Novel Approach for the Isolation and Identification of Sulfur Bacteria by Chemotaxis Assays

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1 Novel Approach for the Isolation and Identification of Sulfur Bacteria by Chemotaxis Assays Diego Giao García University of Aarhus Ny Munkegade , DK- 8000, Aarhus (Denmark) e- mails: / Abstract Chemotaxis behavior was studied in different brackish and marine sediments around Woods Hole, MA, USA. Two different attractants were tested: Thiosulfate, ranged concentrations from 10-1 to 10-5 M, and Sulfide, with concentration from 10-2 to 10-4 M. From all the tested environments/attractants, only the marsh Sipperwisset showed a reproducible chemotactic positive band. However, the technical complications in the recovery of the chemotactic bacteria made impossible to continue with the identification/community analysis, so the results of this project remain inconclusive. 1

2 Introduction Chemotaxis is the ability of bacteria to move towards/away different chemical substances in the environment (attractants versus repellants). The first study about bacterial chemotaxis, by Engelmann and Pfeffer 120 years ago, showed how some bacteria can be attracted or repelled by some compounds (Berg, 1975). Also, directed movement has been observed to a variety of physical conditions as temperature, ph or light (Grebe and Stock, 1998). Bacteria can sense chemical gradients by comparing different concentrations through their body length (direct) or by remembering the environment chemical concentrations and modifying their swimming according to them (indirect).(endres and Wingreen, 2008). This allows them to interact with their environment and react to changes around them. The lowest concentration reported that thrives chemotaxis is 3 nm (Sourjik and Wingreen, 2012). The bacterial chemotactic response consists in two parts: the movement towards a point and tumbling. When moving, the flagella are all propelling the bacteria in the same direction. However, when the bacteria sense a change in the chemical gradient, the movement is stopped and the bacteria tumbles before changing direction (Figure 1). 2

3 Figure 1. Chemotactic response to a increasing concentrations of attractants and repellents. (A) The typical chemotactic movement consist on run towards the attractant/away from the repellent and tumbling, when a change of direction is made. (B) Tendency to move towards a attractant gradient and away from a repellent gradient (Webre et al., 2003). The biochemical mechanism for chemotaxis is carried out by the so- called two- component regulatory system (Webre et al., 2003). They can be imagined as a nano- brain as a homology to a real brain, which translate sensory information into motor activity (Figure 2). The methylation- demethylation of a Methyl- accepting chemotaxis protein (MCP) is the starting point of the general chemotaxis response (Figure 3). Figure 2. The bacterial nanobrain. The protein network responsible of the chemotaxis response is situated in the bacterial pole (Adapted from (Levit et al., 2002)). 3

4 past concentrations and therefore, find the most favorable path for movement. This process is carried out by CheR and CheB respectively (Wadhams and Armitage, 2004). Sulphate- reducing bacteria can oxidize a variety of organic substrates coupled to the reduction of sulphate, with sulfide as the reduced sulfur compound. In marine sediment, this happens mostly in the oxic zone close to the surface, as these bacteria benefit from the presence of oxygen. On the other hand, sulfide- oxidizing bacteria will use sulfide as electron donor and reduce oxygen if possible. Therefore, sulfur bacteria have to react to different gradients of electron acceptors/donors for their metabolic requirements. Figure 3. The transmembrane methyl- accepting chemotaxis protein (MCP) regulates the chemotactic response. In the absence of an attractant, MCP stimulates the phosphorylation of CheA. The phosphate from CheA is then donated to CheY that will make the flagella to turn clockwise, stopping or tumbling the cell. CheZ, however can dephosphorylate the flagella producing long smooth movements. When MCP is attached to any attractant, the levels of phosporylated CheY drop, so the bacteria continue with a smooth directed movement towards the attractant gradient. The progressive methylation- demethilation of the cytoplasmic domain of the protein when MPC is attached to attractant allows the bacteria to remember The purpose of this project is to develop and test a new rapid test to isolate sulfur bacteria directly from environmental samples by means of attraction to different chemical substances, based on the traditional capillary assay (Adler, 1973; Adler and DAHL, 1967). 4

5 Material and Methods Sampling sites Sediment was collected in several places around Woods Hole, USA, between 5 th 10 th July 2013 according to Table 1. Table 1 Sampling sites, showing the name of the site, the location as GPS coordinates and the type of environment (Brackish or Marine) Site Location Type School/st./Marsh 41 /31'/34"/N,/70 /40'/5"/W Brackish Sippewisset 41 /34'/35"/N,/70 /38'/15"/W Marine Naushon/Island 41 /28'/2"/N,/70 /46'/56"/W Marine Trunk/River 41 /32'/5"/N,/70 /38'/28"/W BrackishKMarine Note that different kind of microbial communities were sampled at Trunk River: white mat (referred as white) and white filaments attached to algae in rocks at the mouth of Trunk River (referred as algae). Experimental setup Three different setups were used for the chemotaxis assessment: perforated bottle, microscopic chamber (Overmann, 2005) and stirring Erlenmayer flasks to analyze the interface water- sediment. In each type, different concentrations of attractant were tested in the range 10-1 to 10-5 M (Table 2). The capillaries (0.10 x 1 mm inner diameter, VITROTUBES, Vitrocom, NJ, USA) that showed a positive band of bacteria were centrifuged in PCR Eppendorf tubes at 6600 rpm, sealed with modeling clay to avoid contact between the capillary and its eluted content. 5

6 Table 2. Different attractants used by location, showing the tested concentrations, from 10-1 to 10-5 M for Thiosulfate, and 10-2 to 10-4 for Sulfide. Dashes (- ) indicate that no chemotaxis band was observed in the capillaries, check marks ( ) when they were observed, and N/A when it was not tested. Site Attractant Thiosulfate-(M) Sulfide-(M) 10 #1 10 #2 10 #3 10 #4 10 #5 10 #2 10 #3 10 #4 School-st-Marsh # # # # # # # # Sippewisset # # # # # # # Naushon-Island # # # # # # # # Trunk-River-(white) N/A N/A N/A N/A N/A # # # Trunk-River-(algae) N/A N/A N/A N/A N/A # # # DNA analysis The eluted samples were boiled to lysate the cells. Then, DNA was extracted and purified with a commercial kit using the manufacturer instructions (Wizard PCR Preps DNA Purification System, Promega, USA). Also, a PCR from the original sample was used to compare to the one from the capillary. CARD- FISH DAPI staining was used to check the viability of performing CARD- FISH, considering the low amount of cells containing in capillaries. If DAPI showed enough number of cells, the same filter was used to perform CARD- FISH as indicated by (Pernthaler and Pernthaler, 2007). This was planned to compare the amount and type of bacteria between the sample and the chemotactic positive band. Microscopy Pseudo- dark field microscopy was used to locate the chemotaxis bands in the capillaries (magnification 10x, phase contrast ring 3) using microscopes Zeiss 16 standard phase- contrast and Zeiss Axio Scope.A1 (Carl Zeiss, Germany) connected to a computer for the acquisition of images and videos. 6

7 Results & Discussion The only chemotaxis band appeared in samples from Sippewisset using Thiosulfate 10-3 M as attractant, both when the sediment was mixed with the water and after incubation of 2h with no stirring (Figure 4 and Figure 5). Figure 4. Chemotactic response in Sippewizzet sediment using Thiosulfate 10-3 M as attractant. The bacteria were sampled after mixing sediment with the overlying water. Figure 5. Chemotactic response in Sippewizzet sediment using Thiosulfate 10-3 M as attractant. The bacteria were sampled from the overlying water after 2 h of incubation. None of the other attractants and concentrations seemed to work. The low reproducibility of the chemotaxis experiments led to the failure of the project. 7

8 Moreover, the recovery of the cells inside the capillaries presented a lot of complications. Therefore, only a PCR could be performed and the electrophoresis gel showed a very weak band, indicating the small amount of bacteria that can be recovered from the capillaries. Conclusions and future perspectives The failure to extract a clean sample from the capillaries made impossible to carry out a precise identification/community analysis. Therefore, this project should be used as a pre- study of the chemotactic approach for the isolation of sulfur bacteria from marine sediment. Several points of the experimental design should be improved, being the elution of the capillaries the most critical point. The different approaches for this purpose during the last three weeks showed to be not good enough. Bigger capillaries should be tested, which would allow the elution of their content with a small syringe or air bulb like the ones employed with Pasteur pipettes. The identification and characterization of the community could be performed by the same CARD- FISH and by culturing in specific plates, which would allow performing a clone library and finding out the species of the chemotactic bacteria. Acknowledgements I am sincerely thankful to Lars Peter Nielsen for encourage me to attend this course and by his financial support. To HHMI MD for their financial aid. To Verena Salman for the good moments and guidance throughout this project. To Sara Kleindienst for having patience during the FISH experiment. To Ederson Jesus Conciençao and Piotr Starnawski for providing stinky sulfidic sediment to work with. To Vinh Nguyen for bringing joy during the darkest moments of this project. 8

9 References Adler, J. (1973). A method for measuring chemotaxis and use of the method to determine optimum conditions for chemotaxis by Escherichia coli. Journal of General Microbiology 74, Adler, J., and DAHL, M.M. (1967). A method for measuring the motility of bacteria and for comparing random and non- random motility. Journal of General Microbiology 46, Berg, H.C. (1975). Chemotaxis in bacteria. Annual Review of Biophysics and Bioengineering 4, Endres, R.G., and Wingreen, N.S. (2008). Accuracy of direct gradient sensing by single cells. Proceedings of the National Academy of Sciences 105, Grebe, T.W., and Stock, J. (1998). Bacterial chemotaxis: the five sensors of a bacterium. Current Biology 8, R154 R157. Levit, M.N., Grebe, T.W., and Stock, J.B. (2002). Organization of the receptor- kinase signaling array that regulates Escherichia coli chemotaxis. Journal of Biological Chemistry 277, Overmann, J. (2005). Chemotaxis and Behavioral Physiology of Not- Yet- Cultivated Microbes. In Methods in Enzymology, (Elsevier), pp Pernthaler, A., and Pernthaler, J. (2007). Fluorescence In Situ Hybridization for the Identification of Environmental Microbes. In Methods in Molecular Biology, E. Hilario, and J. Mackay, eds. (Humana Press), pp Sourjik, V., and Wingreen, N.S. (2012). Responding to chemical gradients: bacterial chemotaxis. Current Opinion in Cell Biology 24, Wadhams, G.H., and Armitage, J.P. (2004). Making sense of it all: bacterial chemotaxis. Nat Rev Mol Cell Biol 5, Webre, D.J., Wolanin, P.M., and Stock, J.B. (2003). Bacterial chemotaxis. Current Biology 13, R47 R49. 9