Live cell imaging methods provide important insights into the dynamics of cellular processes that cannot be derived easily from population-averaged datasets.In the bioenergy field,much research is focused on fermentat...Live cell imaging methods provide important insights into the dynamics of cellular processes that cannot be derived easily from population-averaged datasets.In the bioenergy field,much research is focused on fermentation of cellulosic biomass by thermophilic microbes to produce biofuels;however,little effort is dedicated to the development of imaging tools to monitor this dynamic biological process.This is,in part,due to the experimental challenges of imaging cells under both anaerobic and thermophilic conditions.Here an imaging system is described that integrates confocal microscopy,a flow cell device,and a lipophilic dye to visualize cells.Solutions to technical obstacles regarding suitable fluorescent markers,photodamage during imaging,and maintenance of environmental conditions during imaging are presented.This system was utilized to observe cellulose colonization by Clostridium thermocellum under anaerobic conditions at 60℃.This method enables live cell imaging of bacterial growth under anaerobic and thermophilic conditions and should be widely applicable to visualizing different cell types or processes in real time.展开更多
Galacturonosyltransferase 1 （GAUT1） is an α1,4-D-galacturonosyltransferase that transfers galacturonic acid from uridine 5＇-diphosphogalacturonic acid onto the pectic polysaccharide homogalacturonan （Sterling et ...Galacturonosyltransferase 1 （GAUT1） is an α1,4-D-galacturonosyltransferase that transfers galacturonic acid from uridine 5＇-diphosphogalacturonic acid onto the pectic polysaccharide homogalacturonan （Sterling et al., 2006）. The 25-member Arabidopsis thaliana GAUT1-related gene family encodes 15 GAUT and 10 GAUT-like （GATL） proteins with, respectively, 56-84 and 42-53% amino acid sequence similarity to GAUT1. Previous phylogenetic analyses of AtGAUTs indicated three clades： A through C. A comparative phylogenetic analysis of the Arabidopsis, poplar and rice GAUT families has sub-classified the GAUTs into seven clades： clade A-1 （GAUTs 1 to 3）; A-2 （GAUT4）; A-3 （GAUTs 5 and 6）; A-4 （GAUT7）; B-1 （GAUTs 8 and 9）; B-2 （GAUTs 10 and 11）; and clade C （GAUTs 12 to 15）. The Arabidopsis GAUTs have a distribution comparable to the poplar orthologs, with the exception of GAUT2, which is absent in poplar. Rice, however, has no orthologs of GAUTs 2 and 12 and has multiple apparent orthologs of GAUTs 1, 4, and 7 compared with eitherArabidopsis or poplar. The cell wall glycosyl residue compositions of 26 homozygous T-DNA insertion mutants for 13 of 15 Arabidopsis GAUTgenes reveal significantly and reproducibly different cell walls in specific tissues of gaut mutants 6, 8, 9, 10, 11, 12, 13, and 14 from that of wild-type Arabidopsis walls. Pectin and xylan polysaccharides are affected by the loss of GAUT function, as demonstrated by the altered galacturonic acid, xylose, rhamnose, galactose, and arabinose composition of distinct gaut mutant walls. The wall glycosyl residue compositional phenotypes observed among the gaut mutants suggest that at least six different biosynthetic linkages in pectins and/or xylans are affected by the lesions in these GAUTgenes. Evidence is also presented to support a role for GAUT11 in seed mucilage expansion and in seed wall and mucilage composition.展开更多
Several genes in Arabidopsis, including PARVUS/AtGATL1, have been implicated in xylan synthesis. However, the biosynthesis of xylan in woody plants, where this polysaccharide is a major component of wood, is poorly un...Several genes in Arabidopsis, including PARVUS/AtGATL1, have been implicated in xylan synthesis. However, the biosynthesis of xylan in woody plants, where this polysaccharide is a major component of wood, is poorly understood. Here, we characterize two Populus genes, PdGATL 1.1 and PdGATL 1.2, the closest orthologs to the Arabidopsis PARVUS/GATL1 gene, with respect to their gene expression in poplar, their sub-cellular localization, and their ability to complement the parvus mutation in Arabidopsis. Overexpression of the two poplar genes in the parvus mutant rescued most of the defects caused by the parvus mutation, including morphological changes, collapsed xylem, and altered cell wall monosaccharide composition. Quantitative RT-PCR showed that PdGATL1.1 is expressed most strongly in developing xylem of poplar. In contrast, PdGATL1.2 is expressed much more uniformly in leaf, shoot tip, cortex, phloem, and xylem, and the transcript level of PdGATL1.2 is much lower than that of PdGATL 1.1 in all tissues examined. Sub-cellular localization experiments showed that these two proteins are localized to both ER and Golgi in comparison with marker proteins resident to these sub-cellular compartments. Our data indicate that PdGATLI.1 and PdGATL1.2 are functional orthologs of PARVUS/ GATL1 and can play a role in xylan synthesis, but may also have role（s） in the synthesis of other wall polymers.展开更多
Virus-induced gene silencing （VIGS） is a powerful genetic tool for rapid assessment of plant gene functions in the post-genomic era. Here, we successfully implemented a Tobacco Rattle Virus （TRV）-based VlGS system...Virus-induced gene silencing （VIGS） is a powerful genetic tool for rapid assessment of plant gene functions in the post-genomic era. Here, we successfully implemented a Tobacco Rattle Virus （TRV）-based VlGS system to study functions of genes involved in either primary or secondary cell wall formation in Nicotiana benthamiana plants. A 3-week post- VIGS time frame is sufficient to observe phenotypic alterations in the anatomical structure of stems and chemical composition of the primary and secondary cell walls. We used cell wall glycan-directed monoclonal antibodies to demonstrate that alteration of cell wall polymer synthesis during the secondary growth phase of VIGS plants has profound effects on the extractability of components from woody stem cell walls. Therefore, TRV-based VlGS together with cell wall component profiling methods provide a high-throughput gene discovery platform for studying plant cell wall formation from a bioenergy perspective.展开更多
Cellulases are important glycosyl hydrolases （GHs） that hydrolyze cellulose polymers into smaller oligosaccharides by breaking the cellulose β （1→4） bonds, and they are widely used to produce cellulosic ethanol ...Cellulases are important glycosyl hydrolases （GHs） that hydrolyze cellulose polymers into smaller oligosaccharides by breaking the cellulose β （1→4） bonds, and they are widely used to produce cellulosic ethanol from the plant biomass. N-linked and O-linked glycosylations were proposed to impact the catalytic efficiency, cellulose binding affinity and the stability of cellulases based on observations of individual cellulases. As far as we know, there has not been any systematic analysis of the distributions of N-linked and O-linked glycosylated residues in cellulases, mainly due to the limited annotations of the relevant functional domains and the glycosylated residues. We have computationally annotated the functional domains and glycosylated residues in cellulases, and conducted a systematic analysis of the distributions of the N-linked and O-linked glycosylated residues in these enzymes. Many N-linked glycosylated residues were known to be in the GH domains of cellulases, but they are there probably just by chance, since the GH domain usually occupies more than half of the sequence length of a cellulase. Our analysis indicates that the O-linked glycosylated residues are significantly enriched in the linker re- gions between the carbohydrate binding module （CBM） domains and GH domains of cellulases. Possible mechanisms are discussed.展开更多
The availability of a large number of sequenced bacterial genomes allows researchers not only to derive functional and regulation information about specific organisms but also to study the fundamental properties of th...The availability of a large number of sequenced bacterial genomes allows researchers not only to derive functional and regulation information about specific organisms but also to study the fundamental properties of the organization of a genome. Here we address an important and chal- lenging question regarding the global arrangement of operons in a bacterial genome： why operons in a bacterial genome are arranged in the way they are. We have previously studied this question and found that operons of more frequently activated pathways tend to be more clustered together in a genome. Specifically, we have developed a simple sequential distance-based pseudo energy func- tion and found that the arrangement of operons in a bacterial genome tend to minimize the clus- teredness function （C value） in comparison with artificially-generated alternatives, for a variety of bacterial genomes. Here we extend our previous work, and report a number of new observations： （a） operons of the same pathways tend to group into a few clusters rather than one; and （b） the global arrangement of these operon clusters tend to minimize a new ＂energy＂ function （C＋ value） that reflects the efficiency of the transcriptional activation of the encoded pathways. These obser- vations provide insights into further study of the genomic organization of genes in bacteria.展开更多
The availability of a large number of sequenced bacterial genomes facilitates in-depth studies about why genes(operons)in a bacterial genome are globally organized the way they are.We have previously discovered that(t...The availability of a large number of sequenced bacterial genomes facilitates in-depth studies about why genes(operons)in a bacterial genome are globally organized the way they are.We have previously discovered that(the relative)transcription-activation frequencies among different biological pathways encoded in a genome have a dominating role in the global arrangement of operons.One complicating factor in such a study is that some operons may be involved in multiple pathways with different activation frequencies.A quantitative model has been developed that captures this information,which tends to be minimized by the current global arrangement of operons in a bacterial(and archaeal)genome compared to possible alternative arrangements.A study is carried out here using this model on a collection of 52 closely related Escherichia coli genomes,which revealed interesting new insights about how bacterial genomes evolve to optimally adapt to their environments through adjusting the(relative)genomic locations of the encoding operons of biological pathways once their utilization and hence transcription activation frequencies change,to maintain the above energy-efficiency property.More specifically we observed that it is the frequencies of the transcription activation of pathways relative to those of the other encoded pathways in an organism as well as the variation in the activation frequencies of a specific pathway across the related genomes that play a key role in the observed commonalities and differences in the genomic organizations of genes(and operons)encoding specific pathways across different genomes.展开更多
This article addresses how the functionalities of the cellular machinery of a bacterium might have constrained the genomic arrangement of its genes during evolution and how we can study such problems using computation...This article addresses how the functionalities of the cellular machinery of a bacterium might have constrained the genomic arrangement of its genes during evolution and how we can study such problems using computational approaches, taking full advantage of the rapidly increasing pool of the sequenced bacterial genomes, potentially leading to a much improved understanding of why a bacterial genome is organized in the way it is. This article discusses a number of challenging computational problems in elucidating the genomic structures at multiple levels and the information that is encoded through these genomic structures, gearing towards the ultimate understanding of the governing rules of bacterial genome organization.展开更多
基金supported by the BioEnergy Science Center (BESC),which is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science
文摘Live cell imaging methods provide important insights into the dynamics of cellular processes that cannot be derived easily from population-averaged datasets.In the bioenergy field,much research is focused on fermentation of cellulosic biomass by thermophilic microbes to produce biofuels;however,little effort is dedicated to the development of imaging tools to monitor this dynamic biological process.This is,in part,due to the experimental challenges of imaging cells under both anaerobic and thermophilic conditions.Here an imaging system is described that integrates confocal microscopy,a flow cell device,and a lipophilic dye to visualize cells.Solutions to technical obstacles regarding suitable fluorescent markers,photodamage during imaging,and maintenance of environmental conditions during imaging are presented.This system was utilized to observe cellulose colonization by Clostridium thermocellum under anaerobic conditions at 60℃.This method enables live cell imaging of bacterial growth under anaerobic and thermophilic conditions and should be widely applicable to visualizing different cell types or processes in real time.
文摘Galacturonosyltransferase 1 （GAUT1） is an α1,4-D-galacturonosyltransferase that transfers galacturonic acid from uridine 5＇-diphosphogalacturonic acid onto the pectic polysaccharide homogalacturonan （Sterling et al., 2006）. The 25-member Arabidopsis thaliana GAUT1-related gene family encodes 15 GAUT and 10 GAUT-like （GATL） proteins with, respectively, 56-84 and 42-53% amino acid sequence similarity to GAUT1. Previous phylogenetic analyses of AtGAUTs indicated three clades： A through C. A comparative phylogenetic analysis of the Arabidopsis, poplar and rice GAUT families has sub-classified the GAUTs into seven clades： clade A-1 （GAUTs 1 to 3）; A-2 （GAUT4）; A-3 （GAUTs 5 and 6）; A-4 （GAUT7）; B-1 （GAUTs 8 and 9）; B-2 （GAUTs 10 and 11）; and clade C （GAUTs 12 to 15）. The Arabidopsis GAUTs have a distribution comparable to the poplar orthologs, with the exception of GAUT2, which is absent in poplar. Rice, however, has no orthologs of GAUTs 2 and 12 and has multiple apparent orthologs of GAUTs 1, 4, and 7 compared with eitherArabidopsis or poplar. The cell wall glycosyl residue compositions of 26 homozygous T-DNA insertion mutants for 13 of 15 Arabidopsis GAUTgenes reveal significantly and reproducibly different cell walls in specific tissues of gaut mutants 6, 8, 9, 10, 11, 12, 13, and 14 from that of wild-type Arabidopsis walls. Pectin and xylan polysaccharides are affected by the loss of GAUT function, as demonstrated by the altered galacturonic acid, xylose, rhamnose, galactose, and arabinose composition of distinct gaut mutant walls. The wall glycosyl residue compositional phenotypes observed among the gaut mutants suggest that at least six different biosynthetic linkages in pectins and/or xylans are affected by the lesions in these GAUTgenes. Evidence is also presented to support a role for GAUT11 in seed mucilage expansion and in seed wall and mucilage composition.
文摘Several genes in Arabidopsis, including PARVUS/AtGATL1, have been implicated in xylan synthesis. However, the biosynthesis of xylan in woody plants, where this polysaccharide is a major component of wood, is poorly understood. Here, we characterize two Populus genes, PdGATL 1.1 and PdGATL 1.2, the closest orthologs to the Arabidopsis PARVUS/GATL1 gene, with respect to their gene expression in poplar, their sub-cellular localization, and their ability to complement the parvus mutation in Arabidopsis. Overexpression of the two poplar genes in the parvus mutant rescued most of the defects caused by the parvus mutation, including morphological changes, collapsed xylem, and altered cell wall monosaccharide composition. Quantitative RT-PCR showed that PdGATL1.1 is expressed most strongly in developing xylem of poplar. In contrast, PdGATL1.2 is expressed much more uniformly in leaf, shoot tip, cortex, phloem, and xylem, and the transcript level of PdGATL1.2 is much lower than that of PdGATL 1.1 in all tissues examined. Sub-cellular localization experiments showed that these two proteins are localized to both ER and Golgi in comparison with marker proteins resident to these sub-cellular compartments. Our data indicate that PdGATLI.1 and PdGATL1.2 are functional orthologs of PARVUS/ GATL1 and can play a role in xylan synthesis, but may also have role（s） in the synthesis of other wall polymers.
文摘Virus-induced gene silencing （VIGS） is a powerful genetic tool for rapid assessment of plant gene functions in the post-genomic era. Here, we successfully implemented a Tobacco Rattle Virus （TRV）-based VlGS system to study functions of genes involved in either primary or secondary cell wall formation in Nicotiana benthamiana plants. A 3-week post- VIGS time frame is sufficient to observe phenotypic alterations in the anatomical structure of stems and chemical composition of the primary and secondary cell walls. We used cell wall glycan-directed monoclonal antibodies to demonstrate that alteration of cell wall polymer synthesis during the secondary growth phase of VIGS plants has profound effects on the extractability of components from woody stem cell walls. Therefore, TRV-based VlGS together with cell wall component profiling methods provide a high-throughput gene discovery platform for studying plant cell wall formation from a bioenergy perspective.
基金supported in part by the National Science Foundation of USA (Grants DBI-0354771,ITR-IIS-0407204,DBI-0542119 and CCF0621700),the grant for the BioEnergy Science Center,and a Distinguished Scholargrant from the Georgia Cancer Coalition
文摘Cellulases are important glycosyl hydrolases （GHs） that hydrolyze cellulose polymers into smaller oligosaccharides by breaking the cellulose β （1→4） bonds, and they are widely used to produce cellulosic ethanol from the plant biomass. N-linked and O-linked glycosylations were proposed to impact the catalytic efficiency, cellulose binding affinity and the stability of cellulases based on observations of individual cellulases. As far as we know, there has not been any systematic analysis of the distributions of N-linked and O-linked glycosylated residues in cellulases, mainly due to the limited annotations of the relevant functional domains and the glycosylated residues. We have computationally annotated the functional domains and glycosylated residues in cellulases, and conducted a systematic analysis of the distributions of the N-linked and O-linked glycosylated residues in these enzymes. Many N-linked glycosylated residues were known to be in the GH domains of cellulases, but they are there probably just by chance, since the GH domain usually occupies more than half of the sequence length of a cellulase. Our analysis indicates that the O-linked glycosylated residues are significantly enriched in the linker re- gions between the carbohydrate binding module （CBM） domains and GH domains of cellulases. Possible mechanisms are discussed.
基金supported in part by National Science Foundation (Grant No. NSF DEB-0830024 and NSF MCB-0958172)by Grant from the BioEnergy Science Center(BESC) of US Department of Energy through the Office of Biological and Environmental Research
文摘The availability of a large number of sequenced bacterial genomes allows researchers not only to derive functional and regulation information about specific organisms but also to study the fundamental properties of the organization of a genome. Here we address an important and chal- lenging question regarding the global arrangement of operons in a bacterial genome： why operons in a bacterial genome are arranged in the way they are. We have previously studied this question and found that operons of more frequently activated pathways tend to be more clustered together in a genome. Specifically, we have developed a simple sequential distance-based pseudo energy func- tion and found that the arrangement of operons in a bacterial genome tend to minimize the clus- teredness function （C value） in comparison with artificially-generated alternatives, for a variety of bacterial genomes. Here we extend our previous work, and report a number of new observations： （a） operons of the same pathways tend to group into a few clusters rather than one; and （b） the global arrangement of these operon clusters tend to minimize a new ＂energy＂ function （C＋ value） that reflects the efficiency of the transcriptional activation of the encoded pathways. These obser- vations provide insights into further study of the genomic organization of genes in bacteria.
基金supported in part by National Science Foundation (#NSF DEB-0830024 and NSF MCB-0958172)the US Department of Energy’s BioEnergy Science Center grant through the Office of Biological and Environmental Research+1 种基金The BioEnergy Science Center is a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of ScienceFunding for open access charge: US Department of Energy’s BioEnergy Science Center
文摘The availability of a large number of sequenced bacterial genomes facilitates in-depth studies about why genes(operons)in a bacterial genome are globally organized the way they are.We have previously discovered that(the relative)transcription-activation frequencies among different biological pathways encoded in a genome have a dominating role in the global arrangement of operons.One complicating factor in such a study is that some operons may be involved in multiple pathways with different activation frequencies.A quantitative model has been developed that captures this information,which tends to be minimized by the current global arrangement of operons in a bacterial(and archaeal)genome compared to possible alternative arrangements.A study is carried out here using this model on a collection of 52 closely related Escherichia coli genomes,which revealed interesting new insights about how bacterial genomes evolve to optimally adapt to their environments through adjusting the(relative)genomic locations of the encoding operons of biological pathways once their utilization and hence transcription activation frequencies change,to maintain the above energy-efficiency property.More specifically we observed that it is the frequencies of the transcription activation of pathways relative to those of the other encoded pathways in an organism as well as the variation in the activation frequencies of a specific pathway across the related genomes that play a key role in the observed commonalities and differences in the genomic organizations of genes(and operons)encoding specific pathways across different genomes.
基金supported in part by the NSF of USA (Grant Nos. DBI-0354771, ITR-IIS-0407204, DBI-0542119, CCF0621700)NIHof USA (Grant Nos. 1R01GM075331 and 1R01GM081682)the grant for the BioEnergy Science Center
文摘This article addresses how the functionalities of the cellular machinery of a bacterium might have constrained the genomic arrangement of its genes during evolution and how we can study such problems using computational approaches, taking full advantage of the rapidly increasing pool of the sequenced bacterial genomes, potentially leading to a much improved understanding of why a bacterial genome is organized in the way it is. This article discusses a number of challenging computational problems in elucidating the genomic structures at multiple levels and the information that is encoded through these genomic structures, gearing towards the ultimate understanding of the governing rules of bacterial genome organization.
