Genomic organization of multiple genes coding for rhodotorucine A,a lipopeptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides

Rhodotorucine A, a lipopeptide mating pheromone, is secreted from mating type A cells of Rhodosporidium toruloides and induces sexual differentiation of the opposite mating type a cells. Genome of A-type cells contains three homologous genes (RHA1, RHA2, and RHA3) encoding rhodotorucine A. Genomic Southern blot analysis using RHA1 DNA as a probe showed that RHA1 strongly hybridize with A-type genomic DNA but weakly with a-type, suggesting that the sequences of RHA genes were dissimilar in the opposite a-type genome. The range of dissimilar regions in a-type genome was searched using RHA-flanking DNA segments as probes. The result suggests that a-type genome lacks sequences coding for rhodotorucine A and its 5 upstream but contains its 3 non-coding sequences. The absence of mating pheromone genes in the opposite mating type genome suggests that the expression of mating-type-specific genes in R. toruloides is not controlled trans-criptionally, as shown in the yeast Saccharomyces cerevisiae.     

香菇B交配型位点的分子遗传学结构研究II.一个香菇单核体的B位点结构的分子解析

在先前的工作中,曾经运用简并PCR和染色体步行的方法从香菇中获得了1个信息素受体编码基因和1个信息素前体编码基因。根据香菇135菌株的原生质体单核体的全基因组测序信息,设计了4对引物,用于扩增香菇苏香菌株的原生质体单核体SUP2中的信息素受体编码基因STE-3的同源物及其侧翼保守基因。实验结果共获得了33,655bp的DNA序列,运用BlastX搜索对所获得的序列进行同源性分析后,发现了7个推定基因,其中有3个为信息素受体编码基因。再根据信息素前体所具有的保守基序特征,在2个信息素受体编码基因附近发现了4个信息素前体编码基因。首次对香菇的B交配型位点的分子遗传学结构有了比较全面的了解。     

Identification of the cAD1 sex pheromone precursor in Enterococcus faecalis

The Enterococcus faecalis virulence plasmid pAD1 encodes a mating response induced by exposure to an octapeptide sex pheromone, cAD1, secreted by plasmid-free enterococci. The determinant for the pheromone in E. faecalis FA2-2, designated cad, was found to encode a 309-amino-acid lipoprotein precursor with the last 8 residues of its 22-amino acid signal sequence representing the cAD1 moiety. The lipoprotein moiety contained two 77-amino-acid repeats (70% identity) separated by 45 residues. The nonisogenic E. faecalis strain V583 determinant encodes a homologous precursor protein, but it differs at two amino acid positions, both of which are located within the pheromone peptide moiety (positions 2 and 8). Construction of a variant of strain FA2-2 containing the differences present in V583 resulted in cells that did not produce detectable cAD1. The mutant appeared normal under laboratory growth conditions, and while significantly reduced in recipient potential, when carrying pAD1 it exhibited a normal mating response. A mutant of FA2-2 with a truncated lipoprotein moiety appeared normal with respect to recipient potential and, when carrying plasmid DNA, donor potential. A gene encoding a protein designated Eep, believed to be a zinc metalloprotease, had been previously identified as required for pheromone biosynthesis and was believed to be involved in the processing of a pheromone precursor. Our new observation that the pAD1-encoded inhibitor peptide, iAD1, whose precursor is itself a signal sequence, is also dependent on Eep is consistent with the likelihood that such processing occurs at the amino terminus of the cAD1 moiety.     

Role of metabolism of the mating pheromone in sexual differentiation of the heterobasidiomycete Rhodosporidium toruloides.

A trypsin-type endopeptidase (Kamiya et al., Biochem. Biophys. Res. Commun. 94:855-860, 1980) responsible for the metabolism of rhodotorucine A, the farnesyl undecapeptide mating pheromone secreted by mating type A cells of Rhodosporidium toruloides, was biologically characterized. Metabolic activity was found to be present exclusively on the cell surface of the pheromone target cell. The activity was highly specific to the pheromone, and a biologically inactive analog which has the complete amino acid sequence of rhodotorucine A but lacks the farnesyl residue was not metabolized by intact cells. Pheromone metabolism was inhibited by trypsin substrates such as tosyl-L-arginine methyl ester. The presence of tosyl-L-arginine methyl ester strongly inhibited the sexual differentiation induced by the pheromone at a concentration which did not affect the vegetative growth of R. toruloides. Pheromone-induced sexual differentiation was also strongly inhibited by a metabolizable analog, rhodotorucine A S-oxide, but not by a non-metabolizable one. In mutants defective in early processes of mating, the decrease in the pheromone metabolic activity correlated well with the extent of loss of sensitivity to the pheromone. Both the pheromone metabolism and the capacity for sexual differentiation of a sterile mutant were restored concomitantly with reversion from the sterile to the fertile phenotype. These results suggested that metabolism of the mating pheromone plays an essential role in the process of sexual differentiation in R. toruloides.     

Cloning of the Lentinula edodes B mating-type locus and identification of the genetic structure controlling B mating

During the life cycle of heterothallic tetrapolar Agaricomycetes such as Lentinula edodes (Berk.) Pegler, the mating type system, composed of unlinked A and B loci, plays a vital role in controlling sexual development and resulting formation of the fruit body. L. edodes is produced worldwide for consumption and medicinal purposes, and understanding its sexual development is therefore of great importance. A considerable amount of mating type factors has been indicated over the past decades but few genes have actually been identified, and no complete genetic structures of L. edodes B mating-type loci are available. In this study, we cloned the matB regions from two mating compatible L. edodes strains, 939P26 and 939P42. Four pheromone receptors were identified on each new matB region, together with three and four pheromone precursor genes in the respective strains. Gene polymorphism, phylogenetic analysis and distribution of pheromone receptors and pheromone precursors clearly indicate a bipartite matB locus, each sublocus containing a pheromone receptor and one or two pheromone precursors. Detailed sequence comparisons of genetic structures between the matB regions of strains 939P42, 939P26 and a previously reported strain SUP2 further supported this model and allowed identification of the B mating type subloci borders. Mating studies confirmed the control of B mating by the identified pheromone receptors and pheromones in L. edodes.     

A large pheromone and receptor gene complex determines multiple B mating type specificities in Coprinus cinereus.

Pheromone signaling plays an essential role in the mating and sexual development of mushroom fungi. Multiallelic genes encoding the peptide pheromones and their cognate 7-transmembrane helix (7-TM) receptors are sequestered in the B mating type locus. Here we describe the isolation of the B6 mating type locus of Coprinus cinereus. DNA sequencing and transformation analysis identified nine genes encoding three 7-TM receptors and six peptide pheromone precursors embedded within 17 kb of mating type-specific sequence. The arrangement of the nine genes suggests that there may be three functionally independent subfamilies of genes each comprising two pheromone genes and one receptor gene. None of the nine B6 genes showed detectable homology to corresponding B gene sequences in the genomic DNA from a B3 strain, and each of the B6 genes independently alter B mating specificity when introduced into a B3 host strain. However, only genes in two of the B6 groups were able to activate B-regulated development in a B42 host. Southern blot analysis showed that these genes failed to cross-hybridize to corresponding genes in the B42 host, whereas the three genes of the third subfamily, which could not activate development in the B42 host, did cross-hybridize. We conclude that cross-hybridization identifies the same alleles of a particular subfamily of genes in different B loci and that B6 and B42 share alleles of one subfamily. There are an estimated 79 B mating specificities: we suggest that it is the different allele combinations of gene subfamilies that generate these large numbers.     

Purification, characterization, and amino acid sequence of the mating pheromone Er-10 of the ciliate Euplotes raikovi

The mating pheromone Er-10 from mat-10 homozygous Euplotes raikovi was purified by a three-step purification procedure with an overall yield of 62%. It was identified as a protein of molecular weight 8000 having an isoelectic point of 3.9. Its complete primary structure was determined by automated Edman degradation of the whole protein after performic acid oxidation and of peptides generated by cyanogen bromide and Staphylococcus aureus V8 protease. The proposed sequence is Asp1-Leu-Cys-Glu-Gln-Ser-Ala-Leu-Gln-Cys10-Asn-Glu-Gln-Gly-Cys-His -Asn-Phe-Cys- Ser20-Pro-Glu-Asp-Lys-Pro-Gly-Cys-Leu-Gly-Met30-Val-Trp-Asn- Pro-Glu-Leu-Cys- Pro38. The calculated molecular weight of 4191.7, which is in good agreement with the value of m/z 4190.7 obtained by fission fragment ionization mass spectrometry, suggests that the native structure is a dimer with three intrachain disulfide bonds in each subunit. The amino acid sequence is 43% identical with that of the E. raikovi mating pheromone Er-1, with the identities concentrated in the amino-terminal half. The half-cystine locations are conserved, but Er-10 is two residues shorter than Er-1. Prediction of the secondary structure suggests that Er-10 may also contain a helical structure at the amino terminus. These results indicate that the mating pheromones of E. raikovi form a homologous family.     

应用简并引物扩增黑木耳信息素受体基因片段

Degenerate primers, br1-F and br1-R, designed based on the conserved amino acid sequence of STE3 pheromone receptor in Schizophyllum commune, were used to amplify genomic DNA of monkaryotic parental strains(H2, J3) and fifty-nine monokaryons of their F1 progenies in Auricularia auricula. A fragment of the PCR product 811bp in length were amplified from the parental strain H2, nine monokaryons of the H2 mating –type and fifteen ones of the J3 mating –type of F1 progenies. After cloning , sequencing the fragm…     

The Neurospora crassa pheromone precursor genes are regulated by the mating type locus and the circadian clock

Pheromones play important roles in female and male behaviour in the filamentous ascomycete fungi. To begin to explore the role of pheromones in mating, we have identified the genes encoding the sex pheromones of the heterothallic species Neurospora crassa. One gene, expressed exclusively in mat A strains, encodes a polypeptide containing multiple repeats of a putative pheromone sequence bordered by Kex2 processing sites. Strains of the opposite mating type, mat a, express a pheromone precursor gene whose polypeptide contains a C-terminal CAAX motif predicted to produce a mature pheromone with a C-terminal carboxy-methyl isoprenylated cysteine. The predicted sequences of the pheromones are remarkably similar to those encoded by other filamentous ascomycetes. The expression of the pheromone precursor genes is mating type specific and is under the control of the mating type locus. Furthermore, the genes are highly expressed in conidia and under conditions that favour sexual development. Both pheromone precursor genes are also regulated by the endogenous circadian clock in a time-of-day-specific fashion, supporting a role for the clock in mating.     

JmjC-domain-containing histone demethylases of the JMJD1B type as putative precursors of endogenous DSIP

Delta sleep inducing peptide (WAGGDASGE, DSIP) is a well known multifunctional regulatory peptide. Numerous studies have confirmed its stress-protective and adaptive activity which is independent of the origin or nature of the stress or other harmful factors. However, the biosynthetic origin of DSIP remains obscure, since nothing is known of its protein precursor(s) and their encoding gene(s). We have performed a comprehensive analysis of available gene and protein databases for homologous peptide sites within mammalian resources including man. A family of Jumonji C (JmjC)-domain-containing histone demethylases was shown to contain a sequence fragment closely homologous to DSIP. One type of these ubiquitous and phylogenetically ancient proteins encoded by JMJD1B gene includes the WKGGNASGE sequence that differs from DSIP by only 2 amino acid residues in positions 2 and 5. The respective peptide was synthesized and its biological effects were evaluated in a preliminary way in the forced swimming and antitoxic tests. We suggest that the histone demethylases of the JmjC-group containing DSIP-related region can be considered as possible protein precursors of endogenous peptides with DSIP-like activity.     

Transient increase of Ca2+ uptake as a signal for mating pheromone-induced differentiation in the heterobasidiomycetous yeast Rhodosporidium toruloides.

The role of Ca2+ for the signaling of rhodotorucine A, a mating pheromone of Rhodosporidium toruloides, was investigated. The efficiency with which the target cells responded to the mating pheromone was dependent on the Ca2+ concentration in the medium. The pheromone induced a very rapid and transient increase of Ca2+ uptake in the recipient cell. We concluded that the transient increase in the intracellular Ca2+ concentration could play an essential role in the control of differentiation by the pheromone.     

Isolation and primary structure of the califins, three biologically active egg-laying hormone-like peptides from the atrial gland of Aplysia californica

The atrial gland of the marine mollusk Aplysia californica contains several biologically active peptides that are thought to be important in reproductive function. In the present study, three novel peptides, which we named califin A, B, and C, were purified from extracts of atrial glands by high performance liquid chromatography, and their primary structures were determined. Each consists of a 36-residue subunit bound by a single disulfide bond to an 18-residue subunit. The large subunits differ from each other by one or two residues, whereas the small subunits are identical. The large subunits are 78-83% homologous to egg-laying hormone (ELH), a 36-residue peptide synthesized by the neuroendocrine bag cells of Aplysia. Like ELH, the califins excite LB and LC cells of the abdominal ganglion and cause egg laying when injected into sexually mature animals. Based on previously described DNA sequence data, each califin is likely to be derived from one of several precursor proteins that are encoded by members of the ELH gene family. Califin A is encoded on the peptide A precursor, and califin B may be encoded on the peptide B precursor. No gene encoding califin C has been sequenced. Because peptides A and B are also biologically active, the precursors encoding them and califins A and B are polyproteins. The possible role of atrial gland peptides as pheromones is discussed.     

Isolation of pheromone precursor genes of Magnaporthe grisea.

In heterothallic ascomycetes one mating partner serves as the source of female tissue and is fertilized with spermatia from a partner of the opposite mating type. The role of pheromone signaling in mating is thought to involve recognition of cells of the opposite mating type. We have isolated two putative pheromone precursor genes of Magnaporthe grisea. The genes are present in both mating types of the fungus but they are expressed in a mating type-specific manner. The MF1-1 gene, expressed in Mat1-1 strains, is predicted to encode a 26-amino-acid polypeptide that is processed to produce a lipopeptide pheromone. The MF2-1 gene, expressed in Mat1-2 strains, is predicted to encode a precursor polypeptide that is processed by a Kex2-like protease to yield a pheromone with striking similarity to the predicted pheromone sequence of a close relative, Cryphonectria parasitica. Expression of the M. grisea putative pheromone precursor genes was observed under defined nutritional conditions and in field isolates. This suggests that the requirement for complex media for mating and the poor fertility of field isolates may not be due to limitation of pheromone precursor gene expression. Detection of putative pheromone precursor gene mRNA in conidia suggests that pheromones may be important for the fertility of conidia acting as spermatia.     

Cloning and Analysis of Partial B Mating Gene Pheromone Receptor in Agrocybe salicacola (Strophariaceae)

A 4231bp DNA fragment of B mating type pheromone receptor from strain YAASM0711 of Agrocybe salicacola was obtained by using degenerate PCR and DNA walking techniques. The result of alignment and structure prediction of DNA sequences showed that one 1194bp nucleotides gene ASRcb1 encoding B mating pheromone receptor was found, including four introns(72bp, 49bp, 48bp and 41bp) and five extrons (217bp, 113bp, 67bp, 138bp and 449bp). The spliced open reading frame (ORF) contains 984bp nucleotides encoding 327 amino acid residues, and includes seven transmembrane protein regions as in its similar sequences of Coprinus cinerea and Laccaria bicolor pheromone receptors. The genetic evolution of pheromone receptor showed ASRcb1 was clustered with more receptors, which suggested multiple ways of evolution occurred in fungi.     

Functional characterization of an alpha-factor-like Sordaria macrospora peptide pheromone and analysis of its interaction with its cognate receptor in Saccharomyces cerevisiae

The homothallic filamentous ascomycete Sordaria macrospora possesses genes which are thought to encode two pheromone precursors and two seven-transmembrane pheromone receptors. The pheromone precursor genes are termed ppg1 and ppg2. The putative products derived from the gene sequence show structural similarity to the alpha-factor precursors and a-factor precursors of the yeast Saccharomyces cerevisiae. Likewise, sequence similarity has been found between the putative products of the pheromone receptor genes pre2 and pre1 and the S. cerevisiae Ste2p alpha-factor receptor and Ste3p a-factor receptor, respectively. To investigate whether the alpha-factor-like pheromone-receptor pair of S. macrospora is functional, a heterologous yeast assay was used. Our results show that the S. macrospora alpha-factor-like pheromone precursor PPG1 is processed into an active pheromone by yeast MATalpha cells. The S. macrospora PRE2 protein was demonstrated to be a peptide pheromone receptor. In yeast MATa cells lacking the endogenous Ste2p receptor, the S. macrospora PRE2 receptor facilitated all aspects of the pheromone response. Using a synthetic peptide, we can now predict the sequence of one active form of the S. macrospora peptide pheromone. We proved that S. macrospora wild-type strains secrete an active pheromone into the culture medium and that disruption of the ppg1 gene in S. macrospora prevents pheromone production. However, loss of the ppg1 gene does not affect vegetative growth or fertility. Finally, we established the yeast assay as an easy and useful system for analyzing pheromone production in developmental mutants of S. macrospora.     

Purification and characterization of a Ca2+-dependent membrane peptidase involved in the signaling of mating pheromone in Rhodosporidium toruloides.

A mating-type-specific, membrane thiol peptidase (referred to as trigger peptidase) that seems to play a key role in the transmembrane signaling of the lipopeptidyl mating pheromone rhodotorucine A at the cell surface of mating type a cells of Rhodosporidium toruloides (T. Miyakawa, M. Kaji, T. Yasutake, Y.K. Jeong, E. Tsuchiya, and S. Fukui, J. Bacteriol. 162:294-299, 1985) was purified to homogeneity and characterized. The following lines of evidence support the contention that the enzyme we purified was the trigger peptidase: the identical specificity of hydrolysis at the Arg-Asn sequence of rhodotorucine A and the sensitivity of the reaction to sulfhydryl-blocking reagents; the identical specificity for the substrate, with a strict requirement for the presence of the lipid moiety; and the absence of the corresponding activity in the pheromone-producing strain (mating type A) and in a sterile mutant strain, M-39 (type a), that lacks trigger peptidase activity in vivo. The apparent molecular weight of trigger peptidase was estimated to be 68,000 by Sepharose 6B gel filtration in the presence of octylglucoside and 63,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Trigger peptidase alone was inactive but exhibited enzymatic activity with the simultaneous addition of Ca2+, membrane phospholipids, and a nonionic detergent such as octylglucoside. The concentration of Ca2+ required for maximum activation was approximately 1 mM. Only Mn2+ could replace Ca2+ at comparable concentrations. Among the phospholipids tested, only phosphatidylserine and phosphatidylethanolamine supported trigger peptidase activation. Solubilized trigger peptidase was strongly inhibited by antipain and phosphoramidon.     

杨柳田头菇B交配型基因信息素受体片段的克隆与分析

利用简并PCR及DNA步移法,从杨柳田头菇Agrocybe salicacola YAASM0711菌株中扩增得到了一个4 231 bp的核酸片段.经过比对及序列预测,所获得序列中含有杨柳田头菇交配型编码基因中的信息素受体部分,其序列长度为1194 bp,包含4个内含子,5个外显子的长度分别为217 bp,113 bp,67 bp,138bp,449 bp.拼接后的ORF全长984 bp,编码327个氨基酸残基.该序列与灰盖鬼伞Coprinus cinerea、双色蜡蘑Laccaria bicolor信息素受体氨基酸序列较为相似,含有7个跨膜区.信息素受体遗传进化分析显示,其与多个物种信息素受体聚集在一起,可能与真菌信息素受体的多种起源有关.     

ccfA, the Genetic Determinant for the cCF10 Peptide Pheromone in Enterococcus faecalis OG1RF

The nosocomial pathogen Enterococcus faecalis has a unique pheromone-inducible conjugative mating system. Conjugative transfer of the E. faecalis plasmid pCF10 is specifically induced by the cCF10 peptide pheromone (LVTLVFV). Genomic sequence information has recently allowed the identification of putative structural genes coding for the various enterococcal pheromones (D. B. Clewell et al., Mol. Microbiol. 35:246-247, 2000). The cCF10 pheromone sequence LVTLVFV was found within an open reading frame designated ccfA, encoding a putative lipoprotein precursor. Several other pheromone sequences were found in similar locations within other predicted lipoproteins. CcfA shows significant sequence relatedness to the Escherichia coli protein YidC, an inner membrane protein translocase, as well as to a large number of homologs identified in gram-positive and in gram-negative bacteria. Analysis of the deduced CcfA amino acid sequence suggested that mature cCF10 peptide could be formed from the proteolytic degradation of its signal peptide. Expression of the cloned ccfA gene with an inducible expression vector dramatically increased cCF10 production by E. faecalis and also resulted in cCF10 production by Lactococcus lactis, a non-pheromone producer. Site-directed mutagenesis of the ccfA sequence encoding the cCF10 peptide confirmed that ccfA was a functional genetic determinant for cCF10.