Is Natural Competence Used to Source Phosphates in Acinetobacter Bacteria?

Marissa Zintel Author

07/26/2021 Added

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DNA-supplementation experiments were performed in Acinetobacter nosocomialis mutant strains to determine if the ability to take up DNA to use as a phosphate source was due to the possession of natural competence.

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[00:00:02.210]Hi, I'm Marissa Zintel
[00:00:03.710]and this summer I've been working through the bioenergy REU to answer the
[00:00:06.890]question of whether natural competence is involved in the uptake of DNA as a
[00:00:10.700]source of phosphates in Acinetobacter.
[00:00:14.210]So the model organism that we're using to answer
[00:00:17.360]this question is Acinetobacter nosocomialis,
[00:00:19.010]but we anticipate that this research will have broad implications for the Acinetobacter
[00:00:22.280]genus as a whole. Acinetobacter species are often used as model
[00:00:26.390]organisms to study type IV filaments,
[00:00:28.670]which are called type IV pili in Acinetobacter,
[00:00:31.400]and which you can see enlarged here on the right.
[00:00:33.770]These are the structures that take up DNA from the environment in natural
[00:00:37.220]competence. Acinetobacter species are also used to study biofilms,
[00:00:41.360]which can be particularly interesting for bioremediation: Acinetobacter
[00:00:44.870]has been proposed as an anchoring agent to use in bio-reactor systems where
[00:00:48.890]Acinetobacter forms a biofilm matrix,
[00:00:51.440]which can then be inoculated with other microbial remediators and then have
[00:00:55.410]wastewater run through that resulting biofilm like a filter.
[00:00:59.930]Acinetobacter are also bioremediators just by themselves because they can metabolize
[00:01:04.280]contaminating hydrocarbons,
[00:01:06.170]so they've been used in the past to clean up oil spills. In a medical context,
[00:01:10.640]Acinetobacter baumannii is well known as an opportunistic pathogen
[00:01:14.210]that's a huge problem, because
[00:01:16.470]since Acinetobacter is naturally competent
[00:01:18.350]it can easily acquire antibiotic resistance genes in hospital environments and
[00:01:22.490]infect immunocompromised patients,
[00:01:24.350]which quickly becomes a big challenge to treat.
[00:01:27.920]We are more interested in directly studying the natural competence abilities of
[00:01:31.580]Acinetobacter. So these are two depictions of the type IV pili,
[00:01:35.330]which are responsible for taking up DNA. Very generally, first,
[00:01:39.050]the pili bind DNA,
[00:01:40.880]then DNA is brought through the outer membrane through some type of channel,
[00:01:45.410]then a single strand is translocated through the inner membrane where it can
[00:01:48.770]be integrated into the cell's genome through horizontal gene transfer,
[00:01:53.570]or it can be broken down in its component parts, such as phosphates.
[00:01:58.460]And these pilin structures are composed primarily of the PilA subunit,
[00:02:01.580]in blue here, which makes up about 99% of the pilin.
[00:02:07.440]So one of the reasons that we're interested in linking natural competence and
[00:02:10.260]phosphate uptake is because the evolution of natural competence is not clear.
[00:02:14.620]Horizontal gene transfer makes sense for the proliferation of natural competence,
[00:02:18.290]because organisms that are naturally competent can easily take up antibiotic
[00:02:21.732]resistance genes and then survive in environments where antibiotics are used.
[00:02:25.955]However, this doesn't make sense for the early evolution of natural competence, because
[00:02:29.345]transformation events are very rare and they're not specific for beneficial
[00:02:32.720]genes - meaning naturally competent organisms are more likely to pick up random
[00:02:37.110]non-coding bits of DNA or genes that they already have rather than specific,
[00:02:41.240]useful genes like antibiotic resistance.
[00:02:44.150]So some researchers proposed that maybe DNA could be used for all of the
[00:02:47.570]component parts: carbon, nitrogen, phosphorus. However,
[00:02:51.080]it takes a lot of energy to make type IV pili, and overall more energy is
[00:02:54.960]lost making this natural competence machinery than a cell could possibly gain from
[00:02:59.700]a strand of DNA.
[00:03:01.570]So our theory is that perhaps these naturally competent organisms evolved from
[00:03:05.890]someplace where phosphate was scarce and they took up DNA to use as an alternate
[00:03:10.480]phosphate source, which enabled them to survive.
[00:03:13.660]So in order to determine if this was a valid theory,
[00:03:16.180]we tested this claim using ΔPilA and +PilA mutant strains,
[00:03:20.360]The PilA gene encodes
[00:03:21.810]that major subunit of type IV Pili and ΔPilA is a knockout mutant where
[00:03:27.070]the PilA gene has been deleted.
[00:03:28.600]so that the bacteria cannot make type IV pili, while
[00:03:31.840]+PilA is a complement mutant that has the PilA gene reinserted elsewhere
[00:03:35.490]in the genome, meaning it has functioning pili and is naturally competent.
[00:03:40.560]So in order to go through this process,
[00:03:42.710]we based our experiments on a recent paper that showed that Haloarchaea
[00:03:45.360]could live on DNA as a source of phosphates.
[00:03:48.643]Our first step then was to adapt the Haloarchaea media to Acinetobacter,
[00:03:52.553]then we extracted genomic DNA from E. coli and determined the exact DNA
[00:03:56.764]concentration that would be required for Acinetobacter to grow.
[00:04:00.300]And we determined this for liquid media,
[00:04:02.180]which was used in those Haloarchaea experiments,
[00:04:04.434]but also solid media because Acinetobacter grows more naturally on the solid
[00:04:08.640]media. Then once we had these concentrations determined,
[00:04:11.670]we could compare the effect of natural competence using our strains. M2,
[00:04:15.900](which is the wild type strain) ΔPilA
[00:04:18.200](which is not naturally competent) and +PilA (which is naturally competent).
[00:04:23.460]This project is still ongoing,
[00:04:24.913]due to limitations of time and access to ordering materials,
[00:04:27.886]but we've made significant progress through those methods. As you can see,
[00:04:31.400]our liquid media experiments have been the most successful.
[00:04:34.219]We see that bacteria can grow when media is supplemented with phosphates,
[00:04:37.380]but cannot grow when the media is not supplemented with phosphates.
[00:04:40.524]And we've determined that the wild type M2 strain,
[00:04:42.969]which we've been using as our initial test strain can grow on at least 300
[00:04:47.030]nanograms per microliter of DNA as a source of phosphates.
[00:04:50.653]In order to validate those bacterial growths that we observed in liquid media,
[00:04:54.763]we performed growth curves, measuring the optical density of each culture,
[00:04:58.420]condition, testing a concentration of DNA,
[00:05:00.628]along with our positive and negative phosphate controls,
[00:05:03.740]which I'll go through one by one and then circle back to those tables.
[00:05:08.070]In our positive control,
[00:05:09.720]we can see the standard logistic curve that bacterial cultures follow, showing
[00:05:13.790]that when phosphates were added
[00:05:15.260]the bacteria grew normally. Then in our negative control
[00:05:18.788]we see the optical density drop to zero,
[00:05:21.017]so there was no growth when phosphates were not added.
[00:05:24.810]And in our DNA experiment,
[00:05:26.130]we saw that bacteria could not grow on 150 nanograms per
[00:05:29.910]microliter of DNA,
[00:05:31.401]meaning that the bacteria did not get enough phosphates from this concentration of DNA.
[00:05:37.310]So here's our 150 nanograms per microliter band that we tested. However,
[00:05:41.900]this M2 box is yellow because in a previous, overnight culture
[00:05:45.570](without a growth curve)
[00:05:47.132]we did see growth in the M2 strain with 150 nanograms per microliter of DNA.
[00:05:52.240]So more replicates are required to resolve this contradiction,
[00:05:55.740]to see if this is just a borderline concentration for growth,
[00:05:58.740]or if the growth curve initial inoculum was too high,
[00:06:01.950]or maybe just the growth curve data collection process was too harsh on the
[00:06:05.500]bacteria. Then in solid media,
[00:06:07.710]we're still working to optimize the initial phosphate controls and have not yet
[00:06:11.630]moved on to DNA concentration trials.
[00:06:15.400]The next steps for this project are to try the growth curves again,
[00:06:18.530]using a higher concentration of 300 nanograms per microliter of DNA.
[00:06:23.160]Then also to extract more high-quality DNA as it takes lots of DNA to run these
[00:06:27.560]high concentration experiments.
[00:06:29.350]And we want to be sure that we're using DNA that has no chance of being
[00:06:32.690]somehow contaminated with phosphates. Then finally,
[00:06:36.030]we'd like to optimize the solid media by finding a way to consistently
[00:06:39.120]supplement with phosphates so that we can then test DNA concentrations with our
[00:06:43.620]mutant strains, since as you can see here,
[00:06:46.080]our outer ring of positive controls is growing just as poorly as our inner ring
[00:06:50.550]of negative controls.
[00:06:52.774]And if we can get these experiments to work and we see that natural competence
[00:06:56.330]is involved and allows bacteria to survive on DNA as a phosphate source,
[00:07:00.152]then our next steps would be to investigate this in a biofilm setting.
[00:07:04.009]Mature biofilms are usually made up of about 1-10% DNA,
[00:07:08.264]so we'd like to grow a mature biofilm and then remove phosphates from the
[00:07:11.855]media and see if the biofilm can use its own DNA as a source of phosphates.
[00:07:16.700]Then knowing if that DNA is available as a phosphate source can inform how
[00:07:20.850]Acinetobacter is used in bioreactor systems for bioremediation like this one
[00:07:24.930]on the right.
[00:07:26.402]One of the ways to optimize this system is to determine which nutrients not to
[00:07:30.366]add, and it would be very useful to know that the biofilm can sustain itself on its
[00:07:34.299]own DNA instead of needing to add phosphates to the system,
[00:07:38.089]so we can determine the most efficient way to run an Acinetobacter bioreactor.
[00:07:43.880]I'd like to thank Ben Sidner,
[00:07:45.410]Leslie Ronish and Ben Glazer for their very helpful insights and discussions,
[00:07:49.640]as well as the NSF for funding this research. And thank you for listening.

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Is Natural Competence Used to Source Phosphates in Acinetobacter Bacteria?

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