Serine proteases from two cell types target different components of a complex that governs regula... more Serine proteases from two cell types target different components of a complex that governs regulated intramembrane proteolysis of pro-σ K during Bacillus subtilis development
Intramembrane proteases regulate diverse processes by cleaving substrates within a transmembrane ... more Intramembrane proteases regulate diverse processes by cleaving substrates within a transmembrane segment or near the membrane surface. Bacillus subtilis SpoIVFB is an intramembrane metalloprotease that cleaves Pro-K during sporulation. To elucidate features of Pro-K important for cleavage by SpoIVFB, coexpression of the two proteins in Escherichia coli was used along with cell fractionation. In the absence of SpoIVFB, a portion of the Pro-K was peripherally membrane associated. This portion was not observed in the presence of SpoIVFB, suggesting that it serves as the substrate. Deletion of Pro-K residues 2 to 8, addition of residues at its N terminus, or certain single-residue substitutions near the cleavage site impaired cleavage. Certain multiresidue substitutions near the cleavage site changed the position of cleavage, revealing preferences for a small residue preceding the cleavage site N-terminally (i.e., at the P1 position) and a hydrophobic residue at the second position following the cleavage site C-terminally (i.e., P2=). These features appear to be conserved among Pro-K orthologs. SpoIVFB did not tolerate an aromatic residue at P1 or P2= of Pro-K. A Lys residue at P3= of Pro-K could not be replaced with Ala unless a Lys was provided farther C-terminally (e.g., at P9=). ␣-Helix-destabilizing residues near the cleavage site were not crucial for SpoIVFB to cleave Pro-K. The preferences and tolerances of SpoIVFB are somewhat different from those of other intramembrane metalloproteases, perhaps reflecting differences in the interaction of the substrate with the membrane and the enzyme.
bioRxiv (Cold Spring Harbor Laboratory), Jun 29, 2020
In response to environmental changes, vegetative cells of Anabaena cylindrica can differentiate i... more In response to environmental changes, vegetative cells of Anabaena cylindrica can differentiate into two other cell types: a heterocyst for oxic N2-fixation, and an enlarged spore called akinete for stress survival. Akinetes normally differentiate from vegetative cells adjacent to heterocysts. Heterocysts inhibit nearby cells from differentiating into heterocysts but can induce adjacent cells to become akinetes, a rare embryogenetic induction in prokaryotes. The mechanism for a patterned differentiation in A. cylindrica has been little studied. Here, we isolated three types of cells from A. cylindrica to identify their proteomes using LC-MS/MS. A total of 1395 proteins were identified, including 664 proteins from akinetes, 751 proteins from heterocysts, and 1236 proteins from vegetative cells. There were 45 proteins (33 novel proteins) found exclusive to akinetes, 57 heterocyst-specific proteins (33 novel proteins), including nif gene products, and 485 proteins exclusively in vegetative cells. Our proteomic data suggest that akinetes, unlike the typical spores of bacteria, perform unique biochemical functions that collaborate with both heterocysts and vegetative cells. A HAVe model for collaboration among heterocysts, akinetes and vegetative cells is proposed to illustrate the metabolic network of cyanophycin and carbohydrates based on the distribution of their biosynthesis related proteins in three types of cells. Interestingly, cell division proteins, DNA replication proteins, some carboxysomal proteins including RuBisCO and proteins in photosystems I, II were found abundant in heterocysts, the non-dividing cells dedicated exclusively to oxic N2-fixation. The identification of the akinete and heterocyst proteomes enables the pursuit of genetic studies into the patterned differentiation of akinetes and heterocysts.
Regulated intramembrane proteolysis (RIP) involves cleavage of a transmembrane segment of a prote... more Regulated intramembrane proteolysis (RIP) involves cleavage of a transmembrane segment of a protein, releasing the active form of a membrane-anchored transcription factor (MTF) or a membrane-tethered signaling protein in response to an extracellular or intracellular signal. RIP is conserved from bacteria to humans and governs many important signaling pathways in both prokaryotes and eukaryotes. Proteases that carry out these cleavages are named intramembrane cleaving proteases (I-CLips). To date, little is known about I-CLips in cyanobacteria. In this study, five putative site-2 type I-Clips (Ava_1070, Ava_1730, Ava_1797, Ava_3438, and Ava_4785) were identified through a genome-wide survey in Anabaena variabilis. Biochemical analysis demonstrated that these five putative A. variabilis site-2 proteases (S2Ps Av) have authentic protease activities toward an artificial substrate pro-K , a Bacillus subtilis MTF, in our reconstituted Escherichia coli system. The enzymatic activities of processing pro-K differ among these five S2Ps Av. Substitution of glutamic acid (E) by glutamine (Q) in the conserved HEXXH zinccoordinated motif caused the loss of protease activities in these five S2Ps Av , suggesting that they belonged to the metalloprotease family. Further mapping of the cleaved peptides of pro-K by Ava_4785 and Ava_1797 revealed that Ava_4785 and Ava_1797 recognized the same cleavage site in pro-K as SpoIVFB, a cognate S2P of pro-K from B. subtilis. Taking these results together, we report here for the first time the identification of five metallo-intramembrane cleaving proteases in Anabaena variabilis. The experimental system described herein should be applicable to studies of other RIP events and amenable to developing in vitro assays for I-CLips.
2014 ASABE Intersectional Meeting South Dakota, 2014
Abstract Photosynthetic microorganisms are endowed with the unique ability to utilize light energ... more Abstract Photosynthetic microorganisms are endowed with the unique ability to utilize light energy to convert inorganic carbon into biomass, offering an opportunity to use metabolically-designed cyanobacteria to synthesize biofuels and high value chemicals for societal benefits. Farnesene, a C 15 -hydrocarbon, has a similar energy density to diesel and jet fuels, and is a sought-after compound for large-scale production using engineered cyanobacteria. Here, we report the photosynthetic production of farnesene by the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 using CO 2 , water, and light. A codon-optimized farnesene synthase (FaS) gene from Norway spruce was expressed in the cyanobacterium, giving it the ability to synthesize farnesene using its endogenous MEP pathway. Volatized farnesene was emitted from the engineered strain into the flask headspace, allowing for quick separation of the target compound from the culture biomass. Thus, the engineered Anabaena serves as a solar-driven, living cellular factory for continuous farnesene production. Total farnesene yield was measured to be 305.4±17.7 µg·L -1 during a 15-day production trial, with a maximum productivity of 69.1±1.8 µg·L -1 ·O.D. -1 ·d -1 . We envision the use of metabolically-engineered cyanobacteria as a biosolar factory for production of a wide range of biofuels and commodity chemicals.
The Journal of American Science is an international journal with a purpose to enhance our natural... more The Journal of American Science is an international journal with a purpose to enhance our natural and scientific knowledge dissemination in the world. Any valuable paper that describes natural phenomena and existence or any reports that convey scientific research and pursuit is welcome. Papers submitted could be reviews, objective descriptions, research reports, opinions/debates, news, letters, and other types of writings that are nature and science related. All the manuscripts will be processed in a professional peer review. After the peer review, the journal will make the best efforts to publish all the valuable works as soon as possible.
Nowadays, food safety is one of the biggest challenges in global markets. Therefore, there is a c... more Nowadays, food safety is one of the biggest challenges in global markets. Therefore, there is a critical need to develop a simple, affordable, and environmentally friendly color indicator that can rapidly and conveniently monitor and indicate the quality of packaged food products.
Superoxide dismutase (Sod) plays an important role in all aerobic organisms. The sodB gene of a h... more Superoxide dismutase (Sod) plays an important role in all aerobic organisms. The sodB gene of a heterocystous cyanobacterium Anabaena sp. PCC 7120 was cloned and sequenced. The Sod protein is predicted to have 199 amino acids and a molecular mass of 22.5 kDa. Sequence comparison among SodB from cyanobacteria and chloroplasts revealed that the sodB gene indeed encodes an iron-Sod. Northern blot analysis showed that the sodB gene of Anabaena sp. PCC 7120 is transcribed as a single gene and its expression was up-regulated when the cells were subjected to a shift from a nitrogen repletion condition to a nitrogen depletion condition. fl 2000 Elsevier Science B.V. All rights reserved.
La presente invention concerne, selon des modes de realisation, des cyanobacteries spp. qui ont e... more La presente invention concerne, selon des modes de realisation, des cyanobacteries spp. qui ont ete genetiquement modifiees pour une production augmentee de produits a base de carbone d'interet. Ces hotes genetiquement modifies convertissent efficacement le dioxyde de carbone et la lumiere en produits a base de carbone d'interet tels que des hydrocarbures a longue chaine. Plusieurs constructions contenant des polynucleotides codant pour des enzymes actives dans les voies metaboliques des cyanobacteries sont decrites. Dans de nombreux cas, les souches de cyanobacteries ont ete davantage genetiquement modifiees pour optimiser la production de produits a base de carbone d'interet. L'optimisation comprend la regulation positive et la regulation negative de genes particuliers.
Mauremys reevesii is an endangered freshwater turtle that symbolizes longevity in Chinese culture... more Mauremys reevesii is an endangered freshwater turtle that symbolizes longevity in Chinese culture. Despite its importance, genetic studies of this species remain limited, with no genomic sequence reported to date. Here, we report a high‐quality, chromosome‐level genomic sequence of M. reevesii obtained using a combination of Nanopore and Hi‐C sequencing technologies. The 2.37 Gb M. reevesii genome was assembled from a total of ~226.80 Gb of Nanopore sequencing data. The M. reevesii genome contig N50 is 34.73 Mb, the highest value in published turtle genomes. In total, 18,238 genes were functionally annotated. The contigs were clustered and ordered onto 27 pseudochromosomes covering ~96.55% of the genome assembled with Hi‐C data. To explore genome evolution, synteny analysis was performed between M. reevesii (freshwater turtle) and Gopherus evgoodei (terrestrial turtle) genomes. In general, each chromosome of M. reevesii corresponded to one chromosome of Gopherus evgoodei, but some interchromosomal rearrangements occurred between the two species based on the assembled genomes. These interchromosomal rearrangements were further confirmed by mapping of the long‐read nanopore data to the assembly. The reconstructed demographic history showed varied effective population size among freshwater, marine and terrestrial turtles. We also discovered expansion of genes related to the innate immune system in M. reevesii that may provide defence against freshwater pathogens. The high‐quality genomic sequence provides a valuable genetic resource for further studies of genetics and genome evolution in turtles.
Serine proteases from two cell types target different components of a complex that governs regula... more Serine proteases from two cell types target different components of a complex that governs regulated intramembrane proteolysis of pro-σ K during Bacillus subtilis development
Intramembrane proteases regulate diverse processes by cleaving substrates within a transmembrane ... more Intramembrane proteases regulate diverse processes by cleaving substrates within a transmembrane segment or near the membrane surface. Bacillus subtilis SpoIVFB is an intramembrane metalloprotease that cleaves Pro-K during sporulation. To elucidate features of Pro-K important for cleavage by SpoIVFB, coexpression of the two proteins in Escherichia coli was used along with cell fractionation. In the absence of SpoIVFB, a portion of the Pro-K was peripherally membrane associated. This portion was not observed in the presence of SpoIVFB, suggesting that it serves as the substrate. Deletion of Pro-K residues 2 to 8, addition of residues at its N terminus, or certain single-residue substitutions near the cleavage site impaired cleavage. Certain multiresidue substitutions near the cleavage site changed the position of cleavage, revealing preferences for a small residue preceding the cleavage site N-terminally (i.e., at the P1 position) and a hydrophobic residue at the second position following the cleavage site C-terminally (i.e., P2=). These features appear to be conserved among Pro-K orthologs. SpoIVFB did not tolerate an aromatic residue at P1 or P2= of Pro-K. A Lys residue at P3= of Pro-K could not be replaced with Ala unless a Lys was provided farther C-terminally (e.g., at P9=). ␣-Helix-destabilizing residues near the cleavage site were not crucial for SpoIVFB to cleave Pro-K. The preferences and tolerances of SpoIVFB are somewhat different from those of other intramembrane metalloproteases, perhaps reflecting differences in the interaction of the substrate with the membrane and the enzyme.
bioRxiv (Cold Spring Harbor Laboratory), Jun 29, 2020
In response to environmental changes, vegetative cells of Anabaena cylindrica can differentiate i... more In response to environmental changes, vegetative cells of Anabaena cylindrica can differentiate into two other cell types: a heterocyst for oxic N2-fixation, and an enlarged spore called akinete for stress survival. Akinetes normally differentiate from vegetative cells adjacent to heterocysts. Heterocysts inhibit nearby cells from differentiating into heterocysts but can induce adjacent cells to become akinetes, a rare embryogenetic induction in prokaryotes. The mechanism for a patterned differentiation in A. cylindrica has been little studied. Here, we isolated three types of cells from A. cylindrica to identify their proteomes using LC-MS/MS. A total of 1395 proteins were identified, including 664 proteins from akinetes, 751 proteins from heterocysts, and 1236 proteins from vegetative cells. There were 45 proteins (33 novel proteins) found exclusive to akinetes, 57 heterocyst-specific proteins (33 novel proteins), including nif gene products, and 485 proteins exclusively in vegetative cells. Our proteomic data suggest that akinetes, unlike the typical spores of bacteria, perform unique biochemical functions that collaborate with both heterocysts and vegetative cells. A HAVe model for collaboration among heterocysts, akinetes and vegetative cells is proposed to illustrate the metabolic network of cyanophycin and carbohydrates based on the distribution of their biosynthesis related proteins in three types of cells. Interestingly, cell division proteins, DNA replication proteins, some carboxysomal proteins including RuBisCO and proteins in photosystems I, II were found abundant in heterocysts, the non-dividing cells dedicated exclusively to oxic N2-fixation. The identification of the akinete and heterocyst proteomes enables the pursuit of genetic studies into the patterned differentiation of akinetes and heterocysts.
Regulated intramembrane proteolysis (RIP) involves cleavage of a transmembrane segment of a prote... more Regulated intramembrane proteolysis (RIP) involves cleavage of a transmembrane segment of a protein, releasing the active form of a membrane-anchored transcription factor (MTF) or a membrane-tethered signaling protein in response to an extracellular or intracellular signal. RIP is conserved from bacteria to humans and governs many important signaling pathways in both prokaryotes and eukaryotes. Proteases that carry out these cleavages are named intramembrane cleaving proteases (I-CLips). To date, little is known about I-CLips in cyanobacteria. In this study, five putative site-2 type I-Clips (Ava_1070, Ava_1730, Ava_1797, Ava_3438, and Ava_4785) were identified through a genome-wide survey in Anabaena variabilis. Biochemical analysis demonstrated that these five putative A. variabilis site-2 proteases (S2Ps Av) have authentic protease activities toward an artificial substrate pro-K , a Bacillus subtilis MTF, in our reconstituted Escherichia coli system. The enzymatic activities of processing pro-K differ among these five S2Ps Av. Substitution of glutamic acid (E) by glutamine (Q) in the conserved HEXXH zinccoordinated motif caused the loss of protease activities in these five S2Ps Av , suggesting that they belonged to the metalloprotease family. Further mapping of the cleaved peptides of pro-K by Ava_4785 and Ava_1797 revealed that Ava_4785 and Ava_1797 recognized the same cleavage site in pro-K as SpoIVFB, a cognate S2P of pro-K from B. subtilis. Taking these results together, we report here for the first time the identification of five metallo-intramembrane cleaving proteases in Anabaena variabilis. The experimental system described herein should be applicable to studies of other RIP events and amenable to developing in vitro assays for I-CLips.
2014 ASABE Intersectional Meeting South Dakota, 2014
Abstract Photosynthetic microorganisms are endowed with the unique ability to utilize light energ... more Abstract Photosynthetic microorganisms are endowed with the unique ability to utilize light energy to convert inorganic carbon into biomass, offering an opportunity to use metabolically-designed cyanobacteria to synthesize biofuels and high value chemicals for societal benefits. Farnesene, a C 15 -hydrocarbon, has a similar energy density to diesel and jet fuels, and is a sought-after compound for large-scale production using engineered cyanobacteria. Here, we report the photosynthetic production of farnesene by the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 using CO 2 , water, and light. A codon-optimized farnesene synthase (FaS) gene from Norway spruce was expressed in the cyanobacterium, giving it the ability to synthesize farnesene using its endogenous MEP pathway. Volatized farnesene was emitted from the engineered strain into the flask headspace, allowing for quick separation of the target compound from the culture biomass. Thus, the engineered Anabaena serves as a solar-driven, living cellular factory for continuous farnesene production. Total farnesene yield was measured to be 305.4±17.7 µg·L -1 during a 15-day production trial, with a maximum productivity of 69.1±1.8 µg·L -1 ·O.D. -1 ·d -1 . We envision the use of metabolically-engineered cyanobacteria as a biosolar factory for production of a wide range of biofuels and commodity chemicals.
The Journal of American Science is an international journal with a purpose to enhance our natural... more The Journal of American Science is an international journal with a purpose to enhance our natural and scientific knowledge dissemination in the world. Any valuable paper that describes natural phenomena and existence or any reports that convey scientific research and pursuit is welcome. Papers submitted could be reviews, objective descriptions, research reports, opinions/debates, news, letters, and other types of writings that are nature and science related. All the manuscripts will be processed in a professional peer review. After the peer review, the journal will make the best efforts to publish all the valuable works as soon as possible.
Nowadays, food safety is one of the biggest challenges in global markets. Therefore, there is a c... more Nowadays, food safety is one of the biggest challenges in global markets. Therefore, there is a critical need to develop a simple, affordable, and environmentally friendly color indicator that can rapidly and conveniently monitor and indicate the quality of packaged food products.
Superoxide dismutase (Sod) plays an important role in all aerobic organisms. The sodB gene of a h... more Superoxide dismutase (Sod) plays an important role in all aerobic organisms. The sodB gene of a heterocystous cyanobacterium Anabaena sp. PCC 7120 was cloned and sequenced. The Sod protein is predicted to have 199 amino acids and a molecular mass of 22.5 kDa. Sequence comparison among SodB from cyanobacteria and chloroplasts revealed that the sodB gene indeed encodes an iron-Sod. Northern blot analysis showed that the sodB gene of Anabaena sp. PCC 7120 is transcribed as a single gene and its expression was up-regulated when the cells were subjected to a shift from a nitrogen repletion condition to a nitrogen depletion condition. fl 2000 Elsevier Science B.V. All rights reserved.
La presente invention concerne, selon des modes de realisation, des cyanobacteries spp. qui ont e... more La presente invention concerne, selon des modes de realisation, des cyanobacteries spp. qui ont ete genetiquement modifiees pour une production augmentee de produits a base de carbone d'interet. Ces hotes genetiquement modifies convertissent efficacement le dioxyde de carbone et la lumiere en produits a base de carbone d'interet tels que des hydrocarbures a longue chaine. Plusieurs constructions contenant des polynucleotides codant pour des enzymes actives dans les voies metaboliques des cyanobacteries sont decrites. Dans de nombreux cas, les souches de cyanobacteries ont ete davantage genetiquement modifiees pour optimiser la production de produits a base de carbone d'interet. L'optimisation comprend la regulation positive et la regulation negative de genes particuliers.
Mauremys reevesii is an endangered freshwater turtle that symbolizes longevity in Chinese culture... more Mauremys reevesii is an endangered freshwater turtle that symbolizes longevity in Chinese culture. Despite its importance, genetic studies of this species remain limited, with no genomic sequence reported to date. Here, we report a high‐quality, chromosome‐level genomic sequence of M. reevesii obtained using a combination of Nanopore and Hi‐C sequencing technologies. The 2.37 Gb M. reevesii genome was assembled from a total of ~226.80 Gb of Nanopore sequencing data. The M. reevesii genome contig N50 is 34.73 Mb, the highest value in published turtle genomes. In total, 18,238 genes were functionally annotated. The contigs were clustered and ordered onto 27 pseudochromosomes covering ~96.55% of the genome assembled with Hi‐C data. To explore genome evolution, synteny analysis was performed between M. reevesii (freshwater turtle) and Gopherus evgoodei (terrestrial turtle) genomes. In general, each chromosome of M. reevesii corresponded to one chromosome of Gopherus evgoodei, but some interchromosomal rearrangements occurred between the two species based on the assembled genomes. These interchromosomal rearrangements were further confirmed by mapping of the long‐read nanopore data to the assembly. The reconstructed demographic history showed varied effective population size among freshwater, marine and terrestrial turtles. We also discovered expansion of genes related to the innate immune system in M. reevesii that may provide defence against freshwater pathogens. The high‐quality genomic sequence provides a valuable genetic resource for further studies of genetics and genome evolution in turtles.
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