Microbial Cross-Feeding: Extracellular Metabolism

Definition of Cross-Feeding:

Cross-feeding is the transfer of material from a producer to a recipient, which is assimilated by the recipient or participates in energy transformation in the recipient and/or producer, and alters the fitness of the producer and/or the recipient. Cross-feeding must involve different species or genotypically or phenotypically distinct populations.

Introduction:

• Abundance of Microbes: The staggering number of microbes on Earth, estimating around 10^30 individual bacterial and archaeal cells. This immense microbial diversity allows them to inhabit diverse environments.
• Microbial Communities: Microbes are often found in complex communities. Notably, the human gut and cow rumen host incredibly dense microbial populations of around 10^14 and 10^15 microbes, respectively, with the human colon containing approximately 10^11 cells per ml. In contrast, seawater communities are comparatively dilute, ranging from 10^4 to 10^6 cells per ml.
• Competition for Nutrients: The existence of microbes in these communities means that they often have to compete for limited resources, leading to physiological and ecological consequences. This competition for scarce nutrients is a fundamental aspect of microbial life.
• Dormant Microbes: Most microbes experience periods of nutrient starvation and may enter a state of dormancy or growth arrest. Dormant microbes can be a significant portion of the microbial population in various environments.
• Maintenance Metabolism: Even in a dormant state, microbes still exhibit a low metabolic rate. They maintain critical activities like DNA repair, protein and lipid turnover, osmoregulation, and nutrient transport. This low-level metabolic activity ensures their survival and prepares them for potential growth when nutrients become available.
• Extracellular Behaviors: Microbes combat nutrient limitation by releasing molecules into the extracellular environment. These molecules can include a wide range of substances like metabolites, exoenzymes, siderophores, toxins, signals, and cell surface-associated factors. These released molecules are actively secreted or passively excreted into the environment or even transferred directly between cells.
• Effects of Released Molecules: The molecules released by microbes have various effects:
• They can influence the survival and proliferation of the producer, helping them compete for resources.
• These molecules can impact neighboring microbes, either through competition or cooperation, depending on the specific substances involved.
• In some cases, these molecules can even affect host cells, highlighting the complex interactions between microbes and their environments.
• Significance of Cross-Feeding: The text introduces the idea that microbial cooperation through cross-feeding has significant implications:
• Cross-feeding can affect community-wide processes of global and societal importance. For example, it plays a role in biogeochemical cycles that influence Earth's climate and can impact the progression of polymicrobial infections.

Cross Feeding:

• Material Transfer: Cross-feeding involves the transfer of various materials. This can include not only molecules but also subatomic particles like electrons and protons. An example is the sharing of a proton motive force between cells, where the electrochemical gradient is crucial.
• Assimilation or Energy Transformation: For cross-feeding to occur, the material transferred must be assimilated by the recipient or used for energy transformation in both the producer and recipient. This is essential for the mutual benefit of the interaction.
• Altered Fitness: The fitness of both the producer and recipient must change as a result of assimilation or energy derived from the transferred material. This altered fitness is a key outcome of cross-feeding interactions.
• Involvement of Different Species/Populations: Cross-feeding typically involves different species or genotypically/phenotypically distinct populations. This diversity is a practical consideration for studying and understanding the nature and consequences of cross-feeding interactions.

EXTRACELLULAR MOLECULES THAT PROMOTE CROSS-FEEDING

1. Metabolites
2. Exoenzymes
3. Siderophores
4. Toxins
5. Quorum-Sensing Signals
6. Extracellular Matrix Components of Biofilms
7. Extracellular Vesicles
8. Nanotubes
9. Nanowires

1. Metabolites:

• Microbes release a wide range of small molecules, known as metabolites.
• Metabolites are not toxins or signals for cell-cell communication; instead, they primarily serve as biosynthetic intermediates.
• Examples of cross-fed metabolites include sugars, organic acids, amino acids, vitamins, gases, and inorganic elements.
• Metabolite cross-feeding is a common occurrence in microbial communities and is found across all three domains of life.
• Cross-feeding relationships can often be established through various means, including genetic engineering, experimental evolution, and the careful design of environmental conditions.
• Example where no genetic engineering or evolution was needed to establish cross-feeding between NH4+-excreting Chlamydomonas reinhardtii and CO2-producing yeast Saccharomyces cerevisiae. This exemplifies how some cross-feeding interactions can naturally occur based on environmental conditions.
• Metabolite cross-feeding can take different forms, including unidirectional (from one microbe to another), bidirectional, or multidirectional, where metabolites are reciprocally exchanged between partners.
• Cross-fed metabolites can serve different purposes: they can be either metabolic waste, which no longer benefits the producer after excretion, or they can be communally valuable compounds, where both the producer and recipient benefit from their exchange.
• In a microbial community, metabolite cross-feeding interactions can be numerous, resulting in a complex network involving both waste and communally valuable metabolites. This complexity underscores the dynamic and intricate nature of cross-feeding within microbial ecosystems.

Waste Metabolites:

• Microbial metabolism results in the production of various waste products.
• These waste products, such as O2, CO2, and reduced compounds, can be essential for the growth and survival of other microbes in the community.
• For instance, O2 waste from photosynthetic cyanobacteria is crucial for the aerobic respiration of diverse microbes.
• CO2, a waste product of many heterotrophic lifestyles, provides essential carbon for various photo- and litho-autotrophs.
• Fermentative microbes excrete reduced compounds that serve as important carbon and/or electron sources for diverse lifestyles.
• Anaerobic respiration leads to cross-feeding of various inorganic metabolites like H2S, NH4+, and Fe2+.
• These cross-feeding interactions contribute to global biogeochemical cycles that regulate Earth's climate.

Syntrophy:

• Syntrophy is a form of cross-feeding where an energetically unfavorable metabolism of a producer is coupled to the metabolism of a recipient partner.
• The producer is often a fermentative bacterium relying on electron-rich carbon sources.
• As fermentation products like H2, CO2, formate, and acetate accumulate, syntrophic partners, such as archaeal methanogens, remove these products.
• This removal makes the metabolism of the producer thermodynamically favorable.
• Syntrophy is crucial in the carbon cycle and anaerobic degradation of electron-rich pollutants.
• In some cases, syntrophic relationships have been established and evolved in laboratories, illustrating their importance.

Example:

Syntrophy is a form of microbial cooperation where one microbe relies on the metabolic activity of another microbe to survive. This cooperation is crucial when the metabolic process of one microbe produces waste products that are harmful or inhibitory to it. So, by working together, they can overcome these challenges and both benefit.

Desulfovibrio vulgaris is a sulfate-reducing bacterium. It typically consumes organic compounds and produces sulfate as a waste product. However, in certain conditions where sulfate is not available (deprived of sulfate), Desulfovibrio vulgaris can switch to a fermentative lifestyle.

Fermentation is a metabolic process where organic compounds like lactate are broken down without the use of external electron acceptors like sulfate. During fermentation, waste products, such as hydrogen gas (H2), carbon dioxide (CO2), and organic acids, are generated.

Now, fermentative lifestyles are energetically unfavorable when these waste products, especially hydrogen gas, accumulate. The buildup of hydrogen gas can inhibit the growth of Desulfovibrio vulgaris. This is where Methanococcus maripaludis, a methanogenic archaeon, comes into play.

Methanococcus maripaludis is capable of consuming hydrogen gas and carbon dioxide. In a syntrophic relationship, Desulfovibrio vulgaris and Methanococcus maripaludis cooperate. The hydrogen gas produced by Desulfovibrio vulgaris during fermentation becomes a valuable resource for Methanococcus maripaludis, which consumes it. In return, Methanococcus maripaludis helps remove the inhibitory hydrogen gas and allows Desulfovibrio vulgaris to continue its fermentation process.

This mutual support creates a thermodynamically favorable environment for both species. Over time, the populations of Desulfovibrio vulgaris often lose the ability to respire sulfate, indicating they've specialized in obligate syntrophic growth with Methanococcus maripaludis. In other words, they've evolved to depend on this cooperative partnership.

Communally Valuable Metabolites:

• Microbes exchange metabolites that are valuable to their growth and survival, and this can occur naturally or be engineered synthetically.
• Valuable compounds shared through cross-feeding include ammonia/ammonium (NH3/NH4+), amino acids, nucleobases, and vitamins.
• Many microbes have specific needs and cannot produce certain essential metabolites on their own, a condition known as auxotrophy. This means they require these compounds to grow and reproduce.
• Auxotrophy, especially for amino acids and vitamins, is common in microbial populations.

Evolution of Auxotrophies and Cross-Feeding:

• The evolution of auxotrophy and cross-feeding is closely intertwined. Cross-feeding environments can provide a surplus of costly metabolites, which encourages the adaptive loss of genes responsible for biosynthesis.
• This drives the emergence of auxotrophic microbes that rely on the metabolite-producing microbes to provide essential nutrients.
• A genome-scale survey of 800 microbial communities found a high potential for metabolic interdependencies between different subcommunities, emphasizing the prevalence of cross-feeding in nature.

Examples of Cross-Feeding:

Example 1 - Amino Acid Cross-Feeding:

• Many microbes have specific needs for essential amino acids, and some of them cannot produce these amino acids on their own.
• This inability to synthesize certain amino acids is referred to as amino acid auxotrophy.
• To address this need, microbes engage in cross-feeding, where one microbe, the producer, is capable of synthesizing these amino acids and releases them into the environment.
• Other microbes, the auxotrophic partners, rely on the producer for these essential amino acids for their growth.
• Even a single producer microbe can support a community of auxotrophic partners by providing the necessary amino acids, allowing them to thrive.
• This cross-feeding strategy helps microbes in the community cooperate and grow efficiently.

Example 2 - Vitamin B12 Exchange in Marine Environments:

• Another example of cross-feeding involves the exchange of vitamin B12 in marine ecosystems.
• Vitamin B12 is a crucial and energetically costly compound to produce, and not all marine microbes can synthesize it.
• In this case, heterotrophic bacteria that can produce vitamin B12 share it with major algal primary producers like diatoms in the oceans.
• Diatoms are essential primary producers, contributing significantly to oceanic primary production.
• By sharing vitamin B12, the heterotrophic bacteria support the growth and productivity of diatoms.
• This cooperation between different microbial groups has a direct impact on oceanic ecosystems and primary production, influencing the marine food web and carbon cycling.

Example 3 - Cross-Feeding of Metabolically Expensive Compounds:

• Cross-feeding can extend to more complex and metabolically expensive compounds like heme and quinones, which are essential for respiratory processes in certain microbes.
• Sometimes, subpopulations of microbes cooperate to provide each other with these missing respiratory cofactors.
• This cooperation enhances their ability to perform crucial metabolic activities, particularly respiration.
• The synergistic cross-feeding of heme and quinones can also lead to an increase in the virulence or disease-causing potential of certain microbial populations.
• This cooperative behavior can have complex effects on the interactions between microbes and their host organisms.

Mechanisms of Metabolite Release:

• It's not always clear why microbes release valuable compounds in sufficient quantities to support other species.
• Excretion may be accidental due to leaky cell membranes. This can be particularly true for compounds like ammonia/ammonium where charge equilibrium depends on pH.
• Another potential mechanism of nutrient transfer is through cell lysis, where cells die and release their valuable contents.
• Selection for excretion may occur if the release of valuable compounds stimulates reciprocation from recipient microbes, leading to mutualism.

Directed Reciprocation and Positive Selection:

• Reciprocal cross-feeding is thought to drive rapid metabolism and excretion of metabolites like pyruvate and glycolate by microbes like Prochlorococcus, one of the most abundant marine cyanobacteria.
• This metabolic exchange may have been reinforced through the coevolution of associated heterotrophic partners that reciprocate by excreting complementary compounds.
• The mechanisms behind the passive or active excretion of costly metabolites are diverse and not fully characterized.

Conditions for Cross-Feeding Evolution:

• To evolve cooperative cross-feeding of valuable metabolites, the conditions must allow for reciprocation to be confined to cooperative partners and avoid being outcompeted by exploitative community members.
• The phylogenetic conservation of positive or negative effects of one microbe on another suggests that some mechanism of directed reciprocation was involved in the evolution of cross-feeding.

Example 1 - Enhanced Ammonia Acquisition:

• In this scenario, we have a population of Escherichia coli (E. coli) bacteria.
• Some of these E. coli mutants have been genetically modified or naturally evolved to be really good at taking up ammonia (NH4+), which is a source of nitrogen.
• Ammonia is essential for their growth, and these mutants rely on it for their nitrogen needs.
• However, there are also other E. coli bacteria in the same environment that aren't as efficient at producing ammonia.
• These two groups of E. coli, the ammonia-enhanced mutants and the ammonia-poor ones, are grown together.
• The ammonia-enhanced mutants, because they're so good at acquiring ammonia, can help their poor partners by taking in extra ammonia and sharing it.
• This cooperation benefits both groups, as the ammonia-poor E. coli can grow better with a more abundant source of nitrogen, and the ammonia-enhanced mutants can grow better because they receive something they need, too.
• Essentially, this is an example of cross-feeding where one group helps the other by sharing an essential nutrient, and both benefit.

Example 2 - Division of Labor:

• Here, in a population of E. coli bacteria, a single group of E. coli cells has divided into two subpopulations with different roles.
• One subpopulation specializes in consuming acetate, a type of organic compound.
• The other subpopulation focuses on growing faster when they have access to glucose, which is another energy source.
• When glucose is available, the subpopulation that's good at using glucose grows quickly.
• Meanwhile, the acetate-consuming subpopulation is better at breaking down acetate and can thrive in its presence.
• This is like a division of labor within the same community of E. coli.
• The glucose-specialists take advantage of glucose when it's around, and the acetate-specialists consume acetate efficiently.
• Together, they maximize their use of different energy sources in the environment.

2. Exoenzymes (Extracellular Enzymes)

• Exoenzymes are extracellular enzymes that break down large polymers into smaller, transportable monomers.
• They have diverse substrates, including glycosidases (for breaking down carbohydrates), proteases (for breaking down proteins), nucleases (for breaking down nucleic acids), lipases (for breaking down lipids), lignin-cleaving laccases, and peroxidases (for breaking down lignin and other complex organic compounds).
• Exoenzymes are important in cross-feeding because they release readily consumable nutrients into the extracellular environment, which can be taken up by neighboring cells.
• Microbes that secrete exoenzymes are often considered keystone species and primary colonizers in establishing microbial communities on polymeric substrates.

Key Role in Cross-Feeding:

• Exoenzymes are essential for cross-feeding, which is a cooperative strategy among microorganisms in a community.
• When microorganisms release exoenzymes into their surroundings, they break down complex polymers present in the environment into simpler components.
• These simpler components, or monomers, are then readily consumable nutrients that can be taken up by neighboring microorganisms in the community.
• In essence, exoenzymes help convert complex, hard-to-use resources into easily accessible nutrients, promoting cooperation and resource sharing among different microbial species.

Keystone Species and Primary Colonizers:

• Microbes that secrete exoenzymes are often considered "keystone species" in microbial communities.
• Keystone species are those whose presence has a disproportionately large impact on the community's structure and function. In the case of exoenzyme-producing microbes, they create a favorable environment for other microorganisms by making nutrients more accessible.
• Exoenzyme-producing microorganisms are often the primary colonizers on polymeric substrates, as they kickstart the breakdown of complex organic materials, making it possible for other species to establish and thrive in the community.

Examples of Exoenzyme-Mediated Cross-Feeding:

1. Cellulase-Secreting Microbes:

• Host-associated cellulase-secreting microbes sustain communities of gut bacteria and cellulose-consuming animal hosts (e.g., ruminants and termites).
• These animals lack the native ability to access carbon in cellulose, but cellulase-secreting microbes provide them with this capability.
• The breakdown of cellulose and lignin via exoenzymes is essential in the global carbon cycle, as it drives the mineralization of plant polymers.
• Industries also utilize lignocellulose-degrading enzymes to lower the cost of liberating sugars for biofuel production.

In depth:

1. Cellulase-Secreting Microbes in Host-Associated Ecosystems:

• Cellulase-secreting microbes are microorganisms that produce cellulase enzymes, which are essential for breaking down cellulose, a complex carbohydrate found in plant cell walls.
• In host-associated ecosystems like the digestive systems of ruminants (e.g., cows) and termites, cellulase-secreting microbes play a crucial role.
• Ruminants and termites lack the native ability to directly access the energy stored in cellulose. However, they have evolved to host cellulase-secreting microbes that provide them with the capability to digest cellulose.
• These microbes break down cellulose into simpler sugars (monomers), which can then be used by the host animals as an energy source. In essence, these microbes act as "digestive helpers" for their hosts.

2. Contribution to the Global Carbon Cycle:

• The breakdown of cellulose and lignin by cellulase-secreting microbes is vital in the global carbon cycle.
• Plants store a significant amount of carbon in cellulose and lignin. When these complex polymers are broken down into simpler compounds (monomers), such as glucose, by cellulase enzymes, the stored carbon is released.
• This carbon release is essential as it contributes to the cycling of carbon in ecosystems. It allows carbon to be used as an energy source by various organisms and eventually returned to the environment.

3. Industrial Applications for Biofuel Production:

• The cellulase enzymes produced by cellulase-secreting microbes have practical applications in various industries, particularly in the production of biofuels.
• Lignocellulose-degrading enzymes, including cellulases, are used to break down plant biomass into simple sugars, such as glucose.
• These sugars can then be fermented by microorganisms to produce biofuels like ethanol. The use of cellulase enzymes in this process is crucial for efficiently liberating sugars from plant materials.
• This industrial application not only supports the production of biofuels but also contributes to the sustainable utilization of plant resources and reducing the cost of biofuel production.

2. Invertase-Producing Microbes:

• Exoenzymes, such as invertase (β-Fructofuranosidase), can release glucose and fructose from sucrose.
• Invertase (β-Fructofuranosidase) also known as saccharase and sucrase.
• However, under well-mixed conditions, an estimated 99% of these monosaccharides diffuse away from the producer yeast cell before they can be imported.
• While exoenzyme production can be cooperative, it also makes these microbes vulnerable to exploitation by other species and "cheater" subpopulations.
• Cheaters do not produce exoenzymes but benefit from nearby exoenzyme-secreting cells without the cost of synthesis.

In depth:

1. Invertase and Sucrose Digestion:

• Invertase, also known as β-Fructofuranosidase, is an exoenzyme produced by certain microorganisms, including yeast.
• Invertase plays a vital role in breaking down sucrose, a common sugar found in various natural sources.
• When sucrose is digested by invertase, it is split into two simpler sugars: glucose and fructose. These monosaccharides can be used as an energy source by the producing microorganism or potentially other nearby microorganisms.

2. Diffusion of Monosaccharides:

• One critical aspect to consider is the behavior of the released glucose and fructose monosaccharides.
• Under well-mixed conditions, it's estimated that approximately 99% of these monosaccharides tend to diffuse away from the microorganism that produced them before they can be taken up or imported back into the producer's cell.
• This means that most of the sugars the microorganism worked hard to break down end up being lost to the environment or potentially utilized by other microorganisms in the vicinity.

3. Cooperative Nature of Exoenzyme Production:

• The production of exoenzymes like invertase can be considered a cooperative behavior in microbial communities.
• By breaking down complex sugars into simpler forms, microorganisms can make nutrients available for themselves and, to some extent, for other microbes in the same environment.
• This cooperation can contribute to the overall well-being of the microbial community by providing accessible nutrients.

4. Vulnerability to Cheating:

• While exoenzyme production is cooperative, it also creates a vulnerability for the producing microorganisms. This vulnerability comes in the form of "cheaters."
• Cheaters are microorganisms that take advantage of the cooperative behavior of exoenzyme producers without incurring the cost of producing exoenzymes themselves.
• Cheaters benefit from the nearby exoenzyme-secreting cells without the energy expenditure required for enzyme synthesis. This gives them a competitive advantage, as they reap the benefits without the costs.

3. Chitinase Producers:

• Vibrio cholerae, for example, produces chitinase to degrade insoluble chitin into N-acetylglucosamine oligomers and monomers.
• Under well-mixed conditions, mutants of V. cholerae that do not produce chitinase can acquire extracellular N-acetylglucosamine released by chitinase-producing strains and outcompete the producers.
• Exoenzyme-secreting populations must avoid being overrun by exploitative neighbors.

Strategies to Prevent Exploitation:

• Some polymer-degrading enzymes, like bacterial cellulases, are often attached to the cell surface. This requires the exoenzyme-secreting cell to be in close contact with the polymer substrate, increasing the chances of substrate acquisition after depolymerization.
• Security can also come from the use of exoenzymes that release soluble oligomers instead of monomers. Oligomers are then imported into the periplasm before further depolymerization.
• This oligomer-cleaving and import strategy was shown to allow Bacteroides
• thetaiotaomicron from the human gut to utilize mannan without supporting the growth of a mannose monomer-utilizing cocultured strain.
• Not all microbes can degrade oligomers, giving an advantage to the oligomer-cleaving enzyme producers.
• In some cases, the control of related cheaters is achieved through group behaviors, including quorum sensing (QS) and the clustering of producers in biofilms.

In depth:

1. Chitinase Production by Vibrio cholerae:

• Chitinase is an exoenzyme produced by Vibrio cholerae, a bacterium known for causing cholera.
• Chitinase serves the purpose of degrading chitin, which is a complex and insoluble carbohydrate found in the exoskeletons of arthropods (insects, crustaceans) and the cell walls of some fungi.
• Chitinase breaks down chitin into simpler compounds, including N-acetylglucosamine oligomers and monomers. These breakdown products can serve as a source of nutrients for microorganisms.

2. Competition and Exploitation:

• Under well-mixed conditions, certain mutants of Vibrio cholerae have emerged that do not produce chitinase.
• These non-chitinase-producing mutants can still benefit from the presence of chitinase-producing strains in their environment.
• The chitinase-producing strains break down chitin, releasing N-acetylglucosamine compounds into the environment. These compounds can then be utilized by the non-chitinase-producing mutants without incurring the metabolic cost of chitinase production.
• As a result, these mutants can outcompete the chitinase producers for the available nutrients. This situation illustrates the exploitative aspect of their interaction.

3. Balancing Cooperation and Competition:

• Exoenzyme-secreting populations, like those producing chitinase, face the challenge of avoiding being overrun by exploitative neighbors.
• While cooperation, in the form of exoenzyme production, benefits the microbial community by making nutrients accessible, it also creates the potential for exploitation.
• The balance between cooperation (releasing nutrients into the environment) and competition (competing for those same nutrients) is a fundamental aspect of microbial ecology.

Strategies to Prevent Exploitation:

1. Cell-Surface Attachment of Enzymes:

• Some polymer-degrading enzymes, like bacterial cellulases, are anchored or attached to the cell surface of the exoenzyme-secreting microorganisms.
• This attachment ensures that the producing cells remain in close contact with the polymer substrate they are breaking down. It increases the chances of effectively acquiring the breakdown products (oligomers or monomers) after depolymerization.
• By remaining attached to the substrate, the producing microorganisms can prevent cheaters from taking away the released products, ensuring that the benefits of enzyme production are retained by the producer.

2. Release of Soluble Oligomers:

• Another strategy to prevent exploitation involves the use of exoenzymes that release soluble oligomers, rather than monomers, during polymer degradation.
• Oligomers are intermediate products with multiple smaller subunits. Oligomers are released into the environment but are not broken down completely into monomers.
• These oligomers can be imported into the periplasm (the space between the cell membrane and cell wall) before further depolymerization occurs. This import step can act as a security measure against cheaters.

3. Advantage of Oligomer-Cleaving Enzymes:

• Not all microorganisms have the necessary enzymes to degrade oligomers effectively. This gives an advantage to the microorganisms that produce enzymes capable of cleaving oligomers into monomers.
• By having this capability, the oligomer-cleaving enzyme producers can fully utilize the breakdown products, preventing cheaters from benefiting from the oligomers.

4. Group Behaviors and Quorum Sensing (QS):

• In some cases, the control of related cheaters is achieved through group behaviors, including quorum sensing (QS).
• QS is a microbial communication system that coordinates gene expression based on population density. When the population reaches a certain threshold (quorum), specific genes are activated.
• QS can control the timing of exoenzyme production and other cooperative behaviors. This can help ensure that the benefits of cooperation are only extended to cooperative members when their numbers are sufficient to deter cheaters.
• Additionally, microbial communities may form biofilms, where microorganisms cluster together. In biofilms, the proximity of producers can help protect against exploitation by cheaters.

Example:

The example involving Bacteroides thetaiotaomicron from the human gut illustrates how the strategy of cleaving oligomers and importing them can lead to a competitive advantage, particularly when dealing with complex polysaccharides like mannan.

1. Mannan as a Complex Polysaccharide:

• Mannan is a type of complex polysaccharide found in various plant materials and cell walls.
• It consists of long chains of mannose monomers linked together.

2. Bacteroides thetaiotaomicron's Strategy:

• Bacteroides thetaiotaomicron is a type of gut bacterium that has evolved a strategy to effectively utilize mannan as a nutrient source.
• Instead of directly breaking down mannan into mannose monomers, Bacteroides thetaiotaomicron produces an enzyme that cleaves mannan into soluble oligomers.
• These oligomers are not fully broken down into mannose monomers but remain as intermediate products, which are still valuable as an energy source.

3. Competitive Advantage:

• The key advantage of this strategy is that not all microorganisms in the gut have the capability to efficiently degrade these mannan-derived oligomers.
• When Bacteroides thetaiotaomicron releases these oligomers into the gut environment, it can quickly import and utilize them for growth.
• Meanwhile, a co-cultured strain of bacteria in the gut that primarily relies on mannose monomers as a nutrient source may not be able to fully utilize the oligomers.
• As a result, Bacteroides thetaiotaomicron gains a competitive advantage in accessing and utilizing mannan, without significantly supporting the growth of the mannose monomer-utilizing strain.

3. Siderophores

• Siderophores are small molecules secreted by microorganisms that chelate essential metals, particularly iron, but also metals like copper, manganese, and zinc. These metals serve as crucial cofactors for numerous enzymes.
• In the oceans, siderophores play a significant role in global iron cycling, impacting ocean productivity and nitrogen cycling.
• Iron is often scarce in various environments, including within host organisms, where there is competition for iron between host cells and resident microbiota, as well as among different microbial species.
• Siderophore production and access to iron through these molecules can determine the course of infections. Some hosts actively try to block siderophore function as part of their innate immune defense, and pathogens have evolved "stealth siderophores" to evade detection by the immune system.
• Siderophores are secreted molecules that can participate in cross-feeding by enabling iron acquisition not only by their producers but also by unrelated members of microbial communities.
• Examples of siderophore-mediated iron cross-feeding include interactions between different bacterial taxa and even between bacteria and eukaryotic microbes.
• Some microbes may specialize in scavenging siderophores produced by other organisms.
• It's important to note that the ability of a microbe to use a foreign siderophore is an ongoing molecular arms race, as siderophore synthesis permits a wide range of structural permutations.

In depth:

1. Chelation of Essential Metals:

• Siderophores are small molecules produced and secreted by microorganisms, and their primary role is to chelate essential metals, with a particular emphasis on iron.
• Essential metals like iron, copper, manganese, and zinc serve as crucial cofactors for numerous enzymes in living organisms. Thus, acquiring these metals is essential for microbial growth and metabolic processes.

2. Impact on Ocean Productivity and Nitrogen Cycling:

• In the oceans, siderophores play a significant role in global iron cycling.
• Iron is often scarce in seawater, and the ability of microorganisms to produce and secrete siderophores helps them acquire iron.
• This, in turn, can impact ocean productivity, as iron is a limiting nutrient for phytoplankton growth. Increased iron availability due to siderophore activity can influence the entire marine food web and carbon cycling, including nitrogen cycling.

3. Competition for Iron:

• In various environments, including within host organisms, there is intense competition for iron between host cells and resident microbiota, as well as among different microbial species.
• Siderophore production and the ability to access iron through these molecules can determine the outcome of infections and microbial interactions.

4. Immune Defense and "Stealth Siderophores":

• Some hosts have evolved mechanisms to block siderophore function as part of their innate immune defense. This is a defense strategy to limit the growth of potential pathogens by restricting their access to iron.
• In response, some pathogens have developed "stealth siderophores" that evade detection by the host's immune system. These stealth siderophores allow pathogens to access iron without alerting the host's defense mechanisms.

"Stealth siderophores" are a clever adaptation developed by certain pathogens to evade detection by the host's immune system while still enabling the pathogen to access iron, a crucial nutrient for their growth. Let's delve into how these stealth siderophores work:

A. Siderophores and the Immune System:

• Siderophores are small molecules produced by microorganisms to chelate and acquire essential metals, particularly iron.
• In a host's body, the immune system recognizes the presence of foreign invaders, including pathogenic microorganisms. One of the immune responses to infection is to restrict the availability of iron, as it limits the growth of many pathogens.

B. The Challenge for Pathogens:

• For pathogens, acquiring iron in a host's body is a challenging task, as they must contend with the host's immune defenses, which work to sequester iron and restrict its availability to limit the pathogen's growth.
• Traditional siderophores produced by pathogens can be detected by the host's immune system, triggering a response to sequester or limit iron availability.

C. Evolution of Stealth Siderophores:

• In response to the host's immune system, some pathogens have evolved stealth siderophores, which are modified or specialized siderophores designed to avoid detection by the immune system.
• These stealth siderophores have structural variations that reduce their recognition by the host's iron-sequestration mechanisms and immune defenses.

D. Evasion of Host Immune Surveillance:

• Stealth siderophores may have altered chemical structures that make them less recognizable as foreign molecules by the host's immune system.
• This evasion strategy allows the pathogen to access iron without triggering a robust host immune response aimed at limiting iron availability.

E. Iron Acquisition and Pathogen Growth:

• With stealth siderophores, pathogens can effectively scavenge iron from the host's tissues, such as the bloodstream or specific sites of infection, without being subjected to the same level of immune surveillance.
• This enables the pathogens to secure the iron they need for essential metabolic processes, including growth and proliferation, within the host's body. 

5. Cross-Feeding and Microbial Interactions:

• Siderophores participate in cross-feeding by enabling iron acquisition not only by their producers but also by unrelated members of microbial communities.
• This cross-feeding can involve interactions between different bacterial taxa and even between bacteria and eukaryotic microbes, such as fungi.
• Some microbes have specialized in scavenging siderophores produced by other organisms, and this can be a strategy to gain access to essential iron resources.

how siderophores enable cross-feeding between different microbes:

1. Siderophores as Iron-Scavenging Molecules:

• Siderophores are molecules produced by microorganisms to chelate and acquire essential metals, especially iron, which is often a limiting nutrient in various environments.
• These siderophores are secreted into the environment, where they can capture iron ions from the surroundings.

2. Cross-Feeding among Microbes:

• Siderophores play a crucial role in cross-feeding, which is the exchange of nutrients between different members of microbial communities.
• This cross-feeding involves interactions not only between different bacterial species but also between bacteria and eukaryotic microbes, such as fungi or other microorganisms.

3. Role of Siderophores in Cross-Feeding:

• Siderophores facilitate cross-feeding by enabling the acquisition of iron not only by their producers but also by unrelated members of the microbial community.

Here's how it works:

• Producer Microbes: Some microbes are proficient siderophore producers, and they secrete siderophores into the environment to scavenge iron. These producer microbes might be capable of utilizing the iron themselves.
• Non-Producer Microbes: Other microbes within the community may not produce their own siderophores or may produce them less efficiently. These non-producer microbes face challenges in acquiring iron from the environment directly.
• Cross-Feeding: In a cross-feeding scenario, non-producer microbes can take advantage of the siderophores released by producer microbes. The siderophores capture iron and make it available in a form that is accessible to a wider range of microorganisms.
• Benefit to Non-Producers: Non-producer microbes benefit from this arrangement by accessing essential iron resources without the metabolic cost of siderophore production. They essentially "borrow" the iron-capturing capabilities of the producers.
• Benefit to Producers: Producers also benefit, as their siderophores, while initially capturing iron for themselves, can indirectly support the growth of other community members, fostering a cooperative dynamic.

4. Specialized Iron Scavengers:

• Some microorganisms have specialized in scavenging siderophores produced by other organisms. These scavengers have evolved to efficiently recognize and utilize siderophores from the environment.
• This specialization allows these microbes to thrive by accessing iron resources even in iron-limited conditions.

6. Molecular Arms Race:

• It's important to note that the ability of a microbe to use a foreign siderophore is part of an ongoing molecular arms race. Siderophore synthesis permits a wide range of structural permutations, and microorganisms continually evolve strategies to acquire and utilize these molecules effectively.

Siderophore Specificity:

• Cell surface receptors that recognize siderophores can be specific to certain species, limiting the uptake of siderophores by other community members. This specificity restricts iron acquisition by non-producers and contributes to the competition for iron within microbial communities.
• Siderophore specificity serves to protect against iron theft from unrelated organisms, ensuring that iron is accessed primarily by the bacteria that produce the corresponding siderophores.
• However, despite siderophore specificity, siderophore-secreting bacteria are still susceptible to exploitation by cheaters within their own species or closely related taxa.
• Cheaters, which do not invest in siderophore production, can benefit from the siderophores released by others, gaining access to iron without the cost of synthesizing siderophores themselves.

In depth:

1. Cell Surface Receptors and Siderophores:

• Cell surface receptors on the outer membranes of microorganisms play a crucial role in recognizing and binding to siderophores, which are small molecules secreted to chelate and capture iron ions.

2. Specificity of Siderophore Receptors:

• These cell surface receptors can exhibit specificity, meaning they are tailored to recognize and bind to specific siderophores produced by particular microbial species.
• Specificity of these receptors ensures that a microorganism primarily takes up the siderophores it produces or those that are compatible with its receptor.

3. Limiting Iron Acquisition by Non-Producers:

• Siderophore specificity places limitations on the uptake of siderophores by other community members. This restricts iron acquisition by non-siderophore-producing microorganisms and contributes to the competition for iron within microbial communities.
• In other words, non-producer microbes that do not have matching receptors for a specific siderophore are unable to efficiently utilize that siderophore to access iron.

4. Protection Against Iron Theft:

• Siderophore specificity serves as a protective mechanism against iron theft from unrelated organisms. It ensures that iron is primarily accessed by the bacteria that produce the corresponding siderophores.
• This protection mechanism helps to maintain the integrity of microbial communities and prevents iron from being exploited by microorganisms that did not invest in siderophore production.

5. Vulnerability to Cheaters:

• Despite siderophore specificity, siderophore-secreting bacteria are still susceptible to exploitation by "cheaters" within their own species or closely related taxa.
• Cheaters are microbial variants that have evolved not to invest in siderophore production. They benefit from the siderophores released by the producers, gaining access to iron without the metabolic cost of synthesizing siderophores themselves.

6. Balancing Cooperation and Competition:

• The presence of cheaters adds a layer of complexity to the dynamics of microbial communities. It highlights the constant balance between cooperation (siderophore production) and competition (cheating) in the quest for essential nutrients like iron.
• This balance may involve mechanisms like quorum sensing, which allows microorganisms to coordinate the timing of siderophore production based on population density.

Siderophore Cheaters:

• Siderophore cheating, where certain bacteria exploit the siderophores produced by others to access iron, has been observed in various environments, including soil, freshwater, and marine bacterial communities.
• Despite the existence of siderophore cheaters, microbial communities have developed mechanisms to mitigate or police this form of cheating.
• One example of mitigation involves the secondary role of pyoverdine siderophore produced by Pseudomonas aeruginosa, which goes beyond iron acquisition.
• When the environment experiences oxidative stress, Pseudomonas aeruginosa downregulates pyoverdine secretion, allowing pyoverdine to accumulate in the periplasm of the bacterial cells.
• In the periplasm, pyoverdine sequesters iron and prevents Fe3+ from generating harmful hydroxyl radicals through the Fenton reaction.
• Cheater strains that do not produce pyoverdine lose the protective role of pyoverdine and can be removed from the population during periods of oxidative stress.

In depth:

1. Siderophore Cheating:

• Siderophores are molecules produced by microorganisms to capture iron, a critical nutrient. In some cases, certain bacteria, referred to as "siderophore cheaters," exploit the siderophores produced by others to acquire iron without investing in siderophore production themselves.
• Siderophore cheating has been observed in various environments, including soil, freshwater, and marine bacterial communities, highlighting its ecological significance.

2. Mitigation of Siderophore Cheating:

• While siderophore cheating can be advantageous for the cheaters, it poses challenges to the microbial communities, as it disrupts the balance of iron acquisition strategies.
• Microbial communities have evolved mechanisms to mitigate or police siderophore cheating, ensuring a level of fairness and cooperation within the community.

3. Pyoverdine Siderophore in Pseudomonas aeruginosa:

• One example of mitigation involves the secondary role of the pyoverdine siderophore produced by the bacterium Pseudomonas aeruginosa.
• Pyoverdine is primarily used for iron acquisition, but it has a secondary function that goes beyond simply capturing iron.

4. Role of Pyoverdine During Oxidative Stress:

• When the environment experiences oxidative stress, which can lead to the formation of harmful hydroxyl radicals through the Fenton reaction, Pseudomonas aeruginosa responds by downregulating pyoverdine secretion.
• This downregulation allows pyoverdine to accumulate in the periplasm of Pseudomonas aeruginosa bacterial cells.

5. Protective Role of Pyoverdine:

• In the periplasm, pyoverdine sequesters iron and prevents Fe3+ from generating harmful hydroxyl radicals through the Fenton reaction.
• Essentially, pyoverdine acts as a protective molecule, safeguarding the bacterial cells from the damaging effects of oxidative stress by controlling the availability of iron.

6. Consequences for Cheater Strains:

• Cheater strains within the same microbial community that do not produce pyoverdine lose the protective role of pyoverdine during oxidative stress.
• As a result, they can be disadvantaged during periods of oxidative stress, as they lack the protective mechanism provided by pyoverdine.

a. The Fenton Reaction

The Fenton reaction is a chemical reaction involving the conversion of hydrogen peroxide (H2O2) into highly reactive hydroxyl radicals (•OH) in the presence of ferrous iron (Fe2+). This reaction is responsible for the generation of harmful hydroxyl radicals, which can cause oxidative damage to biomolecules such as proteins, lipids, and DNA. The Fenton reaction can be represented as follows:

H2O2 + Fe2+ → •OH + OH- + Fe3+

Here's an explanation of each component:

• H2O2 (Hydrogen Peroxide): Hydrogen peroxide is a relatively stable molecule that can serve as a source of reactive oxygen species (ROS) under certain conditions. It can be produced within cells as a natural byproduct of metabolism.
• Fe2+ (Ferrous Iron): Ferrous iron is the reduced form of iron (Fe2+) that can catalyze the Fenton reaction. It can be found in cells and can be released during various physiological processes.
• •OH (Hydroxyl Radical): The hydroxyl radical is an extremely reactive and damaging ROS. It can initiate chain reactions that result in oxidative damage to biomolecules. It is highly toxic to cells.

b. Oxidative Stress:

Oxidative stress refers to a condition where there is an imbalance between the production of ROS (reactive oxygen species), including hydroxyl radicals, and the ability of an organism to detoxify or repair the resulting damage. This imbalance can be induced by various factors, including:

• Exposure to Toxins: Exposure to environmental toxins, pollutants, or chemicals can lead to the production of ROS and oxidative stress.
• Infection and Inflammation: Infection and inflammation can stimulate the immune system to produce ROS as a defense mechanism against pathogens, resulting in oxidative stress.
• Radiation: Ionizing radiation, such as X-rays and gamma rays, can directly or indirectly generate ROS and induce oxidative stress.
• Metabolic Processes: Normal cellular metabolism, including the electron transport chain in mitochondria, can produce ROS as byproducts, contributing to oxidative stress.
• Metal Ions: The presence of transition metal ions, particularly iron, can catalyze the generation of ROS through reactions like the Fenton reaction.

Oxidative stress can lead to damage to cellular components, including proteins, lipids, and DNA, and is associated with various diseases and aging processes. Cells and organisms have developed antioxidant defense mechanisms to counteract the effects of oxidative stress and prevent damage caused by ROS.

Mutualistic Cross Feeding with Siderophores:

• While siderophore exploitation is well-documented, there are fewer known examples of siderophores forming the basis for mutualistic cross-feeding in microbial communities.
• In one such example, bacteria associated with marine algae produce siderophores that enhance the algal host's iron acquisition. This mutualistic relationship benefits both the bacteria and the algae, potentially through the reciprocal release of consumable metabolites by the algae.
• Another intriguing exception is the discovery that hosts, such as Caenorhabditis elegans and mammalian cells, can derive nutritional benefits from siderophores secreted by their microbiota.
• For instance, the siderophore enterobactin, produced by Escherichia coli, was found to promote iron uptake within the mitochondria of these host cells, leading to improved iron acquisition. In contrast, siderophores from potential pathogens, such as pyoverdine, did not have the same iron-promoting effect.

In depth:

Mutualistic cross-feeding involving siderophores is a fascinating aspect of microbial interactions. Here's an in-depth exploration of these interactions and their implications:

1. Mutualistic Cross-Feeding with Siderophores:

• Siderophores, as small molecules secreted by microorganisms to chelate and capture essential metals like iron, are often associated with competition and exploitation. However, there are intriguing instances where siderophores foster mutualistic cross-feeding in microbial communities.

2. Siderophores Enhancing Iron Acquisition in Marine Algae:

• One example of mutualistic cross-feeding involving siderophores occurs in the marine ecosystem. Bacteria associated with marine algae produce siderophores that enhance the iron acquisition of the algal hosts.
• Iron is a critical nutrient for algal growth, and these mutualistic bacteria help the algae acquire iron efficiently. In return, the bacteria potentially benefit from the release of consumable metabolites by the algae.

3. Nutritional Benefits for Hosts:

• Siderophores can also provide nutritional benefits to hosts, such as Caenorhabditis elegans (a nematode) and mammalian cells. In these cases, the siderophores are produced by the microbiota associated with the hosts.
• For instance, enterobactin, a siderophore produced by Escherichia coli, was found to play a mutualistic role by promoting iron uptake within the mitochondria of host cells. This improved iron acquisition benefits the host.
• It's important to note that not all siderophores have the same effect. Siderophores produced by potential pathogens, such as pyoverdine, may not promote iron acquisition to the same extent.

4. Implications of Mutualistic Siderophore Cross-Feeding:

• These mutualistic interactions challenge the traditional view of siderophores as molecules involved in competitive iron acquisition. Instead, they demonstrate the versatility of siderophores as molecules that can facilitate mutually beneficial relationships.

5. Ecological and Biomedical Significance:

• Mutualistic cross-feeding with siderophores has ecological significance in the marine environment, where it contributes to nutrient cycling and algal growth.
• In a biomedical context, understanding how host-associated microbiota can provide nutritional benefits through siderophores sheds light on the intricate relationships between microbes and their hosts.

4. Toxins

• Microbial toxins are secreted compounds that have evolved for their inhibitory and often lethal effects on other organisms, including host cells or other microbes.
• Toxins come in various chemical structures and sizes, ranging from small molecules like antibiotics and cyanide to large multisubunit proteins such as cholera toxin.
• Toxin delivery methods vary, including passive excretion or active secretion into the extracellular environment, as well as direct injection into host or microbial cells.
• Some small molecule toxins can serve as cross-fed metabolites when present at subinhibitory concentrations or when the recipient microbe can tolerate the toxin.
• For example, certain bacteria can use cyanide as a nitrogen source for growth, and there have been observations of some microbes consuming antibiotics, although this is met with some skepticism.
• Toxins can also indirectly facilitate cross-feeding by promoting nutrient acquisition. Toxins often damage or lyse cells, releasing their intracellular contents, which can benefit neighboring microbes. This makes toxins similar to exoenzymes in their potential to support cross-feeding.
• In polymicrobial infections, pathogens can interact synergistically, possibly involving toxin-mediated cross-feeding. However, the exact role of toxin-mediated cross-feeding in these interactions is not yet well understood.
• Contrary to expectations, there is evidence suggesting that toxins may lead to increased competition between microbial populations. Some studies have shownseveral TnSeq experiments that more genes are essential for a pathogen during coinfection compared to monoinfection, indicating intensified competition and potential nutrient limitation.
• The extent to which toxin-mediated cross-feeding occurs in microbial communities remains unclear and warrants further investigation.

In depth:

1. Diverse Nature of Microbial Toxins:

• Microbial toxins are compounds produced and secreted by microorganisms, and they have evolved for their inhibitory and often lethal effects on other organisms. These effects can target host cells or other microbes within the community.
• Toxins come in various chemical structures and sizes, ranging from small molecules like antibiotics and cyanide to large multisubunit proteins such as cholera toxin.

2. Varied Delivery Methods:

• Toxins can be delivered to their targets through different methods, including passive excretion or active secretion into the extracellular environment. Some toxins can also be injected directly into host or microbial cells.

Passive excretion and active secretion are two different methods by which toxins are released into the extracellular environment in microbial communities:

Passive Excretion:

• Passive excretion refers to the process by which toxins are released into the extracellular environment without the active involvement of the microorganism producing them.
• In this case, toxins are typically diffused or leaked from the producing cell into the surrounding environment.
• Passive excretion can occur due to various factors, such as cell lysis or damage, changes in cell membrane permeability, or the natural release of toxins during normal cell growth and metabolism.

Active Secretion:

• Active secretion involves a more controlled and deliberate process by which microorganisms actively release toxins into the extracellular environment.
• Microbes have specialized mechanisms, including secretion systems, to export toxins and other molecules out of the cell.
• Active secretion allows microorganisms to target specific sites or organisms with their toxins, making it a more precise method of toxin delivery.

3. Toxins as Cross-Fed Metabolites:

• Interestingly, some small molecule toxins can serve as cross-fed metabolites when present at subinhibitory concentrations or when the recipient microbe can tolerate the toxin.
• For example, certain bacteria can utilize cyanide as a nitrogen source for growth, and there have been observations of some microbes consuming antibiotics, although the latter is still a subject of debate.

Subinhibitory concentrations refer to the levels or concentrations of a substance, such as a toxin or antimicrobial agent, that are present at a concentration lower than the minimum inhibitory concentration (MIC) required to completely inhibit the growth or activity of the target microorganism.

Significance of Subinhibitory Concentrations:

• Subinhibitory concentrations can have various effects on microorganisms:
• They may not kill the microorganism but could slow down its growth.
• They can potentially induce changes in the microorganism's behavior, such as the development of resistance mechanisms or the production of specific proteins or metabolites in response to the stress caused by the substance.
• Subinhibitory concentrations can also lead to phenomena like "tolerance," where the microorganism becomes less responsive to the substance over time.

4. Toxins as Facilitators of Cross-Feeding:

• Toxins can indirectly facilitate cross-feeding in microbial communities by promoting nutrient acquisition. When toxins damage or lyse cells, they release intracellular contents, including valuable nutrients.
• This mechanism makes toxins similar to exoenzymes in their potential to support cross-feeding within the community.

5. Toxins in Polymicrobial Infections:

• In polymicrobial infections, where multiple pathogens or microorganisms interact within a host, toxins can play a complex role. Pathogens may interact synergistically, and toxin-mediated cross-feeding is a possibility.
• However, the exact role and extent of toxin-mediated cross-feeding in these interactions are not yet fully understood.

6. Competition and Nutrient Limitation:

• Contrary to expectations, there is evidence suggesting that toxins may lead to increased competition between microbial populations. Some studies have shown that during coinfection (involving multiple pathogens), more genes become essential for a given pathogen compared to monoinfection.
• This indicates intensified competition and the potential for nutrient limitation in coinfection scenarios.

Increased Competition in Coinfection:

• When multiple pathogens or microbial populations infect the same host simultaneously, they often compete for limited resources within the host's environment. Resources can include nutrients, space, and host factors that are essential for their growth and survival.
• Toxins produced by these pathogens can play a role in this competition by various means:
• Resource Competition: Toxins can damage or kill host cells, releasing intracellular contents, including nutrients. Different pathogens can then compete to utilize these released nutrients for their growth.
• Induction of Host Immune Responses: Toxins may trigger the host's immune response, leading to inflammation and immune defenses. This can result in competition between the pathogens as they strive to evade or counteract the host's immune system.
• Niche Competition: Toxins can create niches or microenvironments within the host's body that are more favorable for specific pathogens. Different pathogens may compete for dominance in these niches.
• Coinfection often intensifies competition, as multiple pathogens are vying for the same resources, which can lead to a higher level of stress and competition than in monoinfection.

Essential Genes and Competition:

• Studies have shown that during coinfection, more genes become essential for a given pathogen compared to when the pathogen is in a monoinfection scenario.
• This indicates that in coinfection, pathogens may need to adapt and utilize a broader range of genetic functions and metabolic pathways to compete effectively for resources and overcome host defenses.
• The need for a larger set of essential genes reflects the heightened competition and potential nutrient limitation in coinfection scenarios.

7. Ongoing Research:

• The extent to which toxin-mediated cross-feeding occurs in microbial communities is still an active area of research. Researchers are working to unravel the precise mechanisms and outcomes of toxin interactions within microbial ecosystems.

5. Quorum-Sensing Signals in Microbial Communities:

• Quorum sensing (QS) is a microbial cell-cell communication system that coordinates gene expression based on population density through the production and recognition of diffusible signal molecules.
• In QS systems, when a threshold population density or quorum is reached, bacterial populations synchronize the expression of specific gene subsets.
• Key components of known QS systems include 
• a signal synthase (produces the signal molecule) and 
• a cytoplasmic receptor (binds the signal and acts as a transcriptional regulator to modulate gene expression).
• QS signals, also called autoinducers, can potentially serve as cross-fed metabolites and even be used as a sole energy source in monoculture by some bacteria. However, the catabolism of QS signals in communities may have more relevance in quorum quenching, reducing the signaling effects of these molecules.
• The extent to which QS signals are cross-fed between different organisms has not been extensively studied. Instead, QS is better known for its role in regulating the production of exoenzymes, siderophores, toxins, and other group behaviors in microbial communities.
• In cross-feeding scenarios, QS often serves as an auxiliary factor, controlling access to extracellular nutrients at the population level. A population is more likely to benefit from exoenzymes, siderophores, or toxins if these molecules are produced in high concentrations due to QS.
• QS frequently coordinates multiple and sometimes contrasting activities, potentially limiting the exploitation of extracellular nutrients by cheaters.
• An example of this is the case of:
• the opportunistic human pathogen Pseudomonas aeruginosa, where the expression of the casein-hydrolyzing exoenzyme elastase is regulated by the QS transcriptional regulator LasR. lasR-null mutants that do not produce elastase can quickly emerge and cheat elastase-producing strains when casein is the sole carbon source.
• However, the addition of adenosine to a casein-containing medium reduces the relative fitness of lasR mutants in competition with the elastase-secreting strain because LasR also activates adenosine consumption pathways.
• Coupled expression of exoenzymes with other physiological activities can help prevent cheaters from benefiting from the loss of exoenzyme secretion. In the case of LasR, it regulates not only elastase production but also other activities, like cyanide production and resistance in P. aeruginosa, creating a dynamic and multifaceted control system against cheaters.

In depth:

Quorum sensing (QS) is like a language that bacteria use to talk to each other. When a group of bacteria grows and reaches a certain number, they start producing chemical signals. These signals act like a message that tells the bacteria in the group that there are many of them around.

When enough of these chemical messages build up, all the bacteria in the group can hear them. This is a sign that there are lots of bacteria nearby. In response, the bacteria change the way they behave. They might turn on specific genes or do certain things that are only useful when they work together as a group. It's like a team huddle where they make a plan based on how many of them are there.

So, quorum sensing helps bacteria cooperate and act together when they're in a big enough group, and it's a way for them to communicate with each other.

Background:

Pseudomonas aeruginosa is a versatile bacterium that uses QS to regulate the expression of various virulence factors and enzymes, including elastase, an exoenzyme that hydrolyzes casein. Elastase is important for nutrient acquisition, as it breaks down casein into smaller, more easily assimilable compounds. In this example, the QS system of P. aeruginosa involves a transcriptional regulator called LasR.

Example Scenario:

• Elastase Production and Cheaters: In a scenario where P. aeruginosa is growing on a medium with casein as the sole carbon source, elastase-secreting strains are favored because they can efficiently hydrolyze casein and access the nutrients. However, there is a potential for "cheaters" to emerge within the population. Cheaters are mutants that do not produce elastase themselves but benefit from the nearby elastase-secreting cells' activity without incurring the cost of enzyme synthesis.
• Adenosine Addition: To counteract the rise of cheaters, an additional factor comes into play. The addition of adenosine to the casein-containing medium has an interesting effect. Adenosine is another carbon source that can be utilized by P. aeruginosa. However, LasR, the QS regulator, is not just responsible for controlling elastase production; it also plays a role in activating adenosine consumption pathways.
• Complex Regulatory Network: LasR's involvement in the activation of multiple pathways, including elastase production, adenosine utilization, and other physiological activities, makes the system multifaceted and dynamic. LasR also has regulatory control over cyanide production and resistance in P. aeruginosa.
• Multifaceted Regulation: The multifaceted regulation by LasR ensures that the presence of adenosine has an impact on the competition between elastase-producing strains and cheaters. LasR-null mutants, which do not produce elastase, have a competitive advantage when casein is the sole carbon source. However, the addition of adenosine decreases the relative fitness of these lasR mutants in competition with the elastase-secreting strain.
• Counteracting Cheaters: The addition of adenosine changes the competitive dynamics by reducing the benefit gained by the loss of elastase secretion in lasR mutants. In this way, the coordinated expression of exoenzymes like elastase with other physiological activities, regulated by LasR, helps prevent cheaters from exploiting the system.

Simplifying:

1. LasR-Null Mutants: These are Pseudomonas aeruginosa bacteria with a genetic mutation (LasR-null) that prevents them from producing elastase, an enzyme needed to break down casein.
2. Competitive Advantage: When these LasR-null mutants find themselves in an environment where the only available food is casein, they have a competitive advantage. This is because they don't waste resources producing elastase, which is unnecessary in this case.
3. Adenosine Added: However, when adenosine (another type of food) is introduced into the environment, things change. LasR is not only responsible for elastase production but also for helping the bacteria use adenosine as a food source.
4. Reduced Fitness: In the presence of adenosine, the LasR-null mutants are not as competitive as they were when only casein was available. This is because regular Pseudomonas bacteria, which can produce elastase and utilize adenosine, have an advantage due to their ability to access a variety of food sources. The mutants lose their competitive edge when multiple food options are present.

In simpler terms, LasR-null mutants are good at surviving when the only food is casein. However, when other food options like adenosine are introduced, they struggle to compete with the regular Pseudomonas bacteria, which can make use of different food sources. This illustrates how the availability of different food sources can impact bacterial competition in a community.

Role of Autoinducers:

Autoinducers play a key role in quorum sensing (QS) by controlling access to nutrients and enabling communication among bacteria. Here are some important points:

1. Autoinducers are signaling molecules that bacteria use for QS.
2. There are two main categories of autoinducers: 
• Gram-positive bacteria typically use oligopeptide autoinducers.
• Gram-negative bacteria mainly produce acyl homoserine lactone autoinducers.
3. Each category of bacteria can have diverse types of autoinducers with different chemical structures.
4. Normally, the bacteria that produce a specific autoinducer can detect and respond to that particular autoinducer.
5. Some QS receptors can detect multiple types of autoinducers to some extent.
6. There is another type of autoinducer called autoinducer-2 (AI-2), which is produced as a by-product during the methionine synthesis process. Because many bacteria can make methionine, they also have the potential to produce AI-2.
7. AI-2 is unique because it's suggested to be an interspecies signal, meaning different types of bacteria can use it for communication.
8. However, not all bacteria that can produce AI-2 have the receptors needed to detect it, so the use of AI-2 for interspecies communication varies.

In summary, autoinducers are the molecules that help bacteria talk to each other, and there's a lot of diversity in the types of autoinducers and their detection mechanisms among different bacteria.

In depth:

1. Categories of Autoinducers: Autoinducers can be categorized into two main groups based on the type of bacteria that produce them.
• Gram-Positive Bacteria: Gram-positive bacteria typically produce oligopeptide autoinducers. These are small peptides that serve as signaling molecules to communicate within the bacterial community.
• Gram-Negative Bacteria: Gram-negative bacteria generally use acyl homoserine lactone autoinducers. These are small organic molecules that act as QS signals.
2. Diversity within Categories: Even within these categories, there is significant chemical and structural diversity among autoinducers. Different species of bacteria can produce slightly different autoinducers, allowing for specificity in their communication.
3. Signal Selectivity: Typically, the ability to detect and respond to a specific autoinducer is limited to the bacteria that produce the corresponding receptor for that autoinducer. However, there can be variations in how different QS receptors recognize these signals. Some receptors may exhibit a higher degree of selectivity, while others might be more flexible in what signals they can detect.
4. Autoinducer-2 (AI-2): In addition to the two main categories mentioned above, there is another type of autoinducer called autoinducer-2 (AI-2). AI-2 is unique because it's a by-product of the activated methyl cycle in the methionine synthesis pathway, which is widespread among various bacteria. This makes AI-2 a potential interspecies QS signal.
5. AI-2 Receptors: While many bacteria are capable of producing AI-2, far fewer bacteria possess genuine AI-2 receptors. AI-2 receptors are proteins that can specifically detect and respond to AI-2 signals. The presence or absence of these receptors determines which bacteria can participate in AI-2-mediated QS.

6. Extracellular Matrix Components of Biofilms

• Microbes form biofilms by attaching to surfaces and each other, with biofilm formation mediated by the secretion of an extracellular matrix composed of polysaccharides, nucleic acids, and/or proteins.
• The matrix components can potentially contribute to cross-feeding when they are degraded into accessible monomers by exoenzymes.
• For example, some cyanobacteria store carbon in their extracellular matrix and later reacquire it. This stored material is also accessible to other bacteria within microbial mats.
• Biofilms play a role in controlling access to externalized nutrients, similar to quorum sensing (QS), and the two are often interconnected.
• QS coordinates biofilm development by regulating the biosynthesis of the biofilm matrix.
• Biofilm-residing cells are densely packed, allowing smaller populations to reach quorum more easily compared to dispersed planktonic populations.
• The spatial structure provided by biofilms and dense aggregations promotes cooperative cross-feeding of costly metabolites.
• Clustering of partners is important for the evolution of cooperative cross-feeding relationships.
• Spatially structured environments can select for the evolution of mutualistic cross-feeding by favoring local retention of costly nutrients, directing them to reciprocating neighbors, and limiting diffusion to noncooperative populations.
• However, the impact of cheaters in biofilms is nuanced. Close spatial proximity can limit their access to resources, but there are cases where cheaters exploit producer cells at the edges of aggregates or biofilms.
• For example, in populations of Vibrio cholerae, the production of a biofilm matrix concentrates chitinase-secreting cells on chitin, limiting diffusion and restricting access of a cheater population to released monomers.
• In a different context, spatially structured environments on agar plates were shown to select for costly methionine-excretion by a spontaneous Salmonella enterica mutant, enabling the growth of nearby E. coli methionine auxotrophs. In return, E. coli evolved costly galactose secretion during lactose consumption when grown together with S. enterica as colonies on agar.

In summary, biofilms and spatial organization play a significant role in cross-feeding dynamics and the balance between cooperation and cheating in microbial communities.

In depth:

Biofilms are multicellular communities of microbes that attach to surfaces and are held together by an extracellular matrix. This matrix can consist of various components such as polysaccharides, nucleic acids, and proteins. Here's an in-depth look at the role of extracellular matrix components of biofilms in microbial communities:

• Biofilm Formation: Microbes often attach to surfaces and each other to form biofilms. Biofilm formation is a complex process, and one of the key elements is the secretion of an extracellular matrix. This matrix provides structural support to the biofilm and is essential for its formation and stability.
• Composition of Matrix Components: The extracellular matrix can contain a variety of components, including polysaccharides, nucleic acids (e.g., DNA), and proteins. These components contribute to the overall architecture of the biofilm.
• Contribution to Cross-Feeding: Matrix components can potentially contribute to cross-feeding in combination with exoenzymes. Some microbes in biofilms have the ability to degrade matrix components into accessible monomers. These monomers can serve as nutrients for other members of the biofilm.
• Storage and Reacquisition of Carbon: Some cyanobacteria have been shown to store carbon within their extracellular matrix and later reacquire it. This stored carbon can also be accessible to other bacteria within the microbial community, indicating a potential role in cross-feeding.
• Control of Nutrient Access: Biofilms play a crucial role in controlling access to externalized nutrients. This function is similar to quorum sensing (QS), and in fact, QS and biofilm formation are often interconnected. QS helps coordinate the biosynthesis of the biofilm matrix.
• Spatial Structure and Cooperation: Biofilm-residing cells are densely packed, allowing smaller populations to reach a quorum. The spatial structure provided by biofilms promotes cooperative cross-feeding of costly metabolites. Clustering of partners within biofilms is important for the evolutionary trajectory of certain cooperative cross-feeding relationships.
• Effect on Cheaters in Biofilms: Within biofilms, the way microbes are structured spatially can impact whether "cheater" microbes can take advantage of the nutrients released by cooperative microbes or not.
• Benefiting Cooperators: In some cases, this spatial structure benefits the microbes that are cooperating. It does so by limiting the diffusion of released nutrients and protecting them from being easily exploited by cheaters. This is because the nutrients are retained within the biofilm matrix.
• Examples of Cheaters: However, there are situations where high concentrations of cooperative microbes within the biofilm can be vulnerable to invasion by cheaters. For instance, in marine Vibrio populations, nonproducing strains that don't make siderophores (iron-chelating molecules) can cheat the siderophore production by cooperative microbes.
• How Cheaters Benefit: In this specific example, the nonproducing strains exploit the siderophores produced by the cooperative microbes. Siderophores are molecules that scavenge iron. By invading the dense aggregations of siderophore-producing microbes, the nonproducers can take advantage of the siderophores to access iron without having to produce siderophores themselves.

In summary, extracellular matrix components of biofilms play a multifaceted role in microbial communities. They provide structural support, contribute to cross-feeding, control nutrient access, and impact the dynamics of cooperation and competition within biofilms. The spatial structure within biofilms is a critical factor in shaping these interactions.

7. Extracellular Vesicles

Occurrence Across Various Organisms

• Extracellular vesicles are found in a wide range of organisms, including microscopic and multicellular eukaryotes, archaea, Gram-negative bacteria, and Gram-positive bacteria.

Gram-Negative Bacteria and Vesicle Release

• In Gram-negative bacteria, the release of vesicles is linked to a reduction in cross-linking between peptidoglycan and outer membrane proteins.

Mechanism in Gram-Positive Bacteria

• The precise mechanism of vesicle release in Gram-positive bacteria is not fully understood but may involve weak points in the cell wall or facilitated channels.

Role in Toxin Delivery

• Extracellular vesicles can participate in toxin delivery, with instances like Myxococcus xanthus packaging exoenzymes in vesicles to degrade macromolecules in E. coli prey cells.

Contributing to Biofilm Matrix

• Extracellular vesicles are also part of biofilm matrixes, contributing to the structural integrity of biofilms.

Facilitating Quorum Sensing (QS)

• Some extracellular vesicles facilitate quorum sensing (QS) by enabling the diffusion of hydrophobic QS signals that might be ineffective in aqueous environments.

Siderophores and Immune Responses

• Siderophores, iron-chelating molecules, can be secreted in association with extracellular vesicles. This may help siderophores evade host immune responses targeting bacterial iron acquisition. However, extracellular vesicles themselves can elicit immune responses in some cases.

Direct Nutrient Cross-Feeding

• Extracellular vesicles can serve as a direct cross-fed nutrient. For example, vesicles released by Prochlorococcus cyanobacteria support the growth of heterotrophic marine bacteria.

Significance in Marine Ecosystems

• Prochlorococcus, one of the most abundant marine bacteria, releases vesicles that could contribute significantly to carbon transfer in the oceans.

Protective Role

• Extracellular vesicles serve a protective role, helping to combat unwanted cross-feeding by reducing or delaying acquisition by competing microbes.

These findings highlight the versatile roles of extracellular vesicles in microbial interactions, encompassing nutrient exchange, cargo protection, biofilm formation, quorum sensing, and toxin delivery.

In depth:

Extracellular vesicles are another element that can influence cross-feeding behavior in microbial communities. These vesicles have been identified in various microscopic and multicellular organisms, including eukaryotes, archaea, and both Gram-negative and Gram-positive bacteria.

In Gram-negative bacteria, the release of extracellular vesicles is associated with a reduction in the cross-linking between peptidoglycan and outer membrane proteins. The composition of lipids and lipopolysaccharides might also play a role in vesicle release.

The mechanisms behind the release of vesicles from Gram-positive bacteria are not fully understood, but it's possible that they are expelled through weak points in the cell wall or facilitated via channels.

Extracellular vesicles are sometimes released passively and accidentally. For instance, there are instances where vesicles are involved in delivering toxins. In one case, exoenzymes were packaged in extracellular vesicles by Myxococcus xanthus, and these vesicles were used to degrade macromolecules in E. coli prey cells.

These vesicles can also be components of biofilm matrices, contributing to the structural support of biofilms.

Extracellular vesicles can have an impact on quorum sensing (QS) by facilitating the diffusion of signals that would otherwise be too hydrophobic to be effective in aqueous environments. Siderophores, molecules used for iron acquisition, can also be secreted in association with extracellular vesicles. This might help siderophores evade host immune responses that target bacterial iron acquisition. However, it's important to note that extracellular vesicles themselves can elicit immune responses in some cases.

Extracellular vesicle-packaged siderophores could potentially offer an additional layer of specificity for the uptake of sequestered iron, similar to what was observed with QS signals.

Moreover, extracellular vesicles can directly serve as a source of cross-fed nutrients. For instance, vesicles released from Prochlorococcus cyanobacteria support the growth of heterotrophic marine bacteria. Given that Prochlorococcus is one of the most abundant marine bacteria, extracellular vesicles from these cyanobacteria alone could account for the release of 10^4 to 10^5 metric tons of carbon into the oceans each day.

While vesicles and their cargo can likely serve as cross-fed nutrients, they also have another role: protecting their cargo. Extracellular vesicles might combat unwanted cross-feeding by decreasing or delaying the acquisition of their cargo by competitors. This duality makes extracellular vesicles an intriguing element in the complex dynamics of microbial cross-feeding.

Contact-Dependent Cross-Feeding

• While quorum sensing (QS), biofilms, and extracellular vesicles play key roles in microbial interactions, contact-dependent mechanisms also influence the action of externalized molecules.
• Contact-dependent interactions can be both antagonistic (involving harmful toxins delivered directly into neighboring cells) and beneficial (supporting cross-feeding).
• Some cases of contact-dependent cross-feeding involve remarkably intimate cell interactions where one cell resides within another.
• In some instances, cells share a periplasm, potentially leading to a shared electrochemical gradient or proton motive force.
• For example, cable bacteria consist of filaments that share a periplasm and have electrically conductive appendages of unknown composition. These filaments span redox gradients in sediments, allowing separate cells to contribute oxidation and reduction reactions to a combined metabolism, despite being centimeters apart.
• Certain filamentous cyanobacteria also share a periplasm. These cyanobacteria exhibit significant phenotypic differentiation into vegetative cells and non-vegetative heterocysts specialized for CO2 and N2 fixation (diazotrophy). Specialized connective structures facilitate the transfer of nitrogen and carbon at the junction between vegetative cells and heterocysts.

These examples demonstrate the extent to which microbial communities can evolve highly specialized mechanisms for direct cell-to-cell interactions that foster cross-feeding and cooperative behaviors.

In depth:

Contact-Dependent Cross-Feeding:

In addition to quorum sensing (QS), biofilms, extracellular vesicles, contact-dependent mechanisms have been reported in microbial communities. Contact-dependent interactions involve direct physical connections between cells, and they can either be antagonistic, where harmful toxins are delivered to neighboring cells, or beneficial, where cross-feeding occurs.

Some contact-dependent interactions are so intimate that one cell resides within another. Others involve shared periplasm, a region enclosed by a membrane, and typically associated with an electrochemical gradient or proton motive force.

One remarkable example is cable bacteria, which form filaments that share a periplasm and an electrically conductive intercellular appendage of unknown composition. These filaments span redox gradients in sediments, allowing separate cells to contribute oxidation and reduction reactions to a combined metabolism, even though they can be centimeters apart.

Filamentous cyanobacteria provide another example. They share a periplasm and exhibit phenotypic differentiation into vegetative cells and non-vegetative heterocysts. Heterocysts are specialized for CO2 and N2 fixation (diazotrophy). In this case, specialized connective structures participate in the transfer of nitrogen and carbon at the junction between vegetative cells and heterocysts.

Contact-dependent mechanisms introduce a level of intimacy and efficiency in cross-feeding that can overcome spatial and physiological barriers, making them an important facet of microbial cross-feeding dynamics.

Cable Bacteria

Cross-Feeding Mechanisms by Direct Contact

• In microbial ecosystems, there are instances of cross-feeding between different microorganisms facilitated by direct contact. These mechanisms allow for the exchange of essential nutrients and metabolites between different species.

Unexplained Cross-Feeding

• Some forms of cross-feeding by direct contact are not yet fully understood. For example, observations of stable isotope transfer between cyanobacterial heterocysts and attached Rhizobia, both of which are environmental isolates, hint at a transfer mode that remains poorly defined.

Contact-Dependent Microbial Interactions

• Investigations into these contact-dependent interactions can reveal unique and fascinating physiological traits in microbial partnerships.

The Contact-Dependent Pairing of Ignicoccus hospitalis and Nanoarchaeum equitans

An Intriguing Microbial Interaction

• One example of contact-dependent cross-feeding involves two archaea, Ignicoccus hospitalis and Nanoarchaeum equitans, working together to exchange various molecules.

Transfer of Essential Molecules

• The interaction between I. hospitalis and N. equitans likely involves the transfer of molecules, such as phospholipids, amino acids, and possibly even ATP.

Unique Physiological Traits

• N. equitans lacks the necessary genes to synthesize a complete ATP synthase, leading to questions about its energy acquisition.
• I. hospitalis, in contrast, is the only known organism with an energized outer membrane and generates ATP in the periplasm through an outer membrane ATP synthase.

Role of ATP Cross-Feeding

• The periplasmic generation of ATP in I. hospitalis is thought to be important for cross-feeding ATP to N. equitans, highlighting the intriguing ways in which direct contact-dependent interactions can lead to metabolic collaboration and nutrient transfer between different microbial partners.

These examples highlight the complexities and unique mechanisms involved in cross-feeding interactions where different microorganisms exchange essential resources, such as ATP and nutrients, through direct physical contact.

In depth:

Cable Bacteria:

Cable bacteria are a fascinating example of interspecific cross-feeding involving direct contact between microorganisms. These unique microorganisms form long filaments and are known for their ability to span redox gradients in sediments. The filaments can be centimeters apart, but they still engage in metabolic cooperation.

Cable bacteria share a periplasm and an electrically conductive intercellular appendage of unknown composition. This intercellular connection enables them to conduct electrons along their filaments, facilitating the transfer of reducing equivalents from one cell to another.

These filaments play a crucial role in sediment biogeochemistry. The cells in the filament work together to oxidize sulfide from deeper sediments and transport the electrons to the surface, where they reduce oxygen. This cable-like behavior allows the cable bacteria to efficiently couple the oxidation of sulfide with the reduction of oxygen, enhancing sediment respiration and nutrient cycling.

Cable bacteria are a striking example of how direct physical connections and metabolic cooperation enable microorganisms to thrive in environments with steep gradients and limited resources.

8. Nanotubes

Nanotube Structure and Occurrence

• Nanotubes are membranous connections between cells, allowing intimate interactions both within species (intraspecific) and between different species (interspecific).
• They have been observed among various microbial combinations, including interactions between Gram-negative and Gram-positive bacteria and potentially between bacteria and mammalian cells.

Nanotube-Mediated Molecule Transfer

• Nanotubes enable the transfer of a range of molecules, including small metabolites, large proteins, and DNA molecules, between connected cells.

Mechanistic Details and Formation

• The mechanistic details of how nanotubes bridge the inner membranes of bacterial cells through the peptidoglycan layers are not yet fully understood.
• Peptidoglycan hydrolases are suggested to play a role in nanotube formation, particularly in Bacillus subtilis.
• Nanotube formation may share similarities with the mechanisms involved in extracellular vesicle formation.

Genetic Factors and Regulation

• Only a few genetic factors have been implicated in nanotube formation, including a phosphodiesterase, sigma factor in B. subtilis, and components of the pathogenic E. coli injectosome.
• The regulation of nanotubes appears inconsistent; in some cases, nutrient deprivation stimulates nanotube formation, suggesting a role in nutrient acquisition. However, nanotube formation has also been observed in rich media.
• Recent findings suggest that nanotubes might be a by-product of cell death in various Gram-positive bacteria, with implications for molecule transfer.

Challenges and Controversies

• Observations of molecule transfer via nanotubes are often based on correlations, and frequencies of nanotube connections and transfer events within a population are rarely reported.
• There are challenges and questions regarding the validity of traditional microbiological practices, particularly the transfer of plasmids in bacteria not naturally competent via nanotubes.
• The co-occurrence of cell death with nanotube formation raises questions about the role of cell lysis and its impact on cross-feeding.

Ongoing Research and Uncertainties

• Despite being an intriguing mechanism for cross-feeding, nanotubes continue to be a subject of ongoing research and debate, with many mechanistic and functional details yet to be fully understood.

In depth:

Nanotubes are intercellular membranous connections that enable close contact between cells. These structures have been reported to form between cells of various types, including both intraspecific and interspecific interactions. For example, nanotubes can connect Gram-negative and Gram-positive bacteria, and there are even suggestions of potential connections between bacteria and mammalian cells. They allow for the transfer of various molecules, including small metabolites, large proteins, and DNA.

Despite their intriguing potential, the mechanistic details of nanotube formation remain unclear. It is uncertain how the inner membrane of one bacterium might be passed through the peptidoglycan layers of both the donor and recipient cells. Some research has implicated peptidoglycan hydrolases in the process, but a comprehensive understanding is lacking.

Nanotube formation might share mechanistic features with the formation of extracellular vesicles. Beyond peptidoglycan hydrolases, other genetic factors have been implicated in nanotube formation, including a phosphodiesterase and a sigma factor in Bacillus subtilis, as well as components of an injectosome in pathogenic Escherichia coli.

The function of nanotubes remains mysterious and has sparked some controversy. Evidence for the intercellular transfer of molecules via nanotubes is often correlative, with nanotube structures present, but molecule transfer via other mechanisms cannot be ruled out. While intercellular transfer of fluorescent signatures has been observed, the frequencies of connections and transfer events within populations are rarely reported.

There are inconsistencies in the regulation of nanotubes. In some cases, nanotube formation was stimulated by nutrient deprivation, suggesting a role in nutrient acquisition from neighboring cells. However, nanotube formation in pathogenic E. coli and B. subtilis has been observed in rich media.

Recent findings have implicated nanotubes as a by-product of cell death in various bacteria, including E. coli and B. subtilis. While fluorescent proteins were observed in B. subtilis nanotubes, DNA transfer through nanotubes required the involvement of well-described competence proteins, indicating that nanotubes were not directly responsible for DNA transfer.

The potential for nanotube-mediated transfer of proteins and DNA between living cells raises questions about experimental results that rely on distinguishing genetically distinct populations using drug resistance markers or fluorescent reporters. The co-occurrence of cell death with nanotube formation also suggests that cell lysis could explain some correlative observations of cross-feeding.

In summary, nanotubes represent an intriguing mechanism for cross-feeding, but they remain a subject of debate and further investigation. Critical mechanistic and functional details are yet to be fully elucidated, and their role in microbial communities remains a topic of ongoing study and discussion.

Genetic factors involved in nanotube formation:

1. Bacillus subtilis (B. subtilis):

• In B. subtilis, the formation of nanotubes involves several genetic factors, including a phosphodiesterase and a sigma factor.
• Phosphodiesterases are enzymes involved in breaking down phosphodiester bonds in molecules like nucleotides. Their role in nanotube formation is not entirely clear, but they may be involved in regulating the process.
• Sigma factors are proteins that control the initiation of transcription in bacteria. In the context of nanotube formation, the specific sigma factor in question likely plays a role in coordinating the expression of genes involved in creating these structures.

2. Pathogenic Escherichia coli (E. coli):

• In pathogenic E. coli, nanotube formation is associated with the presence of components of an injectosome.
• The injectosome is a specialized structure that some pathogenic bacteria use to deliver proteins or molecules directly into host cells. In the context of nanotube formation, it's possible that these injectosome components are adapted for creating nanotubes to facilitate interactions between cells.

9. Nanowires

Nanowires as Electron Transfer Structures

• Nanowires facilitate cross-feeding by allowing the transfer of electrons from the nanowire-producing microbe to extracellular terminal electron acceptors that are typically challenging to reduce within the cell.
• They play a role in anaerobic respiration, enabling the utilization of terminal electron acceptors that are too large, insoluble, or toxic for intracellular reduction.

Nanowire-Mediated Syntrophic Partnerships

• Direct electron transfer via nanowires sustains syntrophic partnerships between microbial species by overcoming thermodynamic limitations.
• This is similar to the function of organic acids, hydrogen (H2), and other reduced compounds acting as intercellular electron shuttles in syntrophic relationships.
• Examples include syntrophy between Geobacter metallireducens (ethanol fermentation) and Geobacter sulfurreducens (fumarate respiration).
• These partnerships depend on the formation of multicellular aggregates and specific nanowire components, such as pili and outer membrane cytochromes.
• Natural aggregates between sulfate-reducing bacteria and archaeal anaerobic methanotrophs (ANME) also involve nanowire-mediated syntrophic electron transfer.

Nanowire Composition and Diversity

• The composition of nanowires is a subject of debate, and it appears to be diverse.
• Some studies suggest that electrons are transferred between aromatic amino acids in the pilus, while other research indicates that a pilus composed entirely of cytochrome subunits may be involved in electron transfer.
• Different microbial species may have distinct nanowire compositions; for example, Shewanella oneidensis nanowires resemble mechanisms seen in nanotubes or extracellular vesicles.

Other Mechanisms of Direct Electron Transfer

• Apart from nanowires, various mechanisms of direct electron transfer exist, including cell surface cytochromes, transfer through abiotic surfaces, and conductive structures in filaments of cable bacteria.
• Cable bacteria exemplify how intimate contacts between cells can enable the exchange of electrons in redox reactions.

Applications and Significance

• Microbes capable of externalizing electron flow have applications in electricity generation, bioremediation, and electricity-driven production of reduced compounds.
• Understanding nanowires and related structures enhances our knowledge of microbial interactions, biogeochemical cycles, and potential biotechnological applications.

In depth:

1. Direct Electron Transfer via Nanowires:

• Nanowires are specialized structures involved in direct electron transfer between microbial cells.
• They enable the transfer of electrons from one microbe to another, which can be essential for various metabolic processes.

2. Role in Anaerobic Respiration:

• Nanowires are particularly important in anaerobic respiration. They allow the microbe producing the nanowires to deposit electrons on extracellular terminal electron acceptors.
• These acceptors are often too large, insoluble, or toxic to reduce within the microbial cell.

3. Cross-Feeding via Waste Metabolites:

• Nanowires, along with soluble extracellular electron shuttles, participate in cross-feeding by generating waste metabolites.
• The reduced metals produced by this process can serve as electron sources for other microbes with different metabolic lifestyles, such as photo- or litho-autotrophs.

4. Direct Coupling of Redox Metabolisms:

• Nanowires enable the direct coupling of redox metabolisms between microbial cells.
• This direct electron transfer can sustain syntrophic partnerships, where microbes rely on each other for specific metabolic processes.

5. Examples of Nanowire-Mediated Syntrophy:

• Geobacter metallireducens and Geobacter sulfurreducens: A syntrophic relationship was evolved experimentally between these two Geobacter species. Electrons were transferred from G. metallireducens to G. sulfurreducens, supporting ethanol fermentation and fumarate respiration.
• Sulfate-Reducing Bacteria and Anaerobic Methanotrophs: Natural aggregates formed between these microbes in ocean sediments show syntrophic electron transfer.

6. Composition of Nanowires:

• The composition of nanowires remains a topic of ongoing research and debate.
• Some studies suggest that electrons are transferred between aromatic amino acids in the pilus, while others suggest that the pilus itself does not transfer electrons.
• Nanowires are likely compositionally diverse; for example, Shewanella oneidensis nanowires were found to be extensions of the outer membrane containing electrically conductive cytochromes.

7. Applications of Microbes with Externalized Electron Flow:

• Microbes capable of externalizing electron flow through nanowires are of interest for various applications, including electricity generation, bioremediation, and electricity-driven production of reduced compounds.