Genetic basis of chemical communication in eusocial insects.

Social behavior is one of the most fascinating and complex behaviors in humans and animals. A fundamental process of social behavior is communication among individuals. It relies on the capability of the nervous system to sense, process, and interpret various signals (e.g., pheromones) and respond with appropriate decisions and actions. Eusocial insects, including ants, some bees, some wasps, and termites, display intriguing cooperative social behavior. Recent advances in genetic and genomic studies have revealed key genes that are involved in pheromone synthesis, chemosensory perception, and physiological and behavioral responses to varied pheromones. In this review, we highlight the genes and pathways that regulate queen pheromone-mediated social communication, discuss the evolutionary changes in genetic systems, and outline prospects of functional studies in sociobiology.

A fatty acid anabolic pathway in specialized-cells sustains a remote signal that controls egg activation in Drosophila.

Egg activation, representing the critical oocyte-to-embryo transition, provokes meiosis completion, modification of the vitelline membrane to prevent polyspermy, and translation of maternally provided mRNAs. This transition is triggered by a calcium signal induced by spermatozoon fertilization in most animal species, but not in insects. In Drosophila melanogaster, mature oocytes remain arrested at metaphase-I of meiosis and the calcium-dependent activation occurs while the oocyte moves through the genital tract. Here, we discovered that the oenocytes of fruitfly females are required for egg activation. Oenocytes, cells specialized in lipid-metabolism, are located beneath the abdominal cuticle. In adult flies, they synthesize the fatty acids (FAs) that are the precursors of cuticular hydrocarbons (CHCs), including pheromones. The oenocyte-targeted knockdown of a set of FA-anabolic enzymes, involved in very-long-chain fatty acid (VLCFA) synthesis, leads to a defect in egg activation. Given that some but not all of the identified enzymes are required for CHC/pheromone biogenesis, this putative VLCFA-dependent remote control may rely on an as-yet unidentified CHC or may function in parallel to CHC biogenesis. Additionally, we discovered that the most posterior ventral oenocyte cluster is in close proximity to the uterus. Since oocytes dissected from females deficient in this FA-anabolic pathway can be activated in vitro, this regulatory loop likely operates upstream of the calcium trigger. To our knowledge, our findings provide the first evidence that a physiological extra-genital signal remotely controls egg activation. Moreover, our study highlights a potential metabolic link between pheromone-mediated partner recognition and egg activation.

Recent Advances in Behavioral (Epi)Genetics in Eusocial Insects.

Eusocial insects live in societies in which distinct family members serve specific roles in maintaining the colony and advancing the reproductive ability of a few select individuals. Given the genetic similarity of all colony members, the diversity of morphologies and behaviors is surprising. Social communication relies on pheromones and olfaction, as shown by mutants of orco, the universal odorant receptor coreceptor, and through electrophysiological analysis of neuronal responses to pheromones. Additionally, neurohormonal factors and epigenetic regulators play a key role in caste-specific behavior, such as foraging and caste switching. These studies start to allow an understanding of the molecular mechanisms underlying social behavior and provide a technological foundation for future studies of eusocial insects. In this review, we highlight recent findings in eusocial insects that advance our understanding of genetic and epigenetic regulations of social behavior and provide perspectives on future studies using cutting-edge technologies.

Multisite imaging of neural activity using a genetically encoded calcium sensor in the honey bee.

Understanding of the neural bases for complex behaviors in Hymenoptera insect species has been limited by a lack of tools that allow measuring neuronal activity simultaneously in different brain regions. Here, we developed the first pan-neuronal genetic driver in a Hymenopteran model organism, the honey bee, and expressed the calcium indicator GCaMP6f under the control of the honey bee synapsin promoter. We show that GCaMP6f is widely expressed in the honey bee brain, allowing to record neural activity from multiple brain regions. To assess the power of this tool, we focused on the olfactory system, recording simultaneous responses from the antennal lobe, and from the more poorly investigated lateral horn (LH) and mushroom body (MB) calyces. Neural responses to 16 distinct odorants demonstrate that odorant quality (chemical structure) and quantity are faithfully encoded in the honey bee antennal lobe. In contrast, odor coding in the LH departs from this simple physico-chemical coding, supporting the role of this structure in coding the biological value of odorants. We further demonstrate robust neural responses to several bee pheromone odorants, key drivers of social behavior, in the LH. Combined, these brain recordings represent the first use of a neurogenetic tool for recording large-scale neural activity in a eusocial insect and will be of utility in assessing the neural underpinnings of olfactory and other sensory modalities and of social behaviors and cognitive abilities.

A non-pheromone GPCR is essential for meiosis and ascosporogenesis in the wheat scab fungus.

Meiosis is essential for generating genetic diversity and sexual spores, but the regulation of meiosis and ascosporogenesis is not clear in filamentous fungi, in which dikaryotic and diploid cells formed inside fruiting bodies are not free living and independent of pheromones or pheromone receptors. In this study, Gia1, a non-pheromone GPCR (G protein-coupled receptor) with sexual-specific expression in Fusarium graminearum, is found to be essential for ascosporogenesis. The gia1 mutant was normal in perithecium development, crozier formation, and karyogamy but failed to undergo meiosis, which could be partially rescued by a dominant active mutation in GPA1 and activation of the Gpmk1 pathway. GIA1 orthologs have conserved functions in regulating meiosis and ascosporogenesis in Sordariomycetes. GIA1 has a paralog, GIP1, in F. graminearum and other Hypocreales species which is essential for perithecium formation. GIP1 differed from GIA1 in expression profiles and downstream signaling during sexual reproduction. Whereas the C-terminal tail and IR3 were important for intracellular signaling, the N-terminal region and EL3 of Gia1 were responsible for recognizing its ligand, which is likely a protein enriched in developing perithecia, particularly in the gia1 mutant. Taken together, these results showed that GIA1 encodes a non-pheromone GPCR that regulates the entry into meiosis and ascosporogenesis via the downstream Gpmk1 MAP kinase pathway in F. graminearum and other filamentous ascomycetes.

A tale of two copies: Evolutionary trajectories of moth pheromone receptors.

Pheromone communication is an essential component of reproductive isolation in animals. As such, evolution of pheromone signaling can be linked to speciation. For example, the evolution of sex pheromones is thought to have played a major role in the diversification of moths. In the crop pests Spodoptera littoralis and S. litura, the major component of the sex pheromone blend is (Z,E)-9,11-tetradecadienyl acetate, which is lacking in other Spodoptera species. It indicates that a major shift occurred in their common ancestor. It has been shown recently in S. littoralis that this compound is detected with high specificity by an atypical pheromone receptor, named SlitOR5. Here, we studied its evolutionary history through functional characterization of receptors from different Spodoptera species. SlitOR5 orthologs in S. exigua and S. frugiperda exhibited a broad tuning to several pheromone compounds. We evidenced a duplication of OR5 in a common ancestor of S. littoralis and S. litura and found that in these two species, one duplicate is also broadly tuned while the other is specific to (Z,E)-9,11-tetradecadienyl acetate. By using ancestral gene resurrection, we confirmed that this narrow tuning evolved only in one of the two copies issued from the OR5 duplication. Finally, we identified eight amino acid positions in the binding pocket of these receptors whose evolution has been responsible for narrowing the response spectrum to a single ligand. The evolution of OR5 is a clear case of subfunctionalization that could have had a determinant impact in the speciation process in Spodoptera species.

Genetic and Structural Analyses of RRNPP Intercellular Peptide Signaling of Gram-Positive Bacteria.

Bacteria use diffusible chemical messengers, termed pheromones, to coordinate gene expression and behavior among cells in a community by a process known as quorum sensing. Pheromones of many gram-positive bacteria, such as Bacillus and Streptococcus, are small, linear peptides secreted from cells and subsequently detected by sensory receptors such as those belonging to the large family of RRNPP proteins. These proteins are cytoplasmic pheromone receptors sharing a structurally similar pheromone-binding domain that functions allosterically to regulate receptor activity. X-ray crystal structures of prototypical RRNPP members have provided atomic-level insights into their mechanism and regulation by pheromones. This review provides an overview of RRNPP prototype signaling; describes the structure-function of this protein family, which is spread widely among gram-positive bacteria; and suggests approaches to target RRNPP systems in order to manipulate beneficial and harmful bacterial behaviors.

Competence shut-off by intracellular pheromone degradation in salivarius streptococci.

Competence for DNA transformation is a major strategy for bacterial adaptation and survival. Yet, this successful tactic is energy-consuming, shifts dramatically the metabolism, and transitory impairs the regular cell-cycle. In streptococci, complex regulatory pathways control competence deactivation to narrow its development to a sharp window of time, a process known as competence shut-off. Although characterized in streptococci whose competence is activated by the ComCDE signaling pathway, it remains unclear for those controlled by the ComRS system. In this work, we investigate competence shut-off in the major human gut commensal Streptococcus salivarius. Using a deterministic mathematical model of the ComRS system, we predicted a negative player under the control of the central regulator ComX as involved in ComS/XIP pheromone degradation through a negative feedback loop. The individual inactivation of peptidase genes belonging to the ComX regulon allowed the identification of PepF as an essential oligoendopeptidase in S. salivarius. By combining conditional mutants, transcriptional analyses, and biochemical characterization of pheromone degradation, we validated the reciprocal role of PepF and XIP in ComRS shut-off. Notably, engineering cleavage site residues generated ultra-resistant peptides producing high and long-lasting competence activation. Altogether, this study reveals a proteolytic shut-off mechanism of competence in the salivarius group and suggests that this mechanism could be shared by other ComRS-containing streptococci.

Use of Rgg quorum-sensing machinery to create an innovative recombinant protein expression system in Streptococcus thermophilus.

Streptococcus thermophilus holds promise as a chassis for producing and secreting heterologous proteins. Used for thousands of years to ferment milk, this species has generally recognized as safe (GRAS) status in the USA and qualified presumption of safety (QPS) status in Europe. In addition, it can be easily genetically modified thanks to its natural competence, and it secretes very few endogenous proteins, which means less downstream processing is needed to purify target proteins, reducing costs. Extracellular degradation of heterologous proteins can be eliminated by introducing mutations that inactivate the genes encoding the bacterium's three major surface proteases. Here, we constructed an inducible expression system that utilizes a peptide pheromone (SHP1358) and a transcriptional regulator (Rgg1358) involved in quorum-sensing regulation. We explored the functionality of a complete version of the system, in which the inducer is produced by the bacterium itself, by synthesizing a luciferase reporter protein. This complete version was assessed with bacteria grown in a chemically defined medium but also in vivo, in the faeces of germ-free mice. We also tested an incomplete version, in which the inducer had to be added to the culture medium, by synthesizing luciferase and a secreted form of elafin, a human protein with therapeutic properties. Our results show that, in our system, protein production can be modulated by employing different concentrations of the SHP1358 inducer or other SHPs with closed amino acid sequences. We also constructed a genetic background in which all system leakiness was eliminated. In conclusion, with this new inducible expression system, we have added to the set of tools currently used to produce secreted proteins in S. thermophilus, whose myriad applications include the delivery of therapeutic peptides or proteins.

Sexy fingers: Pheromones in the glands of male dendrobatid frogs.

Many animals exchange chemicals during courtship and mating. In some amphibians, sexual chemical communication is mediated by pheromones produced in male breeding glands that are transferred to the female's nostrils during mating. This has been mostly studied in salamanders, despite frogs having similar glands and courtship behaviours suggestive of chemical communication. In Neotropical poison frogs (Dendrobatidae and Aromobatidae), males of many species develop breeding glands in their fingers, causing certain fingers to visibly swell. Many also engage in cephalic amplexus, whereby the male's swollen fingers are placed in close contact with the female's nares during courtship. Here, we investigate the possible roles of swollen fingers in pheromone production using whole-transcriptome sequencing (RNAseq). We examined differential gene expression in the swollen versus non-swollen fingers and toes of two dendrobatid species, Leucostethus brachistriatus and Epipedobates anthonyi, both of which have specialised mucous glands in finger IV, the latter of which has cephalic amplexus. The overwhelming pattern of gene expression in both species was strong upregulation of sodefrin precursor-like factors (SPFs) in swollen fingers, a well-known pheromone system in salamanders. The differentially expressed SPF transcripts in each species were very high (>40), suggesting a high abundance of putative protein pheromones in both species. Overall, the high expression of SPFs in the swollen fingers in both species, combined with cephalic amplexus, supports the hypothesis that these traits, widespread across members of the subfamilies Colostethinae and Hyloxalinae (ca. 141 species), are involved in chemical signalling during courtship.

Muchos animales intercambian sustancias químicas durante el cortejo y el apareamiento. En algunos anfibios, la comunicación química sexual está mediada por feromonas producidas en las glándulas reproductoras de los machos que se transfieren a las hembras durante el apareamiento. Esto se ha estudiado sobre todo en salamandras, a pesar de que las ranas tienen glándulas similares y comportamientos de cortejo que sugieren una comunicación química. En las ranas venenosas neotropicales (Dendrobatidae y Aromobatidae), los machos de muchas especies desarrollan glándulas en los dedos, lo que hace que algunos dedos se vean hinchados. Asimismo, varias especies presentan amplexo cefálico, comportamiento de cortejo en el cual los dedos hinchados entran en estrecho contacto con las narinas y boca de la hembra. En este estudio investigamos las posibles funciones de los dedos hinchados en la producción de feromonas mediante la secuenciación del transcriptoma completo (RNAseq). Examinamos la expresión génica diferencial en los dedos hinchados y no hinchados de dos especies de dendrobátidos, Leucostethus brachistriatus y Epipedobates anthonyi, ambos con glándulas mucosas especializadas en el dedo IV, y esta última especie, con amplexo cefálico. El patrón abrumador de expresión génica en ambas especies fue la alta expression de Sodefrin Precursor­Like Factor (SPF) en los dedos hinchados, un sistema de feromonas ampliamente conocido en las salamandras. El número de transcritos SPF expresados diferencialmente en cada especie fue muy elevado (>40), lo que sugiere una gran abundancia de feromonas proteicas putativas en ambas especies. En general, la elevada expresión de SPF en los dedos hinchados en ambas especies, combinada con el amplexo cefálico, apoya la hipótesis de que estos rasgos, muy extendidos entre los miembros de las subfamilias Colostethinae e Hyloxalinae (aprox 141 especies), están implicados en la señalización química durante el cortejo.

Evolutionary shifts in pheromone receptors contribute to speciation in four Helicoverpa species.

Male moths utilize their pheromone communication systems to distinguish potential mates from other sympatric species, which contributes to maintaining reproductive isolation and even drives speciation. The molecular mechanisms underlying the evolution of pheromone communication systems are usually studied between closely-related moth species for their similar but divergent traits associated with pheromone production, detection, and/or processing. In this study, we first identified the functional differentiation in two orthologous pheromone receptors, OR14b, and OR16, in four Helicoverpa species, Helicoverpa armigera, H. assulta, H. zea, and H. gelotopoeon. To understand the substrate response specificity of these two PRs, we performed all-atom molecular dynamics simulations of OR14b and OR16 based on AlphaFold2 structural prediction, and molecular docking, allowing us to predict a few key amino acids involved in substrate binding. These candidate residues were further tested and validated by site-directed mutagenesis and functional analysis. These results together identified two hydrophobic amino acids at positions 164 and 232 are the determinants of the response specificity of HarmOR14b and HzeaOR14b to Z9-14:Ald and Z9-16:Ald by directly interacting with the substrates. Interestingly, in OR16 orthologs, we found that position 66 alone determines the specific binding of Z11-16:OH, likely via allosteric interactions. Overall, we have developed an effective integrated method to identify the critical residues for substrate selectivity of ORs and elucidated the molecular mechanism of the diversification of pheromone recognition systems.

Structure and number of mating pheromone genes is closely linked to sexual reproductive strategy in Huntiella.

BACKGROUND: Huntiella resides in the Ceratocystidaceae, a family of fungi that accommodates important plant pathogens and insect-associated saprotrophs. Species in the genus have either heterothallic or unisexual (a form of homothallism) mating systems, providing an opportunity to investigate the genetic mechanisms that enable transitions between reproductive strategies in related species. Two newly sequenced Huntiella genomes are introduced in this study and comparative genomics and transcriptomics tools are used to investigate the differences between heterothallism and unisexuality across the genus. RESULTS: Heterothallic species harbored up to seven copies of the a-factor pheromone, each of which possessed numerous mature peptide repeats. In comparison, unisexual Huntiella species had only two or three copies of this gene, each with fewer repeats. Similarly, while the heterothallic species expressed up to 12 copies of the mature α-factor pheromone, unisexual species had up to six copies. These significant differences imply that unisexual Huntiella species do not rely on a mating partner recognition system in the same way that heterothallic fungi do. CONCLUSION: While it is suspected that mating type-independent pheromone expression is the mechanism allowing for unisexual reproduction in Huntiella species, our results suggest that the transition to unisexuality may also have been associated with changes in the genes governing the pheromone pathway. While these results are specifically related to Huntiella, they provide clues leading to a better understanding of sexual reproduction and the fluidity of mating strategies in fungi more broadly.

Ratiometric quorum sensing governs the trade-off between bacterial vertical and horizontal antibiotic resistance propagation.

Plasmid-mediated horizontal gene transfer of antibiotic resistance and virulence in pathogenic bacteria underlies a major public health issue. Understanding how, in the absence of antibiotic-mediated selection, plasmid-bearing cells avoid being outnumbered by plasmid-free cells is key to developing counterstrategies. Here, we quantified the induction of the plasmidial sex pheromone pathway of Enterococcus faecalis to show that the integration of the stimulatory (mate-sensing) and inhibitory (self-sensing) signaling modules from the pCF10 conjugative plasmid provides a precise measure of the recipient-to-donor ratio, agnostic to variations in population size. Such ratiometric control of conjugation favors vertical plasmid transfer under low mating likelihood and allows activation of conjugation functions only under high mating likelihood. We further show that this strategy constitutes a cost-effective investment into mating effort because overstimulation produces unproductive self-aggregation and growth rate reduction. A mathematical model suggests that ratiometric control of conjugation increases plasmid fitness and predicts a robust long-term, stable coexistence of donors and recipients. Our results demonstrate how population-level parameters can control transfer of antibiotic resistance in bacteria, opening the door for biotic control strategies.

Two Putative Pheromone Receptors, but Not Their Cognate Pheromones, Regulate Female Fertility in the Atypical Mating Fungus Colletotrichum fructicola.

Colletotrichum fungi are a group of damaging phytopathogens with atypical mating type loci (harboring only MAT1-2-1 but not MAT1-1-1) and complex sexual behaviors. Sex pheromones and their cognate G-protein-coupled receptors are conserved regulators of fungal mating. These genes, however, lose function frequently among Colletotrichum species, indicating a possibility that pheromone signaling is dispensable for Colletotrichum sexual reproduction. We have identified two putative pheromone-receptor pairs (PPG1:PRE2, PPG2:PRE1) in C. fructicola, a species that exhibits plus-to-minus mating type switching and plus-minus-mediated mating line development. Here, we report the generation and characterization of gene-deletion mutants for all four genes in both plus and minus strain backgrounds. Single-gene deletion of pre1 or pre2 had no effect on sexual development, whereas their double deletion caused self-sterility in both the plus and minus strains. Moreover, double deletion of pre1 and pre2 caused female sterility in plus-minus outcrossing. Double deletion of pre1 and pre2, however, did not inhibit perithecial differentiation or plus-minus-mediated enhancement of perithecial differentiation. Contrary to the results with pre1 and pre2, double deletion of ppg1 and ppg2 had no effect on sexual compatibility, development, or fecundity. We concluded that pre1 and pre2 coordinately regulate C. fructicola mating by recognizing novel signal molecule(s) distinct from canonical Ascomycota pheromones. The contrasting importance between pheromone receptors and their cognate pheromones highlights the complicated nature of sex regulation in Colletotrichum fungi.

Pheromone sensing in Drosophila requires support cell-expressed Osiris 8.

BACKGROUND: The nose of most animals comprises multiple sensory subsystems, which are defined by the expression of different olfactory receptor families. Drosophila melanogaster antennae contain two morphologically and functionally distinct subsystems that express odorant receptors (Ors) or ionotropic receptors (Irs). Although these receptors have been thoroughly characterized in this species, the subsystem-specific expression and roles of other genes are much less well-understood. RESULTS: Here we generate subsystem-specific transcriptomic datasets to identify hundreds of genes, encoding diverse protein classes, that are selectively enriched in either Or or Ir subsystems. Using single-cell antennal transcriptomic data and RNA in situ hybridization, we find that most neuronal genes-other than sensory receptor genes-are broadly expressed within the subsystems. By contrast, we identify many non-neuronal genes that exhibit highly selective expression, revealing substantial molecular heterogeneity in the non-neuronal cellular components of the olfactory subsystems. We characterize one Or subsystem-specific non-neuronal molecule, Osiris 8 (Osi8), a conserved member of a large, insect-specific family of transmembrane proteins. Osi8 is expressed in the membranes of tormogen support cells of pheromone-sensing trichoid sensilla. Loss of Osi8 does not have obvious impact on trichoid sensillar development or basal neuronal activity, but abolishes high sensitivity responses to pheromone ligands. CONCLUSIONS: This work identifies a new protein required for insect pheromone detection, emphasizes the importance of support cells in neuronal sensory functions, and provides a resource for future characterization of other olfactory subsystem-specific genes.

Coevolution of the bacterial pheromone ComS and sensor ComR fine-tunes natural transformation in streptococci.

Competence for natural transformation extensively contributes to genome evolution and the rapid adaptability of bacteria dwelling in challenging environments. In most streptococci, this process is tightly controlled by the ComRS signaling system, which is activated through the direct interaction between the (R)RNPP-type ComR sensor and XIP pheromone (mature ComS). The overall mechanism of activation and the basis of pheromone selectivity have been previously reported in Gram-positive salivarius streptococci; however, detailed 3D-remodeling of ComR leading up to its activation remains only partially understood. Here, we identified using a semirational mutagenesis approach two residues in the pheromone XIP that bolster ComR sensor activation by interacting with two aromatic residues of its XIP-binding pocket. Random and targeted mutagenesis of ComR revealed that the interplay between these four residues remodels a network of aromatic-aromatic interactions involved in relaxing the sequestration of the DNA-binding domain. Based on these data, we propose a comprehensive model for ComR activation based on two major conformational changes of the XIP-binding domain. Notably, the stimulation of this newly identified trigger point by a single XIP substitution resulted in higher competence and enhanced transformability, suggesting that pheromone-sensor coevolution counter-selects for hyperactive systems in order to maintain a trade-off between competence and bacterial fitness. Overall, this study sheds new light on the ComRS activation mechanism and how it could be exploited for biotechnological and biomedical purposes.

Origin and Evolution of the Gene Family of Proteinaceous Pheromones, the Exocrine Gland-Secreting Peptides, in Rodents.

The exocrine-gland secreting peptide (ESP)gene family encodes proteinaceous pheromones that are recognized by the vomeronasal organ in mice. For example, ESP1 is a male pheromone secreted in tear fluid that regulates socio-sexual behavior, and ESP22 is a juvenile pheromone that suppresses adult sexual behavior. The family consists of multiple genes and has been identified only in mouse and rat genomes. The coding region of a mouse ESP gene is separated into two exons, each encoding signal and mature sequences. Here, we report the origin and evolution of the ESP gene family. ESP genes were found only in the Muridea and Cricetidae families of rodents, suggesting a recent origin of ESP genes in the common ancestor of murids and cricetids. ESP genes show a great diversity in number, length, and sequence among different species as well as mouse strains. Some ESPs in rats and golden hamsters are expressed in the lacrimal gland and the salivary gland. We also found that a mature sequence of an ESP gene showed overall sequence similarity to the α-globin gene. The ancestral ESP gene seems to be generated by recombination of a retrotransposed α-globin gene with the signal-encoding exon of the CRISP2 gene located adjacent to the ESP gene cluster. This study provides an intriguing example of molecular tinkering in rapidly evolving species-specific proteinaceous pheromone genes.

Candida albicans MTLa2 regulates the mating response through both the a-factor and α-factor sensing pathways.

The diploid fungal pathogen Candida albicans has three configurations at the mating type locus (MTL): heterozygous (a/α) and homozygous (a/a or α/α). C. albicans MTL locus encodes four transcriptional regulators (MTLa1, a2, α1, and α2). The conserved a1/α2 heterodimer controls not only mating competency but also white-opaque heritable phenotypic switching. However, the regulatory roles of MTLa2 and α1 are more complex and remain to be investigated. MTLa/a cells often express a cell type-specific genes and mate as the a-type partner, whereas MTLα/α cells express α-specific genes and mate as the α-type partner. In this study, we report that the MTLa2 regulator controls the formation of mating projections through both the a- and α-pheromone-sensing pathways and thus results in the bi-mater feature of "α cells" of C. albicans. Ectopic expression of MTLa2 in opaque α cells activates the expression of not only MFA1 and STE3 (a-pheromone receptor) but also MFα1 and STE2 (α-pheromone receptor). Inactivation of either the MFa-Ste3 or MFα-Ste2 pheromone-sensing pathway cannot block the MTLa2-induced development of mating projections. However, the case is different in MTLα1-ectopically expressed opaque a cells. Inactivation of the MFα-Ste2 but not the MFa-Ste3 pheromone-sensing pathway blocks MTLα1-induced development of mating projections. Therefore, MTLa2 and MTLα1 exhibit distinct regulatory features that control the mating response in C. albicans. These findings shed new light on the regulatory mechanism of bi-mating behaviors and sexual reproduction in C. albicans.

Asymmetric diversification of mating pheromones in fission yeast.

In fungi, mating between partners depends on the molecular recognition of two peptidyl mating pheromones by their respective receptors. The fission yeast Schizosaccharomyces pombe (Sp) has two mating types, Plus (P) and Minus (M). The mating pheromones P-factor and M-factor, secreted by P and M cells, are recognized by the receptors mating type auxiliary minus 2 (Mam2) and mating type auxiliary plus 3 (Map3), respectively. Our recent study demonstrated that a few mutations in both M-factor and Map3 can trigger reproductive isolation in S. pombe. Here, we explored the mechanism underlying reproductive isolation through genetic changes of pheromones/receptors in nature. We investigated the diversity of genes encoding the pheromones and their receptor in 150 wild S. pombe strains. Whereas the amino acid sequences of M-factor and Map3 were completely conserved, those of P-factor and Mam2 were very diverse. In addition, the P-factor gene contained varying numbers of tandem repeats of P-factor (4-8 repeats). By exploring the recognition specificity of pheromones between S. pombe and its close relative Schizosaccharomyces octosporus (So), we found that So-M-factor did not have an effect on S. pombe P cells, but So-P-factor had a partial effect on S. pombe M cells. Thus, recognition of M-factor seems to be stringent, whereas that of P-factor is relatively relaxed. We speculate that asymmetric diversification of the two pheromones might be facilitated by the distinctly different specificities of the two receptors. Our findings suggest that M-factor communication plays an important role in defining the species, whereas P-factor communication is able to undergo a certain degree of flexible adaptation-perhaps as a first step toward prezygotic isolation in S. pombe.

Bisphenols Promote the Pheromone-Responsive Plasmid-Mediated Conjugative Transfer of Antibiotic Resistance Genes in Enterococcus faecalis.

The enrichment and spread of antibiotic resistance genes (ARGs) induced by environmental chemical pollution further exacerbated the threat to human health and ecological safety. Several compounds are known to induce R plasmid-mediated conjugation through inducing reactive oxygen species (ROS), increasing cell membrane permeability, enhancing regulatory genes expression, and so forth. Up to now, there has been no substantial breakthrough in the studies of models and related mechanisms. Here, we established a new conjugation model using pheromone-responsive plasmid pCF10 and confirmed that five kinds of bisphenols (BPs) at environmentally relevant concentrations could significantly promote the conjugation of ARGs mediated by plasmid pCF10 in E. faecalis by up to 4.5-fold compared with untreated cells. Using qPCR, gene knockout and UHPLC, we explored the mechanisms behind this phenomenon using bisphenol A (BPA) as a model of BPs and demonstrated that BPA could upregulate the expression of pheromone, promote bacterial aggregation, and even directly activate conjugation as a pheromone instead of producing ROS and enhancing cell membrane permeability. Interestingly, the result of mathematical analysis showed that the pheromone effect of most BPs is more potent than that of synthetic pheromone cCF10. These findings provide new insight into the environmental behavior and biological effect of BPs and provided new method and theory to study on enrichment and spread of ARGs induced by environmental chemical pollution.