Acetylene reduction assay for nitrogenase activity in root nodules under salinity stress.

Nitrogenase, an enzyme present in a select group of prokaryotes reduces inert N2 into NH3 that can be utilized through biological pathways. This process, termed biological nitrogen fixation, plays a crucial role in the biogeochemical N cycle. The ability of nitrogenase to reduce acetylene to ethylene has been exploited to develop a reliable and accessible biochemical assay to measure this enzyme's activity. Biological nitrogen fixation by rhizobia bacteria that occupy root nodules of legume crops is a major source of sustainable nitrogen nutrition in agriculture. Environmental stresses exacerbated by climate change necessitate the need to evaluate nitrogen fixation in root nodules under various stress conditions. Here, we provide a detailed step-by-step protocol for nitrogenase activity measurements using acetylene reduction assay in field pea plants under saline stress. The protocol can be easily adapted for use with other biological systems.

Single Crossover to Inactivate Target Gene in Cyanobacteria.

Anabaena sp. PCC 7120 (hereafter Anabaena 7120) is a model cyanobacterium for studying pathways such as photosynthesis and nitrogen fixation along with many other metabolic pathways common to plants. In addition, since Anabaena 7120 forms specialized N2-fixing cells, called heterocysts, to perform uniquely solar-powered, oxic nitrogen fixation under fixed-nitrogen depleted conditions, this cyanobacterium provides the unique opportunity to study cellular differentiation in bacteria. Since more than 155,810 sequenced prokaryotic genomes are currently available (Zhang et al., Microbiome 8(1):134, 2020), target gene inactivation, combined with analyses of the corresponding mutant's phenotype, has become a powerful tool to assess gene function through detecting a loss-of-function in the knockout mutant. In the method described here, a single crossover approach is used to knockout a target gene in Anabaena 7120. The method requires inserting an internal fragment of the target gene into the cyanobacterial integration vector pZR606 to create a knockout plasmid, and then is introduced to Anabaena 7120 via conjugative transformation. A single crossover, occurring via homologous recombination, disrupts the target gene, creating 3'- and 5'-deleted fragments (Fig. 1). The mutant containing the inactivated gene can then be studied to determine any loss of function, thereby defining the gene's function. This gene inactivation approach is based on an integrative vector pZR606 (Chen et al., Appl Microbiol Biotechnol 99:1779-1793, 2015), which may be broadly applied to gene inactivation in other cyanobacterial species as well as other prokaryotic organisms.

Double Crossover Approach to Inactivate Target Gene in Cyanobacteria.

Anabaena sp. PCC7120 (hereafter Anabaena 7120) is a nitrogen-fixing, filamentous cyanobacterium. Given its diverse metabolism, it serves as an excellent model organism, particularly for studying cell differentiation, nitrogen fixation, photosynthesis, production of high-value chemicals, and synthetic biology. Gene knockout is a common approach to assess the function of gene products through assessing phenotypic loss of function. In the method described here, a double crossover approach is used to inactivate a target gene or target genes in Anabaena 7120. This method involves replicating the gene(s) from the wild-type genomic DNA and inserting them into an integrative plasmid vector. An internal portion of the genes may be removed and replaced with a GFP-Spectinomycin (gfp-sp) cassette. The plasmid is then introduced into Anabaena 7120 where a double crossover event occurs between the wild-type chromosome and the cargo plasmid, effectively replacing the wild-type gene with the disrupted gene from the plasmid. The gfp-sp cassette combined with the sacB gene serve as positive selection to identify double crossover mutants (Cai and Wolk (1990), 172(6):3138-3145, J. Bacteriol). Finally, the functional genes are cloned into another replicating plasmid vector to produce a cargo plasmid, which is conjugatively introduced into the mutant for a complementation test. By comparing the phenotypes among the wild-type, mutant, and complement, one should see a loss of function in the mutant which is recovered in the complement, thereby defining the function of the target gene. The double crossover approach described here for Anabaena PCC 7120 may be broadly applicable to the study of gene function in cyanobacteria and other prokaryotic organisms.

Chromosome-level genome assembly of the Chinese three-keeled pond turtle (Mauremys reevesii) provides insights into freshwater adaptation.

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.

Development of a polylactic acid-coated nanocellulose/chitosan-based film indicator for real-time monitoring of beef spoilage.

Food safety is one of the biggest challenges in global markets. There is a critical need to develop a simple, affordable, and environmentally friendly color indicator that can quickly and conveniently monitor and indicate the quality of packaged food products in the home, supermarkets, shops, etc. This study aimed to develop a nanocellulose/chitosan-based film coated with polylactic acid (PLA) to monitor beef spoilage in real-time. This film named PLA/NCM was fabricated by casting a suspension of a nanocellulose/chitosan mixture doped with methyl red, followed by a coating of PLA on the film surface. The film displayed a visible color change in response to different pH buffer solutions (2-10). The PLA/NCM film was applied to monitor the spoilage of beef under a refrigeration condition of 4 °C and showed an apparent color change after 5 days as a threshold for beef spoilage. The color modulation of the PLA/NCM films was processed each time via a colorimetric device and revealed substantial color difference values (ΔE) after 5 days of beef spoilage. The total viable microbial counts (TVC) and pH of the beef sample were determined, and the findings showed that the TVC and pH increased simultaneously during the beef spoilage. Although further research is necessary, the PLA/NCM film has the potential to be a color indicator for application in both smart food packaging and real-time monitoring of spoilage of beef and other meat products.

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.

Identification of surface polysaccharides in akinetes, heterocysts and vegetative cells of Anabaena cylindrica using fluorescein-labeled lectins.

In response to environmental changes, Anabaena cylindrica differentiate three cell types: vegetative cells for photosynthesis, heterocysts for nitrogen fixation, and akinetes for stress survival. Cell-surface polysaccharides play important roles in cyanobacterial ecophysiology. In this study, specific cell-surface sugars were discovered in heterocysts, akinetes and vegetative cells of A. cylindrica using 20 fluorescein-labeled lectins. Both N-acetylglucosamine-binding lectins WGA and succinylated WGA bound specifically to the vegetative cells. Akinetes bound to three mannose-binding lectins (LCA, PSA, and ConA), and one of the galactose-binding lectins (GSL-I). Heterocyst also bound to ConA. However, the heterocysts in all4388 mutant of Anabaena sp. PCC 7120, in which the putative polysaccharide export protein gene all4388 was disrupted, exhibited diminished binding to ConA. Identification of distinct cell-surface sugar helped us to understand the role of polysaccharide for each cell type. Fluorescence-activated cell sorting may be applicable in isolating each cell type for comparative "omics" studies among the three cell types.

Identification of two genes required for heptadecane production in a N2-fixing cyanobacterium Anabaena sp. strain PCC 7120.

Cyanobacteria photosynthetically produce long-chain hydrocarbons, which are considered as infrastructure-compatible biofuels. However, native cyanobacteria do not produce these hydrocarbons at sufficient rates or yields to warrant commercial deployment. This research sought to identify specific genes required for photosynthetic production of alkanes to enable future metabolic engineering for commercially viable production of alkanes. The two putative genes (alr5283 and alr5284) required for long-chain hydrocarbon production in Anabaena sp. PCC 7120 were knocked out through a double crossover approach. The knockout mutant abolished the production of heptadecane (C17H36). The mutant is able to be complemented by a plasmid bearing the two genes along with their native promoters only. The complemented mutant restored photosynthetic production of heptadecane. This combined genetic and metabolite (alkanes) profiling approach may be broadly applicable to characterization of knockout mutants, using N2-fixing cyanobacteria as a cellular factory driven by solar energy to produce a wide range of commodity chemicals and drop-in-fuels from atmospheric gases (CO2 and N2 gas) and mineralized water.

Photobioreactor cultivation strategies for microalgae and cyanobacteria.

The current burden on fossil-derived chemicals and fuels combined with the rapidly increasing global population has led to a crucial need to develop renewable and sustainable sources of chemicals and biofuels. Photoautotrophic microorganisms, including cyanobacteria and microalgae, have garnered a great deal of attention for their capability to produce these chemicals from carbon dioxide, mineralized water, and solar energy. While there have been substantial amounts of research directed at scaling-up production from these microorganisms, several factors have proven difficult to overcome, including high costs associated with cultivation, photobioreactor construction, and artificial lighting. Decreasing these costs will substantially increase the economic feasibility of these production processes. Thus, the purpose of this review is to describe various photobioreactor designs, and then provide an overview on lighting systems, mixing, gas transfer, and the hydrodynamics of bubbles. These factors must be considered when the goal of a production process is economic feasibility. Targets for improving microalgae and cyanobacteria cultivation media, including water reduction strategies will also be described. As fossil fuel reserves continue to be depleted and the world population continues to increase, it is imperative that renewable chemical and biofuel production processes be developed toward becoming economically feasible. Thus, it is essential that future research is directed toward improving these processes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:811-827, 2018.

Molecular genetic improvements of cyanobacteria to enhance the industrial potential of the microbe: A review.

The rapid increase in worldwide population coupled with the increasing demand for fossil fuels has led to an increased urgency to develop sustainable sources of energy and chemicals from renewable resources. Using microorganisms to produce high-value chemicals and next-generation biofuels is one sustainable option and is the focus of much current research. Cyanobacteria are ideal platform organisms for chemical and biofuel production because they can be genetically engineered to produce a broad range of products directly from CO2 , H2 O, and sunlight, and require minimal nutrient inputs. The purpose of this review is to provide an overview on advances that have been or could be made to improve strains of cyanobacteria for industrial purposes. First, the benefits of using cyanobacteria as a platform for chemical and biofuel production are discussed. Next, an overview of cyanobacterial strain improvements by genetic engineering is provided. Finally, mutagenesis techniques to improve the industrial potential of cyanobacteria are described. Along with providing an overview on various areas of research that are currently being investigated to improve the industrial potential of cyanobacteria, this review aims to elucidate potential targets for future research involving cyanobacteria as an industrial microorganism. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1357-1371, 2016.

Optimizing cyanobacteria growth conditions in a sealed environment to enable chemical inhibition tests with volatile chemicals.

Cyanobacteria are currently being engineered to photosynthetically produce next-generation biofuels and high-value chemicals. Many of these chemicals are highly toxic to cyanobacteria, thus strains with increased tolerance need to be developed. The volatility of these chemicals may necessitate that experiments be conducted in a sealed environment to maintain chemical concentrations. Therefore, carbon sources such as NaHCO3 must be used for supporting cyanobacterial growth instead of CO2 sparging. The primary goal of this study was to determine the optimal initial concentration of NaHCO3 for use in growth trials, as well as if daily supplementation of NaHCO3 would allow for increased growth. The secondary goal was to determine the most accurate method to assess growth of Anabaena sp. PCC 7120 in a sealed environment with low biomass titers and small sample volumes. An initial concentration of 0.5g/L NaHCO3 was found to be optimal for cyanobacteria growth, and fed-batch additions of NaHCO3 marginally improved growth. A separate study determined that a sealed test tube environment is necessary to maintain stable titers of volatile chemicals in solution. This study also showed that a SYTO® 9 fluorescence-based assay for cell viability was superior for monitoring filamentous cyanobacterial growth compared to absorbance, chlorophyll α (chl a) content, and biomass content due to its accuracy, small sampling size (100µL), and high throughput capabilities. Therefore, in future chemical inhibition trials, it is recommended that 0.5g/L NaHCO3 is used as the carbon source, and that culture viability is monitored via the SYTO® 9 fluorescence-based assay that requires minimum sample size.

Testing a dual-fluorescence assay to monitor the viability of filamentous cyanobacteria.

Filamentous cyanobacteria are currently being engineered to produce long-chain organic compounds, including 3rd generation biofuels. Because of their filamentous morphology, standard methods to quantify viability (e.g., plate counts) are not possible. This study investigated a dual-fluorescence assay based upon the LIVE/DEAD® BacLight™ Bacterial Viability Kit to quantify the percent viability of filamentous cyanobacteria using a microplate reader in a high throughput 96-well plate format. The manufacturer's protocol calls for an optical density normalization step to equalize the numbers of viable and non-viable cells used to generate calibration curves. Unfortunately, the isopropanol treatment used to generate non-viable cells released a blue pigment that altered absorbance readings of the non-viable cell solution, resulting in an inaccurate calibration curve. Thus we omitted this optical density normalization step, and carefully divided cell cultures into two equal fractions before the isopropanol treatment. While the resulting calibration curves had relatively high correlation coefficients, their use in various experiments resulted in viability estimates ranging from below 0% to far above 100%. We traced this to the apparent inaccuracy of the propidium iodide (PI) dye that was to stain only non-viable cells. Through further analysis via microplate reader, as well as confocal and wide-field epi-fluorescence microscopy, we observed non-specific binding of PI in viable filamentous cyanobacteria. While PI will not work for filamentous cyanobacteria, it is possible that other fluorochrome dyes could be used to selectively stain non-viable cells. This will be essential in future studies for screening mutants and optimizing photobioreactor system performance for filamentous cyanobacteria.

Characterization of five putative aspartate aminotransferase genes in the N2-fixing heterocystous cyanobacterium Anabaena sp. strain PCC 7120.

Aspartate and glutamate are two key amino acids used in biosynthesis of many amino acids that play vital role in cellular metabolism. Aspartate aminotransferases (AspATs) are required for channelling nitrogen (N(2)) between Glu and Asp in all life forms. Biochemical and genetic characterization of AspATs have been lacking in N(2)-fixing cyanobacteria. In this report, five putative AspAT genes (alr1039, all2340, alr2765, all4327 and alr4853) were identified in the N(2)-fixing heterocystous cyanobacterium Anabaena sp. PCC 7120. Five recombinant C-terminal hexahistidine-tagged AspATs (AspAT-H(6)) were overexpressed in Escherichia coli and purified to homogeneity. Biochemical analysis demonstrated that these five putative AspATs have authentic AspAT activity in vitro using aspartate as an amino donor. However, the enzymic activities of the five AspATs differed in vitro. Alr4853-H(6) showed the highest AspAT activity, while the enzymic activity for the other four AspATs ranged from 6.5 to 53.7 % activity compared to Alr4853 (100 %). Genetic characterization of the five AspAT genes was also performed by inactivating each individual gene. All of the five AspAT knockout mutants exhibited reduced diazotrophic growth, and alr4853 was further identified to be a Fox gene (requiring fixed N(2) for growth in the presence of oxygen). Four out of five P(aspAT)-gfp transcriptional fusions were constitutively expressed in both diazotrophic and nitrate-dependent growth conditions. Quantitative reverse transcriptase PCR showed that alr4853 expression was increased by 2.3-fold after 24 h of N(2) deprivation. Taken together, these findings add to our understanding of the role of AspATs in N(2)-fixing within heterocystous cyanobacteria.

Simultaneous gene inactivation and promoter reporting in cyanobacteria.

Determining spatiotemporal gene expression and analyzing knockout mutant phenotypes have become powerful tools in elucidating the function of genes; however, genetic approaches for simultaneously inactivating a gene and monitoring its expression have not been reported in the literature. In this study, we designed a dual-functional gene knockout vector pZR606 that contains a multiple cloning site (MCS) for inserting the internal fragment of a target gene, with a gfp gene as its transcriptional marker located immediately downstream of the MCS. By using this gene knockout system, we inactivated ava_2679 from Anabaena variabilis ATCC 29413, as well as all2508, alr2887, alr3608, and all4388 from Anabaena sp. strain PCC 7120. The ava_2679 knockout mutant fails to grow diazotrophically. Morphological analysis of ava_2679 knockout mutant after nitrogen step-down revealed defective junctions between heterocysts and adjacent vegetative cells, and the heterocyst was 1.53-fold longer compared to wild-type heterocysts. The alr2887, all4388, and alr3608 mutant colonies turned yellow and showed lack of protracted growth when deprived of fixed nitrogen, consistent with the previous reports that alr2887, all4388, and alr3608 are Fox genes. The all2508 encodes a GTP-binding elongation factor (EF4/LepA), and its knockout mutant exhibited reduced diazotrophic growth. The heterocyst development of all2508 knockout was significantly delayed, and only about 4.0 % of vegetative cells differentiated to heterocysts after nitrogen deprivation for 72 h, decreased 49.6 % compared to wild-type. Thus, we discovered that All2508 may regulate heterocyst development spatiotemporally. Concurrently, the GFP reporter revealed that all five target gene expressions were up-regulated in response to nitrogen deprivation. We demonstrated that the pZR606-based specific gene knockout approach worked effectively for the five selected genes, including four previously identified Fox genes or Fox gene homolog, and a previously unknown function of gene all2508. Thus, gene expression and phenotypic analysis of mutants can be achieved simultaneously by targeted gene inactivation using the pZR606-based system. This combined approach for targeted gene inactivation and its promoter reporting with GFP may be broadly applicable to the study of gene function in other prokaryotic organisms.

Genetically engineering cyanobacteria to convert CO2, water, and light into the long-chain hydrocarbon farnesene.

Genetically engineered cyanobacteria offer a shortcut to convert CO2 and H2O directly into biofuels and high value chemicals for societal benefits. Farnesene, a long-chained hydrocarbon (C15H24), has many applications in lubricants, cosmetics, fragrances, and biofuels. However, a method for the sustainable, photosynthetic production of farnesene has been lacking. Here, we report the photosynthetic production of farnesene by the filamentous cyanobacterium Anabaena sp. PCC 7120 using only CO2, mineralized water, and light. A codon-optimized farnesene synthase gene was chemically synthesized and then expressed in the cyanobacterium, enabling it to synthesize farnesene through its endogenous non-mevalonate (MEP) pathway. Farnesene excreted from the engineered cyanobacterium volatilized into the flask head space and was recovered by adsorption in a resin column. The maximum photosynthetic productivity of farnesene was 69.1 ± 1.8 µg·L(-1)·O.D.(-1)·d(-1). Compared to the wild type, the farnesene-producing cyanobacterium also exhibited a 60 % higher PSII activity under high light, suggesting increased farnesene productivity in such conditions. We envision genetically engineered cyanobacteria as a bio-solar factory for photosynthetic production of a wide range of biofuels and commodity chemicals.

Residues in conserved loops of intramembrane metalloprotease SpoIVFB interact with residues near the cleavage site in pro-σK.

Intramembrane metalloproteases (IMMPs) control critical biological processes by cleaving membrane-associated proteins within a transmembrane segment or at a site near the membrane surface. Phylogenetic analysis divides IMMPs into four groups. SpoIVFB is a group III IMMP that regulates Bacillus subtilis endospore formation by cleaving Pro-σ(K) and releasing the active sigma factor from a membrane. To elucidate the enzyme-substrate interaction, single-cysteine versions of catalytically inactive SpoIVFB and C-terminally truncated Pro-σ(K)(1-126) (which can be cleaved by active SpoIVFB) were coexpressed in Escherichia coli, and proximity was tested by disulfide cross-linking in vivo. As expected, the results provided evidence that catalytic residue Glu-44 of SpoIVFB is near the cleavage site in the substrate. Also near the cleavage site were two residues of SpoIVFB in predicted conserved loops; Pro-135 in a short loop and Val-70 in a longer loop. Pro-135 corresponds to Pro-399 of RseP, a group I IMMP, and Pro-399 was reported previously to interact with substrate near the cleavage site, suggesting a conserved interaction across IMMP subfamilies. Val-70 follows a newly recognized conserved motif, PXGG (X is a large hydrophobic residue), which is in a hydrophobic region predicted to be a membrane reentrant loop. Following the hydrophobic region is a negatively charged region that is conserved in IMMPs of groups I and III. At least two residues with a negatively charged side chain are required in this region for activity of SpoIVFB. The region exhibits other features in IMMPs of groups II and IV. Its possible roles, as well as that of the short loop, are discussed. New insights into IMMP-substrate interaction build toward understanding how IMMPs function and may facilitate manipulation of their activity.

Features of Pro-σK important for cleavage by SpoIVFB, an intramembrane metalloprotease.

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-σ(K) during sporulation. To elucidate features of Pro-σ(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-σ(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-σ(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-σ(K) orthologs. SpoIVFB did not tolerate an aromatic residue at P1 or P2' of Pro-σ(K). A Lys residue at P3' of Pro-σ(K) could not be replaced with Ala unless a Lys was provided farther C-terminally (e.g., at P9'). α-Helix-destabilizing residues near the cleavage site were not crucial for SpoIVFB to cleave Pro-σ(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.

Identification and characterization of five intramembrane metalloproteases in Anabaena variabilis.

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-σ(K), a Bacillus subtilis MTF, in our reconstituted Escherichia coli system. The enzymatic activities of processing pro-σ(K) differ among these five S2Ps(Av). Substitution of glutamic acid (E) by glutamine (Q) in the conserved HEXXH zinc-coordinated 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-σ(K) by Ava_4785 and Ava_1797 revealed that Ava_4785 and Ava_1797 recognized the same cleavage site in pro-σ(K) as SpoIVFB, a cognate S2P of pro-σ(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.

Intramembrane proteolytic cleavage of a membrane-tethered transcription factor by a metalloprotease depends on ATP.

Regulated intramembrane proteolysis (RIP) involves cleavage of a transmembrane segment of a protein. RIP governs diverse processes in a wide variety of organisms and is carried out by different types of intramembrane proteases (IPs), including a large family of metalloproteases. The Bacillus subtilis SpoIVFB protein is a putative metalloprotease that cleaves membrane-tethered Pro-sigma(K), releasing sigma(K) to direct transcription of genes necessary for spore formation. By attaching an extra transmembrane segment to the N terminus of SpoIVFB, expression in E. coli was improved more than 100-fold, facilitating purification and demonstration of metalloprotease activity, which accurately cleaved purified Pro-sigma(K). Uniquely for IPs examined so far, SpoIVFB activity requires ATP, which binds to the C-terminal cystathionine-beta-synthase (CBS) domain of SpoIVFB. Deleting just 10 residues from the C-terminal end of SpoIVFB nearly eliminated cleavage of coexpressed Pro-sigma(K) in E. coli. The CBS domain of SpoIVFB was shown to interact with Pro-sigma(K) and ATP changed the interaction, suggesting that ATP regulates substrate access to the active site and renders cleavage sensitive to the cellular energy level, which may be a general feature of CBS-domain-containing IPs. Incorporation of SpoIVFB into preformed liposomes stimulated its ability to cleave Pro-sigma(K). Cleavage depended on ATP and the correct peptide bond was shown to be cleaved using a rapid and sensitive mass spectrometry assay. A system for biochemical studies of RIP by a metalloprotease in a membrane environment has been established using methods that might be applicable to other IPs.

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 sigma(F), sigma(E), sigma(G), and sigma(K) coordinate much of the developmental process. The C. perfringens cpe gene, encoding CPE, is transcribed from three promoters, where P1 was proposed to be sigma(K) dependent, while P2 and P3 were proposed to be sigma(E) 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 sigma(E)-associated RNA polymerase and sigma(K)-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 sigma(E)-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.