DMS SYNTHESIS

Introduction

Don't you think it is fantastic if the small organism, E. coli, create something big, like cloud? You might think we are daydreamers, but we found a clue in some scientists' words. In our project, for making cloud, we try to make E. coli which can produce dimethyl sulfide (DMS), the source of Cloud Condensation Nuclei (CCN). In wetlands or sea, specific marine bacteria and coral produce DMS, so we try to get the genes related to this synthesis pathway and introduce the genes into E. coli.

How cloud is produced from DMS?

DMS is a simple volatile material formed through multi-step reactions by some marine organisms. After formed in the ocean, it is volatilized, decomposed by being exposed to ultraviolet rays in the sky. Then it is converted to sulfate aerosol. In nature this sulfate aerosol plays a role as one of Cloud Condensation Nuclei (CCN), which are tiny particles around which water vapor condenses to form cloud (Fig. 1) [1].

There exists Methionine (Met) - DimethylSulfidePropionate (DMSP) - DMS route as one of DMS biosynthetic pathway. This route consists of Met-DMSP synthetic pathway of certain diatoms and corals and DMSP-DMS metabolic pathway of marine bacteria [2] (Fig. 1).

The structure of each intermediate chemical had been already clarified.(Fig. 2)

They are 4-Methylthio-2-oxobutyrate(MTOB), 4-Methylthio-2-hydroxybutyrate(MTHB) and 4-Dimethylsulfonio-2-hydroxy-butyrate(DMSHB). However, genes responsible for each intermediate reaction of Met-DMSP pathway are still unknown (Fig. 2). And only candidates in Fragilariopsis cylindrus are suggested by Barbara R. Lyon et al [5]. This organism is a model sea-ice diatom and can produce DMS in Met-DMSP synthetic pathway.

Their osmotic pressure is controlled by DMSP density (Fig. 3-1). So, the authors of the paper hypothesized that proteins whose amounts increase under a condition when F. cylindrus produces a lot of DMSP seem to be enzymes that catalyze the DMSP biosynthetic pathway. They compared the proteome of F. cylindrus cultured under high-salinity with under general salinity by using 2-dimensional electrophoresis and found that the amounts of some proteins increased under high salinity condition compared to general condition (Fig. 3-2). Taking advantage of the mass spectrometry to these proteins, chemical reaction types in the Met-DMSP biosynthesis pathway and knowledge on substances of known enzymes, they speculated 5 candidate genes (AT, REDOX, SAMmt, DECARB, DiDECARB ) might assigned to each step.

On the other hand, a gene concerned with DMSP-DMS pathway is identified from another organism. The enzyme which catalyzes the one-step reaction is encoded by dddD gene. It was reported that dddD gene of Marinomonas sp., one of marine bacteria, is functional in E. coli. [3]

Depending on these previous works, we speculate that introducing the 5 genes of F. cylindrus and dddD could enable E. coli to produce DMS. But it is still unclear whether each of these genes finely expresses in E. coli. So we tried to introduce these genes separately into E. coli and verify its expression. In order to know whether the candidate proteins properly catalyze each reaction step, we used High Performance Liquid Chromatography (HPLC) to detect each reaction product of Met-DMSP synthetic route. To detect DMS, we used DMS detecting tube.

Experiments

As described in the Introduction section, there are 5 candidate genes (AT, REDOX, SAMmt, DECARB, and DiDECARB) which are considered to be involved in the Met-DMSP synthesis pathway. Also, one gene, DddD, is known to be involved in the DMSP-DMS metabolic pathway. We planed to introduce each gene to E. coli, to check the function of each gene one by one (Fig. 4).

1. Cloning of 5 genes from genomic DNAs

We obtained the target genes from genomic DNA of each organism. For the Met-DMSP biosynthesis pathway, we purchased the genomic DNA of F. cylindrus, which is used in the preceding study [Barbara et al., 2011]. With respect to dddD gene, which is involved in the final step of DMS synthesis, the function of dddD homolog from Marinomonas sp. MWYL1 strain was verified in E. coli in the preceding study [Jonathan et al., 2007]. However, we used Ruegeria pomeroyi genome for the source of dddD cloning [Jonathan et al., 2007], since the genome of Marinomonas sp. MWYL1 was less commonly available.

The function of dddD homolog from R. pomeroyi has not been experimentally verified so far. So we decided to start our project from the confirmation of this gene. We first compared the protein seaquence of dddD gene from R. pomeroyi and the one from Marinomonas sp. These proteins show high homology each other based on the global alignment (41 % identity, 59 % similarity).

Moreover, as a common feature, these proteins share a domain structure with CaiB protein, which is a predicted acyl-CoA transferases/carnitine dehydratase.

From the genomic DNAs, we amplified the selected genes (AT, REDOX, SAMmt, DECARB, DiDECARB, and DddD) by PCR. Because F. cylindrus is an eucaryote, genes of AT, REDOX, SAMmt, DECARB, and DiDECARB contain introns. To remove these introns, we performed Overlap Extension PCR (OE-PCR) (Fig. 7).

2. Plasmid construction

We constructed the expression plasmids using the amplified PCR products. To date, we completed the DddD generator and the AT generator (Fig. 8).

We chose T7 promoter as a promoter, so that we can overproduce AT and dddD gene products using E. coli BL21(DE3).

3. Examination of the AT generator

Initially, we were planning to verify the function of AT generator by directly detecting MTOB, the product of AT reaction, by the UV absorption with HPLC. However, it turned out that E. coli produces too many chemicals with similar characteristics to MTOB, making the direct detection of MTOB difficult. We searched for alternative method and found a protocol for fluorescent labeling of MTOB.

DMB (1,2-diamino-4,5-methylenedioxybenzene) reacts with α-keto acid specifically and give strong fluorescence (Fig. 9)[6][7][8]. MTOB is an α-keto acid that should be a good substrate of the labeling. Fortunately, there seem to be few α-keto acid in E. coli lysate, making our MTOB detection assay easy.

For the E. coli lysate preparation, we grow the AT generator transformants in a rich media containing methionine, IPTG and 2-oxoglutarate (necessary for the reaction from Met to MTOB [Summers et al., 1998]).

4. Examination of DddD generators

In order to verify the production of DMS, we used the "TMB-DMS Detector tube" (Tokyo Gas Engineering Co.,Ltd). The Detector tube detects gas condensation of DMS by reduction of permanganate by DMS.

E. coli transformants with DddD generator is grown in a rich media supplemented with IPTG and DMSP at final 10 mM concentration.

Results and Discussion

1. Cloning of genes

Using genomic DNA as a template, we performed OE-PCR to remove introns from F. cylindrus genes for AT, SAMmt, DECARB, DiDECARB. OE-PCR products were checked by electrophoresis (Fig. 10). For all samples, the left is the PCR product from each gene (negative control) and the right is the OE-PCR product.

Compared to the PCR products, the bands of the OE-PCR products showed a shorter length, indicating that the introns were removed. Thus, we attained our parts composed of only exons from each gene. We sequenced these parts and confirmed that the introns were precisely removed.

2. Assay

As is described in Experiment section, we cannot directly detect MTOB by HPLC. So it is necessary to establish alternative assay system. We try to establish an MTOB detection system which use fluorescent labeling by DMB (Fig. 11). In this assay, pure chemicals (sample 1 and 2) or E. coli crude extract (sample 3) were separated by C-18 column with acetonitrile gradient.

In Sample 1, a single strong peak at 35.233 minutes is observed, indicating that this peak represents DMB. Similarly, in addition to the DMB peak, there is another strong peak at 34.070 minutes in Sample 2. This peak represents MTOB labeled by DMB, since free MTOB is not fluorescent. Note that the peak for free DMB (35.233 minutes) is less abundant in Sample 2 when compared to Sample1, suggesting the conversion of free DMB to DMB-MTOB conjugates.

The peak of MTOB-DMB conjugate was also observed in Sample 3 (35.228 minutes). In this experiment, samples were prepared from E. coli grown in a rich media supplemented with IPTG, 2-oxoglutarate and methionine. The cell pellets were disrupted and the supernatant was obtained and filtered by an ultrafiltration column (MWCO 3,000). The collected lysate was treated by DMB. As clearly observed, the peak around 34.070 is missing in this sample, demonstrating that all the component of MTOB formation without AT generator produce no significant signal at this specific retention time. These results indicate that MTOB labeling by DMB is an ideal method to detect MTOB synthesis by AT generator transformants.

3. AT reaction

Starting from the genomic DNA of F. cylindrus, we have completed the construction of the AT generator. However, unfortunately, we did not use the generator in our detection system described above, as the we could not prepare the generator until very recently for some reasons. So, the much-anticipated results of the AT reaction assay will be obtained in the future. We believe that we can present the detailed results in the coming jamboree this year.

4. DddD reaction

Next, we transformed the DddD generator into E. coli and cultivated the cells in a DMSP and IPTG containing medium, to verify the conversion of DMSP to DMS by dddD gene product. The cells were grown in a sealed test tube to retain vapor from the culture, as DMS is a highly-volatile compound. The gaseous phase of the culture containing DMS was collected and examined by the DMS Detection tube.

Conclusion

We have completed two plasmids for the partial reconstitution of DMS synthesis in E. coli. We have also established the effective system for the detection of MTOB, a intermediate of DMSP synthesis, by using fluorescent labeling of MTOB in E. coli lysates. For the final step of DMS synthesis, we have successfully detected the conversion of DMSP to DMS in our DddD generator-dependent manner. (we examined the function of DddD homolog in a crude extract of E. coli. Unfortunately, we did not detect significant amount of DMS produced by our DddD generator.)

Future Work

The examination of the AT generator is finally possible and we believe that it works. Also, the step of DddD can be improved as discussed in this study. The next step of this project is to introduce all of the four candidate genes hypothetically involved in this pathway to E. coli. We will try to detect DMS formation directly by adding excess quantity of methionine to the growth media.

What can we do if we achieve the creation of DMS producing E. coli? In nature, biologically produced DMS is evaporated and converted to sulfate aerosol by UV irradiation, which forms cloud condensation nuclei (CCN). What we can try first is to emulate this process in vitro. Experimental demonstration of these reactions will become an intriguing show for the understanding of the global climate system.

Taking a long view, it might become possible for us to spread the engineered bacteria, to manipulate the local climate. However, it is absolutely necessary to establish effective security system before we use engineered organism in nature.

Reference

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