Ruanbao Zhou

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 others 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.

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.

Engineering cyanobacteria for the production of a cyclic hydrocarbon fuel from CO2 and H2O

Cyclic hydrocarbons are a critical component of petroleum fuels. However, biofuels produced by current biochemical and thermochemical processes contain small amounts of cyclic hydrocarbons, and can only provide the requisite performance characteristics with the addition of petroleum fuels to them. Limonene (C10H16) is a cyclic monoterpene that possesses attractive characteristics as a biodiesel and a jet fuel. Current strategies for harvesting limonene from plant biomass require arable land, high energy input, inefficient multiple-step processes, and the release of CO2 as a greenhouse gas. This research focuses on a direct photons-to-product approach for biofuel production by metabolically engineering a cyanobacterium as the cellular machinery to over-produce and secrete valuable compounds using CO2, mineralized H2O, and light. As a proof of concept, we have engineered the filamentous, nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 to synthesize and secrete limonene by introducing a plant limonene synthase gene (lims) from Sitka spruce. Our data revealed that limonene produced by the engineered cyanobacterium was secreted across the cell membrane and volatilized into the headspace, allowing for easy separation of the target compound from the culture biomass. Furthermore, a synthetic DXP operon (dxs-ipphp-gpps) encoding three rate-limiting enzymes from the MEP pathway was co-expressed with lims to re-route carbon flux from the Calvin cycle into limonene synthesis. Under higher light (150 μE m−2 s−1), we observed a 6.8-fold increase in limonene yield and an 8.8-fold increase in the maximum limonene production rate when expressing the DXP operon in conjunction with lims, compared to lims alone, and achieved a maximum production rate of 3.6 ± 0.5 μg limonene L−1 O.D.−1 h−1. This limonene-producing Anabaena has about three times higher photosystem II activity than its wild-type. These results demonstrated that increasing the light intensity and metabolic flux improves limonene productivity in the engineered cyanobacterium. We envision that the method of using N2-fixing cyanobacteria as a cellular factory and CO2 and N2 as sustainable feedstock can be applied for the production of a wide range of commodity chemicals and drop-in-fuels.

Identification of an Akinete Marker Gene in Anabaena variabilis

Cyanobacteria that form akinetes as well as heterocysts present a rare opportunity to investigate the relationships between alternative differentiation processes and pattern formation processes in a single bacterium. Because no akinete marker gene has been identified, akinete formation has been little studied genetically. We report the first identification of an akinete marker gene.

Serine proteases from two cell types target different components of a complex that governs regulated intramembrane proteolysis of pro-σK during Bacillus subtilis development

Upon starvation Bacillus subtilis undergoes a developmental process involving creation of two cell types, the mother cell and forespore. A signal in the form of a serine protease, SpoIVB, is secreted from the forespore and leads to regulated intramembrane proteolysis (RIP) of pro‐σK, releasing active σK into the mother cell. RIP of pro‐σK is carried out by a membrane‐embedded metalloprotease, SpoIVFB, which is inactive when bound by BofA and SpoIVFA. We have investigated the mechanism by which this complex is activated. By expressing components of the signalling pathway in Escherichia coli, we reconstructed complete inhibition of pro‐σK RIP by BofA and SpoIVFA, and found that SpoIVB serine protease activity could partially restore RIP, apparently by targeting SpoIVFA. Pulse‐chase experiments demonstrated that SpoIVFA synthesized early during B. subtilis sporulation is lost in a SpoIVB‐dependent fashion, coincident with the onset of pro‐σK RIP, supporting the idea that SpoIVB targets SpoIVFA to trigger RIP of pro‐σK. Loss of BofA depended not only on SpoIVB, but also on CtpB, a serine protease secreted from the mother cell. CtpB appeared to cleave BofA near its C‐terminus upon coexpression in E. coli, and purified CtpB degraded BofA. We propose that RIP of pro‐σK involves a three‐step proteolytic cascade in which SpoIVB first cleaves SpoIVFA, CtpB then cleaves BofA and finally SpoIVFB cleaves pro‐σK.

Anabaena sp. strain PCC 7120 gene devH is required for synthesis of the heterocyst glycolipid layer.

In response to deprivation for fixed nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 provides a microoxic intracellular environment for nitrogen fixation through the differentiation of semiregularly spaced vegetative cells into specialized cells called heterocysts. The devH gene is induced during heterocyst development and encodes a product with characteristics of a trans-acting regulatory protein. A devH mutant forms morphologically distinguishable heterocysts but is Fox(-), incapable of nitrogen fixation in the presence of oxygen. We demonstrate that rearrangements of nitrogen fixation genes take place normally in the devH mutant and that it is Fix(+), i.e., has nitrogenase activity under anoxic conditions. The Fox(-) phenotype was shown by ultrastructural studies to be associated with the absence of the glycolipid layer of the heterocyst envelope. The expression of glycolipid biosynthetic genes in the mutant is greatly reduced, and heterocyst glycolipids are undetectable.

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-σK, releasing σK 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-σK. Uniquely for IPs examined so far, SpoIVFB activity requires ATP, which binds to the C-terminal cystathionine-β-synthase (CBS) domain of SpoIVFB. Deleting just 10 residues from the C-terminal end of SpoIVFB nearly eliminated cleavage of coexpressed Pro-σK in E. coli. The CBS domain of SpoIVFB was shown to interact with Pro-σK 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-σK. 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.

Paired cloning vectors for complementation of mutations in the cyanobacterium Anabaena sp. strain PCC 7120.

The clones generated in a sequencing project represent a resource for subsequent analysis of the organism whose genome has been sequenced. We describe an interrelated group of cloning vectors that either integrate into the genome or replicate, and that enhance the utility, for developmental and other studies, of the clones used to determine the genomic sequence of the cyanobacterium, Anabaena sp. strain PCC 7120. One integrating vector is a mobilizable BAC vector that was used both to generate bridging clones and to complement transposon mutations. Upon addition of a cassette that permits mobilization and selection, pUC-based sequencing clones can also integrate into the genome and thereupon complement transposon mutations. The replicating vectors are based on cyanobacterial plasmid pDU1, whose sequence we report, and on broad-host-range plasmid RSF1010. The RSF1010- and pDU1-based vectors provide the opportunity to express different genes from either cell-type-specific or -generalist promoters, simultaneously from different plasmids in the same cyanobacterial cells. We show that pDU1 ORF4 and its upstream region play an essential role in the replication and copy number of pDU1, and that ORFs alr2887 and alr3546 (hetFA) of Anabaena sp. are required specifically for fixation of dinitrogen under oxic conditions.

Evidence That the Bacillus subtilis SpoIIGA Protein Is a Novel Type of Signal-transducing Aspartic Protease

The bacterium Bacillus subtilis undergoes endospore formation in response to starvation. σ factors play a key role in spatiotemporal regulation of gene expression during development. Activation of σ factors is coordinated by signal transduction between the forespore and the mother cell. σE is produced as pro-σE, which is activated in the mother cell by cleavage in response to a signal from the forespore. We report that expression of SpoIIR, a putative signaling protein normally made in the forespore, and SpoIIGA, a putative protease, is necessary and sufficient for accurate, rapid, and abundant processing of pro-σE to σE in Escherichia coli. Modeling and mutational analyses provide evidence that SpoIIGA is a novel type of aspartic protease whose C-terminal half forms a dimer similar to the human immunodeficiency virus type 1 protease. Previous studies suggest that the N-terminal half of SpoIIGA is membrane-embedded. We found that SpoIIGA expressed in E. coli is membrane-associated and that after detergent treatment SpoIIGA was self-associated. Also, SpoIIGA interacts with SpoIIR. The results support a model in which SpoIIGA forms inactive dimers or oligomers, and interaction of SpoIIR with the N-terminal domain of SpoIIGA on one side of a membrane causes a conformational change that allows formation of active aspartic protease dimer in the C-terminal domain on the other side of the membrane, where it cleaves pro-σE.

Substrate Requirements for Regulated Intramembrane Proteolysis of Bacillus subtilis Pro-σK

During sporulation of Bacillus subtilis, pro-sigmaK is activated by regulated intramembrane proteolysis (RIP) in response to a signal from the forespore. RIP of pro-sigmaK removes its prosequence (amino acids 1 to 20), releasing sigmaK 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-sigmaK to serve as a substrate for RIP were investigated by producing C-terminally truncated pro-sigmaK 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-sigmaK (amino acids 1 to 117), including part of sigma factor region 2.4, was required for RIP of pro-sigmaK-GFP chimeras in sporulating B. subtilis. Likewise, pro-sigmaK-hexahistidine chimeras demonstrated that the N-terminal 117 amino acids of pro-sigma(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-sigmaK-GFP chimera. Addition or deletion of five amino acids near the N terminus allowed accurate processing of pro-sigmaK, 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-sigmaK and prevented detectable RIP by SpoIVFB. These results elucidate substrate requirements for RIP of pro-sigmaK by SpoIVFB and may have implications for substrate recognition by other S2P family members.