Manuel Varela: Who knows what goes on in the lives of those bacteria?

An Interview with Manuel Varela: Who knows what goes on in the lives of those bacteria?


Michael F. Shaughnessy –


1)  Bacteria seem to be all around us- and there are good bacteria and bad bacteria. Frederick Griffith seemed to discover something going on with bacteria and DNA. Can you first tell us why some bacteria are good, and others not so good?

This is a very good question! Bacteria are single-celled living microbes which are indeed present around us, virtually everywhere. An entire bacterial organism consists of a single living cell. The bacteria do not have an internal nucleus, a situation that is called prokaryotic. In general, there are two main domains of prokaryotes, those called the archaea, for ancient bacteria (previously designated as archaeobacteria and archaebacteria) and the eubacteria, for true bacteria.

There certainly may be what one may call, as you say, good versus bad bacteria.

Although estimates vary widely between 90 and 99.9%, many microbiology textbooks denote that the vast majority of the bacteria are considered “good” in the sense that they are harmless, or perhaps simply just benign, or maybe even useful in some way.  On the other hand, then, between 0.1 and 10% of the known bacteria are thought to be “bad” in the sense that they are, for instance, pathogenic, i.e., disease-causing, or perhaps they could very well be pathogenic, given the right set of circumstances.

Examples of ways in which bacteria may be thought of as “good” are briefly considered here.

First, it is widely thought by microbiologists that the archaea were the first living organisms on Earth. This is considered good in that evidence shows these ancient beings conducted activities that made it possible for other life to be possible, as well. For example, the archaea may have “invented” photosynthesis, allowing a good energy making mechanism from light, for later higher plants to use in order to live.

Additionally, the ancient bacteria developed enzymes that neutralized the degradative activity of oxygen, called oxidation. In short, because the archaea were the first living beings, they made it possible for other organisms to live, too.

Second, the good bacteria (and other microbes) are necessary for all other life on Earth to continue. One way in which this is possible is that the bacteria recycle many of the elements needed for life, like carbon, oxygen, nitrogen, sulfur, etc. Without these elemental cycles, human life on Earth would be impossible.

Third, good bacteria make substances we may like, desire, or need. For example, bacteria help make foods, like sauerkraut, pickles, cheeses, breads, or chocolate. Bacteria may help produce beverages, like coffee and wine. Bacteria are quite often used in biotechnology to make diagnostic tests for diagnosis of infectious diseases. Bacteria in the human gut may make our vitamin K. Some bacteria produce antibiotics which we can use to treat infectious diseases.

Fourth, bacteria may also constitute part of the overall microbiome on a person or in some environmental niche. As part of the human microbiome, for instance, many species of bacteria keep certain pathogenic bacteria from gaining a foothold and causing disease.

Fifth, some bacteria may live on the roots of plants, helping the plants to acquire important nutrients, like nitrogen, from soil in order to permit the plants to synthesize protein or nucleic acids for plant and crop growth.

Lastly, some bacteria, called probiotics, are considered so beneficial that they are packaged up in foods, like yogurt, or in pill form; these bacteria are meant to provide health benefits in the human gut. Some recent studies have shown that certain gut bacteria affect moods, with certain bacterial types associated with happiness and others with depression, etc.

On the other hand, bacteria that we may consider to be bad are those in which pain and suffering are caused during infectious disease. As a whole, bacteria that are pathogens have been responsible for more death and disease than any other microorganisms.  The term ‘pathos’ means ‘to suffer.’ Pathogenesis means to make suffering or disease, which is considered any deviation from a healthy state. Infectious disease caused by bacteria may vary from mild to moderate to severe to even lethal.

Pathogenic (bad) bacteria may be transmitted to humans and other animals via a variety of modes. Such transmission modes include foodborne, soil-borne, airborne, vector-borne, person-to-person, blood-borne, oral-fecal routes, etc.

In certain circumstances, bacteria that are normally benign may occasionally take advantage of an opportunity to become pathogenic. These so-called opportunistic bacteria may then grow and predominate, causing pathological consequences in a patient. As one example, harmless bacteria residing on the skin and serving to keep other dangerous bacteria from taking over, might find themselves having become moved to deeper tissue layers or even the blood, from a minor trauma such as a scratch, an insect bite, a needle injection, a surgical incision, etc., and now this previously harmless, helpful bacterium grows unchecked in the blood or internal tissue wreaking havoc and causing disease.

Another way in which so-called bad bacteria mediate their effects is through environmental contamination and pollution. Bacteria play a key role in causing the production of foul-smelling organic compounds that are indicative of polluted environments. Such environments are non-productive and even toxic to human health.

One last example includes bacterial contamination of commercial products, like pharmaceutical items, plastics, blood products, eukaryotic cell cultures, medical and dental equipment, industrial equipment, spacecraft, and commonly used items like cell phones, etc. These types of bacterial contaminations may affect the quality of the products and of the resulting profits, and of commerce, in general.

2) How do bacteria change and grow and mutate and develop over time?

Interestingly, when bacteria grow, they exponentially increase in their numbers rather than get bigger or older. Yet, their generation times, i.e., the time it takes to double their bacterial cell numbers, are quite shorter than, let’s say, that of humans. Bacterial growth generations may be as short as 20 minutes. Other bacteria may take a few days to grow, depending on the species or growth conditions. Bacteria often change and mutate when they grow.

Bacteria have developed several ways to change and mutate. Bacteria will change by behaviorally responding to their environment and by mutation. Behaviorally speaking, if bacteria come across a new nutrient, they may turn on cellular machinery that’s specialized to acquire it and metabolize it for energy to grow. Sometimes, the nutrient utilizing cellular machinery is already turned on, staying on, especially in cases where there may be a universal nutrient that is available, like the sugar glucose.

A mutation is defined as an alteration in the nucleotide base sequence along a string of double-stranded DNA within, e.g., a genome or a plasmid molecule. The organism harboring the mutated DNA is called a mutant. When bacteria change by mutation, they have developed several ways to do so, some of which are clever but simple.

One simple way in which DNA is mutated is called spontaneous mutation. In this process, the biological machinery that makes DNA just simply makes a mistake when inserting the new nucleotides into the new DNA chains. These types of mutations happen randomly once or twice in every 100,000 or in a million nucleotide incorporations, which is very accurate but not perfect, of course.

Other ways include physical and chemical means of generating mutations in DNA. Physical modes of DNA mutation might include radiation or ultra-violet light. Chemical means of DNA mutagenesis involve changes to the chemistry properties of the DNA that in turn alter the base sequences along DNA.

Importantly, once a bacterium has had its DNA mutated and has, thus, become a mutant, the affected DNA will be transferred to the next generation of bacteria (called vertical DNA or gene transfer) or can be transferred to completely different microbial species (called horizontal DNA or gene transfer).

There are several kinds of these DNA transfer systems. One is called conjugation in which DNA from one generation to the next is propagated into the next generation by mating. The bacterial host receiving the DNA is called a recipient or a conjugant.

Another DNA transfer system is transduction, in which a virus called a bacteriophage (or phage for short) carrying the DNA does the transfer by infecting the bacterium. The infected bacterium in turn propagates the DNA producing by many new viral progeny, each new phage in turn can move on to a new bacterial host and start the infection cycle all over again. The bacterial host getting the new DNA by this method is referred to as a transductant.

Another mode for the transfer of DNA is called transposition. During this process, a piece of DNA called a transposon or “jumping gene” will separate from its location and either move to another location along the genome, to a plasmid, or actually leave the host to find a new host whereupon the transposon DNA will insert itself into the genome or plasmid of another bacterial species; such a recipient bacterium is called a transposant.

The last DNA transfer mechanism considered here involves the one that Dr. Griffith discovered. It’s called transformation. In this system, a host cell becomes competent in order to allow the uptake of the foreign DNA, becoming a transformant.

Each of these DNA or gene transfer methods will manifest their new properties to the new host cells that take up the new foreign genetic elements. It is through these systems that bacteria mutate and change. Since the generation times are short bacteria can be altered just as quickly.

3) Frederick Griffith lived in the 1920’s (probably long before the electron microscope).  How did he do his research and what did he learn?

Dr. Griffith’s famous 1928 experiment is routinely included in a fair amount of detail in most major textbooks dealing with Biology, Biochemistry, Cell Biology, Genetics, Microbiology, Microbial Physiology, and Molecular Biology. Griffith studied two versions of a bacterium that investigators at the time had called Pneumococcus but is now known as Streptococcus pneumoniae. The two bacterial versions were distinct variants of each other.

The first version was a non-virulent and non-lethal kind of the bacterium which he called Type II or the R form. The R form bacteria were called as such because they formed rough-looking colonies on culture media in Petri plates. The R form was harmless because it lacked a specialized covering called a capsule—it was non-encapsulated, and macrophages and phagocytes could readily protect the body from the R forms of the bacteria; thus the R type was non-lethal.

The second version Griffith called type III or the S form. The S form bacteria were referred to as such because they formed smooth-looking colonies on cultured Petri plates. The S form was harmful because it contained the outer covering capsule. The encapsulated S form was protected from the macrophages and phagocytes. Thus, the S form bacteria survived in the body, permitting them to grow virtually unchecked throughout the lungs and other organs, causing a severe pneumonia from which the body often could not tolerate, killing the animal or human host. Thus, the S form was lethal.

Griffith injected live mice with the lethal S bacteria, and within days the mice were dead. When autopsied, the dead mice had numerous S bacteria. When Griffith injected living mice with the non-lethal R bacteria, the mice lived, and no R or S bacteria were found in the mice. When Griffith injected into living mice the lethal S bacteria that were first killed by heating, the mice lived—this was not terribly surprising that a dangerous but dead bacterium cannot cause disease.

It is the next step, however, that surprised everyone, including Griffith. He injected into the mice a mixture of live harmless R bacteria with dangerous but dead (heat-killed) S bacteria, and the mice died! Remember, each one alone (i.e., live R and dead S bacteria) did not kill any mice—but together the live R and dead S mixture did kill the mice. When autopsied, the dead mice all had living S bacteria in their blood and organs! But these latter dead mice hadn’t been injected with any of the S bacteria, yet there they were in large numbers, living in the dead mice. These data were surprising and unsettling.

How could it possibly be that mice injected with a mixture of two seemingly harmless entities (i.e., live but non-virulent R plus virulent but dead S) be so acutely lethal?  Neither entity alone was lethal, but together they were potently lethal. Many explanations were put forth to explain these startling results.

Somehow, something, an “S Substance,” as Griffith called it, from the dead S bacteria “transformed” the live R to become lethal S bacteria! What, then, was the nature of this so-called “transforming principle?”

On the face of it, one could simply argue that the live R bacteria stole the capsules from the dead S bacteria. That is to say perhaps the transformation principle consisted of stolen capsules. The problem with this explanation is that Griffith had already established that the S Substance was sensitive to heat but the capsules were not—the capsules were heat resistant. Yet, the S bacteria themselves, and hence the transforming principle, were sensitive to the heat.

Unfortunately, Griffith himself incorrectly reasoned that the transforming principle, the S Substance, was protein; and this protein then made sugar, which then made up part of the capsules—it was known that the capsules contained protein and sugar.

To further confuse the situation, Griffith had then referred to the S Substance as the “S Antigen.” In this way, Griffith and others then argued that the R form disassembled the parts (a process called catabolism) from the dead S bacteria (or took their already disassembled S parts) and used the parts to reassemble (a process called anabolism or biosynthesis) the capsules onto themselves to become S bacteria. This explanation was based on the newly emerged field of metabolism—the breaking up of foodstuffs to make new stuff that a bacterium might need for other purposes.

Next, Griffith then argued that perhaps the R bacteria simply mutated to the S form, a process called reversion—the R bacteria reverted back to the S bacteria. He based this explanation on the observation that in hospitals, patients with the pneumonia had several types of the bacteria—so, did the patients get infected with multiple bacterial types or did the bacteria convert to other types?  Griffith reasoned that the bacteria may have reverted by mutation from the R to the S forms.

The problem with all of these explanations is that they contradicted the incorrect notion that that bacteria species were unable to switch between sub-types, like R versus S. Therefore, primary opponents of the idea of bacterial transformation, Dr. Oswald Avery and colleagues Drs. Maclyn McCarty and Colin MacLeod, ironically became the first to provide definitive experimental data about the true nature of Griffith’s transforming principle: DNA.

Distilled down to the essential and simple basics, in 1944, Avery, McCarty and MacLeod repeated Griffith’s shocking experiments, except that this time they included protease (digests protein), RNase (digests RNA) or DNase (digests DNA) in their protocol. They found that the group with DNase failed to transform live R to the S type when live R cells were mixed with heat-killed S cells. With the dead S bacteria DNA being chewed up by the DNase, there was no principle (no DNA) by which to conduct the transformation of the R cells into the S cells. In so doing, Avery and colleagues were the first to identify DNA as the transforming agent. Later, equally famous studies in 1952 performed by Alfred Hershey and Martha Chase using bacteriophages and Escherichia coli bacteria to perform transduction, these investigators convincingly confirmed that DNA was indeed Griffith’s S Substance, the transformation principle.

4) How do bacteria “transform” (and does heat and cold have any impact on bacteria?)

Bacterial transformation may be defined as the uptake of foreign free DNA that can be incorporated into the bacteria, which in turn can then go on to acquire novel properties conferred by the newly acquired DNA. Bacteria that can acquire DNA are said to be “competent” to do so.

Today, the biological mechanism for bacterial transformation has been worked out for a variety of bacterial species, including that for Griffith’s Streptococcus pneumoniae. The transformation process for Griffith’s bacteria follows below.

First, the bacterium makes a protein called competence factor (CF) that’s secreted from the bacterial cell. As the cells grow, the CF proteins accumulate, and, in a process called quorum sensing, CF binds to another protein called ComD that’s present on the cell walls of the grown bacteria. Next, the ComD uses ATP to attach a phosphate molecule to itself, and in a process called a phosphorylation cascade, transfers the phosphate to another protein called ComE, and then on to SigH. The phosphate-SigH complex then turns on a set of genes that code for a large molecule called the “transformasome complex.” Once a bacterium makes this transformasome, the cell is said to be competent to take up foreign DNA. The transformasome then binds DNA that’s nearby the cells and brings it to the inside of the cell, where the DNA can then be expressed, altering the transformed bacterium.

Regarding your question about heat and cold, investigators can instigate transformation artificially in the laboratory using calcium chloride, cold, and heat, in order to have bacteria take up DNA, for cloning or other purposes. Investigators grow bacteria, add calcium chloride, and place on ice, in ice buckets. This artificially makes the cells competent. Next, the investigators add DNA. The DNA enters some of the cells. Next, the cells with DNA are briefly warmed up in a process called heat-shocking; the cells are not killed, but it possibly closes up the cells walls to lock-in the DNA inside the cell. Then the cells are cultured on Petri plates containing culture media to obtain transformants. Others argue that the calcium chloride and cold merely lets the bacteria bind DNA and that the heat-shocking permits the DNA entry into the cells. Whatever the case is, transformation in the laboratory is an important molecular biology technique.

5) What do we know about the life and times of Frederick Griffith?

Unfortunately, details of Griffith’s early life are not entirely clear. Regarding his birthdate, for example, sources vary between 1877, 1879, and 1881. Undoubtedly, he was born in Hale, England. His parents were Joseph and Emily Griffith. Later, Griffith studied medicine at Victoria University in Liverpool, England, and graduated in 1901, with his M.B. degree. He was then a physician and surgeon at the Royal Infirmary in Liverpool. He was awarded an Alexander Fellowship in the area of pathology at a private institute called the Thompson Yates Laboratory. While there, Griffith studied the Great Consumption as a member of the Tuberculosis Commission as a bacteriologist. Acquiring a degree in public health (D.P.H.) at Oxford in 1910, Griffith then became a medical officer in 1911 in London, England, at the Ministry of Health. Tragically, Griffith was killed during an air raid blitz by German bombers on April 17, 1941, during World War II. He was never to know the acclaim bestowed in his honor.

6) In your opinion, why is his work so important?

I think that Griffith’s discovery of bacterial transformation led ultimately and directly to the elucidation of DNA as the heredity material and later to the structure of DNA by James Watson, Francis Crick, Rosalind Franklin, Raymond Gosling, and Maurice Wilkins. The focus on DNA as being central to life and being the blueprint for living beings then led to the development of the fields of molecular biology and biotechnology, and later to genomics, proteomics, and now bioinformatics. These are all considered important avenues of study.

7) What have I neglected to ask?

Just so it is clear to the reader, the term bacterial transformation as we have discussed here has a completely different meaning when compared to transformation of non-bacterial cells from eukaryotic organisms. As such, transformation in this other case is taken to mean the conversion of a normally dividing cell into a tumorigenic cell, which may be considered either benign or cancerous. Transformation in this particular case involves an uncontrolled cell growth. This is also an extremely active field of study. When one considers the introduction of new DNA into cells that are eukaryotes, such as plant cells, or fungi, or even human cells in culture, the term is often called transfection.