5G and the mcirobiome

Wang 2024 was a collaboration between four military related schools in China. This group commented on different mobile phone providers operating on slightly different bans. Prior work showed that one caused depression like symptoms in mice. This follow up study tested the hypothesis that the RF PEMF caused changes in the microbiome as well as well as the metabolites. Escherichia species were wiped out and Bacillus species were promoted by 5 G PEMF. Anaerobes wiped out, aerobes favored? Can these differences be explained by enzymes with cofactors with unpaired electrons that act as antenna? The fumarate/nitrate reductase transcription regulator is present in both albeit with scant percentage sequence identity. Electron paramagnetic resonance, which uses ~9 GHz microwave PEMF to study the unpaired electrons of metal ligands in proteins. This post ends with an hypothesis concerning 5 GHz PEMF.

Wang 2024 [1], just a few stand out

The animals were whole body exposed to 4.9 GHz RF field for 21 consecutive days (1 h/day). Previous studies by this group demonstrated that 4.9 GHz RF caused depression like behavior. This study also performed an exhaustive metabolic analysis of the GI contents to address the gut-brain axis. The average power density (PD) was 50 ± 2.5 W/m². For the Sham group, the animals were processed in the same condition as the RF group. The mice were reported to not have been stressed by the protocol. This post will focus on Table 2 of some major changes in the microbiome in terms of percent representation of the whole. In compiling this table the bar graph of Fig 2D was enlarged and some estimates were. I tired to determine if the bacteria were aerobes, facultative aerobes, or obligate anaerobes. Is there a commonality in simple identifiers like Gram Stain? Lactobacillus and a larger family, Muribaculaeceae, made up the bulk of bacteria in the feces and the cecal samples. It is assumed for now that the two were combined. for analysis.

bug & tax ID	RF PEMF rel to sham	% relative abundance	p value	FNR transcriptin factor	FrdABCD operon	narGHJI operon
Lactobacillus, :91061 also a Streptococcus group	2x	18/40	0.04057	no widespread FNR	nothing for the Lactobacillus genus	Lactobacillus plantarum WCFS1 NarGHJI Brooijamens 2009
nonank_f_ Muribaculaeceae 2005473	0.5	30/16	0.04057	6 hits Crp/Fnr family transcriptional regulator 22-30% ident.	Many hits for L-aspartate oxidase 30% ident. Also known as fumarate reductase	gb|MDE6176758.1| molybdopterin-dependent oxidoreductase Paramuribaculum sp. 60% ident
Allobaculum 174708	0.2	2.5/0.5	0.013	nothing	nothing	no hits
Escherichia/ Shigella		2/wiped out	0.00122	ref FNR	ref FrdA	NarG Blastp ref
Bifidobacter 1678	10x	?/2	0.0312	Three hits 22-27% identity	Many 35% identity FAD binding oxo-reductases.	B breve 80% ident. few hits
Bacillus 1389	4x	?/1	0.01813	Top hit B amylo amyloliquefaciens 97% indent. Nothing else above 40% ident.	Top hit B amylo amyloliquefaciens 94% indent. Many more ~40% identity FAD succinate oxo-reductaases	Bacillus subtillis NarGHIJ Hoffman 1995

The first thought that the ones being spared are not anaerobes, facultative or obligate. In preparing this post it was discovered just how divergent bacterial proteins really are.

Is this bug an anaerobe?, tidbits of info

These are some interesting observations that are a bit divergent from the observation that anerobic, as well as aerobic growth, requires terminal electron acceptors that that have metal cofactors, that are paramagnetic, and can respond to the right PEMF. 9 GHz has historically been used a lot in electron paramagnetic resonance (EPR) spectroscopy to study structure. This overview mentions a few very low ferqeuncy PEMF studies.

Lactobacilli Flavoproteins and Heme Yuji Yamamoto 2024

This review covers flavoproteins and heme proteins involved in electron transport in the Lactobacilli. [2] Mokhamed Tirono of the Muslim University of Indonesia studied the effect of 50 Hz (room electricity) PEMF of mT field strength on Lactobacillus acidophilus on cow milk fermentation. [3] Optimum results were obtained with a 5 minute exposure at 0.2 mT whereas longer exposure for 15 minutes and 0.3 mT slowed growth. If FAD is an antenna, there are plenty of them in Lactobacilli. We do have some sort of experimental support that some form of PEMF supports growth under yogurt making conditions. .

Muribaculaeceae producers of short chain fatty acids [4]

Some artificial “intelligence” noise associated with Google claims this family are obligate anaerobes without citing a reference or describing what it means to be an obligate anaerobe biochemically. The Zhu 2024 publication does a good job of reviewing preferred carbohydrate sources and short chain fatty acid products of the family Muribaculaeceae that are members of the Order Bacteroidetes. Figure 1 Metabolic pathways of Muribaculaceae to produce short-chain fatty acids. Muribaculaceae do the initial breakdown of dietary fibers. Lactobacillus and Bifidobacterium take those carbohydrates to phosphoenol pyruvate.

Allobaculum in humans[6]

Ulcerative colitis associated Allobaculum strains 128^T and 539^T were characterized phenotypically with the closest relative being A. stercoricanis based on biochemical tests. Unlike the reference 128^T and 539^T were were weakly positive for nitrate reduction. Based on test results two novel species are proposed, Allobaculum mucilyticum sp. nov. with the type strain 128^T (=NCTC 14626^T=DSM 112815^T) and Allobaculum fili sp. nov. with the type strain 539^T (=NCTC 14627^T=DSM 112814^T).

Escherichia and Shigella

There is no questioning that Shigella is a pathon. Some strains of E coli are pathogenic. O157:H7 is one example. Bayir 2015 [6] looked at E coli O157:H7 exposure to three different frequencies (20, 40, and 50 Hz), and two different magnetic intensities (2 and 4 mT), and 4 different time durations (1, 2, 4 and 6 h) at 25^oC. The plate counts were performed under aerobic conditions. The PEMF exposure might have been under minimal O₂ as the cultures were in capped tubes called Eppendorfs. Exposure was in Mueller–Hinton Broth (MHB) which contains digested casein and beef extract. This translates into some iron, heme, and nitrogen from the protein. There is always the caveat that nutrient availability might affect expression of the potential antenna proteins. At 4 mT and 20 Hz PEMF, 1 hr was enough for 40% killing and 6hours for about 90% killing. [6]

Torgomyan 2011 [7] used wild type E coli K12 grown anaerobically in a broth containing peptone, a pepsin digest of animal tissue. The bacterial suspensions on plates were
irradiated with a coherent electromagnetic field of 70.6 or 73 GHz frequencies. The power flux was 0.06 mW cm.² Control and irradiated bacteria were transferred to a growth medium whose growth could be measured by scattering of light passing through the culture tube. Click here for an image for non scientists. 70 nd 73 GHz prior exposure were found to decrease the growth rate and increase the time spent in lag phase, the time before the bacteria grow at the maximal rate.

The switching of metabolic pathways in E coli [8] and more?

The scientific community knew so much about of the turning of genes on and off in response to O2 in E coli in 1997. Unden and Bongaerts published and amazing review that is still relevant today and the search for RF PEMF antenna. Here is a direct quote [8]

The electron-transport chains of Escherichia coli are composed of many different dehydrogenases and terminal reductases (or oxidases) which are linked by quinones (ubiquinone, menaquinone and demethylmenaquinone). Quinol:cytochrome c oxido-reductase (`bc₁ complex’) is not present. For various electron acceptors (O₂, nitrate) and donors (formate, H₂, NADH, glycerol-3-P) isoenzymes are present. The enzymes show great variability in membrane topology and energy conservation. Energy is conserved by conformational proton pumps, or by arrangement of substrate sites on opposite sides of the membrane resulting in charge separation. Depending on the enzymes and isoenzymes used, the H⁺/e⁻ ratios are between 0 and 4 H⁺/e⁻ for the overall chain. The expression of the terminal reductases is regulated by electron acceptors. O₂ is the preferred electron acceptor and represses the terminal reductases of anaerobic respiration. In anaerobic respiration, nitrate represses other terminal reductases, such as fumarate or DMSO reductases. Energy conservation is maximal with O₂ and lowest with fumarate. By this regulation pathways with high ATP or growth yields are favoured. The expression of the dehydrogenases is regulated by the electron acceptors, too. In aerobic growth, non-coupling dehydrogenases are expressed and used preferentially, whereas in fumarate or DMSO respiration coupling dehydrogenases are essential. Coupling and non-coupling isoenzymes are expressed correspondingly. Thus the rationale for expression of the dehydrogenases is not maximal energy yield, but could be maximal flux or growth rates. Nitrate regulation is effected by two-component signal transfer systems with membraneous nitrate/nitrite sensors (NarX, NarQ) and cytoplasmic response regulators (NarL, NarP) which communicate by protein phosphorylation. O₂ regulates by a two-component regulatory system consisting of a membraneous sensor (ArcB) and a response regulator (ArcA). ArcA is the major regulator of aerobic metabolism and represses the genes of aerobic metabolism under anaerobic conditions. FNR is a cytoplasmic O₂ responsive regulator with a sensory and a regulatory DNA-binding domain. FNR is the regulator of genes required for anaerobic respiration and related pathways. The binding sites of NarL, NarP, ArcA and FNR are characterized for various promoters. Most of the genes are regulated by more than one of the regulators, which can act in any combination and in a positive or negative mode. By this the hierarchical expression of the genes in response to the electron acceptors is achieved. FNR is located in the cytoplasm and contains a 4Fe4S cluster in the sensory domain. The regulatory concentrations of O₂ are 1–5 mbar. Under these conditions O₂ diffuses to the cytoplasm and is able to react directly with FNR without involvement of other specific enzymes or protein mediators. By oxidation of the FeS cluster, FNR is converted to the inactive state in a reversible process. Reductive activation could be achieved by cellular reductants in the absence of O₂. In addition, O₂ may cause destruction and loss of the FeS cluster. It is not known whether this process is required for regulation of FNR function.” [8]

Four terminal electron acceptors and their operons

Figure 1 of this review [8] covers the enzyme names that reduce the terminal electron acceptors. These complexes contain subunits of proteins whose genes reside in operons under the control of bind to regions of the gene upstream of the protein coding parts. If PEMF somehow mucks up the function of FNR, the consequences could be felt on all genes in the operon.

1. FrdABCD (subunits) reduces fumarate to succinate.
2. NarGHI reduces nitrate to nitrite.
3. CyoABCD reduces oxygen to water, one report high and low O₂ conditions
4. and CydAB reduce oxygen to water, controlled by a cAMP binding protein.

FNR turns on alternatives to O₂ terminal electron acceptor reduction

FNR, Fumarate Nitrate Regulon has its own Wikipedia page now. Of course the sequence where FNR binds is called the FNR box. Transcription of whole operons of related genes ensues. Oxidant dependent degradation turns off the FNR switch that controls the transcription of anaerobic respiration genes. Under reducing conditions FNR forms a dimer and specifically binds to DNA. In the presence of O₂ the cluster is degraded causing FNR monomerization and DNA dissociation. A crystal structure of E coli FNR was not found. A marine facultative anaerobe is available.

The bottom line seems to be that FNR is very labile in the presence of oxidants. Breaking up the center is a signal to monomerization and loss of DNA binding.

E coli Frd fumarate reductase

The QFR respiratory complex is composed of four polypeptide chains. Two of these chains, the flavoprotein (FrdA) and the iron protein (FrdB), comprise the soluble domain, which is involved in fumarate reduction, whereas the remaining two subunits (FrdC and FrdD) are membrane-spanning polypeptides involved in the electron transfer with quinones [9]

A PubMed search for “fumarate reductase and electron paramagnetic resonance” yielded 232 peer reviewed publications. Some of these are studies of the human version of he enzyme. The frequency tends to be in the ballpark of 9 G Hz and 300 something mT scans.

E coli Nar nitrate reductase

This image was generated from the NCBI structure database. Details of the flow of electrons through this structure were published by Bertero and coauthors in 2006. In explaining Wang 2024 reordering of the microbiome with 5G, perhaps it is a matter of altering the flow of electrons too. It is hard to ignore the number of metal cofactors with unpaired electrons that might respond to the right combination of frequency and field strength.

A PubMed search on “nitrate reductase and electron paramagnetic resonance” yielded 175 publications.

Concluding remarks and electron paramagnetic resonance [11,12]

For the record, the soil bacterium Bacillus has its own version of FNR, which links to the NCBI sequence that was used for Blastp searches. Reents and coauthors isolated this protein and used mircowave PEMF, aka electron spin resonance spectroscopy, to study its structure. [11] A handful of Bacillus species turned up in the blast search. The same sequence was blasted against the Escherichia database. Hits were recovered from Escherichia coli though the percent identity was low. The featured image is based on the Cutsail publication. [11]

EPR is ideal for studying metal complexes and organic radicals, as per the radical states of FAD covered in previous posts. The ability of a variety of different magnetic fields and frequencies to cause ions to spin, ion cyclotron resonance, was covered in the heavy metal detox post. Wikipedia authors tell us that the preferred frequencies in EPR tend to be in the microwave region of the electromagnetic spectrum, say 9-10 GHz. With a set frequency, the field strength is increases from 300 to 400 mT. The Cutsail 2015 review has some very nice imagers of various Fe-S containing enzymes, bacterial and human, that have been studied with EPR. The selective killing of some bacteria but not others in Wang 2024[1] raises the question of whether some sort of ion cyclotron resonance fine tuning thing is going on that makes one bugs FNR susceptible to a particular ban of RF PEMF. FR PRMF is used to study iron=sulfur center proteins. Are some humans susceptible to dirty electricity and others not simply because of some minor amino acid difference in a paramagnetic metal group electron in a key enzyme? PEMF and Complex II is about an extremely low frequency PEMF emitter.

References

1. Wang X, Zhou G, Lin J, Qin T, Du J, Guo L, Lai P, Jing Y, Zhang Z, Zhou Y, Ding G. Effects of radiofrequency field from 5G communication on fecal microbiome and metabolome profiles in mice. Sci Rep. 2024 Feb 12;14(1):3571. PMC free paper
2. Yamamoto Y. Roles of flavoprotein oxidase and the exogenous heme- and quinone-dependent respiratory chain in lactic acid bacteria. Biosci Microbiota Food Health. 2024;43(3):183-191. PMC free paper
3. Tirono M. The application of extremely low-frequency (ELF) magnetic fields to accelerate the growth of Lactobacillus acidophilus L. bacteria and the milk fermentation process. Acta Sci Pol Technol Aliment. 2022 Jan-Mar;21(1):31-38.
4. Zhu Y, Chen B, Zhang X, Akbar MT, Wu T, Zhang Y, Zhi L, Shen Q. Exploration of the Muribaculaceae Family in the Gut Microbiota: Diversity, Metabolism, and Function. Nutrients. 2024 Aug 12;16(16):2660. PMC free paper
5. van Muijlwijk GH, Rice TA, Flavell RA, Palm NW, de Zoete MR. Allobaculum mucilyticum sp. nov. and Allobaculum fili sp. nov., isolated from the human intestinal tract. Int J Syst Evol Microbiol. 2023 Feb;73(1).
6. Bayır E, Bilgi E, Şendemir-Ürkmez A, Hameş-Kocabaş EE. The effects of different intensities, frequencies and exposure times of extremely low-frequency electromagnetic fields on the growth of Staphylococcus aureus and Escherichia coli O157:H7. Electromagn Biol Med. 2015 Mar;34(1):14-8. Sci-Hub free paper
7. Torgomyan H, Kalantaryan V, Trchounian A. Low intensity electromagnetic irradiation with 70.6 and 73 GHz frequencies affects Escherichia coli growth and changes water properties. Cell Biochem Biophys. 2011 Jul;60(3):275-81.
8. Unden G, Bongaerts J. Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors. Biochim Biophys Acta. 1997 Jul 4;1320(3):217-34. free paper
9. Iverson TM, Luna-Chavez C, Croal LR, Cecchini G, Rees DC. Crystallographic studies of the Escherichia coli quinol-fumarate reductase with inhibitors bound to the quinol-binding site. J Biol Chem. 2002 May 3;277(18):16124-30. free paper
10. Bertero MG, Rothery RA, Palak M, Hou C, Lim D, Blasco F, Weiner JH, Strynadka NC. Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A. Nat Struct Biol. 2003 Sep;10(9):681-7. Sci-Hub free paper
11. Reents H, Gruner I, Harmening U, Böttger LH, Layer G, Heathcote P, Trautwein AX, Jahn D, Härtig E. Bacillus subtilis Fnr senses oxygen via a [4Fe-4S] cluster coordinated by three cysteine residues without change in the oligomeric state. Mol Microbiol. 2006 Jun;60(6):1432-45.. free paper
12. Cutsail GE 3rd, Telser J, Hoffman BM. Advanced paramagnetic resonance spectroscopies of iron-sulfur proteins: Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM). Biochim Biophys Acta. 2015 Jun;1853(6):1370-94. doi: 10.1016/j.bbamcr.2015.01.025. Epub 2015 Feb 14. PMID: 25686535; PMCID: PMC4390514. PMC free paper