Genetically Engineered Cyanobacteria

The disclosed embodiments provide cyanobacteria spp. that have been genetically engineered to have increased production of carbon-based products of interest. These genetically engineered hosts efficiently convert carbon dioxide and light into carbon-based products of interest such as long chained hydrocarbons. Several constructs containing polynucleotides encoding enzymes active in the metabolic pathways of cyanobacteria are disclosed. In many instances, the cyanobacteria strains have been further genetically modified to optimize production of the carbon-based products of interest. The optimization includes both up-regulation and down-regulation of particular genes

Identification and Characterization of Mitogen-Activated Protein Kinase (MAPK) Genes in Sunflower (Helianthus annuus L.)

Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that regulate biotic and abiotic stresses in plants through signaling cascades comprised of three major subfamilies: MAP Kinase (MPK), MAPK Kinase (MKK), and MAPKK Kinase (MKKK). The main objectives of this research were to conduct genome-wide identification of MAPK genes in Helianthus annuus and examine functional divergence of these genes in relation to those in nine other plant species (Amborella trichopoda, Aquilegia coerulea, Arabidopsis thaliana, Daucus carota, Glycine max, Oryza sativa, Solanum lycopersicum, Sphagnum fallax, and Vitis vinifera), representing diverse taxonomic groups of the Plant Kingdom. A Hidden Markov Model (HMM) profile of the MAPK genes utilized reference sequences from A. thaliana and G. max, yielding a total of 96 MPKs and 37 MKKs in the genomes of A. trichopoda, A. coerulea, C. reinhardtii, D. carota, H. annuus, S. lycopersicum, and S. fallax. Among them, 28 MPKs and eight MKKs were confirmed in H. annuus. Phylogenetic analyses revealed four clades within each subfamily. Transcriptomic analyses showed that at least 19 HaMPK and seven HaMKK genes were induced in response to salicylic acid (SA), sodium chloride (NaCl), and polyethylene glycol (Peg) in leaves and roots. Of the seven published sunflower microRNAs, five microRNA families are involved in targeting eight MPKs. Additionally, we discussed the need for using MAP Kinase nomenclature guidelines across plant species. Our identification and characterization of MAP Kinase genes would have implications in sunflower crop improvement, and in advancing our knowledge of the diversity and evolution of MAPK genes in the Plant Kingdom

Photos for Chinese three-keeled pond turtle \u3cem\u3e(M. reevesii)\u3c/em\u3e

This data set zip file contains four images in JPEG format. File size: 57 M

Substrate Requirements for Regulated Intramembrane Proteolysis of Bacillus subtilis Pro-σ(K)

During sporulation of Bacillus subtilis, pro-σ(K) is activated by regulated intramembrane proteolysis (RIP) in response to a signal from the forespore. RIP of pro-σ(K) removes its prosequence (amino acids 1 to 20), releasing σ(K) from the outer forespore membrane into the mother cell cytoplasm, in a reaction catalyzed by SpoIVFB, a metalloprotease in the S2P family of intramembrane-cleaving proteases. The requirements for pro-σ(K) to serve as a substrate for RIP were investigated by producing C-terminally truncated pro-σ(K) fused at different points to the green fluorescent protein (GFP) or hexahistidine in sporulating B. subtilis or in Escherichia coli engineered to coexpress SpoIVFB. Nearly half of pro-σ(K) (amino acids 1 to 117), including part of sigma factor region 2.4, was required for RIP of pro-σ(K)-GFP chimeras in sporulating B. subtilis. Likewise, pro-σ(K)-hexahistidine chimeras demonstrated that the N-terminal 117 amino acids of pro-σ(K) are sufficient for RIP, although the N-terminal 126 amino acids, which includes all of region 2.4, allowed much better accumulation of the chimeric protein in sporulating B. subtilis and more efficient processing by SpoIVFB in E. coli. In contrast to the requirements for RIP, a much smaller N-terminal segment (amino acids 1 to 27) was sufficient for membrane localization of a pro-σ(K)-GFP chimera. Addition or deletion of five amino acids near the N terminus allowed accurate processing of pro-σ(K), ruling out a mechanism in which SpoIVFB measures the distance from the N terminus to the cleavage site. A charge reversal at position 13 (substituting glutamate for lysine) reduced accumulation of pro-σ(K) and prevented detectable RIP by SpoIVFB. These results elucidate substrate requirements for RIP of pro-σ(K) by SpoIVFB and may have implications for substrate recognition by other S2P family members

M. reevesii Chromosomal Genome Annotations

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Cyanobacteria: Harnessing the power of microorganisms for plant growth promotion, stress alleviation, and phytoremediation in the era of sustainable agriculture

In the ever-evolving landscape of sustainable agriculture, cyanobacterium has emerged as a viable option to offer multifaceted benefits to both crops and the environment. This comprehensive review navigates the contributions of cyanobacteria in agriculture and begins with an insightful exploration of their fundamental role in sustainable agricultural systems. This review article explains the mechanisms through which cyanobacterium may foster plant growth and demonstrates their ability to boost crop yields and optimize nutrient utilization. Their capacity to alleviate stresses in plants in drought to salinity conditions, unveils a remarkable feature. Beyond the fields, this bacterium plays a crucial role in protecting our environmental as well as help through phytoremediation by addressing soil pollution and effectively removing heavy metals and organic contaminants. In the context of sustainable agriculture, it represents an environmentally friendly alternative to chemical fertilizers and can be seamlessly integrated into organic farming. Looking forward, this review peers into the future of its applications in agriculture, pragmatically addressing the hurdles of commercialization, regulatory compliance, and public perception. In conclusion, this review consolidates the pivotal contributions of this bacterium to sustainable agriculture, underscoring the imperative of ongoing research and innovation in harnessing their transformative potential for a greener and more productive agricultural practices in future

Sporulation and Enterotoxin (CPE) Synthesis Are Controlled by the Sporulation-Specific Sigma Factors SigE and SigK in Clostridium perfringens▿ ‡

Clostridium perfringens is the third most frequent cause of bacterial food poisoning annually in the United States. Ingested C. perfringens vegetative cells sporulate in the intestinal tract and produce an enterotoxin (CPE) that is responsible for the symptoms of acute food poisoning. Studies of Bacillus subtilis have shown that gene expression during sporulation is compartmentalized, with different genes expressed in the mother cell and the forespore. The cell-specific RNA polymerase sigma factors σF, σE, σG, and σK coordinate much of the developmental process. The C. perfringens cpe gene, encoding CPE, is transcribed from three promoters, where P1 was proposed to be σK dependent, while P2 and P3 were proposed to be σE dependent based on consensus promoter recognition sequences. In this study, mutations were introduced into the sigE and sigK genes of C. perfringens. With the sigE and sigK mutants, gusA fusion assays indicated that there was no expression of cpe in either mutant. Results from gusA fusion assays and immunoblotting experiments indicate that σE-associated RNA polymerase and σK-associated RNA polymerase coregulate each other's expression. Transcription and translation of the spoIIID gene in C. perfringens were not affected by mutations in sigE and sigK, which differs from B. subtilis, in which spoIIID transcription requires σE-associated RNA polymerase. The results presented here show that the regulation of developmental events in the mother cell compartment of C. perfringens is not the same as that in B. subtilis and Clostridium acetobutylicum

Differential Expression and Localization of Mn and Fe Superoxide Dismutases in the Heterocystous Cyanobacterium Anabaena sp. Strain PCC 7120

Superoxide dismutases (Sods) play very important roles in preventing oxidative damages in aerobic organisms. The nitrogen-fixing heterocystous cyanobacterium Anabaena sp. strain PCC 7120 has two Sod-encoding genes: a sodB, encoding a soluble iron-containing Sod (FeSod), and a sodA, encoding a manganese-containing Sod (MnSod). The FeSod was purified and characterized. A recombinant FeSod was also obtained by overproduction in Escherichia coli. Immunoblot study of the FeSod during induction of heterocyst differentiation showed that the cells produced six- to eightfold more FeSod 8 h after a shift from a nitrogen-replete condition to a nitrogen-depleted condition. However, the amount of FeSod protein in filaments with mature heterocysts was the same as that in filaments grown with combined nitrogen. Superoxide anion-generating chemicals such as methyl viologen did not induce upregulation of the sodB gene expression. The predicted preprotein of the sodA gene has a leader peptide and a motif for membrane attachment at the N terminus of the mature protein. Activity staining after gel electrophoresis of the purified thylakoid membranes showed that most of the MnSod in Anabaena sp. strain PCC 7120 was located on thylakoids toward the lumenal side. Expression of the sodA gene in E. coli shows that the leader peptide was required for its activity and the membrane localization of the MnSod. Northern hybridization detected one 0.82-kb transcript of sodA. The sodA gene was upregulated by methyl viologen, whereas its amount was unchanged during heterocyst differentiation. Immunoblotting and activity staining showed that isolated heterocysts contained a lower but still significant amount of FeSod, suggesting that its function is required in heterocysts. No MnSod was observed in isolated heterocysts. These results show that the two different Sod proteins have differentiated roles in Anabaena sp. strain PCC 7120

Evidence that HetR protein is an unusual serine-type protease

The hetR gene plays a very important role in cell differentiation of heterocystous cyanobacteria. To understand the mechanism of the hetR gene product in regulation of heterocyst differentiation, the recombinant HetR protein (rHetR) was overproduced in Escherichia coli. Purified rHetR was unstable and degraded easily in solution. Phenylmethanesulfonyl fluoride, a serine-type protease inhibitor, prevented the degradation and was shown to modify covalently rHetR. Dansyl fluoride (DnsF), another serine-type protease inhibitor, also covalently modifies rHetR as shown by electrophoresis and electroblotting of the labeled rHetR and by MS. The labeling of rHetR with phenylmethanesulfonyl fluoride and DnsF was at the same site of rHetR and required Ca(2+). S179N-rHetR, a mutant protein from strain 216 of Anabaena PCC 7120, which cannot differentiate heterocysts because of the mutation, was also overproduced and characterized. Although S170N-rHetR still can be labeled with DnsF, no proteolysis was observed, suggesting that Ser179 is involved in proteolytic activity. DnsF-labeled rHetR was digested with trypsin, and the labeled peptide was isolated and sequenced. The labeled peptide matches a sequence from HetR. These results show that HetR is a protease