Which Mechanisms do Bacteria use for Polysaccharide Utilization?
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Transcript: English(auto-generated)
00:04
In our research, we're looking at the surface ocean where carbon in form of CO2 is photosynthetically fixed into organic biomass. This organic biomass can then constitute for up to 50% of polysaccharides, which are long-chain sugars, rather structurally complex molecules that can consist of different
00:24
types of monosaccharides that are linked into polymeric chains. Once these polysaccharides are released into the environment, they contribute to a vast pool of organic matter, which is the source of food or nutrition for heterotrophic bacteria. These bacteria are the main recyclers of organic carbon within the ocean.
00:45
And our research questions focus on the degradative potential of bacteria on these complex substrates such as polysaccharides. So far there are two different ways of bacterial polysaccharide utilization known. One is the extracellular hydrolysis, where the polysaccharide is degraded into smaller subunits
01:05
outside of the cell, and only these subunits are then small enough to be taken up by the cells. In contrast, the selfish uptake mechanism means that the whole polysaccharide as such is taken up into the bacterial periplasm, where it is then further degraded.
01:22
We are mainly interested in looking into the balance of which of both mechanisms is used and which bacteria have the capability to use these mechanisms. We can look at both the selfish uptake but also the extracellular hydrolysis of polysaccharides
01:43
within just one experimental setup. And therefore we use fluorescently labeled polysaccharides in short flaps, which are fed and incubated with the bacterial community. Then we can quantify the amount of extracellular hydrolysis. And on the other hand, visualize the selfish uptake based on a fluorescent feature of the
02:05
cells that they gained by taking up the fluorescent polysaccharide. We can then use the fluorescent characteristics of the cells that used the selfish uptake mechanisms and combine it with other tools to combine the function together with identity. Therefore we can use, for example, fluorescence in Z2 hybridization, where bacteria are
02:25
stained based on their characteristics, 16S rRNA, and then look which bacteria have the capability to use the selfish uptake mechanism. This is not yet possible for the extracellular hydrolysis, but with the extracellular hydrolysis
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we have the positive effect that we here have a quantitative measure, which is not yet present for the selfish uptake. So finally we could then measure extracellular hydrolysis rate and on the other hand also look at the proportion of the total community that used the selfish uptake mechanism.
03:03
We were trying to look at the bacterial polysaccharide utilization in a seasonal study where we went to surface ocean water from Helgeland and looked at the balance between the extracellular hydrolysis and the selfish uptake over the course of the year. Our findings were on the one hand side on the balance between both mechanisms and
03:25
on the other hand we could identify some bacteria that used one or the other mechanism. We used a substrate, laminarine, xylan, and chondroitin, which are three polysaccharides of different complexity that all occur in the marine environment and by incubating
03:42
bacteria with these three fluorescently labeled polysaccharides we could see that actually in the very beginning of the year, so in winter and spring, there was little polysaccharide utilization through both mechanisms, but then going on to summer we could see that there's a rapid increase in the selfish uptake of laminarine and xylan.
04:02
It was surprising to see that already 60% of all cells could use laminarine as a substrate in summer and that actually 30% of the cells use xylan through the selfish mechanism. Trying to identify these bacteria, we first looked through a microscope and could see that
04:22
the accumulation of the polysaccharide that was taken up through the selfish mechanism, the accumulation pattern looked actually very diverse over the community, which gave us the impression that we here have a multitude of different bacteria using this mechanism. To identify them, we in first place used fluorescence in situ hybridization where
04:43
we could identify mainly gamma proteobacteria and bacteriodetes to use the selfish mechanism for laminarine and xylan, but surprisingly by sorting bacteria based on their fluorescence laminarine signature, we could also identify that some of these bacteria belong to the
05:03
verrucomicrobial family perispheraceae, a group where we could never see before that they used the selfish uptake mechanism. Going from mainly selfish uptake in summer to autumn where we could mainly see extracellular hydrolysis of xylan and chondroitin, and here based on bulk community analysis we could see that solely in the chondroitin incubation
05:26
there was an increase in abundance of the bacteriodetyl genus Flavicella, and with that we speculate as Flavicella was highest in abundance at the same time where we saw highest chondroitin extracellular hydrolysis rate, that these are actually
05:42
the main bacterial group to hydrolyze this complex substrate. We were really surprised by the finding that within just very few hours in summer about 60% of the whole bacterial community were capable of taking up laminarine through the selfish
06:02
mechanism. This is even more striking when considering that the selfish mechanism was only visualized for the first time very few years ago. Furthermore we could also see that both mechanisms, the selfish uptake and the extracellular hydrolysis are not used in parallel at the same proportions but that there must be conditions that favor either one or the other. And going back
06:26
to the carbon that we are interested in and how the both mechanisms contribute to the carbon availability to the whole bacterial community, we can see that with the selfish uptake mechanism where the whole polysaccharide is taken up as once, the carbon and energy source here is
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removed from the bacterial plankton community, but when looking at the extracellular hydrolysis where the polysaccharide is first degraded into sort of say digestible portions, these can be also then used by other members of the community that sort of say steal away the food from the
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bacteria that actively produce the enzymes to do the first initial hydrolysis. When we now look into the summer results where we have mainly selfish uptake, there must be the right to use the selfish uptake mechanisms for a wide range of the bacteria. Going further to the
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mostly extracellular hydrolysis of chondroitin and xylan in autumn, we could here see a specialist with Flavicella and this finally lets us speculate that here Flavicella was maybe sitting on aggregates or particles, so a place where the polysaccharide could occur in high abundances
07:43
and the diffusive loss of the hydrolysis products might actually be reduced. In our seasonal study, we could now see that selfish uptake is preferentially used in summer whereas we could find mainly extracellular hydrolysis in autumn. When now looking at the
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cost of using one or the other, we need to balance out how much energy and cost is put by bacterium into the production of the enzymes to use this mechanism versus the energy and carbon they gain from utilizing the polysaccharide. This gives us the challenge to also understand
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not only the rate of polysaccharide hydrolysis extracellular but being able to quantify the amount of polysaccharide that is taken up through the selfish mechanism. Furthermore, the question arises, are bacteria switching between the use of both mechanisms? Are they capable to do so
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or do they only specialize on the use of one mechanism? As an outlook, there's still the challenge to really understand the interaction between the bacteria and environmental conditions but also the presence of the substrate and I think with the fluorescently labeled polysaccharide
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incubation, we have a great method to look at both mechanisms within one methodological setup but also combine this method with other tools, molecular tools for example, to get all the different diverse aspects of bacterial polysaccharide utilization which can then not
09:21
only be applied to an environmental complex sample but could for example also be used for individual bacterial isolates.