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A recent study reports that Bacillus subtilis biofilm formation depends upon paracrine signaling where the signal-producing and target-responsive cells are different.
Bacteria rely on precisely coordinated signaling mechanisms to ensure efficient and accurate transmission of chemical messages within a population. During bacterial differentiation, this signaling has been thought to be autocrine — that is, all cells produce and respond to the same signal. However, in a recent paper in Genes and Development, Lopez et al.  report that biofilm formation in Bacillus subtilis involves paracrine signaling. Specifically, they found that, while most cells within the population produce a prenylated peptide, this molecule triggers the production of another signaling molecule — surfactin — only in a small subset of cells. As a consequence, a subpopulation of cells not capable of producing surfactin responds to surfactin to produce the extracellular matrix component of the biofilm.
In autocrine signaling the same cells both produce and respond to a signal, whereas in paracrine signaling the producing and receiving cells are different. While paracrine signaling controls eukaryotic processes dependent on cell–cell signaling, such as neurotransmission, blood clotting, angiogenesis, and embryonic differentiation, cell–cell communication in bacteria has been thought to be autocrine. For example, the phenomenon of quorum sensing involves the detection of a threshold concentration of a signaling molecule by bacteria that also produce these signals (Figure 1A) . In the case of quorum sensing in Vibrio species, AHL autoinducers are detected by cytoplasmic proteins like LuxR, which activate transcription of quorum-sensing genes upon binding to their partner autoinducers . Similarly, the B. subtilis genetic competence regulator ComX is recognized by a sensor histidine kinase that triggers phosphorylation events necessary for proper target gene expression .
While these responses can occur over distances within bacterial populations, signaling that requires cells to be in close proximity to one another can also occur. For example, during fruiting body formation in Myxococcus xanthus, a signal protein is displayed on the surface and interacts with a receptor on an adjacent cell to transmit signal. Both cells express the signaling molecule as well as the receptor (Figure 1B) . Another example is the criss-cross signaling between the mother cell and the forespore during sporulation in B. subtilis where cell–cell communication occurs between cells that are in intimate contact (Figure 1C) .
Other inter-bacterial signals have been described whereby growing cells release muropeptides derived from the cell wall that trigger a developmental switch in dormant cells that are not producing this molecule. Since both populations express the receptor for the muropeptide, the growing cells can, in theory, respond to the signal. However, this signal does not lead to changes in the physiological state of the growing cells, in contrast with dormant cells that exit dormancy in response to the signal. Thus, this system can be viewed as autocrine and paracrine  (Figure 1D).
Unlike the situation in eukaryotic cells whereby developmental processes result in many co-existing cell types, bacterial populations are often thought to consist of singular cell types. However, there is increasing evidence that bacteria have qualities of multicellular organisms — for example, the mycelial structures observed in streptomycetes , fruiting bodies in myxobacteria  or contact-dependent inhibition in Escherichia coli . Biofilm formation in bacteria involves genetically identical cells that undergo differentiation into different types with only a fraction synthesizing the extracellular matrix necessary for the formation of the mature biofilm [11,12].
How this differentiation is generated has been a mystery since most cells generate and respond to ComX, the prenylated peptide that operates extracellularly and stimulates production of the cyclic lipopeptide surfactin . An important clue arose from the observation that surfactin also acted as a signaling molecule capable of inducing a subpopulation of B. subtilis cells within a developing biofilm to become matrix producers . An hypothesis following from this observation was that surfactin-producing cells were unable to respond to surfactin. To examine this possibility, Lopez et al.  monitored gene expression in individual cells that carried transcriptional reporters (PcomQ-yfp, PsrfAA-yfp and PyqxM-cfp) distinguishable using flow cytometry to identify cells producing ComX, surfactin and the matrix, respectively. As expected, most of the cells in the biofilm expressed genes involved in ComX production, and a small fraction of the population responded to ComX by producing surfactin. Furthermore, expression of the matrix protein operon was observed only in a subpopulation of cells. Importantly, however, surfactin-producing and matrix-producing cells were found as distinct populations, an observation that was confirmed by direct microscopy. Thus, Lopez et al.  concluded that surfactin producers are not capable of responding to the surfactin that they themselves generate and thereby provided a plausible explanation for the source of heterogeneity observed in the production of matrix.
As matrix producers do not become surfactin producers, the authors explored the possibility that the extracellular matrix physically prevents ComX action on the matrix producers. Lopez et al.  used different genetic backgrounds to manipulate the synthesis of the extracellular matrix and found that the levels of a ComX reporter were increased in the absence of extracellular matrix. They suggested that the matrix interferes with the ability of ComX to activate ComP, a kinase that phosphorylates a transcription factor required for surfactin production. They tested this interpretation using mutants lacking SinR, a repressor of matrix gene expression, which constitutively produce matrix. As expected, these cells did not produce surfactin, but they did produce functionally active ComX, as cleverly assayed using a heterologous strain that reports ComX activity but cannot synthesize ComX. Therefore, the defect in surfactin signaling does not lie in an inability to make ComX, but, consistent with the hypothesis, the matrix must somehow interfere with the ability of ComX to activate ComP and prevent the production of surfactin in matrix producers.
Thus, Lopez et al.  demonstrate that distinct populations of cells co-exist during B. subtilis biofilm formation, resulting in the formation of multicellular communities composed of genetically identical cells that are signal producers, signal responders or neither (Figure 1E). Surfactin-producing cells may become cells capable of genetic competence, whereas the matrix producers ultimately differentiate to become dormant spores. How these fates are determined by their prior developmental state remains unknown. In addition, an important question is how surfactin producers develop immunity to surfactin. The authors speculate that ComS, a protein produced by all surfactin-producing cells, may indirectly inactivate the transcription factor Spo0A that is required for the response to surfactin.
Cell–cell communication is central to a variety of developmental processes in bacteria. This signaling can be either between species or within species or between organisms that are in close proximity or between organisms that are in communities in which the signal-generating cell may be distant from the receiving cell. Further research will undoubtedly illuminate how these signaling events lead to genome-wide alterations in expression and provide genetically identical bacteria with the capability to exhibit individual phenotypic and communal properties.