisms for effective anaerobic growth with an insoluble extracellular electron donor and may offer unique possibilities as a chassis for the introduction of synthetic metabolic pathways for the production of commodities with electrons derived from electrodes