Investigating electromicrobiology, researchers are learning how microbial cells communicate via nanowires and electronic signals

Gemma Reguera

Gemma Reguera is an assistant professor in the Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing.


Summary

● Quorum sensing enables some bacteria to communicate via signaling molecules, whereas other bacteria produce nanowires through which they communicate electronically.

● Microbial cell-cell communication via nanowires may be widespread in nature.

● Nanowires provide two distinct electron-flow pathways for Geobacter sulfurreducens cells, one for fast electron flow at high voltages and the other a high-resistance path reflecting metabolic status.

● Various types of energy cause microbial cells to vibrate, sometimes changing gene expression and metabolic activity, and also releasing sound energy and other potential physical forms of communication.

Even though bacteria are speechless, they nonetheless communicate with one another, engaging in social behaviors- collaborating, cooperating, and sometimes cheating. These behaviors are important, improving the chances, for example, for bacteria to survive in their natural environments or for some of them to cause infections when introduced into host species. Indeed, how many ways do bacteria communicate with one another or other species that they encounter? If we fail to recognize some of those means, will some bacteria be lost in translation? Although researchers recognized several decades ago that bacteria can communicate by means of chemical signals, we more recently came to know that some bacteria also signal one another electronically.

Some Bacteria Communicate via a Chemical Language

Chemical communication is perhaps the most widespread form of communication in the living world. For example, vertebrates, social invertebrates, and fungi communicate via pheromones, which sometimes are a means to attract mates. Another type of chemical signaling controls cell density functions in fungi, while other types mediate cell-cell communication during tissue development in animals and plants. Researchers who were studying genetic competence in Streptococcus pneumoniae and bioluminescence in Vibrio separately discovered density-dependent collective behaviors in both those species. Thus, for example, S. pneumoniae cells respond to one another when they accumulate signaling molecules, called autoinducers (Fig. 1). This mode of chemical communication, known as quorum sensing, allows these or other bacteria to reprogram gene expression as a function of population density. These changes are consistent with some functions being more effective when the bacterial community rather than an individual bacterial cell performs them. In these instances, cell density- dependent regulation becomes an effective way for the cells to turn off processes that may otherwise become unproductive if started before cell numbers reach a particular "quorum" level. Quorum sensing controls various processes among bacteria, including genetic competence, bioluminescence, virulence, biofilm formation, and sporulation. Bacteria also communicate chemically in an interkingdom mode with their eukaryotic hosts.

The quorum sensing "language" depends on signaling molecules such as acyl-homoserine lactones (AHLs) in gram-negative bacteria and short peptides in gram-positive bacteria. Regardless of the chemical nature of such signals, all forms of chemical communication require mechanisms for making and detecting those signals. In the gram-negative bioluminescent bacterium Vibrio fischeri, for example, the LuxI (autoinducer synthesis)/LuxR (detection) system mediates that communication. As bacteria grow and increase in cell numbers, the autoinducer concentration and the detection signal output increase, thereby triggering bioluminescence as a function of higher cell densities. Although LuxI/LuxR circuits are found in several species of gram-negative bacteria, the chemical nature of the AHL and the cell density thresholds required for its detection are, in general, species specific. This level of specificity ensures the privacy of the bacterial conversation and an adequate response to the quorum size. Gram-positive bacteria communicate chemically mainly via modified oligopeptides or, in the case of streptomycetes, via γ-butyrolactones. The peptide language for these and other gram-positive bacteria also appears to be species-specific. However, it differs from the canonical LuxI/LuxR system in that it depends on two-component signal transduction systems to recognize and then respond to the signaling peptides.

In contrast, free-living, bioluminescent Vibrio harveyi cells communicate via a hybrid language that shares characteristics of both gram-negative and gram-positive quorum sensing mechanisms. V. harveyi cells secrete two different autoinducers (AI-1 and AI-2) that are recognized by separate detection systems yet share the same phospho-relay cascade for cell reprogramming. Chemical communication via AI-2-like autoinducers and signaling circuits is widespread among bacterial species, with some cross-talk between AI-2 "dialects" involving different species.

Thus, the AI-2 chemical language could be considered the universal mode of communication of the bacterial world. However, several sequenced genomes show no homologues of AI-2 signaling circuits and of any other quorum sensing systems, raising the possibility that novel forms of bacterial languages may await discovery.

Some Bacteria Communicate via an Electronic Language

The iron-reducing bacterium Geobacter sulfurreducens produces electrically conductive protein filaments, called nanowires, that transfer electrons to insoluble metal oxides. The nanowires of G. sulfurreducens are type IV pili that are composed of a single structural subunit, pilin or PilA, which polymerizes through hydrophobic interactions into a protein filament. This nanowire pilin is a very small peptide that lacks the carboxy-terminus variable and hypervariable regions found in all other bacterial type IV pilins. Thus, the nanowire pilin is thin, only 4-5 nmin diameter, and it extends more than 20 μm beyond the cell surface. Perhaps because of these structural differences, the nanowires behave unlike other pili, which typically help to attach cells to one another and also twitch to provide motility. The nanowire pilin also differs from other pilins in terms of amino acid composition, and it appears to come from an independent line of descent. These qualitative differences are consistent with these type IV pili playing a specialized role in the life of G. sulfurreducens.

Following the 2005 discovery of the pilus nanowires of G. sulfurreducens, my research group found, for example, that the nanowires bind to and conduct electrons to soluble metals such as uranium. Further, the nanowires reductively precipitate the soluble metal and also protect the cell from the toxic effects of uranium, which would otherwise penetrate the periplasmic space and mineralize the cell envelope. Nanowire expression also increases the rates and extent of uranium precipitation per cell, thus providing substantial energy gain for cell growth. Nanowires are also produced under growth-limiting conditions or when cell densities are low. Under these conditions, the nanowires form electronic networks that interconnect cells, bringing them closer together (Fig. 1 and 2). This behavior contrasts with the high cell density dependence of chemical communication.

With Stuart Tessmer at Michigan State University, we developed a scanning probe technique to measure electronic paths between living G. sulfurreducens cells that are connected via nanowires. We find that the nanowires are electrically active and form a nanopower grid that supports electronic exchanges between cells (Fig. 2). Further, the nanowire-connected cells undergo extensive cell reprogramming, according to gene-expression analyses. These findings suggest that cells are exchanging electronic information through nanowires, with some cells acting as senders while others act as receivers. With our scanning technique, we identified evidence for two electron-flow pathways along the nanowires. One pathway allows fast electron flow at high voltages such as those found between a cell and the Fe(III) oxide mineral surface, whereas the other works in a lower ±300-400 mV range and provides a highresistance path for electrons (Fig. 2). This high resistance pathway controls the electron flow between cells as a function of the differential potential and, therefore, the metabolic status of the connected cells and the nanowire length.

Nanowires Play a Key Role in Managing Metabolic Heterogeneities

Metabolic heterogeneity is intrinsic to cell populations undergoing suboptimal growth. This heterogeneity can produce a variety of phenotypes within the same culture. In G. sulfurreducens, whose metabolism is based on electron transfer from an electron donor to an acceptor, metabolic heterogeneity correlates with a range of cell potentials reflecting a distinct oxidizing or reducing state of each cell within the same culture.

Metabolic inactivity could lead to cell death unless cells overcome their electron transfer limitations- for example, by using the nanowires to connect the cells and dissipate electrons as a function of redox status. Preliminary evidence suggests that the high-resistance nanowire pathway signals the metabolic status of neighboring cells and then dissipates electrons cooperatively to benefit the networked community. The nanowires extend long distances beyond the cell surface to reach neighboring cells and cause the cells to aggregate and agglutinate, suggesting they may retract to bring the cells closer while optimizing electronic flow (Fig. 1 and 2).

Metabolic heterogeneity is also a hallmark of biofilm physiology. Mature biofilms are composed of microenvironments with different osmolarities, nutrient availability, and cell densities. What role do pilus nanowires play during biofilm development? Although some microbiologists consider biofilms to be the predominant mode of growth for bacteria in natural environments, there were questions about whether Geobacter could even form biofilms. All the early evidence suggested that these microorganisms needed to establish intimate contact with electron-accepting surfaces simply to grow, thus preventing cells from stacking in multilayered communities.

However, when we coated coverslips with Fe(III) oxide, we found that G. sulfurreducens cells formed structured, multilayered biofilms on the mineral and that they generate energy for growth by transferring electrons across cell layers (Fig. 3). Further, these biofilms formed on electrodes that are exposed to an optimum potential that does not limit biofilm power production capacity (Fig. 3). Electron flow was readily measured across the electrode-associated biofilms as electricity.

Geobacter biofilms form only when cells can produce pilus nanowires, which take on a structural role in addition to their electronic role. When these biofilms are stained with viability dyes, cells in the upper layers of the biofilm remain viable, according to confocal scanning laser microscopy (CSLM). Furthermore, correlations between the height or mass of the biofilm with the electrons flowing across the biofilms are linear, indicating that the electronic efficiency per cell remains constant as the biofilm develops. Moreover, more cells are found at increasing distances from the electron-accepting surface. Spacings among biofilm cells are consistent with an electronic pathway mediated by pilus nanowires rather than cell-associated electron transport proteins such as c-type cytochromes, which would require intimate contact between cells. Because cells can retract pili, the nanowires may be adjusting spacings between cells to optimize electronic communication and electron flow across the biofilm.

The biofilm matrix potentially contributes to electron flow in biofilms. Some outer membrane c-type cytochromes required for optimum electron transfers are loosely bound to and easily detach from the cell envelope. Because these cytochromes remain trapped in the biofilm matrix, they might provide an electronic pathway for the cells. However, the biological significance of this biofilm component is not known.

Cell-Cell Communication via Nanowires May Be Widespread

The ability of microorganisms to communicate electronically via nanowire-like appendages may not be restricted to Geobacter bacteria. The metal-reducing bacterium Shewanella oneidensis, when grown under conditions that limit electron acceptors, produces nanowire appendages, according to Yuri Gorby of the J. Craig Venter Institute in Rockville, Md. These nanowires appear to conduct electrons via electron transport proteins, such as c-cytochromes, assembled on a filamentous scaffold. Separate atomic force microscopy (AFM) experiments provide physical evidence that Shewanella nanowires are conductive along their length, according to Moh El-Naggar of the University of Southern California. Gorby and his collaborators also find evidence of conductive, nanowire- like appendages in other microorganisms, including cyanobacteria, suggesting that cell-cell communication via nanowires is widespread in nature.

However, these microbial nanowires studies are not the first to suggest a role for electronic signals in cell-cell communication. Other reports suggest that microorganisms may communicate using physical signals such as microwave radiation, magnetic fields, and sound waves. For example, sound waves stimulate Bacillus cells to produce physical signals, possibly sonic in nature, that signal nearby cells to grow, according to Michio Matsuhashi and his collaborators at Tokai University in Numazu, Japan. Furthermore, electroactive materials such as graphite similarly stimulate growth of neighboring cells, perhaps by converting electromagnetic radiation into sonic waves, he and his collaborators speculate.

Other researchers are skeptical about this interpretation, however. For example, those sonic signatures could arise from the vibrational modes of individual cells in a colony, according to Vic Norris of the University of Rouen in France and Gerard Hyland of the University of Warwick in Coventry, United Kingdom. They suggest that all biological systems can be electrically excited to vibrate at particular frequencies.

Further, proteins oscillating within the cytoplasm might cause bacterial or other cells to vibrate, inducing ionic polarization while other cytoplasmic components are also shaking. Applying electromagnetic radiation or changing the metabolic status of the cell could further excite and polarize it. However, elastic forces in the cell membranes or walls help to dampen those oscillatory forces. When cells are growing at high densities, vibrating cells are apt to stimulate their neighbors physically, potentially amplifying these signals and enabling cells to communicate over greater distances, including via transmitted sound waves.

Metabolic energy can make the cell wall of Saccharomyces cerevisae yeast oscillate, according to Andrew Pelling, James Gimzewski, and their collaborators at the University of California, Los Angeles, who used AFM. The frequencies of these vibrations range from 0.8 to 1.6 kHz, putting them in the same acoustic range detected by Matshuhashi and collaborators. Furthermore, the magnitude of the force from that nanomechanical activity, about 10 nN, is high enough to make it operative in cells.

The same energy that causes the yeast cytoskeleton to oscillate also can activate or deactivate enzymes, which can change gene expression. Further, changes in hydration and counterion binding lead DNA to vibrate and change its conformation, affecting protein binding at promoter sites, also changing gene expression. External energy can trigger and enhance intracellular motions. For instance, microwaves with appropriate frequencies affect the growth of Escherichia coli cells. Moreover, weak magnetic fields affect enzyme activity in Rhodobacter sphaeroides, and can reprogram such cells. Meanwhile, exposing S. oneidensis to strong static magnetic fields, such as those in clinical and research laboratories, changes expression of genes involved in iron and energy metabolism.

Because polar oscillations are a fundamental property of biological systems, physical modes of communication may be widespread in the microbial world and may play important roles in microbial colonies and biofilms, where cells grow very near to one another. The same vibrational forces could be transmitted via nanowires, enabling them to serve as electronic conduits between cells of the same or different species. Until we uncover a Rosetta stone for the microbial world, however, we continue to listen for microbial voices whose messages are still mainly lost in translation.

ACKNOWLEDGMENTS

Financial support was provided by grants from NIEHS (R01 ES017052-01) and a Strategic Partnership Grant from the MSU Foundation. I thank current and former members of my lab, particularly Catherine Silva, Dena Cologgi, and Becky Steidl, and my collaborators Stuart Tessmer and Joshua Veazey for their contribution to the nanowire work. This article is based on the author's lecture at the electromicrobiology symposium at the 2008 ASM General Meeting.

SUGGESTED READING

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