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From Micro- to Macromolecular Life: The Microbiome

Microorganisms (or microbes) can be found anywhere on Earth - from a boiling hot Yellowstone Geyser to the lowest point on Earth's surface and even inside your gut!


In fact, from the day we are born, we form a symbiotic relationship (meaning a mutually beneficial relationship) with bacteria. Nonetheless, it comes as a surprise to many to hear that the human body holds as many as ten trillion of these microorganisms. This is around ten times more than all of your body cells combined!


In fact, despite being hidden from the naked eye, scientists have estimated that there are an estimate of nonillion (10^30) microbial cells on Earth, which is more than the estimated number of stars in the Universe!


What constitutes a microbiome?

A microbiome describes a unique network of microorganisms that are occupying a habitat. These different types of microbes range from organisms such as bacteria, protists, archaea, fungi, and algae.


Essentially, you can picture the microbiome as a tiny bustling city, with every road and pavements filled with thousands of different microbial species interacting with each other on a daily basis.


When we are born, our microbiome has a low species diversity and is mainly populated by Proteobacteria and Actinobacteria. Proteobacteria are facultative anaerobes, meaning they can switch between aerobic and anaerobic respiration, depending on the availability of oxygen. They, in turn, encourage the colonization of obligate anaerobes (i.e. organisms that can only live in an environment deprived of oxygen) like Actinobacteria, which are necessary for us to maintain a healthy gut by lowering its redox potential (which is crucial for homeostasis).


As we grow up, the microbiome diversifies. In particular, anaerobic bacteria such as Firmicutes and Bacteroidetes tend to dominate the adult microbiota and they play a role in carbohydrate metabolism. Besides that, Bacteroidetes also contribute to energy production and conversion in addition to the transport and metabolism of amino acids.


What is the role of a microbiome…


...in nature?

The microbes in your body are not the only examples of microbiomes.

For example, microbiomes are essential for the formation of healthy ecological relationships. In the food chain, certain microbes such as the photosynthetic cyanobacteria act as the primary producers of food, whereas others like Pseudomonas fluorescens - which break down dead organic substances in the soil - serve as decomposers. Hence, the presence of both is crucial to sustaining the food chain of entire ecosystems!


Due to the nutrient conversion properties that arise from the microorganisms’ interactions, microbiomes are also crucial to our understanding of ecological cycles (such as the carbon and the nitrogen cycles). In fact, Sergéi Winogradsky (who we will revisit later in this article), the guy who discovered the starting components of the nitrogen cycle, discovered the nitrogen-fixing bacteria (i.e. the bacteria that convert atmospheric nitrogen into inorganic compounds usable by plants) by studying the microbiome. Besides that, other bacteria found in the microbiome, namely denitrifying bacteria, transform nitrogen-compounds secreted by animals (e.g. ammonia) back into atmospheric nitrogen, which thereby initiates a new cycle.


Thus, as we can see, microorganisms can interact within themselves to produce major effects in the food-chain and ecological cycles due to the high variety and proximity of species found in microbiomes.


…in our body?

The microbiome constitutes a large part of what we are. So far, microbiomes have been found in the skin, mammary glands, placenta, seminal fluid, uterus, ovarian follicles, vagina, lungs, oral cavity, eyes, biliary tracts, and the gut.


Microbiomes have been observed to be incredibly advantageous for us. Given that the gut and lung microbiomes are the most well-studied microbiomes so far, we will focus on them in this section.


The gut microbiota

To begin with, the normal gut microbiota has specific roles in host nutrient metabolism.


For example, the enzymes present in our body can only break down sugars with α-linkages. Nonetheless, long-chain hydrocarbons such as cellulose (which are found in vegetables and fruit) have ß-linkages. So, how can we digest foods that constitute a great part of our diet if the main nutrient they’re composed of cannot be digested by our own enzymes?


The answer lies in the microbiome. Bacterial enzymes in the gut microbiome can digest the ß–(1-4’) glycosidic bond of cellobiose (i.e. the monomer of cellulose), which is found in plant-based foods. Some of the by-products formed are then consumed by the bacteria, thereby increasing the size and diversity of the microbiome, whilst others can be converted into fat within our body, and therefore become a major source of energy for us. Thus, eating fruit and vegetables can offer you both a great source of energy and a maintenance plan for your microbiome. All of these, in turn, results in improved gut health, increased movement of substances across the intestine, and the acceleration of your metabolism rate.


Another example of its benefits to digestion includes its xenobiotic metabolism (a.k.a. drug metabolism) abilities, whereby less polar compounds are transformed into excretable polar compounds and the maintenance of the gut mucosal barrier’s structural integrity.


Additionally, the gut microbiota is also involved in supporting immunomodulation (i.e. the regulation of our immune system) and they have the ability to protect us against pathogens. Hence, having a stable gut microbiota correlates with having good health. When this microbial community is unstable, diseases associated with metabolism (e.g. inflammatory bowel disease, obesity, and diabetes) become prevalent.


There are several factors that play a role in shaping a normal gut microbiota, and consequently, good health. They include:


  1. The mode of delivery of the individual, either vaginal or caesarean section (C-section)

  2. Diet during infancy (breast milk or formula feeds) and adulthood (vegan-based or meat-based)

  3. The use of antibiotics or antibiotic-like molecules that are derived from the environment of the gut commensal community


As discussed above, our environment and lifestyle heavily impact our microbiomes across our bodies. Even though diet is an important factor in shaping the microbial population within our body, other habits such as smoking can also affect the microbiome (particularly the microbiome that is found inside our lungs).


The lung microbiome

Maintaining a healthy lung microbiome is a crucial task, as it is involved in preventing excessive inflammation whenever we are exposed to air pollutants.


In fact, were it not for the lung microbiome, exposure to commonly found chemicals in the air, such as common household air pollutants as nitrogen oxides, could lead to an anaphylactic shock, which is an extreme allergic reaction that causes our blood pressure to drop so low that oxygen cannot get to our organs. It is crucial to highlight that that does not make us immune to air pollution – constant exposure to air pollutants may cause populations in our lung microbiome to decay, making us more susceptible to respiratory tract infections, supposing a major threat to our health.


Meanwhile, another interesting application of lung microbiomes is that they also act as biomarkers for disease. That is, its bacterial composition can tell us the health status of our lungs.


But why is this?


The bacterial composition of the lung microbiome has been observed to change within patients suffering from pulmonary diseases like lung cancer. Therefore, the lung microbiome’s composition may reflect the health status of our lungs. As a matter of fact, research in this field goes as far as to suggest that the lung microbiota’s composition plays a role in carcinogenesis.


In fact, did you know that a healthy, stable lung microbiome could be a possible factor of immunity against COVID-19? Maintaining a healthy, stable lung microbiome has been shown to play a role in developing immunity against viral infections, so future research on the lung microbiome might be crucial to understand the factors involved in pathogenesis and immunity of COVID-19.


How does our body maintain the microbiome?

Now that we have looked at the functions of certain microbiomes, one question you may be asking yourself is - how exactly are this relationship between the bacteria from the microbiome and its host maintained? Why does our immune system allow these microbes to co-exist with us, whilst the system initiates an immune response to other pathogenic microbes?


This can be explained by immunology, which you may have come across in your pre-university studies.


The immune system can distinguish between our own cells and foreign invaders because all cells have antigens (i.e. molecules that can induce an immune response) on their surface. On that note, foreign cells have distinct antigens that can be immediately recognised by the immune system (and so they get attacked).


Thinking along those lines, the microbiome has a set of antigens that stimulates the immune system to recognise them as self rather than foreign, thereby limiting any attacks by the immune system against these bacteria.


How could we study microbiomes?


The early experimental techniques

The first time that the concept of microbiomes was conceived was when the field of microbial ecology was first introduced back in the 19th century.


In particular, microbial ecology constitutes the study of microbial interactions within the same microbiome and their contribution to ecological cycles (including the carbon cycle and the nitrogen cycle).


While working with both Beggiatoa and nitrogen-fixing bacteria, Serguéi Winogradsky, the father of microbial ecology, faced a lot of complications when trying to isolate these bacteria into a pure culture. He eventually came to the conclusion that in order to culture microorganisms, the culture has to resemble the original growth environment.


The methodology used by Winogradsky involved collecting small samples of the bacterial populations in their natural environment using glass columns, whereby the input of nutrients can be regulated. The columns are left for a few days and a gradient is formed according to oxygen distribution (from a high to low concentration as we go down the column). Hence, the different types of bacterial populations can be identified by their position in the column. Using this method, Winogradsky was able to study bacterial populations of interest in their natural environment. The distribution of microbes along the column is described below in Figure 1.

Figure 1: Distribution of bacterial populations in a Winogradsky column. As time goes by, an oxygen gradient is formed in the column. When this happens, bacteria that can only grow in aerobic or light conditions float on the top of the liquid, whereas those that can only grow in anaerobic (oxygen-null) conditions deposit themselves at the bottom. On the top of the liquid, there are cyanobacteria, algae, and other light-dependent or obligate anaerobes. As we move down the column from a higher to lower oxygen concentration, non-sulfur photosynthetic bacteria (i.e. bacterial species that cannot use hydrogen sulfide as an electron donor) such as Rhodomicrobium are observed. These are followed by purple sulfur photosynthetic bacteria (such as Chromatium) and green photosynthetic bacteria (such as Chlorobium). Finally, at the very bottom, you’ll find excreted organic compounds, mud, and inorganic salts deposit. This figure was adapted from Plantlet.


Even though this technique allows us to identify the different types of bacterial populations in a Winogradsky column, there are limitations in terms of the extent to which we can study the functions and characteristics of each individual microorganism in the microbiome.


Modern techniques to study the microbiome

Despite the progress made at that time, microbiome studies were limited to observational studies until the late 20th century.


16s rRNA gene sequencing

In fact, it wasn’t until 1977 when Carl Woese started cataloging the differences between 16S ribosomal RNA (one of the RNA components of the ribosome) from different microorganisms, and this immensely helped us to understand microorganisms better.


To provide some context, ribosomes play a critical role in protein synthesis and are made out of two key components, namely the large and small ribosomal subunits.


16S rRNA is one of the components found within the small ribosomal subunit. It was hypothesised that 16S rRNA was likely to be conserved over evolutionary time (as to preserve the ribosome’s functionality). Hence, a meaningful comparison can be made between the 16S rRNA of different microorganisms - organisms that are closely related in the phylogenetic tree would have very similar genetic sequences, whereas unrelated ones would have greater differences in their 16S rRNA sequence.

Figure 2: A diagram of the prokaryotic ribosome’s components, with 16S rRNA highlighted in the top right corner. The ribosome is composed of a large and small subunit, both of which are needed to initiate protein synthesis. Each subunit is composed of protein and ribosomal RNA (rRNA) components. The sizes of the complete ribosome, its subunits and RNA components are indicated in Svedberg (S), which references sedimentation coefficients in ultracentrifugation. More specifically, heavier objects will have a larger sedimentation coefficient. Figure modified from Open University and EZ BioCloud.


Through 16S rRNA gene sequencing, the sequence of 16S rRNA for the different organisms can be solved, and we would be able to find a highly conserved sequence together with some hypervariable regions - all of which could be used to classify the species present within the microbiome.

Figure 3: 16S rRNA sequencing of the skin microbiota. The process first begins with the collection of a biological sample from the skin, whereby DNA and RNA from the microbiome species are isolated. Next, the hypervariable region within the 16S rRNA fragment that we are interested to study is amplified using the polymerase chain reaction (PCR). Then, high-throughput sequencing tools such as amplicon pooling and next-generation sequencing (NGS) are used to identify the fragments of interest and to obtain their DNA sequence. Finally, once the data is processed using bioinformatic tools, the classification of species follows suit. This figure was modified from the Journal of Investigative Dermatology, the JoVE journal, Burst, and Pixabay.


As seen in Figure 3 above, those hypervariable regions can be used to distinguish among taxa. This revolutionised the field of taxonomy. As a result, 16S rRNA sequencing remains the gold standard for taxonomic identification even up to this day.


Nevertheless, a disadvantage of 16S rRNA sequencing is that this technique only works effectively for simple and small DNA samples. This is because 16S rRNA sequencing often has laborious workflows and varying amplification frequencies per organism (i.e. one specific microbe may produce more 16S rRNA copies than another). This makes it harder for researchers to determine the actual proportions of bacterial populations within the microbiome.


Metagenomic sequencing

On a separate note, trying to derive functional information of microbial populations from their genetic sequence opens up a whole new field known as metagenomics, which is defined as “the study of a collection of genomes from a mixed community of organisms (usually microbial communities” using bioinformatic tools (e.g. next-generation sequencing).


In particular, metagenomic studies are helping researchers to widen their knowledge of host-pathogen interactions. This is done through the discovery of genes that permit certain microbes to influence their host. Other than that, we can also use metagenomic studies to investigate host-microbe interactions, and this could reveal novel information about how the microbiome is connected to human diseases.


Moving forward - what could we do with our knowledge?

It is clear that microbes are both essential for our health and maintenance of the environment we live in. However, the extent of their importance has not been tested yet due to limitations of in vivo studies (i.e. experiments involving working with living organisms) in human subjects. Although we might never be able to know what would happen if we completely removed the microbiome from an organism, we can still learn about the many benefits of a healthy microbiome to their host as well as the diseases that are correlated with its malfunctioning.


As we uncovered more about the microbiome and how they affect our health, a major interest arose in the scientific community to sequence the microbiome in order to better understand the overall picture of our physiology, health, and disease. Shortly after the Human Genome Project was finalised, another equally ground-breaking and ambitious project was conceived - this was called the Human Microbiome Project, whereby their goal is to identify microorganisms from the human microbiome that are central to our health and development of diseases. This was finalised in 2016 and you can read more about it here.


Meanwhile, a research team from the Massachusetts Institute of Technology (MIT) have also developed artificial guts that are designed to mirror the human microbiome. This ultimately opens up a new way for the research community to study the microbiome and how it changes when in a diseased state.


Performing fecal microbiota transplantation (FMT) is one of the many examples of how we have managed to exploit our knowledge about the microbiome, and this method has been particularly effective in combating the increasingly prevalent Clostridium difficile infection (CDI). In short, FMT, also known as a stool transplant, involves the administration of microbes found in faeces from a healthy donor into the recipient’s intestinal tract through processes like a colonoscopy or by prescribing oral capsules. The purpose of this treatment is to alter the composition of the patient’s gut microbiome so that positive health status can be restored.


One could argue that this finding is a pretty big deal. The reason for this is due to antibiotic resistance becoming a huge headache. In this case, research data has suggested that metronidazole and vancomycin (first-line drugs used to treat CDI) have gradually lost its efficacy and become extremely costly, respectively. Thus, FMT may be a more cost-effective and viable replacement for antibiotics.


Moving onto the impact of microbiomes in nature, our current knowledge can also be exploited as a tool to improve agricultural productivity. For example, did you know that microbes can be used instead of fertilisers? By developing the right cocktail of bacteria, engineering them, followed by coating the seed with them, we could potentially use our knowledge about the microbiome to reduce the use of fertilizers, especially nitrogen fertilizers such as ammonium nitrate. In fact, companies like Indigo Ag or Joyn Bio are already exploring the full potential of microbes in agriculture to tackle this issue.


Nevertheless, there is still so much more to learn about the microbiome, their roles in nature, and the relationships that they hold with their hosts. Excited to learn more? Do check out the BiomedCentral Journal for Microbiome to read more about this fascinating field of study.


Authors

Catalina Costenco, BSc Biochemistry with Management

Alexandra Hopkins, BSc Biochemistry

Julia Barret Joly, BSc Biochemistry


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