Extracellular Metabolism: Mechanisms and Impacts of Microbial Cross-Feeding

Table of Contents

• Introduction to Cross-Feeding
• Cross-Feeding Concepts
• Extracellular Molecules That Promote Cross-Feeding
• Metabolites
• Exoenzymes
• Siderophores
• Toxins
• Quorum-Sensing Signals
• Extracellular Matrix Components of Biofilms
• Extracellular Vesicles
• Nanotubes
• Nanowires
• Dynamic Aspects of Microbial Cross-Feeding
• Dose-Dependent Effects of Cross-Fed Metabolites
• Cross-Feeding Independent of Growth
• Facultative Cross-Feeding
• Competition within Mutualistic and Synergistic Relationships

Introduction to Cross-Feeding

• The vast abundance of microbes on Earth is remarkable, with an estimated ∼10³⁰ individual bacterial and archaeal cells globally.
• Many eukaryotic organisms, including protists, microalgae, and some fungi, are also unicellular and microscopic in nature.
• This immense microbial abundance can be attributed to their genomic and metabolic diversity, which allows them to occupy nearly every possible ecological niche.
• In natural environments, microbes typically exist as multispecies communities rather than in isolation.
• Extremely dense microbial communities are found in habitats such as the human gut and cow rumen, which harbor approximately ∼10¹⁴ and ∼10¹⁵ microbes, respectively.
• In the human colon, microbial density reaches around 10¹¹ cells per milliliter.
• Comparatively, less dense microbial communities are observed in environments such as seawater, which contain about 10⁴ to 10⁶ cells per milliliter.
• Regardless of density, competition for limited nutrients is a common occurrence in all microbial communities.
• This competition for scarce resources has both physiological and ecological implications for microbial populations.
• Most microbes spend at least a portion of their lifecycle in nutrient-limited conditions, leading them to enter a nongrowing state known as dormancy or growth arrest.
• Dormant microbes make up a significant fraction of the total microbiota in many environments.
• Although dormant or nongrowing microbes are not actively proliferating, they are not entirely metabolically inactive.
• Even during dormancy, microbes maintain a low metabolic rate known as maintenance metabolism to support essential cellular functions.
• These critical activities include DNA repair, protein and lipid turnover, osmoregulation, and nutrient transport.
• While maintaining these vital processes, nutrient-starved microbes also adopt strategies to enhance nutrient acquisition.
• These strategies often involve the production and release of various molecules into the extracellular environment.
• Such molecules include metabolites, exoenzymes, siderophores, toxins, signaling compounds, and cell surface-associated factors.
• The release of these molecules may occur either actively through secretion or passively through excretion, and in some cases, they can even be directly transferred between cells.
• Once released, these extracellular molecules can influence multiple targets, including the survival and growth of the producing cell, neighboring microbes, and sometimes even host cells.
• In many circumstances, the release of these molecules intensifies competition among microbes.
• However, under certain conditions, these externalized molecules can promote beneficial interactions, leading to synergistic cross-feeding among microbial species.
• When favorable conditions persist, cooperative cross-feeding interactions can become stable and reinforced over time.
• Such cooperative relationships can have profound effects on community-wide functions and processes of global and societal importance.
• These impacts extend from regulating biogeochemical cycles that influence Earth’s climate to affecting the development and progression of polymicrobial infections.

Figure 1. Microbes secrete a variety of extracellular molecules that facilitate cross-feeding. These include quorum-sensing signals, exoenzymes, siderophores, toxins, metabolites (such as sugars, organic acids, and amino acids), components of the biofilm matrix, nanowires, extracellular vesicles, and nanotubes. These substances can either be utilized by nearby microorganisms or modulate cross-feeding interactions between cells.

Cross Feeding

1. Cross-feeding involves the transfer of material from a producer organism to a recipient organism. The transferred material can consist of molecules (such as metabolites) or even subatomic particles like electrons and protons. An example of energy cross-feeding is the sharing of a proton motive force between cells, where the crucial factor is the shared electrochemical gradient rather than the transfer of protons themselves.
2. The transferred material must be either assimilated by the recipient organism or participate in energy transformation within the recipient and/or the producer. 
3. As a result of this transfer and utilization, the fitness (growth, survival, or reproductive success) of the producer and/or recipient must be altered.
4. Cross-feeding interactions must occur between different species or between genotypically or phenotypically distinct populations within a species. This distinction is important for studying the mechanisms, nature, and ecological consequences of cross-feeding relationships.

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 produce and release a wide range of small molecules known as metabolites, which are excreted compounds primarily serving as biosynthetic intermediates rather than toxins or signaling molecules for cell-to-cell communication.
• Common cross-fed metabolites include sugars, organic acids, amino acids, vitamins, gases, and reduced or oxidized inorganic elements and molecules.
• Metabolite cross-feeding is ubiquitous across microbial communities and occurs in all three domains of life — Bacteria, Archaea, and Eukarya.
• Stable cross-feeding relationships can be established through genetic engineering, experimental evolution, or simply by designing appropriate environmental conditions.
• A well-known example involves NH₄⁺-excreting Chlamydomonas reinhardtii and CO₂-producing yeast, which established a cross-feeding interaction without requiring any genetic modification or evolutionary adaptation.
• Metabolite cross-feeding can take multiple forms:
• Unidirectional, where metabolites flow from one microbe to another;
• Bidirectional, involving mutual exchange;
• Multidirectional, where metabolites circulate among several partners.
• Cross-fed metabolites may represent either metabolic waste, offering no further benefit to the producer, or communally valuable compounds, which can benefit both the producer and the recipient.
• Within microbial communities, numerous metabolite cross-feeding interactions occur simultaneously, forming complex metabolic networks involving both waste and valuable compounds.

Figure 2. Synthetic microbial consortia designed with essential cross-feeding interactions.
(A) Saccharomyces cerevisiae and Chlamydomonas reinhardtii exchange CO₂ and NH₄⁺ bidirectionally.
(B) Desulfovibrio vulgaris and Methanococcus maripaludis engage in syntrophic cross-feeding of acetate, CO₂, and H₂.
(C) Escherichia coli auxotrophs mutually exchange amino acids.
(D) S. cerevisiae auxotrophs reciprocally cross-feed lysine and adenine.
(E) E. coli, Salmonella enterica, and Methylobacterium extorquens participate in multidirectional exchange of acetate, methionine, and NH₄⁺.
(F) Lobomonas rostrata and Mesorhizobium loti share fixed carbon and cobalamin bidirectionally.
(G) E. coli and Rhodopseudomonas palustris mutually exchange organic acids and NH₄⁺.
(H) Geobacter metallireducens and Geobacter sulfurreducens engage in syntrophic electron transfer.
The “Δ” symbol denotes an auxotrophy requiring the uptake of a specific metabolite.

Waste Metabolites

• The diverse metabolic capabilities of microbes create numerous opportunities for metabolite cross-feeding.
• For example, O₂ produced as a waste product by photosynthetic cyanobacteria is vital for aerobic respiration in many other microorganisms.
• CO₂, a common waste product of heterotrophic metabolisms (including fermentative and photoheterotrophic lifestyles), serves as an essential carbon source for photoautotrophs and lithoautotrophs.
• CO₂ can also act as a conditionally essential electron acceptor for some photoheterotrophic microbes and even as an auxiliary carbon source for chemoheterotrophs such as succinate-excreting fermenters, which exhibit net CO₂ fixation.
• Fermentative microbes partition carbon sources for both biosynthesis and as disposable electron acceptors, releasing reduced compounds that become crucial carbon or electron sources for other microbes.
• Anaerobically respiring microbes show wide variation in electron donor–acceptor pairings, leading to cross-feeding of inorganic metabolites.
• Reduced products of anaerobic respiration, including H₂S, NH₄⁺, and Fe²⁺, are subsequently used by lithoautotrophic and anoxygenic photoautotrophic microbes as electron donors.
• These connections form essential parts of global biogeochemical cycles involving carbon, nitrogen, sulfur, iron, and phosphorus.
• Thus, microbial cross-feeding of metabolic waste significantly influences both the course of life on Earth and the planet’s climate.
• At a physiological level, consumption of waste metabolites often provides minimal benefit to the producer, though in some cases, it is essential for producer survival — as seen in syntrophy.
• Syntrophy refers to a cross-feeding relationship where an energetically unfavorable metabolism in the producer becomes thermodynamically feasible due to the metabolic activity of the recipient.
• Typically, a fermentative bacterium relies on an electron-rich substrate but faces thermodynamic limitations as fermentation products accumulate.
• When recipient microbes (such as methanogenic archaea, acetogens, or sulfate/iron reducers) consume fermentation products like H₂, CO₂, formate, and acetate, the coupled system becomes energetically favorable for both.
• Such syntrophic interactions are vital components of the global carbon cycle and play a major role in anaerobic degradation of electron-rich pollutants.
• Laboratory examples of syntrophy have been observed between Desulfovibrio vulgaris (a sulfate-reducing bacterium) and Methanococcus maripaludis (a methanogen).
• When deprived of sulfate, D. vulgaris ferments lactate, producing H₂ and CO₂ that M. maripaludis consumes.
• Although lactate fermentation alone is thermodynamically infeasible, the coupled metabolism enables both species to thrive.
• Over time, evolved populations of D. vulgaris often lose the ability to respire sulfate, suggesting specialization for obligate syntrophic growth with M. maripaludis.
• These examples illustrate how obligate cross-feeding, initially imposed by environmental constraints, can become genetically fixed through loss of independent metabolic pathways.

Figure 3. Cross-feeding of carbon-containing waste metabolites drives the global carbon cycle. The trophic categories shown represent potential roles in carbon transformations. For instance, chemoheterotrophs primarily break down macromolecules from microbes and multicellular organisms into a variety of organic compounds that can serve as nutrients for other metabolic lifestyles, although they may also contribute to cycles of other elements.

The acetogenesis arrows depict acetate secretion resulting from the conversion of two CO₂ molecules into acetyl-CoA via the energy-conserving Wood–Ljungdahl pathway, rather than representing all possible lifestyles of acetogenic bacteria. Syntrophy arrows indicate fermentative carbon processes that require a partner organism to make the reaction energetically favorable. The diazotrophy arrow highlights a carbon transformation performed by nitrogenase in sufficient amounts to sustain a partner’s growth.

In some cases, only specific organisms within each trophic group produce particular metabolites. The figure does not capture every possible function within each lifestyle category, nor does it include all microbial carbon transformations known to occur.

Communally Valuable Metabolites

• Both natural and synthetic cross-feeding systems involve exchange of costly, communally valuable nutrients such as ammonia/ammonium (NH₃/NH₄⁺), amino acids, nucleobases, and vitamins.
• Many microorganisms exhibit auxotrophy, meaning they lack the ability to synthesize certain essential metabolites.
• Auxotrophies for amino acids and vitamins are widespread in microbial ecosystems.
• The evolution of auxotrophy and cross-feeding are likely interconnected, as the abundance of cross-fed nutrients in the environment may drive loss of biosynthetic genes.
• A genome-scale analysis of 800 microbial communities revealed a high potential for metabolic interdependencies between different subcommunities.
• Amino acid auxotrophy is one of the most frequently employed strategies to engineer synthetic cross-feeding consortia.
• Even a single producer strain can stabilize a mixed community of auxotrophic partners that would otherwise compete.
• There appears to be no energetic limitation on what valuable metabolites can be cross-fed.
• For instance, vitamin B₁₂, one of the most energetically expensive molecules to synthesize, is actively cross-fed among species.
• Vitamin B₁₂ cross-feeding has global ecological importance, as it is transferred from heterotrophic bacteria to marine diatoms, which are responsible for about 40% of oceanic primary production and up to 20% of total global primary productivity.
• Exchange of B vitamins is also common among gut microbiota.
• Similarly, cross-feeding of complex cofactors like heme and quinones occurs in Staphylococcus aureus subpopulations that rely on each other for missing respiratory factors.
• These cross-feeding strains display greater virulence when together compared to the self-sufficient ancestral strain.
• The selective pressures that foster such synergistic cross-feeding relationships and their role in pathogenicity remain open questions.
• The reason microbes release costly metabolites in sufficient amounts to sustain others is not always clear.
• In many cases, excretion is accidental, often due to leaky cell membranes, particularly for molecules like NH₃/NH₄⁺ where charge equilibrium depends on pH.
• Cell lysis can also contribute to nutrient release, as seen in synthetic yeast mutualisms where cell death released amino acids and nucleotides.
• Active selection for excretion may occur if releasing valuable compounds stimulates reciprocation from recipient microbes, creating mutualistic benefits.
• Reciprocal cross-feeding is believed to have driven the evolution of rapid metabolism and organic acid excretion (e.g., pyruvate and glycolate) in Prochlorococcus, one of the most abundant marine cyanobacteria.
• Prochlorococcus may have coevolved with heterotrophic partners that consume its excreted metabolites and reciprocate by releasing malate and citrate, reinforcing mutualism.
• This raises the possibility that nutrient release may sometimes be an active process, potentially involving specific transporters.
• However, mechanisms of metabolite secretion or excretion remain largely uncharacterized and may vary widely among molecules.
• For cooperative cross-feeding of costly metabolites to evolve and persist, environmental conditions must favor reciprocation among cooperative partners while minimizing exploitation by non-cooperative members.
• Evidence that interactions between Prochlorococcus and marine heterotrophs are phylogenetically conserved supports the idea that directed reciprocation mechanisms evolved to stabilize such relationships.
• In some cases, the recipient itself drives cross-feeding by enhancing uptake capacity for the shared metabolite.
• For example, Escherichia coli mutants with higher NH₄⁺ acquisition efficiency showed improved growth in coculture with a weak NH₄⁺ producer.
• Even within E. coli monocultures, genetic and phenotypic differentiation can lead to acetate cross-feeding, an example of division of labor.
• Acetate-consuming subpopulations evolve higher affinity for acetate, while acetate-producing subpopulations grow faster on glucose.
• The mutual benefit likely arises from the removal of inhibitory acetate, demonstrating how cross-feeding can evolve within a single species as an adaptive strategy for community stability.

2. Exoenzymes

Extracellular enzymes, or exoenzymes, are secreted by microorganisms to break down large, complex polymers into smaller monomers that can be transported into the cell.

Major Classes of Exoenzymes

Exoenzymes act on a wide range of substrates. Prominent examples include:

• Glycosidases – degrade polysaccharides (e.g., cellulases, amylases)
• Proteases – break down proteins into peptides and amino acids
• Nucleases – hydrolyze nucleic acids
• Lipases – cleave lipids into glycerol and fatty acids
• Lignin - cleaving enzymes – such as laccases and peroxidases

Ecological Significance

Exoenzymes release readily consumable nutrients into the environment. These nutrients can be utilized not only by the producing microbe but also by neighboring organisms, a process known as cross-feeding.

Therefore, exoenzyme-secreting microbes are often regarded as:

• Keystone species, and
• Primary colonizers on polymeric substrates in microbial communities.

Examples of Exoenzyme-Producing Microbes

1. Cellulase-Secreting Microorganisms

• Cellulase-producing microbes (e.g., Clostridium, Trichoderma, Actinobacteria) support the growth of other gut bacteria and their animal hosts (e.g., ruminants and termites), which lack enzymes to digest cellulose.
• On a global scale, the breakdown of cellulose and lignin via exoenzymes drives the carbon cycle and mineralization of plant material.
• Industrially, lignocellulose-degrading enzymes are researched to lower costs in producing biofuels such as cellulosic ethanol.
• Co-culturing cellulolytic microbes with other species demonstrates cross-feeding mechanisms in biofuel production systems.

2. Invertase-Producing Microorganisms

• The exoenzyme invertase (β-fructofuranosidase)—also known as saccharase or sucrase—hydrolyzes sucrose into glucose and fructose.
• Under well-mixed conditions, up to 99% of glucose and fructose produced by invertase-secreting yeast diffuses away before uptake.
• This diffusion promotes cooperation but also vulnerability to exploitation by:
• Other species, or
• Cheater mutants that do not produce invertase but utilize the products secreted by others.

Example:

• In yeast populations, invertase-deficient mutants can invade producer populations and coexist due to shared resource availability.

3. Chitinase-Producing Microorganisms

• Vibrio cholerae produces chitinase, which degrades insoluble chitin into N-acetylglucosamine (GlcNAc) monomers and oligomers.
• Mutant strains lacking chitinase can still acquire released GlcNAc from producers, outcompeting them under well-mixed conditions.

Avoiding Exploitation by Cheaters

Exoenzyme-secreting populations have evolved strategies to avoid being overrun by exploiters:

1. Cell-Surface-Attached Enzymes:

• Some enzymes, like bacterial cellulases, remain attached to the producer’s cell surface, ensuring that the secreting microbe stays in close contact with the substrate and gains first access to released nutrients.

2. Oligomer Release Strategy:

• Instead of releasing monomers, some microbes release soluble oligomers, which are then imported into the periplasm for further breakdown.
• Fewer organisms can degrade oligomers, making this an effective defense against competitors.
• Example: Bacteroides thetaiotaomicron utilizes mannan through oligomer import without supporting the growth of mannose-utilizing strains.

3. Group Behaviors and Policing Mechanisms:

• Quorum sensing (QS) regulates enzyme secretion and helps maintain cooperation.
• Biofilm formation promotes clustering of producers, limiting resource diffusion and protecting against cheaters.

3. Siderophores

Definition:

• Siderophores are small, high-affinity metal-chelating molecules secreted by microorganisms to capture iron (Fe³⁺) and, in some cases, other metals such as copper (Cu²⁺), manganese (Mn²⁺), and zinc (Zn²⁺).
• These trace metals are vital as enzyme cofactors involved in numerous cellular processes.

Ecological and Environmental Role

• In marine environments, siderophores significantly influence the global iron cycle, which directly affects ocean productivity and nitrogen cycling.
• In host environments, iron is limited due to nutritional immunity — a defense strategy in which hosts sequester iron to inhibit microbial growth.
• Therefore, siderophore production plays a critical role in infection dynamics, microbial competition, and host–pathogen interactions.

Some pathogens produce “stealth siderophores”, which evade detection by host immune proteins, allowing continued iron acquisition.

Cross-Feeding and Competition

• As secreted molecules, siderophores can facilitate cross-feeding, enabling non-producing microbes to access iron captured by siderophore producers.
• This process occurs:
• Between different bacterial species, and
• Even between bacteria and eukaryotic microbes.
• Some microbes may specialize in scavenging siderophores produced by others — a strategy that contributes to complex metal-cycling interactions in microbial communities.
• However, these interactions often represent a molecular arms race, as both siderophore structure and receptor specificity evolve rapidly to balance cooperation and competition.

Siderophore Specificity

• Siderophores display structural diversity — for example, over 60 distinct pyoverdine siderophores have been described.
• Cell surface receptors that recognize specific siderophores can be highly species-specific, limiting their utilization by unrelated microbes.
• This specificity helps protect producers from iron theft, but also restricts community-wide iron access and can slow the growth of other species

An eco-evolutionary model predicts that siderophore specificity reduces overall iron availability in a community, maintaining selective pressure for innovation and cheating resistance.

Siderophore Cheating

• Siderophore cheating occurs when certain microbial strains do not produce siderophores but exploit those secreted by others.
• This phenomenon has been observed in soil, freshwater, and marine environments.

Example: Pseudomonas aeruginosa

• The siderophore pyoverdine plays a dual role:
• Iron acquisition under normal conditions, and
• Iron sequestration within the periplasm during oxidative stress.
• Under oxidative conditions, P. aeruginosa downregulates pyoverdine secretion, retaining it intracellularly to prevent Fenton reaction–mediated hydroxyl radical formation.
• Cheaters that fail to produce pyoverdine lose this protective advantage and are naturally eliminated under stress — an example of self-policing within microbial populations.

Mutualistic Cross-Feeding via Siderophores

While most siderophore-related interactions are competitive or exploitative, a few involve mutualistic cross-feeding:

Marine Alga–Bacteria Mutualism:

• Certain alga-associated bacteria secrete siderophores that enhance algal iron acquisition.
• In return, the algae release metabolites that support bacterial growth — a reciprocal metabolic exchange.

Host–Microbiota Mutualism:

• Siderophore enterobactin, produced by E. coli, promotes iron uptake in mitochondria of both Caenorhabditis elegans and mammalian cells.
• In contrast, siderophores from pathogens (e.g., pyoverdine from Pseudomonas) do not benefit host iron uptake, illustrating selective mutualism between host and commensal microbiota.

3. Toxins

• Microbial toxins are secreted compounds that have likely evolved through natural selection for their inhibitory and sometimes lethal effects on other organisms, including host cells and other microbes.
• Toxins exist in a wide range of chemical structures and molecular sizes, from small molecules such as antibiotics and cyanide to large multisubunit protein toxins like cholera toxin.
• The methods by which toxins are delivered also vary; some toxins are passively excreted or actively secreted into the extracellular environment, while others are injected directly into host or microbial cells.
• Certain small-molecule toxins can directly function as cross-fed metabolites when they are present at subinhibitory concentrations or when the recipient organism possesses sufficient tolerance to the toxin.
• Some bacteria are capable of utilizing cyanide as a nitrogen source for their growth, demonstrating the metabolic flexibility of microbes toward certain toxins.
• Other microbes might even consume antibiotics, though such observations have been met with skepticism and require further validation.
• It is also speculated that toxins can play a broader and more indirect role in promoting cross-feeding within microbial communities.
• Toxins often facilitate nutrient acquisition by damaging or lysing cells, which leads to the release of intracellular contents into the surrounding environment.
• This toxin-mediated release of nutrients can provide readily available resources to neighboring microbes, allowing toxins to function as facilitators of cross-feeding in a manner similar to that of extracellular enzymes (exoenzymes).
• During polymicrobial infections, synergistic interactions between coexisting pathogens can occur, and these interactions are often dependent on the specific toxins characteristic of each infection.
• However, the precise role of toxin-mediated cross-feeding in such polymicrobial interactions remains obscure and not fully understood.
• In contrast to expectations of cooperation, evidence also suggests that toxins can lead to increased competition among microbial populations.
• Findings from several transposon sequencing (TnSeq) experiments have shown that a greater number of genes become essential for a pathogen during coinfection compared to monoinfection.
• These results imply that coinfection may intensify competition rather than promote cooperative interactions, as heightened competition could restrict nutrient availability, thereby making more genes necessary for survival.
• Overall, the extent to which toxin-mediated cross-feeding occurs in microbial systems remains unclear and warrants further detailed investigation to understand its ecological and evolutionary significance.

4. Quorum-Sensing Signals

• Quorum sensing (QS) is a microbial communication mechanism that coordinates gene expression according to population density through the production and detection of diffusible signal molecules.
• When a microbial population reaches a critical density (a quorum), the concentration of signal molecules rises, triggering a synchronized expression of specific genes across the community.
• A QS system typically includes two core components:
• A signal synthase, responsible for producing the signaling molecule.
• A cytoplasmic receptor, which binds to the signal and acts as a transcriptional regulator, modulating gene expression.
• QS signals—also known as autoinducers—can, in some cases, be used as the sole energy source by certain bacteria in monoculture. This suggests that QS signals may sometimes function as cross-fed metabolites.
• However, in microbial communities, catabolism of QS signals is often more relevant to quorum quenching, a process that mitigates or disrupts signaling rather than promoting cross-feeding.
• The degree to which QS signals themselves are cross-fed between different organisms has not yet been comprehensively tested.
• QS plays a well-established role in regulating the production of key extracellular molecules such as exoenzymes, siderophores, and toxins, as well as other community-wide behaviors.
• Therefore, the primary role of QS in cross-feeding is indirect and regulatory, as it helps coordinate and control access to extracellular nutrients on a population level.
• A population benefits most from the products of exoenzymes, siderophores, or toxins when these molecules are produced in sufficiently high concentrations, a condition often achieved through QS regulation.
• QS frequently coordinates multiple and sometimes contrasting activities, which can also serve to limit the exploitation of extracellular public goods by cheaters (non-producing individuals).
• Example:
• In Pseudomonas aeruginosa, the production of the casein-hydrolyzing exoenzyme elastase is regulated by the QS transcriptional regulator LasR.
• lasR-null mutants that fail to produce elastase can act as cheaters, benefiting from nearby producers when casein is the sole carbon source.
• However, when adenosine is added to the casein medium, the fitness of lasR mutants decreases because LasR is also necessary for adenosine metabolism.
• This coupling of exoenzyme production with other vital physiological pathways (like adenosine utilization) minimizes the advantage of cheating and maintains cooperative behavior.
• Furthermore, LasR regulates both cyanide production and cyanide resistance in P. aeruginosa, meaning that mutants lacking LasR (and thus elastase) become susceptible to cyanide toxicity, further balancing population dynamics.

Figure 4. Illustration of AHL-mediated quorum sensing (QS) (A and B) and enzymatic quorum quenching (QQ) (C).
(A) Bacteria possess a LuxI synthase that produces N-acyl homoserine lactones (AHLs) within the cytoplasm. These molecules diffuse freely out of the cell. At low cell densities, extracellular AHL levels remain low, and QS remains inactive.
(B) As the bacterial population grows, extracellular AHL concentrations rise. Once a threshold concentration is reached, AHLs bind to their specific LuxR receptors in the cytoplasm. The AHL–LuxR complex acts as a transcription factor, activating target gene expression—including additional AHL production—leading to coordinated, population-wide changes in gene expression and behavior.
(C) Quorum quenching (QQ) enzymes break down extracellular AHLs, lowering their concentration and preventing the signaling molecules from reaching the threshold required to initiate QS.

Role of Autoinducers:

• QS-mediated control of nutrient access also depends on the diversity of autoinducer molecules.
• Gram-positive bacteria generally produce oligopeptide autoinducers, whereas Gram-negative bacteria typically use acyl homoserine lactone (AHL) autoinducers.
• Within these classes, there is extensive chemical and structural diversity, and receptors differ in their signal selectivity—some are highly specific, while others can detect signals from different species.
• Additionally, many bacteria (both Gram-positive and Gram-negative) produce another signaling molecule known as autoinducer-2 (AI-2).
• AI-2 is a by-product of the activated methyl cycle within the methionine synthesis pathway, which is widespread among microbes.
• Consequently, the ability to produce AI-2 is common across diverse taxa, leading to the hypothesis that AI-2 acts as a form of interspecies QS signal.
• However, while many microbes can generate AI-2, relatively few species possess the specific receptors required to actually detect and respond to AI-2 signaling.

Overall, quorum sensing serves as a central coordinating mechanism that regulates the timing and concentration-dependent production of extracellular factors, indirectly shaping cross-feeding dynamics by managing nutrient accessibility, preventing exploitation, and stabilizing cooperative behaviors within microbial populations.

Figure 5. Overview of the general mechanism of quorum sensing in bacteria. Bacterial cells produce and release signaling molecules (autoinducers) into their environment. As cell density increases, these autoinducers accumulate. Once a critical threshold concentration is reached, the signaling molecules bind to their specific receptors, activating gene expression pathways that coordinate group behaviors across the bacterial population.

6. Extracellular Matrix Components of Biofilms

• Microbes often attach to surfaces and each other to form multicellular biofilms.
• Biofilm formation is facilitated by secretion of an extracellular matrix, which can include polysaccharides, nucleic acids, and/or proteins.
• These matrix components may directly contribute to cross-feeding, especially when combined with exoenzymes that degrade the matrix into accessible monomers.
• Isotopic tracer experiments in cyanobacteria have demonstrated that they can store and later reacquire carbon from their extracellular matrix, and this stored material can also be utilized by other bacteria within microbial mats.
• The biofilm plays a key role in controlling access to externalized nutrients, functioning similarly to quorum sensing (QS) systems.
• QS frequently helps coordinate biofilm development by regulating the biosynthesis of the matrix components.
• The dense packing of biofilm-residing cells allows them to reach quorum with smaller population sizes compared to dispersed planktonic cells.
• The spatial structure of biofilms promotes cooperative cross-feeding of costly metabolites by clustering partners in close proximity.
• Experiments on agar plates have shown that spatially structured environments can select for the evolution of mutualistic cross-feeding:
• A Salmonella enterica mutant evolved costly methionine excretion, enabling growth of E. coli methionine auxotrophs nearby.
• In return, E. coli evolved galactose secretion during lactose consumption when grown with S. enterica, supporting a reciprocal cross-feeding relationship.
• These findings indicate that certain spatially structured environments encourage the evolution of mutualistic cross-feeding by promoting local nutrient retention, directing nutrients to reciprocating partners, and reducing diffusion losses to noncooperative competitors.
• Clustering of cooperative cells also influences the dynamics between cooperators and cheaters:
• In some cases, clustering benefits cooperators by limiting cheater access to shared metabolites.
• In other cases, clustering can benefit cheaters by enabling them to invade dense producer populations.
• Example: In Vibrio cholerae, biofilm formation concentrated chitinase-secreting cells on chitin surfaces, reducing diffusion and limiting cheater access to released monomers.
• Close spatial proximity between cross-feeding partners improves nutrient exchange efficiency and minimizes loss to cheaters or competitors.
• Studies show that producers and cross-feeding partners often cluster together, allowing cheaters to exploit only at the biofilm edges, where diffusion allows limited access.
• However, this interaction is context-dependent; in marine Vibrio populations, nonproducing strains lacking siderophore biosynthesis genes were often found in large natural aggregates with siderophore producers — suggesting cheater invasion of dense cooperative populations.
• Overall, biofilm spatial structure plays a crucial role in balancing cooperation and competition, influencing how cross-feeding and cheating dynamics evolve in microbial communities.

7. Extracellular Vesicles

• Cross-feeding behavior can be influenced by the release of extracellular vesicles (EVs), similar to quorum sensing (QS) and biofilms.
• Extracellular vesicles have been reported in eukaryotes, archaea, Gram-negative, and Gram-positive bacteria.
• In Gram-negative bacteria, EV release is linked to reduced cross-linking between peptidoglycan and outer membrane proteins and may be influenced by lipid and lipopolysaccharide composition.
• Mechanistic details for EV release in Gram-positive bacteria are unclear but may involve weak points in the cell wall or channels.
• Some EV release may be accidental/passive, as observed in vesicle-mediated toxin delivery, e.g., Myxococcus xanthus packages exoenzymes in EVs to degrade E. coli macromolecules.
• EVs are also components of biofilm matrices.
• EVs can participate in QS, facilitating the diffusion of hydrophobic signals in aqueous environments.
• Siderophores can be secreted via EVs, which may help evade host immune responses or provide uptake specificity for sequestered metals.
• EVs can serve as cross-fed nutrients; e.g., Prochlorococcus vesicles support growth of heterotrophic marine bacteria, potentially releasing 10⁴–10⁵ metric tons of carbon daily into the oceans.
• EVs can protect their cargo, potentially reducing or delaying acquisition by competitors, limiting unwanted cross-feeding.

Contact-Dependent Cross-Feeding:

• QS, biofilms, and EVs influence externalized molecules, but contact-dependent mechanisms also occur.
• Many contact-dependent interactions are antagonistic, but beneficial cross-feeding is also observed.
• Some interactions are intimate enough for one cell to be housed within another, or to share a periplasm or proton motive force.
• Filaments of cable bacteria share a periplasm along with electrically conductive appendages, spanning redox gradients in sediments to enable combined metabolism across separate cells.
• Filamentous cyanobacteria share a periplasm and differentiate into vegetative cells and heterocysts for CO₂ and N₂ fixation, transferring carbon and nitrogen at junctions.

Cable Bacteria

• There are examples of interspecific cross-feeding by direct contact.
• In some cases, the mode of transfer is poorly defined, e.g., stable isotope transfer from cyanobacterial heterocysts to attached Rhizobia, both environmental isolates.
• Other cases reveal unique mechanistic traits in contact-dependent cross-feeding.
• The archaeal pairing of Ignicoccus hospitalis and Nanoarchaeum equitans likely involves transfer of phospholipids, amino acids, and possibly ATP from I. hospitalis to N. equitans.
• N. equitans lacks genes to produce a complete ATP synthase.
• I. hospitalis, the only known organism with an energized outer membrane, generates ATP in the periplasm via an outer membrane ATP synthase.
• This periplasmic ATP generation is thought to facilitate ATP cross-feeding to N. equitans.

8. Nanotubes

• Nanotubes are intimate intercellular membranous connections reported both intra- and interspecifically, including between Gram-negative and Gram-positive bacteria, and possibly between bacteria and mammalian cells.
• Nanotubes can enable transfer of small metabolites, large proteins, and DNA molecules.
• The mechanism by which the inner membrane passes through the peptidoglycan layers of donor and recipient cells is unclear, though peptidoglycan hydrolases are implicated in B. subtilis.
• Nanotube formation may share mechanistic features with extracellular vesicle formation.
• Other genetic factors implicated include a B. subtilis phosphodiesterase, a sigma factor, and pathogenic E. coli injectosome components.
• Nanotube function remains mysterious and sometimes controversial; evidence is often correlative, and alternative transfer mechanisms are not always ruled out.
• Intercellular transfer of fluorescent markers has been observed, but frequencies of connections and transfer events are rarely reported.
• Regulation of nanotube formation is inconsistent:
• In E. coli, formation was stimulated by nutrient deprivation, suggesting a role in nutrient acquisition.
• In pathogenic E. coli and B. subtilis, formation occurred in rich media.
• Nanotubes may form as a by-product of cell death in E. coli, B. subtilis, and other Gram-positive bacteria.
• DNA transfer via nanotubes in B. subtilis required competence proteins, suggesting nanotubes may not be directly involved.
• In noncompetent bacteria, plasmid transfer via nanotubes is hard to reconcile with known microbiology techniques, challenging conventional ideas of conjugation and transformation.
• Nanotube-mediated protein or DNA transfer could confound competition assays that rely on genetic markers.
• Co-occurrence of cell death with nanotube formation suggests that some observed cross-feeding may actually result from cell lysis.
• Overall, nanotubes are an intriguing potential mechanism for cross-feeding, but critical mechanistic and functional details remain contentious and under debate.

9. Nanowires

• Nanowires are a well-studied contact-dependent mechanism for cross-feeding via direct electron transfer between cells.
• They facilitate anaerobic respiration by allowing the producer to deposit electrons onto extracellular terminal electron acceptors that are too large, insoluble, or toxic to reduce intracellularly.
• Nanowires, along with soluble extracellular electron shuttles, contribute to cross-feeding by generating reduced metabolites; these reduced metals can serve as electron sources for other organisms, such as photo- or litho-autotrophs.
• They can also directly couple redox metabolisms between cells, sustaining syntrophic partnerships by overcoming thermodynamic limitations, similar to intercellular transfer of organic acids, H₂, or other reduced compounds.
• Nanowire-mediated electron transfer has been observed in both natural and synthetic syntrophies.
• Example: A syntrophy between Geobacter metallireducens (ethanol-fed fermentation) and Geobacter sulfurreducens (fumarate-respiring) involved electron transfer via nanowires, relieving thermodynamic barriers for ethanol fermentation and supporting fumarate respiration.
• This syntrophy depended on G. sulfurreducens nanowire components (pili and outer membrane cytochrome), but not on H₂ oxidation by G. sulfurreducens.
• Natural aggregates: Nanowire-mediated syntrophy occurs between sulfate-reducing bacteria and archaeal anaerobic methanotrophs (ANME), important for regulating methane emissions from ocean sediments.
• Some H₂ cross-feeding syntrophs may grow instead via direct electron transfer to a partner.
• The composition of nanowires is debated:
• Some studies suggest electrons transfer via aromatic amino acids in pili.
• A recent crystal structure showed pili composed entirely of cytochrome subunits, questioning the role of pilin in electron transfer.
• Despite this, G. sulfurreducens pilin is required to receive electrons from G. metallireducens, indicating a role in interspecies electron transfer even if not directly conductive.
• Nanowires are likely compositionally diverse:
• Example: Shewanella oneidensis nanowires are outer membrane extensions containing electrically conductive cytochromes, similar mechanistically to nanotubes or extracellular vesicles.
• Other mechanisms of direct electron transfer include cell surface cytochromes, transfer through abiotic surfaces, and conductive structures in cable bacteria filaments.
• Microbes capable of externalizing electron flow are of interest for applications such as electricity generation, bioremediation, and electricity-driven production of reduced compounds.

Dynamic Aspects of Microbial Cross-Feeding

• The dynamic aspects of microbial cross-feeding can be illustrated by studies on synthetic cross-feeding communities, such as those between E. coli and Rhodopseudomonas palustris.
• Insights from this system reveal how metabolic exchanges are coordinated between species to support mutual growth and survival.
• Observations from this model are supported by studies in other microbial communities, highlighting common principles of resource sharing, cross-feeding regulation, and interspecies interactions.
• Such systems help uncover the temporal and spatial dynamics of metabolite exchange, including how nutrient availability, population density, and environmental factors influence cross-feeding efficiency.
• Synthetic communities serve as a controlled framework to test hypotheses about cooperation, competition, and the evolution of cross-feeding in more complex natural microbial ecosystems.

Cross-Feeding Rates Determine the Relative Benefit or Detriment of a Cross-Fed Metabolite

• Many excreted microbial metabolites can be toxic, making detoxification a key aspect of cooperative microbial interactions. One partner may protect another by consuming or processing toxic intermediates.
• Some metabolites, such as fermentation products (alcohols, organic acids), are central to anaerobic food webs, acting as carbon and electron shuttles, but can also become toxic at high concentrations.
• Cross-feeding levels determine whether a metabolite acts as a nutrient or a toxin.

Example: E. coli and R. palustris coculture

• In a coculture with fermentative E. coli and N2-fixing R. palustris engineered to excrete NH4+, each species depends on the other: E. coli provides organic acids as carbon for R. palustris, and R. palustris provides NH4+ as nitrogen for E. coli.
• When NH4+ excretion is low, E. coli growth matches R. palustris, and organic acids are consumed as fast as they are excreted.
• When NH4+ excretion is high, E. coli grows faster, overproducing organic acids that accumulate, acidify the medium, and inhibit R. palustris growth.
• This phenomenon, called “dose-dependent toxicity,” shows that increased reciprocation can paradoxically harm the producer by turning a nutrient into a toxin.

Factors influencing dose-dependent toxicity

• Local concentration of metabolites
• Metabolite diffusion
• Community spatial structure
• Environmental factors, e.g., flow rate

Other examples

• Nitrite (NO2–) cross-feeding: NO2– toxicity is pH-dependent. Lower pH increases inhibition of Nitrobacter in a denitrifying consortium of Pseudomonas stutzeri.
• During nitrification, Nitrosomonus produces NO2– from NH4+, which can harm NO2– consumers like Nitrobacter if excretion rates are high.
• Overall, the benefit or detriment of cross-fed metabolites depends on the rate of transfer and local environmental context, highlighting the importance of dose in microbial interactions.

Cross-Feeding Is Not Always Coupled to Growth

• For microbes unable to form specialized dormant structures like spores, a low level of metabolic activity is generally essential to generate maintenance energy for survival.
• Few studies have directly examined the role of cross-feeding in survival, but cross-feeding could be an important driver of maintenance metabolism in nutrient-limited environments.
• Respiration is critical for stationary-phase survival of some lactic acid bacteria, including group B Streptococcus (GBS) pathogens, affecting both growth and survival.
• GBS require exogenous sources of heme and naphthoquinones to carry out respiration, as humans do not produce napthoquinones; these cofactors must be acquired from other microbes.
• Quinone cross-feeding may favor GBS survival by offsetting lactic acid accumulation, creating a more favorable pH, and by maintaining a proton motive force to generate maintenance energy under nongrowing conditions.
• Low metabolic activity in nutrient-deprived cells can foster cross-feeding. For example, nongrowing E. coli fermented glucose and excreted organic acids sufficient to support the phototrophic growth of R. palustris.
• This growth-independent cross-feeding from E. coli stimulated reciprocation from R. palustris, ultimately lifting both partners out of starvation.
• An obligate cross-feeding relationship between E. coli and R. palustris can naturally evolve, initially relying on growth-independent organic acid excretion by E. coli.
• Maintenance metabolism is important for both the initiation and maintenance of cross-feeding relationships.
• Growth-independent cross-feeding can have medical relevance. For example, acetoin production by nongrowing S. aureus supported growth and survival of P. aeruginosa isolated from the same cystic fibrosis patient samples.
• In turn, P. aeruginosa facilitated S. aureus survival by preventing acetoin accumulation to toxic levels.
• Cross-feeding between nongrowing partners can therefore support persistence during coinfection.

Cross-Feeding Can Be Facultative

• Cross-feeding is not always essential; it can be facultative, augmenting a recipient’s metabolism rather than fulfilling a strict nutritional requirement.
• Quinone cross-feeding to GBS exemplifies facultative cross-feeding: GBS can grow by fermenting glucose even when its electron transport chain is incomplete.
• Growth yield of GBS improves when aerobic respiration is enabled through quinone cross-feeding.

Mutualistic and Synergistic Relationships Contain Competitive Interactions

• Cooperation between microbial species can appear to conflict with evolutionary theory, as strong competitors are expected to outcompete species that invest in costly traits benefiting neighbors.
• Competition is likely predominant even within cooperative relationships; truly selfless cooperation may be rare or nonexistent.
• Cooperative interactions often arise due to environmental necessity rather than altruism; for example, a host may become dependent on a parasite for a function, making the relationship obligatory.
• Competition can occur between otherwise cooperative partners, as seen in syntrophic cocultures of D. vulgaris and M. maripaludis, where pairing different evolved partners resulted in lower growth rates and yields than expected, indicating antagonistic interactions despite required syntrophic cross-feeding.
• Cooperative cross-feeding can occur while partners compete for the very resource from which a cross-fed metabolite is derived.
• In long-term E. coli cultures, subpopulations specialized in glucose uptake excreted acetate, while another subpopulation fed on that acetate. This is cooperative because acetate sustains the recipient and its removal protects the producer, but competition remains since the recipient can still consume glucose and grows faster on it than the producer in monoculture.
• Competition can be central to cooperative cross-feeding; in cocultures dependent on NH4+ cross-feeding from R. palustris to E. coli, both species competed for NH4+.
• Maintenance of the cooperative relationship required E. coli to be more competitive than R. palustris for excreted NH4+.
• Transcriptomic and proteomic analyses revealed that E. coli’s competitive advantage relied on a nitrogen starvation response.
• If R. palustris outcompeted E. coli for NH4+, it would starve E. coli and lose reciprocal cross-feeding of essential carbon.
• Evolution of competitive NH4+ acquisition by E. coli alone was sufficient to establish cross-feeding with wild-type R. palustris, involving mutations that further enhanced the nitrogen starvation response.

Level of privatization

• The previously discussed example required a competitive bias favoring the recipient in a cross-feeding relationship.
• Competitive bias toward the recipient versus the producer is influenced by the level of privatization of the shared nutrient.
• When there is high privatization, such as for metabolites generated intracellularly, the producer naturally retains most of the metabolite, so competition must favor the recipient to maintain cross-feeding.
• When there is low privatization, such as for metabolites released via exoenzymes or some siderophores, the competitive bias needed to sustain cross-feeding can differ, since the metabolite is more freely accessible in the environment.