Sains Malaysiana 45(6)(2016): 915–931

Diversity and DMS(P)-related Genes in Culturable Bacterial Communities in Malaysian Coastal Waters (Kepelbagaian dan Gen berkaitan-DMS(P) dalam Komuniti Kultur Bakteria di Perairan Pantai Malaysia)

FELICITY W.I. KUEK*, AAZANI MUJAHID, PO-TEEN LIM, CHUI-PIN LEAW & MORITZ MÜLLER

ABSTRACT Little is known about the diversity and roles of microbial communities in the South China Sea, especially the eastern region. This study aimed to expand our knowledge on the diversity of these communities in Malaysian waters, as well as their potential involvement in the breakdown or osmoregulation of dimethylsulphoniopropionate (DMSP). Water samples were collected during local cruises (Kuching, Kota Kinabalu, and Semporna) from the SHIVA expedition and the diversity of bacterial communities were analysed through the isolation and identification of 176 strains of cultured . The bacteria were further screened for the existence of two key genes (dmdA, dddP) which were involved in competing, enzymatically-mediated DMSP degradation pathways. The composition of bacterial communities in the three areas varied and changes were mirrored in physico-chemical parameters. Riverine input was highest in Kuching, which was mirrored by dominance of potentially pathogenic Vibrio sp., whereas the Kota Kinabalu community was more indicative of an open ocean environment. Isolates obtained from Kota Kinabalu and Semporna showed that the communities in these areas have potential roles in bioremediation, nitrogen fixing and sulphate reduction. Bacteria isolated from Kuching displayed the highest abundance (44%) of both DMSP-degrading genes, while the bacterial community in Kota Kinabalu had the highest percentage (28%) of dmdA gene occurrence and the dddP gene responsible for DMS production was most abundant (33%) within the community in Semporna. To the best of our knowledge, this is the first study looking at the diversity of culturable bacteria in coastal waters of East Malaysia and also their potential roles in the DMS(P) cycle.

Keywords: Culturable bacterial communities; dimethylsulphide; dimethylsulphoniopropionate; diversity

ABSTRAK Kepelbagaian dan peranan yang dimainkan oleh komuniti mikrob di Laut China Selatan, khususnya di Wilayah Timur, adalah kurang diketahui. Kajian ini bertujuan untuk mengembangkan pengetahuan tentang kepelbagaian komuniti ini di perairan Malaysia, serta potensi penglibatan mereka dalam penguraian atau pengawalan osmosis dimetilsulfoniopropionat (DMSP). Sampel air diperoleh semasa pelayaran tempatan (Kuching, Kota Kinabalu dan Semporna) daripada ekspedisi SHIVA dan kepelbagaian komuniti bakteria telah dianalisis melalui pengasingan dan pengenalpastian 176 strain kultur bakteria. Bakteria ini seterusnya disaring untuk menentukan kehadiran dua gen utama (dmdA, dddP) yang terlibat dalam dua laluan bersaingan degradasi DMSP secara berenzim. Komposisi komuniti bakteria dalam tiga kawasan ini berbeza dan perbezaan ini boleh dilihat dalam parameter fisiko-kimia. Input sungai paling tinggi di Kuching dan ini ditunjukkan melalui dominasi Vibrio sp., yang berpotensi untuk menjadi patogenik, manakala komuniti Kota Kinabalu adalah petunjuk untuk persekitaran lautan terbuka. Bakteria yang diasingkan dari Kota Kinabalu dan Semporna menunjukkan bahawa komuniti dalam dua kawasan ini berpotensi memainkan peranan dalam bioremediasi, pengikatan nitrogen dan penurunan sulfat. Bakteria yang diasingkan dari Kuching menunjukkan kebanyakan tertinggi (44%) untuk kedua-dua gen pengurai DMSP, manakala komuniti bakteria di Kota Kinabalu menunjukkan peratusan tertinggi (28%) kejadian gen dmdA dan gen dddP yang bertanggungjawab untuk pengeluaran DMS adalah paling banyak (33%) dalam komuniti di Semporna. Sepanjang pengetahuan kami, ini merupakan penyelidikan pertama yang melihat kepelbagaian bakteria yang boleh dikultur di perairan pantai Malaysia timur dan juga potensi penglibatan mereka dalam kitaran DMS(P).

Kata kunci: Dimetilsulfida; dimetilsulfoniopropionat; kepelbagaian; komuniti kultur bakteria

INTRODUCTION the Celebes and Sulu Seas have been reported to display The South China Sea is a marginal sea that is part of the some similarities (Yoshida et al. 2007), not much is Pacific Ocean, encompassing an area from the Karimata known about the diversity and function of the microbial Straits in the south, to the Straits of Taiwan and Luzon in communities in South China Sea, especially regarding the North (Morton & Blackmore 2001). The Celebes Sea the eastern region (Kuching and Kota Kinabalu). Most is connected to the South China Sea through the Sulu Sea studies about bacterial communities focused on regions (Yoshida et al. 2007). While the bacterial communities in near China (Jiang et al. 2007; Liao et al. 2009; Li et 916 al. 2006; Tao et al. 2008; Zhu et al. 2013), with the The present study tries to expand our knowledge on exceptions of Kuek et al. (2015), Lee et al. (2009) and culturable microbial communities in the eastern South Song et al. (in preparation), all of whom sampled from China Sea and identify potential key players in the local the coasts of Malaysia. Aside from Kuek et al. (2015) DMS(P) cycle. and Lee et al. (2009), all the other cited studies used culture-independent techniques to show the community MATERIALS AND METHODS structure and diversity of the predominant bacteria at the sampling environment. The studies by Jiang et al. (2007) and Tao et al. (2008) showed that most lineages within the STUDY SITES, SAMPLE COLLECTION AND represented uncultured microorganisms, INITIAL ISOLATION suggesting that a vast amount of microbial resources in In conjunction with European and Malaysian research the South China Sea are unknown and unexplored. Song partners, the SHIVA (Stratospheric ozone: Halogen Impacts et al. (in preparation) found that Proteobacteria (Alpha- in a Varying Atmosphere, EU call ENV.2008.1.1.2.1) and Gamma-) and Cyanobacteria (Synechococcus sp. and Western Pacific field campaign was performed in the Prochlorococcus sp.) dominated at all study sites and fall of 2011. The core field campaign took place in the that the highest proportion of was South China Sea and along the coastline of Peninsular found in Sarawak. Similarly, Lee et al. (2009) discovered Malaysia and Borneo using the German Research Vessel that Gram-negative bacteria dominated their study of (RV) Sonne during a cruise leading from Singapore to cultured bacteria, with the most prevalent class belonging Manila, Philippines. Local cruises took place in Kuching to the Gammaproteobacteria. on November 19, 2011, Kota Kinabalu on November 23, The ocean is a major source of sulphur (Andreae 2011 and Semporna on November 26, 2011 (Table 1) to 1986) and microorganisms residing in the ocean have provide additional data for coastal input. Samples for this the ability to metabolise organic and inorganic sulphur study were collected during the local cruises. (Sievert et al. 2007). Dimethylsulphoniopropionate Physico-chemical parameters (depth, temperature, (DMSP) represents a major carrier for sulphur transfer pH, salinity, nitrate, phosphate, nitrite and silicate) through microbial food webs and organic sulphur cycling were quantified using a QuAAtro auto-analyser SEAL( in the ocean as it is an abundant component in many Analytical, UK) following protocols provided in the SEAL phytoplankton taxa and prone to microbial degradation analytical operation manual and methods published in (Kiene et al. 2000). The Roseobacter which are part of Grashoff et al. (1999). Sea water samples were streaked on marine agar the Alphaproteobacteria lineage are mainly responsible at half strength (Difco, 2.76% solution, dissolved in for the degradation of DMSP into methanethiol (MeSH) purified water) and incubated under aerobic conditions and have been found in different regions of the world, at 30°C. Bacterial colonies were isolated based on their ranging from the Sargasso Sea to the Black Sea (González morphological differences. Colonies were picked and et al. 2000, 1999). A competing metabolic pathway results purified by repeated streaking on plates. Pure cultures were in the production of dimethylsulphide (DMS) from DMSP preserved as a glycerol suspension (20%, w/v) at -80°C. (González et al. 1999; Johnston et al. 2008). Due to highly efficient bacterial DMSP demethylation and DMS consumption processes, only a small percentage (1-2%) DIVERSITY INDICES of DMSP produced by marine phytoplankton is ventilated Several ecological diversity indices frequently applied to the atmosphere as DMS (Levine et al. 2012). Despite to microbial community profile data were used in order the low percentage, DMS does, however, represent a to compare diversity among microbial communities, major source of biogenic sulphur to the atmosphere, enabling us to quantify diversity within the communities where oxidation products form cloud condensation and describe their numerical structure. Taxonomic nuclei and ultimately influence radiative backscatter classification up to genus was used as someBLAST results (Andreae & Crutzen 1997; Lovelock et al. 1972; Simó could only relate the isolates to strains which have been 2001). The DMSP demethylase gene (dmdA) which identified up to genus level. Five representative stations encodes the first step in the demethylation pathway, is were chosen per sampling site in order to standardise taxonomically diverse and highly abundant, present in the sampling effort and enable us to compare among the over 50% of marine bacterioplankton (Howard et al. sampling sites. 2008). In comparison to dmdA, the genes involved in DMS The Margalef index (DMg) is an accurate index to production (dddD, dddL, dddP, dddQ, dddY and dddW; sample richness which utilises absolute numbers compared all of which mediate the same step of DMSP cleavage) to a density data matrix (Gamito 2010; Magurran 2004). are present in less than 10% of bacteria based on marine Meanwhile, the commonly used Shanon index (H’) metagenomic surveys (Curson et al. 2011a, 2008; Howard considers proportions, ensuring no differences when using et al. 2008; Todd et al. 2012, 2011, 2007). dddP is one either data set (Gamito 2010). The Shannon evenness of the most abundant occurring ddd genes (Levine et al. index (J’) is derived from H’ which therefore makes it 2012; Todd et al. 2009; Varaljay et al. 2012). sensitive to changes in evenness of rare species, thereby 917

TABLE 1. Locations of sampling stations in Kuching, Kota Kinabalu and Semporna

Sampling GPS coordinates stations Kuching Kota Kinabalu Semporna Station 1 1°39’28.81”N, 110°31’24.42”E 6° 3’4.56”N, 116° 5’54.60”E 4°35’15.96”N, 118°32’58.14”E Station 2 1°42’44.24”N, 110°33’23.46”E 6° 3’5.82”N, 116° 4’1.45”E N/A Station 3 1°45’32.93”N, 110°35’16.86”E 6° 3’4.02”N, 116° 0’2.77”E N/A Station 4 1°48’2.16”N, 110°37’51.53”E 6° 2’49.85”N, 115°57’38.26”E N/A Station 5 1°50’54.15”N, 110°40’11.26”E 6° 4’23.64”N, 115°54’36.42”E 4°37’31.26”N, 118°41’5.99”E Station 6 N/A N/A 4°35’56.76”N, 118°43’19.14”E Station 7 N/A N/A 4°35’30.66”N, 118°42’17.10”E Station 8 N/A N/A 4°33’17.83”N, 118°39’22.57”E possibly overestimating its true value (Hill et al. 2003). (Tamura & Nei 1993). The nucleotide sequences obtained

The Smith and Wilson evenness index (Evar), however, is in the present study have been deposited in GenBank known to show greater resolution in reflecting true values database (http://www.ncbi.nlm.nih.gov) under accession (Blackwood et al. 2007). numbers KF373319 to KF373440.

DNA EXTRACTION AND PURIFICATION OF PCR AMPLIFICATION OF BACTERIAL DMSP CLEAVAGE CULTURED BACTERIA (DDDP) AND DEMETHYLATION (DMDA) GENES The isolates were grown in marine broth at half strength at The bacterial DNA were amplified by polymerase 30°C with shaking at 180 rpm. The cells were pelleted by chain reaction (PCR) and PCR products were purified centrifugation at 13,000 rpm for 5 min before re-suspension using PureLink® PCR Purification Kit following the in 50 μL of TE buffer (10 mM Tris-HC pH8.0, 1 mM EDTA). manufacturer’s protocol (Invitrogen Life Technologies). Three cycles of freezing in a -80°C freezer for 3 min and Amplification of dddP genes was performed with thawing in an 85°C water bath for 3 min were conducted degenerate dddP primers dddP_874F and dddP_971R to release DNA from the microbial cells. (Levine et al. 2012) while amplification of dmdA genes was performed with universal dmdA primers dmdAUF160 PCR AMPLIFICATION OF BACTERIAL 16S RRNA GENES and dmdAUR697 (Varaljay et al. 2010). Amplification was performed by using RedTaqMix (Sigma Aldrich) with The bacterial DNA were amplified by polymerase the following cycling conditions: Initial denaturation at chain reaction (PCR) and PCR products were purified 95°C for 5 min, 40 cycles of 95°C for 30 s, 41°C for 30 using PureLink® PCR Purification Kit following the s, extension at 72°C for 30 s and then a final denaturation manufacturer’s protocol (Invitrogen Life Technologies). and annealing for 1 min each. The samples of extracted Amplification of bacterial 16S rRNA genes was performed DNA were analysed on a 1% agarose gel containing 1 μg with broad-specificity primers 8F (Eden et al. 1991) and of ethidium bromide per mL. 519R (Lane et al. 1985). Amplification was performed by RedTaqMix (Sigma Aldrich) using instructions provided by Sigma Aldrich with the following cycling conditions: RESULTS AND DISCUSSION Initial denaturation at 96°C for 4 min, 40 cycles of 96°C for 1 min, 55°C for 1 min, extension at 72°C for 2 min PHYSICO-CHEMICAL PARAMETERS and then a final elongation at 72°C for 4 min.The samples Basic physico-chemical parameters were recorded during of extracted DNA were analysed on a 1% agarose gel sampling in Kuching and Kota Kinabalu (Table 2). containing 1 μg of ethidium bromide per mL. Values for Semporna were not reported as the measuring instruments were not in working order at the time of SEQUENCING AND PHYLOGENETIC ANALYSIS sampling. The sampling stations at Kota Kinabalu stretched Sequences were analysed against the NCBI (USA) database further away from the coastline and displayed average using BLAST program packages and matched to known values of salinity at 31.88 ppt, pH of 8.36 and temperature 16S rRNA gene sequences (Altschul et al. 1990; Zhang of 29.65°C, all indicative of a typical ocean environment et al. 2000). Ambiguous sequences were checked (Raven et al. 2005). The first sampling station at Kuching manually by eye and further edited using MUSCLE (Edgar (KCH-1) was closer to the river mouth of the Sarawak river 2004). Sequences were aligned and phylogenetic trees and displayed a visible influence by riverine water with its reconstructed with MEGA 6 (Tamura et al. 2013) using the surface water displaying a salinity of 28.48 ppt and pH of maximum likelihood method based on Tamura-Nei model 7.90. The riverine input at Kuching was also visible with 918 higher nitrate, phosphate, nitrite and silicate values closer 1 & 4). Our results correlated with existing records of to the river mouth (KCH-1 and KCH-2). Nutrient levels in microbial communities found in coastal and open-ocean Kuching were also generally higher than in Kota Kinabalu. environments (Bernard et al. 2000).

To assess differences in distribution in the upper surface Values for sample richness using both DMg and H’ layers, the samples were also taken from 5 m depth (KCH- indicated that the bacterial communities in Kota Kinabalu 5 and KK-5). Interestingly, the samples for Kota Kinabalu and Semporna were more diverse than the one in Kuching showed consistent values. For Kuching however, silicate (DMg of 3.82 and 3.36 compared to 1.67; Table 3). concentration dropped from 63.64 to 24.00 μM within the Evenness values from both J’ and Evar also indicate that first 5 m, indicative of an active biological pump (Dugdale the communities in Kota Kinabalu and Kuching are more et al. 1995). evenly distributed. In the following, we discuss some highlights of the bacterial diversity found at the three DIVERSITY OF CULTURABLE BACTERIAL COMMUNITIES sampling sites. A total of 36 isolates were obtained from Kuching The cultured Alphaproteobacteria can be found across waters and 89% of the cultured bacteria were clustered all three sampling sites and consists of representatives from within the Gammaproteobacteria and 11% within the Caulobacteraceae, Phyllobacteriaceae, Rhodobacteraceae Alphaproteobacteria (Figures 1 & 2). In Kota Kinabalu and Rhodospirillaceae (Figures 2 to 4). Isolates from this waters, 39 isolates were obtained and the majority group were likely to be involved in the nitrogen cycle and (72% of the cultured bacteria) were clustered within the possibly in the degradation of hydrocarbons (Bell et al. Gammaproteobacteria (Figures 1 & 3). The remaining 1992; Itoh et al. 1989; Labbé et al. 2004; Richardson et isolates were members of the Firmicutes (18%) and al. 1989; Zumft 1997). Alphaproteobacteria (10%). In Semporna waters, 24 The sole Betaproteobacteria that was cultured (Figure isolates were obtained from four phylogenetic groups. In 4) is related to Alcaligenes faecalis (GenBank accession total, 83% of the cultured bacteria were members of the number JF264463; 88% similarity) which was previously Gammaproteobacteria, 9% Alphaproteobacteria and 4% isolated from a coastal aquaculture environment. each in Betaproteobacteria and Firmicutes groups (Figures Alcaligenes faecalis have also been found in salt marsh and

TABLE 2. Physico-chemical parameters measured from Kuching (KCH) and Kota Kinabalu (KK) at depths of 1 and 5 m

Depth Temperature Salinity Nitrate Phosphate Nitrite Silicate Station pH (m) (°C) (ppt) (μM) (μM) (μM) (μM) 1 29.06 7.90 28.48 147.25 6.32 31.74 254.88 KCH-1 5 29.34 8.10 30.59 BD BD BD BD 1 28.98 8.25 30.65 32.58 3.47 13.04 81.23 KCH-2 5 29.11 8.25 30.89 BD BD BD BD 1 29.05 8.33 31.18 13.71 1.58 0.65 32.25 KCH-3 5 29.16 8.30 30.53 BD BD BD BD 1 29.00 8.33 31.07 7.90 1.05 0.00 49.09 KCH-4 5 29.10 8.29 30.52 BD BD BD BD 1 29.27 8.31 31.61 2.42 0.63 0.00 63.64 KCH-5 5 29.40 8.29 31.85 2.42 0.53 0.00 24.00 29.15 8.24 30.74 34.38 2.26 7.57 84.18 KCH mean (±0.14) (±0.14) (±0.92) (±56.42) (±2.26) (±12.91) (±86.16) 1 29.80 8.44 31.85 16.77 1.58 BD 37.68 KK-1 5 29.90 8.37 32.04 BD BD BD BD 1 29.73 8.36 31.44 4.03 1.79 BD 34.86 KK-2 5 29.78 8.33 31.95 BD BD BD BD 1 29.55 8.34 31.88 3.71 1.16 BD 29.00 KK-3 5 29.54 8.33 31.87 BD BD BD BD 1 29.52 8.36 31.93 BD BD BD BD KK-4 5 29.45 8.34 31.91 BD BD BD BD 1 29.68 8.38 32.03 2.10 0.32 BD 29.76 KK-5 5 29.50 8.37 31.92 2.42 0.21 BD 30.30 29.65 8.33 31.88 5.81 1.01 32.32 KK mean BD (±0.15) (±0.03) (±0.17) (±6.18) (±0.72) (±3.77)

*BD denotes values below detection limit 919

FIGURE 1. Diversity of bacterial groups based on partial 16S rRNA gene sequences from bacteria isolated from (a) Kuching, (b) Kota Kinabalu and (c) Semporna

FIGURE 2. 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in Kuching. The phylogenetic tree was generated with distance methods and sequence distances were estimated with the neighbour-joining method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.02 substitution per site. Accession numbers for the reference sequences are indicated 920

FIGURE 3. 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in Kota Kinabalu. The phylogenetic tree was generated with distance methods and sequence distances were estimated with the neighbour-joining method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.05 substitution per site. Accession numbers for the reference sequences are indicated 921

FIGURE 4. 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in Semporna. The phylogenetic tree was generated with distance methods and sequence distances were estimated with the neighbour-joining method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.05 substitution per site. Accession numbers for the reference sequences are indicated

TABLE 3. Indices used to quantify the diversity of bacterial communities at Kuching, Kota Kinabalu and Semporna

Genus Kuching Kota Kinabalu Semporna Total isolates (N) 36 39 48 Total genus (S) 7 15 14

Margalef index (DMg) 1.67 3.82 3.36 Shannon index (H’) 1.14 2.42 2.18 Shannon evenness (J’) 0.59 0.89 0.83

Smith and Wilson evenness (Evar) 0.49 0.69 0.59 *Formulae of diversity indices are from Margalef (1958), Shannon and Weaver (1963) and Smith and Wilson (1996) estuarine waters (Ansede et al. 2001) and has the potential Shewanellaceae, Pseudomonadaceae and Vibronaceae to degrade DMSP to DMS via acrylate metabolism through can be found across all three sampling sites (Figures 2 to the induction of β-hydroxypropionate (Ansede et al. 1999; 4). Isolates from Kuching which were related to strains Yoch et al. 1997). of Pseudoalteromonas ganghwensis (GenBank accession Within the Gammaproteobacteria group, isolates number DQ011614; 99% similarity) have been observed from , Pseudoalteromonadaceae, to possess the ability to form biofilms and may contribute 922 in part to the removal of excess proteineous matters from BACTERIAL STRAINS WITH POTENTIAL TO METABOLISE the sediment sludge of fish farms (Iijima et al. 2009). DMS AND/OR DEMETHYLATE DMSP Pseudoalteromonas lipolytica (GenBank accession To date, there are no available reports on the sulphur number JX173567) has only been recently characterised cycle in the region or of DMSP catabolism from bacterial (Xu et al. 2010) and has the ability to hydrolyse lipids communities of Kuching, Kota Kinabalu and Semporna; and reduce nitrate to nitrite. Kota Kinabalu has isolates neither are any bioinformatics data available on the that were closely related to this particular strain. prevalence of dmdA and dddP genes in bacteria from these Members of Vibrionaceae are common in the marine regions. As part of our effort to understand the importance environment, with species found in hydrothermal vents, of bacteria in the region for the local sulphur cycle, we deep sea, open water, estuaries and marine sediments screened our isolates for the presence of dmdA and dddP (Eilers et al. 2000; Lee & Ruby 1994; Maruyama et genes. al. 2000; Raguénès et al. 1997) and is the most heavily Previously reported bacteria with the ability to represented family within the Gammaproteobacteria. demethylate DMSP and/or metabolise DMS which we also Studies have suggested that some Vibrio can degrade managed to isolate and culture include Rhodobacter and ecologically hazardous compounds, such as polycyclic Roseovarius within the Alphaproteobacteria (Curson aromatic hydrocarbons (Ramaiah et al. 2000) and are et al. 2008; González et al. 2003; Johnston et al. 2008; major decomposers of chitin in the ocean (Hedlund & Kirkwood et al. 2010; Moran et al. 2007; Todd et al. Staley 2001; Nagasawa & Terazaki 1987). They have 2009); the aforementioned Alcaligenes faecalis within also been shown to cause potentially lethal diseases the Betaproteobacteria; , Pseudomonas, in humans and fish (Kusuda & Kawai 1998; McCarter and Vibrio within the Gammaproteobacteria 1999). More recently, studies have shown Vibrio shiloi (Ansede et al. 1999; de Souza & Yoch 1995; Johnston et al. to be a coral pathogen, producing toxins that inhibit 2008; Moran et al. 2007; Raina et al. 2010, 2009; Sievert Bacillus photosynthesis and lyse zooxanthellae resulting in coral et al. 2007; Yoch 2002; Yoch et al. 1997); and within the Firmicutes (Todd et al. 2009). bleaching (Banin et al. 2000a, 2000b). Species such Bacteria isolated from Kuching displayed the highest as Vibrio parahaemolyticus and Vibrio vulnificus have abundance of both DMSP-degrading genes (44% of all been shown to express virulence-related properties such isolates from Kuching) compared to communities isolated as the production of toxR gene (Lin et al. 1993; Okuda from Kota Kinabalu and Semporna (with 13 and 21%, et al. 2001) and production of phenolate siderophore respectively). The bacterial community in Kota Kinabalu (Stelma et al. 1992). Vibrio harveyi and Photobacterium has the highest percentage of dmdA gene occurrence sp. are luminous bacteria which often cause disease in (28% of all isolates from Kota Kinabalu) while the aquaculture (Baticados et al. 1990; Prayitno & Latchford dddP gene responsible for DMS production appears to 1995). While most Vibrio sp. isolated from Kuching be most abundant (33%) within the bacterial community appeared to be related to pathogenic strains, many of the in Semporna (Figure 5). Stefels (2000) has previously isolates from Kota Kinabalu and Semporna have potential hypothesized that DMSP production is an overflow roles in bioremediation, nitrogen fixing and sulphate mechanism for when growth is unbalanced by lack of reduction. nutrients and the need to release excess energy and excess Members of the cultured Firmicutes group consisted reduced sulphur. These carbon-energy overflow substances of members of the Bacillaceae, Bacillaceae Family might evolve through natural selection to be useful in the XII. incertae sedis and Paenibacillaceae. Isolates from cell (through auxiliary structures or defence mechanisms) Bacillaceae were mostly related to Bacillus spp. and (Hill et al. 1998). Based on our findings, it seems likely that Lysinibacillus spp. and are unique to each sampling site. at low nutrient conditions, the distribution of dmdA and Isolates from the Bacillaceae Family XII. incertae sedis dddP genes within the bacterial community becomes more were matched with Exiguobacterium spp. which have specific (more dmdA in KK and more dddP in Semporna) previously been isolated from, or molecularly detected to adapt to a preferred pathway to degrade DMSP. This is in, a wide range of habitats including cold and hot discussed as follows. environments with temperatures ranging from -12 to 55°C The sampling locations at Kuching and Kota Kinabalu (Vishnivetskaya et al. 2009). Interestingly, members of were observed to have heavy shipping traffic which may this family were only isolated from Kota Kinabalu and influence the sulphur concentration in the area. Ship plumes Semporna where recent temperature spikes resulted in emit large amounts of anthropogenic nitrogen and sulphur mass coral bleaching in the region (Tan & Heron 2011). into the atmosphere, particularly within potential transport Of the three sampling sites, Sarawak was the only area distance of land regions (Corbett et al. 1999) which may with no reported bleaching events (Tun et al. 2010). influence the algal production of DMSP (Malin & Erst In conclusion, several species isolated from Kuching 1997). The waters of Kota Kinabalu are known for having waters appear to be related to pathogenic strains, whereas seasonal phytoplankton blooms (Adam et al. 2011). The many of the isolates from Kota Kinabalu and Semporna relative production of DMSP was suggested to depend on have potential roles in bioremediation, nitrogen fixing nitrogen availability (Andreae 1986). Small haptophytes and sulphate reduction. (e.g. coccolithophorids) and many small dinoflagellates are 923 typical of more nitrogen-deficient conditions, so they have with both genes; Figure 6) and they seem well-adapted evolved to produce more DMSP, implying the probability of to the variable environmental conditions. Other key finding higher levels of DMSP is greater under conditions players involved in DMS production seem to be members of nitrogen depletion during phytoplankton blooms of the Pseudomonas and Pseudoalteromonas, whereas (Simó 2001). Nitrate and nitrite concentrations at Kota Citrobacter seem to be involved in DMSP assimilation in Kinabalu are low (5.81 μM on average and not detectable, Kuching waters. respectively; Table 2), especially in comparison with Vibrio is also potential key players in samples from Kuching (up to 147.25 and 31.74 μM nearest to the coast), Kota Kinabalu but seem to serve a slightly different indicating a low nutrient environment and suggesting role. Occurrence of DMSP assimilation genes within the the likelihood of high concentration of DMSP in the area Gammaproteobacteria is again dominated by Vibrio; especially in the event of phytoplankton blooms. The however, Shewanella-related isolates are more likely to bacterial community in the area have possibly evolved to possess both genes (Figure 7). This may be due to the adapt to these conditions and preferred the demethylation different Vibrio species that form the majority in Kuching pathway as the occurrence of dmdA genes is the highest and Kota Kinabalu communities (Vibrio parahaemolyticus within the community at Kota Kinabalu (28% of all and Vibrio splendidus respectively; Figures 2 and 3). isolates; Figure 5). The higher diversity of the community at Kota Kinabalu Due to riverine input, the waters of Kuching showed shows a possible correlation with flexibility of DMSP higher nutrient concentrations compared to Kota Kinabalu degradation pathways with a more even distribution of and Semporna and it is possible that the high nutrient groups possessing both dmdA and dddP genes compared environment at Kuching forces the bacterial community to communities in Kuching and Semporna (Figures 6 & in the area to be more metabolically flexible. This is 8, respectively). highlighted by the fact that the dominant genus, Vibrio, The sampling stations at Semporna were observed to emerged as the key group in these waters with high be surrounded by seaweed farms. Micro- and macroalgae occurrences of both dmdA and dddP genes (representing and halophytic plants are abundant sources of DMSP in the 100% of all isolates within the Gammaproteobacteria marine environment (Yoch 2002) and past studies (Scarratt

FIGURE 5. Relative abundance of dmdA and dddP genes in cultured bacterial communities from the waters of (a) Kuching, (b) Kota Kinabalu and (c) Semporna

FIGURE 6. Relative abundance of dmdA and dddP genes in isolated Gammaproteobacteria from Kuching 924

FIGURE 7. Relative abundance of dmdA and dddP genes in isolated Gammaproteobacteria from Kota Kinabalu

FIGURE 8. Relative abundance of dmdA and dddP genes in isolated Gammaproteobacteria from Semporna et al. 2000) suggested that bacteria growing near algal cells from Kota Kinabalu and Semporna possessing dmdA genes might be exposed to high local levels of DMSP, which would (Figures 7 & 8). lead to DMS yields that are higher than those inferred from Based on our preliminary observations, we believe that bulk seawater measurements. Our results supported this as many of our isolates have the ability to undergo both DMSP- the dddP gene which was responsible for DMS production degradation processes depending on current environmental was most abundant in the bacterial community at Semporna conditions. Considering the observed conditions of the (33% of all isolates; Figure 5). The Alcaligenes faecalis- sampling sites, our data supports the hypothesis of a related isolate was obtained from Semporna (Figure 4) and ‘bacterial switch’. Further studies which include DMS and showed positive results for both dmdA and dddP genes. DMSP measurements as well as a further look into other Its colonies displayed yellow pigmentation indicative of factors controlling bacterial activity (e.g. UV radiation and its potential to produce DMS. dissolved organic matter) (Kirchmann 2000) in these areas Oceanimonas sp. is one of the earliest are recommended to better understand the role of these Gammaproteobacteria to have been studied biochemically bacterial communities in DMSP cycling in the area. for multiple DMSP-degrading genes including dddP (Yoch 2002) and isolates related to them were isolated from Kuching and Semporna (Figures 2 & 4). Studies have ACKNOWLEDGEMENTS indicated that Oceanimonas sp. have multiple DMSP- The authors would like to thank N.M. Levine for her degrading genes, allowing them to play a role in the sulphur assistance with the identification of dddP and dmdA cycle (Curson et al. 2011b). The availability of different genes. We also thank the Sarawak Biodiversity Centre for ddd genes in Oceanimonas sp. implies that DMSP may be their kind permission to conduct the research in Sarawak a key substrate for this bacteria genus, enabling them to waters (Permit No. SBC-RA-0094-MM). F.W.I. Kuek produce DMS from DMSP (Ledyard et al. 1993). They also is funded by the Sarawak Foundation’s Tunku Abdul have a cytoplasmic DMSP lyase (de Souza & Yoch 1995; Rahman scholarship. The research leading to these results Yoch et al. 1997) resembling the periplasmic dddY of has received funding from the European Union’s Seventh Alcaligenes faecalis (de Souza et al. 1996). Our results Framework Programme FP7/2007-2013 under grant showed an interesting feature with Oceanimonas isolates agreement no. 226224 - SHIVA. 925

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APPENDIX 16S rRNA gene sequence analysis of bacterial cultures based on BLAST analysis

GenBank Sequence accession Closest match Identities Phylogenetic division number Vibrio orientalis strain JC97, isolate 465/468 Gammaproteobacteria; Vibrionales; 1911-S1-01-1.2.1 KF373319 Pkl-17 [FR837599] (99%) Vibrionaceae; Vibrio Rhodobacter capsulatus strain PSB- 434/440 Alphaproteobacteria; Rhodobacterales; 1911-S1-01-1.2.2 KF373320 06 [FJ866784] (99%) Rhodobacteraceae; Rhodobacter Rhodobacter capsulatus strain PSB- 440/455 Alphaproteobacteria; Rhodobacterales; 1911-S1-01-2 KF373321 06 [FJ866784] (97%) Rhodobacteraceae; Rhodobacter Rhodobacter capsulatus strain PSB- 433/440 Alphaproteobacteria; Rhodobacterales; 1911-S1-01-3 KF373322 06 [FJ866784] (98%) Rhodobacteraceae; Rhodobacter Gammaproteobacteria; Pseudomonadales; Pseudomonas oleovorans strain 456/457 1911-S1-05-2 KF373323 Pseudomonadaceae; Pseudomonas; HNS030 [JN128264] (99%) Pseudomonas Gammaproteobacteria; Pseudomonadales; Pseudomonas oleovorans strain 459/460 1911-S1-07-1 KF373324 Pseudomonadaceae; Pseudomonas; HNS030 [JN128264] (99%) Pseudomonas Vibrio alginolyticus isolate Va150 476/478 Gammaproteobacteria; Vibrionales; 1911-S2-01-1 KF373325 [EU155497] (99%) Vibrionaceae; Vibrio Vibrio alginolyticus strain HZBC71 471/474 Gammaproteobacteria; Vibrionales; 1911-S2-05-1 KF373326 [JN188402] (99%) Vibrionaceae; Vibrio Vibrio alginolyticus strain HZBC71 473/475 Gammaproteobacteria; Vibrionales; 1911-S2-07-1 KF373327 [JN188402] (99%) Vibrionaceae; Vibrio Vibrio parahaemolyticus isolate 471/473 Gammaproteobacteria; Vibrionales; 1911-S2-07-2 KF373328 Vp481 [EU155540] (99%) Vibrionaceae; Vibrio Pseudoalteromonas ganghwensis 464/465 Gammaproteobacteria; ; 1911-S3-01-1.1.1 KF373329 [DQ011614] (99%) Pseudoalteromonadaceae; Pseudoalteromonas Vibrio parahaemolyticus strain 319/402 Gammaproteobacteria; Vibrionales; 1911-S3-01-1.1.2 KF373330 VPMP55 [JQ663925] (79%) Vibrionaceae; Vibrio Vibrio alginolyticus strain P61224 474/475 Gammaproteobacteria; Vibrionales; 1911-S3-01-1.2 KF373331 [AJ704375] (99%) Vibrionaceae; Vibrio Vibrio diabolicus strain KM30-12-3 475/478 Gammaproteobacteria; Vibrionales; 1911-S3-01-2 KF373332 [JQ670740] (99%) Vibrionaceae; Vibrio Vibrio parahaemolyticus strain 93A- 474/476 Gammaproteobacteria; Vibrionales; 1911-S3-05-1 KF373333 5807 [DQ497398] (99%) Vibrionaceae; Vibrio Vibrio parahaemolyticus strain 93A- 470/473 Gammaproteobacteria; Vibrionales; 1911-S3-05-2 KF373334 5807 [DQ497398] (99%) Vibrionaceae; Vibrio Vibrio harveyi strain IS01 473/474 Gammaproteobacteria; Vibrionales; 1911-S3-10-1.1 KF373335 [GU974342] (99%) Vibrionaceae; Vibrio Vibrio campbellii strain CAIM 886 473/475 Gammaproteobacteria; Vibrionales; 1911-S3-10-1.2 KF373336 [HM584033] (99%) Vibrionaceae; Vibrio Vibrio rotiferianus strain BV1 475/478 Gammaproteobacteria; Vibrionales; 1911-S3-10-2.1 KF373337 [JN391272] (99%) Vibrionaceae; Vibrio Pseudoalteromonas ganghwensis 462/463 Gammaproteobacteria; Alteromonadales; 1911-S4-01-1 KF373338 [DQ011614] (99%) Pseudoalteromonadaceae; Pseudoalteromonas Vibrio alginolyticus strain H050815-1 474/475 Gammaproteobacteria; Vibrionales; 1911-S4-01-1.1 KF373339 [EF219054] (99%) Vibrionaceae; Vibrio Thalassospira xiamenensis strain 411/416 Alphaproteobacteria; Rhodospirillales; 1911-S4-01-2.2 KF373340 PTG4-18 [EU603449] (99%) Rhodospirillaceae; Thalassospira Citrobacter freundii strain AIMST 461/462 Gammaproteobacteria; Enterobacteriales; 1911-S4-05-1.1 KF373341 Ehe5 [JQ312038] (99%) Enterobacteriaceae; Citrobacter Leclercia adecarboxylata strain 461/462 Gammaproteobacteria; Enterobacteriales; 1911-S4-05-1.2 KF373342 AIMST Ehe6 [JQ312039] (99%) Enterobacteriaceae; Leclercia Vibrio azureus strain 41113 452/468 Gammaproteobacteria; Vibrionales; 1911-S4-05-2 KF373343 [HM032787] (97%) Vibrionaceae; Vibrio 929

GenBank Sequence accession Closest match Identities Phylogenetic division number Vibrio alginolyticus strain H050815-1 472/473 Gammaproteobacteria; Vibrionales; 1911-S4-10-2.1 KF373344 [EF219054] (99%) Vibrionaceae; Vibrio Vibrio natriegens strain AUCASVE1 471/472 Gammaproteobacteria; Vibrionales; 1911-S5-01-1 KF373345 [JQ043186] (99%) Vibrionaceae; Vibrio Vibrio natriegens strain AUCASVE1 474/475 Gammaproteobacteria; Vibrionales; 1911-S5-01-2.1 KF373346 [JQ043186] (99%) Vibrionaceae; Vibrio Vibrio natriegens strain AUCASVE1 472/473 Gammaproteobacteria; Vibrionales; 1911-S5-01-2.2 KF373347 [JQ043186] (99%) Vibrionaceae; Vibrio Citrobacter freundii strain AIMST 462/463 Gammaproteobacteria; Enterobacteriales; 1911-S5-05-1.1.2 KF373348 Ehe5 [JQ312038] (99%) Enterobacteriaceae; Citrobacter Vibrio natriegens strain AUCASVE1 472/473 Gammaproteobacteria; Vibrionales; 1911-S5-05-1.2 KF373349 [JQ043186] (99%) Vibrionaceae; Vibrio Vibrio azureus strain F77118 473/473 Gammaproteobacteria; Vibrionales; 1911-S5-05-1.2.1 KF373350 [HQ908716] (100%) Vibrionaceae; Vibrio Vibrio parahaemolyticus strain 448 474/475 Gammaproteobacteria; Vibrionales; 1911-S5-05-2 KF373351 [JN188417] (99%) Vibrionaceae; Vibrio Vibrio natriegens strain AUCASVE1 471/472 Gammaproteobacteria; Vibrionales; 1911-S5-05-3 KF373352 [JQ043186] (99%) Vibrionaceae; Vibrio Vibrio azureus strain 41113 471/474 Gammaproteobacteria; Vibrionales; 1911-S5-10-1 KF373353 [HM032787] (99%) Vibrionaceae; Vibrio Vibrio splendidus strain AP625 469/471 Gammaproteobacteria; Vibrionales; 1911-S5-10-2 KF373354 [GQ254509] (99%) Vibrionaceae; Vibrio Gammaproteobacteria; Pseudomonadales; Pseudomonas oleovorans strain 452/453 2311-S1-01-1.1 KF373355 Pseudomonadaceae; Pseudomonas; HNS030 [JN128264] (99%) Pseudomonas Shewanella haliotis strain MS41 461/461 Gammaproteobacteria; Alteromonadales; 2311-S1-01-1.2 KF373356 [FN997635] (100%) Shewanellaceae; Shewanella Shewanella haliotis strain MS41 469/469 Gammaproteobacteria; Alteromonadales; 2311-S1-01-2.1 KF373357 [FN997635] (100%) Shewanellaceae; Shewanella Shewanella haliotis strain MS41 467/467 Gammaproteobacteria; Alteromonadales; 2311-S1-01-2.2 KF373358 [FN997635] (100%) Shewanellaceae; Shewanella Shewanella haliotis strain MS41 466/466 Gammaproteobacteria; Alteromonadales; 2311-S1-01-3.1 KF373359 [FN997635] (100%) Shewanellaceae; Shewanella Exiguobacterium aurantiacum var. 485/485 Firmicutes; Bacilli; Bacillales; Bacillales 2311-S1-05-1 KF373360 Colo. Road [AY047481] (100%) Family XII. Incertae Sedis; Exiguobacterium Oceanimonas smirnovii strain 31-13 442/461 Gammaproteobacteria; ; 2311-S1-05-2 KF373361 [NR_042963] (96%) Aeromonadaceae; Oceanimonas Vibrio rotiferianus strain 5S 466/470 Gammaproteobacteria; Vibrionales; 2311-S1-10-1 KF373362 [JF792070] (99%) Vibrionaceae; Vibrio Brevibacillus laterosporus strain 472/472 Firmicutes; Bacilli; Bacillales; 2311-S2-01-1 KF373363 GZUB11 [FJ434663] (100%) Paenibacillaceae; Brevibacillus Vibrio splendidus strain AP625 414/453 Gammaproteobacteria; Vibrionales; 2311-S2-10-1 KF373364 [GQ254509] (91%) Vibrionaceae; Vibrio Bacillus sphaericus clone 7-16 431/456 Firmicutes; Bacilli; Bacillales; Bacillaceae; 2311-S3-01-1.1 KF373365 [DQ364585] (95%) Lysinibacillus Shewanella putrefaciens strain R1418 455/461 Gammaproteobacteria; Alteromonadales; 2311-S3-01-1.2 KF373366 [AB208055] (99%) Shewanellaceae; Shewanella Shewanella putrefaciens strain R1418 459/462 Gammaproteobacteria; Alteromonadales; 2311-S3-01-2 KF373367 [AB208055] (99%) Shewanellaceae; Shewanella Vibrio vulnificus strain W045 473/473 Gammaproteobacteria; Vibrionales; 2311-S3-01-3 KF373368 [EF114147] (100%) Vibrionaceae; Vibrio Enterobacter ludwigii strain KW 93 463/463 Gammaproteobacteria; Enterobacteriales; 2311-S3-05-1 KF373369 [JX262395] (100%) Enterobacteriaceae; Enterobacter Pseudomonas plecoglossicida strain 459/459 Gammaproteobacteria; Pseudomonadales; 2311-S3-05-2.1 KF373370 AIMST Aie20 [JQ312025] (100%) Pseudomonadaceae; Pseudomonas 930

GenBank Sequence accession Closest match Identities Phylogenetic division number Thalassospira sp. SKUK MB1005 417/417 Alphaproteobacteria; Rhodospirillales; 2311-S3-10-1 KF373371 [EU907920] (100%) Rhodospirillaceae; Thalassospira Bacillus malacitensis strain TP12 404/408 Firmicutes; Bacilli; Bacillales; Bacillaceae; 2311-S3-10-2.1 KF373372 [FJ887890] (99%) Bacillus Vibrio natriegens strain AUCASVE5 471/472 Gammaproteobacteria; Vibrionales; 2311-S3-10-2.2 KF373373 [JQ277719] (99%) Vibrionaceae; Vibrio Providencia sp. Sam130-9A 456/460 Gammaproteobacteria; Enterobacteriales; 2311-S4-01-1 KF373374 [FJ418577] (99%) Enterobacteriaceae; Providencia Nitratireductor basaltis strain J3 409/409 Alphaproteobacteria; Rhizobiales; 2311-S4-05-1 KF373375 [NR_044414] (100%) Phyllobacteriaceae; Nitratireductor Oceanimonas smirnovii strain 31-13 463/468 Gammaproteobacteria; Aeromonadales; 2311-S4-10-1 KF373376 [NR_042963] (99%) Aeromonadaceae; Oceanimonas Oceanimonas smirnovii strain 31-13 463/468 Gammaproteobacteria; Aeromonadales; 2311-S4-10-2.1.1 KF373377 [NR_042963] (99%) Aeromonadaceae; Oceanimonas Lysinibacillus fusiformis strain R3 476/476 Firmicutes; Bacilli; Bacillales; Bacillaceae; 2311-S4-10-2.1.3 KF373378 [JQ991002] (100%) Lysinibacillus Exiguobacterium aurantiacum var. 489/490 Firmicutes; Bacilli; Bacillales; Bacillales 2311-S4-10-2.2 KF373379 Colo. Road [AY047481] (99%) Family XII. Incertae Sedis; Exiguobacterium Oceanimonas smirnovii strain 31-13 460/465 Gammaproteobacteria; Aeromonadales; 2311-S4-10-2.3 KF373380 [NR_042963] (99%) Aeromonadaceae; Oceanimonas Vibrio vulnificus strain W045 475/475 Gammaproteobacteria; Vibrionales; 2311-S4-18-1.1 KF373381 [EF114147] (100%) Vibrionaceae; Vibrio Oceanimonas smirnovii strain 31-13 436/447 Gammaproteobacteria; Aeromonadales; 2311-S4-18-1.2 KF373382 [NR_042963] (98%) Aeromonadaceae; Oceanimonas Pseudoalteromonas lipolytica strain 464/465 Gammaproteobacteria; Alteromonadales; 2311-S5-01-1.2 KF373383 ZR064 [JX173567] (99%) Pseudoalteromonadaceae; Pseudoalteromonas Pseudoalteromonas lipolytica strain 463/463 Gammaproteobacteria; Alteromonadales; 2311-S5-01-2.1 KF373384 ZR064 [JX173567] (100%) Pseudoalteromonadaceae; Pseudoalteromonas Pseudomonas stutzeri strain UP-1 453/454 Gammaproteobacteria; Pseudomonadales; 2311-S5-01-2.2 KF373385 [AY364327] (99%) Pseudomonadaceae; Pseudomonas Pseudomonas stutzeri strain UP-1 458/459 Gammaproteobacteria; Pseudomonadales; 2311-S5-01-2.3 KF373386 [AY364327] (99%) Pseudomonadaceae; Pseudomonas Brevundimonas diminuta strain c138 405/406 Alphaproteobacteria; Caulobacterales; 2311-S5-01-3.1.1 KF373387 [FJ950570] (99%) Caulobacteraceae; Brevundimonas Exiguobacterium arabatum 438/479 Firmicutes; Bacilli; Bacillales; Bacillales 2311-S5-01-3.1.2 KF373388 [JF758868] (91%) Family XII. Incertae Sedis; Exiguobacterium Brevundimonas diminuta strain KSC_ 407/407 Alphaproteobacteria; Caulobacterales; 2311-S5-01-3.2 KF373389 AK3a [EF191247] (100%) Caulobacteraceae; Brevundimonas Vibrio natriegens strain AUCASVE5 472/472 Gammaproteobacteria; Vibrionales; 2311-S5-01B-1 KF373390 [JQ277719] (100%) Vibrionaceae; Vibrio Vibrio splendidus strain AP625 472/473 Gammaproteobacteria; Vibrionales; 2311-S5-05-1 KF373391 [GQ254509] (99%) Vibrionaceae; Vibrio Vibrio splendidus strain AP625 470/472 Gammaproteobacteria; Vibrionales; 2311-S5-05-2 KF373392 [GQ254509] (99%) Vibrionaceae; Vibrio Alcaligenes faecalis strain 410/465 Betaproteobacteria; Burkholderiales; 2611-S1-01-1.1 KF373393 OCEN2DBT [JF264463] (88%) Alcaligenaceae; Alcaligenes Vibrio communis strain F75216 472/472 Gammaproteobacteria; Vibrionales; 2611-S1-01-1.2 KF373394 [HQ161743] (100%) Vibrionaceae; Vibrio Exiguobacterium lactigenes strain: 483/483 Firmicutes; Bacilli; Bacillales; Bacillales 2611-S1-05-1.1 KF373395 HYS0503-MK66 [AB259161] (100%) Family XII. Incertae Sedis; Exiguobacterium Oceanimonas smirnovii strain 31-13 463/468 Gammaproteobacteria; Aeromonadales; 2611-S1-05-1.2 KF373396 [NR_042963] (99%) Aeromonadaceae; Oceanimonas Pseudidiomarina sediminum strain 423/463 Gammaproteobacteria; Alteromonadales; 2611-S5-01-1 KF373421 c121 [NR_044176] (91%) Idiomarinaceae; Idiomarina 931

GenBank Sequence accession Closest match Identities Phylogenetic division number Gammaproteobacteria; Pseudomonadales; Pseudomonas pseudoalcaligenes 437/438 2611-S5-05A-1 KF373422 Pseudomonadaceae; Pseudomonas; strain K29411 [DQ298030] (99%) Pseudomonas Pseudoalteromonas sp. S187 465/466 Gammaproteobacteria; Alteromonadales; 2611-S5-05B-1.1 KF373423 [FJ457123] (99%) Pseudoalteromonadaceae; Pseudoalteromonas Photobacterium sp. MM14 473/473 Gammaproteobacteria; Vibrionales; 2611-S5-05B-1.2 KF373424 [JN791371] (100%) Vibrionaceae; Photobacterium Shewanella sp. UMS11/10 460/465 Gammaproteobacteria; Alteromonadales; 2611-S5-05B-3.2.1 KF373425 [JQ231163] (99%) Shewanellaceae; Shewanella Shewanella sp. UMS11/10 464/465 Gammaproteobacteria; Alteromonadales; 2611-S5-05B-3.2.2 KF373426 [JQ231163] (99%) Shewanellaceae; Shewanella Photobacterium sp. MM14 477/477 Gammaproteobacteria; Vibrionales; 2611-S5-05C-2 KF373427 [JN791371] (100%) Vibrionaceae; Photobacterium Nitratireductor aquimarinus CL- 404/406 Alphaproteobacteria; Rhizobiales; 2611-S5-10-2 KF373428 SC21 [HQ176467] (99%) Phyllobacteriaceae; Nitratireductor Gammaproteobacteria; Pseudomonadales; Pseudomonas pseudoalcaligenes 347/410 2611-S6-01-1 KF373429 Pseudomonadaceae; Pseudomonas; strain K29411 [DQ298030] (85%) Pseudomonas Gammaproteobacteria; Pseudomonadales; Pseudomonas pseudoalcaligenes 450/450 2611-S6-01-1.1 KF373430 Pseudomonadaceae; Pseudomonas; strain K29411 [DQ298030] (100%) Pseudomonas Vibrio campbellii strain CAIM 886 463/467 Gammaproteobacteria; Vibrionales; 2611-S6-01-1.2 KF373431 [HM584033] (99%) Vibrionaceae; Vibrio Vibrio alginolyticus strain 486 475/475 Gammaproteobacteria; Vibrionales; 2611-S6-01-3 KF373432 [JN188409] (100%) Vibrionaceae; Vibrio Oceanimonas smirnovii strain 31-13 445/449 Gammaproteobacteria; Aeromonadales; 2611-S6-05-1.1 KF373433 [NR_042963] (99%) Aeromonadaceae; Oceanimonas Rhodobacter capsulatus strain PSB- 468/468 Alphaproteobacteria; Rhodobacterales; 2611-S6-05-2 KF373434 06 [FJ866784] (100%) Rhodobacteraceae; Rhodobacter Vibrio parahaemolyticus strain 448 474/475 Gammaproteobacteria; Vibrionales; 2611-S6-09-1 KF373435 [JN188417] (99%) Vibrionaceae; Vibrio Vibrio parahaemolyticus strain 448 471/472 Gammaproteobacteria; Vibrionales; 2611-S6-09-2 KF373436 [JN188417] (99%) Vibrionaceae; Vibrio Vibrio parahaemolyticus strain 448 475/476 Gammaproteobacteria; Vibrionales; 2611-S7-01-1 KF373437 [JN188417] (99%) Vibrionaceae; Vibrio Vibrio parahaemolyticus strain S9- 475/475 Gammaproteobacteria; Vibrionales; 2611-S7-01-2 KF373438 891-B0919354-5-8F [KC520577] (100%) Vibrionaceae; Vibrio Vibrio alginolyticus strain 486 471/472 Gammaproteobacteria; Vibrionales; 2611-S8-01-1.1 KF373439 [JN188409] (99%) Vibrionaceae; Vibrio Vibrio communis strain F75216 474/474 Gammaproteobacteria; Vibrionales; 2611-S8-01-3 KF373440 [HQ161743] (100%) Vibrionaceae; Vibrio