July 28, 2010

The Many Talents of Many-celled Magnetotactic Bacteria

Final Report

Microbial Diversity Course 2010 Marine Biological Laboratory Woods Hole, Ma, USA

Esther Singer University of Southern California, Los Angeles [email protected] Abstract

North-seeking many-celled magnetotactic bacteria (MMBs) were isolated from a salt marsh on Cape Cod, MA, and tested for magnetophototaxis and chemotaxis. MMBs generally showed swimming behavior towards the North Pole, which was reversed and influenced by phototaxis during exposure to sunlight and LED light. It is assumed that coupled magnetophototaxis helps MMBs to find their preferred redox zone while moving vertically within the sediment column while avoiding cell death due to light. Cell counts showed an increased abundance of MMBs in shallow sediments collected during the night compared with day samples, which supports the hypothesis of an upward movement of MMBs linked to a vertical upwards shift of geochemical redox zones in the sediment column during the night. Chemotaxis experiments showed a clear preference of MMBs to acetate over formate and prolonged life duration. Enrichment cultures were further used to construct a clone library and a metagenome, which will be helpful to identify MMB-specific genes involved in metabolism, regulation and the coupling of photo-, chemo- and magnetotaxis.

Introduction Many-celled Magnetotactic Bacteria are sphere-shaped aggregates of clonal gram-negative cells belonging to the Deltaproteobacteria. They vary in size and cell number between 3-15 µm (10-40 cells) and typically occur as spherical aggregates of cocci or rod-shaped cells. Due to their intracellular magnetosomes composed of greigite (Fe3S4) MMBs align to lines of magnetic field and thereby orient their swimming direction. It is assumed that magnetotaxis is used by these organisms to move to their preferred oxygen concentrations within the sediment, and possibly, water column. Greigite-producing magnetotactic bacteria have so far been only observed in oxic-anoxic interfaces of saline or hypersaline habitats with cell abundances of about 104 cells/ml (Bazylinski 2007). The life cycle of MMBs is unique among bacteria because it is believed to be multicellular at all stages, indicating a high level of regulation during cell division, taxis and response to extracellular stimuli (Martins 2009). Disaggregation of the aggregates results in death of all cells. To date, MMBs have not been successfully cultivated, neither is there a full genome sequence is available. However, various studies assessing chemo- and phototaxis have elucidated some characteristics of these organisms. Wenter et al. (2009) showed that MMBs respond chemotactically to a variety of carbon sources. Last year, MBL students Hatzenpichler and Shapiro showed photomagnetotactic behavior in response to exposure of light of various wavelengths. On this basis, this mini project was aimed at constructing 454 metagenomic data and investigating photomagnetotaxis with and without the influence of an artificial magnetic field linked to MMB abundance in the environment during day and night.

Material and Methods Microscopy Microscopy images were taken using Zeiss Epifluorescence, Confocal and Compound Microscopes with unstained, DAPI and membrane (FM 1-43 X) stained cells. Z-stack images on the confocal microscope were used for 3-D visualization of the cell aggregates.

Magnetic Enrichment Mud and pond water were repeatedly collected in small buckets (~600 ml) from a salt marsh in Little Sippewissett, West Falmouth, MA, during day and night at low tide (figure 1). Magnets to enrich for north- and south-seeking MMBs were attached to the outside of the buckets slightly above the sediment-water interface (figure 2). After a few minutes pellets became visible at the magnets on the inside of the buckets (figure 3), which were collected with syringes and transferred to Hungate tubes with sterile-filtered anoxic pond water and magnets attached to their side walls (figure 4). After a pellet formed on the magnet, this was transferred to another tube for secondary magnetic enrichment and purification of the magnetotactic community. In total 4 washing steps were performed.

Figure 1: Satellite image of Little Sippewissett Marsh in Massachusetts, retrieved from Google Maps on July 28, 2010. The red arrow indicates the pond, out of which MMBs were isolated.

Figure 2: Bucket enrichments in the laboratory. Magnets were taped to the outside walls about 1cm above the water-sediment interface. Highest yields were retrieved during enrichments directly after sampling.

Figure 3: Pellet of magnetotactic bacteria formed on the inside of the bucket on the height of the magnet in the water phase. The time it took for pellets to form was dependent on sampling time (day vs. night) and age of the sediment in the bucket. Microscopy Microscopy images were taken using Zeiss Epifluorescence, Confocal and Compound Microscopes with unstained, DAPI and membrane (FM 1-43 X) stained cells. Z-stack images on the Confocal microscope allowed 3-D visualization of the cell aggregates.

Photomagnetotaxis Isolated MMBs were inserted into capillaries (5 cm x 0.5 cm x 0.05 cm) with anoxic sterile-filtered pond water. One half of the capillaries were covered with aluminum foil, and exposed to sunlight as well as LED light without magnets and with magnets pointing their North pole toward the lighted half and perpendicular to the capillary. After a few hours, after MMBs had died and stopped moving, capillaries were examined under the microscope at 40x and aggregate abundances were evaluated in the covered and light exposed parts of the capillaries.

Chemotaxis Chemotaxis towards acetate and formate was tested by injecting 100 µM acetate on one side of the capillary and 100 µM formate on the other side. MMBs were placed in the middle suspended in anoxic sterile-filtered pond water, so that they would be able to decide for one or the other carbon source.

Cell Counts Sediment cores were taken during the day and night and dissected into several increments of ~1 cm. Pore water was squeezed out via centrifugation at 5000 rpm for 10 minutes. Cells were fixed with paraformaldehyde and 1 mL was filtered onto 0.2 µm membranes, which were then stained with DAPI for cell counts under the epifluorescence microscope.

Sequencing Metagenome Construction DNA was extracted from an enriched MMB pellet using the TOPO MO-BIO Kit. Whole Genome Amplification (WGA) was applied to the PCR product and sent to Penn State University for 454 metagenomic sequencing. Sequence analysis was started by searching for key genes of metabolic pathways with the BLASTALL command in Bioperl.

16S Clone Library A 16S clone library was constructed with universal primers. Sequences were compared to the NCBI database (BLASTN) and processed with Geneious (Copyright © 2005-2009 Biomatters Limited). 16S rRNA phylogenies were constructed by aligning with MUSCLE and implementing the PhyML Maximum- likelihood method.

Results and Discussion

Magnetic Enrichment Magnetic Enrichment as described in the methods section yielded mostly North- seeking MMBs. The resulting product used for metagenome and clone library construction was estimated to be about ~80-90% pure as observed in a DAPI- stained sub-sample with epifluorescence microscopy. Further enrichments were used to conduct photomagnetotactic and chemotactic experiments. Figure 5 shows a pellet of MMBs concentrated towards the North Pole of the magnet.

Figure 5: MMBs were collected and stored in sterile-filtered, anoxic pond water for several hours before they started to die. Only alive MMBs swim actively towards their preferred magnetic orientation.

Microscopy Figures 6 and 7 show the 3-D structure of a typical MMB. The center core appears hollow when stained with DAPI and solid when stained with FM 1-43 X. This suggests that the core is potentially made of lipids, which may help to hold the aggregate together and serve as storage. However, it would be useful to use an (exopolysaccharide) EPS stain to further confirm this hypothesis and counter- stain with DAPI.

Figure 6: DAPI-stained MMBs in 3-D as viewed with a confocal microscope. The center of the aggregate appears hollow and is not a cell.

Figure 7: Center slice of an MMB stained with FM 1-43 X. The center part seems to be solid and made of lipid material.

Phototaxis MMBs were exposed to sunlight and LED light of various intensities with and without the influence of a magnetic field. In general, MMBs “decided” to stay within the aluminum foil coated part of the capillary and avoided exposure to light even when they were encouraged to swim into the light following the North Pole of a magnet. Placement of a magnet perpendicular to the capillaries resulted in an accumulation of MMBs on the Northern aluminum coated rim of the capillary. Quantification of photomagnetotaxis response was performed via absolute and average cell counts.

Figure 8: Relative abundance of MMBs in a capillary placed on a window bench without the influence of a magnet. In all capillaries, more than 90% of the inserted MMBs eventually remained in the covered, dark part.

Figure 9: Even under the influence of a magnet encouraging North-seeking MMBs to swim into the bright parts of the capillaries, still >90% remained in the covered halves.

Figure 10: Exposure to the bright, cold light of an LED resulted in similar phototactic responses exposure to sunlight and proves that there was no or very little influence of temperature on the direction of movement of the MMBs within the capillary.

Figure 11: MMB response to the LED light under the influence of a magnet with the North Pole pointing towards the bright side, resulted in a less obvious phototactic behavior at first sight.

The photomagnetotactic response of the MMBs was strongly dependent on the viability of the cells and the intensity of the light. For example, capillaries on slide 5 and 6 contained MMBs that had been isolated a few days prior to the experiment and therefore were not as motile and responsive as “fresh” cells anymore. Moreover, those slides had been placed under relatively low intensity LED light, which may have been less harmful to them. Further inspection of the spatial distribution of MMBs within capillaries on slides 5 and 6 shows that most cells had barely traveled across the dark-bright boundary before they died (figure 12). This supports the hypothesis that the used MMBs were weak at the start of the experiment. On slides 7 and 8, cell abundances appeared higher in the dark part of the capillaries, however, magneto-phototaxis did not appear as clear as in the capillaries located on the window bench. However, more precise evaluation of the capillary showed that almost all MMBs located in the bright part of the capillary were actually located on the very end, which was covered by the plasticine plug and therefore dark (figure 13). These observations show that some MMBs actually travel through the light following the magnetic field line before they sense the lethal harm of the light and revise their magnetic orientation. The response therefore appears to be gradual and involves “decision making”. Moreover, most MMBs in the dark part of the capillary were found on the border to the bright part, which suggests that magnetotaxis and phototaxis is eventually balanced out.

Figure 12: Spatial distribution of MMBs across the capillary on slide 6. Most cells are located at the boundary of the dark-bright transition as determined by total cell counts.

Figure 13: Spatial distribution of MMBs across capillaries 7 and 8. Most MMBs in the dark part are located just on the border of the dark-bright transition zone. Almost all MMBs in the bright part are located at the edge, which was covered by a plasticine plug.

To test this decision making process further and evaluate the power of phototaxis versus magnetotaxis, additional capillaries were prepared and magnets located perpendicular to the slides. MMB abundance distribution within the capillaries showed that most MMBs preferred the Northern rim of the dark capillary sides. This reflects the link between photo- and magnetotaxis and a response behavior, which results from a balance of the two sensation potentials.

Figure 14: MMB magnetophototaxis response in a capillary located on the window bench with a magnet perpendicular to the slide. About 85% of all MMBs swam into the dark field, where they stayed.

Figure 15: Similar to exposure to sunlight, MMBs preferred the dark part of the capillary when exposed to LED light and under the influence of a magnet perpendicular to the slide.

Figure 16: Within the dark part of the capillary, most MMBs were found on the Northern rim of the capillary, which shows that magnetotaxis was not shut down completely, although phototaxis overruled the swimming behavior of the cell aggregates.

Cell Counts To relate photomagnetotaxis to their environment, abundance profiles of MMBs with sediment depth were conducted for a day and a night core via cell counts of DAPI-stained filters. In general, more MMBs were present in the night core sample. Moreover, it seemed that MMB abundance increased with depth in the day core, whereas there was no clear trend visible in the night core, but aggregate numbers were high in the top sediment layers as well as in the deeper layers.

Figure 17: Cell counts of MMBs per mL pore water in a sediment core. On average there are 103 cells/mL in a day core profile and there seem to be an increase in MMB abundance with sediment depth.

Figure 18: Average MMB abundance amounted to 104 cells/ mL pore water. There does not seem to be a clear increase of cell numbers with depth, but cell abundance is rather high in the top sediment layers compared to the day core profile.

Chemotaxis A fresh sample of MMBs were tested for response toward acetate versus formate. About 90% of the cell aggregates accumulated in the acetate-spiked water, although not at the very edge of the capillary. This might suggest that MMBs swam to their preferred concentration of acetate, which was slightly less than 100 µM, or it might have been a response to oxygen diffusing in on the end of the capillary.

Figure 19: Relative MMB abundance in a capillary loaded with acetate on one end and formate on the other end shows that most MMBs preferred acetate over formate.

Figure 20: Most MMBs were not located at the very rim of the capillary in the acetate field, but about 1 cm away from it due to either a preference of a concentration lower than 100 µM or a motile response to diffusing oxygen from the end.

Community Analysis The 16S clone library constructed from the WGA-sample resulted in about 16% MMB-related sequences. Most sequences were identified as (Pseudo- )Alteromonas, common species in marine environments. Although the enriched culture was estimated to be ~80-90% pure, this result suggests that there might have been a post-WGA contamination with (Pseudo-)Alteromonas, a common marine organism or there was a lot of “hitch-hiking” DNA in the sample, which was present as extra-cellular DNA and amplified during WGA, therefore resulting in this skewed representation of sequences in the clone library.

Figure 21: Phylogenetic distribution of 16S sequences as obtained from a clone library.

Figure 22: MMB-related sequences clustered closely with sequences obtained from MMBs isolated from other marine environments, for example in the North Sea. All of the related sequences are Delta-Proteobacteria and many are from within the family of Desulfobacteraceae.

Metagenomics Data The metagenomics dataset contained 132,622 reads, of which ~41% were aligned. 3180 contigs were assembled with an average contig size of 777 bp.

A first glance of the metagenome revealed the presence of various genes, which play a key role in sulfate reduction (dsrAB), carbon utilization (pyruvate dehyrogenase, D-lactate dehydrogenase, formate dehydrogenase), chemotaxis (mcp), and outer membrane electron transport (cytochrome c oxidase). Due to time limitation no further data evaluation was conducted, however, although the coverage was rather low (3-6x), the dataset leaves a large potential to characterize the Deltaproteobacterial sequences and obtain insight into the metabolism, regulation and taxis of MMBs.

Outlook Future research on MMBs should include the construction of a clone library and a full metagenomics plate, constructed from a magnetically enriched sample that is treated with DNA lyase prior to WGA to get rid of extra-cellular DNA. Element composition of MMBs could be conducted on an Element Analyzer available on the MBL campus. However, a cooperation with the responsible people shall be planned well in advance and with patience. Further capillary experiments may be improved by using oxygen-tight caps instead of plasticine to avoid dehydration over time and response to oxygen when other stimuli are tested.

Acknowledgements Thanks to Dan, Steve, Sheri for supporting my ideas and making this project possible. To all you TAs, who drove me to my salt marsh, especially Bekah – we will have wine, when I see you again –, and helped me with everything I needed in and outside the lab. Class of 2010 – you rock! I will miss each and every one of you and am looking forward to seeing you all again!

References

Bazylinski et al. (2007) Modes of Biomineralization of Magnetite by Microbes. Geomicrobiology Journal, Vol 24, Issue 6

Wenter et al. (2009) Ultrastructure, tactic behaviour and potential for sulfate reduction of a novel multicellular magnetotactic prokaryote from North Sea sediments. Environmental Microbiology Reports, Vol 11, Issue 6, p. 1493-1505

Simmons and Edwards (2007) Unexpected diversity in populations of the many- celled magnetotactic prokaryote. Environmental Microbiology, Vol 9, issue 1, p. 206-215

Shapiro et al. (submitted) Multicellular Photo-Magnetotactic Bacteria. Environmental Microbiology Reports