Metabolic Engineering of Saccharomyces cerevisiae for Production of Novel Cyanophycins with an Extended Range of Constituent Amino Acids

Cyanophycin (multi-l-arginyl-poly-l-aspartic acid; also known as cyanophycin grana peptide [CGP]) is a putative precursor for numerous biodegradable technically used chemicals. Therefore, the biosynthesis and production of the polymer in recombinant organisms is of special interest. The synthesis of cyanophycin derivatives consisting of a wider range of constituents would broaden the applications of this polymer. We applied recombinant Saccharomyces cerevisiae strains defective in arginine metabolism and expressing the cyanophycin synthetase of Synechocystis sp. strain PCC 6308 in order to synthesize CGP with citrulline and ornithine as constituents. Strains defective in arginine degradation (Car1 and Car2) accumulated up to 4% (wt/wt) CGP, whereas strains defective in arginine synthesis (Arg1, Arg3, and Arg4) accumulated up to 15.3% (wt/wt) of CGP, which is more than twofold higher than the previously content reported in yeast and the highest content ever reported in eukaryotes. Characterization of the isolated polymers by different analytical methods indicated that CGP synthesized by strain Arg1 (with argininosuccinate synthetase deleted) consisted of up to 20 mol% of citrulline, whereas CGP from strain Arg3 (with ornithine carbamoyltransferase deleted) consisted of up to 8 mol% of ornithine, and CGP isolated from strain Arg4 (with argininosuccinate lyase deleted) consisted of up to 16 mol% lysine. Cultivation experiments indicated that the incorporation of citrulline or ornithine is enhanced by the addition of low amounts of arginine (2 mM) and also by the addition of ornithine or citrulline (10 to 40 mM), respectively, to the medium.Cyanophycin (multi-l-arginyl-poly-[l-aspartic acid]), also referred to as cyanophycin grana peptide (CGP), represents a polydisperse nonribosomally synthesized polypeptide consisting of poly(aspartic acid) as backbone and arginine residues bound to each aspartate (49) (Fig. ​(Fig.1).1). One enzyme only, referred to as cyanophycin synthetase (CphA), catalyzes the synthesis of the polymer from amino acids (55). Several CphAs originating from different bacteria exhibit specific features (2, 7, 5, 32, 50, 51). CphAs from the cyanobacteria Synechocystis sp. strain PCC 6308 and Anabaena variabilis ATCC 29413, respectively, exhibit a wide substrate range in vitro (2, 7), whereas CphA from Acinetobacter baylyi or Nostoc ellipsosporum incorporates only aspartate and arginine (23, 24, 32). CphA from Thermosynechococcus elongatus catalyzes the synthesis of CGP primer independently (5); CphA from Synechococcus sp. strain MA19 exhibits high thermostability (22). Furthermore, two types of CGP were observed concerning its solubility behavior: (i) a water-insoluble type that becomes soluble at high or low pH (34, 48) and (ii) a water-soluble type that was only recently observed in recombinant organisms (19, 26, 42, 50, 56). In the past, bacteria were mainly applied for the synthesis of CGP (3, 14, 18, 53), whereas recently there has been greater interest in synthesis in eukaryotes (26, 42, 50). CGP was accumulated to almost 7% (wt/wt) of dry matter in recombinant Nicotiana tabacum and Saccharomyces cerevisiae (26, 50).Open in a separate windowFIG. 1.Chemical structures of dipeptide building blocks of CGP variants detected in vivo. Structure: 1, aspartate-arginine; 2, aspartate-lysine; 3, aspartate-citrulline; 4, aspartate-ornithine. Aspartic acid is presented in black; the second amino acid of the dipeptide building blocks is shown in gray. The nomenclature of the carbon atoms is given.In S. cerevisiae the arginine metabolism is well understood and has been investigated (30) (see Fig. ​Fig.2).2). Arginine is synthesized from glutamate via ornithine and citrulline in eight successive steps. The enzymes acetylglutamate synthase, acetylglutamate kinase, N-acetyl-γ-glutamylphosphate reductase, and acetylornithine aminotransferase are involved in the formation of N-α-acetylornithine. The latter is converted to ornithine by acetylornithine acetyltransferase. In the next step, ornithine carbamoyltransferase (ARG3) condenses ornithine with carbamoylphosphate, yielding citrulline. Citrulline is then converted to l-argininosuccinate by argininosuccinate synthetase. The latter is in the final step cleaved into fumarate and arginine by argininosuccinate lyase (ARG4). The first five steps occur in the mitochondria, whereas the last three reactions occur in the cytosol (28, 54). Arginine degradation is initiated by arginase (CAR1) and ornithine aminotransferase (CAR2) (10, 11, 38, 39).Open in a separate windowFIG. 2.Schematic overview of the arginine metabolism in S. cerevisiae. Reactions shown in the shaded area occur in the mitochondria, while the other reactions are catalyzed in the cytosol. Abbreviations: ARG2, acetylglutamate synthase; ARG6, acetylglutamate kinase; ARG5, N-acetyl-γ-glutamyl-phosphate reductase; ARG8, acetylornithine aminotransferase; ECM40, acetylornithine acetyltransferase; ARG1, argininosuccinate synthetase; ARG3, ornithine carbamoyltransferase; ARG4, argininosuccinate lyase; CAR1, arginase; CAR2, ornithine aminotransferase.A multitude of putative applications for CGP derivatives are available (29, 41, 45, 47), thus indicating a need for efficient biotechnological production and for further investigations concerning the synthesis of CGP with alternative properties and different constituents. It is not only the putative application of the polymer as a precursor for poly(aspartic acid), which is used as biodegradable alternative for poly(acrylic acid) or for bulk chemicals, that makes CGP interesting (29, 45-47). In addition, a recently developed process for the production of dipeptides from CGP as a precursor makes the synthesis of CGP variants worthwhile (43). Dipeptides play an important role in medicine and pharmacy, e.g., as additives for malnourished patients, as treatments against liver diseases, or as aids for muscle proliferation (43). Because dipeptides are synthesized chemically (40) or enzymatically (6), novel biotechnological production processes are welcome.     

Cloning of a Novel Pyrethroid-Hydrolyzing Carboxylesterase Gene from Sphingobium sp. Strain JZ-1 and Characterization of the Gene Product

A novel esterase gene, pytH, encoding a pyrethroid-hydrolyzing carboxylesterase was cloned from Sphingobium sp. strain JZ-1. The gene contained an open reading frame of 840 bp. Sequence identity searches revealed that the deduced enzyme shared the highest similarity with many α/β-hydrolase fold proteins (20 to 24% identities). PytH was expressed in Escherichia coli BL21 and purified using Ni-nitrilotriacetic acid affinity chromatography. It was a monomeric structure with a molecular mass of approximately 31 kDa and a pI of 4.85. PytH was able to transform p-nitrophenyl esters of short-chain fatty acids and a wide range of pyrethroid pesticides, and isomer selectivity was not observed. No cofactors were required for enzyme activity.Pyrethroid pesticides are now the major class of insecticides used for insect control in agriculture and households as a replacement for more toxic organophosphorus pesticides, and their usage is continuing to grow (10). Although pyrethroid pesticides generally have lower acute oral mammalian toxicity than organophosphate insecticides, exposure to very high levels of pyrethroid pesticides might cause endocrine disruption, lymph node and spleen damage, and carcinogenesis (6, 12). In addition, most pyrethroid pesticides possess acute toxicity to some nontarget organisms, such as bees, fish, and aquatic invertebrates, often at concentrations of less than 0.5 μg/kg (19, 22). Great concerns have been raised about the persistence and degradation of pyrethroid pesticides in the environment.In general, pyrethroid pesticides are degraded by both abiotic and biotic pathways, including photooxidation, chemical oxidation, and biodegradation. Microorganisms play the most important role in degradation of pyrethroids in soils and sediments. Many pyrethroid-degrading microorganisms have been isolated from soils (13, 16, 24, 27).The major routes of pyrethroid metabolism in pyrethroid-resistant insects and pyrethroid-degrading microorganisms include oxidation by cytochrome P450s and ester hydrolysis by carboxylesterases (9). Carboxylesterases are a family of enzymes that are important in the hydrolysis of a large number of endogenous and xenobiotic ester-containing compounds, such as carbamates, organophosphorus pesticides, and pyrethroids. Carboxylesterases from Bacillus cereus SM3 (17), Aspergillus niger ZD11 (13), Nephotettix cincticeps (2), and mouse liver microsomes (23) hydrolyzing the carboxyl ester linkage of the pyrethroids were purified to homogeneity and characterized. Genes encoding the pyrethroid-hydrolyzing carboxylesterases from mouse liver microsomes and Klebsiella sp. strain ZD112 were cloned and functionally expressed (23, 27).Pyrethroids differ from many other pesticides in that they contain one to three chiral centers; the chirality may arise from the acid moiety, the alcohol moiety, or both (Fig. ​(Fig.1).1). A pyrethroid compound therefore consists of two to eight isomers. Isomers of a chiral compound often differ from each other in biological properties. Isomer selectivity has been widely observed in insecticidal activity for the isomers of a pyrethroid compound. Recently, studies have shown that biodegradation of pyrethroids also exhibits significant isomer selectivity (15, 23).Open in a separate windowFIG. 1.Molecular structures of pyrethroids tested. Chiral centers are indicated by black dots.In this study, we described the isolation and identification of the pyrethroid-degrading Sphingobium sp. strain JZ-1, the cloning and expression of the gene pytH encoding a novel pyrethroid-hydrolyzing carboxylesterase, and the characterization of the purified enzyme.     

Improved Squalene Production via Modulation of the Methylerythritol 4-Phosphate Pathway and Heterologous Expression of Genes from Streptomyces peucetius ATCC 27952 in Escherichia coli

Putative hopanoid genes from Streptomyces peucetius were introduced into Escherichia coli to improve the production of squalene, an industrially important compound. High expression of hopA and hopB (encoding squalene/phytoene synthases) together with hopD (encoding farnesyl diphosphate synthase) yielded 4.1 mg/liter of squalene. This level was elevated to 11.8 mg/liter when there was also increased expression of dxs and idi, E. coli genes encoding 1-deoxy-d-xylulose 5-phosphate synthase and isopentenyl diphosphate isomerase.Squalene, an industrially important compound obtained primarily from the liver oil of deep-sea sharks and whales, is an important ingredient in skin cosmetics due to its photoprotective role (2, 7). The decreased cancer risk associated with high olive oil consumption could result from high squalene content (12, 16). Squalene has a chemopreventive effect on colon cancer (14). Moreover, squalene has wide applications in fine chemicals, magnetic tape, and low-temperature lubricants and as an additive in animal feed (1).The use of shark liver oil is limited, due to the presence of environmental pollutants, such as polychlorinated biphenyls, heavy metals, and methylmercury residues, as well as an unpleasant fishy odor and taste (17, 19). Moreover, the presence of similar compounds, such as cholesterol, in the oils from marine animal liver can make squalene purification difficult. In addition, squalene production is limited by uncertain availability because of international concern for the protection of marine animals. Squalene has also been obtained from plant sources (4, 10, 11, 18), but very few methods can produce sufficient quantities at the desired purity level for pharmaceutical and industrial applications (6). The use of engineered microbial cell factories for the biosynthesis of squalene may be a suitable alternative to address these issues.In the genome project for Streptomyces peucetius ATCC 27952, a cluster of genes which comprises five open reading frames, encoding hopanoid biosynthesis, has been detected and annotated. Even though these open reading frames share sequence homology with genes involved in hopanoid biosynthesis, no plausible hopanoid products have been isolated from S. peucetius in all laboratory cultures. Therefore, the hopanoid biosynthetic gene cluster of S. peucetius was considered “cryptic” in the present study. We were interested in activating the so-called “cryptic” hopanoid biosynthetic gene cluster of S. peucetius to produce pharmaceutically important compounds by using genetic engineering tools. Isoprenoid production in Escherichia coli has been extensively studied and reviewed (5, 8, 9, 15, 20, 21), but very few reports detail squalene formation in E. coli by the use of exogenous genes (13). In the present study, we introduced three cryptic genes (hopABD) from the hopanoid biosynthesis gene cluster from S. peucetius that catalyzed squalene production and also modulated the 2-C-methyl-d-erythritol 4-phosphate pathway in E. coli to enhance squalene production (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Schematic representation of the engineered squalene biosynthetic pathway in E. coli BL21(DE3). DXS and IDI were overexpressed for the modulation of the MEP pathway to increase and balance the IPP pool, HopD (farnesyl diphosphate synthase) was overexpressed to increase FPP, and HopA and HopB (HopAB) (squalene synthases) were overexpressed to overproduce squalene. The recombinant enzymes overexpressed in E. coli are boxed.     

Exploiting the Natural Diversity of Microviridin Gene Clusters for Discovery of Novel Tricyclic Depsipeptides

Microviridins are ribosomally synthesized tricyclic depsipeptides produced by different genera of cyanobacteria. The prevalence of the microviridin gene clusters and the natural diversity of microviridin precursor sequences are currently unknown. Screening of laboratory strains and field samples of the bloom-forming freshwater cyanobacterium Microcystis via PCR revealed global occurrence of the microviridin pathway and an unexpected natural variety. We could detect 15 new variants of the precursor gene mdnA encoding microviridin backbones that differ in up to 4 amino acid positions from known isoforms of the peptide. The survey not only provides insights into the versatility of the biosynthetic enzymes in a closely related group of cyanobacteria, but also facilitates the discovery and characterization of cryptic microviridin variants. This is demonstrated for microviridin L in Microcystis aeruginosa strain NIES843 and heterologously produced variants.Bloom-forming freshwater cyanobacteria are a rich source of natural products (30). They flourish in lakes and ponds of different temperate zones, where they can eventually form thick scums at the surface. Microcystis is one of the predominant genera composing these blooms, particularly in lakes suffering from eutrophication (17). Like other bloom-forming species, it is infamous for the production of the hepatotoxin microcystin, a nonribosomal peptide toxin that inhibits protein phosphatases in a broad range of eukaryotes from zooplankton to humans (1, 9). Moreover, Microcystis is known to produce a multitude of other peptides that are considered to be potential drug leads (14, 30).Microviridins form one of the most intriguing classes of peptides, since they feature a cage-like structure (e.g., microviridin B) (Fig. ​(Fig.1A).1A). The highly unusual tricyclic architecture results from ω-ester and ω-amide bonds between amino acid side chains. The past few years have afforded 13 variants of the peptide from both field samples and laboratory strains (5, 8, 13, 15, 21, 22). Most of the variants show inhibitory activities against serine-type proteases, most notably against elastase, which is a target enzyme in the treatment of lung emphysema (28). One of the peptide isoforms, microviridin J, has been shown to inhibit the molting process of Daphnia, leading to death of the animals (23).Open in a separate windowFIG. 1.(A) Representative structure of microviridin B and three-dimensional model. (B) Schematic representation of the microviridin gene cluster of M. aeruginosa NIES298. The positions of primers for the amplification of mdnA, -B, and -C are indicated. GNAT, GCN5-related N-acetyltransferase. (C) PCR gel picture showing the amplification of mdnB from a selection of laboratory strains.Recent studies have revealed a unique biosynthetic mechanism for microviridins in Microcystis and the filamentous cyanobacterial genus Planktothrix (18, 31). The 14-amino-acid (aa) peptide sequence is encoded at the C terminus of the ribosomal precursor peptide MdnA (31). Macrocyclization of the peptide depends on the activities of two ATP grasp-type ligases, MdnB and -C, that are encoded downstream of the precursor gene. Further enzymes encoded by the cluster include the N-acetyltransferase MdnD and the putative transporter-peptidase MdnE (31) (Fig. ​(Fig.1B).1B). The activities of the enzymes MdnB, -C, and -D were confirmed by heterologous production of microviridins in Escherichia coli (31). The MdnB and -C orthologs of the filamentous cyanobacterial strain Planktothrix agardhii CYA126, MvdC and MvdD, were characterized using synthetic precursor peptides in vitro (18). Furthermore, Philmus et al. have shown limited flexibility of MvdD, catalyzing two rounds of microviridin lactonization to accept substrates with altered amino acid compositions and a stringent ring size requirement (19).Recent genome-sequencing projects with cyanobacteria have revealed widespread occurrence of microviridin-related gene clusters in various cyanobacterial genera, although the production of microviridins by these strains remains to be shown (18, 24, 31). Here, we present data showing an unprecedented natural diversity of microviridin precursor genes in a set of closely related Microcystis laboratory strains and field samples. The insights gained from our survey provide lessons about the flexibility of the microviridin ligases in a small group of cyanobacteria and an estimate of the size of the natural microviridin library that still awaits discovery. The detection of novel natural microviridin prepeptides from Microcystis can ultimately guide the heterologous production of novel microviridins in E. coli. As proof of principle, we have identified and characterized the new variant microviridin L from the strain Microcystis aeruginosa NIES843.     

Malonic Semialdehyde Reductase,Succinic Semialdehyde Reductase,and Succinyl-Coenzyme A Reductase from Metallosphaera sedula: Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in Sulfolobales

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates during autotrophic CO₂ fixation in various members of the Crenarchaea. In this cycle, as determined using Metallosphaera sedula, malonyl-coenzyme A (malonyl-CoA) and succinyl-CoA are reductively converted via their semialdehydes to the corresponding alcohols 3-hydroxypropionate and 4-hydroxybutyrate. Here three missing oxidoreductases of this cycle were purified from M. sedula and studied. Malonic semialdehyde reductase, a member of the 3-hydroxyacyl-CoA dehydrogenase family, reduces malonic semialdehyde with NADPH to 3-hydroxypropionate. The latter compound is converted via propionyl-CoA to succinyl-CoA. Succinyl-CoA reduction to succinic semialdehyde is catalyzed by malonyl-CoA/succinyl-CoA reductase, a promiscuous NADPH-dependent enzyme that is a paralogue of aspartate semialdehyde dehydrogenase. Succinic semialdehyde is then reduced with NADPH to 4-hydroxybutyrate by succinic semialdehyde reductase, an enzyme belonging to the Zn-dependent alcohol dehydrogenase family. Genes highly similar to the Metallosphaera genes were found in other members of the Sulfolobales. Only distantly related genes were found in the genomes of autotrophic marine Crenarchaeota that may use a similar cycle in autotrophic carbon fixation.The thermoacidophilic autotrophic crenarchaeum Metallosphaera sedula uses a 3-hydroxypropionate/4-hydroxybutyrate cycle for CO₂ fixation (9, 28, 29, 35) (Fig. ​(Fig.1).1). A similar cycle may operate in other autotrophic members of the Sulfolobales (31) and in mesophilic marine group I Crenarchaea (Cenarchaeum sp., Nitrosopumilus sp.). This cycle uses elements of the 3-hydroxypropionate cycle that was originally discovered in the phototrophic bacterium Chloroflexus aurantiacus (15, 22-25, 41, 42). It involves the carboxylation of acetyl coenzyme A (acetyl-CoA) to malonyl-CoA by a biotin-dependent acetyl-CoA carboxylase (12, 29). The carboxylation product is reduced to malonic semialdehyde by malonyl-CoA reductase (1). Malonic semialdehyde is further reduced to 3-hydroxypropionate, the characteristic intermediate of the pathway (9, 31, 35). 3-Hydroxypropionate is further reductively converted to propionyl-CoA (3), which is carboxylated to (S)-methylmalonyl-CoA by propionyl-CoA carboxylase. Only one copy of the genes encoding the acetyl-CoA/propionyl-CoA carboxylase subunits is present in most Archaea, indicating that this enzyme is a promiscuous enzyme that acts on both acetyl-CoA and propionyl-CoA (12, 29). (S)-Methylmalonyl-CoA is isomerized to (R)-methylmalonyl-CoA, which is followed by carbon rearrangement to succinyl-CoA catalyzed by coenzyme B₁₂-dependent methylmalonyl-CoA mutase.Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle in M. sedula and other autotrophic Sulfolobales. Enzymes: 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonate semialdehyde reductase (NADPH); 4, 3-hydroxypropionate-CoA ligase (AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, propionyl-CoA carboxylase, identical to acetyl-CoA carboxylase; 8, (S)-methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH), identical to malonyl-CoA reductase; 11, succinic semialdehyde reductase (NADPH); 12, 4-hydroxybutyrate-CoA ligase (AMP forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14, crotonyl-CoA hydratase; 15, (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 16, acetoacetyl-CoA β-ketothiolase. The highlighted steps are catalyzed by the enzymes studied here.Succinyl-CoA is converted via succinic semialdehyde and 4-hydroxybutyrate to two molecules of acetyl-CoA (9), thus regenerating the starting CO₂ acceptor molecule and releasing another acetyl-CoA molecule for biosynthesis. Hence, the 3-hydroxypropionate/4-hydroxybutyrate cycle (Fig. ​(Fig.1)1) can be divided into two parts. The first part transforms one acetyl-CoA molecule and two bicarbonate molecules into succinyl-CoA (Fig. ​(Fig.1,1, steps 1 to 9), and the second part converts succinyl-CoA to two acetyl-CoA molecules (Fig. ​(Fig.1,1, steps 10 to 16).The second part of the autotrophic cycle also occurs in the dicarboxylate/4-hydroxybutyrate cycle, which operates in autotrophic CO₂ fixation in Desulfurococcales and Thermoproteales (Crenarchaea) (27, 37), raising the question of whether the enzymes in these two lineages have common roots (37). The first part of the cycle also occurs in the 3-hydroxypropionate cycle for autotrophic CO₂ fixation in Chloroflexus aurantiacus and a few related green nonsulfur phototrophic bacteria (19, 22, 23, 32, 49).The two-step reduction of malonyl-CoA to 3-hydroxpropionate in Chloroflexus is catalyzed by a single bifunctional 300-kDa enzyme (30). The M. sedula malonyl-CoA reductase is completely unrelated and forms only malonic semialdehyde (1), and the enzyme catalyzing the second malonic semialdehyde reduction step that forms 3-hydroxypropionate is unknown. In the second part of the 3-hydroxypropionate/4-hydroxybutyrate cycle a similar reduction of succinyl-CoA via succinic semialdehyde to 4-hydroxybutyrate takes place. The enzymes responsible for these reactions also have not been characterized.In this work we purified the enzymes malonic semialdehyde reductase, succinyl-CoA reductase, and succinic semialdehyde reductase from M. sedula. The genes coding for these enzymes were identified in the genome, and recombinant proteins were studied in some detail. Interestingly, succinyl-CoA reductase turned out to be identical to malonyl-CoA reductase. We also show here that enzymes that are highly similar to succinyl-CoA reductase in Thermoproteus neutrophilus do not function as succinyl-CoA reductases in M. sedula.     

Antibody Recognition Force Microscopy Shows that Outer Membrane Cytochromes OmcA and MtrC Are Expressed on the Exterior Surface of Shewanella oneidensis MR-1

Antibody recognition force microscopy showed that OmcA and MtrC are expressed on the exterior surface of living Shewanella oneidensis MR-1 cells when Fe(III), including solid-phase hematite (Fe₂O₃), was the terminal electron acceptor. OmcA was localized to the interface between the cell and mineral. MtrC displayed a more uniform distribution across the cell surface. Both cytochromes were associated with an extracellular polymeric substance.Shewanella oneidensis MR-1 is a dissimilatory metal-reducing bacterium that is well known for its ability to use a variety of anaerobic terminal electron acceptors (TEAs), including solid-phase iron oxide minerals, such as goethite and hematite (8, 10). Previous studies suggest that S. oneidensis MR-1 uses outer membrane cytochromes OmcA and MtrC to catalyze the terminal reduction of Fe(III) through direct contact with the extracellular iron oxide mineral (2, 8, 10, 15, 16, 20, 21, 23). However, it has yet to be shown whether OmcA or MtrC is actually targeted to the external surface of live S. oneidensis MR-1 cells when Fe(III) serves as the TEA.In the present study, we used atomic force microscopy (AFM) to probe the surface of live S. oneidensis MR-1 cells, using AFM tips that were functionalized with cytochrome-specific polyclonal antibodies (i.e., anti-OmcA or anti-MtrC). This technique, termed antibody recognition force microscopy (Ig-RFM), detects binding events that occur between antibodies (e.g., anti-OmcA) on an AFM tip and antigens (e.g., OmcA) that are exposed on a cell surface. While this is a relatively new technique, Ig-RFM has been used to map the nanoscale spatial location of single molecules in complex biological structures under physiological conditions (5, 9, 11, 13).Anti-MtrC or anti-OmcA molecules were covalently coupled to silicon nitride (Si₃N₄) cantilevers (Veeco or Olympus) via a flexible, heterofunctional polyethylene glycol (PEG) linker molecule. The PEG linker consists of an NHS (N-hydroxysuccinimide) group at one end and an aldehyde group at the other end (i.e., NHS-PEG-aldehyde). AFM tips were functionalized with amine groups, using ethanolamine (6, 7). The active NHS ester of the NHS-PEG-aldehyde linker molecule was then used to form a covalent linkage between PEG-aldehyde and the amine groups on the AFM tips (6, 7). Next, anti-MtrC or anti-OmcA molecules were covalently tethered to these tips via the linker molecule''s aldehyde group. This was accomplished by incubating the tips with antibody (0.2 mg/ml) and NaCNBH₃ as described previously (7). The cantilevers were purchased from Veeco and had spring constant values between 0.06 and 0.07 N/m, as determined by the thermal method of Hutter and Bechhoefer (12).Prior to conducting the Ig-RFM experiments, the specificity of each polyclonal antibody (i.e., anti-OmcA and anti-MtrC) for OmcA or MtrC was verified by Western blot analysis as described previously (24, 28). Proteins were resolved by both denaturing and nondenaturing polyacrylamide gel electrophoresis (PAGE). Briefly, 2.5 μg of purified OmcA or MtrC (23) was resolved by sodium dodecyl sulfate-PAGE or native PAGE, transferred to a polyvinylidene difluoride membrane, incubated with either anti-OmcA or anti-MtrC, and then visualized using the Amersham ECL Plus Western blotting detection kit. Anti-OmcA bound exclusively to OmcA, anti-MtrC bound exclusively to MtrC, and neither antibody showed cross-reactivity with the other cytochrome. Antibody specificities of anti-OmcA and anti-MtrC were also validated by immunoblot analysis of S. oneidensis whole-cell lysate (28).To determine if MtrC or OmcA was expressed on the external surface of live bacteria when Fe(III) served as the TEA, Ig-RFM was conducted on wild-type versus ΔomcA ΔmtrC double mutant cells. For these experiments, bacteria were cultivated anaerobically with Fe(III), in the form of Fe(III) chelated to nitrilotriacetic acid (NTA), serving as the TEA (19, 23). Growth conditions have been described elsewhere (3, 15) and were based on previous studies (3, 15, 16, 18) that suggest that S. oneidensis MR-1 targets OmcA and MtrC to the cell surface when Fe(III) serves as the TEA.An Asylum Research MFP-3D-BIO AFM or a Digital Instruments Bioscope AFM (16, 17) was used for these experiments. The z-piezoelectric scanners were calibrated as described previously (17). Cells were deposited on a hydrophobic glass coverslip and immersed in imaging buffer (i.e., phosphate-buffered saline [pH 7.4]). The hydrophobic glass coverslips were made as described previously (17) using a self-assembling silane compound called octadecyltrichlorosilane (OTS; Sigma-Aldrich). S. oneidensis MR-1 cells readily adsorbed onto OTS glass coverslips and remained attached to the coverslips during the entire experiment. No lateral cell movement was observed during the experiment, consistent with previous studies that used OTS glass to immobilize bacteria (15, 17, 18, 27).The AFM tip was brought into contact with the surface of a bacterium, and the antibody-functionalized tip was repeatedly brought into and out of contact with the sample, “fishing” for a binding reaction with cytochrome molecules that were exposed on the external cell surface. Binding events were observed upon separating anti-OmcA- or anti-MtrC-functionalized tips from wild-type S. oneidensis MR-1 cells (Fig. ​(Fig.1).1). For the wild-type cells, we observed both nonspecific and specific interactions (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Retraction force curves for anti-MtrC-functionalized tips (A) and anti-OmcA-functionalized tips (B) that are being pulled away from the surface of living ΔomcA ΔmtrC double mutant (gray dotted line) or wild-type (solid black line) S. oneidensis MR-1. These bacteria were adsorbed onto OTS glass coverslips. (C) Retraction curves exhibiting nonspecific binding, specific binding, or no binding between the AFM tip and the cell surface.The distinction between “specific” and “nonspecific” adhesion is made by observing the change in slope of the force curve during the retraction process (26). During specific binding (Fig. ​(Fig.1C),1C), the cantilever is initially relaxed as it is pulled away from the sample. Upon further retraction, the ligand-receptor complex becomes stretched and unravels, resulting in a nonlinear force profile as noted in references 26 and 16. On the other hand, nonspecific adhesion (Fig. ​(Fig.1C)1C) maintains the same slope during the retraction process because only the cantilever flexes (26).Figure ​Figure22 summarizes the frequency or probability of observing a binding event for both anti-OmcA and anti-MtrC tips. Each bar in Fig. ​Fig.22 represents one experiment in which 500 to 1,000 force curves were collected between one AFM tip and two to four live bacterial cells. This figure does not make a distinction between specific and nonspecific binding. It simply shows the frequency of observing an attractive interaction as the antibody-functionalized tip was pulled away from the surface of S. oneidensis MR-1. Binding events occurred with roughly the same frequency when wild-type S. oneidensis MR-1 cells were probed with anti-MtrC-functionalized tips as when they were probed with anti-OmcA-functionalized tips (Fig. ​(Fig.22).Open in a separate windowFIG. 2.Histograms showing the frequency of observing a binding event for anti-MtrC-functionalized (blue) or anti-OmcA-functionalized (red) AFM tips on live wild-type S. oneidensis MR-1 (solid bars) or ΔomcA ΔmtrC double mutant (diagonally hatched bars) cells. The downward arrows designate injection of free antibody into the imaging buffer. The solid gray bars correspond to results obtained with unbaited AFM tips.A number of control experiments were performed to verify the detection of OmcA and MtrC on the surface of wild-type S. oneidensis MR-1. First, 0.1 μM of free anti-OmcA (or anti-MtrC) was added to the imaging fluid to block binding between the antibody-functionalized AFM tip and surface-exposed cytochromes (11, 16). This decreased the adhesion that was observed between the antibody-functionalized tip and the cell surface (Fig. ​(Fig.22).Second, we performed force measurements on ΔomcA ΔmtrC double mutant S. oneidensis MR-1 cells. This mutant is deficient in both OmcA and MtrC (19, 23, 24) but produces other proteins native to the outer surface of S. oneidensis MR-1. The resulting force spectra showed a noticeable reduction in binding events for the ΔomcA ΔmtrC double mutant cells (Fig. ​(Fig.2).2). The binding events that were observed for the double mutant were only nonspecific in nature (Fig. ​(Fig.1).1). This indicates that the antibodies on the tip do not participate in specific interactions with other proteins on the surface of S. oneidensis MR-1 cells.As a final control experiment, force measurements were conducted on wild-type S. oneidensis MR-1 cells, using Si₃N₄ tips conjugated with the PEG linker but not functionalized with polyclonal antibody (unbaited tips). Like the results with the double mutant, the unbaited tips were largely unreactive with the surface of the bacteria (Fig. ​(Fig.2).2). Those binding events that were observed were nonspecific in nature. Taken together, these results demonstrate that the antibody-coated tips have a specific reactivity with OmcA and MtrC molecules. Furthermore, these force measurements show that MtrC and OmcA are present on the external cell surface when Fe(III) serves as the TEA.To map the distribution of cytochromes on living cells, Ig-RFM was conducted on living S. oneidensis MR-1 cells that were growing on a hematite (α-Fe₂O₃) thin film. The conditions for these experiments were as follows. A hematite film was grown on a 10-mm by 10-mm by 1-mm oxide substrate via oxygen plasma-assisted molecular beam epitaxy (14, 16). The cells were grown anaerobically to mid-log phase with Fe(III)-NTA serving as the TEA. Cells were deposited onto the hematite thin film along with anaerobic growth medium that lacked Fe(III)-NTA. The cells were allowed to attach to the hematite surface (without drying) overnight in an anaerobic chamber. The following day, the liquid was carefully removed and immediately replaced with fresh anaerobic solution (pH 7.4). Ig-RFM was performed on the cells by raster scanning an antibody-functionalized AFM tip across the sample surface, thereby creating an affinity map (1). Force curves were collected for a 32-by-32 array. The raw pixilated force-volume data were deconvoluted using a regularized filter algorithm. The total time to acquire a complete image was approximately 20 min.As noted above, attractive interactions between an antibody tip and cell resulted in relatively short-range, nonspecific and longer-range, specific adhesive forces (Fig. ​(Fig.1C).1C). To distinguish between these two interactions, we integrated each force curve beginning at >20 nm and ending at the full retraction of the piezoelectric motor (∼1,800 nm). This integration procedure quantifies the work of binding, measured in joules, between the antibody tip and a particular position on the sample. While this integration procedure does not totally exclude nonspecific binding, it does select for those events associated primarily with specific antibody-antigen binding. Figure ​Figure33 is the antibody-cytochrome recognition images for MtrC and OmcA. The corresponding height (or topography) images of the bacterial cells are also shown in Fig. ​Fig.33.Open in a separate windowFIG. 3.Ig-RFM of live S. oneidensis MR-1 cells deposited on a hematite (α-Fe₂O₃) thin film. Height image (A) and corresponding Ig-RFM image (B) for a bare unfunctionalized Si₃N₄ tip. Height and corresponding Ig-RFM image for a tip functionalized with anti-MtrC (C and D) or anti-OmcA (E and F). Each panel contains a thin white oval showing the approximate location of the bacterium on the hematite surface. A color-coded scale bar is shown on the right (height in micrometers [μm], and the work required to separate the tip from the surface in attojoules [aJ]).OmcA molecules were concentrated at the boundary between the bacterial cell and hematite surface (Fig. 3E and F). MtrC molecules were also detected at the edge of a cell (Fig. 3C and D). Some MtrC, unlike OmcA, was observed on the cell surface distal from the point of contact with the mineral (Fig. 3C and D). Both OmcA and MtrC were also present in an extracellular polymeric substance (EPS) on the hematite surface (Fig. 3D and F), which is consistent with previous results showing MtrC and OmcA in an EPS produced by cells under anaerobic conditions (19, 24). This discovery is interesting in light of the research by Rosso et al. (22) and Bose et al. (4), who found that Shewanella can implement a nonlocal electron transfer strategy to reduce the surface of hematite at locations distant from the point of cell attachment. Rosso et al. (22) proposed that the bacteria utilize unknown extracellular factors to access the most energetically favorable regions of the Fe(III) oxide surface. The Ig-AFM results (Fig. ​(Fig.3)3) suggest the possibility that MtrC and/or OmcA are the “unknown extracellular factors” that are synthesized by Shewanella to reduce crystalline Fe(III) oxides at points distal from the cell. Additional experiments showing reductive dissolution features coinciding with the extracellular location of MtrC and/or OmcA would need to be performed to test this hypothesis.It is important to note that these affinity maps were collected on only a few cells because it so challenging to produce large numbers of quality images. Future work should be conducted on a population of cells. Until this time, these affinity maps can be used to provide a crude, lowest-order estimate of the number of cytochromes on the outer surface of living S. oneidensis MR-1. For example, there were 236 force curves collected on the bacterium shown in Fig. ​Fig.3D.3D. Thirty-eight of these curves exhibited a distinct, sawtooth-shaped, antibody-antigen binding event. In other words, MtrC molecules were detected in one out of every six force curves (16%) that were collected on the cell surface.This probability can be compared to other independent studies that estimated the density and size of MtrC and OmcA molecules from S. oneidensis MR-1. Lower et al. (16) estimated that S. oneidensis has 4 × 10¹⁵ to 7 × 10¹⁵ cytochromes per square meter by comparing AFM measurements for whole cells to force curves on purified MtrC and OmcA molecules. Wigginton et al. (25) used scanning tunneling microscopy to determine that the diameter of an individual cytochrome is 5 to 8 nm. These values can be used to create a simple, geometric, close-packing arrangement of MtrC or OmcA molecules on a surface. Using this approach, cytochromes could occupy 8 to 34% of the cell surface.This estimate is consistent with the observed number of putative MtrC molecules shown in Fig. ​Fig.3D.3D. Therefore, it appears that these affinity maps can be used as a lowest-order estimate for the number of cytochromes on S. oneidensis MR-1 even though we do not know a priori the exact configuration of the antibody tip (e.g., the concentration of antibody on the tip, the exact shape of the tip, the binding epitopes within the antibody).In summary, the data presented here show that S. oneidensis MR-1 localizes OmcA and MtrC molecules to the exterior cell surface, including an EPS, when Fe(III) is the TEA. Here, the cytochromes presumably serve as terminal reductases that catalyze the reduction of Fe(III) through direct contact with the extracellular iron-oxide mineral.     

Anaerobic Fermentation of Glycerol in Paenibacillus macerans: Metabolic Pathways and Environmental Determinants

Paenibacillus macerans is one of the species with the broadest metabolic capabilities in the genus Paenibacillus, able to ferment hexoses, deoxyhexoses, pentoses, cellulose, and hemicellulose. However, little is known about glycerol metabolism in this organism, and some studies have reported that glycerol is not fermented. Despite these reports, we found that several P. macerans strains are capable of anaerobic fermentation of glycerol. One of these strains, P. macerans N234A, grew fermentatively on glycerol at a maximum specific growth rate of 0.40 h⁻¹ and was chosen for further characterization. The use of [U-¹³C]glycerol and further analysis of extracellular metabolites and proteinogenic amino acids via nuclear magnetic resonance (NMR) spectroscopy allowed identification of ethanol, formate, acetate, succinate, and 1,2-propanediol (1,2-PDO) as fermentation products and demonstrated that glycerol is incorporated into cellular components. A medium formulation with low concentrations of potassium and phosphate, cultivation at acidic pH, and the use of a CO₂-enriched atmosphere stimulated glycerol fermentation and are proposed to be environmental determinants of this process. The pathways involved in glycerol utilization and synthesis of fermentation products were identified using NMR spectroscopy in combination with enzyme assays. Based on these studies, the synthesis of ethanol and 1,2-PDO is proposed to be a metabolic determinant of glycerol fermentation in P. macerans N234A. Conversion of glycerol to ethanol fulfills energy requirements by generating one molecule of ATP per molecule of ethanol synthesized. Conversion of glycerol to 1,2-PDO results in the consumption of reducing equivalents, thus facilitating redox balance. Given the availability, low price, and high degree of reduction of glycerol, the high metabolic rates exhibited by P. macerans N234A are of paramount importance for the production of fuels and chemicals.Although many microorganisms can metabolize glycerol in the presence of external electron acceptors (respiratory metabolism), few are able to do so fermentatively (i.e., in the absence of electron acceptors). Fermentative metabolism of glycerol has been reported in species of the genera Klebsiella, Citrobacter, Enterobacter, Clostridium, Lactobacillus, Bacillus, Propionibacterium, and Anaerobiospirillum but has been studied more extensively in a few species of the family Enterobacteriaceae, namely, Citrobacter freundii and Klebsiella pneumoniae (6, 9). Glycerol fermentation in these organisms is mediated by a two-branch pathway, which results in the synthesis of the glycolytic intermediate dihydroxyacetone (DHA) phosphate (DHAP) and the fermentation product 1,3-propanediol (1,3-PDO) (Fig. ​(Fig.1A)1A) (6). In the oxidative branch, glycerol is dehydrogenated to DHA by a type I NAD-linked glycerol dehydrogenase (glyDH). DHA is then phosphorylated by ATP- or phosphoenolpyruvate (PEP)-dependent DHA kinases (DHAKs) to generate DHAP. In the parallel reductive branch, glycerol is dehydrated by glycerol dehydratase, and 3-hydroxypropionaldehyde (3-HPA) is formed. 3-HPA is then reduced to the major fermentation product 1,3-PDO by an NADH-linked 1,3-PDO dehydrogenase (1,3-PDODH), thereby regenerating NAD⁺ (Fig. ​(Fig.1A).1A). Organisms that lack the capacity to synthesize 1,3-PDO have been deemed unable to utilize glycerol in a fermentative manner (6, 9, 10). The metabolism of glycerol in these organisms is thought to require an electron acceptor and takes place through a respiratory pathway that involves a glycerol kinase and two respiratory (aerobic and anaerobic) glycerol-3-phosphate dehydrogenases (G3PDHs) (6, 7, 24, 29, 35, 38) (Fig. ​(Fig.1B).1B). A recent development in this area is the finding that Escherichia coli, an organism that is unable to produce 1,3-PDO, can indeed ferment glycerol in the absence of external electron acceptors (15, 26). In this model, synthesis of the fermentation products 1,2-PDO and ethanol enables glycerol fermentation by facilitating redox balance and ATP generation, respectively (Fig. ​(Fig.1C)1C) (15). A type II glyDH and a PEP-dependent DHAK mediate the conversion of glycerol to glycolytic intermediates. glyDH also catalyzes the last step in the synthesis of the key fermentation product 1,2-PDO (Fig. ​(Fig.1C1C).Open in a separate windowFIG. 1.Glycerol metabolism in bacteria. (A) 1,3-PDO model for the fermentative utilization of glycerol. (B) Respiratory metabolism of glycerol (i.e., metabolism in the presence of an electron acceptor). (C) 1,2-PDO-ethanol model for the fermentative utilization of glycerol. Dashed lines indicate multiple steps. glyD, glycerol dehydratase; glyDH-I, type I glyDH; GK, glycerol kinase; ae-G3PDH, aerobic G3PDH; an-G3PDH, anaerobic G3PDH; QH₂, reduced quinone; glyDH-II, type II glyDH; FHL, formate hydrogen lyase; ADH, alcohol/acetaldehyde dehydrogenase.Paenibacillus macerans, previously called Bacillus macerans and Bacillus acetoethylicum, is a gram-positive, spore-forming bacterium belonging to the genus Paenibacillus (17) that is capable of fermentative metabolism of hexoses, deoxyhexoses, pentoses, cellulose, and hemicellulose (33, 39, 40, 41). Glycerol, however, is considered a nonfermentable carbon source for P. macerans. The “nonfermentable status” of glycerol has been used to determine whether certain electron acceptors, such as fumarate, trimethylamine N-oxide, nitrate, and nitrite, can mediate anaerobic respiration in this organism (34).In this study we found that several P. macerans strains are able to ferment glycerol in the absence of external electron acceptors. The fermentation of glycerol by one of these strains, P. macerans N234A, occurred at high metabolic rates and in the absence of an active 1,3-PDO pathway. Therefore, the environmental and metabolic determinants of glycerol fermentation in P. macerans N234A were investigated.     

Enhanced Production of a Plant Monoterpene by Overexpression of the 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Catalytic Domain in Saccharomyces cerevisiae

Linalool production was evaluated in different Saccharomyces cerevisiae strains expressing the Clarkia breweri linalool synthase gene (LIS). The wine strain T₇₃ was shown to produce higher levels of linalool than conventional laboratory strains (i.e., almost three times the amount). The performance of this strain was further enhanced by manipulating the endogenous mevalonate (MVA) pathway: deregulated overexpression of the rate-limiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) doubled linalool production. In a haploid laboratory strain, engineering of this key step also improved linalool yield.Monoterpenes are a class of isoprenoids of increasing industrial and clinical interest usually produced by plants. They are used as aromatic additives in the food and cosmetics industries and are also important components in wine aroma. Moreover, certain monoterpenes display antimicrobial, antiparasitic, and antiviral properties as well as a plethora of promising health benefits (for recent reviews, see references 2, 7, 15, 28, and 30 and references cited therein). To date, many studies have focused on plant metabolic engineering of monoterpene production (for selected reviews, see references 1, 14, 19, 29, and 35 and references cited therein), and few studies have been carried out on microorganisms (9, 21, 22, 34, 38). Efficient microbial production of these metabolites could provide an alternative to the current methods of chemical synthesis or extraction from natural sources. In this regard, a considerable number of studies have shown the utility of Saccharomyces cerevisiae as a valuable platform for sesquiterpene, diterpene, triterpene, and carotene production (references 5, 10, 23, 26, 30, 31, 32, and 33 and references cited therein). However, all the efforts dedicated to the improvement of isoprenoid yields in S. cerevisiae have been performed using conventional laboratory strains, and there are no studies concerning natural or industrially relevant isolates.In recent years, many genes that encode plant monoterpene synthases (MTS), a family of enzymes which specifically catalyze the conversion of the ubiquitous C₁₀ intermediate of isoprenoid biosynthesis geranyl pyrophosphate (GPP) to monoterpenes, have been characterized. Such is the case with the LIS gene (codes for S-linalool synthase) of Clarkia breweri, the first MTS-encoding gene to be isolated (13). In contrast to plants, S. cerevisiae cannot produce monoterpenes efficiently, mainly due to the lack of specific pathways involving MTS. However, GPP is formed as a transitory intermediate in the two-step synthesis of farnesyl pyrophosphate (FPP), catalyzed by FPP synthase (FPPS) (Fig. ​(Fig.1),1), and some natural S. cerevisiae strains have been shown to possess the ability to produce small amounts of monoterpenes (8). Whether this occurs through unspecific dephosphorylation of a more available endogenous pool of GPP and subsequent bioconversions is not known. In addition, it has recently been established that S. cerevisiae has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions (22, 34).Open in a separate windowFIG. 1.Simplified isoprenoid pathway in S. cerevisiae, including the branch point to linalool. Dotted arrows indicate that more than one reaction is required to convert the substrate to the product indicated. Dashed arrows indicate the engineered steps. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase.Here we present the process for selecting and optimizing yeast strains for foreign monoterpene production. We have chosen the C. breweri LIS gene as a prototype because, when heterologously expressed in S. cerevisiae, it specifically results in the production of linalool (3,7-dimethyl-1,6-octadien-3-ol; a floral scent and bioactive acyclic monoterpene identified in numerous fruits and flowers) and no other by-products (22). Two S. cerevisiae strains of different origins have been selected and their endogenous mevalonate (MVA) pathways engineered to enhance the production of linalool. These strategies might be employed to produce any other recombinant monoterpene in S. cerevisiae by expressing the appropriate monoterpene synthase.     

Target Immunity of the Tn3-Family Transposon Tn4430 Requires Specific Interactions between the Transposase and the Terminal Inverted Repeats of the Transposon

Specificity of the Tn4430 target immunity signal was examined by fusing the transposase TnpA to the LacI repressor of Escherichia coli. The resulting chimeric proteins failed to impose immunity to DNA targets carrying copies of the lacO operator, though they were proficient in lacO binding in vivo and remained responsive to wild-type immunity conferred by the Tn4430 inverted repeat end. Intriguingly, the presence of lacO repeats within the target was found to strongly influence target site selection by Tn4430, but in a LacI-independent manner.Tn4430 is a transposon of the Tn3 family that was originally isolated from Bacillus thuringiensis (Fig. ​(Fig.11 A) (12). Transposons of this family exhibit “target immunity,” a mechanism that prevents multiple insertion of the element into the same DNA molecule (9). Immunity has also been described for two other bacterial transposons, the bacteriophage Mu (1) and Tn7 (14). In all cases, the presence of a single copy of the transposon end is sufficient to confer immunity to the target, indicating that specific recognition of the target DNA by the transposase protein plays a central role in the process (2, 6, 8, 10). In the case of Mu and Tn7, target immunity results from the interplay between the transposase (i.e., MuA and TnsAB, respectively) and an ATP-dependent DNA binding protein involved in target capture (i.e., MuB and TnsC, respectively) (3, 4). No equivalent accessory protein is found in Tn3 family transposons, indicating that the transposase is the only transposon-encoded protein involved in immunity. The mechanism of “molecular repulsion,” underlying transposition immunity of this family of transposons, remains poorly understood.Open in a separate windowFIG. 1.(A) Genetic organization of Tn4430. The transposon (4,149 bp) is delineated by two identical inverted repeats (IR) of 38 bp that are specifically contacted by the transposase TnpA. The internal recombination site (IRS; 116 bp) is where the tyrosine recombinase TnpI acts to resolve the replicative intermediates (cointegrates) of transposition (15). (B) Schematic overview of the fusion proteins used in this study. The LacI349 and TnpA coding sequences are shown as shaded and black arrows, respectively. The position of the cMyc epitope is shown as a gray box.In this study, we sought to see whether specific recognition of Tn4430 terminal inverted repeat (IR; 38 bp) by the TnpA transposase is a mandatory step in transposition immunity or whether TnpA binding to unrelated DNA sequences is sufficient to reorient target site selection. To this end, we examined whether fusion proteins between TnpA and the LacI repressor of Escherichia coli could confer transposition immunity to target molecules containing copies of the lacO operator.     

Enhancement of the Diversity of Polyoxins by a Thymine-7-Hydroxylase Homolog outside the Polyoxin Biosynthesis Gene Cluster

Polyoxins consist of 14 structurally variable components which differentiate at three branch sites of the carbon skeleton. Open reading frame (ORF) SAV_4805 of Streptomyces avermitilis, showing similarity to thymine-7-hydroxylase, was proved to enhance the diversity of polyoxins at the C-5 site of the 1-(5′-amino-5′-deoxy-β-d-allofuranuronosyl) pyrimidine moiety.The antifungal nucleoside antibiotic polyoxins synthesized naturally by Streptomyces cacaoi subsp. asoensis (S. cacaoi here) consist of a mixture of at least 14 different compounds called polyoxins A to N (Fig. ​(Fig.1)1) (6, 13, 25). Most of the polyoxins have a common nucleoside skeleton that is attached with variable side groups at three different places (polyoxins C, I, and N have a modified skeleton). Polyoxins are grouped into four different classes according to the identity of the side group R1 attached at C-5 of the 1-(5′-amino-5′-deoxy-β-d-allofuranuronosyl) pyrimidine moiety. Different classes of polyoxins differ markedly in their activity spectra against plant pathogenic fungi (1, 10, 20).Open in a separate windowFIG. 1.Chemical structure of polyoxin complex. Class is determined by R1. The × indicates a different skeleton that does not have this particular residue.It was deduced that the nucleoside moiety originated from the condensation of uridine with phosphoenolpyruvate (PEP) to generate octosyl acid as the intermediate (7, 12, 14). Then, a subsequent oxidative elimination of the two terminal carbons would create the nucleoside moiety (12). The detailed biosynthetic pathway of nucleoside moiety of polyoxins remains obscure. Our previous work showed that heterologous expression of the polyoxin biosynthetic gene cluster pol in Streptomyces lividans TK24 produces only thymine-derived polyoxin H (class III) (5). This prompted us to investigate the origin of a gene(s) related to the structural variation of polyoxins at the R1 site, which could be located outside the polyoxin biosynthetic gene cluster in the native producer.The pyrimidine rings of polyoxins correspond one to one to the intermediates of the thymidine salvage pathway, which was characterized only for some fungi (see Fig. S1 in the supplemental material) (22). In the prime pathway of nucleotide metabolism, dUMP could be converted to dTMP under the catalysis of thymidylate synthase (TS) with tetrahydrofolic acid as a methyl donor while dTMP could not be converted back to dUMP reversibly (4, 17). In the 1970s, Shaffer et al. separated two enzymes, thymine-7-hydroxylase (THase; official name thymine dioxygenase) and isoorotate decarboxylase (IDCase), from fungal sources which showed potential ability to convert thymine to uracil (22). THase is a trifunctional oxygenase and catalyzes three enzymatic reactions: thymine to 5-hydroxymethyluracil, 5-hydroxymethyluracil to 5-formyluracil, and 5-formyluracil to uracil-5-carboxylic acid (also designated isoorotate) (18, 26). IDCase catalyzes the subsequent decarboxylation reaction, and uracil-5-carboxylic acid is converted to uracil in the end (21). This process is catalyzed by THase, and IDCase is the core step of the thymidine salvage pathway in fungi.BLAST analysis results with the amino acid sequence of THase from Rhodotorula glutinis and IDCase from Neurospora crassa as queries in the 25 sequenced Streptomyces strains (5 finished and 20 in process) of the genome database (from NCBI GenBank, accessed 24 July 2010) showed that the THase and IDCase gene homologs are distributed extensively in Streptomyces, such as open reading frame (ORF) SAV_4805 (sharing 27% identity and 45% similarity with the THase gene) from S. avermitilis strain MA4680 and SCO_6305 (sharing 25% identity and 44% similarity with the IDCase gene) from S. coelicolor strain A3(2) (2, 11, 23). The results indicated a potential thymidine salvage pathway in Streptomyces, and the salvage pathway provides alternative nucleoside monophosphates as precursors for polyoxin biosynthesis.     

Genome-Scale Phylogenetic Analyses of Chikungunya Virus Reveal Independent Emergences of Recent Epidemics and Various Evolutionary Rates

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has traditionally circulated in Africa and Asia, causing human febrile illness accompanied by severe, chronic joint pain. In Africa, epidemic emergence of CHIKV involves the transition from an enzootic, sylvatic cycle involving arboreal mosquito vectors and nonhuman primates, into an urban cycle where peridomestic mosquitoes transmit among humans. In Asia, however, CHIKV appears to circulate only in the endemic, urban cycle. Recently, CHIKV emerged into the Indian Ocean and the Indian subcontinent to cause major epidemics. To examine patterns of CHIKV evolution and the origins of these outbreaks, as well as to examine whether evolutionary rates that vary between enzootic and epidemic transmission, we sequenced the genomes of 40 CHIKV strains and performed a phylogenetic analysis representing the most comprehensive study of its kind to date. We inferred that extant CHIKV strains evolved from an ancestor that existed within the last 500 years and that some geographic overlap exists between two main enzootic lineages previously thought to be geographically separated within Africa. We estimated that CHIKV was introduced from Africa into Asia 70 to 90 years ago. The recent Indian Ocean and Indian subcontinent epidemics appear to have emerged independently from the mainland of East Africa. This finding underscores the importance of surveillance to rapidly detect and control African outbreaks before exportation can occur. Significantly higher rates of nucleotide substitution appear to occur during urban than during enzootic transmission. These results suggest fundamental differences in transmission modes and/or dynamics in these two transmission cycles.Chikungunya virus (CHIKV; Togaviridae: Alphavirus) is an arbovirus (arthropod-borne virus) vectored by Aedes mosquitoes to humans in tropical and subtropical regions of Africa and Asia (Fig. ​(Fig.1;1; reviewed in references 26 and 46). CHIKV has a single-stranded, positive-sense RNA genome of ∼12 kb and causes chikungunya fever (CHIK), a febrile illness associated with severe arthralgia and rash (2, 15, 31, 35); the name is derived from a Bantu language word describing the severe arthritic signs (32), which can persist for years. Thus, CHIK has enormous economic costs in addition to its public health impact (9). Because the signs and symptoms of CHIK overlap with those of dengue and because CHIKV is transmitted sympatrically in urban areas by the same mosquito vectors, it is grossly underreported in the absence of laboratory diagnostics (10, 37).Open in a separate windowFIG. 1.Distribution of the CHIKV strains used in this study. The map, based on a world map template from http://www.presentationmagazine.com, was edited with permission.CHIKV was first isolated during a 1953 outbreak in present-day Tanzania by Ross (48, 49). Since then, outbreaks have been documented in Africa and Asia, including the Indian subcontinent (Fig. ​(Fig.1)1) (1, 4). In 2005, CHIKV emerged from East Africa to cause an explosive urban epidemic in popular tourist island destinations in the Indian Ocean (Fig. ​(Fig.1;1; reviewed in reference 31). In late 2005, CHIKV spread into the Indian subcontinent, where millions of people have been affected (5). However, the geographic source of spread into India, from the mainland of Africa or from the Indian Ocean Islands, has not been delineated. India had seen large epidemics of CHIK in the past (reviewed in reference 30), but CHIKV apparently disappeared during the 1970s (5). Since 2006, CHIKV has been imported into Europe and the western hemisphere (including the United States) via many viremic travelers, and an epidemic was initiated in Italy by a traveler from India (4, 11, 47). The dramatic spread since 1980 of dengue viruses (DENV) throughout tropical America, via the same vectors, portends the severity of the public health problem if CHIKV becomes established in the western hemisphere.The first phylogenetic analysis of CHIKV (45) identified three geographically associated genotypes: the West African (WAf), East/Central/South African (ECSA), and Asian genotypes. More recent analyses indicate that the recent Indian Ocean and Indian strains form a monophyletic group within the ECSA lineage (5, 12, 14, 27, 40, 51, 52). However, most CHIKV phylogenetic studies (1, 14, 28, 29, 38, 40, 41, 47, 52) have utilized only partial sequences from the envelope glycoprotein E1 gene, preventing a robust assessment of some of the relationships among strains and of their evolutionary dynamics.The CHIKV strains represented in different geographic lineages apparently circulate in different ecological cycles. In Asia, CHIKV appears to circulate primarily in an urban transmission cycle involving the peridomestic mosquitoes Aedes aegypti and A. albopictus, as well as humans (25, 45). Asian epidemics typically infect thousands-to-millions of people over the course of several years (46). In contrast, African CHIKV circulates primarily in a sylvatic/enzootic cycle, transmitted by arboreal primatophilic Aedes mosquitoes (e.g., A. furcifer and A. africanus) and probably relies on nonhuman primates as reservoir hosts (reviewed in reference 16). Epidemics in rural Africa usually occur on a much smaller scale than in Asia, likely a result of the lower human population densities, and possibly more stable herd immunity. Although the assignments of “urban” and “sylvatic/enzootic” are based on the most common mode of transmission, CHIKV strains of African origin are capable of urban transmission by A. aegypti and A. albopictus, as evidenced by outbreaks in the Democratic Republic of the Congo (41), Nigeria (36), Kenya (27), and Gabon (42). The ecological differences between the sylvatic/enzootic (henceforth called enzootic) and urban/endemic/epidemic transmission cycles (henceforth called epidemic) such as seasonality of vector larval habitats, vertebrate host abundance and herd immunity, and vector host preferences, prompted us to hypothesize that the evolutionary dynamics of CHIKV may differ between the two transmission cycles. To test this hypothesis, to provide more robust estimates of the evolutionary relationships among the CHIKV strains including the sources of the recent epidemics, and to elucidate the temporal and spatial history of CHIKV evolution, we performed an extensive, genome-scale phylogenetic analysis, utilizing complete open reading frame (ORF) sequences of a large collection of 80 isolates with broad temporal, spatial, and host coverage.     

Identification and Analysis of the Biosynthetic Gene Cluster Encoding the Thiopeptide Antibiotic Cyclothiazomycin in Streptomyces hygroscopicus 10-22

Thiopeptide antibiotics are an important class of natural products resulting from posttranslational modifications of ribosomally synthesized peptides. Cyclothiazomycin is a typical thiopeptide antibiotic that has a unique bridged macrocyclic structure derived from an 18-amino-acid structural peptide. Here we reported cloning, sequencing, and heterologous expression of the cyclothiazomycin biosynthetic gene cluster from Streptomyces hygroscopicus 10-22. Remarkably, successful heterologous expression of a 22.7-kb gene cluster in Streptomyces lividans 1326 suggested that there is a minimum set of 15 open reading frames that includes all of the functional genes required for cyclothiazomycin production. Six genes of these genes, cltBCDEFG flanking the structural gene cltA, were predicted to encode the enzymes required for the main framework of cyclothiazomycin, and two enzymes encoded by a putative operon, cltMN, were hypothesized to participate in the tailoring step to generate the tertiary thioether, leading to the final cyclization of the bridged macrocyclic structure. This rigorous bioinformatics analysis based on heterologous expression of cyclothiazomycin resulted in an ideal biosynthetic model for us to understand the biosynthesis of thiopeptides.The thiopeptides are a family of highly modified, sulfur-containing macrocyclic peptides, such as thiostrepton, thiocillins, and micrococcinic acid (3, 11). Their structures have several common features: a tri- or tetrasubstituted nitrogen heterocycle central domain, a macrocyclic framework, and heavily modified amino acid residues, including thiazoles, oxazoles, and dehydroamino acids (Fig. ​(Fig.1)1) (3). Members of this family exhibit various biological properties, such as inhibition of ribosomal protein synthesis (24), rennin inhibitory activity (2), and induction of TipA (20). Moreover, many thiopeptide antibiotics show bioactivity against some bacterial strains resistant to most conventional treatments, including methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae (PRSP), and vancomycin-resistant enterococci (VRE) (3, 17).Open in a separate windowFIG. 1.Structures of thiostrepton, thiocillin, microncoccinate, and cyclothiazomycin (3).Early in vitro investigations of these special heterocycles of thiopeptides were performed by organic chemists and included stereoselective synthesis of a γ-lactam acidic hydrolysate of cyclothiazomycin (4) and total synthesis of thiostrepton (21). Extensive research on microccins (5, 19) and lantibiotics (6, 29) described biosynthesis of the thiazole- and dehydroamino acid-containing polypeptides that were derived from ribosomally synthesized prepeptides. Similarly, Lee et al. described a widely conserved gene cluster for toxin biosynthesis and suggested a ribosome biosynthetic pathway for the modified polypeptide containing thiazoles and oxazoles (16).Recently, Wieland Brown et al. (28) and Kelly et al. (14) identified the gene clusters encoding thiocillin and thiostrepton, respectively, with a probe that targeted hypothetic prepeptide genes, whereas Liao and coworkers (17) took advantage of the conservation of one putative cyclodehydratase. Although most of the biochemical reactions involved in the biosynthetic pathway remain obscure, it is clear that thiopeptides are synthesized ribosomally and then there is a series of posttranslational modifications.Cyclothiazomycin, which was isolated as a novel selective inhibitor of human plasma rennin, is a unique bridged macrocyclic thiopeptide (2) whose stereo structure was recently revealed by degradation experiments and spectroscopic analysis (10). It contains a dehydroserine, two dehydrothreonine residues, three thiazolines, three thiazoles, and a trisubstituted pyridine. Compared with common thiopeptides, it lacks the characteristic 2- and 3-azole substituent on the central pyridine domain; instead, it has an alanine-derived heterocyclic residue with the (R) configuration, a quaternary sulfide, and two macrocyclic peptide loops (Fig. ​(Fig.1).1). Moreover, a pair of convertible isomers of cyclothiazomycin B, the cyclothiazomycin analogues produced by Streptomyces sp. strain A307 (10), were identified as Z and E configurations caused by the tautomerization of the dehydrated threonine. These structural traits may indicate that some new genetic elements are likely involved in posttranslational modification.Here we reported cloning and sequencing of the cyclothiazomycin biosynthetic gene cluster of Streptomyces hygroscopicus 10-22 (23). In addition, an analysis of heterologous expression in Streptomyces lividans 1326 and a deletion analysis were also performed, which indicated that a gene cluster at least 22.7 kb long is required for biosynthesis. A bioinformatics-based approach to analysis of this gene cluster postulated that there is a posttranslational modification pathway in which eight proteins are involved in the biosynthetic machinery.     

Oleate Hydratase Catalyzes the Hydration of a Nonactivated Carbon-Carbon Bond

The hydration of oleic acid into 10-hydroxystearic acid was originally described for a Pseudomonas cell extract almost half a century ago. In the intervening years, the enzyme has never been characterized in any detail. We report here the isolation and characterization of oleate hydratase (EC 4.2.1.53) from Elizabethkingia meningoseptica.The ability of cells to convert oleic acid (OA) into 10-hydroxystearic acid (10-HSA) was discovered by Wallen et al. in Pseudomonas sp. strain 3266 in 1962 (Fig. ​(Fig.1)1) (12). In the following years, many other strains were identified that were also able to convert OA into 10-HSA or to further oxidize it to 10-ketostearic acid (2, 3, 5, 7). The Pseudomonas cells generally start to produce optically pure d-10-HSA in the stationary growth phase, and they do not seem to metabolize it any further, since levels of product accumulate in the fermentation broth. The putative enzyme for this conversion is referred to as oleate hydratase (EC 4.2.1.53); however, so far it has not been purified or characterized in any detail.Open in a separate windowFIG. 1.Reaction catalyzed by oleate hydratase; the conversion of OA into 10-HSA.Kinetic studies have been performed with cell extracts, giving some insight into the stereospecificity and the possible mechanism of the reaction. Studies with ¹⁸O-labeled water reported the incorporation of ¹⁸O at the C-10 position of 10-HSA, confirming a hydration mechanism (7). The reaction was shown to be reversible; however, the detected concentration ratio at equilibrium was always in the range of 85:15 in favor of 10-HSA (9).Here we report the isolation and first biochemical characterization of the oleate hydratase protein from Elizabethkingia meningoseptica (formerly known as Pseudomonas sp. strain 3266).The primer set GM3 and GM4 (8) was used for PCR amplification of the Pseudomonas sp. strain 3266 16S-rRNA genes. The product (1,444 bp) was sequenced, and 16S phylogeny analysis resulted in a unanimous determination of the species as Elizabethkingia (Chryseobacterium) meningoseptica with a >99.8% resemblance.