E. coli Up Close and Personal: Scientific Rockstar and Public Enigma

It seems nothing puts fear in the hearts of the masses like mentioning E. coli. Most think of the disease-causing germ that contaminates everything from spinach to beef. I agree the strain Escherichia coli O157:H7 and its cousins O26, O145, STEC O104:H4, and others, are a wretched bunch that give the whole species a bad reputation. What makes these strains so vile are the extra proteins encoded within their genome. For example, E. coli O157:H7 has a larger genome coding for 5561 proteins while the parent strain E. coli W codes for 4739 proteins. Thus is the life of a bacterium. The fact there are so many bacteria means they are usually in close proximity to each other. Physical contact between bacteria, not just those of the same species, allows for the transfer of genetic material between two cells (horizontal gene transfer); the closest thing to sexual reproduction you will find for prokaryotes. If the genes transferred to the recipient give it an advantage or new ability that helps it compete and thrive in its environment, they will remain in the genome. Otherwise, they will be discarded after genome compaction.

Most E. coli are completely harmless and, in fact, beneficial. If the general public knew more than what was broadcasted on the 24 hour news channels, they would see the tiny rockstar scientists have known about for some time now. Beginning in earnest in the 1950s, E. coli is easily cultured in laboratories and very cheaply. Its quick generation time (20 min. at optimum temperature) made it a great model organism to study in many fields of science and medicine. This organism is the work horse of biotechnology due to the relative ease of manipulating its genome or adding complete genetic circuits into the cell using plasmids.

Sidebar

Even after 50 years of intense research, E. coli still holds many unknowns out of the reach of our knowledge. Like all other sequenced genomes, there are a number of “hypothetical proteins” and “proteins of unknown function”. This means by our best abilities, we can locate parts of the genome that code for proteins, however, this doesn’t mean we are able to understand the function of a particular protein.

Image courtesy of Predrag Radivojac. Thanks, Pedra.

The above shows just how much work is left to understand the biological capabilities of Mother Nature. Short version: over 40 gene sequences in databases, but the number of which that we know what the function is holding steady around 500,000 and the number of solved protein structures is over 100,000. This is a growing gap between the known and unknown.

 Where would we be without E. coli?

One advantage of E. coli is their effect on our immune system. Some may find this counter-intuitive, but E. coli can lower the workload of our immune system when pathogens are present, especially in the intestine. When E. coli attach to the GI wall, it changes the acidity of the lining thus making infection from other bacteria less likely. Another benefit is in overall digestion. E. coli promotes better breakdown of food thus preventing accumulation of waste which is a major cause of bloating and constipation.

Many outside the scientific community may not be aware of how integral E. coli are to the advancement of many fields including medicine, pharmacology, biology, and even human physiology. Another reason to not believe the hype.

Soil: an under-appreciated dynamic consortium of communities.

Azospirillum brasilense electron micrograph

Quick fact: the amount of data generated by analyzing the genetic make-up of 1 gram of soil would surpass the total for the entire Human Genome Project. That is because a gram of soil may contain between 2,000 and 18,000 different genomes comprised within roughly 40,000,000 to 2,000,000,000 bacteria cells (1) and (2). Soil; we all walk on it, but do we ever think about what might be lurking in it? My daughter does, for instance, because she looks for tiny black snails to bring into our house and put in potted plants. However, I’m referring to things much smaller and much more influential to the overall ecosystem. Bacteria and fungi have mostly beneficial impacts on the lives of plants, but we know only a fraction of a fraction of the total species present. Many of us think of soil as dirt; dry and inorganic, but soil is a dynamic matrix of clay, sand, and silt particles with a mix of decomposing matter and living organisms. The surfaces of these particles make good niches for bacteria to live if they can survive the extreme variation in water and nutrient availability due to the wet/dry cycle.

Soil is a vast reserve of organic carbon, but only a fraction is usable due to decomposition of most carbon into hummus. So, bacteria and other microorganisms are in eternal competition with each other for precious nutrients in their microenvironments. This makes life arduous for soil organisms that are loners or isolated from good sources of nutrients. This is one reason the root zones of plants are like retirement communities to bacteria and fungi; and the plants know it.

Plants are like shipwrecked sailors stranded on a desert island. They have no where to go if conditions change. To help themselves out, they recruit bacteria and fungi to live on, or within, their roots by excreting valuable nutrients into the soil. Surrounding microorganisms take to this like sharks to blood which is what the plants want and need. These bacteria and fungi offer several advantages to plants. First, they can simply take up space; space that plant pathogens would like to inhabit. On top of this, the good bacteria can readily produce antibiotics to kill off any pathogens that might kill their food source. Second, many of these bacteria have the genetic machinery to produce a class of plant hormones called auxins, derivatives of the amino acid tryptophan. Auxin is like human growth hormone. When the bacteria excrete auxin, the plants take it in because it is a cue to increase water and nutrient uptake. So, plants increase nutrient uptake, become healthier and bigger, and by default excrete larger amounts of nutrients for the bacteria. Genius.

The third reason plants attract bacteria is because plants have a big problem; they can’t make useful sources of nitrogen out of thin air. Luckily, many soil bacteria can, and it’s called nitrogen fixation. These bacteria are able to take nitrogen gas from the air and convert it into a form useful to both plant and bacterium: ammonium. This is an energetically expensive process for the bacterial cell. So, in order to make it as easy as possible on the bacteria, plants will protect the cells from nitrogen fixation inhibitors like oxygen and provide essential carbon in the form of amino acids. For this to happen, the bacterial cell literally crawls inside the root cell and becomes a bacteroid encapsulated within a special structure, a nodule, and ultimately becoming an endosymbiont.

And you thought only mammals had beneficial internal bacterial ecosystems. Just like humans, plants would be in a sorry state if it were not for the bacteria that they associate with. I haven’t even touched upon the benefits of fungi, but I’m definitely not a fungal expert. Any takers?

References

(1) How Deep Is Soil? Daniel D. Richter and Daniel Markewitz BioScience , Vol. 45, No. 9 (Oct., 1995), pp. 600-609

(2) Paul, E. A. & Clark, F. E. Soil microbiology and biochemistry (Academic Press, San Diego,1989).

The Microbiome: Can We Please Consider the Human Body an Ecosystem Now?

It has long been thought the type and amount of microbes using the human body as a home shape the way we live and behave. The microbiome as it is known is shown to have a greater and greater impact in our daily lives.

A new study published in Nature (paywall) provides evidence demonstrating the artificial sweeteners we all love and consume to control weight leads to increased blood glucose levels. How can something used to replace sugar in consumables raise the amount of sugar in the blood?

Like many other answers regarding human health, look no further than the microbiome. Consuming artificial sweeteners alters the composition of the intestinal microbes leading to a growing glucose intolerance. The researchers linked artificial sweetener use to altering metabolic pathways within the microbiome that leads to increased susceptibility to metabolic disease.

To verify their findings, researchers gave antibiotics to the mice used as models thus reversing the effects of artificial sweeteners. Results were also verified by using fecal transplantation in the mice to reverse glucose intolerance.

The Human Microbiome: Our Ecosystem

We already knew the microbes outnumbered our human cells 10 to 1 and that the microgenome outnumbered our human genome 100 to 1. The evidence is growing suggesting our normal flora govern more of our lives than we naively assumed for decades. We are not individuals but individual incubators for the microbial overlords that we could not live without. Just like other ecosystems, changing our lifestyles have a complicated effect on system as a whole. Small alterations to the microbiome can have major impacts and be the difference between health and disease.

Future posts will hopefully provide evidence demonstrating how we are shaped into individual ecosystems. Thank you, microbiome.

 

Abstract 2.0 Is On: Help Wanted

I have sat on this long enough. It’s not like a have anything else going on right now (except the birth of a son in a  month, syllabus to write, classes to prepare, evaluations to do, data to journal, …). Introducing:

Abstract 2.0

Here are the details presently. I and anyone willing to help will scour the journals of our respective fields and choose those we feel need to be disseminated to the larger public. In a short synopsis (abstract if you will), an overview of the article and why it is important will be written and deposited here. Details will be worked out on how to submit the abstracts in the near future.

Now is the time to act (or later if now is not convenient)!

A STEM Book Explaining Bacteria to Kids: Begging Without the Cardboard Sign

I was recently approached about developing a children’s book to educate about bacteria in hopes of clarifying misconceptions many have about ‘nasty germs’. I must say how amazed and honored by the invitation I am. The company is small without a lot of capital to produce such a book at will. So, I was asked if I had contacts that would graciously sponsor the production of the book. This to me is bittersweet. I would love to be a part of something that would be so helpful for the public regarding the reality of microbes (they tend to get bad press in general). However, I’m not one to ask for money…ever. 

This has sparked questions in my head about the state of educational media production. S.T.E.M. is all the rage these days and rightly so. As our society progresses, the need for a workforce trained for technical and scientific positions is essential. One example…billboard signs. Growing up, I used to get excited and amazed when I saw a person putting up a new billboard sign. Taking the old one off, applying the new one in its place. However, now these signs are replaced by digital billboards. Who is going to change the billboard advertisement? Someone trained to tear down the old and glue the new one on? Someone with a background in electrical engineering? If there is a problem with the billboard, who will fix it? A carpenter or an engineer? This is just one example. 

The STEM push is necessary and welcome in my opinion. However, a quite fitting phrase comes to mind: show me the money. We are throwing money into public school systems that are fueled by bureaucracy and inefficiency. Yet we still have to cut out box tops to support local schools and have several fundraisers a year for a new gym floor. Anyone see the irony?

Put the money where it can be useful. Put it in projects that will encourage our children to pursue a career that will promote curiosity and critical thinking. This has been my soapbox, today sponsored by the letters S, T, E, and M.

Bacterial Chemotaxis and Human Memory: Pete and Repeat

Many do not place ‘bacteria’ and ‘memory’ in the same sentence. Normal human perception does not connect the two concepts. However, Mother Nature seems to have a more profound perception. The past 50 years or so of scientific investigation has shown how our uniqueness as humans is actually commonplace across all forms of life on Earth. Case in point, how closely associated molecular memory is between bacteria and human.

Bacteria use adaptation to signals as memory

Swimming bacteria do not move randomly in their environment. This behavior would be futile and counterproductive. Instead, bacteria are constantly monitoring their environment in search of food and poisons. Moving towards the former and away from the latter. This observation was first published in the late 19th century. Bacteria, like the famous and infamous E. coli, use molecular antennae to receive these important ‘signals’ as the basis in the decision of which direction to swim. What if the bacteria find a great place to reside with lots of food but still need to receive signals to ensure they remain there? The antennae have sections that can be modified easily and reversibly. These modifications, in the form of methylation, alter the sensitivity of the antenna protein to subsequent signals. Methylation allows these antennae not to receive the number of absolute signals but relative signals. In other words, the antenna protein through fine-tuned methylation detects changes in the number of signals now versus some time in the past. This is the basis of molecular memory.

These antennae are proteins called methyl-accepting chemotaxis proteins, or MCPs. MCPs accept methyl groups from the essential cofactor S-adenosylmethionine (aka SAM or AdoMet). AdoMet is essential to both prokaryotes and eukaryotes like humans. The methyl groups are added by a protein called CheR (pronounced ‘key R’) which transfers the methyl from AdoMet to very specific amino acid side groups of glutamate. The process, called O-methylation adds the methyl group to the single-bonded oxygen on the carboxyl.

O-methylation reaction
O-methylation reaction. Courtesy of www.brenda-enzymes.org.

The length of a bacterium’s molecular memory is very short in comparison to how we perceive memory at only a few seconds. But, to bacteria it is long enough to successfully navigate the environment with similar precision when concentrations of food or poison vary (up to several orders of magnitude, or ~1000x).

Does the basis of molecular memory in humans mimic bacteria?

Eukaryotes, including humans, use a very similar mechanism in signal transduction to bacteria. Phosphorylation (transferring a phosphate group from ATP or GTP to a protein amino acid) is the basis of all signal transduction and cell regulation. Bacteria use histidine kinases and response regulators, as do plants to some degree. However, the majority of regulation through signal transduction in eukaryotes is through two types of proteins, RAS proteins and the heterotrimeric G-proteins. G-proteins interact with membrane receptors that regulate their activity. What determines which surface receptors G-proteins interact with? Isoprenylcysteine methyltransferase, or ICMT, is one of two methyltransferases that regulate signal transduction activity. ICMT is a membrane protein that uses AdoMet to add methyl groups to isoprenylcysteine, a post-translationally modified cysteine residue on both heterotrimeric and RAS-related G proteins. Methylation regulates which receptors the G-proteins interact with, thus playing a major role in connecting the initial signal to downstream regulatory pathways. The carboxyl methylation essentially modulates G-protein signalling globally.

G-protein carboxyl methylation is regulated by GPCR signaling and, as seen above, GPCR signaling is regulated by G-protein carboxyl methylation. This feedback/feed forward loop could be seen as a form of molecular memory stored in methylation patterns. Within the brain, ICMT activity is almost exclusively found in the region controlling coordination of movement. Thus, methylation could be used to modulate certain neuronal signaling pathways which result in learned patterns of sensory-motor skills.

The only other major methyltransferase is from a protein known as PPMT. PPMT interacts with a major enzyme in signal termination, the protein phosphatase PP2A. PPMT adds methyl groups to the backbone carboxyl of a specific leucine in PP2A. This carboxyl methylation helps determine which B subunit PP2A interacts with and where in the cell PP2A can be found. PPMT structurally resembles CheR in bacterial memory. Moreover, the enzyme that removes the methyl group from PP2A, PME, structurally resembles the bacterial enzyme that removes methyls from MCPs, CheB.

PP2A is one of the major regulators of pathway coordination to maintain synaptic plasticity in the brain. Interestingly, methylation defects and PP2A-PME complexes are suggested to play a role in the cause of Alzheimer’s Disease and memory loss. Methylation defects leading to defective phosphatase activity of PP2A leads to accumulation of a phosphorylated subunit of the structural protein microtubule. In this phosphorylated form, the filaments used to keep axons structurally sound collapse and lead to loss of normal synapses. Therefore, molecular memory in the form of methylation plays a vital role in promoting normal brain activity and its disruption can ultimately lead to dementia. 

Chicken, meet egg. Egg, meet chicken.

So, from bacteria to human, carboxyl methylation is necessary for memory. Did these pathways evolve individually in parallel, or did the memory we have today originate in the predominant lifeforms found within us?

 

Suggested Reading

Li and Stock. (2009) Biol. Chem. 390: 1067-1096. DOI 10.1515/BC.2009.133

STORYTELLING IN SCIENCE: THE CELL AS YOUR FAVORITE RESTAURANT PART III

<img alt="Storytelling in science visualized through bacteria"      src="open-for-business.png">
Storytelling in Science visualized

Perhaps a running list of metaphors so far:

Restaurant: bacterial cell

Building: cell membrane

Doors: channels and transporters

Patrons: metabolites/compounds/substrates and products

Employees: proteins/enzymes

Managers: two-component proteins to regulate gene transcription

Employee list: genome

Copy machine: DNA replication machinery

So, in the last part our restaurant was going great and we opened up a new restaurant with the same employee list among other things. The two restaurants are now independent of each other and are free to act accordingly.

What if things change and times are not going as well? The overall number of patrons drastically decreases, not enough electricity (ATP) to run the restaurant or running water (redox potential)? What if disaster is about to strike? How can the restaurant prepare all the managers, employees, the building, the doors, the patrons for it?

Luckily the restaurant has a monitoring system that can quickly make sure the restaurant will be ready for anything that comes its way. The monitoring system can take snapshots of all data generated by the restaurant: power supply, water supply, patron count, employee count, conditions outside the restaurant like weather or competing restaurants. The monitoring system is the bacterial second messenger systems. With the support of the managers, the monitoring system can instantaneously keep track of all variables and make changes as needed.

The system is detecting the start of a drought. This drought will lower the number of patrons coming and going from the restaurant. The drought will also change the available electricity and water supply of the restaurant. The monitoring system sounds the alarm, a message is sent over the intercom for all the managers and employees to hear and react to. The intercom message alerts some managers to call in additional employees while telling others to stop their work. Some employees take on a new job in preparation for the drought. The intercom message is the bacterial second messenger cyclic-di-GMP. The entire restaurant begins preparations for the drought so it can survive until better times are present. Other than changes to managers and employees, some new employees are called in to prepare the building itself. Perhaps to change the number of doors. The employees may also change the exterior of the building to better withstand the drought like changing a wood exterior to a brick or stucco one. The brick or stucco are the exopolysaccharides, complex sugars on the exterior of the cell that can serve as protection or to help cells adhere to each other to ride out the hard times together. 

When times change, the restaurant has to be able to change with them. That is why these restaurants have been in business for ~3 billion years and still going strong.