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Research Project BIO-6019Y

Characterization of a novel DMSP-producing

alphaproteobacterium, Novosphingobium sp. MBES04.

Carolina Pereira Godinho 100039990

Under the supervision of Professor Jonathan Todd

University of East Anglia

May 6th, 2016

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TABLE OF CONTENTS

ABSTRACT 3

INTRODUCTION 4

MATERIALS AND METHODS 8

RESULTS 10

DISCUSSION 17

ACKNOWLEDGEMENTS 21

REFERENCES 22

APPENDIX 25

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ABSTRACT

Dimethylsulfoniopropionate (DMSP) is one of the Earth’s most abundant organosulfur molecules, and is produced at around 109 tons per year. It is the main precursor for the volatile organic sulfur compound, dimethylsulfide (DMS), which plays a key role in the global sulfur cycle and local climate regulation. For years it was believed that marine eukaryotes were the sole producers of DMSP, however, recent work revealed that this process also occurs in bacteria, which produce DMSP through the same pathway as marine algae. This work also identified the first gene associated with DMSP production, mmtB. To attempt to demonstrate the significance of bacterial DMSP production in the environment, culture dependent techniques were used to identify DMSP producing bacteria from Stiffkey saltmarsh. Among these, a Novosphingobium sp, MBES04 was identified that did not appear to contain mmtB. Here we show for the first time that an alphaproteobacterium from the order Sphingomonadales produces DMSP, not through the same pathway previously shown in bacteria, but instead, following the pathway used by higher plants from the Compositae family; the methylation pathway. Our characterization of DMSP production by this bacterium shows that this molecule likely serves as an osmoprotectant within the organism, and is also produced at greater amounts in low nitrogen conditions. This characterization is the next step in understanding the role of bacteria in environmental DMSP production globally. !!

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INTRODUCTION

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! The!sulfur!molecule!dimethylsulfoniopropionate (DMSP) is an environmentally significant molecule predominantly found in the marine environment. It was first isolated in the red algae Polysiphonia fastigiated and Polysiphonia nigrescens, and for many years it was thought that its production was limited to marine eukaryotes, including phytoplankton (diatoms, haptophytes and dinoflagellates), macroalgae, a few higher plants and by some corals (Dickschat et al., 2015). It is catabolized primarily by marine bacteria, specifically those in the phylum proteobacteria, and by some non-DMSP producing phytoplankton (Stefels, 2000). DMSP is an environmentally important molecule in several ways (figure 1). When it is degraded, it provides essential nutrients specifically sulfur and carbon, to marine bacteria. In DMSP-producing algae, DMSP-sulfur can make up between 50-100% of the total organic sulfur in the cell, and it contributes to ~10% of the fixed carbon in the ocean (Stefels, 2000). DMSP has been reported to have multiple cellular functions. It is a known osmolyte- organisms use its osmotic potential to regulate cell volume, and its osmoprotectant properties have shown to enhance salinity tolerance in Escherichia coli even at nanomolar concentrations (Cosquer et al.,1999). In marine algae, DMSP and the products of its breakdown serve as an antioxidant system by scavenging harmful reactive oxygen species released by oxidative stresses, as demonstrated by studies where CO2 and Fe limitation, as well as the presence of increased solar ultraviolet radiation, appear to increase intracellular DMSP levels (Sunda et al., 2002). It has been proposed that DMSP also serves as a cryoprotectant in polar algae and as a grazing deterrent to zooplankton via its cleavage by DMSP lyase enzymes to acrylate, due to its antimicrobial properties (Sunda et al., 2002; Wolfe et al., 1997). Recent studies also show that DMSP is a strong chemoattractant, and that the alphaproteobacterium Silicibacter sp. and !-proteobacterium Pseudoalteromonas haloplanktis exhibit strong chemotaxis towards high DMSP concentrations, using highly directional swimming to migrate towards it (Seymour et al., 2010). The importance of DMSP lies not only in its availability as a source of reduced sulfur and carbon for marine microbes, but also because DMSP is the precursor for the volatile organic sulfur compound dimethylsulfide (DMS) (Reisch et al., 2011). DMSP catabolism is responsible for the production of ~300 million tones of DMS in the ocean every year (Levine et al., 2012). It predominantly occurs via DMSP-dependent DMS (ddd) genes. Six different ddd gene products have been identified (DddD, DddL, DddP, DddQ, DddY and DddW), and these are responsible for generating DMS from DMSP. While DddD produces DMS and 3-hydroxypropionate, the other five DMSP-cleavage enzymes produce DMS and acrylate (Todd et al., 2007; Reisch et al., 2011). DMS is the natural sulfur compound which fills the role originally assigned to H2S of transferring sulfur from the sea to the atmosphere and subsequently to land (Stefels, 2000). In the atmosphere, DMS is oxidized by free radicals such as OH and NO3, to form a variety of products including sulfate and methane sulfonate aerosols, which act as a cloud condensation nuclei (CCN). The number and density of the CCN is a major determinant in cloud formation, which has an albedo effect cooling the ocean by reducing the amount of solar radiation that it absorbs (Levine et al., 2012). These clouds are also part of the sulfur cycle and are responsible for the transfer of sulfur from the atmosphere to land by acid rain (Sievert et al., 2007). Ultimately, the degradation of DMSP to DMS is the only route through which biogenic sulfur is moved from ocean to land (figure 1).

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In addition to these cleavage pathways, DMSP can also be catabolized in a different manner, without releasing DMS. The initial step involves the demethylation of DMSP to methylmercaptopropionate (MMPA), which is then catabolized into methanethiol (MeSH) and acetate (Todd et al., 2012). The gene responsible for the initial demethylation, dmdA, is abundant in two groups of marine ∝-protobacteria namely Roseobacters and SAR11 clade. Even though this pathway does not liberate DMS, it is responsible for most of the global DMSP catabolic flux (Howard et al., 2006). Around 109 tons of DMSP are produced per year globally (Curson et al., 2011). Three pathways for DMSP biosynthesis have currently been established (figure 2); a methylation pathway in higher plants, a transamination pathway in marine algae, and a decarboxylation pathway found in one dinoflagellate so far (figure 2) (Stefels, 2000; Uchida et al., 1996). The initial molecule used for all biosynthesis of DMSP via these three pathways is L-methionine (Met), and there is strong evidence that the biochemical pathway from methionine to DMSP has evolved at least three independent times through different intermediates (Stefels, 2000). Methionine is formed from cysteine, a product of environmental sulfate assimilation (Leustek & Saito, 1999). The thiol group of cysteine is transferred to O-phosphohomoserine de novo, forming homocysteine, which is then methylated to make methionine. Methionine is subsequently used in various transmethylation reactions via its methyl group, and the molecule S-adenosylmethionine (AdoMet), a methyl donor (Stefels, 2000).

Figure 1- Schematic representation of the DMSP cycle. DMSP is produced my marine eukaryotes, and is released when these are killed or die. DMSP is primarily cleaved by DMSP lyase enzymes produced by marine bacteria, releasing the volatile organic sulfur compound, dimethylsulfide (DMS), and acrylate of 3- hydroxypropionate (3-HP). DMS serves a carbon source for marina bacteria, and once in the atmosphere it serves as a chemoattractant to marine mammals and seabirds. In the atmosphere, DMS is oxidized forming the cloud condensation nuclei, which reflect UV radiation, causing local cooling. Ultimately, biogenic sulfur is returned to land by rain.

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The higher plants known to produce DMSP are Wollastonia biflora, some Spartina species and sugar canes (Stefels, 2000). They do this through the methylation pathway (figure 2). The initial methylation step involves the methylation of L-methionine to S-methyl-methionine (SMM), which is dependent of AdoMet (Otte et al., 2004). In W. biflora of the Compositae family, the methylation reaction occurs in the cytosol, and SMM is then transported to the chloroplast (Stefels, 2000). The pathway proceeds through the production of DMS-aldehyde via a decarboxylation reaction, forming an unstable intermediate still to be identified (Dickschat et al., 2015). Ultimately, an oxidation reaction catalyzed by a dehydrogenase using NAD as a cofactor, forms DMSP from DMSP-ald (Trossat et al., 1996). A second route via this pathway has been identified in S. alterniflora; in this grass, DMSP-amine was identified as an intermediate between SMM and DMSP-ald. The enzymes that catalyze the production of DMSP-amine from SMM, and the conversion of DMSP-amine to DMSP-ald are yet to be identified, but it is believed that they involve a decarboxylase and oxidase respectively (Stefels, 2000). The production of DMSP-amine in S.alterniflora shows that the DMSP-specific pathway from SMM to DMS-ald has evolved independently among different families of higher plants, specifically, Gramineae and Compositae.

Another pathway for DMSP biosynthesis, the decarboxylation pathway has been seen in one dinoflagellate (Dickschat et al., 2015). Dinoflagellates are one of the major DMSP producers, containing up to 0.5 M of intracellular DMSP. This production pathway was first shown in the marine dinoflagellate, Crypthecodinium cohnii (Uchida et al., 1996), but it is not yet fully understood, with only one of the steps currently identified (figure 2). In C. cohnii, the first step is the decarboxylation of L-methionine to produce 3-(methylthio)propylamine (MTPA), via a PLP-

Figure 2- Three different DMSP biosynthesis pathways have been identified among different marine eukaryotes (intermediates in bold, reactions in italic). The decarboxylation pathway (red) has been identified in one dinoflagellate (stripped lines represent the steps still to be identified and the intermediate yet to be confirmed). The transamination pathway has been identified in marine algae (black), and the methylation pathway has been identified in higher plants. The methylation pathway varies among families of higher plants, namely the Compositae (green) and the Gramineae (blue) family.

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dependent L-methionine decarboxylase (Kitaguchi et al., 1999). This is the only identified intermediate, however the following steps are believed to be a deamination, oxidation and methylation, which will involve the formation of methylmercaptopropionate (MMPA), and ultimately, the synthesis of DMSP (Stefels, 2000). A third key pathway, the transamination pathway, was identified in a green macroalgae, Enteromorphaintestinalis (now classified as Ulva intestinalis), and is used by algae as well as diatoms (Summer et al., 1998; Gage et al., 1997). This pathway begins with the transamination of methionine to form 4-methylthio-2-oxobutyrate (MTOB), instead of the methylation seen in higher plants (figure 2). The transamination reaction is followed by an NADPH-linked reduction to 4-methylthio-2-hydroxybutyrate (MTHB), which is then methylated to 4-dimethylsufonio-2-hydroxybutyrate (DMSHB) via the methyl group donor S-Adenosyl-L-methionine (SAM) (Dickschat et al., 2015). Ultimately, DMSP is formed through an oxidative decarboxylation of DMSHB. While the first two steps of this pathway appear reversible and are found among a variety of higher and lower plants (even though much higher activities are found in DMSP-producers), the conversion of MTHB to DMSHB appears to be the committing step in this pathway. It seems to be specific for DMSP synthesis, and DMSP production is defined by levels of DMSHB synthesis (Ito et al., 2011). It was long thought that DMSP was only produced by marine eukaryotes. However, a recent study by Curson et al, (Unpublished), showed that a marine heterotrophic bacteria, namely Labrenzia aggregata LZB033, also produces DMSP (Curson et al., Unpublished). LZB033 was found to produce levels of 8.4 pmol DMSP µg protein-1 (Curson et al., Unpublished). In this study, it was also shown that LZB033 uses the same pathway as marine algae to synthesize DMSP; the transamination pathway. Following this discovery, the first DMSP synthesis gene in any organisms, mmtB, was also identified. This gene encodes the enzyme MTHB methyltransferase (MTT), present in three orders of alphaproteobacteria; Rhizobiales, Rhodobacterales and Rhodospirallales (Curson et al., Unpublished). MTT is responsible for catalyzing the methylation of MTHB to DMSHB, the rate-limiting and committing step in the transamination DMSP synthesis pathway (Curson et al., Unpublished). Now that marine bacteria were known to produce DMSP, a study was conducted by Williams et al (unpublished) to investigate the importance of bacterial DMSP production in marine environments. The environment chosen was Stiffkey saltmarsh on the North Norfolk Coast. This is a DMSP-rich environment and is an ideal study site for bacterial DMSP production due to its high levels of salinity, and the ease of studying communities within sediment. Isolates from the sediment were analyzed and 26% of the bacterial isolates grown on marine minimal media were confirmed to produce DMSP between 1-200 nmol DMSP mg protein-1. Using degenerative primers designed to identify the mmtB gene, 21 DMSP-producing bacterial isolates were tested and screened for the presence of mmtB. While many of the isolates were found to contain mmtB, 12 did not. Among those isolated, it was found that an alphaproteobacterium from the order Sphingomonadales, Novosphingobium sp. MBES04 (MBES04), produced high levels of DMSP without the presence of mmtB. This is particularly interesting, as until now only bacteria containing this gene have been shown to produce DMSP. Furthermore, this is the first species outside of the Rhodobacterales, Rhizobiales and Rhodospirillales orders, able to carry out this process. In this study, we aim to characterize DMSP production by this MBES04 species in various environmental conditions, and attempt to discover the pathway used to produce DMSP by testing which pathway intermediates enhance DMSP production. We also estimate whether this organism has the capacity to generate DMS from DMSP.

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MATERIALS AND METHODS

Bacterium growth and media Novosphingobium sp. MBES04 was grown in MBM minimal medium (Baumann & Baumann, 1981) (with a mixed carbon source containing 2 mM of succinate, glucose, sucrose, pyruvate and glycerol) at 30ºC, to an OD600 of ~0.5. In experiments requiring variations of this medium, the salinity of MBM was kept at 35 psu by adjusting the amount of sea salt (Sigma-Aldrich) added. The growth curve was obtained by inoculating 100 ml MBM minimal medium with 2 ml OD600~ 0.6 culture in triplicate and incubating at 30ºC. After 3 hours, the first OD600 measurement was taken, and then repeated hourly until stationary phase was reached. Quantification of Environmental conditions To characterize DMSP production by Novosphingobium sp. MBES04 under different environmental conditions, the bacterium was grown in triplicate in high/low salt and low nitrogen. The salinity was varied using either 5 g or 50 g of sea salts (Sigma-Aldrich), giving salinities of 5 and 50 practical salinity units (PSU). The nitrogen supply was limited be making 35 PSU MBM with no added nitrogen source, and supplementing with 0.5 mM NH4Cl after autoclaving. The cultures were grown shaking at 30ºC for 16h, and were monitored spectrophotometrically by optimal density at wavelength 600 nm (OD600). The DMSP content was tested using gas chromatography (see below). DMS and DMSP by gas chromatography Gas chromatography (GC) assays involved measurement of headspace DMS either produced directly, or via alkaline lysis of DMSP. A flame photometric detector (Agilent 7890A GC fitted with a 7693 autosampler) and a HP-INNOWax 30 m x 0.320 mm capillary column (Agilent Technologies J&W Scientific) were used to measure the headspace of 2 ml vials containing 300 !l liquid. An eight-point calibration curve of DMSP standards (7.5!!M, 75 !M, 750 !M and 7.5mM) was run and the detection limit for headspace DMS was 0.015 nmol. To measure DMS production from DMSP, Novosphingobium sp. MBES04 was grown in MBM minimal medium to OD600 ~0.5, then diluted 1:10 into 300 ml of MBM media containing 5 mM DMSP, with a control MBM with no added DMSP, and incubated for 48 hours at 30ºC before assaying by GC. For measurements of DMSP in Novosphingobium sp. MBES04, cultures were grown in MBM minimal media for 16 hours at 30ºC. An aliquot of 500 !l was centrifuged for 1 min at (14,000rpm, Eppendorf Centrifuge 5424) and resuspended in 200 !l of MBM minimal media. This was sealed in a 2 ml vial with 100 !l of 10 M NaOH to lyse the DMSP. These were incubated for 24 hours at 30ºC before assaying by GC. Protein Estimations To quantify protein concentration in DMSP production assays within cultures, 1 ml of culture was centrifuged for 2 min at 14,000 rpm, and the pellet was resuspended in 500 !l Tris-HCl buffer (50 mM, pH 7). The mix was sonicated for 3 x 10 s with a Markson GE50 Ultrasonic Processor to break open the cells, and centrifuged for 5 min at 14,000 rpm to remove cell debris. A 20 !l aliquot of the supernatant was mixed with 980 !l Bradford reagent. This was repeated with 20 !l of standards of 0, 100, 200 and 400 !g/!!l BSA. These were measured spectrophotometrically at an absorbance of 595 nm (OD595). The standards were used to create a standard curve from which the concentration of protein in the samples could be calculated, and this was used to normalize DMSP production per mg protein.

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Intermediate induction experiments The induction of DMSP was best measured at exponential phase, so 100 ml MBM minimal medium was inoculated with Novosphingobium sp. MBES04 and grown at 30ºC for 11 hours, before being separated into 5 ml aliquots. To measure the effect of pathway intermediates on DMSP production 0.5 mM of either Met, MTOB, MTHB, DMSHB, MMPA, MTPA, SMM and DMSP-amine was added. Control samples were set up containing MBM and culture with no additional intermediates. These were incubated at 30ºC for 4 hours. Subsequently, 1 ml of MBM with the intermediate was centrifuged for 1 min at full speed (4580 x). It was then resuspended in 200 !l of distilled water, and 200 !l of cell-free extract was added to a vial and 100 !l of 100 M NaOH was added to lyse DMSP, and vials were immediately sealed and incubated at 30ºC for 6 hours to allow release of DMS into the headspace before assaying by GC. DMSP estimates measurements were taken prior to addition of the intermediates, and then repeated at 30, 60, 120 and 20 min. To study the effect of oxidative stress, the same process was conducted using 500 !M of H2O2. Chemical syntheses DMSP was generated from DMS (Sigma-Aldrich) and acrylic acid (Sigma-Aldrich) as described in (Todd et al., 2010). DMSHB, DMSP-amine and SMM were synthesized as described in (Curson et al., Unpublished). Met, MTOB, MTHB and MTPA are commercially available and were obtained from Sigma-Aldrich. Polymerase chain reaction (PCR) Amplifications using the mmtB degenerate primers consisted of a 25 !l mix per reaction; 12.5 !l of MyFi polymerase, 11 !l dH20, 0.5 !l forward (CATGGGSTCSAAGGCSCTKTT) and reverse (GCAGRTARTCGCCGAAATCGTA) primers, and 0.5!!l of template. The templates were Novosphingobium sp. MBES04, Labrenzia aggregata LZB033 (positive control) and Ruegeria pomeroyi (negative control). These were run on this program: 95ºC for 5 min, 95ºC for 30 s, 61ºC for 1 min, 72ºC for 15 s, repeat 30 times, 72ºC for 5 min, hold at 4ºC in a TECHNE TC-512 Thermocycler. Amplification was confirmed using gel electrophoresis in a 1% agarose gel. Microscopy To image Novosphingobium sp. MBES04, 5 !l of culture at an OD600 of ~0.5 was placed on a glass slide, and a cover slip was laid over it. This was photographed using the Zeiss-AxioCam microscope with an MRm camera, and images were recorded at 100 x oil immersion magnification. Bioinformatics- BLAST analysis The genome sequence of MBESO4 was sequenced by MicrobesNG, and BioEdit was used to make this sequence into a protein database. The full amino acid sequence for MmtB was blasted in the database using blastn analysis to confirm the presence of absence. A blastp was carried out using ratified mmtB, ddd (D, L, P, Q, Y, W) and dmdA sequences against the generated theoretical proteome of Novosphingobium sp. MBES04.

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RESULTS

Isolation and confirmation of DMSP production by Novosphingobium sp. MBES04 Novosphingobium sp. MBES04, an alphaproteobacterium from the order Sphingomonadales was isolated on MBM minimal medium and checked by microscopy for purity (figure 3).

After confirmation of purity, Novosphingobium sp. MBES04 was found to produce 93.29 nmol DMSP mg protein-1. Confirming mmtB absence The mmtB gene encodes for the MmtB protein, belonging to a family of S-adenosyl-methionine-dependent methyltransferases. Close homologues have been found in 40 sequenced alphaproteobacteria, the majority of which were isolated from hypersaline or marine environments. mmtB has been identified in only three orders of alphaproteobacteria - Rhodobacterales, Rhizobiales and Rhodospirillales. Therefore, it was of interest to test whether Novosphingobium sp. MBES04, from the order, Sphingomonadales contained this gene. Genomic DNA was isolated from Novosphingobium sp. MBES04 and this was used as a template in PCR using the mmtB degenerate primers as described in methods. Genomic DNA from Ruegeria pomeroyi, which lacks mmtB and does not produce DMSP, was used as a negative control. Labrenzia aggregata that has mmtB and produces DMSP was used as a negative control.

Figure 3- Microscope image of Novosphingobium sp. MBES04, confirming its purity.

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As can be seen in figure 4, Novosphingobium sp. MBES04 much like Rugeria pomeroyi gave no PCR product at the expected 246 bp size for mmtB. In contrast, when Labrenzia aggregata genomic DNA was used, a clear 246 bp band corresponding to mmtB was visible, confirming that the PCR was carried out successfully, suggesting that Novosphingobium sp. MBES04 lacks mmtB. To further demonstrate this, the sequenced genome of this bacterium was probed with ratified mmtB sequences and was found to contain no significant matches above the e-50 cut off for functional mmtB (Curson et al., Unpublished). Together, these results confirm that mmtB is absent for Novosphingobium sp. MBES04. Novosphingobium sp. MBES04 growth curve In order to further characterize Novosphingobium sp. MBES04 DMSP production phenotype, it was necessary to gain a basic understanding of its growth characteristics. To do this, Novosphingobium sp. MBES04 was incubated in MBM minimal medium and its growth phases monitored by OD600 in a spectrophotometer (figure 5). We were specifically interested in the identification of its exponential growth phase, where it likely to be most metabolically active.

Figure 4- PCR results with degenerate primers optimized to identify mmtB using genomic DNA from different bacteria. Three organisms were tested, Novosphingobium sp. MBES04 (Novo), Labrenzia LZB033 (+), the positive control conferring mmtB and Rugeria pomeroyi (-), the negative control. The presence of mmtB is denoted by a band at 246bp.

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Figure 5 – Novosphingobium sp. MBES04 Growth curve. Growth curve of Novosphingobium sp. MBES04 grown in MBM minimal media according to its absorbance at OD 600 (n=3). From inoculation, Novosphingobium sp. MBES04 has a lag phase of ~7 hours before reaching an exponential phase between 7-16 hours, where cells are most rapidly diving. After 16 hours, the stationary phase emerges and the cell density plateaus. Our further studies were conducted during the exponential phase, thus samples were used once they had been grown for 11 hours, and reached an OD600 of ~0.6 DMSP production by Novosphingobium sp. MBES04 varies with growth in different intermediates of the DMSP synthesis pathway In an attempt to identify the bacterial DMSP synthesis pathway used by Novosphingobium sp. MBES04, this bacterium was grown in MBM minimal medium to exponential phase and then amended with transamination, methylation and decarboxylation pathway intermediates, and their effect on DMSP production was monitored over time using gas chromatography and normalized to protein levels (figure 6). Microscopy was used to confirm the purity of the samples after the experiment.

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The methylation pathway intermediates, namely methionine and SMM (figure 6) significantly enhanced DMSP production by Novosphingobium sp. MBES04 (239,24±!13,96!and 453,07±3,04 nmol DMSP mg protein-1 produced respectively). In contrast, the specific transamination intermediates, MTOB, MTHB and DMSHB, and the decarboxylation pathway intermediates MTPA and MMPA, had almost no effect on DMSP accumulation in Novosphingobium sp. MBES04 compared to the control, suggesting that these molecules are unlikely to be intermediates of the DMSP synthesis pathway used by this alphaproteobacterium. Furthermore, among the methylation pathway intermediates, DMSP-amine does not increase DMSP production by Novosphingobium sp. MBES04 suggesting that it follows the same DMSP production pathway used by the Compositae family of plants (see discussion). DMSP production by Novosphingobium sp. MBES04 varies with growth in different environmental conditions To identify the potential role of DMSP in Novosphingobium sp. MBES04, DMSP production was monitored in response to changes in environmental conditions that have been previously shown to alter DMSP production in DMSP-producers including changes in salinity and low nitrogen (Curson et al., Unpublished) (figure 7).

Figure 6- Intracellular DMSP production by Novosphingobium sp. MBES04 grown with DMSP pathway intermediates. Average levels of DMSP mg protein-1 (±!!") produced by Novosphingobium sp. MBES04 at certain time points over 240 minutes when incubated with 0.5mM of various intermediates of the DMSP biosynthesis pathways, and under control conditions (no intermediate added) (n=3). DMSP production by Novosphingobium sp. MBES04 in control conditions was significantly different from DMSP production in the presence of SMM (t6= 134.1, ! < !.!!!"), Met (t6= 14.62, ! < !.!!!") and MTOB (t6= 21.27, ! < !.!!!").

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Figure 7- Intracellular DMSP production by Novosphingobium sp. MBES04 in different environmental conditions. Average levels of DMSP mg protein-1 (±!!") DMSP produced by Novosphingobium sp. MBES04 when grown in MBM minimal media of different conditions- low nitrogen (0.5mM of NH4Cl), low salinity (5 psu), high salinity (50 psu) and control (35 psu, 13 mM of NH4Cl) (n=3). ! Increased intracellular DMSP production was observed most significantly under low nitrogen conditions, 229,56 ±15,52 nmol DMSP mg protein-1; this is likely to do with the availability of nitrogen for the production of nitrogenous osmolytes (see discussion). Under high salinity, there was a 2.75 fold increase in DMSP production compared with the control, 90,81±5,59 nmol DMSP mg protein-1 was produced. Low salinity conditions showed barely detectable DMSP production compared with the control. This further supports DMSP having a role in salinity stress response. Intracellular DMSP-production by Novosphingobium sp. MBES04 decreases in response to oxidative stress To further investigate the role of DMSP production in Novosphingobium sp. MBES04, DMSP production was monitored in response to oxidative stress over time, using hydrogen peroxide (H2O2) to stimulate oxidative stress conditions (figure 8).

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H2O2 was added to the MBM minimal media, and it was found that exposure to H2O2 caused a small decrease in DMSP production by Novosphingobium sp. MBES04 at each time point over 2h. After 2h, the mean intracellular DMSP produced by Novosphingobium sp. MBES04 grown with H2O2 was 2.32±1,85 nmol DMSP mg protein-1 lower compared to the control, 30,93±0,64 nmol DMSP mg protein-1 produced. However, this is surprising as DMSP has been speculated to have antioxidant properties within organisms and we would have expected these levels to increase. However, it is possible that DMSP reacts with the reactive oxygen species and is degraded (see discussion). Novosphingobium sp. MBES04 does not catabolize DMSP In alphaproteobacteria that contain mmtB and produce DMSP, the vast majority also contain DMSP catabolic enzymes, either DMSP lyse enzymes or demethylase enzymes. To test whether the same was true for Novosphingobium sp. MBES04, its ability to demethylase or lyse DMSP was monitored. To do this, Novosphingobium sp. MBES04 was incubated with 5mM of DMSP and DMS and MeSH production was monitored by gas chromatography (figure 9).

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Figure 8- Intracellular!DMSP production by Novosphingobium sp. MBES04 in response to oxidative stress. Average levels of DMSP mg protein-1 (±!!") by Novosphingobium when under oxidative stress induced by 100 !M hydrogen peroxide (H202), compared with control, at time points over 240 minutes (n=3).!

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Our results indicate that Novosphingobium sp. MBES04 does not have the ability to catabolize DMSP into DMS. The intracellular DMS produced was 14,3±1,82 nmol of DMS, which is considerably lower than 385±34 nmol of DMS produced by Sulfitobacter EE-36, which is known to lyse DMSP, and was only slightly greater than the amount produced by E. coli (1,7±0,5 nmol of DMS) known to be unable to degrade DMSP into DMS. Furthermore no methanethiol, the product of the DMSP demethylation pathway, was detected in the headspace. Moreover, blastp analysis carried out with all known DMSP lyase proteins, ddd (D, L, P, Q, Y, W) and the DMSP demethylase protein, dmdA, against the Novosphingobium sp. MBES04 predicted proteome, yield no significant homologues above e-50. These data both support the conclusion that Novosphingobium sp. MBES04 does not catabolize DMSP via the DMSP demethylation or lysis pathways. This is quite distinct from bacteria that make DMSP via the mmtB mediated transamination pathway.

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Figure 9-!DMSP-dependent production of DMS in Novosphingobium sp.MBES04. Average levels of DMS mg protein-1 (±!!") produced by Novosphingobium, Sulfitobacter (positive control) and Escherichia coli (negative control) when supplied with DMSP to observe DMSP-dependent production of DMS (n=3).!

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DISCUSSION

The discovery of DMSP biosynthesis in bacteria, specifically in Labrenzia aggregata LZB033 was an entirely novel finding (Curson et al., Unpublished). In this bacterium, DMSP production occurs via the transamination pathway, and the first gene responsible for DMSP production in any organism was identified: mmtB. This gene has only been found in specific orders of alphaproteobacteria, and Novosphingobium sp. MBES04 does not fall under any of them, yet it was observed to produce DMSP. Our amplification with degenerate mmtB primers, which have been optimized to find mmtB in multiple species, suggests that Novosphingobium sp. MBES04 does not contain mmtB. Using BLAST it was confirmed that mmtB is not found in the genome, as there were no significant matches above e-50, and in all tests done on mmtB-like proteins, matches above e-50 to mmtB of Labrenzia aggregata have no mmtB function (Curson et al., Unpublished). Therefore, this study presents the first known bacterium to produce DMSP without the presence of mmtB in its genome. Continuing from this discovery, it was logical to look at the pathway used by this bacterium to produce DMSP. If mmtB is not present, then it is possible that the pathway it is associated with is also not used. SMM caused a significant increase in DMSP production by Novosphingobium sp. MBES04 compared to the other intermediates of the DMSP biosynthesis pathways. This led us to conclude that this bacterium may be producing DMSP via the same pathway as higher plants, the methylation pathway. DMSP-amine did not cause an increase in DMSP production, indicating that Novosphingobium sp. MBES04 is producing DMSP via the same methylation pathway used by W.biflora, in the Compositae family of higher plants (Stefels, 2000). This is unlike the cord grass, Spartina alterniflora from the Gramineae family, which uses DMSP-amine as an intermediate between SMM and DMSP aldehyde (Stefels, 2000). As expected, both methionine and MTOB also increased DMSP production but to a much lower level (figure 6). The methionine is likely enhancing DMSP production in two ways: methionine is the precursor to all DMSP biosynthesis pathways. Since the three pathways known to produce DMSP have methionine as the initial compound, regardless of which pathway is used by Novosphingobium sp. MBES04 to generate DMSP, supplying methionine to this bacterium would always increase DMSP production (Stefels, 2000). The second way in which methionine is increasing DMSP production is because every DMSP synthesis pathway involves a methyltransferase enzyme that utilizes a methyl group from methionine via AdoMet. Methionine is an intermediate of the AdoMet synthesis pathway; therefore, addition of methionine could also be stimulating this pathway, increasing the amount of AdoMet available to the organism (Ravanel et al., 1998). Lastly, MTOB is closely related to methionine; it is the deaminated form of methionine (Albers, 2009). Many organisms including Novosphingobium sp. MBES04, likely have the capacity to recycle MTOB and use it to generate methionine via the methionine salvage pathway (Albers, 2009). Ultimately, we conclude that MTOB does not increase DMSP production because it is an intermediate of the DMSP production pathway used by Novosphingobium sp. MBES04, but because it is generating higher levels of methionine via the methionine salvage pathway, which is then used by this bacterium to produce DMSP via the methylation pathway. This could be easily confirmed using LC-MS to see if there is a higher accumulation of methionine in the samples containing MTOB, or labeling experiments to track the passage of the MTOB within the organism. In the study presented here, production of DMSP by Novosphingobium sp. MBES04 varied greatly in response to differences in salinity. When grown in 50 PSU MBM, Novosphingobium sp. MBES04 produced considerably higher amounts of DMSP compared to the control (35 PSU), and when grown in 5 PSU, the levels of DMSP were much lower even than the control; this indicates

!

! 18!

that DMSP is mainly produced under conditions of high salinity, most likely to contribute to osmotic stress protection due to its function as an organic osmolyte. In order for single-celled organisms to maintain optimal growth and an active metabolism, cellular conditions such as ionic composition, pH and metabolite pools must be kept within a narrow range (Bisson & Kirst, 1995). This is achieved by adjusting the osmotic potential of the cell through the production of osmotically active compounds, including DMSP, which are not involved in growth (Stefels, 2000). Organic osmolytes such as DMSP act not only through their osmotic potential, but also as compatible solutes, having little or no inhibitory effects on metabolic functions, while protecting proteins and stabilizing membranes against the adverse effects of high ionic strength, which is found in saline conditions (Bisson & Kirst, 1995). Compatible solutes generally accumulate in response to increased salinity; in environments of high salinity, organisms must produce these at concentrations high enough to be osmotically active, explaining the considerable increase of DMSP production at 50 PSU (Stefels, 2000). Furthermore, when the salinity is low (such as 5 PSU), it would not be necessary to have large amounts of an osmoprotectant being synthesized, as there is no need for this protection, and the nutrients involved in producing it could be used elsewhere for growth. Our results are similar to several studies previously published. One study, carried out by Dickson & Kirst (1987), found that DMSP production in the marine algae Phaedactylum tricornutum, increased with increasing salinity in the medium, reinforcing its function as an osmolyte (Dickson & Kirst, 1987). In another study conducted in two marine algae, Scrippsiella trochoidea and Prorocentrum minimum, these results were again replicated and it was shown that under water stress due to increased salinity, intracellular DMSP accumulated within the algae tissues to maintain favorable osmotic tensions and positive turgor (Guangchao et al., 2010). In this study, DMSP appears to have the properties of a compatible solute, however in vitro assays have shown that DMSP may be less effective compared to other compatible solutes such as glycine, betaine and proline, since not all have the same protective properties (Kirst, 1996). This explains why it is often glycine, betaine and proline, not DMSP, that are found at higher levels in algae living in high saline enviornments (Bisson & Kirst, 1995). Of the different enviornmental conditions in which Novosphingobium sp. MBES04 was grown, nitrogen limitation caused the greatest increase in DMSP production. There have been various reports of intracellular DMSP increasing in concentration under N-limitation, such as in the coastal diatom Thalassiosira pseudonana (Keller et al., 1999). DMSP shares a similar structure and properties with its nitrogen analogue, glycine betaine (GBT). It has been suggested that in algae, when nitrogen is limited and required for vital processes, DMSP may replace GBT and other nitrogen-containing osmolytes as the major protection against osmotic stress (Stefels, 2000). This would exaplain why such an increase in DMSP production by Novosphingobium sp. MBES04 is observed under such conditions. The last step of the methylation pathway, the oxidation of DMSP-ald to DMSP, is catalyzed by 3-dimethylsulfoniopropionaldehyde dehydrogenase (DDH) (Trossat et al., 1996). This step is similar to the one in betaine synthesis catalyzed by betaine aldehyde dehydrogenase (BADH), and it has been shown that these enzymes are relatively similar in shape, since 1 mM of betaine aldehyde, the substrate for the last reaction in the betaine synthesis pathway, is able to inhibit DDH activity (Trossat et al., 1996). Therefore, since the last step in both pathways and the enzyme that catalyzes both these reactions is similar, interactive production likely occurs and could be causing the intracellular increase in DMSP production that we observed in low nitrogen conditions. Conversely, there are also studies that report a significant reduction in DMSP levels in high nitrogen conditions in plants such as S.alterniflora, which contain lower DMSP concentrations and greater glycine betaine levels (Dacey et al., 1987), and further supports our hypothesis. It has also been proposed that DMSP and its enzymatic cleavage products (DMS, acrylate) serve as an antioxidant system, by readily scavenging reactive oxidants and hyroxyl radicals in the

! 19!

cells (Sunda et al., 2002). It was therefore expected that DMSP levels would increase in response to oxidative stress. However, this was not the result that our experiment produced. Some studies have also shown that DMSP production decreases when stimulated by oxidative stress (Husband & Kiene, 2007), but there are aso many that show otherwise. Sunda et al., (2002) found that intracellular DMSP concentrations significantly increased under oxidative stress induced by CO2 and Fe limitation, in the diatom Thalassiosira pseudonana, and in the coccolithophore Emilania huxleyi. Similar results have been reported in leaves of S.alterniflora when oxidative stress was applied using herbicides, namely Paraquat (methyl viologen) and dichlorophenyl-dimethylurea (DCMU) (Husband & Kiene, 2007). It may be that DMSP only functions as an antioxidant in algal species, and not in bacteria. A decrease in intracellular DMSP levels might be a result of DMSP being an effective cellular scavenger and rapidly reacting with reactive oxygen species generated by hydrogen peroxide. As shown by Spiece (2010), the reaction of DMSP with reactive oxygen species and hydroxyl radicals results in the formation of another degradation product of DMSP, dimethylsulfoxide (DMSO), which serves as as an antioxidant in marine phytoplankton (Spiese, 2010). Therefore, it is possible that DMSP production increases in response to oxidative stress protecting Novosphingobium sp. MBES04 from oxidative stress, but this increase is masked by its oxidation to DMSO when it reacts with hyroxyl radicals. Perhaps the next step could be to also monitor the levels of DMSO produced during this experiment, or to measure the expression of DMSP producing genes, instead of the amount of product detected. DMSP is mainly catabolised by marine bacteria, and marine organisms capable of breaking down DMSP use one of two pathways; one route is the cleavage of DMSP into DMS and acrylate or 3-hydroxypropionate using DMSP-dependent DMS (ddd) genes, and the other is known as the demethylation pathway, via the gene dmdA. Novosphingobium sp. MBES04 was tested for its ability to degrade DMSP into DMS, however as far as our results show, this bacterium is unable to degrade DMSP to DMS, as the levels of DMS produced by Novosphingobium sp. MBES04 were similar to those of E.coli, which is incapable of degrading DMSP. In addition, our results showed that there was no detectable methanethiol production, suggesting that Novosphingobium sp. MBES04 does not contain the gene responsible for the demthylation pathway. BLAST searches were used to confirm whether any of the ddd genes were present in the genome of Novosphingobium sp. MBES04. We can say with confidence that none of the known ddd genes are present, further suggesting that this alphaproteobacterium does not catabolize DMSP into DMS. The presence of dmdA was also searched for and not found, which was unsurprising, as most dmdA genes have only been found in Roseobacters (Varalijay et al., 2012). Although the diversity of DMSP-cleavage enzymes identified to data is impressive, and suggests that there may be more unidentified genes out there (Reisch et al., 2011), and it may be the case that a gene involved in the degradation of DMSP in Novosphingobium sp. MBES04 is yet to be identified, there still does not appear to be evidence of high levels of DMSP catabolism when tested. All this suggests that Novosphingobium sp. MBES04 does not possess the ability to catabolize DMSP. Perhaps one reason for this is that DMSP synthesis is an energy expensive process compared to many other compatible solutes, and so is the production of AdoMet; (Stefels, 2000; Thomas & Surdin-Kerjan, 1991), so it would seem counterproductive to break it down after the energy expended to produce it. Furthermore, if, as our previous results suggest, DMSP is not being used as an antioxidant, there is less need for this bacterium to be able to break it down to its more highly antioxidant products. Ultimately, these findings are very interesting because they show that Novosphingobium sp. MBES04 is distinct to the majority of DMSP-producing bacteria, since these not only contain mmtB and produce DMSP via the transamination pathway, but they also have DMSP lyase genes and some have demethylation genes.

!

! 20!

In this study we have shown that this Novosphingobium sp. MBES04 isolate is the first alphaproteobacterium known to produce DMSP in the absence of mmtB. Like higher plants of the family Compositeae, DMSP biosynthesis in this organism most likely occurs through the methylation pathway. In this bacterium, de novo DMSP synthesis has been shown to play a major role in osmoprotection, and also, under conditions of nitrogen limitation, in order to protect the cells from damage. It is possible that DMSP may play other roles in this bacterium under different environmental conditions, which have not been replicated in this experiment. The absence of DMSP cleavage enzymes suggests that the presence of intracellular DMSP may be of more importance to the survival of this bacterium than the ability to catabolize it to its breakdown products. Based on these findings, it would be of interest to confirm in future experiments that Novosphingobium sp. MBES04 is using the methylation pathway by growing it in the presence of DMSP-aldehyde, and using other diagnostic method such as LC-MS to search for the presence of all these intermediates in the organism. The next step in this story will be to identify the gene responsible for the production of DMSP in Novosphingobium sp. MBES04 using genomic libraries and screening. This novel DMSP-producing gene could be used to identify other DMSP-producing bacteria, and, in the same was that mmtB has been used to measure bacterial DMSP-producing activity within and environment, it will improve our ability to model and monitor environmental production of DMSP specifically in prokaryotes. It would now appear that the bacterial contribution to global DMSP flux, specifically its production, may be much more significant than previously thought. With this in mind, identifying the key genes and species involved in this production plays a very important role in our understanding of the environment in which we live in.

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ACKNOWLEDGEMENTS

Thank you to Jonathan Todd’s Laboratory and specifically Beth Williams who accompanied and helped me throughout.

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Appendix 1

Reflective Statement

My research project was on characterizing a DMSP-producing bacteria. I was very excited to work on this project since the beginning since not much research has been conducted in this area; in fact, I might have been one of the first people to characterize an alphaproteobacterium from the order sphingomonadales, Novosphingobium sp, MBES04 that produces DMSP. In addition, another aspect of this project that interested me was the fact that on a global scale, these DMSP producing bacteria can be impacting global climate change. Since an early age I have always been very interested and concerned with global warming and climate change, therefore doing a research project which can be relevant, even though in a tiny bit to such a big issue, is something that I really enjoyed.

This coursework was an eye-opener in terms of how much work and dedication is required to carry out a research project. I had never conducted an eight-week research project before so I had no idea. In the end, it was worth it because I learned things about this area which I would have never known without carrying this project, while learning how to be more organized and better at time managing.

Throughout this experiment, I had many different tasks. I kept a lab book with me the whole time to make sure that I had records of everything, as well as the instructions and advice I was given by the PhD student, Beth Williams. In the beginning, I spent a couple of weeks working in the lab, getting confortable with the equipment, as well as practicing how to conduct the experiments I would have to carry out in the future. I started out by doing simple procedures such as preparing MBM minimal medium, making solutions, preparing dilutions, making the gel for gel electrophoresis, conducting a PCR, working with the GC vials, amongst many others. This way, when it came to my experiment I was super confortable with what I was doing. The help of Beth Williams was crucial however.

After my weeks in the laboratory, it was very interesting to actually understand what it was that my results meant. I had already an idea from the background information I had already done, but with the results I had specific things to look for in different pieces of literature. I think for me this was one of the most interesting things about the project; discovering the reasoning behind the results. Ultimately, being able to carry this research project was a great opportunity. I got a taste of what a future in this field would involve, while researching a really interesting and important area.