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Barry P. Rosen - One of the best experts on this subject based on the ideXlab platform.
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Identification of Steps in the Pathway of Arsenosugar Biosynthesis
Environmental science & technology, 2018Co-Authors: Xi-mei Xue, Barry P. Rosen, Georg Raber, Kevin A. Francesconi, Chan Xiong, Zhe Zhu, Christopher Rensing, Yong-guan ZhuAbstract:ArsenosugArs are Arsenic-containing ribosides that play a substantial role in Arsenic biogeochemical cycles. ArsenosugArs were identified more than 30 yeArs ago, and yet their mechanism of biosynthesis remains unknown. In this study we report identification of the ArsS gene from the cyanobacterium Synechocystis sp. PCC 6803 and show that it is involved in Arsenosugar biosynthesis. In the Synechocystis sp. PCC 6803 Ars Operon, ArsS is adjacent to the ArsM gene that encodes an As(III) S-adenosylmethionine (SAM) methyltransferase. The gene product, ArsS, contains a characteristic CX3CX2C motif which is typical for the radical SAM superfamily. The function of ArsS was identified from a combination of ArsS disruption in Synechocystis sp. PCC 6803 and heterologous expression of ArsM and ArsS in Escherichia coli. Both genes are necessary, indicating a multistep pathway of Arsenosugar biosynthesis. In addition, we demonstrate that ArsS orthologs from three other freshwater cyanobacteria and one picocyanobacterium...
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Identification of Steps in the Pathway of Arsenosugar Biosynthesis
2018Co-Authors: Xi-mei Xue, Barry P. Rosen, Georg Raber, Chan Xiong, Zhe Zhu, Christopher Rensing, Kevin Francesconi, Yong-guan ZhuAbstract:ArsenosugArs are Arsenic-containing ribosides that play a substantial role in Arsenic biogeochemical cycles. ArsenosugArs were identified more than 30 yeArs ago, and yet their mechanism of biosynthesis remains unknown. In this study we report identification of the ArsS gene from the cyanobacterium Synechocystis sp. PCC 6803 and show that it is involved in Arsenosugar biosynthesis. In the Synechocystis sp. PCC 6803 Ars Operon, ArsS is adjacent to the ArsM gene that encodes an As(III) S-adenosylmethionine (SAM) methyltransferase. The gene product, ArsS, contains a characteristic CX3CX2C motif which is typical for the radical SAM superfamily. The function of ArsS was identified from a combination of ArsS disruption in Synechocystis sp. PCC 6803 and heterologous expression of ArsM and ArsS in Escherichia coli. Both genes are necessary, indicating a multistep pathway of Arsenosugar biosynthesis. In addition, we demonstrate that ArsS orthologs from three other freshwater cyanobacteria and one picocyanobacterium are involved in Arsenosugar biosynthesis in those microbes. This study represents the identification of the first two steps in the pathway of Arsenosugar biosynthesis. Our discovery expands the catalytic repertoire of the diverse radical SAM enzyme superfamily and provides a basis for studying the biogeochemistry of complex organoArsenicals
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Bacterial resistance to Arsenic protects against protist killing.
Biometals : an international journal on the role of metal ions in biology biochemistry and medicine, 2017Co-Authors: Xiuli Hao, Barry P. Rosen, Yong-guan Zhu, Chandan Pal, Jon L. Hobman, D. G. Joakim Larsson, Quaiser Saquib, Hend A. Alwathnani, Christopher RensingAbstract:Protists kill their bacterial prey using toxic metals such as copper. Here we hypothesize that the metalloid Arsenic has a similar role. To test this hypothesis, we examined intracellular survival of Escherichia coli (E. coli) in the amoeba Dictyostelium discoideum (D. discoideum). Deletion of the E. coli Ars Operon led to significantly lower intracellular survival compared to wild type E. coli. This suggests that protists use Arsenic to poison bacterial cells in the phagosome, similar to their use of copper. In response to copper and Arsenic poisoning by protists, there is selection for acquisition of Arsenic and copper resistance genes in the bacterial prey to avoid killing. In agreement with this hypothesis, both copper and Arsenic resistance determinants are widespread in many bacterial taxa and environments, and they are often found together on plasmids. A role for heavy metals and Arsenic in the ancient predator–prey relationship between protists and bacteria could explain the widespread presence of metal resistance determinants in pristine environments.
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Expression of Arsenic resistance genes in the obligate anaerobe Bacteroides vulgatus ATCC 8482, a gut microbiome bacterium.
Anaerobe, 2016Co-Authors: Goutam Mandal, Barry P. RosenAbstract:The response of the obligate anaerobe Bacteroides vulgatus ATCC 8482, a common human gut microbiota, to Arsenic was determined. B. vulgatus ATCC 8482 is highly resistant to pentavalent As(V) and methylArsenate (MAs(V)). It is somewhat more sensitive to trivalent inorganic As(III) but 100-fold more sensitive to methylArsenite (MAs(III)) than to As(III). B. vulgatus ATCC 8482 has eight continuous genes in its genome that we demonstrate form an Arsenical-inducible transcriptional unit. The first gene of this Ars Operon, ArsR, encodes a putative ArsR As(III)-responsive transcriptional repressor. The next three genes encode proteins of unknown function. The remaining genes, ArsDABC, have well-characterized roles in detoxification of inorganic Arsenic, but there are no known genes for MAs(III) resistance. Expression of each gene after exposure to trivalent and pentavalent inorganic and methylArsenicals was analyzed. MAs(III) was the most effective inducer. The ArsD gene was the most highly expressed of the Ars Operon genes. These results demonstrate that this anaerobic microbiome bacterium has Arsenic-responsive genes that confer resistance to inorganic Arsenic and may be responsible for the organism's ability to maintain its prevalence in the gut following dietary exposure to inorganic Arsenic.
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Characterization of the extremely Arsenic-resistant Brevibacterium linens strain AE038-8 isolated from contaminated groundwater in Tucumán, Argentina
International Biodeterioration & Biodegradation, 2016Co-Authors: Daniela Maizel, Barry P. Rosen, Jodi Switzer Blum, Marcela Alejandra Ferrero, Sagar M. Utturkar, Steven D. Brown, Ronald S. OremlandAbstract:Abstract Brevibacterium linens AE038-8, isolated from As-contaminated groundwater in Tucuman (Argentina), is highly resistant to Arsenic oxyanions, being able to tolerate up to 1 M As(V) and 75 mM As(III) in a complex medium. Strain AE038-8 was also able to reduce As(V) to As(III) when grown in complex medium but paradoxically it could not do this in a defined minimal medium with sodium acetate and ammonium sulfate as carbon and nitrogen sources, respectively. No oxidation of As(III) to As(V) was observed under any conditions. Three copies of the Ars Operon comprising Arsenic resistance genes were found on B. linens AE038-8 genome. In addition to the well known ArsC, ACR3 and ArsR, two copies of the ArsO gene of unknown function were detected.
Sueharu Horinouchi - One of the best experts on this subject based on the ideXlab platform.
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direct transfer of starter substrates from type i fatty acid synthase to type iii polyketide synthases in phenolic lipid synthesis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Akimasa Miyanaga, Nobutaka Funa, Takayoshi Awakawa, Sueharu HorinouchiAbstract:Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the Ars Operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the Ars Operon, are responsible for the biosynthesis of C22–C26 fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C22–C26 fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the Ars Operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer.
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phenolic lipid synthesis by type iii polyketide synthases is essential for cyst formation in azotobacter vinelandii
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Nobutaka Funa, Hiroki Ozawa, Aiko Hirata, Sueharu HorinouchiAbstract:Cysts of Azotobacter vinelandii are resting cells that are surrounded by a protective coat, conferring resistance to various chemical and physical agents. The major chemical components of the cyst coat are alkylresorcinols, which are amphiphilic molecules possessing an aromatic ring with a long aliphatic carbon chain. Although alkylresorcinols are widely distributed in bacteria, fungi, plants, and animals, no enzyme systems for their biosynthesis are known. We report here an Ars Operon in A. vinelandii that is responsible for the biosynthesis of the alkylresorcinols in the cysts. The Ars Operon consisted of four genes, two of which encoded a type III polyketide synthase, ArsB and ArsC. In vitro experiments revealed that ArsB and ArsC, sharing 71% amino acid sequence identity, were an alkylresorcinol synthase and an alkylpyrone synthase, respectively, indicating that ArsB and ArsC are not isozymes but enzymatically distinct polyketide synthases. In addition, ArsB and ArsC accepted several acyl-CoAs with various lengths of the side chain as a starter substrate and gave corresponding alkylresorcinols and alkylpyrones, respectively, which suggests that the mode of the ring folding is uninfluenced by the structure of the starter substrates. The importance of the alkylresorcinols for encystment was confirmed by gene inactivation experiments; the lack of alkylresorcinols synthesis caused by Ars mutations resulted in the formation of severely impaired cysts, as observed by electron microscopy.
Weiping Shi - One of the best experts on this subject based on the ideXlab platform.
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Bacteria-based chemiluminescence sensing system using β-galactosidase under the control of the ArsR regulatory protein of the Ars Operon
Analytica Chimica Acta, 1998Co-Authors: Sridhar Ramanathan, Barry P. Rosen, Weiping Shi, Sylvia DaunertAbstract:Abstract A highly sensitive and selective sensing system for antimonite and Arsenite was developed based on genetically engineered bacteria harboring the plasmid pBGD23. In this plasmid, ArsR , the gene encoding for the ArsR regulatory protein of the Ars Operon, is fused to lacZ , the gene encoding for the reporter enzyme β-galactosidase. The expression of β-galactosidase in E. coli strains bearing pBGD23 is controlled by ArsR, and this can be related to the concentration of antimonite/Arsenite employed to induce the production of β-galactosidase in the bacteria. ArsR has a high specificity for antimonite/Arsenite, thus conferring the developed sensing system with high selectivity. This was demonstrated by evaluating several oxoanions and soft metals as potential interferents. The concentration of β-galactosidase expressed in the bacteria was monitored by chemiluminescence. Using this sensing system, antimonite can be detected at concentrations as low as 10 −15 M. The importance of the E. coli chromosomal Ars Operon on the observed response was evaluated by employing a strain of E. coli where the chromosomal Ars Operon has been deleted.
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Genetically engineered bacteria: electrochemical sensing systems for antimonite and Arsenite.
Analytical Chemistry, 1997Co-Authors: Donna L. Scott, Barry P. Rosen, Sridhar Ramanathan, Weiping Shi, Sylvia DaunertAbstract:A bacterial sensing system that responds selectively to antimonite and Arsenite has been investigated. The bacteria used in these studies have been genetically engineered to produce the enzyme β-galactosidase in response to these ions. This is accomplished by using a plasmid that incorporates the gene for β-galactosidase (reporter gene) under the control of the promoter of the Ars Operon. This plasmid also encodes for the ArsR protein, a regulatory protein of the Ars Operon, which, in the absence of antimonite or Arsenite, restricts the expression of β-galactosidase. In the presence of antimonite or Arsenite the ArsR protein is released from the operator/promoter region of the Ars Operon and β-galactosidase is expressed. The activity of this enzyme was monitored electrochemically using p-aminophenyl β-d-galactopyranoside as the substrate. The bacterial sensing system responds selectively to Arsenite and antimonite (and to a lesser extent Arsenate) and shows no significant response to phosphate, sulfate, n...
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The Role of Arsenic-Thiol Interactions in Metalloregulation of the Ars Operon
The Journal of biological chemistry, 1996Co-Authors: Weiping Shi, Jun Dong, Robert A. Scott, Marina Yu. Ksenzenko, Barry P. RosenAbstract:Abstract The Ars Operon of the Escherichia coli plasmid R773 that confers Arsenical and antimonial resistance is negatively regulated by the ArsR repressor. ArsR residues Cys-32 and Cys-34 were previously identified as involved in induction by Arsenite and antimonite, suggesting coordination between As(III) and the two cysteine thiolates. However, in small molecule thiolate-As(III) complexes, Arsenic is frequently three-coordinate. A site-directed mutagenic approach was employed in a search for a third Arsenic ligand. ArsR proteins with C32G, C34G, and C32G/C34G substitutions were active repressors, but were not inducible in vivo. In vitro, the altered repressor-Ars DNA complexes could not be dissociated by inducers. Alteration of Cys-37 and Ser-43, residues located in or near the putative helix-turn-helix DNA-binding region of the protein, had no effect on the inducibility of the Operon. While these results indicated that neither the thiolate of Cys-37 nor the hydroxyl oxygen of Ser-43 is required for induction, they did not eliminate either atom as a potential Arsenic ligand. Another approach involved reaction with an alternative inducer, phenylArsine oxide, which can form only two coordinations. PhenylArsine oxide was shown to be as effective as or more effective than Arsenite or antimonite in induction in vivo. In vitro, the organic Arsenical was more effective than either Arsenite or antimonite in dissociating the repressor-promoter complex. Thus, two ArsR-Arsenic bonds are sufficient for induction. The interaction of ArsR proteins with As(III) was examined using a phenylArsine oxide affinity resin. ArsR proteins containing any two of the three cysteine residues Cys-32, Cys-34, and Cys-37 bound to the resin. Alteration of any two of the three resulted in loss of binding. Arsenic x-ray absorption spectroscopy of ArsR treated stoichiometrically with Arsenite confirmed the average Arsenic coordination as AsS. These results suggest that all three cysteine thiolates are Arsenic ligands, but binding to only two, the Cys-32 and Cys-34 thiolates, is required to produce the conformational change that results in release of the repressor from the DNA and induction.
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The Chromosomal ArsR Gene of Escherichia coli Encodes a trans-acting Metalloregulatory Protein
The Journal of biological chemistry, 1996Co-Authors: Weiping Shi, Barry P. RosenAbstract:Abstract Plasmid-encoded Arsenical resistance (Ars) Operons confer high level resistance to Arsenicals and antimonials, while the chromosomally encoded Ars Operon of Escherichia coli bestows low level resistance. The transcriptional start site of the chromosomal Ars mRNA was mapped by primer extension, and putative −10 and −35 promoter recognition sites were identified. The ArsR gene, the first gene in this Operon, was cloned using polymerase chain reaction. The ArsR gene product, the ArsR repressor, was expressed and purified. The results of gel mobility shift assays indicated that the repressor is a DNA binding protein that binds to a fragment of DNA containing the chromosomal Ars promoter. The specific binding site, as determined by DNase I footprint analysis, spans 33 nucleotides in the promoter region, including the putative −35 promoter element. By construction and expression of a series of in-frame fusions between truncated ArsR genes and the coding region for the mature form of β-lactamase (blaM′), it was shown that ArsR is a trans-acting repressor that regulates expression of the chromosomal Ars Operon. In addition, the chromosomally-encoded repressor can regulate expression of the Ars Operon of plasmid R773, and the R773 repressor can cross-regulate expression from the chromosomal Operon.
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The Ars Operon of Escherichia coli confers Arsenical and antimonial resistance.
Journal of bacteriology, 1995Co-Authors: Arthur M. Carlin, Weiping Shi, Saibal Dey, Barry P. RosenAbstract:The chromosomally encoded Arsenical resistance (Ars) Operon subcloned into a multicopy plasmid was found to confer a moderate level of resistance to Arsenite and antimonite in Escherichia coli. When the Operon was deleted from the chromosome, the cells exhibited hypersensitivity to Arsenite, antimonite, and Arsenate. Expression of the Ars genes was inducible by Arsenite. By Southern hybridization, the Operon was found in all strains of E. coli examined but not in Salmonella typhimurium, Pseudomonas aeruginosa, or Bacillus subtilis.
Sylvia Daunert - One of the best experts on this subject based on the ideXlab platform.
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Bacteria-based chemiluminescence sensing system using β-galactosidase under the control of the ArsR regulatory protein of the Ars Operon
Analytica Chimica Acta, 1998Co-Authors: Sridhar Ramanathan, Barry P. Rosen, Weiping Shi, Sylvia DaunertAbstract:Abstract A highly sensitive and selective sensing system for antimonite and Arsenite was developed based on genetically engineered bacteria harboring the plasmid pBGD23. In this plasmid, ArsR , the gene encoding for the ArsR regulatory protein of the Ars Operon, is fused to lacZ , the gene encoding for the reporter enzyme β-galactosidase. The expression of β-galactosidase in E. coli strains bearing pBGD23 is controlled by ArsR, and this can be related to the concentration of antimonite/Arsenite employed to induce the production of β-galactosidase in the bacteria. ArsR has a high specificity for antimonite/Arsenite, thus conferring the developed sensing system with high selectivity. This was demonstrated by evaluating several oxoanions and soft metals as potential interferents. The concentration of β-galactosidase expressed in the bacteria was monitored by chemiluminescence. Using this sensing system, antimonite can be detected at concentrations as low as 10 −15 M. The importance of the E. coli chromosomal Ars Operon on the observed response was evaluated by employing a strain of E. coli where the chromosomal Ars Operon has been deleted.
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sensing antimonite and Arsenite at the subattomole level with genetically engineered bioluminescent bacteria
Analytical Chemistry, 1997Co-Authors: Sridhar Ramanathan, Barry P. Rosen, Sylvia DaunertAbstract:A highly sensitive and selective optical sensing system for antimonite has been developed using genetically engineered bacteria. The basis of this system is the ability of certain bacteria to survive in environments that are contaminated with antimonite, Arsenite, and Arsenate. The survival is conferred to the bacteria by the Ars Operon, which consists of five genes that code for three structural proteins, ArsA, ArsB, and ArsC, and two regulatory proteins, ArsD and ArsR. ArsA, ArsB, and ArsC form a protein pump system that extrudes antimonite, Arsenite, and Arsenate once these anions reach the cytoplasm of the bacterium. A method was developed for monitoring antimonite and Arsenite by using a single plasmid that incorporates the regulatory gene of the extrusion system, ArsR, and the genes of bacterial luciferase, luxA and luxB. In the designed plasmid, ArsR regulates the expression of bacterial luciferase in a manner that is dependent on the concentration of antimonite and Arsenite in the sample. Thus, th...
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Genetically engineered bacteria: electrochemical sensing systems for antimonite and Arsenite.
Analytical Chemistry, 1997Co-Authors: Donna L. Scott, Barry P. Rosen, Sridhar Ramanathan, Weiping Shi, Sylvia DaunertAbstract:A bacterial sensing system that responds selectively to antimonite and Arsenite has been investigated. The bacteria used in these studies have been genetically engineered to produce the enzyme β-galactosidase in response to these ions. This is accomplished by using a plasmid that incorporates the gene for β-galactosidase (reporter gene) under the control of the promoter of the Ars Operon. This plasmid also encodes for the ArsR protein, a regulatory protein of the Ars Operon, which, in the absence of antimonite or Arsenite, restricts the expression of β-galactosidase. In the presence of antimonite or Arsenite the ArsR protein is released from the operator/promoter region of the Ars Operon and β-galactosidase is expressed. The activity of this enzyme was monitored electrochemically using p-aminophenyl β-d-galactopyranoside as the substrate. The bacterial sensing system responds selectively to Arsenite and antimonite (and to a lesser extent Arsenate) and shows no significant response to phosphate, sulfate, n...
Nobutaka Funa - One of the best experts on this subject based on the ideXlab platform.
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direct transfer of starter substrates from type i fatty acid synthase to type iii polyketide synthases in phenolic lipid synthesis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Akimasa Miyanaga, Nobutaka Funa, Takayoshi Awakawa, Sueharu HorinouchiAbstract:Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the Ars Operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the Ars Operon, are responsible for the biosynthesis of C22–C26 fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C22–C26 fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the Ars Operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer.
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phenolic lipid synthesis by type iii polyketide synthases is essential for cyst formation in azotobacter vinelandii
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Nobutaka Funa, Hiroki Ozawa, Aiko Hirata, Sueharu HorinouchiAbstract:Cysts of Azotobacter vinelandii are resting cells that are surrounded by a protective coat, conferring resistance to various chemical and physical agents. The major chemical components of the cyst coat are alkylresorcinols, which are amphiphilic molecules possessing an aromatic ring with a long aliphatic carbon chain. Although alkylresorcinols are widely distributed in bacteria, fungi, plants, and animals, no enzyme systems for their biosynthesis are known. We report here an Ars Operon in A. vinelandii that is responsible for the biosynthesis of the alkylresorcinols in the cysts. The Ars Operon consisted of four genes, two of which encoded a type III polyketide synthase, ArsB and ArsC. In vitro experiments revealed that ArsB and ArsC, sharing 71% amino acid sequence identity, were an alkylresorcinol synthase and an alkylpyrone synthase, respectively, indicating that ArsB and ArsC are not isozymes but enzymatically distinct polyketide synthases. In addition, ArsB and ArsC accepted several acyl-CoAs with various lengths of the side chain as a starter substrate and gave corresponding alkylresorcinols and alkylpyrones, respectively, which suggests that the mode of the ring folding is uninfluenced by the structure of the starter substrates. The importance of the alkylresorcinols for encystment was confirmed by gene inactivation experiments; the lack of alkylresorcinols synthesis caused by Ars mutations resulted in the formation of severely impaired cysts, as observed by electron microscopy.