Geosmin Synthesis Essay

1. Introduction

Taste and odor (T & O) problems mainly caused by secondary metabolites of microorganisms in aquatic ecosystems have been frequently reported worldwide [1,2,3,4]. Geosmin (trans-1, 10-dimethyl-trans-9-decalol), a sesquiterpene derivative with an earthy/musty smell, is a common odor compound in surface water. Since its discovery in Streptomyces griseus LP-16 [5], geosmin has been regarded to cause frequent T & O incidents in water supplies, because of its strong earthy smell and extraordinarily low sensory threshold of 4–20 ng·L−1 [6]. Cyanobacteria is the main phytoplankton group in many eutrophic waters; members of this group, along with myxobacteria, actinomycetes and fungi, are the principal producers of geosmin [4,7,8].

A 726-amino acid cyclase encoded by sco6073 in Streptomyces coelicolor A3 (2) catalyzes the Mg2+-dependent cyclization of farnesyl diphosphate (FPP) to geosmin [9]. According to Jiang et al. [10], this type of cyclase is a bifunctional enzyme composed of two similar domains at the N-terminal and C-terminal positions, and FPP is converted to geosmin through the catalysis of these two domains. In cyanobacteria, two putative geosmin synthase genes (geoA1 and geoA2) homologous to sco6073 were identified from the geosmin-producing cyanobacterium, Phormidium sp., by using degenerating primers for PCR and reverse PCR [11]. Agger et al. [12] and Giglio et al. [13] cloned and successfully expressed the germacrene/germacradienol and geosmin synthase genes (npunmod) from the geosmin-producing cyanobacterium, Nostoc punctiforme PCC 73102, in Escherichia coli. Apart from the benthic/periphytic groups represented above, planktonic (bloom-forming) cyanobacterium species are also likely to produce geosmin [2,7]. However, only Giglio et al. [14] have analyzed the expression of geosmin synthase in Anabaena circinalis. Further in-depth studies on the genetics of geosmin synthesis from bloom-forming cyanobacteria have yet to be conducted.

Water blooms have become a frequent occurrence in Chinese waters, particularly in large shallow lakes. T & O episodes that result from these water blooms have become a major environmental problem, with the worst case recorded in Lake Taihu in May, 2007 [15]. Two plateau lakes (i.e., Dianchi and Erhai) in Yunnan Province have experienced severe odor problems caused by water blooms in the past decade [3]. Our previous work found that the planktonic heterocystous species, Anabaena ucrainica (Schhorb.) Watanabe, is involved in the odor problems of these two lakes (unpublished data). In the present study, two strains of A. ucrainica with high geosmin productivities were isolated from Dianchi and Erhai lakes and used to investigate the genes responsible for geosmin synthesis. The present study aimed to identify and characterize geosmin synthase genes from the bloom-forming A. ucrainica strains, to explore whether or not other genes are structurally or functionally related to this gene and to preliminarily investigate the origin and evolution of these genes.

2. Materials and Methods

2.1. Isolation of Cyanobacterial Strains

Two A. ucrainica strains, CHAB (Collection of Harmful Algal Biology) 1432 and CHAB 2154, were isolated from Dianchi and Erhai lakes, respectively, by using the Pasteur micropipette method and were maintained in screw-capped tubes that contained 5 mL of liquid CT medium [16]. For geosmin analysis and further molecular experiments, the strains were cultured in 150 mL of CT medium in 300 mL flasks under a light:dark regime of 12:12 at a photon density of approximately 25 μmol·m−2·s−1 and a temperature of 25 ± 1 °C. Other geosmin producers used in this study were cultured in CT medium and then deposited in the CHAB, Institute of Hydrobiology, Chinese Academy of Sciences. For the determination of geosmin productivities, experiments were performed for 16 days in 250-mL flasks that contained 120 mL of A. ucrainica cultures with an initial concentration of approximately 1.6 × 106 cells·mL−1. All experiments were performed in triplicate.

2.2. Analysis of Geosmin

The geosmin produced by A. ucrainica strains was analyzed by headspace solid phase micro-extraction coupled with gas chromatography (GC), as described by Watson et al. [6] and Li et al. [3]. Solid phase micro-extraction (SPME) fiber (Polydimethylsiloxane/Divinylbenzene (PDMS/DVB), 65 μm, Supelco, Sigma-Aldrich, St. Louis, MO, USA), GC with an flame ionization detector (FID) detector (GC-2014C, Shimadzu, Tokyo, Japan) and a capillary column (TC series, WondaCap 5, 0.25 mm × 30 m × 0.25 μm, Shimadzu) were used in the analyses. The oven temperature program was set as described by Li et al. [3]. The geosmin standard solution (100 ng·μL−1, Supelco) was used to verify the analysis results of samples through the external standard method. Peaks corresponding to the geosmin standard were also confirmed by gas chromatography-mass spectrometer (GC-MS) (HP6890GC-5973MSD, HP, Palo Alto, CA, USA).

2.3. General Molecular Techniques and Genome Walking

The genomic DNAs of the two strains were prepared using a DNA Mini Spin kit (Tiangen, Beijing, China). Two primers, G2f (5'-GCAACGACCTCTTCTCCTAC-3') and G2r (5'-CACCCAACTGTCAGTCATATCCT-3'), were designed on the basis of the geosmin synthase genes of N. punctiforme PCC 73102 and Phormidium sp. to amplify the corresponding sequence (943 bp) of A. ucrainica strains. PCR was carried out using the LA Taq polymerase kit (Takara, Dalian, China) and performed in a Bio-Rad MJ mini personal thermal cycler (MJ Research, Foster, CA, USA) with the following program: 3 min at 94 °C for denaturation, followed by 35 cycles of 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 60 s.

Genome walking experiments were performed to amplify the flanking regions by using a genome walking kit (Takara). The primers used in the genome walking are listed in Table S1. All of the PCR and genome walking products were cloned to the PMD18-T vector (Japan) and then sequenced.

Primer sets GTC1F(R) and GTC2F(R) (listed in Table S1) covering the intergenic spacers between neighboring genes were designed in this study and used to confirm the gene composition and arrangement of the geosmin operon in seven geosmin-producing species by using the same PCR program as mentioned above.

2.4. RNA Extraction and Reverse Transcriptional PCR

Fresh cells of the two A. ucrainica strains were collected by centrifugation (12,000× g, 4 °C, 5 min) and then transferred into 2-mL centrifuge tubes. Mini-beadbeater (0.5 mL) and TRIzol reagent (1 mL, Invitrogen, Carlsbad, CA, USA) were added into tubes for cell resuspension. Total RNA was extracted using TRIzol reagent in accordance with the manufacturer’s instructions, and the RNA sample was treated with DNase I (Promega, Madison, WI, USA) to remove the mixed genomic DNA. Reverse transcription to cDNA using a transcriptase kit (Generay, Shanghai, China) was carried out.

Primers RNC1F with RNC1R and RNC2F with RNC2R (Table S1) were used to examine the integrity of the geosmin synthase gene with flanking genes by PCR using cDNA as a template. Control PCR was conducted using the RNA that underwent DNase digestion as the template. The elongation time was modified to 30 s, and the other steps were identical to the above description.

2.5. Bioinformatics Analysis

Homologous genes of the geo gene in A. ucrainica CHAB 1432 were searched by BLAST [17]. The Conserved Domain Database (CDD; www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) [18] was used to identify the conserved motifs and functional sites. Neighbor-joining (NJ) and maximum parsimony (MP) phylogenetic trees of the geosmin gene were constructed by Mega 4.0 [19] with a bootstrap value of 1000.

Geosmin synthesis operons and 16S rDNA sequences of A. ucrainica CHAB 1432 and CHAB2155 were deposited in the NCBI nucleotide sequence database. The accession numbers are HQ404996 and HQ404997 for the geosmin operons and GU197649 and GU197642 for the 16S rDNA.

3. Results and Discussion

3.1. Isolation of a Bloom-Forming Cyanobacterium Responsible for Geosmin Production

Two A. ucrainica strains that exude an earthy/musty odor were obtained from Dianchi and Erhai lakes, and chemical analysis showed geosmin as their main volatile component (Figure S1). The features of geosmin production in A. ucrainica strains were studied (Figure 1). Strains CHAB 1432 and 2155 similarly showed the following pattern in geosmin production: A slight decrease in rapid growth period (0–6 days), a rapid increase in stagnant period (approximately 14 days) and a significant decrease in the last period of cultivation. The maximum total geosmin production of these two strains were 2.20 × 10−5 ng·cell−1 and 2.60 × 10−5 ng·cell−1, and the total production was more than 1.20 × 10−5 ng·cell−1 in the entire growth cycle.

Among documented cyanobacterial geosmin/2-methylisoborneol (MIB) producers, more than 13 planktonic species were identified [2,7]. These planktonic taxa can form unsightly or highly visible surface blooms, and numerous T & O episodes have been attributed to bloom-forming genera, such as Anabaena, Aphanizomenon and Planktothrix [4,20,21,22]. A. ucrainica has been historically implicated as the main source of geosmin in a reservoir in Japan [21]. Results of the chemical analysis in the present study showed A. ucrainica as a geosmin producer and probably the principal producer of geosmin in Dianchi and Erhai lakes. On the basis of the total geosmin productivity determined in these experiments (1.20–2.60 × 10-5 ng·cell−1 ) and the sensory threshold of geosmin, water bodies are at risk for T & O events when the abundance of geosmin-producing cyanobacteria is greater than 5 × 105 cells/L (10 ng·L−1 total geosmin according to the average productivity).

Figure 1. Cell growth (A) and geosmin productions (B) of A. ucrainica CHAB 1432 and 2155 in the test cycle.

Figure 1. Cell growth (A) and geosmin productions (B) of A. ucrainica CHAB 1432 and 2155 in the test cycle.

3.2. Identification and Characterization of the Geosmin Synthase Genes of A. ucrainica

Two 943-bp identical fragments were amplified from A. ucrainica strains CHAB 1432 and CHAB 2155 by using primers G2f and G2r. Subsequently, the whole geosmin synthase gene (geo gene) in A. ucrainica was successfully cloned through genome walking-PCR (Figure 2). The geo gene of CHAB 1432 and CHAB 2155 is 2256 bp in length. Only one base pair difference at the 442 bp site (T for 1432 and C for 2155) was found between these two strains. The BLAST search revealed that the geo genes from these two strains were 76.9%, 65.4%, 81.0% and 80.6% identical in DNA sequences and 82.9%, 61.4%, 86.8% and 87.1% identical in deduced amino acid sequences with the well-elucidated geosmin synthase genes, npunmod (N. punctiforme PCC 73102), geoA1 (Phormidium sp.), geoA2 (Phormidium sp.) and that from Oscillatoria sp., respectively. The high conservation of the geosmin gene in cyanobacteria could provide essential information for the development of molecular monitoring methods of geosmin-producing cyanobacteria. A recent study has reported a quantification method for potential geosmin-producing Anabaena in freshwater on the basis of geosmin gene sequences [20,23].

Figure 2. Geosmin synthesis genes of A. ucrainica. (A) Strain CHAB 1432; (B) strain CHAB 2155.

Figure 2. Geosmin synthesis genes of A. ucrainica. (A) Strain CHAB 1432; (B) strain CHAB 2155.

The functional sites of Geo were identified by CDD search (specific hits with terpene cyclase non-plant C1) and amino acid alignment; these sites include NPUNMOD (N. punctiforme PCC 73102), GeoA1 (Phormidium sp.), GeoA2 (Phormidium sp.), SAV2163 (Streptomyces avermitilis MA-4680) and SCO6073 (S. coelicolor A3 (2)) (Figure S2). In general, the N-terminal domains of all of the analyzed proteins are more conserved than the C-terminal domains and contain two typical Mg2+-binding motifs, namely, DDHFLE and RNDLFSYQRE sequences (Figure S2) [9,10]. Two less conserved Mg2+-binding motifs, namely, DDY(F/Y)(P/H) and ND(I/V)(F/V)SY(Q/R)KE, were also found in the C-terminus. Interestingly, the N-domains and C-domains of Geo and NPUNMOD were homologous to each other and shared identical motifs, such as the Mg2+-binding sites (Figure S3). The DNA sequences of geo and npunmod in the N- and C-domains shared 50.9% and 44.2% similarities, respectively.

3.3. Structure and Features of the Geosmin Operon

Two putative cyclic nucleotide-binding protein genes (cnb) that are 1398 and 1407 bp in length were identified downstream of the geo gene through genome walking (Figure 2). These two cnb genes have the same transcriptional orientation and are homologous, with 73% DNA similarity. Further BLAST and CDD searches suggest that this type of cyclic nucleotide-binding gene belongs to the Crp–Fnr regulator family. The flanking regions of the geosmin synthase gene were also analyzed in the released genomic data (from NCBI) of N. punctiforme PCC 73102, Oscillatoria sp. PCC 6506, Cylindrospermum stagnale PCC 7417, four representative species of Actinomycetes and Myxococcus xanthus DK 1622. As shown in Figure 3, the organization of the geosmin synthase genes in N. punctiforme PCC 73102, Oscillatoria sp. PCC 6506, C. stagnale PCC 7417 and M. xanthus DK 1622 are similar to that of the A. ucrainica strains. No cnb genes were found in adjacent regions of the geosmin synthases of S. coelicolor A3 (2) and S. avermitilis MA-4680. The array of the geosmin synthase with cnb genes in Saccharopolyspora erythraea NRRL2338 is different from that in A. ucrainica, and only one cnb

Brown University chemists have figured out precisely how the warm, slightly metallic scent of freshly turned soil is made. In Nature Chemical Biology, the team describes how geosmin, the organic compound responsible for the scent, is produced by an unusual bifunctional enzyme.

PROVIDENCE, R.I. [Brown University] — Brown University chemists have found the origins of an odor – the sweet smell of fresh dirt. In Nature Chemical Biology, the Brown team shows that the protein that makes geosmin – source of the good earth scent – has two similar but distinct halves, each playing a critical role in making this organic compound.

“Everyone is familiar with the wonderful smell of warm earth,” said David Cane, professor of chemistry at Brown who oversaw the research. “Now we know precisely how it is made.”

Geosmin, which literally translates to “earth smell,” was scientifically identified more than 100 years ago. In soil, bacteria produce the chemical compound. In water, blue-green algae make it. Along with the pleasant scent of warm, moist soil, geosmin is also responsible for the muddy “off” taste in some drinking water. That is why the substance is of interest to water purification experts and even vintners, who want to keep the benign but pungent substance out of their wine.

Until recently, scientists knew little about how geosmin is made. Then, a few years ago, Cane found the gene responsible for geosmin formation in Streptomyces coelicolor, a strain of plant-munching bacteria found in soil. Last year, the team discovered that a single protein converts farnesyl diphosphate to geosmin.

In their new work, Cane and his lab team found that this protein, called germacradienol/geosmin synthase, folds into two distinct but connected parts, similar to a dumbbell. One piece is responsible for the first half of the reaction, cranking out a chemical that wafts over to the companion bit of protein, which then produces geosmin.

“We found that geosmin is created by this bifunctional enzyme,” Cane said. “The two steps of the process that forms geosmin are metabolically related. This finding was a real surprise. This is the first bifunctional enzyme found for this type of terpene, the class of chemicals geosmin belongs to.”

Jiaoyang Jiang, a Brown graduate student in the Department of Chemistry and lead author of the journal article, said microbiologists working in water purification plants will be most interested in knowing the origins of geosmin. By understanding precisely how the substance is synthesized, Jiang said, these experts may find a way to block it – avoiding the foul taste that keeps people away from the tap.

“Geosmin may smell good in the garden, but not in the glass,” she said.

Xiaofei He, a former Brown graduate student, contributed to the research. The work was funded by the National Institute of General Medical Sciences.

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