Stuhrmann, T. Studying New Eukaryotic Niches of Bacterial Life

Studying new eukaryotic niches of bacterial life

Marine Biological Laboratory Microbial Diversity Course 2005

Torben Stührmann Max Planck Institute for Marine Microbiology Department of Molecular Ecology Celsisus Str. 1 28379 Bremen-Germany

Abstract

Biologists are becoming increasingly aware that a variety of animals and plants bears symbiotic microorganisms. The formation of alliances between animals and microorganisms contribute to their nutrition or defence against predators and parasites. This study has the aim to search for new eukaryotic niches of bacterial live (symbiosis or commensalisms). As study object two different eukaryotic species from varying environments were taken. For the first study a brown insect larva was obtained from the Sippewissett Salt marshes which was not further characterized. The second study object was the jellyfish Cyanea capillata (the Lion’s Mane). This species was highly abundant in the Cape Cod area during the course period (June-August 2005). Symbiotic interactions between the cnidarians and Cyanobacteria are known but there is nothing known about bacterial interactions among the Scyphozoa. Microscopic- (Scanning electron microscopy, Light microscopy), cultivation- and molecular- techniques (16S rRNA clone libraries, Fluorescence in situ hybridization FISH) were applied for studying bacterial-eukaryotic interactions. Only few indications were found with FISH suggesting a eukaryotic niche for bacteria within the Scyphozoa. A rod shaped morphotype detected with a FISH-probe specific for Gammaproteobacteria was detected in the mouth tissue. Other approaches were not applied or failed. The 16S rRNA library of brown larvae revealed a high predominance of Gammaproteobacteria (Thiomicrospira, Halomonas, Marinobacter and Vibrio spp.). Epsilon-, alpha- proteobacteria, Bacteriodetes and Cyanobacteria were only less frequent. These results were confirmed by FISH. A coccoid highly motile morphotype was enriched from the gut tissue with artificial seawater media, yielding an almost pure isolate. This is the first known report surveying bacterial interactions in C. capillata. To reveal the secret of bacterial interactions with jellyfish further attention to this topic is needed (e.g. 16S rRNA libraries, cultivation). The results from the brown larvae taken at Sippewissett marshes indicate a niche of bacterial live. For verification of a new eukaryotic niche for bacteria a more detailed comparison of environmental and intestinal clone libraries is needed. But the results are promising to get further insights into bacterial/host interactions.

Introduction

Inspired by J. Leadbetters research on bacterial interactions in the termite gut (e.g. Leadbetter, J.R. & J.A. Breznak, 1996), this study has the aim to search for new eukaryotic niches of bacterial live (symbiosis or commensalisms). As study object two different eukaryotic species from varying environments were taken. The first study object was found in the Sippewissett Saltmarshes. It is a brown insect larva which was not further characterized during this study (see figure 1). After a few days in the laboratory the Sippewissett marshes become highly sulfidic and the brown larvae were moving out of the sediment. These brown larvae were studied with molecular methods for bacterial associations. The second species was the Lion’s Mane Jellyfish (Cyanea capillata). This species was highly abundant in the Cape Cod area during the course period (June-August 2005). Cnidaria are among the earliest evolved metazoan animal phyla with representatives found in fossils from the Precambrian 550 million years ago (Chen et al., 2002; Wood et al., 2002). Since Scyphozoa often form high biomass in the oceans, the degradation of dead individuals contaminated with antimicrobial and neurotoxins is of special interest. Nothing is known about microorganisms degrading those jellyfish. Using molecular 16S rRNA based methods, microorganisms involved in this process were identified. The phylum Cnidaria comprises benthic and pelagic aquatic animals including the classes Anthozoa (hard corals, soft corals, sea pens, and sea anemones), Hydrozoa (hydroids, fire corals), Scyphozoa (jellyfish) and Cubozoa (box jellyfish). The body plan of Cnidaria is diploblastic, i.e. the body consists of two cell layers, the ectoderm (epidermis) covering the outer surface of the body and the endoderm (gastrodermis) lining the body cavity. There is homogeneous elastic material (mesoglea) between these layers. This gelatinous material is a distinct feature of the bell of jellyfish. Nematocysts are stinging capsules characteristic of Cnidaria. Nematocysts contain and fire harpoon-like microscopic structures (cnida) that penetrate the surface layer of the victim and deliver a mixture of highly toxic substances. Cnidarians are known for their antimicrobial activity (Bhosale et al. 2002). However, there are also known symbiotic interactions between the cnidarians (i.e. the Caribbean coral M. cavernosa) and cyanobacteria (Lesser et al., 2004). This study addresses the question of whether these interactions are limited to corals or if interactions are also detectable in Scyphozoa. For this different types of tissues from Jellyfish were studied. Both topics are not very well studied; in fact there is no publication dealing with bacterial interactions with Scyphozoa. This study wants to contribute new insights into the symbiotic interactions between Scyphozoa and bacteria. Further time was spent on Scanning electron microscopy (SEM) of different cultures enriched during the course.

Materials and Methods

Sample collection Cyanea capillata (Lion’s mane) species were caught in waters of the Cape Cod area. The C. capillata species were maintained at room temperature with a continuous flow of seawater. The brown larvae were obtained from Sippewisset Marshes Mats and were not further characterized. Samples for Scanning Electron microscopy were enriched during the course from Sippewisset marshes (GSB-, H2/CO2-Enrichment, and Azotobacter-media) enrichments mats as described in the course manual (Microbial Diversity course 2005).

Sample preparation Samples from C. capillata were washed three times in filtered seawater (0.2 µm), for relaxation of species a 0.15 M MgCl2 was used. Alternatively a 4% agarose solution was used to preserve the structure of the Jellyfish, but is not recommended for sectioning. Two different samples of jellyfish were analyzed. One species died during maintenance and was fixed in agarose after several hours. The formaldehyde fixation of these samples was directly carried out on the filter sections. The second species was killed during the fixation process. The brown larvae were washed 3 times in 1x PBS. They were sectioned and opened, the white internal substance was diluted in 1x PBS. The brown capsule was discarded. The sample was fixed with 2% Formaldehyde for 30 min and filtrated through a 0.2 µm Millipore GTTP Filter. Otherwise the sample was diluted in 1 ml 1x PBS for the enrichment of intestinal bacteria and spread on agar plates in different dilutions (see Media and Growth conditions).

FISH Fluorescence in-situ-hybridization (FISH) was performed as described in the course manual (Microbial Diversity course 2005). For investigation of different tissues the following probes were used with 35% Formamide at 46°C:

Table 1: Conditions for the FISH probes used in the experiments probe specificity sequence formamide target competitor ALF968 alpha-group of GGTAAGGTTCTGCGCGTT 35 16S

Proteobacteria beta-group of BET42a GCCTTCCCACTTCGTTT 35 23S Gam42a Proteobacteria Cytophaga/Flavo- CF319a TGGTCCGTGTCTCAGTAC 35 16S bacterium cluster most Bacteria, no EUB338 GCTGCCTCCCGTAGGAGT 0-35 16S Planctomycetales Gamma-group of GAM42a GCCTTCCCACATCGTTT 35 23S Bet42a Proteobacteria nonsense probe, for Non338 detection of non ACTCCTACGGGAGGCAGC 0-35 16S specific binding

Light Microscopy The samples were observed under phase-contrast and epifluorescence on a Zeiss AXIOPlan Imager.M1 compound light microscope with AxioVision v4.4 capture software. Dissected jellyfish tissue was stained in DAPI (1 µg/mL) for 10 minutes and washed with sterile MQ water prior to observation.

Scanning Electron Microscopy Samples were fixed from the different enrichments (2% glutaraldehyde, 1.5% formaldehyde in 0.1M sodium cacodylate buffer, pH 7.0) for 4 hours, dehydrated in acidulated 2,2- dimethoxypropane, and kept in absolute ethanol. Specimens were critical-point dried with liquid

CO2 and sputter-coated with gold. Images and micrographs were captured on a JEOL JSM-840 scanning electron microscope.

Media and growth conditions E. coli used in clone libraries was grown at 37° C on Luria-Bertani (LB) with kanamycin supplemented at 50 µg/mL and 40 µg/mL X-gal as appropriate. LB was solidified with 1.8% (wt/vol) Bacto agar. Intestinal larvae bacteria were cultivated on artificial seawater (ASW) at 30° C containing

1 mM Na2SO4, 1.5 mM K2PO4, 5 mM NaNO3, 5 mM MOPS (pH 7.2), and 1 mL of HCl- dissolved trace elements solution per liter seawater base (20 g NaCl, 3 g MgCl2•6H2O, 0.15 g

CaCl2•2H2O per liter distilled water) and 1-mL of the 12-vitamin solution. ASW-media was solidified with 1.8% (wt/vol) prewashed Bacto agar.

HCl-dissolved trace elements solution contained 2.1 g FeSO4•7H2O, 30 mg H3BO3, 100 mg MnCl2•4H2O, 190 mg CoCl2•6H2O, 24 mg NiCl2•6H2O, CuCl2•2H2O, 144 mg

ZnSO4•7H2O, 36 mg Na2MoO4•2H2O, 25 mg NaO3V and 6 mg Na2SeO3•5H2O in 987 mL distilled water. The 12-vitamin solution contained 10 mg riboflavin, and 100 mg each of riboflavin, thaimine•HCl, L-ascorbic acid, D-calcium-pantothenate, folic acid, niacinamide, nicotinic acid, para-aminobenzoate, pyridoxine•HCl, lipoic acid, NAD, and thiamine pyrophosphate in 100 mL of 10 mM phosphate buffer (pH 7.2).

16S rDNA clone library construction DNA was extracted from five larvae of Sippewisset marshes sediment using a MoBio DNA extraction kit for soil according to manufacturer’s directions. 16S rDNA was PCR amplified using universal eubacterial primers 8F and 1492 in 25 µL volume with 0.5 to 2 µL of extracted DNA and a Promega PCR kit on an Eppendorf Mastercycler using the following program: 95° C, 5 minutes, denaturation 95° C 30 seconds, annealing 50° C 30 seconds, amplification 72° C (30 cycles total), final extension 72° C, 4° C infinity. Proper products were confirmed (~1.5 kb) and purity checked by gel electrophoresis on 1.3% agarose TBE gels. PCR products were cloned with a TOPO-TA cloning kit according to the manufacturer’s instructions (Invitrogen).

Sequencing and phylogenetic analysis 56 E. coli clones containing plasmid 16S rDNA inserts were picked and inoculated in 96-well blocks containing 1-mL LB-Kan. Plasmids were mini-prepped and sequenced using M13 forward and reverse primers at the High Throughput Sequencing Center at the Josephine Bay Paul Center in Comparative Molecular Biology and Evolution. Phylogenetic analyses were performed with the ARB software package (www.arb-home.de). Trees were calculated by using neighbor joining (termini filter, bacterial position variation filter, Jukes-Cantor correction)

implemented in ARB. Taxonomic nomenclature was used according to Bergey’s Manual of Systematic Bacteriology (Garrity, 2001).

Results and Discussion

Results and Discussion of FISH in Cyanea capillata In the first species which died during maintenance the FISH-technique was used with the EUB338, GAM42a + Comp., and NON338. Rod shaped morphotypes were detectable with EUB338 (see figure 2a) and GAM42a probe. Bacteria were only observed in the mouth tissue. No fluorescence signal was found with the NON338 probe. The second species was analyzed in more detail, all FISH probes (see table 1) were applied for different tissue types (bell, mouth, filaments and gonads). Only autofluorescence was detectable in all types of tissue with the applied probes see (figure 2b). The results suggest that there is no significant commensalism or symbiotic interactions in living C. capillata species. For further studies, thin-sectioning methods should be applied. The results of FISH from “dead” C. capillata species giving a first hint that Gammaproteobacteria are maybe involved in degrading of jellyfish-tissue. A pre-enrichment of dead C. capillata species was removed by someone during the course. There was no time to start a new enrichment during this course so that the question which bacteria can degrade jellyfish tissue remains so far unclear.

Results from the intestinal brown larvae bacteria

16S rRNA clone libraries from brown larvae The results of the clone library from the brown larvae showed high predominance of Gammaproteobacteria (71% or 40 clones). Most of the sequences within this group belong to Thiomicrospira spp. (32% or 18 clones) and Halomonas spp. (25% or 14 clones). Marinobacter spp. and Vibrio spp. are represented each with 7% (4 clones) in the Gammaproteobacteria. Arcobacter (Epsilonproteobacteria) is also represented with 7% in the library. Other species are only low represented in the clone library (Bacteriodetes, Roseobacter, Cyanobacteria, 1 or 2 clones). For corresponding phylogenetic trees see figures 3a and b.

Enrichment of intestinal brown larvae bacteria Overnight incubation revealed growth of white punctiform colonies, differing in size. In all dilutions (10, 50, and 100 µl) the colony density allowed discrimination of single colonies. Light microscopy showed highly motile coccoid cell morphologies (see figure 4). No other cell morphologies were detected in these enrichments, suggesting an almost pure enrichment.

FISH of intestinal brown larvae bacteria For detection of bacteria from the prepared filter the EUB 338, ALF 968, BET42a and the GAM42a probe were used. The EUB 338 probe showed a bright fluorescence signal but also a high background signal. Rod shaped morphologies dominated these samples (see figure 5a). The coccoid morphologies identified by light microscopy in the primary enrichment (see figure 5) were not detected with EUB 338. Alphaproteobacteria probe showed fluorescence signals but were low represented in the examined samples (see figure 5b). Proteobacteria of the Beta subclass (Bet42a probe) were even less represented. A fluorescence signal was only rarely found in long filaments (see figure 5c). The Gammaproteobacteria were predominant in these samples (see figure 5d). The morphology was mainly rod shaped.

Discussion for the intestinal brown larvae bacteria Interestingly the EUB 338 probe did not target the coccoid bacteria from the primary enrichment. There no sequence data available for this enrichment. However, the EUB 338 does not target all bacteria (Daims et al., 1999). Usually, a set of three different probes is used (EUBI-III) for detection of all bacteria. The ARB probe match function showed several sequences that were not matched by the EUB 338 probe (e.g. Bacteriodetes, Pseudoalteromonas). Comparing the morphologies of these identified species with the coccoid ones from the enrichment no similarities were obtained. With the available dataset no further phylogenetic characterization of this enrichment is possible. Sequencing of the 16S rRNA gene followed by a physiological characterization would clarify of Microorganisms in this enrichment. The data from the clone sequences suggested that the ALF 968 probe targeted bacteria belonging to the Roseobacter group. No sequences were targeted by the BET 42a probe. The predominance of the Gammaproteobacteria in the 16S rDNA clone libraries (71% of all clones) was in

accordance with the FISH results for the GAM42a probe. More specific probes should be applied to reveal the abundance of the different phyla (e.g. Thiomicrospira or Halomonas) within the Gammaproteobacteria. For verification of a new eukaryotic niche of bacterial live a more detailed comparison of environmental and intestinal clone libraries is needed. But the results are promising for new insights into bacterial/host interactions.

Results and Discussion for Electron microscopy of different enrichments

The enrichments of Azotobacter sp., green sulphur bacteria and on H2/CO2 (for media see course manual Microbial Diversity course 2005) were used for scanning electron microscopy. Figures 6a-d show different pictures from the Azotobacter enrichment, indicating an almost pure culture of Azotobacter. The size was 0,5x 2µM on average.

The enrichment on H2/CO2 showed clustered bacteria surrounded by a matrix. There were different morphotypes inside this matrix. The enrichment for green sulphur bacteria revealed some unique prosthecate morphologies. It is dominated by star- or flower- like bacteria (see figures 8a, b). Based on the early findings of Staley in 1968 this unique morphology gave evidence that these bacteria are affiliated with Prosthecomicrobium.

References

Bhosale SH, Nagle VL, Jagtap TG, Antifouling potential of some marine organisms from India against species of Bacillus and Pseudomonas. Mar Biotechnol 2 (2002) pp. 111-8

J.Y. Chen, P. Oliveri, F. Gao, S.Q. Dornbos, C.W. Li, D.J. Bottjer and E.H. Davidson, Precambrian animal life: probable developmental and adult cnidarian forms from Southwest China, Dev. Biol. 248 (2002), pp. 182–196.

Daims H., Brühl A., Amann R., Schleifer K.-H. and Wagner M. (1999). The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: Development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22: 434-444

Garrity, G.M, M. Winters, and D.B. Searles (ed.). 2001. Bergey’s manula of systematic bacteriology, 2nd ed. [Online.] Springer-Verlag, New York, N.Y. http://www.cme.msu.edu/bergeys.

Lesser MP, Mazel CH, Gorbunov MY, Falkowski PG, Discovery of symbiotic nitrogen-fixing cyanobacteria in corals. Science 305 (2004) pp. 997-1000.

Leadbetter, J.R. and J.A. Breznak. (1996). Physiological ecology of Methanobrevibacter cuticularis sp. nov. and Methanobrevibacter curvatus sp. nov., isolated from the hindgut of the termite Reticulitermes flavipes. Appl. Environ. Microbiol. 62:3620-3631.

J.T. Staley. (1968) Prosthecomicrobium and Ancalomicrobium: new prosthecate freshwater bacteria. J Bacteriol. 95(5):1921-42.

R.A. Wood, J.P. Grotzinger and J.A. Dickson, Proterozoic modular biomineralized metazoan from the Nama group, Namibia, Science 296 (2002), pp. 2383–2386.

Figures

Figure 1: Brown larvae from Sippewisset marshes, moving out of the sediment after several days in the laboratory

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Figure 2a and b: FISH on different jellyfish samples using EUB 338 probe.

Halomonas spp.

Marinobacter spp. Gammaproteo- bacteria

Vibrio spp.

Alphaproteo- Roseobacter spp. bacteria

Figure 3a: Phylogenetic Neighbour joining subtree showing 31 out of 59 16S rDNA sequences recovered from the brown larvae clone library. Positional-Variability and Termini Filter were applied.

Thiomicrospira spp. Gammaproteo- bacteria

Arcobacter spp. Epsilonproteo- bacteria

Bacteriodetes spp.

Cyanobacteria sp.

Figure 3b: Phylogenetic Neighbour joining sub-tree showing 25 out of 56 16S rDNA sequences recovered from the brown larvae clone library. Positional-Variability and Termini Filter were applied.

Figure 4: Phase contrast microscopy of primary enrichment of intestinal brown larvae bacteria with seawater media

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C D

Figure 5a, b, c and d: FISH on intestinal of brown larvae bacteria. A: EUB 338, B: ALF 968, C: BET42a + Competitor, D: GAM42a + Competitor

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5 µm

C D

Figure 6a, b, c and d: Lightmicroscopy and SEM of Azotobacter. A: Lightmicroscopy at 1000x magnification, B: SEM overview of Azotobater Isolate, C: Detailed view into a colony of Azotobacter, D: Detailed Azotobacter SEM picture of single cells

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Figure 7a and b: Light microscopy and SEM of H2/CO2 Enrichment. A: Light microscopy at 1000x magnification from an Agar shake culture, B: SEM of a dense packed bacterial colony of different morphotypes

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Figure 8a and b: SEM of the GSB Enrichment. A: Overview of some prosthecate bacteria from the GSB enrichment, B: Detailed SEM-picture of prosthecate single cells.