The Whatley Lab: From Happy Biofilm Partners to the Dynamics of Antimicrobials

Elli Vickers ‘20

Bacteria are small microbes that are usually studied in the planktonic state (free-floating cells). But in nature, bacteria often aggregate into structures called biofilms, clumps of bacteria that stick to a surface and secrete a protective matrix of sugars and proteins. These biofilms protect the bacteria from antibiotics, which is a huge issue for human health! Many drug-resistant infections are due to biofilms. Our lab explores how biofilms form, interact, and communicate in the hopes of better understanding how to treat biofilm-related infections.Screen Shot 2018-07-22 at 1.45.19 PM

Scanning electron microscopy images of planktonic (left) and biofilm (right) cells. On the left are planktonic (free-floating) Mycoplasma bovis cells from Chen et al. 2018. On the right is a biofilm with Microbacterium oxydans and Chryseobacterium hispalense, taken by our very own Sarah DiDomenico! We can see how complex and impenetrable the biofilm is compared to the planktonic cells.

Our lab previously identified a novel biofilm between Microbacterium oxydans and Chryseobacterium hispalense. These two microbes are found in the skin and gut microbiomes, in soil, and in water systems. In fact, we swabbed a drinking water fountain (in Science Center!) and isolated these two microbes growing in a synergistic biofilm. This interaction is synergistic because while neither partner forms much biofilm alone, the two partners together form tons of biofilm! This synergism is sustained for over 200 hours.

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That’s a water fountain in Science Center. We swabbed the fountain, streaked onto R2A agar (yummy bacteria nutrients), and found these orange and yellow colonies of bacteria! The whiter colonies are Microbacterium, while Chryseobacterium produces that orange pigment. Yes, those bacteria came from your water fountain.

We want to explore this biofilm synergism because while Microbacterium and Chryseobacterium alone are not pathogenic to humans, they are often found in polymicrobial biofilms (often containing pathogens) that can contaminate hospital water systems and lead to infections in surgical patients. Understanding how these two microbes interact and communicate may help us fight these stubborn aquatic biofilms.

My goal this summer is to quantify the expression of different genes in Chryseobacterium when it is alone versus paired with Microbacterium. Our genes are encoded by DNA but are expressed by RNA, which is then translated into proteins that make us who we are! Earlier this summer, I isolated the total RNA from Chryseobacterium alone and partnered with Microbacterium. We sent this off to be sequenced. The results will compare the RNA levels (gene expression) between the two conditions (Chryseobacterium alone versus with Microbacterium). This will give us a sense of what genes are differentially expressed when Chryseobacterium meets Microbacterium.

Once we have a sense of what systems are important for this partner synergism, I will use quantitative PCR to absolutely quantify the expression of genes of interest to us. PCR (polymerase chain reaction) is basically replicating DNA in a test tube instead of in our cells! Quantitative PCR incorporates fluorescence into each strand of DNA that is replicated – more fluorescence builds up as the DNA is replicated with each cycle of qPCR. We can detect that fluorescence and use it to quantify gene expression! If we take RNA from Chryseobacterium alone versus paired with Microbacterium, we can amplify genes of interest in Chryseobacterium and use qPCR to compare their expression in the presence and absence of Microbacterium.

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This is a plot of qPCR amplification. With every cycle (X axis), more DNA is replicated, so more fluorescence builds up (Y axis). The colorful lines represent all of the samples that I am running in a single qPCR reaction! Some samples amplify earlier, indicating that there is more RNA of these genes because they are more highly expressed under these conditions.

Currently, I am working on quantifying expression of the Type IX Secretion System (T9SS). Bacteria have many systems to move proteins from the cell to the environment, and the T9SS is a novel secretion system only found in relatives of Chryseobacterium. Its main function is in bacterial motility, but it also has links to colonization and biofilm formation! Our lab has previously identified that our Chryseobacterium isolate contains many T9SS genes. My current project is to use qPCR to quantify expression of these T9SS genes when Chryseobacterium is alone versus paired with Microbacterium to see if the T9SS is involved in our partner synergism.


Sarah DiDomenico ‘19

Exploring the role of dinB in the bacterial response to quinolones

Quinolones are a class of antibiotics used in pharmacology and agriculture that act as topoisomerase inhibitors. We are interested in studying how quinolones kill cells because a greater understanding will lead to better development of antibiotics in the future. This is critical especially with the rise of antibiotic resistance.

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Figure 1. Schematic representation of how SCCs lead to cell death.

We know that quinolones kill bacteria by binding to topoisomerases, a protein found on actively replicating DNA that helps to relax the DNA as it unwinds. The quinolone bound to the topoisomerase creates a stabilized cleavage complex (SCC). SCCs lead to the generation of double strand breaks (DSBs) which leads to cell death. However, we do not know how exactly SCCs cause double strand breaks. Some scientists believe that SCCs can cause double strand breaks without DNA replication occuring. We believe that SCCs can only cause double strand breaks when new DNA is being synthesized.

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Figure 2. Subunits of Polymerase III. Arrow indicates the subunit interaction of interest.

Previous research (by Dr. Whatley) explored the role of the epsilon subunit of Polymerase III, the protein responsible for synthesizing new DNA, in DSB generation. Traditionally, the epsilon subunit was believed to only be responsible for proofreading newly synthesized DNA. In other words, if a T was matched with a C, epsilon could cut out the C and replace it with the correct match, A. Dr. Whatley found that epsilon is also responsible for stabilizing the interaction of the alpha and beta subunits of Polymerase III (Figure 2). To explain how SCCs caused DSBs she developed the Replication Run-Off model. We believe that the processive replisome encounters the SCC causing stalled replication, dissociation of the complex, and release of double stranded DNA.

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Figure 3. Comparing the growth of E. coli mutants to Wildtype in the presence of norfloxacin.

To test this model, Dr. Whatley created a weak and a strong mutant, which had a weaker or stronger epsilon-beta interaction compared to Wildtype. These mutants were treated with norfloxacin, a quinolone, and allowed to grow. The growth of the mutants in the presence of norfloxacin aligns with the predictions based on the replication run-off model. The weak mutant is less sensitive to norfloxacin compared to Wild type due to the weak epsilon-beta interaction causing Pol III to encounter fewer SCCs and create fewer DSBs. The strong mutant is more sensitive to norfloxacin compared to Wild type due to the strong epsilon-beta interaction causing Pol III to encounter more SSCs and generate more more DSBs.

This summer, I am expanding on this project by exploring the role of dinB in the bacterial response to quinolones. dinB is a gene that encodes for Polymerase IV, an error prone polymerase that performs Translesion DNA Synthesis (TLS). This polymerase is able to replicate past damage that Pol III can not. To test if dinB contributes to the survival of our epsilon mutants, we deleted dinB to test how the weak and strong mutants would act without it. The mutants without dinB were treated with norfloxacin and allowed to grow. These results were compared to the results of the growth assay performed on the mutants with dinB. The weak mutant without dinB grew much less than the weak mutant with dinB, which was expected because the damage experienced by the strain without dinB could not be rescued by Pol IV. Additionally, the strong mutant without dinB survived less than the strong mutant with dinB, which was not expected due to the hypothesized inaccessibility of the B-clamp in this strong mutant. Thus,we predicted that Pol IV could have an additional role other than TLS, or may access the clamp differently in these mutants.

I tested for other roles of dinB in the bacterial response to quinolones. One experiment I performed was a B-galactosidase assay to measure SOS response. The SOS response is the cell’s reaction to DNA damage. We expect dinB to prevent the SOS response from occurring because it is able to rescue endogenous damage. I treated the bacteria with norfloxacin and let it grow. Then I added a chemical that is converted to a yellow color by an enzyme present during the SOS response. I use the amount of yellow produced over time as a direct measure of levels of SOS response. Comparing the levels of SOS produced by the different mutants will reveal other possible roles of Pol IV (dinB).


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