Vampirococcus sucking on Chromatium

Transcription

1 July 1993 MBL Microbial Diversity (n ni-i-i-i showed that not only hroniatiunz but also Thiocapsa served as a host. The goal of this study was to enrich and if possible characterize further Vampirococcus or enrichment cultures from the Great Sippewisset Salt Marsh as well as from Oyster Pond. Although confirmed by epifluorescence and scanning electron microscopy. Transmission electron microscopy plaques as indication of predatory activity could not be observed, clear attachment to the host cells was Vampirococcus--lilce bacteria, which use purple sulfur bacteria as their hosts. Predators were observed in ABSTRAT Margret More (mini2@cnix2.citcomel1.edu) Vampirococcus sucking on hromatium

2 nor identified. sulfur bacteria occur in high cell densities. This presents a good environment for a predator preying on hrornatium, but this organism, described as Vampirococcus, has never been brought into pure culture larke et al, 1993 and Guerrero et al., 1987 describe a predator or epibiont preying on these bacteria. buffered glutaraldehyde, washed 3 times in.1 M cacolylate, stained for 1 h in 1% osmium tetroxide and electron microscope. three times in propylene oxide (15 miii each), followed by washing with propylene oxide/epon 1:1 for at 6 for 48 h. The embedded cells were thin sectioned (9 nm) and viewed with a transmission 3 nñn and with epon for 1 h and 1.5 h. The cells were embedded as a pellet in epon, which polymerized For transmission electron microscopy (TEM) 1 ml culture was pelleted, fixed for 1.5 h in 2% washed three times with water. The cells were dehydrated in ethanol (see above), then dehydrated further coated slide. incubated at room temperature in the dark, Sandra Nirzwicki-Bauer) were used in order to identify the predator. The probes were applied as before the dehydration with ethanol. This created the problem that the cells came loose from the filter easily, so that the sample had to be handled with great care. Besides, the extra staining also did not improve to quality of the image, dehydrated in 5%, 7%, 85%, 95%, 1%, 1%, 1% successively for each 1 minutes. Then they filter was additionally stained with 1% osmium tetroxide for I h and then washed tree times with water to produce an electrical connection between the sample and the stub. In an alternative preparation the minutes. The cell pellet was resuspended in 15 ul of the supematant and 1 ul were observed on an agar washing. To demonstrate plaques sample material was filtered onto.2 urn membrane ifiters, which were were critical point dried, placed onto the stubs and gold/palladium sputtered. A silver emulsion was used Pure cultures of hrornatiun7 vinosum (2 ml) were inoculated with.1,.75 and 1.75 ml of a predator 1 ml culture was used for staining. The cells were then concentrated by spinning in a microfuge for 2 epifluorescence microscope with dim bright field illumination. A volume of 1 ul of 1 ug./ml DAPI per temperature without further feeding of H2S to obtain plaques in the sedimented hromatiaceae lawn. containing enrichment culture to show the effect of this addition to the pure culture. natural sample followed by incubation under JR light (absorption of bacteriochlorophyll a and b). Turbid The filter was fixed in 2% buffered glutaraldehyde for 1.5 h. Then the filter was washed with.1 M Liquid hromatiaceae medium as described in the course handout was inoculated with 1-5 ml Rhodamine labeled nucleotide probes of various big phylogenetic groups (kindly provided by proposed in her handout but the cells were spotted onto the slides in their original rnedium without For scanning electron microscopy (SEM) 1.5 ml of culture was collected on a.2 urn nucleopore cacodylate 3 times and cut into pieces which would fit onto SEM stubs (7 mm 2). The pieces were and berries from Sippewisset Marsh. filter, followed by filtering 2 ml 2% glutaraldehyde (.1 M acodylate buffered) through the same filter. Inocula for enrichments were purple sand from Oyster Pond as well as purple sand, purple fluff placed into bottle plates, overlaid with 1% agar and gassed with N2/ Liquid cultures in bottle plate flasks containing predators were placed in the dark at room 2. These bottle plates were METHODS cultures were fed with a neutralized H2S solution every day. Predators were visualized in an preying on hromatiurn as well as Thiocapsa. This study describes the occurrence of Vampirococcus or a Vampirococcus--like bacterium In several natural environments such as the Great Sippewisset Salt Marsh and Oyster Pond, purple INTRODUTION

3 days predators were observed by light microscopy in all cultures, with highest concentrations in the carrying several prcclatorçhains. The enrichment cultures underwent stages of succession: DAPL They attached to their host bacteria forming chains of up to 12 cells. Often one host cell was Enrichment cultures of rornatiaceae together with their predators were prepared. After a few enrichments from Oyster Pond purple sand and Sippewisset fluff. The predators stained brightly with mi-i-a The rhodamine labeled probes did not even light up the predators in the positive controls, predators and their association to the host cells. The scanning electron microscopy showed predators attached to the host cells: bacteria were incubated in the dark. However, during 3 weeks no plaques could be observed. suggesting problems with the probing procedure or the reagents. Both scanning and transmission electron microscopy were used to have a closer look at the stayed diffuse. However predators could not be observed. cells due to bacteria living in close association to hromatium cells, whereas the uninoculated control predator containing sample was added to pure hromatium cultures. This resulted in clumping of the lawn of sedimented purple sulfur bacteria could be detected after 1 days. the dark and no longer fed after predators had been observed microscopically. However no plaques in the Liquid ultures containing predators were also raised in bottle plate flasks, which were placed in In order to show plaques caused by the predators, filters with samples rich in purple sulfur In order to study the effect of a predator containing sample on a pure hromatium culture, fr 69, Lii , 5. RESULTS

4 ) Vampirococcus on Thiocapsa Vampirococcus on hromatiuni With transmission electron microscopy predators could be detected on hromatiunz vinosum as well as on Thiocapsa pfennigii: 4 nfl-i-v

5 DISUSSION flfl-j-c larke, K.3., Finlay, B.J., Vicente, E., Miracle, M.R. (1993) Arch.Microbiol. 159, Guerrero, R., Esteve, I., Pedros-Alio,., aju, N. (1987) Annals New York Academy of Sciences 53, LITERATURE Thiocapsa is new and shows that the host spectrum is wider than previously thought membrane in host and predator. While the predator has been described on homatium, its presence on clearly the presence of sulfur globules and membrane structures in the host cells, as well as outer also at other places where they might have fallen off during preparation. The ThM micrographs show the opposite. From the micrographs it can be derived that the observed bacteria are identical or at least closely related to the epibionts or predators described in larke et al. (1993) and Guerrero et al. (1987). The SEM micrographs show sucking points at the host cells where predators are attached, and et al, 1993) that the described cells are not predators but only epibionts. This study was not able to prove However lysis of the host cell and plaque formation could not be observed. It has been suggested (larke The observed predators obviously gain something from their attachment to the host cell.