Dilution-to-extinction culturing of SAR11 members and other marine bacteria from the Red Sea
Thesis written by
Roslinda Mohamed
In Partial Fulfillment of the Requirements
For the Degree of
Master of Science (MSc.) in Marine Science
King Abdullah University of Science and Technology
Thuwal, Kingdom of Saudi Arabia
December 2013 2
The thesis of Roslinda Mohamed is approved by the examination committee.
Committee Chairperson: Ulrich Stingl
Committee Co-Chair: NIL
Committee Members: Pascal Saikaly
David Ngugi
King Abdullah University of Science and Technology
2013 3
Copyright © December 2013
Roslinda Mohamed
All Rights Reserved 4 ABSTRACT Dilution-to-extinction culturing of SAR11 members and other marine bacteria from the Red Sea
Roslinda Mohamed
Life in oceans originated about 3.5 billion years ago where microbes were the only life form for two thirds of the planet’s existence. Apart from being abundant and diverse, marine microbes are involved in nearly all biogeochemical processes and are vital to sustain all life forms. With the overgrowing number of data arising from culture-independent studies, it became necessary to improve culturing techniques in order to obtain pure cultures of the environmentally significant bacteria to back up the findings and test hypotheses. Particularly in the ultra-oligotrophic Red Sea, the ubiquitous
SAR11 bacteria has been reported to account for more than half of the surface bacterioplankton community. It is therefore highly likely that SAR11, and other microbial life that exists have developed special adaptations that enabled them to thrive successfully. Advances in conventional culturing have made it possible for abundant, unculturable marine bacteria to be grown in the lab. In this study, we analyzed the effectiveness of the media LNHM and
AMS1 in isolating marine bacteria from the Red Sea, particularly members of the SAR11 clade. SAR11 strains obtained from this study AMS1, and belonged to subgroup 1a and phylotype 1a.3. We also obtained other interesting strains which should be followed up with in the future. In the long run, results from this study will enhance our knowledge of the pelagic ecosystem and allow the impacts of rising temperatures on marine life to be understood. 5 ACKNOWLEDGEMENTS
First and foremost, I would like to express my sincere gratitude to my supervisor Dr Ulrich Stingl who has supported me throughout this thesis. I appreciate his support and guidance, and value the freedom he gives to work independently. I attribute the completion of this thesis to his encouragement and effort, and I could not have wished for a better supervisor.
I am also truly grateful to Dr Saikaly and Dr Ngugi for accepting the invitation to being part of my thesis defense committee and appreciate his valuable time and feedbacks.
I would also like to express my appreciation to the Coastal Marine Resources Core Lab (CMOR) crew at KAUST for their assistance in getting the seawater samples used in this study. Principally, Ioannis Georgakakis, for his help in making physical measurements of the water each time of sampling.
My special thanks goes out to the RSRC family, especially to members of the Stingl team, for creating a warm working environment throughout my stay in KAUST. I give special thanks to Francy Jimenez for her time and help in various laboratory experiments, and for her patience in guiding me throughout this project. Also, I am truly grateful to Zahraa Alhashem for her contributions in looking at the nitrogen-fixers. I would definitely not have been able to complete this study single-handedly.
Finally, I would like to thank my family and friends for their endless encouragement throughout the years, especially to my parents for supporting me emotionally and understanding of my visions in life. 6 TABLE OF CONTENTS
EXAMINATION COMMITTEE APPROVALS ______2 COPYRIGHT ______3 ABSTRACT ______4 ACKNOWLEDGEMENTS ______5 TABLE OF CONTENTS ______6 LIST OF ABBREVIATIONS ______8 LIST OF FIGURES ______9 LIST OF TABLES ______10 1. Introduction ______11 1.1 The Red Sea ______11 1.2 Composition of Marine Microbial Communities ______13 1.3 Microbes and Their Roles in the Marine Food Web ______14 1.4 The Ubiquitous SAR11 Clade ______17 1.5 Microdiversity Within the SAR11 Clade ______19 1.6 Microbial Communities and SAR11 in the Red Sea ______20 1.7 Proteorhodopsin ______22 1.8 Significance of Culturing Marine Bacteria ______23 1.9 Dilution-to-extinction Culturing ______24 1.10 Research Objectives ______26
2. Material and Methods ______27 2.1 Collection of Seawater ______27 2.2 Preparation of Low Nutrient Heterotrophic Media (LNHM) ______27 2.3 Preparation of Artificial Seawater Medium (AMS1) ______28 2.4 Preparation of Vessels for Dilution-to-extinction Culturing ______29 2.5 Inoculation of Media ______30 2.6 Screening of Cell Cultures for Growth ______31 2.7 DNA Extraction and Gene-specific Sanger Sequencing ______32 2.8 Long-term Storage ______33 2.9 Sequence Assembly and BLAST Searches ______33 7 2.10 Phylogenetic Analysis ______34
3. Results ______35 3.1 Physical Properties of the Water Column ______35 3.2 Identification of Isolates Grown in LNHM and AMS1 ______35 3.2.1 Growth Behaviors ______35 3.2.2 DNA Extraction and Gene Amplifications ______36 3.2.3 Total Isolates Identified ______38 3.2.4 SAR11 Isolates ______40 3.2.5 Phylogeny of SAR11 Isolates Based on 16S rRNA Gene ______42 3.2.6 Phylogeny of SAR11 Isolates Based on ITS Sequences ______44 3.2.7 Phylogeny of SAR11 Strains Based on PR Gene ______46 3.2.8 Growth Profile of the SAR11 Isolate- SAR08 ______47 3.3 Identification of Isolates Grown In Nitrogen-deficient Media ______48 3.3.1 Growth Behaviors ______48 3.3.2 Total Isolates Identified ______51
4. Discussion ______52 4.1 Effects of Media, Sampling Depth and Cell Densities on Cell Culture Growth ______52 4.2 Isolation of DNA from Cell Cultures ______55 4.3 Red Sea Bacteria Isolates and Their Possible Functions ______56
5. Conclusion and Future Directions ______59
6. References ______60
Appendices ______67
8 LIST OF ABBREVIATIONS
3-PGA 3-phosphoglyceric acid ABI Application binary interface Ac-CoA acetyl CoA AMS1 artificial seawater media ATP adenosine triphosphate BLAST Basic Local Alignment Search Tool DMSO dimethyl sulfoxide DMSP dimethylsulfoniopropionate DNA deoxyribonucleic acid DOC dissolved organic carbon DOM dissolved organic matter GBT glycine betaine Gly glycine GOS Global Ocean Sampling HNF heterotrophic nanoflagellates ITS internal transcribed spacer IUPAC International Union of Pure and Applied Chemistry KAUST King Abdullah University of Science and Technology LAS linear alkylbenzenesulfonate LNHM low nutrient heterotrophic media LTP living tree project Mbp mega basepairs MPN most probable count technique NCBI National Centre for Biotechnology Information NJ neighbour-joining PR proteorhodopsin rRNA ribosomal RNA RSRC Red Sea Research Centre SINA SILVA Incremental Aligner TCA tricarboxylic acid THF tetrahydrofolate TMAO trimethylamine oxide UV ultra-violet
9 LIST OF FIGURES
Figure 1 Map of the Red Sea 11
Figure 2 Schematic diagram of the microbial loop 15
Central carbon, sulfur and glycine-serine Figure 3 18 metabolism in SAR11
Schematic plan for our 10 m, 100 m and 500 m Figure 4 30 cell cultures
Optimization of PCR amplification cycles of Figure 5 38 deep-sea DNA.
Figure 6 Taxonomic classification of cultured isolates 39
Phylogenetic tree based on 16S rRNA gene Figure 7 42 sequences
Phylogenetic tree based on ITS gene Figure 8 43 sequences
Figure 9 Growth curve of SAR08 47
10 LIST OF TABLES
List of compounds and their final concentrations in Table 1 28 AMS1
List of primers and conditions used in amplifying Table 2 32 genesof interest
Number of strains exhibiting growth relative to total Table 3 35 number of wells per cell density, per depth
Number of strains successfully amplified by 16S Table 4 rRNA amplification relative to the total number of 37 strains where DNA were extracted
Table 5 List of all 8 strains of SAR11 isolated from this study 40
Table 6 Results from PR-based BLAST search 46
Number of strains exhibiting growth after 2 weeks of Table 7 48 growing in nitrogen-deficient media
Number of strains exhibiting growth after second Table 8 49 transfer to light and dark
Number of strains exhibiting growth in media with Table 9 50 and without vitamins
List of strains obtained from cell cultures grown in Table 10 51 nitrogen-deficient media
11 1. Introduction
1.1 The Red Sea
Figure 1| Map of the Red Sea, a water body formed as a result of African-Arabian plate separation. Image adapted from: An ABC escapade through Egypt (http://www.bernadettesimpson.com/redsea.html)
The Red Sea (22°N, 38°E) is a land-locked water body located in an arid, tropical zone between Africa and Saudi Arabia. It is bordered by the
Mediterranean Sea and the Indian Ocean, and extends from the Gulf of Suez 12 and Gulf of Aqaba (Eilat) in the north, and terminates in the strait of Bab al
Mandeb in the south (Figure 1). Due to its geographical location, the Red Sea experiences high solar irradiance with surface temperature exceeding 30 °C in summer and fall (F J Edwards 1987). Additionally, its salinity of 36 to 41 psu clearly surpasses the world’s average of 35 psu (F J Edwards 1987;
Chiffings 1994). The deep-water mass in the Red Sea is also unique from other water bodies, as it is both isothermal (21-22 °C) and isohaline all year- round (F J Edwards 1987).
The Red Sea may be regarded as ultra-oligotrophic since total organic carbon
(TOC) concentrations is considerably lower than TOC in the oligotrophic
Sargasso Sea, and primary production was reported to be less than 100 mg C m-2 d-1 (Weisse 1989; Hansell & Peltzer 1998; Hansell et al. 1995).
Furthermore, concentrations of inorganic nutrients are generally below the limit of detection, owing to the lack of riverine inputs into the sea (Bethoux
1988; Sommer 2000; Sofianos et al. 2002; Smeed 2004). The following nutrient concentrations were reported on the Red Sea: 0.23-1.21 µmol/L of
- + nitrate (NO3 ); 0.06-0.98 µmol/L of ammonium (NH4 ); 1.08-1.17 µmol/L of silicon (Si); 0.08-0.12 µmol/L of phosphate (P); and 0.15-0.36 µg/L of chlorophyll a (Berninger & Wickham 2005).
As one of the warmest and saltiest water body in the world, the Red Sea offers a unique environment that simulates a worst-case scenario for global warming. This will enable the impacts of climate change on marine life to be studied. Having evolved with Earth and being the most abundant organism to exist, marine microbes in the Red Sea will most likely be different from others 13 in tropical marine environments given the unique physiochemical conditions here. Surely, the diversity, functions and/or dynamics of the microbial community will be altered in response to the environmental change.
1.2 Composition of Marine Microbial Communities
Oceans are giant reservoirs of microscopic life. Within this magnificent environment, microbes (viruses, bacteria, archaea, fungi and protists) have been found to live in various marine habitats with an estimated global population of more than 1030 cells (Whitman et al. 1998). Microbes occur widely across the water column, which is divided into epipelagial (0-200 m), mesopelagial (200-1000 m), bathypelagial (1000-2000 m), abysopelagial
(4000-6000 m) and hadalpelagial (6000-11000 m) zones (DL Kirchman 2008).
Marine microbes are known to occur most near the seawater surface where organic matter from primary producers is abundant. Microbes typically dominate the biomass and metabolic variations of life in oceans and they include members from all three domains of life- Bacteria, Archaea and
Eukarya (Pomeroy et al. 1998; Karl 2007).
Marine microbes are classified based on their sizes. Microbes can be categorized as femtoplankton (0.02-0.2 µm), picoplankton (0.2-2 µm), nanoplankton (2-20 µm) and microplankton (20-200 µm) (DL Kirchman 2008).
Femtoplanktons are largely made up of viruses, which constitute the most abundant group in the oceans with numbers up to 1010 particles per litre in surface waters (J a Fuhrman 1999). Viruses control bacterial and phytoplankton populations through infection and lysis, so their abundance is 14 directly correlated to prokaryotic abundance. Bacteria, archaea and some flagellates make up the picoplankton population. Prokaryotes in the upper 200 m of the ocean has been estimated to account for about 3.6 x 1028 cells, of which 2.9 x 1027 are identified as autotrophs (Whitman et al. 1998).
Heterotrophic bacteria remain as the most abundant bacteria in oceans, where they dominate dissolved organic matter (DOM) assimilation owing to their large surface-to-volume ratio, which permits the uptake of nutrients even when present at very low concentrations in the water (Pomeroy et al. 1998).
On the other hand, the nanoplankton community include flagellates, diatoms and dinoflagellates, of which heterotrophic nanoflagellates (HNF) are the primary grazers of heterotrophic bacteria, cyanobacteria and picoeukaryotes
(F Azam & Graf 1983; DL Kirchman 2008; Fenchel 2008). Larger organisms grouped as microplankton graze on phytoplanktons and small ciliates, thereby linking the microbial food web to the classic food chain (DL Kirchman 2008).
1.3 Microbes and Their Roles in the Marine Food Web
Having evolved together with Earth since 3.5 billion years ago (Sogin et al.
2006), the roles of this unseen majority is indeed not trivial. The roles of microbes in oceans is best explained by the microbial loop- a cycle of how carbon and energy are channeled from bacteria, nanoflagellates and ciliates, and ultimately to higher organisms (Figure 2) (Weisse 1989; Pomeroy et al.
1998). 15
Figure 2| Schematic diagram of the microbial loop. Continuous lines represent major fluxes of carbon and energy, while dotted lines represent fluxes of lesser magnitude. Green boxes indicates photosynthetic organisms, yellow boxes indicate heterotrophic organisms, blue boxes indicate higher organisms and pink boxes indicate types of organic matter in seawater. Image adapted from Pomeroy et al, 1998.
Primary productivity in oceans is governed by phytoplanktons, which includes
photosynthetic and chemosynthetic bacterioplanktons. In the presence of
light, these photosynthetic organisms are able to fix carbon, nitrogen and
phosphorus from the atmosphere then introducing them into the seawater as
DOM. Conversely, photochemical oxidation of DOM to CO2 and CO also
occurs at seawater surface. Most marine DOM is produced and consumed in
the upper oceanic layer (upper 1000 m). Phytoplankton releases DOM as part
of their growth process, during predation by grazers and during lysis by 16 viruses. Dissolved organic carbon (DOC), as a result of the primary production, is not lost from the marine ecosystem, but is mediated by bacteria to higher organisms via the microbial loop (Figure 2). This is owing to the exceptional ability of bacteria to adapt to varying physiochemical conditions of the seawater (Farooq Azam & Malfatti 2007), thereby ensuring a continuous supply of food for other organisms despite low nutrient levels. Previous studies have reported that more than half of the total carbon fixed by photosynthesis are passed through as DOM (Chróst & Faust 1980), while about 30% of DOC from the DOM pool is incorporated into heterotrophic bacteria and gets respired and released as CO2 (S Bertilsson & Jones 2003).
As portrayed in the microbial loop (Figure 2), nanoflagellates are believed to be the primary feeders of heterotrophic bacteria, which in turn get eaten by ciliates, which is the staple food for copepods and other mesozooplankton that are the food for larval fishes. Not much is known about DOM cycling in deeper oceanic layers (below 1000 m). However, ocean mixing along with the photochemical and biological processes described above is believed to enhance the oxidation of DOM from the deep ocean, although these occur on a millennial timescale.
Principally, microbes are crucial in transforming many essential elements, in the breaking down of organic matter and in the recycling of nutrients, all of which supports and maintains other life forms on Earth (Munn 2011).
Particularly in oligotrophic water such as the Red Sea, accelerated mineralization of nutrients is desired, and the role of the microbial loop becomes unquestionably vital (Fenchel 2008). 17 1.4 The Ubiquitous SAR11 Clade
In 1990, 16S ribosomal RNA (rRNA) survey on the Sargasso Sea has led to the discovery of a novel clade within Alphaproteobacteria named as SAR11, which remained uncultured until 2002 (S J Giovannoni et al. 1990; M S Rappé et al. 2002). The clade consists of a group of abundant Alphaproteobacteria which had less than 82% sequence similarity to other cultivated members of
Alphaproteobacteria and are also highly distinct from all cultured heterotrophic bacteria (M S Rappé et al. 2002; S J Giovannoni et al. 1990).
The ubiquitous SAR11 clade covers more than half of the total surface (upper
200 m) and one-quarter of the sub-euphotic (200-3000 m) microbial communities in oceans, with an estimated global population of 2.4 x 1028 cells
(Robert M Morris et al. 2002). Members of SAR11 were found in both tropical
(>20°C) and polar regions (<10°C) as well as in coastal, freshwaters and brackish waters (Zwart et al. 2002; Logares et al. 2010; Mark V Brown et al.
2012). In addition, their distribution is also depth-specific (K G Field et al.
1997) and their abundances are influenced by seasonal changes and deep ocean mixing events (Robert M Morris et al. 2005; Carlson et al. 2009).
SAR11 members are typically small and genetically simple organisms with the first isolated strain, Pelagibacter ubique strain HTCC1062, having only 1.3
Mbp genome (S J Giovannoni, H J Tripp, et al. 2005). The cells were described as being curved rods, which doubles every 2-3 days to densities of about 105-106 cells/ml (M S Rappé et al. 2002).
18 Culture-independent studies have also provided us a glimpse into the unique metabolism of SAR11 (H James Tripp 2013). Unlike most other free-living organisms, members of SAR11 are unable to reduce sulfate to sulfide (S J
Giovannoni, H J Tripp, et al. 2005) and have a special preference for pyruvate as their main source of carbon (M S Schwalbach et al. 2010; Carini et al.
2013). They are also able to oxidize various methylated and one-carbon compounds for energy and posses an unusual form of conditional glycine auxotrophy that is best described in Figure 3 (H James Tripp et al. 2009; Sun et al. 2011; Carini et al. 2013).
Figure 3| Central carbon, sulfur and glycine-serine metabolism in SAR11. Red circles with slashes indicate missing functions in members of SAR11. Green structures indicate glycine-activated riboswitches that enable expression of the enzyme they control. +Gly and ++Gly labels on the riboswitches indicate relative intracellular level required to activate them at 50% efficiency. Red carbon (C) atoms depict carbon flow to the tricarboxylic acid (TCA) cycle. Ac-CoA, acetyl CoA; DMSP, dimethylsulfoniopropionate; Gly, glycine; HCOH, formaldehyde; HCOOH, formate; 3-PGA, 3- phosphoglyceric acid; THF, tetrahydrofolate; TMAO, trimethylamine oxide. Image adapted from Tripp et al, 2013. 19
Most recently, viruses that infect members within the clade (pelagiphages) have been described by Zhao et al (2013). Sequence data from four pelagiphages indicated that they are similar to viruses affecting cyanobacteria and, like its host, they are also extremely abundant in marine metagenomes.
With respect to viral defense, SAR11 were placed as being a competition strategist, fully-utilizing its small cell-size and streamlined genome to outnumber competitors (Våge et al. 2013). These speculations however remained questionable and in-depth studies on pelagiphages will be essential to give us a comprehensive understanding of the pelagic ecosystem.
1.5 Microdiversity Within the SAR11 Clade
The success of SAR11 to dominate world’s oceans were said to be due to 3 factors: genome streamlining, generation of ecotypes and the lack of mismatch repair genes (Mark V Brown et al. 2012). While a streamlined genome will help minimize nutrient requirements and resuming cell replication even in harsh conditions, SAR11 members also thrive by diverging into several subgroups/phylotypes that suit their specific niche. The latter is also termed as ‘ecotypes’.
Classification within the SAR11 clade has been perplexing. Comparison of their 16S rRNA gene sequences exhibited only 89.8-99.3% similarity between its members (K G Field et al. 1997). Further analysis of their 16S rRNA has classified members of this clade into at least 6 subgroups so far, namely subgroups 1a, 1b, 2, 3, 4 and 5 (García-Martínez & Rodríguez-Valera 2000; 20 Robert M Morris et al. 2005; Mark V Brown et al. 2005, 2012; Grote et al.
2012). Subgroup 1a include open ocean strains that often show highest abundance and are broadly distributed in the surface (upper 50 m) while subgroup 1b is typically found below 50 m depth. Subgroups 2, 3 and 4 are coastal, brackish and freshwater strains respectively while subgroup 5 composed of all other strains including alphaproteobacterium HIMB59.
More recent studies on the distribution of the SAR11 clade were done to include phylogenetic analysis of the internal transcribed spacer (ITS) – the sequence region between the 16S and 23S rRNA (Mark V Brown et al. 2012;
Grote et al. 2012; David Kamanda Ngugi & Stingl 2012). ITS-based analysis further refines the taxonomic classification within SAR11 and grouped members of the clade into phylotypes (Mv Brown & Ja Fuhrman 2005).
Although the functionality of different SAR11 subgroups/phylotypes in marine ecosystems has not been clearly defined, their abundance and global distribution hints to us of their possible role in the exchange of organic matter in the oceans (Malmstrom et al. 2005).
1.6 Microbial Communities and SAR11 in the Red Sea
Within the ultra-oligotrophic Red Sea, bacterial biomass, growth and grazing rates are relatively high in the central region, with bacterial abundance in the epipelagial zone reported to be from 5.2 to 8.8 x 105 cells/ml (Weisse 1989). A
16S rRNA-based survey of the Red Sea found Cyanobacteria and
Proteobacteria to make up the majority with the SAR11 subgroups 1a and 1b 21 to dominate more than half of the surface bacterioplankton community, and subgroup 2 to dominate the deeper waters (200-1500 m) (David Kamanda
Ngugi et al. 2012; David Kamanda Ngugi & Stingl 2012). Later, ITS-based analyses found SAR11 phylotypes 1a.3, 1b.3 and RS1-4 to dominate the upper euphotic zone. The same study also found SAR11 phylotypes 2.3 and
NA2 to dominate the mesopelagic and bathypelagic zones, while phylotype
1b.1 was consistent throughout the water column (David Kamanda Ngugi &
Stingl 2012). The divergence of SAR11 members into these Red Sea ecotypes were most likely attributable by the isothermal and isohaline character of the deep sea water mass.
Metagenomic analysis on the Red Sea found genes for all enzymes involved in osmolyte conversion and phosphorus acquisition (Thompson et al. 2013).
Osmolytes such as glycine betaine (GBT) and DMSP typically provide energy, sulfur and nitrogen, and members of SAR11 are believed to have obtained these previously from Prochlorococus and other phytoplanktons. This also means that the Red Sea ecotypes are able to use osmolytes as an alternative energy source. Furthermore, genomic data on single cells and cultured isolates of SAR11 from the Red Sea revealed conserved hypothetical proteins, which encode for both core functions and environment-specific functions (unpublished). To this end, we could thus infer that adaptations of these Red Sea ecotypes are most likely due to the acquisition and/or up- regulation of these conserved hypotheticals.
22 1.7 Proteorhodopsin
Proteorhodopsin (PR) is a retinal-containing integral membrane protein, which works as a proton pump that generates energy in the form of adenosine triphosphate (ATP) (O Béjà et al. 2001; Gómez-Consarnau et al. 2010;
Miyake & Stingl 2011). By using light energy to translocate protons across the cell membrane, PR creates an additional source of energy that will help maintain an organism’s vitality and enhance locomotive and solute transport processes (a Martinez et al. 2007). PR was first found in the uncultured marine γ-proteobacteria SAR86 (O. Beja 2000) and has been proven to be extensively distributed, phylogenetically diverse and active in oceans (Sabehi et al. 2003; de la Torre et al. 2003; Sabehi et al. 2004). Classification of PR based on DNA sequence comparison alone has by far sorted the protein into
11 separate groups (Sabehi et al. 2003). Although not much is known of the metabolic effect of PR, they are thought to aid in DOC assimilation in heterotrophic cells (S J Giovannoni, Bibbs, et al. 2005).
The first PR gene in cultured bacteria was found in P. ubique strain
HTCC1062, which is also the first cultured representative of the SAR11 clade
(M S Rappé et al. 2002; S J Giovannoni, Bibbs, et al. 2005). HTCC1062 was found to possess approximately 10 000 PR molecules that occupies almost
20% of its inner membrane surface area (S J Giovannoni, Bibbs, et al. 2005).
The same study by Giovannoni et al (2005) also proved that PR genes are expressed all the time, and that cell growth and growth rates remained unchanged in both, light and dark conditions. Still, an extended cell viability was observed during stationary phase when the cells were grown in a 23 light/dark cycle (Gómez-Consarnau et al. 2010; Laura Steindler et al. 2011).
This therefore implies of the significant role of PR in nutrient savings when cells are nutrient-deprived.
Overall, proteorhodopsin appears to provide bacterioplankton a mechanism to survive in an environment where nutrient availability fluctuates markedly.
Understanding the role of this exceptional photochemistry is therefore necessary particularly because their diversity has hinted of their possible importance towards surface water microbiology worldwide.
1.8 Significance of Culturing Marine Bacteria
Due to their importance in maintaining and sustaining life on Earth, obtaining pure cultures of environmentally relevant marine microbes will be essential when their roles and functions in the marine ecosystems is to be elucidated.
Previously, studies on marine bacteria often focused on obtaining pure cultures of the bacteria, but this method has its limitation, for only less than
1% of bacteria are cultivable on standard growth media (Staley & Konopka
1985). Molecular phylogeny emerged later on, as an alternative tool in identifying and assessing microbial diversity in oceans (Woese & Fox 1977).
These methods not only eliminate the challenges faced in classical culturing, but are also able to identify and classify microbes based on their evolutionary relatedness to other microbes using homologous genes such as the rRNA.
Although such analyses may be useful in unveiling hidden properties of an organism, reliable methods to reconstruct genomes from whole-genome shotgun sequences is still unavailable. In that case, having cultured 24 representatives becomes increasingly necessary as they will provide more complete genomes, while allowing physiological hypotheses previously derived from the genomic data to be tested out (Stephen Giovannoni & Stingl
2007). Additionally, these genomic data may also be exploited in designing culture conditions for isolating the organism of interest.
1.9 Dilution-to-extinction Culturing
In culturing marine heterotrophic bacteria, most researchers relied heavily on the use of media containing high organic carbon (170 times more DOC than in oceans) such as Zobell’s Marine Medium (Zobell 1941). However, cultivation using such media only yields a small proportion of the marine microbial community, as most marine microbes are oligotrophs, which thrives in very low organic carbon concentrations (less than 1 mg/l), just like in the oceans.
The first oligotroph was isolated in 1981 from Lake Biwa in Japan via cultivation in sterilized lake water without any addition of nutrients (Ishida &
Kadota 1981). This study proved the minimal nutrient requirement of oligotrophs, and their strong preference to grow at low cell densities. The result of this study prompted researchers to establish the dilution to extinction method of culturing, which involves diluting microbial population in a 106 fold in order to ultimately obtain a minimum cell concentration of less than 10 cells/ml (Frits Schut et al. 1993; D K Button et al. 1993). Additionally, autoclaved seawater is used when preparing the growth media. In this way, competitors are expectantly removed from the desired bacterial community, while the natural environment is closely mimicked to suit their growth 25 preferences. To date, dilution to extinction culturing has successfully isolated many oligotrophic bacteria including members of the SAR11 clade (M S
Rappé et al. 2002). Variations to this culturing technique have since derived to high-throughput procedures which uses microtitre plates and include thorough pre-treatment of all culturing equipment (Stephanie A Connon & Stephen J
Giovannoni 2002; Stingl et al. 2007). In addition, a defined seawater medium
(AMS1) had proven to be successful in cultivating C. Pelagibacter ubique recently (Carini et al. 2013). AMS1 was formulated based on whole genome sequence information from C. Pelagibacter ubique and include pyruvate or precursors of pyruvate. Studies to evaluate the value of AMS1 as an alternative to the former low-nutrient media is actively underway. To this end,
52 whole genome sequences have been generated from bacterial strains isolated by this technique, of which 15 are isolates from the SAR11 clade
(Michael S Rappé 2013).
26 1.10 Research Objectives
In this study, we aim to culture and isolate members of SAR11 from the Red
Sea using high-throughput, dilution-to-extinction cell culturing in both low nutrient heterotrophic media (LNHM) and artificial seawater media (AMS1).
We hope to be able to find novel strains of SAR11 from our cultured isolates, including deep-water strains, which have often been reported in culture- independent studies but have yet to be cultured anywhere in the world. We also aim to isolate novel nitrogen-fixing bacteria using identical dilution-to- extinction culturing and growing the cultures in both light and dark conditions.
We hypothesize that since nitrogen fixation is an extensively energy- consuming process, PR is most likely the best-known mechanism to make up for the energy requirement. We think that they might not be abundant in the ocean and their presence is most likely dependable on interactions with other microorganisms. Moreover, presence of PR in nitrogen-fixers has never been looked into. Therefore, findings from this experiment will help shed light to our current understanding of PR and their potential roles in the marine ecosystem.
27 2. Material and Methods
2.1 Collection of Seawater
Water samples to be serve as inocula were collected at central Red Sea, approximately 20 miles offshore from the KAUST campus. Several 5L Niskin bottles were deployed to collect the seawater at 10 m, 100 m and 500 m below sea level on 5 November 2012. At the point of sampling, the depth, conductivity of water and water temperature were monitored by CTD probes mounted to a large metal frame that is cast together with the Niskin bottles.
2.2 Preparation of Low Nutrient Heterotrophic Media (LNHM)
Seawater for making LNHM were also collected on 6 November 2012 and 30
April 2013 from the same sampling sites mentioned in Section 3.1. The respective seawater were filtered through a 0.22 µm-pore-diameter Supor membrane (Pall Corporation, USA), autoclaved and sparged for at least 6 hours with CO2 and subsequently with sterile air for 12 hours or until its pH is similar to its initial pH (Stephanie A Connon & Stephen J Giovannoni 2002).
The media were altered with KH2PO4 (1 µM), NH4CL (10 µM), a mixture of carbon compounds such as D-glucose, succinic acid and pyruvic acid
(0.001% each), a mixture of Gamborg’s 1000X vitamins solution (M S Rappé et al. 2002), Glycine (1.5 µM), DMSP/DMSO (100 nM) and L-methionine (100 nM) (Stingl et al. 2007). Prior to use, the sterility of the media was verified using Guava EasyCyte cell counter (Guava Technologies, USA). 28
2.3 Preparation of Artificial Seawater Medium (AMS1)
Compound Formula Final concentration Base salts Sodium chloride NaCl 481 mM
Magnesium chloride MgCl 2!6H2O 27 mM
Calcium chloride CaCl2 !2H2O 10 mM Potassium chloride KCl 9 mM
Sodium bicarbonate NaHCO3 6 mM
Magnesium sulfate MgSO 4!7H2O 2.8 mM
Trace metals
Iron (III) chloride FeCl3 !6H2O 117 nM
Manganese (III) chloride MnCl 2!4H2O 9 nM
Zinc sulfate ZnSO4!7H2O 800 pM
Cobalt (III) chloride CoCl2!6H2 O 500 pM
Sodium molibdate Na2MoO4 !2H2O 300 pM
Sodium selenite Na2O3Se 1 nM
Nickel (II) chloride NiCl2 !6H20 1 nM
Macronutrients
Ammonium sulfate (NH4) 2SO4 400 uM
Monosodium phospate NaH2PO4 !2H2O 50 uM
Vitamins
B1 C12H18Cl2N4OS 6 uM
B3 C6H5NO2 800 nM
B5 C9H17NO5 425 nM
B6 C8H11 NO3 500 nM
B7 C10H16N2O3S 4 nM
B9 C19H19N7O6 4 nM
B12 C63H88CoN14O14P 700 pM
PABA C7H7NO2 6 uM
Myo-Inositol C6H12O6 60 nM
Others
L-Methionine C5H11 NO2 S 1 uM
Pyruvate C3H3NaO3 5 uM
Glycine C2H5NO2 5 uM
Sodium glycolate C2H3NaO3 2.5 uM
Table 1| List of compounds and their final concentrations in AMS1
29 AMS1 was prepared by following the protocol of Carini et al (2013) using standard Milli-Q water (Millipore, USA) that was supplemented with base salts, trace metals, macronutrients, vitamins and other compounds at the final concentrations listed on Table 1. The base salts were added first and autoclaved with Milli-Q water. The autoclaved Milli-Q water was sparged with
CO2 for at least 5 h and subsequently with sterile air for 10 h or until its pH is similar to its initial pH (Stephanie A Connon & Stephen J Giovannoni 2002).
Vitamins and organic compounds were added later. The nitrogen-deficient media that we use to tease out the nitrogen-fixers were prepared by excluding above-mentioned compounds that contained nitrogen. These compounds include ammonium sulfate, L-methionine, glycine and sodium glycolate. Prior to use for culturing, the sterility of each media were verified using Guava
EasyCyte cell counter (Guava Technologies, USA).
2.4 Preparation of Vessels for Dilution-to-extinction Culturing
Both 96-wells teflon-coated microplates (V&P Scientific, USA) and Nalgene® polycarbonate clear flasks (Sigma Aldrich, USA) were used for growing our cell cultures. These culturing vessels were cleaned by following the Stingl et al protocol (Stingl et al. 2007) which involved acid-washing, microwaving and
UV irradiation. After autoclaving, the culture vessels were rinsed at least five times with Milli-Q water (Millipore, USA) and soaked overnight in 10% HCl.
The culture vessels were rinsed again then filled up with Milli-Q water and microwaved at maximum power for 15 min or until boil. The boiling water was 30 discarded and the culture vessels were exposed to UV for at least 60 min before subsequent uses.
2.5 Inoculation of Media
10 m 100 m 500 m
NDM AMS1 LNHM AMS1 LNHM AMS1 LNHM
* # a. 10 cells/ml a. 1 cells/ml * a. 1 cell/ml * a. 1 cell/ml * # b. 100 cells/ml b. 5 cells/ml * b. 5 cells/ml * b. 5 cells/ml c. 400 ul/100 ml # c. 10 cells/ml #
Light Dark
Incubation at 28 °C Incubation at 24 °C Incubation at 22 °C
Figure 4| Schematic plan for our 10 m, 100 m and 500 m cell cultures. NDM, AMS1 and LNHM represents for nitrogen-deficient media, artificial seawater media and low nutrient heterotrophic media respectively. * indicate cultures are set in triplicates; # indicate cultures that are set in quadruplicate.
The cell densities in our 10 m, 100 m and 500 m seawater samples were determined using Guava EasyCyte cell counter (Guava Technologies, CA,
USA). Guava is a flow cytometer that accepts 96 well plates and utilizes small volumes of sample, making it a fast and reliable choice for counting cells such as marine microbes (H. J Tripp 2008). Prior counting, 200 µl of each seawater sample were incubated with SYBR Green for 60 min in the dark (final dilution
1:2000, Invitrogen, OR, USA). Based on the counts obtained from Guava, the seawater samples were diluted respectively and incorporated into both LNHM and AMS1 to give final concentrations of 1, 5 and 10 cells/ml (Figure 4). Both media that have been inoculated with the 10 m, 100 m and 500 m seawater were distributed into several 96-wells teflon-coated microplates for cultivation 31 at 28°C, 24°C and 22°C respectively, in the dark- mimicking the temperatures along the water column at the time of sampling. On the contrary, cultures that were growing in the nitrogen-deficient media were inoculated to final cell concentrations of 10 cells/ml and 100 cells/ml (Figure 4). In addition to the above, culture flasks filled with 100 ml of the media and 400 µl of the stock inoculum were also put to incubate at 27°C, in both light and dark conditions.
The cool-white light provided irradiance at 160 µmol photons m-2 s-1.
2.6 Screening of Cell Cultures for Growth
The microplate cultures were measured for growth after 4, 8 and 12 weeks of incubation, using the Guava EasyCyte cell counter (Guava Technologies,
USA) like mentioned in Section 3.5. Each sample was run for 15 s at 700 V using the green photomultiplier and at 400 V using two other photomultipliers.
In Guava, cell counts (cells/ml) from each well are reported in Excel format
(csv file). In this study, wells exhibiting more than 5x104 cells/ml were selected for identification by Sanger sequencing. 32 2.7 DNA Extraction and Gene-specific Sanger Sequencing
PCR conditions
Primer name Sequencea (5'-3') Target gene Denaturation Annealing Extension Reference 94°C, 45 s AGAGTTTGATC 95°C, 3 min 48°C, 45 s 72°C, 10 min Hongoh et al,2003 72°C, 1 min 27-F MTGGCTCAG General, 16S rRNA 94°C, 45 s TACGGYTACCTT 95°C, 3 min 48°C, 45 s 72°C, 10 min Lane, 1991 1492-R GTTACGACTT General, 16S rRNA 72°C, 1 min 94°C, 20 s CGATGTGTGYTA Garcia-Martinez and 95°C, 1 min 54°C, 20 s 72°C, 10 min GACGTTGGAAA Rodriguez-Valera, 2000 72°C, 2 min SAR11_844-F TTTA SAR11, ITS 94°C, 20 s Garcia-Martinez and 95°C, 1 min 54°C, 20 s 72°C, 10 min AGGTGGGTTTC Rodriguez-Valera, 2000 AP23S_117-R CCCATTC SAR11, ITS 72°C, 2 min 94°C, 30 s THGGWGGATAY 94°C, 3 min 54°C, 30 s 72°C, 7 min Campbell et al, 2008 72°C, 30 s 125-F TTAGGWGAAGC General, Proteorhodopsin
CCCAACCWAYW 94°C, 30 s 94°C, 3 min 54°C, 30 s 72°C, 7 min Campbell et al, 2008 GTWACRATCATT 288-R CT General, Proteorhodopsin 72°C, 30 s
GCIWTHTAYGGI 94°C, 45 s AARGGIGGIATH 95°C, 3 min 58°C, 45 s 72°C, 10 min Gaby and Buckley, 2012 IGK3-F GGIAA General, nifH 72°C, 1 min 94°C, 45 s ATIGCRAAICCIC 95°C, 3 min 58°C, 45 s 72°C, 10 min Gaby and Buckley, 2012 DVV-R CRCAIACIACRTC General, nifH 72°C, 1 min 94°C,45 s TCCGGTTGATC 95°C, 3 min 52°C, 45 s 72°C, 10 min Reysenbach, 2000 ARC21-F CYGCCGG Archaeal, 16S rRNA 72°C, 1 min
a Sequence are in a 5' to 3' direction, IUPAC characters are used TableKey to symbols: 2| List R= A+G, of Y=C+T, primers M=A+C, K=G+T,and S=G+C,conditions W=A+T, H=A+T+C, used B=G+T+C, in amplifying D=G+A+T, N=A+C+G+T, genes V=G+A+C of interest. F and R following primer name indicate forward and reverse primers respectively. a Sequence are in a 5’ to 3’ direction, IUPAC characters are used. Key to symbols: R=A+G, Y=C+T, M=A+C, K=G+T, S=G+C, W=A+T, H=A+T+C, B=G+T+C, D=G+A+T, N=A+C+G+T, V=G+A+C
Prior identification, genomic DNA from each cell culture were extracted using
DNAeasy® Blood and Tissue kit (Qiagen, USA) according to the manufacturer’s protocol. Several genes were targeted for the purpose of this study and the PCR were run for 30-40 cycles using respective primers and conditions listed on Table 2. In general, each PCR reaction contained PCR
Master Mix, respective forward and reverse primers (10 mM each) and 2-5 µl 33 of the DNA template. The PCR Master Mix (Promega, USA) contained the following components: 2X PCR buffer, dNTPs (400 µM), Taq DNA polymerase (50 U) and MgCl2 (3 mM). Subsequently, quality of the PCR products were evaluated on 1% agarose gel, viewed under a UV transilluminator. To ensure high-quality DNA, the PCR products were processed further using MinElute PCR purification kit (Qiagen, USA) and their final DNA concentrations were determined by Nanodrop (Thermo Scientific,
USA) before submitting for Sanger sequencing at the Genomics Core Lab facility in KAUST (Thuwal, Saudi Arabia).
2.8 Long-term Storage
In preparation for future use, each isolate (200 µl each) were stored in 2-ml
Nalgene® cryovials with 5% dimethyl sulfoxide (DMSO) and 10% glycerol respectively. The vials were arranged into Nalgene® Mr Frosty containers
(Thermo Scientific, USA) and immersed in 100% isopropyl alcohol before being placed in a freezer. In this way, the cells will be preserved at the optimal rate of -1°C/min, and may subsequently be revived, their DNA extracted and amplified if required later on.
2.9 Sequence Assembly and BLAST Searches
Raw gene sequences of 16S rRNA, ITS, PR and nifH obtained from KAUST
Genomic Core Lab facility were in ABI Chromatogram format (.ab1). The qualities of each sequence were screened and modified using Codoncode
Aligner- version 3.7.1.2 (Dedham, USA). The ‘cleaned’ sequences were later 34 assembled to form contigs and analyzed using NCBI’s BLAST tool
(http://blast.ncbi.nlm.nih.gov/) to determine their taxonomy.
2.10 Phylogenetic Analysis
Multiple SAR11 isolate sequences were aligned using SILVA Incremental
Aligner (SINA) (v1.2.11) (Pruesse et al. 2012) and their misaligned basepairs were manually refined. Our aligned sequences were later introduced into the dataset of SILVA’s 16S rRNA-based living tree project (LTP) (release 111) database to generate maximum likelihood trees, which arranged our SAR11 isolates into their respective taxonomical grouping, relative to sequences of other SAR11 strains already in the database.
35 3. Results
3.1 Physical Properties of the Water Column
Water sampling on the Red Sea was performed at sampling station
22°45.429’ North, 39°03.232’ East. The physical properties of the water column during the time of sampling is summarized in Appendix I. pH was constant at around 8.5 while salinity remained uniform at 40 psu throughout the depths that were sampled. Temperature, oxygen concentrations and conductivity of the water column declined gradually with increasing depth.
Temperatures at 10 m, 100 m and 500 m depth during the time of sampling were recorded to be 30 °C, 24 °C and 22 °C respectively.
3.2 Identification of Isolates Grown in LNHM and AMS1
3.2.1 Growth Behaviors
AMS1 LNHM 10 m 100 m 500 m 10 m 100 m 500 m
1 cell/ml 4 / 288 1 / 288 10 / 384 62 / 288 49 / 288 7 / 384
5 cells/ml 18 / 288 1 / 288 14 / 384 205 / 288 178 / 288 38 / 384
10 cells/ml NIL NIL 0 / 384 NIL NIL 33 / 384
Table 3| Number of strains exhibiting growth relative to the total number of wells per cell density, per depth .
36 Among the three depths that were sampled, more strains were detected in cell cultures from 10 m, while cell cultures from 500 m showed the least number of strains. In total, strains were detected in 289 wells of the 10 m cell cultures;
229 wells of the 100 m cell cultures; and 102 wells of the 500 m cell cultures after 12 weeks of incubation (Table 3). The increase in the number of strains detected was slow in the 500 m cell cultures; occurring only after more than 4 weeks of growing in the lab. When the two media were compared, cell cultures that were growing in LNHM generally yielded more strains after just 4 weeks of incubation. Initial cell concentration of the cultures had an exponential effect on the final number of strains detected. Meaning, more wells showed growth when initially seeded with 10 cells/ml concentration than those that started out with 1 cells/ml concentration. From some of these cell cultures where growth was detected, DNA was extracted and their 16S rRNA,
ITS and PR genes were targeted and amplified via PCR.
3.2.2 DNA Extraction and Gene Amplifications
In order to identify each isolate, DNA was extracted from each cell culture using the commercial extraction kit mentioned in Section 2.7. Genomic DNA concentrations from the cell cultures are usually very little and cannot be screened conventionally using an agarose gel. Attempts to quantify and verify the quality of genomic DNA using the Nanodrop spectrophotometer was also unsuccessful. Therefore, all the samples were directly preceded with PCR and those that were not amplifying were considered as having low-quality
DNA. PCR amplification of 16S rRNA, ITS and PR genes generated products 37 of sizes around 1400 bp, 1200 bp and 400 bp respectively (results not shown).
AMS1 LNHM 10 m 100 m 500 m 10 m 100 m 500 m
1 cell/ml 3 / 3 0 / 1 5 / 8 4 / 12 6 / 17 2 / 7
5 cells/ml 11 / 15 0 / 1 7 / 10 8 / 13 8 / 13 15 / 32
10 cells/ml NIL NIL 0 / 0 NIL NIL 9 / 30
Table 4| Number of strains successfully amplified by 16S rRNA amplification relative to the total number of strains where DNA were extracted per cell density, per depth.
Most of the DNA extracted from the 10 m and 100 m isolates amplified well
(Table 4). However, initial attempts to amplify the 16S rRNA genes from the
DNA of some 500 m isolates were unsuccessful (data not shown). As a result, several factors were changed to resolve the issue. These changes include: increasing start-up volume used for DNA extraction; performing freeze-thaw extraction; increasing number of amplification cycles; and performing nested
PCR. The latter proved to be most successful and the result is shown in
Figure 5. PCR was also performed using archaeal primers ARC-F and 1492-R
(primer details outlined in Table 2) but no amplification of archaeal genes were detected in any of our 500 m isolates. 38
Figure 5| Optimization of PCR amplification cycles on 500 m cultures. Products from the PCR were verified on 1% agarose gel after (a) 40 cycles of amplification (b) 40 cycles of amplification on the first PCR run, then 30 cycles on the second PCR run (c) after 40 cycles of amplification on the first PCR run, then 20 cycles on the second PCR run. M, DNA ladder (refer to Appendix II for size range); 1-14, PCR products; N, negative control; P, positive control.
3.2.3 Total Isolates Identified
A total of 78 isolates were successfully amplified, sequenced and identified via BLAST search based on their 16S rRNA gene sequences (Table 4 and
Appendix III). The contigs ranged between 105 to 1411 bp long and generally had almost 100% sequence similarity to sequences in the database
(Appendix III). Majority of our isolates were obtained from cell cultures of initial concentration of 5 cells/ml and were growing in LNHM. A few of these isolates were obtained from 1 cell/ml and 10 cells/ml initial concentrations, particularly those from 100 m and 500 m cultures. Based on taxonomy, the isolates were explored at order and family levels (Figure 6). 39 90" ORDER
80"
70"
60"
50"
40" No. of strainsNo.of 30"
20"
10"
0"
Cytophagales SAR11 clade ActinomycetalesAlteromonadales OceanospirillalesRhodobacterales Rhodospirillales Sphingomonadales
10 m 100 m 500 m
90" FAMILY 80"
70"
60"
50"
40"
No. of strainsNo.of 30"
20"
10"
0"
SAR11"clade" Alcanivoracaceae" Halomonadaceae" Alteromonadaceae" Flammeovirgaceae" Oceanospirillaceae" Rhodobacteraceae"Rhodospirillaceae" Erythrobacteraceae" Propionibacterineae" Sphingomonadaceae"
10"m" 100"m" 500"m"
Figure 6| Taxonomic classification of strains isolated from this study at order (top) and family (below) levels. 40 Bacteria from the order Alteromonadales, Oceanospirillales and
Rhodobacterales were isolated from all depths. Isolates from 10 m cultures were distinctive by the presence of Cytophagales, Rhodospirillales and members of the SAR11 clade. At the family level, we observed the presence of Erythrobacteraceae and Oceanospirillaceae in 100 m and 500 m cell cultures. 10 m and 500 m isolates are unique in that the latter was characterized with high abundance of Halomonadaceae,
Sphingomonadaceae and Alcanivoraceae. Interestingly, we also found bacteria from the OM60 clade (Appendix III) among our cultured isolates.
3.2.4 SAR11 Isolates
Taxonomy based on SILVA/RDP 16S rRNA- ITS Strain based sequence Max Total Query Maximum Accession Top Blast Hits E-value ID contig contig score score cover identity no. length (bp) length (bp) Uncultured SAR11 cluster alpha proteobacterium2335 clone2335 98 16S100% ribosomal RNA0 gene,99% partial sequenceJN547477.1 SAR01 1267 1120 Uncultured bacterium clone 6C232398 16S2335 ribosomal2335 RNA gene,100% partial sequence0 99% EU804467.1 Uncultured marine microorganism clone2329 HOT157_25m712329 16S100% ribosomal RNA0 gene,99% partial sequenceJN166160.1 Uncultured bacterium clone 6C232521 16S2034 ribosomal2034 RNA gene,100% partial sequence0 99% EU804572.1 SAR02 1104 1129 Uncultured bacterium clone S23_1210 16S2026 ribosomal2026 RNA gene,100% partial sequence0 99% EF573111.1 Unidentified marine bacterioplankton clone2006 P5-1B_692006 16S ribosomal100% RNA 0gene, partial99% sequenceKC002074.1 Uncultured bacterium clone S73_003 16S2001 ribosomal2001 RNA gene,100% partial sequence0 99% KC874331.1 SAR03 1092 1125 Unidentified marine bacterioplankton clone2001 E410B_742001 16S ribosomal100% RNA0 gene, partial99% sequenceKC003205.1 Uncultured bacterium clone 6C233257 16S2001 ribosomal2001 RNA gene,100% partial sequence0 99% EU805257.1 Proteobacteria; Uncultured bacterium clone 6C232820 16S2255 ribosomal2255 RNA gene,100% partial sequence0 99% EU804854.1 Alphaproteobacteria; SAR04 1227 1108 Uncultured bacterium clone S23_1707 16S2255 ribosomal2255 RNA gene,100% partial sequence0 99% EF573608.1 SAR11 clade; Uncultured bacterium clone 6C232740 16S2250 ribosomal2250 RNA gene,100% partial sequence0 99% EU804779.1 Uncultured bacterium clone Reef_C09 16S510 ribosomal510 RNA gene,99% partial2.E-141 sequence 99% GU119315.1 Surface 1 SAR05 280 1127 Uncultured bacterium clone CEP-5m-89 16S510 ribosomal510 RNA gene,99% partial2.E-141 sequence99% GU061710.1 Uncultured bacterium clone 6C233257 16S510 ribosomal510 RNA gene,99% partial2.E-141 sequence 99% EU805257.1 Uncultured bacterium clone 6C233049 16S2357 ribosomal2357 RNA gene,100% partial sequence0 99% EU805066.1 SAR06 1288 1128 Uncultured bacterium clone 6C232399 16S2357 ribosomal2357 RNA gene,100% partial sequence0 99% EU804468.1 Uncultured bacterium clone 6C232137 16S2357 ribosomal2357 RNA gene,100% partial sequence0 99% EU804237.1 Uncultured bacterium clone 6C232398 16S2366 ribosomal2366 RNA gene,99% partial sequence0 100% EU804467.1 SAR07 1282 1122 Uncultured bacterium clone S23_1135 16S2366 ribosomal2366 RNA gene,99% partial sequence0 100% EF573036.1 Uncultured bacterium clone A0_20 16S ribosomal2361 RNA2361 gene, partial99% sequence0 99% JQ731907.1 Uncultured bacterium clone HglFeb003422141 16S ribosomal2141 RNA100% gene, partial0 sequence99% JX016543.1 SAR08 1165 1130 Uncultured bacterium clone 6C232734 16S2141 ribosomal2141 RNA gene,100% partial sequence0 99% EU804774.1 Uncultured bacterium clone S23_1211 16S2141 ribosomal2141 RNA gene,100% partial sequence0 99% EF573112.1
Table 5| List of all 8 strains of SAR11 isolated from this study and their identities based on 16S rRNA similarity search with other cultured sample sequences using online BLAST search tool.
We had successfully isolated 8 strains of SAR11 from the Red Sea (Table 5 and Appendix III). For ease of identification and for future works, these strains 41 were renamed SAR01 to SAR08. All of the SAR11 strains were obtained from
10 m cultures that were growing at 5 cells/ml density in AMS1. The lengths of the assembled 16S rRNA contigs of these isolates ranged between 280 to
1288 bp. Additionally, contig lengths based on their ITS sequences were between 1108 to 1130 bp. Phylogenetic analyses was performed on these 8 strains to determine their taxonomic grouping among other SAR11 strains found in the Red Sea and other parts of the world.
42 3.2.5 Phylogeny of SAR11 Isolates Based on 16S rRNA Gene
Subgroup 1a
Subgroup 1b
Subgroup 2
Subgroup 3a
Subgroup 3b Subgroup 4
Subgroup 5
Figure 7| Phylogenetic tree based on 16S rRNA gene sequences using maximum likelihood approach (BS= 1000). Purple indicate strains isolated from this study; and green indicate strains isolated from other studies in our lab.
We used the terms ‘subgroup’ to describe the branches within the SAR11 clade based on the 16S rRNA gene tree defined by Brown et al. (2012). A maximum likelihood phylogenetic tree was generated using PAUP (Figure 7).
The confidence values based on Felsenstein’s bootstrap method (1000 bootstrap replications) are represented by numbers beside the tree branches. 43 Names were labeled purple to indicate SAR11 strains isolated from this study; green to indicate SAR11 strains isolated from other studies in the Stingl lab; and black to indicate other SAR11 sequences available in the SILVA database.
The sequence of SAR11_05 was excluded from the phylogenetic analysis as the sequence was too short (280 bp), and will give rise to misleading trees. As seen in Figure 7, all of our SAR11 isolates were classified in subgroup 1a within the SAR11 clade. SAR11_08 in particular, was closely related to the
Hawaii strain HIMB5, whose whole genome has been sequenced. Rickettsia ricketsii, from the family Rickettsiaceae, was chosen to be the outgroup that was used to root the branch of our phylogenetic tree.
44 3.2.6 Phylogeny of SAR11 Isolates Based on ITS Sequences
EJ974778
EJ172023 FJSAR11_39 RMSAR11_02 RMSAR11_03 RMSAR11_06 RMSAR11_05 RMSAR11_04 EJ696292 ER418206 RMSAR11_01 ER334923 S2_M60051 Phylotype 1a.3 S2_M60053 RMSAR11_08 NA_I3K096 S2_M20253 S2_M30503 RMSAR11_07 S2_M30509 HTCC7211 EF616631 EF616620 EF616632 EF616627 EF616630 S2_APR5m14 S1a_FEB70m S3_APR14m14 S3_JUL015mF3 S3_OCT5m93 S3_APR5m220 Phylotype 2.1 S3_DEC5m15 S3_OCT5m28 S3_APR5m199 S3_OCT5m66 S3a_A20212_1 100 Phylotype 2.2 S3a_A20215 3 72 Deep_I3K0242 Deep_MAY500m Deep_M305010 Deep Deep_M34502 20 Deep_G2K016 Deep_I3K022 19 HIMB114 Phylotype 3.1 99 IMCC9063 Phylotype 3.2 1 33 FJSAR11_05 34 NA_FEB70m13 31 NA_G2K140 Phylotype 1b/NA1/NA2 27 NA_G2K0122 NA_G2K0152 EF616623 S1a_JUL015mB1 EF616626 S1a_APR14m57 Phylotype 1a.2 EF616625 S1a_APR5m222 S1a_MB11F01 EF616622 EF616621 HTCC1002 S1_G2K279 Phylotype 1a.1 S1_A22004 HTCC1062 S1a_OCT5m74 S1a_G50049 EcoliITS
0.4
Figure 8| Phylogenetic tree based on ITS gene sequences using maximum likelihood approach (BS= 1000). Purple indicate strains isolated from this study; and green indicate strains isolated from other studies in our lab.
45 We used the terms ‘phylogroup’ to describe the branches within the SAR11 clade based on the 16S rRNA gene tree defined by Brown et al. (2012). A maximum likelihood phylogenetic tree was generated using PAUP (Figure 8).
The confidence values based on Felsenstein’s bootstrap method (1000 bootstrap replications) are represented numbers beside the tree branches.
Names were labeled purple to indicate SAR11 strains isolated from this study; green to indicate SAR11 strains isolated from other studies in the Stingl lab; and black to indicate other SAR11 sequences available in the SILVA database.
In contrast to the 16S rRNA classification, the taxonomic grouping of all our isolates were further refined after looking at their ITS sequences. As seen in
Figure 8, all of our isolates were classified in phylogroup 1a.3. Along with a previously isolated strain from our lab (FJSAR11_39), we also found that our cultured isolates are classified in the same phylogroup as other uncultivated members of SAR11 detected from the Great Ocean Sampling (GOS) and in the Sargasso Sea.
46 3.2.7 Phylogeny of SAR11 Strains Based on PR Gene
The PR gene was amplified in all of our isolates and their assembled contigs ranged between 135 to 667 bp only (Table 6). Effort to obtain PR gene sequence for SAR08 is still in progress. Due to their very short sequences, phylogenetic trees based on PR gene were not generated. However, BLAST similarity search against sequences of cultured representative in the NCBI database revealed a close relation between the PR gene of SAR07 with those of strain HTCC7214 (Table 6). Additionally, PR gene of SAR03 also showed relatively high similarity to those of HIMB5.
Contig Strain Maximum length Top Blast Hits Accession ID identity (bp) Candidatus Pelagibacter ubique strain HTCC1062 proteorhodopsin gene, partial cds 87% EF640911.1 SAR01 172 Candidatus Pelagibacter ubique strain HTCC1061 proteorhodopsin gene, partial cds 87% EF640910.1 Candidatus Pelagibacter ubique strain HTCC1051 proteorhodopsin gene, partial cds 87% EF640908.1 SAR02 135 No significant similarity found Candidatus Pelagibacter ubique clone fosmid 01-003783, complete sequence 95% EU410957.1 SAR03 667 Alpha proteobacterium HIMB5, complete genome 93% CP003809.1
Pelagibacter ubique strain HTCC1051 16S ribosomal RNA, partial sequence; 16S-23S intergenic spacer region, complete sequence; tRNA-Ile and tRNA-Ala genes, complete sequence; and 23S ribosomal RNA, partial sequence 92% AF510193.1 Candidatus Pelagibacter ubique strain HTCC1062 proteorhodopsin gene, partial cds 87% EF640911.1 SAR04 167 Candidatus Pelagibacter ubique strain HTCC1061 proteorhodopsin gene, partial cds 87% EF640910.1 Candidatus Pelagibacter ubique strain HTCC1051 proteorhodopsin gene, partial cds 87% EF640908.1 Candidatus Pelagibacter ubique strain HTCC1062 proteorhodopsin gene, partial cds 86% EF640911.1 SAR05 171 Candidatus Pelagibacter ubique strain HTCC1061 proteorhodopsin gene, partial cds 86% EF640910.1 Candidatus Pelagibacter ubique strain HTCC1051 proteorhodopsin gene, partial cds 86% EF640908.1 Candidatus Pelagibacter ubique strain HTCC1062 proteorhodopsin gene, partial cds 87% EF640911.1 SAR06 159 Candidatus Pelagibacter ubique strain HTCC1061 proteorhodopsin gene, partial cds 87% EF640910.1 Candidatus Pelagibacter ubique strain HTCC1051 proteorhodopsin gene, partial cds 87% EF640908.1 Bacterium HTCC7214 putative proteorhodopsin gene, partial cds 93% EF616634.1 SAR07 162 Bacterium HTCC7216 putative proteorhodopsin gene, partial cds 92% EF616635.1 Bacterium HTCC7211 putative proteorhodopsin gene, partial cds 92% EF616633.1
Table 6| Results from our PR-based BLAST search to look at similarities between PR sequences in our strains and PR sequences of other cultured sample sequences using online BLAST search tool.
47 3.2.8 Growth Profile of the SAR11 Isolate- SAR08
1.00E+08
1.00E+07
# of cells 1.00E+06
1.00E+05 0 5 10 15 20 25 30 35 40 45 50
Time (days)
Figure 9| Scatter-plot of SAR08 population over 50 days
We monitored the growth of strain SAR08 for a period of more than 40 days and recorded their abundance on a scatter-plot as seen in Figure 9. We determined the doubling time (G) for strain SAR08 using the formula below:
G = t
3.3 log (b / B)
where t is the time interval in days; and B and b is number of bacteria at the
beginning and end of the time interval respectively. Our calculation predicted
the doubling time of SAR08 growing in AMS1 media to be 24 hours. 48 3.3 Identification of Isolates Grown In Nitrogen- deficient Media
3.3.1 Growth Behaviors
Total light/dark Light Dark with no growth 10 cells/ml (n= 288) 24 wells 14 wells 538 wells 100 cells/ml (n= 288) 97 wells 100 wells 379 wells 400 ul/ 100 ml (n= 6) 6 flasks 6 flasks No flasks
Table 7| Number of strains exhibiting growth after 2 weeks of growing in nitrogen-deficient media. n refers to the total number of wells or flasks per cell density, in light or dark.
We detected potential signs of growth in all of the cell cultures that were growing in nitrogen-deficient media after just 2 weeks of growing in the lab.
The number of strains exhibiting growth was relative to the initial cell densities of the cultures. All of the flask cultures were growing in density, while the number of strains from 100 cells/ml concentration was higher than those that started out with 10 cells/ml concentration (Table 7). However, the effects of light on the number of strains obtained were not apparent when the cultures were seeded at too high concentrations.
Therefore, we decided to compare the growths of cultures in 10 cells/ml concentrations only. We found that the number of strains that grew in light was more than those that grew in the dark (Table 7). In these cultures, we 49 observed that 24 strains exhibiting growth in the light and 14 strains exhibiting growth in the dark after just 2 weeks of growing in the lab.
Condition after transfer Condition Total with no before Light Dark growth transfer Light (n=24) 16 flasks 16 flasks 16 flasks
Dark (n=14) 13 flasks 13 flasks 2 flasks
Table 8| Number of strains exhibiting growth after second transfer to both light and dark conditions. n refers to the total number of flasks per growth condition before and after transfer
To verify whether light really have an influences on the number of strains obtained,, we followed up this analysis by transferring all the 38 cultures and subject them to both light and dark again. This means, cultures that were initially grown in light were transferred and subsequently grown in both light and dark; likewise for those that were initially grown in the dark. From this test, we found that previously ‘dark cultures’ had more strains having growth than the previously ‘light cultures’ (Figure 8). Hence, we could not justify that light was an influence for growth in these cultures.. 50
Total light/dark Light Dark with no growth With vitamins (n= 288) 24 wells 14 wells 538 wells W/out vitamins (n= 288) 31 wells 23 wells 522 wells
Table 9| Number of strains exhibiting growth in media with and without vitamins. n refers to the total number of wells containing media with or without vitamins, growing in light or dark conditions
Since the vitamins used in the media contained traces of nitrogen, we also investigated the effects of including and excluding the vitamins from the growth media. We obtained more strains from media that does not contain the vitamins (Table 9). Additionally, light also seemed to stimulate more growth in density among these strains.
51 3.3.2 Total Isolates Identified
Taxonomy based on SILVA/RDP Contig Strain Growth Max Total Query E- Maximum Accession length Top Blast Hits Phylum Class Order Family Genus ID condition score score cover value identity no. (bp) Parvibaculum indicum strain P31 16S ribosomal RNA gene, partial sequence 2361 2361 99% 0 99% FJ182044.1 Parvibaculum sp. psc10 16S ribosomal RNA gene, partial sequence 2294 2294 99% 0 99% EU930870.2 N23 Light 1292 Proteobacteria Alphaproteobacteria Rhizobiales Rhodobiaceae Parvibaculum Parvibaculum hydrocarboniclasticum strain EPR92 16S ribosomal RNA gene, partial sequence 2257 2257 98% 0 99% GU574708.1 Marinobacter salsuginis strain KJ-W11 16S ribosomal RNA gene, partial sequence 2215 2215 98% 0 98% JQ799108.1 N35 Dark 1281 Marinobacter salsuginis strain K-W5 16S ribosomal RNA gene, partial Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Marinobacter sequence 2215 2215 98% 0 98% JQ799060.1 Marinobacter sp. M4 gene for 16S rRNA, partial sequence 2215 2215 98% 0 98% AB435571.1 Parvibaculum indicum strain P31 16S ribosomal RNA gene, partial sequence 2200 2200 95% 0 99% FJ182044.1 Parvibaculum sp. psc10 16S ribosomal RNA gene, partial sequence 2134 2134 95% 0 98% EU930870.2 N37 Dark 1281 Proteobacteria Alphaproteobacteria Rhizobiales Rhodobiaceae Parvibaculum Parvibaculum hydrocarboniclasticum strain EPR92 16S ribosomal RNA gene, partial sequence 2117 2117 95% 0 98% GU574708.1 Parvibaculum indicum strain P31 16S ribosomal RNA gene, partial sequence 2239 2239 95% 0 99% FJ182044.1 Parvibaculum sp. psc10 16S ribosomal RNA gene, partial sequence 2172 2172 95% 0 99% EU930870.2 Proteobacteria Alphaproteobacteria Rhodobiaceae N38 Dark 1276 Parvibaculum hydrocarboniclasticum strain EPR92 16S ribosomal RNA gene, Rhizobiales Parvibaculum partial sequence 2156 2156 95% 0 99% GU574708.1 Table 10| List of strains obtained from cell cultures grown in nitrogen-deficient media.
We extracted DNA from all 38, 10-cells/ml isolates that were grown in light
and dark. 4 of these were successfully amplified and identified via their 16S
rRNA gene sequence (Table 8). Isolate N23 was grown under lighted
condition, while isolates N35, N37 and N38 were grown in the dark. The
assembled contigs from these cultured isolates were around 1200 bp long.
Results from our BLAST search suggest that N35 belonged to the genus
Parvibaculum while the rest of the isolates are most likely from the genus
Marinobacter. So far, we have have not detected the nifH gene in any of our
cultured isolates.
52 4. Discussion
4.1 Effects of Media, Sampling Depth and Cell Densities on Cell Culture Growth
The dilution-to-extinction method was successful in yielding isolates of heterotrophic bacteria from the Red Sea. Combining this method of culturing with the use of LNHM yield many strains of bacteria, especially for surface water cultures. When grown in the lab, cell cultures from deeper ocean layer not only took a longer time to grow, but the number of strains obtained was not comparable as the number obtained from surface water. We think the inadequacy to identify strains from 500 m depth was partly due to this. We reasoned that the failure to amplify the targeted genes were perhaps due to lower abundance of bacteria in the deep sea as compared to the surface, following the domination of archaeal community with increasing ocean depths
(Edward F DeLong et al. 2006a). We first verified this by screening for archaeal genes using PCR but the outcome was negative. In retrospect, we optimized the parameters of the 16S rRNA PCR to eventually obtain sufficient, detectable PCR products that could later be quantified, sequenced and identified (Figure 5).
Although the number of strains identified from LNHM cultures were generally more, and the cell densities increased within a shorter period as compared to those that grew in AMS1, all of the SAR11 strains that were isolated from this study were all from AMS1 cultures. At this point, we are not certain why these strains had a strong preference for AMS1. Although both media were made from autoclaved seawater/water and contained glycine and methionine, AMS1 53 was formulated to include pyruvate or pyruvate precursors following the unusual metabolic requirement of C. Pelagibacter ubique (H James Tripp
2013). As proven by Carini et al (2013), glycine and methionine alone, without pyruvate is insufficient to sustain growth of SAR11 as the cells were seen to have incomplete cell division when deprived of pyruvate. Additionally, AMS1 also included glycolate as an alternative to glycine, enabling growth to be restored in case glycine is overused by other organisms. We viewed the media (AMS1) as an excellent gateway for testing out the physiology of the isolates in the future. Given the versatility of AMS1 and the greater flexibility to control every content of the media, one could easily re-formulate the media and test out physiological hypotheses that may have been previously derived from genome information in culture-independent studies. For example, the rate at which methionine is taken up by two isolated strains of SAR11 may be compared by monitoring their growth performance both when the nutrient is first excluded, and when later added into the growing media prepared.
In this study, we adopted a similar controlled set up when attempting to isolate nitrogen fixers that might be harboring the euphotic zone of the Red Sea. We hoped that the culture media that was designed for this experiment would be able to stimulate the growth of a consortium of bacteria that have developed strategies to obtain nitrogen independently. Biological nitrogen fixation is a very energy-consuming reaction that spends 16 moles of ATP for the conversion just one mole of dinitrogen gas (DL Kirchman 2008). Hence, we think that bacteria that are involved in nitrogen fixation in the oligotrophic Red
Sea would have developed special mechanisms that could source for an alternative energy source. Therefore, we think that the presence of PR in the 54 nitrogen-fixing commmunity in the Red Sea could be probable. If the survival of these organisms truly depends on energy generation by PR, the high annual solar irradiance in the Red Sea will indefinitely be ideal for them. One way to verify this in the lab is to monitor the growth of the cell cultures under light and dark conditions while growing them in a nitrogen-deficient media. In our cell cultures, we seeded these cells at high concentration to mimic their assembly in the natural settings where nitrogen fixers are generally found in a mixed-species community, like those in soils (Deacon). Our preliminary analysis found that light is in fact an important driver for growth in these cell cultures. However, subsequent transfer of these previously ‘light cultures’ did not justify our first observation. Nonetheless, it is good to note that aerobic, heterotrophic bacteria that fixes nitrogen in marine ecosystems is not many.
Bacteria that performs this function are often Cyanobacteria, which fixes nitrogen in the presence of light. Therefore, further work to identity this isolates will be necessary as it could potentially lead to the discovery of novel strains of marine bacteria that fixes nitrogen in the dark. A quick step to this is to check whether or not these cells are Cyanobacteria using fluorescent microscopy. Further analyses to verify whether all isolates obtained thus far are truly nitrogen-fixers and whether or not they posses PR are still in progress.
55 4.2 Isolation of DNA from Cell Cultures
The primary challenge when studying heterotrophic bacteria cultivated via the dilution-to extinction method is in the inability to directly detect growth. Unlike visible plaques on an agar plate, or significant changes in turbidity/colour in liquid media, cell cultures grown via this method usually display an undisturbed appearance. Although flow cytometry is a really fast and convenient way to detect cell growth, it is not able give a preliminary indication of the isolate’s identity. Therefore, extraction, gene amplification and sequencing of the isolate’s DNA are required to eventually identify them.
Often, we found that high cell counts displayed by Guava is not relative to the quality and quantity of the DNA we get afterwards. For example, a sample with a low count on the Guava may amplify really well and produce good quality sequences, while a sample with a high count may exhibit otherwise.
We think that the issue could possibly lie on the DNA isolation protocol that was used. Although the bulk of both methods were similar, the protocol set by
Connon and Giovannoni (2002) included freezing and thawing of the samples as a means to lyse cells. This was performed briefly on our deep-sea isolates but the effect was not apparent. In the future, other methods of extracting better quality and amplifiable DNA from these cell cultures need to be explored.
56 4.3 Red Sea Bacteria Isolates and Their Possible Functions
Despite its abundance in the Red Sea, members of the SAR11 clade remained to be hard to cultivate in the lab. By adopting high-throughput dilution-to-extinction technique, we managed to isolate 8 strains of SAR11 members from the Red Sea. Even then, only 1 out of the 8 strains isolated continued growing after transfer to fresh media. To this point, strain SAR08 has had two successful rounds of transfer and we hope to be able to obtain a pure culture of the strain to subsequently proceed with sequencing its whole genome. For future study, SAR08 was stored individually in DMSO and glycerol, and archived in the RSRC culture collection.
Ideally, a high-resolution SEM or TEM image of SAR08 should be captured and compared to known morphologies of the other cultured members of
SAR11 like the isolate HTCC1062 (M S Rappé et al. 2002). Moreover, our isolates, like most SAR11 sequences retrieved from the Red Sea, were classified into subgroup 1a (Figure 7). Comparison based on their ITS sequences further categorized our isolates into phylotype 1a.3 (Figure 8), hinting a possible difference in adaptation or functionality of our strain relative to HTCC1062, which fall under phylotype 1a.1 instead. In fact, phylotypes
1a.1 and 1a.2 were reported to be completely absent from the Red Sea and has been found only in cold, oligotrophic coastal environments (David
Kamanda Ngugi & Stingl 2012; M S Rappé et al. 2002; Mv Brown & Ja
Fuhrman 2005). Ngugi and Stingl (2012) suggested that temperature and nutrient concentrations are most likely the main drivers of speciation for 57 members of SAR11 throughout the globe. Essentially, this was the main reason why our study had instigated culturing of samples from different depths. In that way, we hoped to be able to isolate subgroup or phylotype representations from the upper euphotic (10 m), lower euphotic (100 m), and deep, aphotic zones (500 m) of Red Sea, and help us better understand the effects of temperature and nutrients on their physiology and genetic makeup.
Since our current isolates were retrieved only from a single depth, molecular techniques used to culture, isolate and identify members of SAR11 needs to be improved in order to isolate more strains from the Red Sea in the future.
Along with the 8 strains of SAR11 we isolated, we also obtained supposedly
“less abundant” strains of marine bacteria in the majority of our cultured strains. Most of these isolates are marine Gammaproteobacteria that includes
Alteromonas and Marinobacter (S J Giovannoni & Rappe 2000). It is however not odd to obtain this group of marine bacteria, which are said to be readily culturable and grow at a fast rate. As such, feast and famine within this group of bacteria is also common (DL Kirchman 2008).
Interestingly, we also found an isolate of OM60 among our cultures from 500 m. Like SAR11, taxonomic grouping within the OM60 clade is also vague
(Yan et al. 2009). Two members of this clade (HTCC2148 and HTCC2080) were recently isolated from 10 m sample near to the coast of Sargasso Sea and 16S rRNA-based survey have reported them in many ocean surfaces, but never at >130 m (Thrash et al. 2010; Edward F DeLong et al. 2006b). This thereby makes our 500 m isolate of the OM60 member an imperative if the complexity of clade is to be explored. Unfortunately, sustaining this strain in culture was unsuccessful after just the first transfer. Nonetheless, like our 58 strain, HTCC2148 and HTCC2080 were also previously isolated from LNHM and have been found to posses complete glycolysis and TCA cycles (Thrash et al. 2010).
We also found another unique marine bacteria among our isolate. The novel genus Parvibaculum was obtained in our nitrogen-deficient set-up. Although the first isolated member of this genus, Parvibaculum lavamentivorans DS-1 has been said to posses unique metabolic capabilities that enabled the bacteria to degrade a toxic laundry surfactant- linear alkylbenzenesulfonate
(LAS), DS-1 is not of marine origin (Schleheck et al. 2004).
It seemed rather peculiar why the above “less-abundant” strains are outnumbering the total number of SAR11 isolates we obtained. We think that this might be the result of competition for nutrients among the bacteria community within the culture. On top of these strains growing much faster, it might also be possible that SAR11 members in the Red Sea grew much slower than we expected. Therefore, we suggest that the screening for growth be extended to longer than 12 weeks in order to increase our chances of getting more SAR11 isolates. To this time, the growth media should constantly be replenished to ensure that nutrient availability is not compromised.
59 5. Conclusion and Future Directions
Here I report the cultivation and isolation of marine heterotrophic bacteria from the Red Sea using high-throughput, dilution-to-extinction culturing. The result of this study revealed that heterotrophic bacteria from the Red Sea grew faster in LNHM than in AMS1 and that SAR11 members in particular, grew only in AMS1. We showed that members of SAR11 from deep ocean zone remained recalcitrant to cultivation and further optimization of DNA isolation strategies and PCR protocols should be looked into.
Along with the 8 strains of SAR11 that we isolated, numerous readily culturable, yet “less abundant” strains of marine bacteria were obtained.
Among them, we found two novel strains of bacteria where further studies may be channeled to in the future. Members of the SAR11 clade that were isolated from this study were classified under subgroup 1a and phylotype 1a.3 akin to other SAR11 sequences previously retrieved from culture-independent surveys of the Red Sea. Future work to cultivate SAR11 in the lab should look into a system that will provide nutrients at a continuous rate such as the use of a chemostat. In this way, the rate of growth of cells in the culture may be controlled opening avenues to future transcriptomic studies of SAR11 in the Red Sea.
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Appendices
68 Appendix I: Physical ocean profiles at time of sampling (10 November 2012)
69 Appendix II: New England BioLabs® 2-Log DNA ladder used as indicator for band positions in all agarose gel used in this study
Appendix III: List of all isolates obtained from this study based on 16S rRNA sequence BLAST results (N.B. Sample names are labeled to indicate: growth media/ cell density/ depth/ well on microtitre plate)
Taxonomy based on SILVA/RDP
SAR11 Contig Max Total Query E- Maximum Accession Sample isolate length Top Blast Hits Phylum Class Order Family Genus score score cover value identity no. ID (bp) Alcanivorax sp. JAM-GA18 gene for 16S rRNA, partial sequence 2588 2588 100% 0 100% AB526341.1 L5B-500M-F12 - 1411 Alcanivorax venustensis strain PR54-10 16S ribosomal RNA gene, partial sequence 2588 2588 100% 0 100% EU440996.1 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Alcanivorax sp. IW8-2CT gene for 16S rRNA, partial sequence 2588 2588 100% 0 100% AB305315.1 Alcanivorax sp. IW6-2CT gene for 16S rRNA, partial sequence 2584 2584 99% 0 100% AB305314.1 A5B-500M-C8 - 1409 Uncultured Alcanivorax sp. clone II21-30 16S ribosomal RNA, partial sequence 2582 2582 99% 0 99% GU108546.1 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Uncultured Oceanospirillales bacterium clone PRTAB7641 small subunit ribosomal RNA gene, partial sequence 2580 2580 100% 0 99% HM798602.1 Alcanivorax sp. TK-23 16S ribosomal RNA gene, partial sequence 2490 2490 100% 0 99% KC161579.2 L5D-500M-C4 - 1408 Alcanivorax sp. RHS-str.303 partial 16S rRNA gene, strain RHS-str.303 2490 2490 100% 0 99% HE586882.1 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Uncultured Oceanospirillales bacterium clone PRTAB7641 small subunit ribosomal RNA gene, partial sequence 2490 2490 100% 0 99% HM798602.1 Alcanivorax sp. IW6-2CT gene for 16S rRNA, partial sequence 2586 2586 99% 0 100% AB305314.1 A5B-500M-C9 - 1408 Uncultured Alcanivorax sp. clone II21-30 16S ribosomal RNA, partial sequence 2584 2584 99% 0 99% GU108546.1 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Uncultured Oceanospirillales bacterium clone PRTAB7641 small subunit ribosomal RNA gene, partial sequence 2582 2582 100% 0 99% HM798602.1 Alcanivorax sp. TK-23 16S ribosomal RNA gene, partial sequence 2486 2486 100% 0 99% KC161579.2 A5B-500M-D5 - 1407 Alcanivorax sp. RHS-str.303 partial 16S rRNA gene, strain RHS-str.303 2486 2486 100% 0 99% HE586882.1 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Uncultured Oceanospirillales bacterium clone PRTAB7641 small subunit ribosomal RNA gene, partial sequence 2486 2486 100% 0 99% HM798602.1 Uncultured Alcanivorax sp. clone II21-30 16S ribosomal RNA, partial sequence 2590 2590 99% 0 99% GU108546.1 A1C-500M-G3 - 1407 Alcanivorax sp. IW6-2CT gene for 16S rRNA, partial sequence 2590 2590 99% 0 100% AB305314.1 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Uncultured Oceanospirillales bacterium clone PRTAB7641 small subunit ribosomal RNA gene, partial sequence 2588 2588 99% 0 99% HM798602.1 Alcanivorax sp. IW6-2CT gene for 16S rRNA, partial sequence 2586 2586 99% 0 100% AB305314.1 A5B-500M-D10 - 1406 Alcanivorax sp. RHS-str.303 partial 16S rRNA gene, strain RHS-str.303 2584 2584 99% 0 99% HE586882.1 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Uncultured Oceanospirillales bacterium clone PRTAB7641 small subunit ribosomal RNA gene, partial sequence 2584 2584 100% 0 99% HM798602.1 Uncultured Oceanospirillales bacterium clone PRTAB7641 small subunit ribosomal RNA gene, partial sequence 2590 2590 100% 0 99% HM798602.1 A5B-500M-C10 - 1406 Alcanivorax sp. JAM-GA18 gene for 16S rRNA, partial sequence 2590 2590 100% 0 99% AB526341.1 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Alcanivorax venustensis strain PR54-10 16S ribosomal RNA gene, partial sequence 2590 2590 100% 0 99% EU440996.1 Uncultured Alcanivorax sp. clone II21-30 16S ribosomal RNA, partial sequence 2590 2590 100% 0 99% GU108546.1 A1A-500M-H5 - 1406 Alcanivorax sp. IW6-2CT gene for 16S rRNA, partial sequence 2590 2590 99% 0 100% AB305314.1 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Uncultured Oceanospirillales bacterium clone PRTAB7641 small subunit ribosomal RNA gene, partial sequence 2586 2586 99% 0 99% HM798602.1 Uncultured Alcanivorax sp. clone II21-30 16S ribosomal RNA, partial sequence 2590 2590 100% 0 99% GU108546.1 L5D-500M-B9 - 1405 Alcanivorax sp. IW6-2CT gene for 16S rRNA, partial sequence 2590 2590 99% 0 100% AB305314.1 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Uncultured Oceanospirillales bacterium clone PRTAB7641 small subunit ribosomal RNA gene, partial sequence 2586 2586 99% 0 99% HM798602.1 Alcanivorax sp. IW6-2CT gene for 16S rRNA, partial sequence 2551 2551 100% 0 100% AB305314.1 L10A-500M-C10 - 1401 Alcanivorax sp. RHS-str.303 partial 16S rRNA gene, strain RHS-str.303 2545 2545 100% 0 99% HE586882.1 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Uncultured Oceanospirillales bacterium clone PRTAB7641 small subunit ribosomal RNA gene, partial sequence 2545 2545 100% 0 99% HM798602.1 Halomonas aquamarina gene for 16S rRNA, partial sequence, strain: NBRC 101894 2534 2534 100% 0 99% AB681582.1
L5C-500M-C4 - 1400 Halomonas aquamarina gene for 16S rRNA, partial sequence, strain: NBRC 101895 >dbj|AB681586.1| Halomonas aquamarina gene for 16S rRNA, partial sequence, strain: NBRC 101898 2534 2534 100% 0 99% AB681583.1 Proteobacteria Gammaproteobacteria Oceanospirillales Halomonadaceae Halomonas Bacterium L188S.607 16S ribosomal RNA gene, partial sequence 2534 2534 100% 0 99% EU935272.1 Uncultured bacterium clone VH-PA7-26 16S ribosomal RNA gene, partial sequence 2193 2193 98% 0 96% EF379619.1 A5A-500M-A4 - 1400 Gamma proteobacterium 25 gene for 16S rRNA, partial sequence 2150 2150 99% 0 96% AB435573.1 Proteobacteria Gammaproteobacteria Oceanospirillales Oceanospirillaceae Litoribacillus peritrichatus strain JYr12 16S ribosomal RNA gene, partial sequence 2122 2122 99% 0 95% GU391221.2
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Taxonomy based on SILVA/RDP
SAR11 Contig Max Total Query E- Maximum Accession Sample isolate length Top Blast Hits Phylum Class Order Family Genus score score cover value identity no. ID (bp) Alcanivorax sp. IW6-2CT gene for 16S rRNA, partial sequence 2562 2562 100% 0 100% AB305314.1 L10A-500M-E4 - 1397 Uncultured Oceanospirillales bacterium clone PRTAB7641 small subunit ribosomal RNA gene, partial sequence 2556 2556 100% 0 99% HM798602.1 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Alcanivorax sp. JAM-GA18 gene for 16S rRNA, partial sequence 2556 2556 100% 0 99% AB526341.1 Alteromonadales bacterium 3tb13 16S ribosomal RNA gene, partial sequence 2523 2523 98% 0 99% FJ952789.1 L1A-100M-A9 - 1396 Uncultured bacterium clone Past_I11 16S ribosomal RNA gene, partial sequence 2453 2453 99% 0 99% GU119170.1 Proteobacteria Gammaproteobacteria Oceanospirillales Oceanospirillaceae Neptuniibacter Neptuniibacter sp. KD355-21 16S ribosomal RNA gene, partial sequence 2218 2218 99% 0 95% FJ906746.1 Alcanivorax sp. IW6-2CT gene for 16S rRNA, partial sequence 2580 2580 100% 0 100% AB305314.1 L5D-500M-E2 - 1395 Uncultured Oceanospirillales bacterium clone PRTAB7641 small subunit ribosomal RNA gene, partial sequence 2575 2575 100% 0 99% HM798602.1 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Alcanivorax sp. JAM-GA18 gene for 16S rRNA, partial sequence 2575 2575 100% 0 99% AB526341.1 Alcanivorax sp. IW6-2CT gene for 16S rRNA, partial sequence 2531 2531 100% 0 100% AB305314.1 L5D-500M-A8 - 1395 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Uncultured Oceanospirillales bacterium clone PRTAB7641 small subunit ribosomal RNA gene, partial sequence 2525 2525 100% 0 99% HM798602.1 Uncultured bacterium clone RESET_113B10 16S ribosomal RNA gene, partial sequence 2401 2401 100% 0 98% JN873946.1 L10A-500M-G6 - 1384 Uncultured bacterium clone RESET_213D05 16S ribosomal RNA gene, partial sequence 2386 2386 100% 0 98% JN874357.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae OM60(NOR5) clade Halioglobus japonicus gene for 16S rRNA, partial sequence 2331 2331 96% 0 98% AB602427.1 2130 2130 99% 0 95% KC161579.2 Alcanivorax sp. TK-23 16S ribosomal RNA gene, partial sequence L5D-500M-C7 - 1366 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Alcanivorax sp. Ar-25B 16S ribosomal RNA gene, partial sequence 2130 2130 99% 0 95% JX844441.1 Alcanivorax sp. RHS-str.303 partial 16S rRNA gene, strain RHS-str.303 2130 2130 99% 0 95% HE586882.1 Alcanivorax sp. TK-23 16S ribosomal RNA gene, partial sequence 2423 2423 99% 0 100% KC161579.2 L5C-500M-E9 - 1366 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Uncultured Oceanospirillales bacterium clone PRTAB7641 small subunit ribosomal RNA gene, partial sequence 2423 2423 99% 0 100% HM798602.1 Alteromonas macleodii str. 'Balearic Sea AD45' strain Balearic Sea AD45 16S ribosomal RNA, complete sequence 2475 2475 100% 0 99% NR_074797.1 L5A-500M-H4 - 1363 Alteromonas macleodii str. 'Balearic Sea AD45', complete genome 2475 12350 100% 0 99% CP003873.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Alteromonas Alteromonas macleodii str. 'Black Sea 11', complete genome 2475 12367 100% 0 99% CP003845.1 Marinobacter salsuginis strain K-W5 16S ribosomal RNA gene, partial sequence 2501 2501 99% 0 99% JQ799060.1 L5A-10M-A1 - 1360 Uncultured bacterium clone AND_GV0309_C0.25.2_3S3 16S ribosomal RNA gene, partial sequence 2501 2501 99% 0 99% JQ032446.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Uncultured bacterium clone 2216_GV0309_IH8.2_4S1 16S ribosomal RNA gene, partial sequence 2501 2501 99% 0 99% JQ032138.1 Marinobacter salsuginis strain K-W5 16S ribosomal RNA gene, partial sequence 2501 2501 100% 0 99% JQ799060.1
L1A-10M-C11 - 1357 Uncultured bacterium clone AND_GV0309_C0.25.2_3S3 16S ribosomal RNA gene, partial sequence 2501 2501 100% 0 99% JQ032446.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae
Uncultured bacterium clone 2216_GV0309_IH8.2_4S1 16S ribosomal RNA gene, partial sequence 2501 2501 100% 0 99% JQ032138.1 L5D-500M-D12 - 1354 Sphingomonas sp. NBRC 101716 gene for 16S rRNA, partial sequence 2490 2490 99% 0 99% AB681540.1 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingobium Erythrobacter sp. H301 16S ribosomal RNA gene, partial sequence 2370 2370 100% 0 99% HQ622544.1 L5B-500M-C4 - 1352 Uncultured Sphingomonadales bacterium clone PRTBB8673 small subunit ribosomal RNA gene, partial sequence 2370 2370 100% 0 99% HM799069.1 Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacteraceae Erythrobacter Uncultured Sphingomonadales bacterium clone PRTBB8544 small subunit ribosomal RNA gene, partial sequence 2370 2370 100% 0 99% HM798971.1 A1C-500M-D6 - 1352 Sphingomonas sp. NBRC 101716 gene for 16S rRNA, partial sequence 2490 2490 99% 0 99% AB681540.1 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingobium Sphingomonas sp. NBRC 101716 gene for 16S rRNA, partial sequence 2490 2490 100% 0 99% AB681540.1 A5A-500M-D3 - 1351 Sphingomonas sp. Zp1 16S ribosomal RNA gene, partial sequence 2484 2484 100% 0 99% DQ659593.1 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingobium Uncultured Sphingomonadales bacterium clone PRTAB7811 small subunit ribosomal RNA gene, partial sequence 2462 2462 100% 0 99% HM798716.1 Marinobacter salsuginis strain K-W5 16S ribosomal RNA gene, partial sequence 2486 2486 100% 0 99% JQ799060.1 L1A-10M-E5 - 1350 Uncultured bacterium clone AND_GV0309_C0.25.2_3S3 16S ribosomal RNA gene, partial sequence 2486 2486 100% 0 99% JQ032446.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Uncultured bacterium clone 2216_GV0309_IH8.2_4S1 16S ribosomal RNA gene, partial sequence 2486 2486 100% 0 99% JQ032138.1 Marinobacter salsuginis strain KJ-W11 16S ribosomal RNA gene, partial sequence 2484 2484 100% 0 99% JQ799108.1 A5A-10M-G2 - 1348 Marinobacter salsuginis strain K-W5 16S ribosomal RNA gene, partial sequence 2484 2484 100% 0 99% JQ799060.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Marinobacter sp. M4 gene for 16S rRNA, partial sequence 2484 2484 100% 0 99% AB435571.1
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Taxonomy based on SILVA/RDP
SAR11 Contig Max Total Query E- Maximum Accession Sample isolate length Top Blast Hits Phylum Class Order Family Genus score score cover value identity no. ID (bp) Erythrobacter citreus strain LAMA 967 16S ribosomal RNA gene, partial sequence 2403 2403 99% 0 100% KC583244.1
L5B-500M-B9 - 1345 Uncultured Sphingomonadales bacterium clone PRTBB8673 small subunit ribosomal RNA gene, partial sequence 2403 2403 99% 0 100% HM799069.1 Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacteraceae Erythrobacter Uncultured bacterium clone CE2-DCM-13 16S ribosomal RNA gene, partial sequence 2403 2403 99% 0 100% GU061599.1 Erythrobacter sp. JA748 partial 16S rRNA gene, strain JA748, isolate A3 >emb|HE805095.1| Erythrobacter sp. JC136 partial 16S rRNA gene, strain JC136, isolate A3 2459 2459 100% 0 100% HE680095.1 L10A-500M-E3 - 1345 Bacterium BW3PhG11 16S ribosomal RNA gene, partial sequence 2453 2453 100% 0 99% KC012857.1 Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacteraceae Erythrobacter Uncultured bacterium clone CEP-5m-52 16S ribosomal RNA gene, partial sequence 2453 2453 100% 0 99% GU061698.1 2446 2446 99% 0 100% HQ622544.1 L1A-500M-F9 - 1344 Erythrobacter sp. H301 16S ribosomal RNA gene, partial sequence Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacteraceae Erythrobacter Uncultured Sphingomonadales bacterium clone PRTBB8673 small subunit ribosomal RNA gene, partial sequence 2446 2446 99% 0 100% HM799069.1
Erythrobacter sp. JA748 partial 16S rRNA gene, strain JA748, isolate A3 >emb|HE805095.1| Erythrobacter sp. JC136 partial 16S rRNA gene, strain JC136, isolate A3 2459 2459 100% 0 100% HE680095.1 L10A-500M-D3 - 1344 Bacterium BW3PhG11 16S ribosomal RNA gene, partial sequence 2453 2453 100% 0 99% KC012857.1 Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacteraceae Erythrobacter Bacterium 2H301 16S ribosomal RNA gene, partial sequence 2453 2453 100% 0 99% JF411501.1 Erythrobacter sp. H301 16S ribosomal RNA gene, partial sequence 2444 2444 100% 0 100% HQ622544.1 A1D-500M-B12 - 1344 Uncultured Sphingomonadales bacterium clone PRTBB8673 small subunit ribosomal RNA gene, partial sequence 2444 2444 100% 0 100% HM799069.1 Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacteraceae Erythrobacter Uncultured Sphingomonadales bacterium clone PRTBB8544 small subunit ribosomal RNA gene, partial sequence 2444 2444 100% 0 100% HM798971.1 Erythrobacter flavus strain LAMA 944 16S ribosomal RNA gene, partial sequence 2444 2444 100% 0 100% KC583223.1 L10A-500M-C8 - 1343 Bacterium BW3PhG11 16S ribosomal RNA gene, partial sequence 2444 2444 100% 0 100% KC012857.1 Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacteraceae Erythrobacter Erythrobacter sp. JA748 partial 16S rRNA gene, strain JA748, isolate A3 >emb|HE805095.1| Erythrobacter sp. JC136 partial 16S rRNA gene, strain JC136, isolate A3 2444 2444 100% 0 100% HE680095.1 Erythrobacter sp. H301 16S ribosomal RNA gene, partial sequence 2414 2414 100% 0 99% HQ622544.1 L5D-500M-B8 - 1342 Uncultured Sphingomonadales bacterium clone PRTBB8673 small subunit ribosomal RNA gene, partial sequence 2414 2414 100% 0 99% HM799069.1 Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacteraceae Erythrobacter Uncultured organism clone ctg_NISA313 16S ribosomal RNA gene, partial sequence 2414 2414 100% 0 99% DQ396256.1 Uncultured bacterium clone AND_GV0508_IH8.1_1G5 16S ribosomal RNA gene, partial sequence 2446 2446 100% 0 99% JQ032618.1 A5B-10M-D8 - 1339 Uncultured bacterium clone AND_GV0508_IH4.1_1G5 16S ribosomal RNA gene, partial sequence 2446 2446 100% 0 99% JQ032614.1 Bacteroidetes Cytophagia Cytophagales Flammeovirgaceae Roseivirga Uncultured bacterium clone AND_GV0309_IH6.1_1S7 16S ribosomal RNA gene, partial sequence 2446 2446 100% 0 99% JQ032559.1 Uncultured bacterium clone AND_GV0508_IH8.1_2G0 16S ribosomal RNA gene, partial sequence 2457 2457 100% 0 99% JQ032581.1 A1A-10M-E11 - 1336 Thalassospira sp. ZUMI 95 gene for 16S rRNA, partial sequence 2457 2457 100% 0 99% AB548215.1 Proteobacteria Alphaproteobacteria Rhodospirillales Rhodospirillaceae Thalassospira Rhodospirillaceae bacterium EZ54 16S ribosomal RNA gene, partial sequence 2457 2457 100% 0 99% EU704115.1 Uncultured bacterium clone RESET_18C11 16S ribosomal RNA gene, partial sequence 2442 2442 99% 0 99% JN874120.1 A1C-500M-D9 - 1338 Bacterium 1H117 16S ribosomal RNA gene, partial sequence 2409 2409 99% 0 99% JF411456.1 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae Shimia Rhodobacteraceae bacterium 1tc1 16S ribosomal RNA gene, partial sequence 2374 2374 98% 0 99% FJ952830.1 Erythrobacter sp. H301 16S ribosomal RNA gene, partial sequence 2274 2274 99% 0 98% HQ622544.1 L10A-500M-G2 - 1331 Erythrobacter sp. PaH2.09b 16S ribosomal RNA gene, partial sequence 2274 2274 99% 0 98% GQ391951. Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacteraceae Erythrobacter Erythrobacter citreus strain PR52-9 16S ribosomal RNA gene, partial sequence 2274 2274 99% 0 98% EU440970.1 Alcanivorax sp. 2LA10 gene for 16S rRNA, partial sequence 2447 2447 100% 0 99% AB435644.1 A5A-10M-B4 - 1328 Alcanivorax sp. 2PR511-6 16S ribosomal RNA gene, partial sequence 2447 2447 100% 0 99% EU440954.1 Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Alcanivorax sp. CBF L53 gene for 16S rRNA, partial sequence 2447 2447 100% 0 99% AB166953.1 Alteromonas sp. NJSS10 16S ribosomal RNA gene, partial sequence 2447 2447 100% 0 100% EF061411.1 L1A-100M-F8 - 1325 Alteromonas sp. JZ11IS72 16S ribosomal RNA gene, partial sequence 2446 2446 99% 0 100% KC429939.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Alteromonas Alteromonas macleodii str. 'Balearic Sea AD45' strain Balearic Sea AD45 16S ribosomal RNA, complete sequence 2446 2446 99% 0 100% NR_074797.1 Uncultured bacterium clone RESET_18C11 16S ribosomal RNA gene, partial sequence 2414 2414 100% 0 99% JN874120.1 L5A-500M-H1 - 1325 Bacterium 1H117 16S ribosomal RNA gene, partial sequence 2381 2381 100% 0 99% JF411456.1 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae Shimia Rhodobacteraceae bacterium 1tc1 16S ribosomal RNA gene, partial sequence 2372 2372 99% 0 99% FJ952830.1
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Taxonomy based on SILVA/RDP
SAR11 Contig Max Total Query E- Maximum Accession Sample isolate length Top Blast Hits Phylum Class Order Family Genus score score cover value identity no. ID (bp) Uncultured bacterium clone RESET_18C11 16S ribosomal RNA gene, partial sequence 2405 2405 100% 0 99% JN874120.1 L1B-500M-G7 - 1312 Bacterium 1H117 16S ribosomal RNA gene, partial sequence 2372 2372 100% 0 99% JF411456.1 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae Shimia Rhodobacteraceae bacterium 1tc1 16S ribosomal RNA gene, partial sequence 2364 2364 99% 0 99% FJ952830.1 Uncultured bacterium clone RESET_18C11 16S ribosomal RNA gene, partial sequence 2289 2289 99% 0 99% JN874120.1 L10A-500M-G1 - 1308 Rhodobacteraceae bacterium 1tc1 16S ribosomal RNA gene, partial sequence 2266 2266 99% 0 99% FJ952830.1 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae Shimia Shimia marina strain CL-TA03 16S ribosomal RNA, partial sequence >gb|AY962292.1| Shimia marina strain CL-TA03 16S ribosomal RNA gene, partial sequence 2261 2261 99% 0 99% NR_043300.1 Marinobacter salsuginis strain KJ-W11 16S ribosomal RNA gene, partial sequence 2366 2366 100% 0 99% JQ799108.1 L1A-10M-D11 - 1290 Uncultured bacterium clone AND_GV0309_C0.25.2_3S3 16S ribosomal RNA gene, partial sequence 2366 2366 100% 0 99% JQ032446.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Marinobacter Marinobacter sp. SCSWB23 16S ribosomal RNA gene, partial sequence 2366 2366 100% 0 99% FJ461441.1 Uncultured bacterium clone 6C233049 16S ribosomal RNA gene, partial sequence 2357 2357 100% 0 99% EU805066.1 A5B-10M-A6 SAR06 1288 Uncultured bacterium clone 6C232399 16S ribosomal RNA gene, partial sequence 2357 2357 100% 0 99% EU804468.1 Proteobacteria Alphaproteobacteria SAR11 clade Surface 1 Uncultured bacterium clone 6C232137 16S ribosomal RNA gene, partial sequence 2357 2357 100% 0 99% EU804237.1 Uncultured bacterium clone 6C232398 16S ribosomal RNA gene, partial sequence 2366 2366 99% 0 100% EU804467.1 A5C-10M-D5 SAR07 1282 Uncultured bacterium clone S23_1135 16S ribosomal RNA gene, partial sequence 2366 2366 99% 0 100% EF573036.1 Proteobacteria Alphaproteobacteria SAR11 clade Surface 1 Uncultured bacterium clone A0_20 16S ribosomal RNA gene, partial sequence 2361 2361 99% 0 99% JQ731907.1 Marinobacter salsuginis strain K-W5 16S ribosomal RNA gene, partial sequence 2338 2338 100% 0 99% JQ799060.1 L1B-10M-F1 - 1276 Marinobacter sp. M4 gene for 16S rRNA, partial sequence 2338 2338 100% 0 99% AB435571.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Marinobacter Uncultured bacterium clone AND_GV0309_C0.25.2_3S3 16S ribosomal RNA gene, partial sequence 2338 2338 100% 0 99% JQ032446.1 Rhodovulum sp. KU27F3 gene for 16S rRNA, partial sequence 2348 2348 100% 0 99% AB636145.1 L5A-10M-A2 - 1274 Rhodovulum sp. 1R7 gene for 16S rRNA, partial sequence 2342 2342 100% 0 99% AB435652.1 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae Bacterium BW4SW7 16S ribosomal RNA gene, partial sequence 2165 2165 100% 0 97% KC012896.1 Uncultured SAR11 cluster alpha proteobacterium clone 98 16S ribosomal RNA gene, partial sequence 2335 2335 100% 0 99% JN547477.1 A1A-10M-H6 SAR01 1267 Uncultured bacterium clone 6C232398 16S ribosomal RNA gene, partial sequence 2335 2335 100% 0 99% EU804467.1 Proteobacteria Alphaproteobacteria SAR11 clade Surface 1 Uncultured marine microorganism clone HOT157_25m71 16S ribosomal RNA gene, partial sequence 2329 2329 100% 0 99% JN166160.1 Uncultured bacterium clone 2-21 16S ribosomal RNA gene, partial sequence 2296 2296 100% 0 99% KC424777.1 A1A-100M-H2 - 1262 Propionibacterium acnes KPA171202 strain KPA171202 16S ribosomal RNA, complete sequence 2296 2296 100% 0 99% NR_074675.1 Actinobacteria Actinobacteridae Actinomycetales Propionibacterineae Propionibacteriaceae Propionibacterium acnes C1, complete genome 2296 6889 100% 0 99% CP003877.1 Erythrobacter sp. MON004 16S ribosomal RNA gene, partial sequence 2298 2298 100% 0 99% KF042026.1 L1A-100M-A6 - 1248 Erythrobacter flavus strain LAMA 944 16S ribosomal RNA gene, partial sequence 2298 2298 100% 0 99% KC583223.1 Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacteraceae Erythrobacter Bacterium BW3PhG11 16S ribosomal RNA gene, partial sequence 2298 2298 100% 0 99% KC012857.1 L1C-100M-E6 - 1243 Ruegeria sp. 1444 16S ribosomal RNA gene, partial sequence 2296 2296 100% 0 100% JN679843.1 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae Ruegeria L5B-100M-E4 - 1230 Rhodobacteraceae bacterium 1tc1 16S ribosomal RNA gene, partial sequence 2272 2272 100% 0 100% FJ952830.1 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae Shimia Uncultured bacterium clone 6C232820 16S ribosomal RNA gene, partial sequence 2255 2255 100% 0 99% EU804854.1 A5A-10M-G9 SAR04 1227 Uncultured bacterium clone S23_1707 16S ribosomal RNA gene, partial sequence 2255 2255 100% 0 99% EF573608.1 Proteobacteria Alphaproteobacteria SAR11 clade Surface 1 Uncultured bacterium clone 6C232740 16S ribosomal RNA gene, partial sequence 2250 2250 100% 0 99% EU804779.1 Shimia sp. EF3B-CB115 16S ribosomal RNA gene, partial sequence 2121 2121 99% 0 98% KC545296.1 L10A-500M-G4 - 1204 Shimia marina strain CL-TA03 16S ribosomal RNA, partial sequence >gb|AY962292.1| Shimia marina strain CL-TA03 16S ribosomal RNA gene, partial sequence 2115 2115 99% 0 98% NR_043300.1 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae Shimia Bacterium 1H117 16S ribosomal RNA gene, partial sequence 2117 2117 99% 0 98% JF411456.1 Uncultured bacterium clone FE_2_39 16S ribosomal RNA gene, partial sequence 2174 2174 99% 0 99% KC294764.1 Roseobacter L5A-100M-C3 - 1181 Thalassobius mediterraneus gene for 16S ribosomal RNA, partial sequence, strain: RO7 2141 2141 99% 0 99% AB607866.1 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae clade CHAB-I-5 Thalassobius mediterraneus gene for 16S ribosomal RNA, partial sequence, strain: RO8 2141 2141 99% 0 99% AB607867.1 lineage Uncultured bacterium clone HglFeb00342 16S ribosomal RNA gene, partial sequence 2141 2141 100% 0 99% JX016543.1 A5C-10M-B2 SAR08 1165 Uncultured bacterium clone 6C232734 16S ribosomal RNA gene, partial sequence 2141 2141 100% 0 99% EU804774.1 Proteobacteria Alphaproteobacteria SAR11 clade Surface 1 Uncultured bacterium clone S23_1211 16S ribosomal RNA gene, partial sequence 2141 2141 100% 0 99% EF573112.1 Uncultured bacterium clone 6C232521 16S ribosomal RNA gene, partial sequence 2034 2034 100% 0 99% EU804572.1 A5C-10M-G5 SAR02 1104 Uncultured bacterium clone S23_1210 16S ribosomal RNA gene, partial sequence 2026 2026 100% 0 99% EF573111.1 Proteobacteria Alphaproteobacteria SAR11 clade Surface 1 Unidentified marine bacterioplankton clone P5-1B_69 16S ribosomal RNA gene, partial sequence 2006 2006 100% 0 99% KC002074.1 Uncultured bacterium clone S73_003 16S ribosomal RNA gene, partial sequence 2001 2001 100% 0 99% KC874331.1 A5C-10M-C5 SAR03 1092 Unidentified marine bacterioplankton clone E410B_74 16S ribosomal RNA gene, partial sequence 2001 2001 100% 0 99% KC003205.1 Proteobacteria Alphaproteobacteria SAR11 clade Surface 1 Uncultured bacterium clone 6C233257 16S ribosomal RNA gene, partial sequence 2001 2001 100% 0 99% EU805257.1 Continued on next page… 74 Taxonomy based on SILVA/RDP
SAR11 Contig Max Total Query E- Maximum Accession Sample isolate length Top Blast Hits Phylum Class Order Family Genus score score cover value identity no. ID (bp) Marinobacter sp. U1371-101202-SW223 16S ribosomal RNA gene, partial sequence 665 665 100% 0 99% JQ082177.1 L5A-10M-G7 - 364 Gamma proteobacterium SY258 partial 16S rRNA gene, strain SY258 665 665 100% 0 99% HE589590.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Marinobacter Marinobacter salsuginis strain KUAC3038A cb 2 16S ribosomal RNA gene, partial sequence 665 665 100% 0 99% JN202614.1 Marinobacter sp. U1371-101202-SW223 16S ribosomal RNA gene, partial sequence 619 619 100% 4.E-174 99% JQ082177.1 L5A-10M-G5 - 338 Gamma proteobacterium SY258 partial 16S rRNA gene, strain SY258 619 619 100% 4.E-174 99% HE589590.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Marinobacter Uncultured bacterium clone BJGMM-3s-247 16S ribosomal RNA gene, partial sequence 619 619 100% 4.E-174 99% JQ800976.1 Ruegeria mobilis strain SBU5 16S ribosomal RNA gene, partial sequence 621 621 100% 1.E-174 100% KF052993.1 L5C-100M-C11 - 336 Ruegeria sp. EF3B-B100 16S ribosomal RNA gene, partial sequence 621 621 100% 1.E-174 100% KC545263.1 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae Ruegeria Uncultured Ruegeria sp. clone CF27 16S ribosomal RNA gene, partial sequence 621 621 100% 1.E-174 100% JX523669.1 Erythrobacter sp. MON004 16S ribosomal RNA gene, partial sequence 608 608 100% 9.E-171 100% KF042026.1 L1A-100M-A6 - 329 Erythrobacter flavus strain SCSIO 04225 16S ribosomal RNA gene, partial sequence 608 608 100% 9.E-171 100% KC893982.1 Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacteraceae Unidentified marine bacterioplankton clone P5-3B_88 16S ribosomal RNA gene, partial sequence 608 608 100% 9.E-171 100% KC002211.1 Uncultured bacterium clone BBD-Aug08-6BB-77 16S ribosomal RNA gene, partial sequence 587 587 100% 2.E-164 100% GU472164.1 Roseobacter L5A-100M-F1 - 325 Thalassobius mediterraneus gene for 16S ribosomal RNA, partial sequence, strain: RO7 585 585 99% 7.E-164 100% AB607866.1 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae clade CHAB-I-5 Uncultured Thalassobius sp. clone T534deg56 16S ribosomal RNA gene, partial sequence 585 585 99% 7.E-164 100% JN210864.1 lineage Shimia sp. DW3-2 16S ribosomal RNA gene, partial sequence 575 575 100% 8.E-161 100% JN979553.1 Roseobacter L5A-100M-G6 - 311 Thalassobius mediterraneus gene for 16S ribosomal RNA, partial sequence, strain: RO7 575 575 100% 8.E-161 100% AB607866.1 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae clade CHAB-I-5 Uncultured Thalassobius sp. clone T534deg56 16S ribosomal RNA gene, partial sequence 575 575 100% 8.E-161 100% JN210864.1 lineage Shimia sp. DW3-2 16S ribosomal RNA gene, partial sequence 520 520 100% 4.E-144 98% JN979553.1 Roseobacter L1C-100M-B1 - 297 Thalassobius mediterraneus gene for 16S ribosomal RNA, partial sequence, strain: RO7 520 520 100% 4.E-144 98% AB607866.1 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae clade CHAB-I-5 Uncultured Thalassobius sp. clone DOM07 16S ribosomal RNA gene, partial sequence 520 520 100% 4.E-144 98% HQ012272.1 lineage Uncultured bacterium clone Reef_C09 16S ribosomal RNA gene, partial sequence 510 510 99% 2.E-141 99% GU119315.1 A5A-10M-H6 SAR05 280 Uncultured bacterium clone CEP-5m-89 16S ribosomal RNA gene, partial sequence 510 510 99% 2.E-141 99% GU061710.1 Proteobacteria Alphaproteobacteria SAR11 clade Surface 1 Uncultured bacterium clone 6C233257 16S ribosomal RNA gene, partial sequence 510 510 99% 2.E-141 99% EU805257.1 Uncultured bacterium clone Mi5E10 16S ribosomal RNA gene, partial sequence 479 479 99% 5.E-132 99% HQ837605.1 L5A-100M-H11 - 263 Alteromonas sp. UDC534 16S ribosomal RNA gene, partial sequence 475 475 98% 7.E-131 99% JQ895012.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Alteromonas Bacterium 3D701 16S ribosomal RNA gene, partial sequence 473 473 99% 3.E-130 99% JF411507.1 Rhodovulum sp. AK3 partial 16S rRNA gene, strain AK3 462 462 100% 5.E-127 99% FN995237.1 L5A-10M-C3 - 256 Rhodobacteraceae bacterium enrichment culture clone TBNAP74 16S ribosomal RNA gene, partial sequence 462 462 100% 5.E-127 99% JN166983.1 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae Salipiger sp. JLT2016 16S ribosomal RNA gene, partial sequence 457 457 100% 2.E-125 99% JX397932.1 Alteromonas macleodii strain SCSIO 04629 16S ribosomal RNA gene, partial sequence 379 379 100% 4.E-102 100% KC893945.1 L5A-10M-H3 - 205 Uncultured Alteromonas sp. AD1000-357-C11 genomic sequence 379 379 100% 4.E-102 100% JX888784.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Alteromonas Pseudoalteromonas sp. V4 16S ribosomal RNA gene, partial sequence 379 379 100% 4.E-102 100% JX407220.1 Marinobacter sp. U1371-101202-SW223 16S ribosomal RNA gene, partial sequence 665 665 100% 0 99% JQ082177.1 L5A-10M-H1 - 183 Gamma proteobacterium SY258 partial 16S rRNA gene, strain SY258 665 665 100% 0 99% HE589590.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Marinobacter Marinobacter salsuginis strain KUAC3038A cb 2 16S ribosomal RNA gene, partial sequence 665 665 100% 0 99% JN202614.1 Marinobacter adhaerens strain S20-1 16S ribosomal RNA gene, partial sequence 283 283 100% 2.E-73 99% KC420687.1 L5A-10M-G1 - 156 Marinobacter sp. U1371-101202-SW223 16S ribosomal RNA gene, partial sequence 283 283 100% 2.E-73 99% JQ082177.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Marinobacter Gamma proteobacterium SY258 partial 16S rRNA gene, strain SY258 283 283 100% 2.E-73 99% HE589590.1 Uncultured bacterium clone BBD-Aug08-6BB-77 16S ribosomal RNA gene, partial sequence 587 587 100% 2.E-164 100% GU472164.1 Roseobacter L5B-100M-B1 - 137 Thalassobius mediterraneus gene for 16S ribosomal RNA, partial sequence, strain: RO7 585 585 99% 7.E-164 100% AB607866.1 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae clade CHAB-I-5 Uncultured Thalassobius sp. clone T534deg56 16S ribosomal RNA gene, partial sequence 585 585 99% 7.E-164 100% JN210864.1 lineage Alteromonas macleodii str. 'Ionian Sea UM4b', complete genome 195 975 100% 7.E-47 100% CP004855.1 L5A-100M-F2 - 105 Alteromonas macleodii strain LAMA 973 16S ribosomal RNA gene, partial sequence 195 195 100% 7.E-47 100% KC583248.1 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Alteromonas Uncultured Alteromonas sp. clone fosmid AD1000-249-G9 genomic sequence 195 195 100% 7.E-47 100% JX826389.1