Polymer-degrading bacteria associated with the brown algae Fucus spp.

Transcription

1 Polymer-degrading bacteria associated with the brown algae Fucus spp. Elke Allers Carried out as an Individual Project within the Microbial Diversity Course 2006 at the Marine Biological Laboratory in Woods Hole

2 Abstract The attempt to isolate polymer-degrading bacteria from the brown algae Fucus spp. resulted in 20 strains which, according to 16S rrna gene sequence analysis, were members of the Alpha- and Gammaproteobacteria and the Bacteroidetes. The isolate collection was dominated by strains closely related to Vibrio alginolyticus. In order to compare growth kinetics and C-source utilization 5 strains were chosen for further analysis. The gammaproteobacterial strains Pseudoalteromonas sp., Alteromonas sp. and a relative of Cellvibrio reached higher cell yields than the Bacteroidetes strains Cellulophaga sp. 2 and 5c, no matter whether they were grown on polysaccharides or simple sugars. Pseudoalteromonas sp. was identified as the most successful utilizer of fucoidan (an algal-derived polysaccharide), had the shortest lag phase (3 h) and the shortest doubling time (0.3 h on Glucose). In contrast, both Cellulophaga strains displayed lag phases of at least 12 h on any substrate studied, and moreover did not start doubling at all on fucoidan before the experiment was over. The results suggest, that within the strains isolated two different ecological strategies have been observed: The gammaproteobacterial strains being ready to take available C-sources, and the Cellulophaga strains rather taking time to adapt

3 1. Introduction The Cytophagales are a very diverse group within the Bacteroidetes. They inhabit many different types of habitats, ranging from soil to water, from fresh water to sea water, from animal dung to decaying plant-material, from free-living to an attached live style. Especially for the marine world they are known for being able to degrade complex substrates like agar (Balows et al. 1992). Another natural and in the same time biotechnically relevant polymer is fucoidan. It is derived from seaweed, e.g. Fucus spp., and consists a fucose backbone. Since it is a natural compound, one would expect bacteria to exist which can degrade and utilize the fucoidan. These bacteria and their fucanase activity would provide a source of the degrading enzyme and thereby a way to produce bioactive compounds, which are e.g. involved in blocking infections by certain viruses like herpes simplex and HIV (Descamps et al. 2006). The idea of this study was to attempt the isolation of polymer-degrading bacteria and to compare their metabolic traits in terms of polymer and simple sugar utilization. 2. Material & Methods Isolation. The algal thalli were all collected at the same day, either directly at Garbage Beach, Woods Hole (decaying material; anaerobic and aerobic) or further out at the dock (living material; aerobic). Each thallus was put into sterile Seawater Base (see below) and blended until everything was a slurry. One hundred µl of this slurry were plated onto spread-plates either pure or in dilutions 1:10, 1:100, and 1:1000. As soon as growth became apparent on the plates, colonies were re-streaked for isolation. They were transferred at least 3 times before they were considered a pure isolate. The media. 1 x Seawater Base was the basis of all media used in this study (course hand out 2006). According to the Modular Medium Approach presented during the course (J. Leadbetter) nutrients and substrates were added (see Tab. Aa.1 and Aa.2) adjusted to the needs. The isolation was carried out on plates, whereas growth experiments were all done in liquid culture in tubes set up on a shaker at 30 C. 5 ml of the Medium basis were dispensed into culture tubes. Agar and fucoidan were added - 2 -

4 to the tubes before autoclaving, the simple sugars were sterile-filtered through 0.2 µ and then added to the already autoclaved medium. Colony-PCR. The analysis of the 16S rrna gene sequences of all isolates was carried out according to the course hand out MD2006 (chapter 11. Phylogentic analysis of bacterial isolates, see Appendix). Colonies were either picked directly into the PCRmix or into 10 µl of PCR water. The latter was vortexed and 1 µl was added to the PCR. For full sequences 3 different primers were run in the sequencing reaction for the same PCR product: 8F, 519F and 1492R. Sequence data was checked for next relatives using the Blastn tool ( and phylogenetic analysis was done within the software package ARB. Growth kinetics. Growth in general and the exponential growth phase in particular were monitored by OD600 measurements on a photometer against a medium blank. Doubling times were calculated according to the following equation: Td = ln2/µ, where Td is the doubling time, and µ is the slope of the trend line added to a density/time plot in exponential phase. Degradation and utilization of polymers and utilization of sugars. HPLC analysis was carried out according to the course hand out (see Appendix). CARD-FISH on environmental samples. The bacteria present in the algal slurry were studied by applying CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization) according to Pernthaler et al. (2004). The following probes were used: EUBI-III (Amann et al. 1990, Daims et al. 1999) to test the overall detection rate, CF319a (Manz et al. 1996) specific for Cytophacga-Flavobacteria, ALT1413 (Eilers et al. 2000) targeting representatives of the Alteromonadales. A nonsense probe NON338 (Wallner et al. 1993) was applied as a control for specificity. 3. Results During this study 21 bacterial strains were isolated and identified by 16S rrna gene sequence analysis (Tab. Aa.3, see Appendix). Most of the isolates belong to the Gammaproteobacteria and within these they find their closest relatives in representatives of the Alteromonas, Pseudoalteromonas and Vibrio. One isolate showed a similarity of 100% to the Alphaproteobacterium Stappia - 3 -

5 aggregata. Another two isolates were identified as close relatives of Cellulophaga lytica. Five isolates were picked for further analysis. They are indicated in bold. Microscopic images are provided in the Appendix, too (Fig. Ab.1). All isolates displayed specific substrate usage. In terms of utilization of the tested polysaccharides, it became obvious that agar as a C-source as opposed to fucoidan leads to higher yields in cell density or to growth, at all. Generally, all strains isolated in this study grew well with agar as sole carbon source. However, there were distinct differences in the isolates affinity to this substrate. Pseudoalteromonas sp. was the fasted to respond with growth and reached alltogether with the two other gammaproteobacterial isolates the highest yields ranging between 0.4 and 0.5 OD600 (Tab. Aa.4 and Fig. Ab.2, Appendix). On the contrary, the Cellulophaga isolates reached densities of ~0.25 and ~0.1, respectively. Moreover, their lag phase as well as the one of Alteromonas sp. lasted for at least 12 hours. Unfortunately, at this point of time the HPLC data has not been analyzed yet. By applying CARD-FISH it could be shown that a) the detection rate is poor and further method optimization is necessary, b) CF319a- and ALT1413-targeted organisms are associated with Fucus spp.. 4. Discussion The idea of this study was stimulated by the fact that marine Cytophaga are capable of polymer-degradation (Balows et al. 1992). It was shown by this non-directed isolation approach, that, in addition to Cytophaga, representatives of the Alpha- and Gammaproteobacteria inhabit the brown algae Fucus spp.. Most of the isolates belong to the Gammaproteobacteria, which matches findings of previous studies, in which the term opportunistic bacteria was suggested for the fast-responding representatives of Alteromonas sp., Pseudoalteromonas sp., and Vibrio sp.. The isolates of this study were obtained from colonies which were the first to appear on spread plates. It is therefore not surprising that supposed fast-responding and/or fastgrowing organisms dominate the picture. Technically, all testing for C-sources was carried out with single C-sources at a time. However, to bring cells to grow at all, trace amounts of yeast extract (YE) and - 4 -

6 tryptone (T) had to be added to the medium. The sole addition of YE + T to the basic medium without any other C-source did not result in significant growth. This was tested for Alteromonas sp. and Pseudoalteromonas sp. (data not shown). In comparison to agar, fucoidan has not been used for building biomass by most of the isolates. Cellulophaga sp. strain 5c showed not a slightest trace of growth after 44 hrs. Inspite of being technically a well available compound, it appears to be not as utilizable as agar. Enzymes for the degradation process might be missing. The fucose, on the contrary, triggered growth, even though not always as pronounced as galactose or glucose. The most efficient - given you consider the doubling time Td as a measure of efficient substrate utilization isolate was Pseudoalteromonas sp. with a Td of 0.3 h in Glucose and 0.7 h and 0.6 h in Galactose and Glucose, respectively. This again, supports other observations of Pseudoalteromonas spp. being a bacterium with an opportunistic life style. The attempt to check for different substrate and nutrient condition preferences in North Sea bacterioplankton in dilution enrichments ended in all treatments in communities dominated by Pseudoalteromonas (unpublished). Cellulophaga spp. on the contrary might prefer an attached-living lifestyle and is thus in the given experimental setup not growing under optimal conditions. 5. References Amann R, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990): Combination of 16S rrna-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. AEM 56 (6) Balows A, Trüpfer HG, Dwarkin M, Harder W, Schleifer K-H (1992): The Prokaryotes. Springer Verlag, New York Daims H, Bruhl A, Amann R, Schleifer K-H, Wagner M (1990): The domain-specific probe EUB338 is insufficient fort he detection of all bacteria: Development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22,

7 Descamps V, Colin S, Lahaye M, Jam M, Richard C, Potin P, Barbeyron T, Yvin J-C, Kloareg B (2006): Isolation and culture of a marine bacterium degrading the sulfated fucans from marine brown algae. Mar. Biotech. 8, Eilers H, Pernthaler J, Gloeckner FO, Amann R (2000): Culturabilty and in situ abundance of pelagic bacteria from the North Sea. AEM 66 (7) Manz W, Amann R, Ludwig, W, Vancanneyt M, Schleifer K-H(1996): Application of a suite of 16S rrna-specific oligonucleotide probes designed to investigate bacteria of the phylum cytophaga-flavobacter-bacteroidetes in the natural environment. Microbiology 142, Microbial Diversity 2006 course hand out Pernthaler A, Pernthaler J, Amann, R (2004): Sensitive multi-color fluorescence in situ hybridization for the identification of environmental microorganisms. In G. Kowalchuk (ed.), Molecular Microbial Ecology Manual. Kluwer Academic Press, Dordrecht / Boston / London. Wallner G, Amann R (1993): Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure. AEM 59,

8 Appendix Tab. Aa.1: General overview - Media used in this study. "Agar-Agar" "Fucoidan/Gel-rite" SW agar Tryptone (Difco) See SW agar, instead of agar Yeast extract (Difco) use 0.3% fucoidan Agar, washed 1,5 % (or without for liquid) and 1,0% gelrite in seawater base adjust ph to 7.2 autoclave cool to 60 degree C add 50 µg/ml cycloheximide and pour plates 1 x Seawater base water 20 l NaCl 400 g MgCl 2 *6H 2 O 60 g CaCl 2 *2H 2 O 3 g KCl 10 g keep in clean Nalgene platic bottle, not sterile C-sources polysaccharides agar or fucoidan 10 mm galactose or fucose or glucose buffer 1 M MOPS, ph 7,2 final: 5 mm N Ammonium Chloride final: 5 mm, from 100 x stock P 100 x Phosphate Solution, 150 mm, ph 7,2 Potassium Phosphate S 1 M Sodium Sulfate final: 0.25 mm finfal: 1.5 mm, from 150 mm stock TE 1000 x HCl-Dissolved Trace Elemts Stock Solution to 1 l add 0.1 ml

9 Tab Aa.2: The medium used without any C-sources. Medium in 1 x Seawater base 1 x sea water base 800 ml Tryptone (T) 0,1 g Yeast extract (YE) 0,1 g adjust ph to M MOPS, ph 7,2 5 ml 5 M NH4Cl 1 ml 150 mm KPO4 10 ml 1 M H2SO4 0,25 ml 1000x TE solution 0,1 ml filll up to 1 l with 1 x Seawater base autoclave and cool down to 60 C 1000x Cyclohexamide 1 ml

10 Tab. Aa.3: Isolates and their closest relatives according to distance matrix analysis. Species in bold were chosen for further analysis. # of closest relative # Habitat Cultiv. nucleotides isolate described isolate a 1223 Vibrio chagasii 98,6 6-3a 2 b 602 Vibrio sp. SR2 99, a 946 Vibrio alginolyticus 100,0 6-3b 2 b ,0 Vibrio alginolyticus 100,0 6-1a 1 b ,7 Vibrio alginolyticus 99,6 6-3c 2 b 1393 Vibrio midae 98, a a 99,6 Vibrio natriegens 99, b 1188 Vibrio sp. HB-8 99,6 Vibrio fortis 99,9 6-5b 3 b ,0 Alteromonas stellaepolaris 99, b b 100,0 6-5a 3 b 1354 Alteromonas sp. R10SW13 99,3 6-1b 1 b 1372 Pseudoalteromonas sp. NJ345 99, b 701 Pseudoalteromonas sp. NJ345 99, b 1373 Cobetia sp ,0 6-7b 1 b b 776 Halomonas sp. 100,0 Alteromonas stellaepolaris 99,9 Alteromonas macleodii 99,0 Pseudoalteromonas atlantica 97,9 Pseudoalteromonas atlantica 99,0 Cobetia marina 100,0 Halomonas elongata 94,8 6-4b 2 b 657 gammaproteobacterium 97,1 close to Cellvibrio 6-7a 1 b 1312 Stappia sp. 100,0 Stappia aggregata 100,0 6-5c 3 a 698 Cellulophaga lytica 99, b 1339 Cellulophaga lytica 99,9 1 anaerobically decaying brown algae 2 aerobically decaying brown algae 3 intact brown algae a anaerobic

11 Tab. Aa.4: Cell densities of 5 isolates as measured in growth experiments on the polymers agar and fucoidan. Agar Fucoidan time [h] Pseudo- Cellulo- Cellvibrio' Altero- Cellulo- Pseudo- Cellulo- Cellvibrio' Altero- Celluloalteromonas phaga monas phaga alteromonas phaga monas phaga 0 0,011 0,004 0,045 0,033-0,027 0,003 0,016 0,015 0,008 0, ,001 0,005 0,007 0,010-0,021 0,004 0,016 0,012 0,008 0, ,006 0,001 0,006 0,010-0,021 0,007 0,015 0,011 0,009 0, ,013-0,001 0,006 0,007-0,024 0,019 0,015 0,014 0,011 0, ,071 0,002 0,009 0,010-0,021 0,078 0,015 0,013 0,008 0, ,141 0,002 0,013 0,009-0,021 0,140 0,015 0,013 0,006 0, ,263 0,002 0,017 0,007-0,019 0,186 0,016 0,018 0,009 0, ,332 0,000 0,025 0,006-0,023 0,181 0,016 0,014 0,008 0, ,366-0,001 0,048 0,004-0,019 0,173 0,015 0,013 0,006 0, ,375 0,001 0,077 0,006-0,012 0,167 0,015 0,018 0,008 0, ,374 0,003 0,104 0,005-0,011 0,173 0,018 0,019 0,008 0, ,372 0,012 0,132 0,009 0,002 0,170 0,016 0,022 0,012 0, ,374 0,020 0,154 0,008 0,009 0,170 0,016 0,029 0,008 0, ,367 0,037 0,180 0,011 0,018 0,163 0,014 0,022 0,009 0, ,360 0,049 0,201 0,014 0,027 0,163 0,012 0,028 0,008 0, x 0,077 0,255 0,043 0,043 x 0,018 0,036 0,007 0, x 0,109 0,312 0,071 0,058 x 0,017 0,039 0,007 0, x 0,135 0,350 0,118 0,067 x 0,016 0,044 0,007 0, x 0,154 0,377 0,215 0,075 x 0,013 0,058 0,010 0, x 0,176 0,405 0,351 0,089 x 0,018 0,051 0,009 0, x 0,192 0,421 0,417 0,092 x 0,016 0,053 0,009 0, x 0,210 0,426 0,435 0,103 x 0,017 0,058 0,009 0, x 0,246 0,455 0,462 0,108 x 0,017 0,046 0,029 0, x x x x x 0,119 0,024 0,065 0,025 0, x x x x x x 0,033 0,064 x x

12 Tab. Aa.5: Cell densities of 5 isolates as measured in growth experiments on the sugar galactose, fucose, and glucose. Galactose Fucose Glucose time [h] Pseudo- Cellulo- Cellvibrio' Altero- Cellulo- Pseudo- Cellulo- Cellvibrio' Altero- Cellulo- Pseudo- Cellulo- Cellvibrio' Altero- Celluloalteromonas phaga monas phaga alteromonas phaga monas phaga alteromonas phaga monas phaga 0,0 0,010 0,006 0,006 0,003 0,003 0,001 0,001 0,001 0,005-0,001-0,003-0,002 0,006 0,001-0,003 1,3 0,013 0,007 0,005 0,009 0,002 0,010 0,005 0,005 0,018 0,006 0,003 0,001 0,009 0,006-0,001 2,2 0,054 0,010 0,006 0,018 0,009 0,035 0,001 0,002 0,012-0,002 0,047 0,009 0,020 0,025 0,009 2,7 0,110 0,010 0,006 0,031 0,006 0,076 0,001 0,003 0,022-0,001 0,106 0,009 0,019 0,039 0,007 3,2 0,185 0,009 0,010 0,052 0,003 0,112 0,002 0,007 0,031 0,000 0,158 0,007 0,024 0,058 0,006 3,7 0,258 0,007 0,017 0,066 0,006 0,146 0,006 0,011 0,042 0,002 0,199 0,010 0,035 0,078 0,005 4,4 0,355 0,010 0,023 0,112 0,003 0,158 0,008 0,017 0,075 0,006 0,242 0,011 0,057 0,126 0,006 5,1 0,444 0,010 0,045 0,185 0,003 0,171 0,008 0,035 0,101 0,005 0,259 0,009 0,101 0,219 0,004 5,8 0,496 0,015 0,068 0,294 0,007 0,164 0,004 0,043 0,135 0,004 0,290 0,009 0,158 0,315 0,009 6,8 0,558 0,013 0,103 0,433 0,003 0,165 0,006 0,066 0,158 0,004 0,323 0,007 0,320 0,474 0,008 7,8 0,599 0,013 0,184 0,519 0,008 0,165 0,007 0,086 0,164 0,008 0,304 0,011 0,428 0,580 0,006 11,8 0,709 0,017 0,516 0,825 0,008 0,168 0,012 0,116 0,178 0,014 0,425 0,017 0,554 0,814 0,018 13,0 0,741 0,018 0,570 0,893 0,010 0,157 0,015 0,126 0,179 0,017 0,429 0,028 0,597 0,887 0,023 14,2 0,750 0,032 0,613 0,953 0,014 0,155 0,025 0,134 0,181 0,025 0,414 0,033 0,611 0,936 0,037 15,3 0,753 0,048 0,634 1,004 0,026 0,160 0,034 0,139 0,180 0,045 0,442 0,057 0,623 0,995 0,057 33,3 0,518 0,076 0,546 1,128 0,046 0,130 0,103 0,147 0,178 0,129 0,615 0,151 0,593 0,852 0,113

13 Tab. Aa.6: Doubling times Td of all 5 isolates. Isolate C-source Td [h] Alteromonas sp. agar 1,4 Galactose 0,9 Fucose 1,0 Glucose 1,0 Pseudoalteromonas sp. Agar 0,9 Fucoidan 0,8 Galactose 0,7 Fucose 0,6 Glucose 0,3 Cellvibrio-like Agar 1,8 Fucoidan 3,9 Galactose 0,9 Fucose 0,7 Glucose 1,1 Cellulophaga sp. 5c Agar 1,1 Fucoidan b.d. Galactose b.d. Fucose b.d. Glucose b.d. Cellulophaga sp. 2 Agar 1,6 2 Fucoidan b.d. Galactose b.d. Fucose b.d. Glucose b.d. b.d. below detection

14 0,90 0,70 Galactose Fucose Glucose Pseudoalteromonas 1,20 1,00 0,80 Galactose Fucose Glucose Alteromonas OD 600 0,50 0,30 OD 600 0,60 0,40 0,10 0,20-0, , time [h] time [h] OD 600 0,80 0,60 0,40 Galactose Fucose Glucose 'Cellvibrio relative' Fig. Ab.3: Growth curves of the 5 isoaltes growing on either galactose, fucose or glucose. 0,20 0, time [h] 0,16 0,12 Galactose Fucose Glucose Cellulophaga (2) 0,14 0,12 0,10 Galactose Fucose Glucose Cellulophaga (5c) OD 600 0,08 OD 600 0,08 0,06 0,04 0,04 0,02 0, time [h] 0, ,02 time [h]

15 Fig. Ab.2: Growth curves of 5 isolates growing on the polymers agar or fucoidan, respectively. 0,400 0,300 Agar Fucoidan 0,500 0,400 Agar Fucoidan OD600 0,200 0,100 OD600 0,300 0,200 Alteromonas Pseudoalteromonas 0, ]time [h 0,100 0, ] time [h OD600 0,500 0,400 0,300 0,200 Agar Fucoidan 'Cellvibrio relative' OD600 0,300 0,200 0,100 Agar Fucoidan Cellulophaga (2) 0,100 0, ]time [h 0, ,100 ]time [h 0,200 0,100 Agar Fucoidan Cellulophaga (5c) OD600 0, ,100 ]time [h

16 A Fig. Ab.1: The isolates which were chaosen for further analysis. A Alteromonas sp., B Pseudoalteromonas sp. C Cellvibrio-like, D + E Cellulophaga spp. B C ll D E E

17 Appendix C Protocols

18 I would like to thank the faculty and the TAs for making this course the great experience it was. Especially, Jean and Kou-San were more than patient whenever I chose the complicated way to think about the simplest things. Thank you Team 2 you are special, you are different, and you put up with me! Last but not least, thank you to the whole class Microbial Diversity It was a challenging and rewarding experience meeting all of you! I appreciate the funding through the Gordon and Betty Moore Foundation and the Daniel and Edith Grosch Fund. Without them I would not have had this special summer in Woods Hole.