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The Xanthomonas group of phytopathogens causes economically important diseases that lead to severe yield loss in major crops. Some Xanthomonas species are known to have an epiphytic and in planta lifestyle that is coordinated by several virulence-associated functions, cell-to-cell signaling (using diffusible signaling factor [DSF]), and environmental conditions, including iron availability. In this review, we described the role of cell-to-cell signaling by the DSF molecule and iron in the regulation of virulence-associated functions. Although DSF and iron are involved in the regulation of several virulence-associated functions, members of the Xanthomonas group of plant pathogens exhibit atypical patterns of regulation. Atypical patterns contribute to the adaptation to different lifestyles. Studies on DSF and iron biology indicate that virulence-associated functions can be regulated in completely contrasting fashions by the same signaling system in closely related xanthomonads.

Starving Myxococcus xanthus bacteria use short-range C-signaling to coordinate their movements and construct multicellular mounds, which mature into fruiting bodies as rods differentiate into spherical spores. Differentiation requires efficient C-signaling to drive the expression of developmental genes, but how the arrangement of cells within nascent fruiting bodies (NFBs) affects C-signaling is not fully understood. Here, we used confocal microscopy and cell segmentation to visualize and quantify the arrangement, morphology, and gene expression of cells near the bottom of NFBs at much higher resolution than previously achieved. We discovered that “transitioning cells” (TCs), intermediate in morphology between rods and spores, comprised 10 to 15% of the total population. Spores appeared midway between the center and the edge of NFBs early in their development and near the center as maturation progressed. The developmental pattern, as well as C-signal–dependent gene expression in TCs and spores, were correlated with cell density, the alignment of neighboring rods, and the tangential orientation of rods early in the development of NFBs. These dynamic radial patterns support a model in which the arrangement of cells within the NFBs affects C-signaling efficiency to regulate precisely the expression of developmental genes and cellular differentiation in space and time. Developmental patterns in other bacterial biofilms may likewise rely on short-range signaling to communicate multiple aspects of cellular arrangement, analogous to juxtacrine and paracrine signaling during animal development.

Self-incompatibility (SI) is a mechanism that many plant families employ to prevent self-fertilization. In the Brassicaceae, the S-haplotype-specific interaction of the pollen-borne ligand, and a stigma-specific receptor protein kinase triggers a signaling cascade that culminates in the rejection of self-pollen. While the upstream molecular components at the receptor level of the signaling pathway have been extensively studied, the intracellular responses beyond receptor activation were not as well understood. Recent research has uncovered several key molecules and signaling events that operate in concert for the manifestation of the self-incompatible responses in Brassicaceae stigmas. Here, we review the recent discoveries in both the compatible and self-incompatible pathways and provide new perspectives on the early stages of Brassicaceae pollen–pistil interactions.

Plant cells are connected by plasmodesmata (PD), nanoscopic channels in cell walls that allow diverse cytosolic molecules to move between neighboring cells. PD transport is tightly coordinated with physiology and development, although the range of signaling pathways that influence PD transport has not been comprehensively defined. Several plant hormones, including salicylic acid (SA) and auxin, are known to regulate PD transport, but the effects of other hormones have not been established. In this study, we provide evidence that cytokinins promote PD transport in leaves. Using a green fluorescent protein (GFP) movement assay in the epidermis of Nicotiana benthamiana, we have shown that PD transport significantly increases when leaves are supplied with exogenous cytokinins at physiologically relevant concentrations or when a positive regulator of cytokinin responses, ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 5 (AHP5), is overexpressed. We then demonstrated that silencing cytokinin receptors, ARABIDOPSIS HISTIDINE KINASE 3 (AHK3) or AHK4 or overexpressing a negative regulator of cytokinin signaling, AAHP6, significantly decreases PD transport. These results are supported by transcriptomic analysis of mutants with increased PD transport (ise1–4), which show signs of enhanced cytokinin signaling. We concluded that cytokinins contribute to dynamic changes in PD transport in plants, which will have implications in several aspects of plant biology, including meristem patterning and development, regulation of the sink-to-source transition, and phytohormone crosstalk.

Pigment patterns in adult fish show astonishing diversity, reflecting their likely roles in camouflage, kin- and mate-selection, and display. Adult pigment pattern formation has only been explored extensively in the zebrafish, Danio rerio, and here we summarize the experimental studies that help to define a myriad of cell–cell interactions that result in this pattern. We begin by summarizing zebrafish stripe morphology and the general process whereby the adult pattern forms. We then provide a detailed summary of the genetic, cell biology, and ablation studies that inform our knowledge of the biological bases for stripe formation.

Bacteria are highly interactive and possess an extraordinary repertoire of intercellular communication and social behaviors, including quorum sensing (QS). QS has been studied in detail at the molecular level, so mechanistic details are well understood in many species and are often involved in virulence. The use of different animal host models has demonstrated QS-dependent control of virulence determinants and virulence in several human pathogenic bacteria. QS also controls virulence in several plant pathogenic species. Despite the role QS plays in virulence during animal and plant laboratory-engineered infections, QS mutants are frequently isolated from natural infections, demonstrating that the function of QS during infection and its role in pathogenesis remain poorly understood and are fruitful areas for future research. We discuss the role of QS during infection in various organisms and highlight approaches to better understand QS during human infection. This is an important consideration in an era of growing antimicrobial resistance, when we are looking for new ways to target bacterial infections.

Cell-cell signaling in bacteria allows a synchronized and coordinated behavior of a microbial community. LuxR solos represent a subfamily of proteins in proteobacteria which most commonly detect and respond to signals produced exogenously by other microbes or eukaryotic hosts. Here, we report that a plant-beneficial bacterial endophyte belonging to the novel genus of Kosakonia possesses two LuxR solos; one is involved in the detection of exogenous N-acyl homoserine lactone quorum sensing signals and the other in detecting a compound(s) produced by the host plant. These two Kosakonia LuxR solos are therefore most likely involved in interspecies and interkingdom signaling., Endophytes are microorganisms that live inside plants and are often beneficial for the host. Kosakonia is a novel bacterial genus that includes several species that are diazotrophic and plant associated. This study revealed two quorum sensing-related LuxR solos, designated LoxR and PsrR, in the plant endophyte Kosakonia sp. strain KO348. LoxR modeling and biochemical studies demonstrated that LoxR binds N-acyl homoserine lactones (AHLs) in a promiscuous way. PsrR, on the other hand, belongs to the subfamily of plant-associated-bacterium (PAB) LuxR solos that respond to plant compounds. Target promoter studies as well as modeling and phylogenetic comparisons suggest that PAB LuxR solos are likely to respond to different plant compounds. Finally, LoxR is involved in the regulation of T6SS and PsrR plays a role in root endosphere colonization. IMPORTANCE Cell-cell signaling in bacteria allows a synchronized and coordinated behavior of a microbial community. LuxR solos represent a subfamily of proteins in proteobacteria which most commonly detect and respond to signals produced exogenously by other microbes or eukaryotic hosts. Here, we report that a plant-beneficial bacterial endophyte belonging to the novel genus of Kosakonia possesses two LuxR solos; one is involved in the detection of exogenous N-acyl homoserine lactone quorum sensing signals and the other in detecting a compound(s) produced by the host plant. These two Kosakonia LuxR solos are therefore most likely involved in interspecies and interkingdom signaling.

The water-borne bacterium Legionella pneumophila replicates in environmental protozoa and upon inhalation destroys alveolar macrophages, thus causing a potentially fatal pneumonia termed 'Legionnaires' disease'. L. pneumophila employs the Legionella quorum sensing (Lqs) system to control its life cycle, pathogen-host cell interactions, motility and natural competence. Signaling through the Lqs system occurs through the α-hydroxyketone compound Legionella autoinducer-1 (LAI-1) and converges on the prototypic response regulator LqsR, which dimerizes upon phosphorylation of the conserved aspartate, D108 . In this study, we determine the high-resolution structure of monomeric LqsR. The structure reveals a receiver domain adopting a canonical (βα)5 fold, which is connected through an additional sixth helix and an extended α5-helix to a novel output domain. The two domains delineate a mainly positively charged groove, and the output domain adopts a five-stranded antiparallel β-sheet fold similar to nucleotide-binding proteins. Structure-based mutagenesis identified amino acids critical for LqsR phosphorylation and dimerization. Upon phosphorylation, the LqsRD172A and LqsRD302N/E303Q mutant proteins dimerized even more readily than wild-type LqsR, and no evidence for semi-phosphorylated heterodimers was obtained. Taken together, the high-resolution structure of LqsR reveals functionally relevant amino acid residues implicated in signal transduction of the prototypic response regulator.

Stomata are structures on the surfaces of most land plants that are required for gas exchange between plants and their environment. In Arabidopsis thaliana, stomata comprise two kidney bean-shaped epidermal guard cells that flank a central pore overlying a cavity in the mesophyll. These guard cells can adjust their shape to occlude or facilitate access to this pore, and in so doing regulate the release of water vapor and oxygen from the plant, in exchange for the intake of carbon dioxide from the atmosphere. Stomatal guard cells are the end product of a specialized lineage whose cell divisions and fate transitions ensure both the production and pattern of cells in aerial epidermal tissues. The stomatal lineage is dynamic and flexible, altering stomatal production in response to environmental change. As such, the stomatal lineage is an excellent system to study how flexible developmental transitions are regulated in plants. In this Cell Science at a Glance article and accompanying poster, we will summarize current knowledge of the divisions and fate decisions during stomatal development, discussing the role of transcriptional regulators, cell–cell signaling and polarity proteins. We will highlight recent work that links the core regulators to systemic or environmental information and provide an evolutionary perspective on stomata lineage regulators in plants.

Many microbial communities contain organized patterns of cell types, yet relatively little is known about the mechanism or function of this organization. In colonies of the budding yeast Saccharomyces cerevisiae, sporulation occurs in a highly organized pattern, with a top layer of sporulating cells sharply separated from an underlying layer of nonsporulating cells. A mutant screen identified the Mpk1 and Bck1 kinases of the cell-wall integrity (CWI) pathway as specifically required for sporulation in colonies. The CWI pathway was induced as colonies matured, and a target of this pathway, the Rlm1 transcription factor, was activated specifically in the nonsporulating cell layer, here termed feeder cells. Rlm1 stimulates permeabilization of feeder cells and promotes sporulation in an overlying cell layer through a cell-nonautonomous mechanism. The relative fraction of the colony apportioned to feeder cells depends on nutrient environment, potentially buffering sexual reproduction against suboptimal environments.

Cell-cell communication mediated by peptide pheromones (cCF10 [CF]) is essential for high-frequency plasmid transfer in vitro in Enterococcus faecalis. To examine the role of pheromone signaling in vivo, we established either a CF-producing (CF+) recipient or a recipient producing a biologically inactive variant of CF (CF− recipient) in a germfree mouse model 3 days before donor inoculation and determined transfer frequencies of the pheromone-inducible plasmid pCF10. Plasmid transfer was detected in the upper and middle sections of the intestinal tract 5 h after donor inoculation and was highly efficient in the absence of antibiotic selection. The transconjugant/donor ratio reached a maximum level approaching 1 on day 4 in the upper intestinal tract. Plasmid transfer was significantly lower with the CF− recipient. While rescue of the CF− mating defect by coculture with CF+ recipients is easily accomplished in vitro, no extracellular complementation occurred in vivo. This suggests that most pheromone signaling in the gut occurs between recipient and donor cells in very close proximity. Plasmid-bearing cells (donors plus transconjugants) steadily increased in the population from 0.1% after donor inoculation to about 10% at the conclusion of the experiments. This suggests a selective advantage of pCF10 carriage distinct from antibiotic resistance or bacteriocin production. Our results demonstrate that pheromone signaling is required for efficient pCF10 transfer in vivo. In the absence of CF+ recipients, a low level of transfer to CF− recipients occurred in the gut. This may result from low-level host-mediated induction of the donors in the gastrointestinal (GI) tract, similar to that previously observed in serum., IMPORTANCE Horizontal gene transfer is a major factor in the biology of Enterococcus faecalis, an important nosocomial pathogen. Previous studies showing efficient conjugative plasmid transfer in the gastrointestinal (GI) tracts of experimental animals did not examine how the enterococcal sex pheromone response impacts the efficiency of transfer. Our study demonstrates for the first time pheromone-enhanced, high-frequency plasmid transfer of E. faecalis plasmid pCF10 in a mouse model in the absence of antibiotic or bacteriocin selection. Pheromone production by recipients dramatically increased plasmid transfer in germfree mice colonized initially with recipients, followed by donors. The presence of a coresident community of common gut microbes did not significantly reduce in vivo plasmid transfer between enterococcal donors and recipients. In mice colonized with enterococcal recipients, we detected plasmid transfer in the intestinal tract within 5 h of addition of donors, before transconjugants could be cultured from feces. Surprisingly, pCF10 carriage provided a competitive fitness advantage unrelated to antibiotic resistance or bacteriocin production.

Conserved morphogenetic signaling proteins disperse across tissues to generate signal and signaling gradients, which in turn are considered to assign positional coordinates to the recipient cells. Recent imaging studies in Drosophila model have provided evidence for a "direct-delivery" mechanism of signal dispersion that is mediated by specialized actin-rich signaling filopodia, named cytonemes. Cytonemes establish contact between the signal-producing and target cells to directly exchange and transport the morphogenetic proteins. Although an increasing amount of evidence supports the critical role of these specialized signaling structures, imaging these highly dynamic 200 nm-thin structures in the complex three-dimensional contour of living tissues is challenging. Here, we describe the imaging methods that we optimized for studying cytonemes in Drosophila embryos.

Stigmatella aurantiaca fruiting body development depends on cell-cell interactions. One type of the signaling molecule stigmolone isolated from S. aurantiaca cells acts to help cells to stay together in the aggregation phase. Another gene product involved in intercellular signaling in S. aurantiaca is the csgA homolog of Myxococcus xanthus. In close relative M. xanthus C signal the product of the csgA gene is required for rippling, aggregation and sporulation. Isolation of homologous gene in S. aurantiaca implicates a probable role of CsgA in intercellular communication. Inactivation of the gene by insertion mutagenesis caused alterations in S. aurantiaca fruiting. The motility behavior of the cells during development was changed as well as their ability to stay more closely together in the early stages of development. Inactivation of the csgA gene completely abolished rippling of the cells. This indicates the crucial role of the CsgA protein in regulating this rhythmic behavior.

Cell-cell signaling is subject to variability in the extracellular volume, cell number, and dilution that potentially increase uncertainty in the absolute concentrations of the extracellular signaling molecules. To direct cell aggregation, the social amoebae Dictyostelium discoideum collectively give rise to oscillations and waves of cyclic adenosine 3',5'-monophosphate (cAMP) under a wide range of cell density. To date, the systems-level mechanism underlying the robustness is unclear. By using quantitative live-cell imaging, here we show that the magnitude of the cAMP relay response of individual cells is determined by fold change in the extracellular cAMP concentrations. The range of cell density and exogenous cAMP concentrations that support oscillations at the population level agrees well with conditions that support a large fold-change-dependent response at the single-cell level. Mathematical analysis suggests that invariance of the oscillations to density transformation is a natural outcome of combining secrete-and-sense systems with a fold-change detection mechanism.

Many Proteobacteria use N -acyl-homoserine lactone (acyl-HSL) quorum sensing to control specific genes. Acyl-HSL synthesis requires unique enzymes that use S -adenosyl methionine as an acyl acceptor and amino acid donor. We developed and executed an enzyme-coupled high-throughput cell-free screen to discover acyl-HSL synthase inhibitors. The three strongest inhibitors were equally active against two different acyl-HSL synthases: Burkholderia mallei BmaI1 and Yersinia pestis YspI. Two of these inhibitors showed activity in whole cells. The most potent compound behaves as a noncompetitive inhibitor with a K i of 0.7 µM and showed activity in a cell-based assay. Quorum-sensing signal synthesis inhibitors will be useful in attempts to understand acyl-HSL synthase catalysis and as a tool in studies of quorum-sensing control of gene expression. Because acyl-HSL quorum-sensing controls virulence of some bacterial pathogens, anti–quorum-sensing chemicals have been sought as potential therapeutic agents. Our screen and identification of acyl-HSL synthase inhibitors serve as a basis for efforts to target quorum-sensing signal synthesis as an antivirulence approach.

Many lophotrochozoans (i.e., molluscs, annelids, nemerteans, and polyclad flatworms) display a well-conserved early developmental program called spiral cleavage that contrasts with the high diversity of adult body forms present in this group. Due to this stereotypical development, each cell can be uniquely identified and its lineage history known following intracellular injection of lineage tracers. Cell deletion experiments performed mainly in molluscs have demonstrated that one or two cells associated with the endomesodermal lineage represent an embryonic organizer of subsequent development and are causally involved in cell fate and body patterning. Utilizing the published fate map of the spiral-cleaving annelid Capitella teleta, we used infrared laser cell deletions to dissect the role of individual cells on the patterning of the larval body. Thirteen uniquely identifiable individual blastomeres and two double cell combination deletions were studied to assess larval phenotypes by scoring multiple morphological structures and cell type-specific molecular markers differentially expressed along the antero-posterior and dorso-ventral axes. Surprisingly, our results show that in C. teleta, the cellular identity of the "organizing cell" and the timing of the organizing activity are different from that of other spiralians. retain--In C. teleta, the ectodermal primary somatoblast, 2d, is the key cell responsible for organizing activity during early embryonic development, and is necessary for bilateral symmetry and dorso-ventral axis organization of the head as well as neural, foregut and mesoderm tissue formation. Furthermore, we show that the ERK/MAPK signaling pathway does not appear to be involved in organizing activity in retain--C. teleta. This contrasts with data from molluscs and the molecular mechanism suggested for another polychaete, Hydroides elegans, highlighting likely molecular level variation among spiralian embryos. These results reinforce the idea that an embryonic organizing activity is present across spiralians. Our data also emphasize the developmental variation within lophotrochozoans, and may ultimately provide insight into the role of developmental processes in the evolution of diverse body forms in metazoans.

The Streptococcus Invasion Locus (Sil) was first described in Streptococcus pyogenes and Streptococcus pneumoniae, where it has been implicated in virulence. The two-component peptide signaling system consists of the SilA response regulator and SilB histidine kinase along with the SilCR signaling peptide and SilD/E export/processing proteins. The presence of an associated bacteriocin region suggests this system may play a role in competitive interactions with other microbes. Comparative analysis of 42 Streptococcus Anginosus/Milleri Group (SAG) genomes reveals this to be a hot spot for genomic variability. A cluster of bacteriocin/immunity genes is found adjacent to the sil system in most SAG isolates (typically 6-10 per strain). In addition, there were two distinct SilCR peptides identified in this group, denoted here as SilCRSAG-A and SilCRSAG-B, with corresponding alleles in silB. Our analysis of the 42 sil loci showed that SilCRSAG-A is only found in Streptococcus intermedius while all three species can carry SilCRSAG-B. In S. intermedius B196, a putative SilA operator is located upstream of bacteriocin gene clusters, implicating the sil system in regulation of microbe-microbe interactions at mucosal surfaces where the group resides. We demonstrate that S. intermedius B196 responds to its cognate SilCRSAG-A, and, less effectively, to SilCRSAG-B released by other Anginosus group members, to produce putative bacteriocins and inhibit the growth of a sensitive strain of S. constellatus.

Bacteria synthesize and sense low molecular weight signaling molecules, termed autoinducers, to measure their population density and community complexity. One class of autoinducers, the α-hydroxyketones (AHKs), is produced and detected by the water-borne opportunistic pathogens Legionella pneumophila and Vibrio cholerae, which cause Legionnaires' disease and cholera, respectively. The "Legionella quorum sensing" (lqs) or "cholera quorum sensing" (cqs) genes encode enzymes that produce and sense the AHK molecules "Legionella autoinducer-1" (LAI-1; 3-hydroxypentadecane-4-one) or cholera autoinducer-1 (CAI-1; 3-hydroxytridecane-4-one). AHK signaling regulates the virulence of L. pneumophila and V. cholerae, pathogen-host cell interactions, formation of biofilms or extracellular filaments, expression of a genomic "fitness island" and competence. Here, we outline the processes, wherein AHK signaling plays a role, and review recent insights into the function of proteins encoded by the lqs and cqs gene clusters. To this end, we will focus on the autoinducer synthases catalysing the biosynthesis of AHKs, on the cognate trans-membrane sensor kinases detecting the signals, and on components of the down-stream phosphorelay cascade that promote the transmission and integration of signaling events regulating gene expression.

Platelet-activating factor (PAF) is a signaling phospholipid with a significant physiological role in multicellular and unicellular organisms, including fermentative organisms such as yeast. Zymomonas mobilis is an ethanologenic α-proteobacterium currently studied for bioethanol production. In order to examine the presence of PAF and/or PAF inhibitors in Z. mobilis, a new one-step high performance liquid chromatography (HPLC) separation procedure of total lipids was performed, using a C8 reversed-phase semi-preparative column. According to this method and to bioassays based on washed rabbit platelet aggregation, two lipid molecules with PAF-like activity and same retention times as those of standard PAF were detected; electron-spray ionization MS and MS/MS analysis revealed that they share similar structure with 16:0 and 18:0 PAF. Furthermore, other lipids extracted from Z. mobilis were found to exhibit a potent anti-PAF activity. Enzyme activities indicative of key PAF biosynthetic enzymes, such as dithiothreitol-insensitive cholinephosphotransferase (PAF-CPT) and lyso-PAF acetyltransferase were detected in Z. mobilis homogenates. As for PAF degradation, activity similar to that of PAF acetylhydrolase was also discovered. Overall, the presence of PAF, PAF-specific inhibitors, and enzyme activities relating to PAF metabolism, suggests that PAF may play an intrinsic role in this biotechnological organism. Practical applications:Z. mobilis is a platform microorganism for bioethanol production and a potential source of high-value chemicals of interest to the food and healthcare industries. Further investigation of PAF's role is bound to affect applications involving this and other biotechnological organisms. The finding that Z. mobilis lipids exhibit potent anti-PAF activity opens up prospects for their identification, overproduction and pharmaceutical use. The presented HPLC method for lipid fractionation accomplishes a one-step separation of lipids from dense samples, which may be successfully employed to other lipid-rich sources such as blood.

Many bacteria grow attached to a surface as biofilms. Several factors dictate biofilm formation, including responses by the colonizing bacteria to their environment. Here we review how bacteria use cell-cell signaling (also called quorum sensing) and motility during biofilm formation. Specifically, we describe quorum sensing and surface motility exhibited by the bacterium Pseudomonas aeruginosa, a ubiquitous environmental organism that acts as an opportunistic human pathogen in immunocompromised individuals. P. aeruginosa uses acyl-homoserine lactone signals during quorum sensing to synchronize gene expression important to the production of polysaccharides, rhamnolipid, and other virulence factors. Surface motility affects the assembly and architecture of biofilms, and some aspects of motility are also influenced by quorum sensing. While some genes and their function are specific to P. aeruginosa, many aspects of biofilm development can be used as a model system to understand how bacteria differentially colonize surfaces.

Cyclic-di-GMP [bis-(3′-5′)-cyclic diguanosine monophosphate] controls a wide range of functions in eubacteria, yet little is known about the underlying regulatory mechanisms. In the plant pathogen Xanthomonas campestris, expression of a subset of virulence genes is regulated by c-di-GMP and also by the CAP (catabolite activation protein)-like protein XcCLP, a global regulator in the CRP/FNR superfamily. Here, we report structural and functional insights into the interplay between XcCLP and c-di-GMP in regulation of gene expression. XcCLP bound target promoter DNA with submicromolar affinity in the absence of any ligand. This DNA-binding capability was abrogated by c-di-GMP, which bound to XcCLP with micromolar affinity. The crystal structure of XcCLP showed that the protein adopted an intrinsically active conformation for DNA binding. Alteration of residues of XcCLP implicated in c-di-GMP binding through modeling studies caused a substantial reduction in binding affinity for the nucleotide and rendered DNA binding by these variant proteins insensitive to inhibition by c-di-GMP. Together, these findings reveal the structural mechanism behind a novel class of c-di-GMP effector proteins in the CRP/FNR superfamily and indicate that XcCLP regulates bacterial virulence gene expression in a manner negatively controlled by the c-di-GMP concentrations.

Multicellularity appeared early and repeatedly in life's history; its instantiations presumably required the confluence of environmental, ecological, and genetic factors. Comparisons of several independently evolved pairs of multicellular and unicellular relatives indicate that transitions to multicellularity are typically associated with increases in the numbers of genes involved in cell differentiation, cell-cell communication, and adhesion. Further examination of the DNA record suggests that these increases in gene complexity are the product of evolutionary innovation, tinkering, and expansion of genetic material. Arguably, the most decisive multicellular transition was the emergence of animals. Decades of developmental work have demarcated the genetic toolkit for animal multicellularity, a select set of a few hundred genes from a few dozen gene families involved in adhesion, communication, and differentiation. Examination of the DNA records of the earliest-branching animal phyla and their closest protist relatives has begun to shed light on the origins and assembly of this toolkit. Emerging data favor a model of gradual assembly, with components originating and diversifying at different time points prior to or shortly after the origin of animals.

The Agrobacterium tumefaciens oncogenic Ti plasmids replicate and segregate to daughter cells via repABC cassettes, in which repA and repB are plasmid partitioning genes and repC encodes the replication initiator protein. repABC cassettes are encountered in a growing number of plasmids and chromosomes of the alpha-proteobacteria, and findings from particular representatives of agrobacteria, rhizobia and Paracoccus have began to shed light on their structure and functions. Amongst repABC replicons, Ti plasmids and particularly the octopine-type Ti have recently stood as model in regulation of repABC basal expression, which acts in plasmid copy number control, but also appear to undergo pronounced up-regulation of repABC, upon interbacterial and host-bacterial signaling. The last results in considerable Ti copy number increase and collective elevation of Ti gene expression. Inhibition of the Ti repABC is in turn conferred by a plant defense compound, which primarily affects Agrobacterium virulence and interferes with cell-density perception. Altogether, the above suggest that the entire Ti gene pool is subjected to the bacterium-eukaryote signaling network, a phenomenon quite unprecedented for replicons thought of as stringently controlled. It remains to be seen whether similar copy number variations characterize related replicons or if they are of even broader significance in plasmid biology.

Cell-to-cell signaling peptides play important roles in neurotransmission, neuromodulation, and hormonal signaling. Significant progress has been achieved in qualitative investigations of signaling peptides in the nervous system using single cell matrix-assisted laser desorption/ionization mass spectrometry. However, quantitative information about signaling peptides is difficult to obtain with this approach because only small amounts of analytes are available for analysis. Here we describe several methods for quantitative microanalysis of peptides in individual Aplysia californica neurons and small pieces of tissue. Stable isotope labeling with d0- and d4-succinic anhydride and iTRAQ reagents has been successfully adopted for relative quantitation of nanoliter volume samples containing the Aplysia insulin C beta peptide. Comparative analysis of the C beta peptide release site, the upper labial nerve, and its synthesis location, the F- and C-clusters, shows that the release site possesses almost three times more of this compound. The method of standard addition permits absolute quantitation of the physiologically active neuropeptide cerebrin from small structures, including nerves and neuronal clusters, in the femtomole range with a limit of detection of 19 fmol. The simplicity of these methods and the commercial availability of the reagents allow quantitative measurements from a variety of small-volume biological samples.

Cell-to-cell communications in bacteria mediated by small diffusible molecules termed as autoinducers (AI) are known to influence gene expression and pathogenicity. Oligopeptides and N-acylhomoserine lactones (AHL) are major AI molecules involved in intra-specific communication in Gram-positive and Gram-negative bacteria respectively, whereas boronated-diester molecules (AI-2) are involved in inter-specific communication among both Gram-positive and Gram-negative bacteria. Naturally occurring furocoumarins from grapefruit showed > 95% inhibition of AI-1 and AI-2 activities based on the Vibrio harveyi based autoinducer bioassay. Grapefruit juice and furocoumarins also inhibited biofilm formation by Escherichia coli O157:H7, Salmonella typhimurium and Pseudomonas aeruginosa. These results suggest that grape fruit juice and furocoumarins could serve as a source to develop bacterial intervention strategies targeting microbial cell signaling processes.

Cell–cell signaling in Xylella fastidiosa , a xylem-colonizing plant pathogenic bacterium, mediated by a fatty acid D iffusible S ignaling F actor (DSF), is required to colonize insect vectors and to suppress virulence to grape. Here, we show that a hybrid two-component regulatory protein RpfC is involved in negative regulation of DSF synthesis by RpfF in X. fastidiosa. X. fastidiosa rpfC mutants hyperexpress rpfF and overproduce DSF and are deficient in virulence and movement in the xylem vessels of grape. The expression of the genes encoding the adhesins FimA, HxfA, and HxfB is much higher in rpfC mutants, which also exhibit a hyperattachment phenotype in culture that is associated with their inability to migrate in xylem vessels and cause disease. rpfF mutants deficient in DSF production have the opposite phenotypes for all of these traits. RpfC is also involved in the regulation of other signaling components including rpfG , rpfB , a GGDEF domain protein that may be involved in intracellular signaling by modulating the levels of cyclic-di-GMP, and the virulence factors tolC and pglA required for disease. rpfC mutants are able to colonize the mouthparts of insect vectors and wild-type strains but are not transmitted as efficiently to new host plants, apparently because of their high levels of adhesiveness. Because of the conflicting contributions of adhesiveness and other traits to movement within plants and vectoring to new host plants, X. fastidiosa apparently coordinates these traits in a population-size-dependent fashion involving accumulation of DSF.

The genome of Stenotrophomonas maltophilia encodes a cell-cell signaling system that is highly related to the diffusible signal factor (DSF)-dependent system of the phytopathogen Xanthomonas campestris . Here we show that in S. maltophilia , DSF signaling controls factors contributing to the virulence and antibiotic resistance of this important nosocomial pathogen.

Anther development in flowering plants involves the formation of several cell types, including the tapetal and pollen mother cells. The use of genetic and molecular tools has led to the identification and characterization of genes that are critical for normal cell division and differentiation in Arabidopsis early anther development. We review here several recent studies on these genes, including the demonstration that the putative receptor protein kinases BAM1 and BAM2 together play essential roles in the control of early cell division and differentiation. In addition, we discuss the hypothesis that BAM1/2 may form a positive-negative feedback regulatory loop with a previously identified key regulator, SPOROCYTELESS (also called NOZZLE), to control the balance between sporogenous and somatic cell types in the anther. Furthermore, we summarize the isolation and functional analysis of the DYSFUNCTIONAL TAPETUM1 (DYT1) gene in promoting proper tapetal cell differentiation. Our finding that DYT1 encodes a putative transcription factor of the bHLH family, as well as relevant expression analyses, strongly supports a model that DYT1 serves as a critical link between upstream factors and downstream target genes that are critical for normal tapetum development and function. These studies, together with other recently published works, indicate that cell-cell communication and transcriptional control are key processes essential for cell fate specification in anther development.

The synthesis of virulence factors in the plant pathogen Xanthomonas campestris pathovar campestris is regulated by cell-cell signaling mediated by a diffusible signal factor (DSF), and by the RpfC/RpfG two-component regulatory system. Recent findings have indicated that the perception of the DSF signal requires the RpfC sensor and is linked to the degradation of the intracellular second messenger cyclic di-GMP by the HD-GYP domain regulator RpfG.

The content, synthesis and transport of D-aspartate (D-Asp) in the CNS of Aplysia californica is investigated using capillary electrophoresis (CE) with both laser-induced fluorescence and radionuclide detection. Millimolar concentrations of D-Asp are found in various regions of the CNS. In the cerebral ganglion, three adjacent neuronal clusters have reproducibly different D-Asp levels; for example, in the F- and C-clusters, up to 85% of the free Asp is present in the D-form. Heterogeneous distribution of D-Asp is also found in the individual identified neurons tested, including the optical ganglion top-layer neurons, metacerebral cells, R2 neurons, and F-, C- and G-cluster neurons. The F-cluster neurons have the highest percentage of D-Asp (approximately 58% of the total Asp), whereas the lowest value of approximately 8% is found in R2 neurons. In pulse-chase experiments with radiolabeled D-Asp, followed by CE with radionuclide detection, the synthesis of D-Asp from L-aspartate (L-Asp) is confirmed. Is D-Asp in the soma, or is it transported to distantly located release sites? D-Asp is clearly detected in the major nerves of A. californica, including the pleuroabdominal and cerebrobuccal connectives and the anterior tentacular nerves, suggesting it is transported long distances. In addition, both D-Asp and L-Asp are transported in the pleuroabdominal connectives in a colchicine-dependent manner, whereas several other amino acids are not. Finally, d-Asp produces electrophysiological effects similar to those induced by L-Asp. These data are consistent with an active role for D-Asp in cell-to-cell communication.

Xylella fastidiosa , which causes Pierce's disease of grapevine and other important plant diseases, is a xylem-limited bacterium that depends on insect vectors for transmission. Although many studies have addressed disease symptom development and transmission of the pathogen by vectors, little is known about the bacterial mechanisms driving these processes. Recently available X. fastidiosa genomic sequences and molecular tools have provided new routes for investigation. Here, we show that a diffusible signal molecule is required for biofilm formation in the vector and for vector transmission to plants. We constructed strains of X. fastidiosa mutated in the rpfF gene and determined that they are unable to produce the signal activity. In addition, rpfF mutants are more virulent than the wild type when mechanically inoculated into plants. This signal therefore directs interaction of X. fastidiosa with both its insect vector and plant host. Interestingly, rpfF mutants can still form in planta biofilms, which differ architecturally from biofilms in insects, suggesting that biofilm architecture, rather than a passive response to the environment, is actively determined by X. fastidiosa gene expression. This article reports a cell-cell signaling requirement for vector transmission. Identification of the genes regulated by rpfF should elucidate bacterial factors involved in transmission and biofilm formation in the insect.

In a process called quorum sensing, bacteria monitor their population density via extracellular signaling molecules and modulate gene expression accordingly. This paper describes a one-dimensional model of a growing Pseudomonas aeruginosa biofilm. Quorum sensing has been included in the model by the addition of equations describing the production, degradation, and diffusion of acyl-homoserine lactones in the biofilm. In order for quorum sensing to initiate near the substratum, in accordance with experimental observations, model results suggest that cells in oxygen-deficient regions of the biofilm must still be synthesizing the signal compound. This result highlights the importance of careful study of the relationship between metabolic activity of the bacterium and signal synthesis.

Background: Starving amoebae of Dictyostelium discoideum communicate by relaying extracellular cAMP signals, which direct chemotactic movement, resulting in the aggregation of thousands of cells into multicellular aggregates. Both cAMP relay and chemotaxis require the activation of PI3 kinase signaling. The spatiotemporal dynamics of PI3 kinase signaling can be followed in individual cells via the cAMP-induced membrane recruitment of a GFP-tagged PH domain-containing protein, CRAC, which is required for the activation of adenylylcyclase. Results: We show that polarized periodic CRAC-GFP translocation occurs during the aggregation and mound stages of development in response to periodic cAMP signals. The duration of CRAC translocation to the membrane is determined by the duration of the rising phase of the cAMP signal. The system shows rapid adaptation and responds to the rate of change of the extracellular cAMP concentration. When the cells are in close contact, it takes 10 s for the signal to propagate from one cell to the next. In slugs, all cells show a permanent polarized PI3 kinase signaling in their leading edge, which is dependent on cell-cell contact. Conclusions: Measuring the redistribution of GFP-tagged CRAC has enabled us to study the dynamics of PI3 kinase-mediated cell-cell communication at the individual cell level in the multicellular stages of Dictyostelium development. This approach should also be useful to study the interactions between cell-cell signaling, cell polarization, and movement in the development of other organisms.

It has been 100 years since Agrobacterium tumefaciens was demonstrated to cause crown gall tumors at plant wound sites. This chapter first presents background information relevant to quorum sensing in A. tumefaciens and then focuses on our current knowledge of the molecular biology of the TraR-TraI system. Regulation of TraR activity is complex and occurs at the levels of transcription, protein folding, resistance to proteolysis, and the formation of quaternary complexes with other TraR subunits or with two different antiactivators of TraR. Control of traR expression by opines therefore has evolved independently in these two types of Ti plasmids. The genes of the arc operon and occ operon are not similar, except for traR. LysR and AccR are also dissimilar, as OccR is a LysR-type transcriptional activator that binds to promoter DNA both in the presence and absence of the inducing signal. Overproduction of TraR can fully overcome inhibition, suggesting that TraM acts by making stoichiometric contacts with TraR. Direct interactions were confirmed by yeast two-hybrid assays and by far Western immunoblots. The author recently used gene arrays to profile the TraR transcriptome and found that all induced genes were Ti plasmid-encoded. These genes include the tra and trb genes, which are involved in conjugal transfer; the rep genes, which are required for vegetative replication and plasmid partitioning into daughter cells; and traM.

The Actinobacteria, including the genus Streptomyces, constitute, on average, about 13% of soil bacterial communities, making them a dominant form of life on Earth. Research has shown that many wild isolates of Streptomyces spp. are capable of intercellular communication, and in one instance, the extracellular compound is a desferrioxamine siderophore. This apparent diversity of intercellular signaling mechanisms suggests that the streptomycetes rely extensively on cell-cell communication to coordinate growth with the production of secondary metabolites and sporulation. As morphological and physiological differentiation can be visually monitored, extracellular rescue of mutant phenotypes can be quite striking. However, the streptomycetes present challenges that need to be considered when investigating cell-cell signaling. The best-understood signaling systems exhibited by the streptomycetes are those mediated by the γ-butyrolactones. In certain species, including Streptomycetes lavendulae and S. coelicolor, the roles of the γ-butyrolactones appear to be restricted to secondary metabolism. To date, three classes of secreted, hydrophobic molecules have been shown to be involved in aerial hyphae formation. In S. coelicolor, these include the chaplins and a small lanthionine-containing peptide, SapB, which has orthologues in S. griseus, S. avermitilis, and S. scabies, as well as a functional homologue, SapT, produced by S. tendae. The pamamycins, a group of macrolide antibiotics produced by S. alboniger, demonstrate that a single molecule may serve more than one distinct function. At subinhibitory concentrations, they stimulate the formation of aerial hyphae, while at higher concentrations they inhibit the growth of nonproducing streptomycetes and other gram-positive bacteria.

▪ Abstract The myxobacterium Myxococcus xanthus has a life cycle that is dominated by social behavior. During vegetative growth, cells prey on other bacteria in large groups that have been likened to wolf packs. When faced with starvation, cells form a macroscopic fruiting body containing thousands of spores. The social systems that guide fruiting body development have been examined through the isolation of conditional developmental mutants that can be stimulated to develop in the presence of wild-type cells. Extracellular complementation is due to the transfer of soluble and cell contact-dependent intercellular signals. This review describes the current state of knowledge concerning cell-cell signaling during development.

▪ Abstract Studies of about 20 maternally expressed genes are providing an understanding of mechanisms of patterning and cell-fate determination in the early Caenorhabditis elegans embryo. The analyses have revealed that fates of the early blastomeres are specified by a combination of intrinsically asymmetric cell divisions and two types of cell-cell interactions: inductions and polarizing interactions. In this review we summarize the current level of understanding of the molecular mechanisms underlying these processes in the specification of cell fates in the pregastrulation embryo.

Specific proteins and peptides, as well as cAMP, are used as intercellular signals in Dictyostelium. Our understanding of the signal transduction pathways activated by these signals has been expanded by inclusion of newly characterized proteins. cAMP-dependent protein kinase (PKA) and its associated phosphodiesterase, RegA, play multiple roles in these pathways.

Lysobacter enzymogenes is a ubiquitous environmental bacterium that is emerging as a potentially novel biological control agent and a new source of bioactive secondary metabolites, such as the heat-stable antifungal factor (HSAF) and photoprotective polyene pigments. Thus far, the regulatory mechanism(s) for biosynthesis of these bioactive secondary metabolites remains largely unknown in L. enzymogenes . In the present study, the diffusible signal factor (DSF) and diffusible factor (DF)-mediated cell-cell signaling systems were identified for the first time from L. enzymogenes . The results show that both Rpf/DSF and DF signaling systems played critical roles in modulating HSAF biosynthesis in L. enzymogenes . Rpf/DSF signaling and DF signaling played negative and positive effects in polyene pigment production, respectively, with DF playing a more important role in regulating this phenotype. Interestingly, only Rpf/DSF, but not the DF signaling system, regulated colony morphology of L. enzymgenes . Both Rpf/DSF and DF signaling systems were involved in the modulation of expression of genes with diverse functions in L. enzymogenes , and their own regulons exhibited only a few loci that were regulated by both systems. These findings unveil for the first time new roles of the Rpf/DSF and DF signaling systems in secondary metabolite biosynthesis of L. enzymogenes .

Eal is an EAL domain protein in Xylella fastidiosa homologous to one involved in resistance to tobramycin in Pseudomonas aeruginosa . EAL and HD-GYP domain proteins are implicated in the hydrolysis of the secondary messenger bis-(3′-5′)-cyclic dimeric GMP (cyclic di-GMP). Cell density-dependent communication mediated by a Diffusible Signal Factor (DSF) also modulates cyclic di-GMP levels in X. fastidiosa , thereby controlling the expression of virulence genes and genes involved in insect transmission. The possible linkage of Eal to both extrinsic factors such as antibiotics and intrinsic factors such as quorum sensing, and whether both affect virulence, was thus addressed. Expression of eal was induced by subinhibitory concentrations of tobramycin, and an eal deletion mutant was more susceptible to this antibiotic than the wild-type strain and exhibited phenotypes similar to those of an rpfF deletion mutant blocked in DSF production, such as hypermotility, reduced biofilm formation, and hypervirulence to grape. Consistent with that, the rpfF mutant was more susceptible than the wild-type strain to tobramycin. Therefore, we propose that cell-cell communication and antibiotic stress can apparently lead to similar modulations of cyclic di-GMP in X. fastidiosa , resulting in similar phenotypes. However, the effect of cell density is dominant compared to that of antibiotic stress, since eal is suppressed by RpfF, which may prevent inappropriate behavioral changes in response to antibiotic stress when DSF accumulates.

Transcription in the mother cell at early stages of sporulation in Bacillus subtilis is controlled by sigma E, a sigma factor that is synthesized in the predivisional cell as an inactive larger precursor, pro-sigma E. Activation of sigma E depends on sigma F, the factor that governs transcription in the forespore. Genetic experiments have indicated that transduction of the activation signal from the forespore to the mother cell requires the products of some genes belonging to the sigma F-controlled regulon. We have identified and characterized a sigma F-dependent gene, csfX, encoding a protein necessary and sufficient for triggering processing of pro-sigma E. The CsfX protein contains a typical amino-terminal signal sequence suggesting that, although synthesized in the forespore, it may act across the septum to activate the membrane-bound enzyme that is responsible for pro-sigma E processing in the mother cell.

Myxobacteria exhibit complex social traits during which large populations of cells coordinate their behaviors. An iconic example is their response to starvation: thousands of cells move by gliding motility to build a fruiting body in which vegetative cells differentiate into spores. Here we review mechanisms that the model species Myxococcus xanthus uses for cellecell interactions, with a focus on developmental signaling and social gliding motility. We also discuss a newly discovered cellecell interaction whereby myxobacteria exchange their outer membrane (OM) proteins and lipids. The mechanism of OM transfer requires physical contact between aligned cells on a hard surface and is apparently mediated by OM fusion. The TraA and TraB proteins are required in both donor and recipient cells for transfer, suggesting bidirectional exchange, and TraA is thought to serve as a cell surface adhesin. OM exchange results in phenotypic changes that can alter gliding motility and development and is proposed to represent a novel microbial interacting platform to coordinate multicellular activities. 2012 Published by Elsevier Masson SAS on behalf of Institut Pasteur.

Animal appendages require a proximodistal (PD) axis, which forms orthogonally from the two main body axes, anteroposterior and dorsoventral. In this review, we discuss recent advances that begin to provide insights into the molecular mechanisms controlling PD axis formation in the Drosophila leg. In this case, two morphogens, Wingless (Wg) and Decapentaplegic (Dpp), initiate a genetic cascade that, together with growth of the leg imaginal disc, establishes the PD axis. The analysis of cis-regulatory modules (CRMs) that control the expression of genes at different positions along the PD axis has been particularly valuable in dissecting this complex process. From these experiments, it appears that only one concentration of Wg and Dpp are required to initiate PD axis formation by inducing the expression of Distal-less (Dll), a homeodomain-encoding gene that is required for leg development. Once Dll is turned on, it activates the medially expressed gene dachshund (dac). Cross-regulation between Dll and dac, together with cell proliferation in the growing leg imaginal disc, results in the formation of a rudimentary PD axis. Wg and Dpp also initiate the expression of ligands for the EGFR pathway, which in turn induces the expression of a series of target genes that pattern the distal-most portion of the leg.

Biofilms of Candida albicans include both yeast cells and hyphae. Prior studies indicated that a zap1 Δ/Δ mutant, defective in zinc regulator Zap1, has increased accumulation of yeast cells in biofilms. This altered yeast-hypha balance may arise from internal regulatory alterations or from an effect on the production of diffusible quorum-sensing (QS) molecules. Here, we develop biosensor reporter strains that express yeast-specific YWP1-RFP or hypha-specific HWP1-RFP , along with a constitutive TDH3-GFP normalization standard. Seeding these biosensor strains into biofilms allows a biological activity assay of the surrounding biofilm milieu. A zap1 Δ/Δ biofilm induces the yeast-specific YWP1-RFP reporter in a wild-type biosensor strain, as determined by both quantitative reverse transcription-PCR (qRT-PCR) gene expression measurements and confocal microscopy. Remediation of the zap1 Δ/Δ zinc uptake defect through zinc transporter gene ZRT2 overexpression reverses induction of the yeast-specific YWP1-RFP reporter. Gas chromatography-mass spectrometry (GC-MS) measurements of known organic QS molecules show that the zap1 Δ/Δ mutant accumulates significantly less farnesol than wild-type or complemented strains and that ZRT2 overexpression does not affect farnesol accumulation. Farnesol is a well-characterized inhibitor of hypha formation; hence, a reduction in farnesol levels in zap1 Δ/Δ biofilms is unexpected. Our findings argue that a Zap1- and zinc-dependent signal affects the yeast-hypha balance and that it is operative in the low-farnesol environment of the zap1 Δ/Δ biofilm. In addition, our results indicate that Zap1 is a positive regulator of farnesol accumulation.

Wild-type strains of the protozoan ciliate Euplotes collected from different locations on the coasts of Antarctica, Tierra del Fuego and the Arctic were taxonomically identified as the morpho-species Euplotes nobilii , based on morphometric and phylogenetic analyses. Subsequent studies of their sexual interactions revealed that mating combinations of Antarctic and Arctic strains form stable pairs of conjugant cells. These conjugant pairs were isolated and shown to complete mutual gene exchange and cross-fertilization. The biological significance of this finding was further substantiated by demonstrating that close homology exists among the three-dimensional structures determined by NMR of the water-borne signaling pheromones that are constitutively secreted into the extracellular space by these interbreeding strains, in which these molecules trigger the switch between the growth stage and the sexual stage of the life cycle. The fact that Antarctic and Arctic E. nobilii populations share the same gene pool and belong to the same biological species provides new support to the biogeographic model of global distribution of eukaryotic microorganisms, which had so far been based exclusively on studies of morphological and phylogenetic taxonomy.

LEAFY ( LFY ) and APETALA1 ( AP1 ) encode unrelated transcription factors that activate overlapping sets of homeotic genes in Arabidopsis flowers. Sector analysis and targeted expression in transgenic plants were used to study whether LFY and AP1 can participate in cell-cell signaling between and within different layers of the floral meristem. LFY signaled equally well from all layers and had substantial long-range action within layers. Nonautonomous action of LFY was accompanied by movement of the protein to adjacent cells, where it directly activated homeotic target genes. In contrast, AP1 had only limited nonautonomous effects, apparently mediated by downstream genes because activation of early target genes by AP1 was cell-autonomous.

Stomata are turgor-driven epidermal valves on the surface of plants that allow for efficient gas and water exchange between the plant and its environment. The Arabidopsis thaliana basic helix-loop-helix (bHLH) protein, MUTE, is a master regulator of stomatal differentiation where it is required for progression through the stomatal lineage and the differentiation of stomata. The genetic control of stomatal spacing across the epidermal surface is variable in different organs. For instance, a distinct suite of genes from those in leaves regulates stomatal patterning in hypocotyls. Here we report that regardless of organ type, MUTE controls downstream events directing stomatal differentiation, specifically the transition from meristemoid to guard mother cell. Ectopic MUTE expression is sufficient to over-ride cell fate specification in cell types that do not normally differentiate stomata. Furthermore, MUTE is required for the production of the structure evolutionarily related to stomata, the hydathode pore. Consistently, MUTE displays expression at the tip of cotyledons and leaves, thus co-localizing with the auxin maxima. However, MUTE itself was not regulated by the auxin, and the absence of hydathode pores in mute did not affect the auxin maxima. Surprisingly, our analysis revealed that the requirement for MUTE could be partially circumvented under conditions of compromised inhibitory signaling.

Diseases caused by Xylella fastidiosa have attained great importance worldwide as the pathogen and its insect vectors have been disseminated. Since this is the first plant pathogenic bacterium for which a complete genome sequence was determined, much progress has been made in understanding the process by which it spreads within the xylem vessels of susceptible plants as well as the traits that contribute to its acquisition and transmission by sharpshooter vectors. Athough this pathogen shares many similarities with Xanthomonas species, such as its use of a small fatty acid signal molecule to coordinate virulence gene expression, the traits that it utilizes to cause disease and the manner in which they are regulated differ substantially from those of related plant pathogens. Its complex lifestyle as both a plant and insect colonist involves traits that are in conflict with these stages, thus apparently necessitating the use of a gene regulatory scheme that allows cells expressing different traits to co-occur in the plant.

During fruiting body development, the product of the csgA gene is necessary for cellular aggregation, for spore differentiation, and for gene expression that is initiated after 6 hr of starvation. From nascent wildtype fruiting bodies we have purified a polypeptide of 17 kd called C-factor, which, at approximately 1 to 2 nM, restores normal development to csgA mutant cells. C-factor activity is not recovered from extracts of unstarved, growing cells or csgA mutant cells. The amino acid sequence from purified C-factor demonstrates that it is the product of the csgA gene. C-factor is active over a narrow range of concentration and has properties of a morphogenetic paracrine signal.

HD-GYP is a protein domain of unknown biochemical function implicated in bacterial signaling and regulation. In the plant pathogen Xanthomonas campestris pv. campestris , the synthesis of virulence factors and dispersal of biofilms are positively controlled by a two-component signal transduction system comprising the HD-GYP domain regulatory protein RpfG and cognate sensor RpfC and by cell–cell signaling mediated by the diffusible signal molecule DSF (diffusible signal factor). The RpfG/RpfC two-component system has been implicated in DSF perception and signal transduction. Here we show that the role of RpfG is to degrade the unusual nucleotide cyclic di-GMP, an activity associated with the HD-GYP domain. Mutation of the conserved H and D residues of the isolated HD-GYP domain resulted in loss of both the enzymatic activity against cyclic di-GMP and the regulatory activity in virulence factor synthesis. Two other protein domains, GGDEF and EAL, are already implicated in the synthesis and degradation respectively of cyclic di-GMP. As with GGDEF and EAL domains, the HD-GYP domain is widely distributed in free-living bacteria and occurs in plant and animal pathogens, as well as beneficial symbionts and organisms associated with a range of environmental niches. Identification of the role of the HD-GYP domain thus increases our understanding of a signaling network whose importance to the lifestyle of diverse bacteria is now emerging.