a Wi-Fi E coli study

This is a very nice study on the effect of a commercially available Wi-Fi device on aerobically grown E coli gene transcription. The authors used some very modern genomics techniques to discover some changes. Reminders of old fashioned technology is brought into this post.

The featured image comes from the KEGG database. Some of the pathways illustrate my questions rather than conclusions of the authors.

Said-Salman, I.H., Jebaii, F.A., Yusef, H.H. et al. Global gene expression analysis of Escherichia coli K-12 DH5α after exposure to 2.4 GHz wireless fidelity radiation. Sci Rep 9, 14425 (2019). free paper

An actual Wi-Fi router was used as an exposure system. Was it the field strength that was being amplified? If so, by how much? Some of this seems to be a way of getting the exposure inside a temperature controlled incubator.

Wi-Fi radiofrequency radiation was generated by a wireless router extended range (TL-WR524G-Tp-Link -China) corresponding to 2.4 GHz frequency, connected to an amplifier and monopole antenna. The mounted system was placed in an incubator at 30 cm from the bacterial culture that was continuously being stirred. The length of exposure was 5 hours. Control bacteria were in a Faraday Cage. See Fig 9. Growth was under aerobic conditions.

Summary of figures

Fig1 is a Volcano plot of minus log base 10 probability of genes being differentially expressed as a function of fold change between the control and the 2.4 G treatment. Genes at the corners of the “U” are the most changed whereas most gene expression is not much affected by 2.4 G. The genes are colored if they pass the thresholds for −log₁₀ P value (P value = 0.05) and log fold change |FC| ≥1.2, yellow if they are up-regulated and blue if they are down-regulated.

Fig 2 shows the so called hierarchal clustering of genes that were shown to be significantly over or under expressed in Fig 1. Clusters are defined by common functions. Making protein subunits of nitrate reductase is an example of a common function of messenger RNAs. Three different experimental replicates are shown in this figure.

Fig. 3 and the database of essential genes (DEG) Uncharacterized seemed to represent 36% of the differentially expressed genes and metabolic processes represented 27%.

Table 1 Summary of functional annotation clustering analysis by DAVID tools.

• Cluster 1 enrichment score of 3.74 for a total of 24 genes associated with moving about.
• Cluster 2 enrichment score of 2.00 for a total of 13 genes related to chemotaxis, moving about in response to chemicals
• Cluster 3 enrichment score of 1.08 for a total of 4 genes associated with adhesion.
• Cluster 4 enrichment score of 0.71 for a total of 8 genes associated with cellular component organization.
• Cluster 5 enrichment score of 0.63 for 10genes associated with DNA repair and metabolism.
• Cluster 6? enrichment score of 0.54, 5 genes “nucleotide biosynthetic process” could be argued to belong to the previous cluster. The sub category “Carbohydrate derivative metabolic process” has 7 genes. The subcategory “organonitrogen compound metabolic process” has 12 genes, the largest for any subcategory.

Fig 4 GO gene lists: Metabolic processes genes were the most up and down regulated genes Chemotaxis and biofilm/cell adhesion genes tended to be up regulated in response to 2.4 GHz.

Fig 5 is a lovely image of a flagella ensemble complete with chemical sensing protein gene names. CheY is one of the genes upregulated. “Involved in the transmission of sensory signals from the chemoreceptors to the flagellar motors. In its active (phosphorylated or acetylated) form, CheY exhibits enhanced binding to a switch component, FliM, at the flagellar motor which induces a change from counterclockwise to clockwise flagellar rotation. Overexpression of CheY in association with MotA and MotB improves motility of a ycgR disruption, suggesting there is an interaction (direct or indirect) between the c-di-GMP-binding flagellar brake protein and the flagellar stator.” MotB ” MotA and MotB comprise the stator element of the flagellar motor complex. Required for the rotation of the flagellar motor. Probably a linker that fastens the torque-generating machinery to the cell wall. Overexpression of this protein with MotA improves motility in a pdeH disruption, (a c-di-GMP phosphodiesterase) suggesting there is an interaction (direct or indirect) between the c-di-GMP-binding flagellar brake protein YcgR and the flagellar stator.”

Fig 6 shows three “nitrogen metabolism” pathways. Since these experiments were performed under aerobic conditions, it is not surprising that FNR regulation transcribed genes are not changed. Formate dehydrogenase genes FdnG-I are down regulated. FdnG “Formate dehydrogenase allows E.coli to use formate as major electron donor during anaerobic respiration, when nitrate is used as electron acceptor. The alpha subunit FdnG contains the formate oxidation site. Electrons are transferred from formate to menaquinone in the gamma subunit (FdnI), through the 4Fe-4S clusters in the beta subunit (FdnH). Formate dehydrogenase-N is part of a system that generates proton motive force, together with the dissimilatory nitrate reductase (Nar).”

Fig 7 is a nice bar graph comparing changes in select gene expression in response to 2.4GHz WiFi PEMF. The added bonus is that everything is normalized to housekeeping genes gyrA and frr. GyrA is DNA gyrase subunit A is involved in the supercoiled nature of the bacterial chromosome. Frr is the ribosome recycling factor. The two housekeeping gene expression may go up and down depending on how much the bacterium is making new proteins and dividing. This is a very nice figure that shows WiFi PEMF is doing something.

Fig 8 is a nice summary of messenger RNA transcripts that go up and down in response to 2.4 GHz for five hours. This image suggests a bacterium that is stressed and wants to leave the toxic environment.

Overall, this is a very nice paper that did not identify the antenna for the 2.4 GHz WiFi emission. What does this mean for the E coli living in our colons? These bugs are living under anaerobic conditions. What I found interesting was the down regulation of the formate dehydrogenase protein subunit transcripts. formate dehydrogenase is supposedly part of anaerobic respiration when nitrate and fumarate are the terminal electron acceptors instead of oxygen. .

NarX, what regultes formate dehydrogenase?

Wang H, Gunsalus RP. Coordinate regulation of the Escherichia coli formate dehydrogenase fdnGHI and fdhF genes in response to nitrate, nitrite, and formate: roles for NarL and NarP. J Bacteriol. 2003 Sep;185(17):5076-85. PMC free paper

NarL and NarP transcription factors are regulated by phosphorylation. The kinase was not identified in this paper

NarL activates the expression of the nitrate reductase (narGHJI) and formate dehydrogenase-N (fdnGHI) operons and represses the transcription of the fumarate reductase (frdABCD) operon in response to a nitrate/nitrite induction signal transmitted by either the nitrite/nitrate sensitive protein kinases NarX or NarQ that can sometimes also act as protein phosphatases against NarL. This is a nice paper published over 20 years ago. How has our knowledge progressed since? The RCSB.org structural database is a good place to start. There is a nitrate binding sit in NarX.

Where would the nitrate be coming from in the 2.4 GHz E coli study of Said-Salmon 2019? Luria Bertani (LB) medium, a microbiology standard, was used in this study. The LB recipe calls for tryptone (a trypsin digest of meat) and yeast extract. No sugars are added to this medium. If the bug is going to make ATP, it will have to do so by breaking down the protein. This could be where the nitrate is coming from. Said-Salmon did discuss metabolic stress and programmed cell death in response to Wi-Fi. Did the WiFi impede the utilization of LB medium? The use of modern technology was amazing. Sometimes modern technology over shadows simple things like measuring the optical density of the culture as an estimate of how fast the bugs are growing. This is very nice study, in my opinion, that deserves a follow up that more resembles what might be happening in our colons under anaerobic conditions and major sources of energy stored in carbon bonds coming from pro-biotics. If our small intestine and stomach are functioning properly, E coli in our colon is not going to see much protein.

Thoughts for future work

LB, like any culture medium used, has its limitations. Use of protein as a source of carbon was cited as one. Lack of divalent cations is another limitation. What are the connections with some of these transcripts that were decreased as they are connected with quorum sensing? As the population of bacteria increases, an individual bug is more likely to encounter a secreted small molecule and alter transcription of appropriate genes accordingly. We do not know if Wi-Fi altered how fast the bacteria were dividing. Since these E coli were growing in a medium based on a protein digest. They may have no need for nitrogen assimilation.