The Lipid Body Protein , PpoA , Coordinates Sexual and Asexual Sporulation in Aspergillus nidulans *

The coexistence of sexual and asexual reproductive cycles within the same individual is a striking phenomenon in numerous fungi. In the fungus Aspergillus nidulans (teleomorph: Emericella nidulans) endogenous oxylipins, called psi factor, serve as hormone-like signals that modulate the timing and balance between sexual and asexual spore development. Here, we report the identification of A. nidulans ppoA, encoding a putative fatty acid dioxygenase, involved in the biosynthesis of the linoleic acid derived oxylipin psiBalpha. PpoA is required for balancing anamorph and teleomorph development. Deletion of ppoA significantly reduced the level of psiBalpha and increased the ratio of asexual to sexual spore numbers 4-fold. In contrast, forced expression of ppoA resulted in elevated levels of psiBalpha and decreased the ratio of asexual to sexual spore numbers 6-fold. ppoA expression is mediated by two developmental regulators, VeA and the COP9 signalosome, such that ppoA transcript levels are correlated with the initiation of asexual and sexual fruiting body formation. PpoA localizes in lipid bodies in these tissues. These data support an important role for oxylipins in integrating mitotic and meiotic spore development.


INTRODUCTION
A unique property of many fungi is their ability to propagate by both sexual and asexual spores. The integration of the teleomorph (meiotic sexual morph) and anamorph (mitotic asexual morph) stages into the fungal life cycle lends great flexibility in dispersal and survival during suboptimal environmental conditions, both very important characteristics for organisms that are essentially sessile in their somatic stage. Aspergillus nidulans (teleomorph: Emericella nidulans) is a homothallic ascomycete with a defined asexual and sexual cycle that has long served as model system for understanding the genetic regulation of asexual development and secondary metabolism in filamentous fungi (1,2). The asexual cycle is characterized by the production of haploid conidiophores that bear single-cell asexual spores called conidia. Sexual development commences with the formation of multinucleate globular cells, called Hülle cells that surround the cleistothecium, the ascocarp that contains sexual spores called ascospores.
The program of asexual and sexual sporulation is characterized by many developmental stages including temporal and spatial regulation of gene expression, cell specialization and intercellular communication (1,2). The A. nidulans asexual reproductive cycle can be divided into at least three different stages: (i) a growth phase required for cells to acquire the ability to respond to induction signals (competence phenomenon), (ii) initiation of the developmental pathway and (iii) execution of the developmentally regulated events leading to sporulation. A development-specific array of transcription factors is activated that control the expression of multiple sets of genes required for conidiophore morphogenesis (1). Sexual fruiting body formation is influenced by several environmental and genetic determinants; however, the molecular pathways for this developmental stage are not well dissected (3). Current studies are focusing on the characterization of mutants defective in sexual reproduction (4). Normal sexual and asexual development in A. nidulans requires the function of the velvet (veA) gene (5). VeA is known to have a role in activating sexual development and/or inhibiting asexual development, since asexual sporulation in the veA1 mutant is promoted and increased, while sexual development is significantly delayed and reduced (5)(6)(7). Furthermore, veA1 mutants do not exhibit light-dependent development of conidia and ascospores in contrast to the wild type where light induces asexual and delays and reduces sexual spore production (7). The veA1 mutant gene differs from that of the wild type by one nucleotide in the initiation codon resulting in a putative truncated protein by 37 amino acids. veA null (∆veA) mutants do not form cleistothecia; by contrast, overexpression of veA gene leads to formation of cleistothecia even in liquid culture and to formation of fewer conidial heads than in a wild type on solid media (5).
Butnick et al. (8) identified two mutants (acoB202, acoC193) in A. nidulans that fail to become competent and are blocked in both sexual and asexual sporulation. These mutants overproduce hormone-like fatty acid derived oxylipins collectively termed psi factor (precocious sexual inducer). Psi factor serves as signals that modulate sexual and asexual sporulation by affecting the timing and balance of asexual and sexual spore development (9)(10)(11). Biochemical analyses revealed that psi factor is composed primarily of hydroxylated oleic (18:1) and linoleic (18:2) moieties called psiβ and psiα respectively (10)(11)(12)(13). The positioning of the hydroxy groups on the fatty acid backbone further designates the psi compounds as psiB (8' hydroxy-), psiC (5', 8' dihydroxy-) and with psiA designated for a lactone ring of psiC at 5' position ( Fig. 1). Studies, only carried out for the linoleic acid derived psiα molecules, showed that psiBα and psiCα stimulated sexual and inhibited asexual spore development whereas psiAα had the opposite effect (9,10). PsiBα (8-hydroxylinoleic acid or 8-HODE) has also been previously isolated from Gaeumannomyces graminis (14) and the basidiomycete Laetisaria arvalis (15), where it was noted for its biocontrol activity against soil plant pathogens including Rhizoctonia solani and Pythium ultimum (16). 8-HODE is produced by a well biochemically characterized cytosolic oxygenase, linoleate 8-dioxygenase, in both G. graminis and L. arvalis (17,18). However, the role of this enzyme in fungal development is not known.
Our interest in psi factor stems from its role as a signal in Aspergillus sexual and asexual sporulation (9)(10)(11). To gain more insight into the relationship between morphological development and psi factor/oxylipin biosynthesis we have initiated studies to identify and characterize fatty acid dioxygenases in A. nidulans responsible for the production of oxylipins.
Here we describe PpoA, a putative dioxygenase localized to lipid bodies in asexual and sexual fruiting structures. Deletion of ppoA reduced the level of the linoleic acid derived psiBα and increased the ratio of asexual to sexual spore development four fold. On the other hand, forced expression of ppoA resulted in elevated levels of psiBα and decreased the ratio of asexual to sexual spore development six fold. Previous studies have shown similar results by adding exogenous pisBα in confluent mycelial cultures (10). Normal expression of ppoA required the two developmental regulators, VeA and COP9 signalosome. This is the first study that shows at a genetic level the direct involvement of oxylipins in spore development in A. nidulans.

Fungal strains and growth conditions
Aspergillus nidulans strains used in this study are listed in Table I. Strains were grown on glucose minimum medium (GMM) 1 (11) with appropriate supplements for the corresponding auxotrophies, at 37 ºC in continuous dark or in continuous white light. Illumination was carried out in an incubator equipped with General Electric 15-W broad-spectrum fluorescent light bulbs (F15T12CW) placed 50 cm below the plates. Developmental cultures were grown on GMM and asexual and sexual induction was performed as previously described (4). Sexual crosses of A.
nidulans strains were conducted according to Pontecorvo et al. (19). Fungal transformation was carried out as previously described (20) with the modification of embedding the protoplasts in 0.75% top agar rather than spreading them by a glass rod on solid media.

Nucleic acid manipulations
Standard methods were used for construction, maintenance, and isolation of recombinant plasmids (21). Fungal chromosomal DNA was isolated and analyzed from lyophilized mycelia using previously described techniques (22). Total RNA was extracted from lyophilized mycelia using Trizol reagent (Invitrogen Co.) according to manufacturer's recommendations.
Oligonucleotides used in this study are listed in Table II. The PCR product obtained with primers ppoA-F2 and ppoA-R2, using pWFI16 as template, and the XbaI fragment from pDIT1.1, were used as DNA probes for Southern and northern hybridizations. Nucleotide sequences were analyzed and compared using Sequencher (Gene Codes Co. MI, USA.) and ClustalW (http://www.ebi.ac.uk/clustalw/) software programs. The ppoA deletion plasmid pDIT3.11 included the metG marker gene and ppoA flanking sequences without the ppoA encoding region, was created as follows: First, the primers ppoA-5DF3 and ppoA-5DR1 were used to PCR amplify a 1100-bp flanking region at the 5' UTR of the ppoA ORF using the cosmid pWFI16 as template. ppoA-5DF3 and ppoA-5DR1 were designed to introduce XbaI and XmaI restriction sites at either side of the PCR fragment respectively. The amplified XbaI-XmaI product was subcloned into pBluescript SKyielding the vector pDIT2.

Plasmid and strain construction
Next, the primers ppoA-3DF1 and ppoA-3DR1 were used to amplify the flanking region at the 3'UTR of the ppoA ORF using the cosmid pWFI16 as template. ppoA-3DF1 and ppoA-3DR1 were designed to introduce SphI and KpnI restriction sites at either side of the PCR fragment strain yielding the transformant TDIT9.14 that used as control in the GFP experiments.

Lipid extraction and fatty acid analysis
Wild type, ∆ppoA and ppoA overexpression strains were grown in 25 ml of liquid GMM in Petri dishes under stationary conditions at 37°C in the dark. Mycelial mats were collected after 72h, freeze-dried, weighed and homogenized mechanically using Ultra-TurraxT25 dispenser (Ika Werke GmbH & Co. KG). Lipids were extracted and converted into fatty acid methyl esters (FAME) derivatives using 2% sulphuric acid in methanol as described elsewhere (27). FAME were converted into corresponding trimethylsilyl ether (OTMSi) derivatives using BSTFA (Supelco, 33154-U) as a silylation agent according to the method reported previously (28).

Physiological studies
All strains used for physiological studies were prototrophic. The wild type RDIT9.32 was a prototrophic meiotic progeny of a sexual cross between WIM126 and RDIT1.7. Asexual and sexual spore production studies were performed on plates containing 30 ml of solid 1.5% GMM or YGT media. GMM is a medium known to promote the asexual spore stage and YGT medium has been used in previous research to promote sexual development in A. nidulans (9,10,31).
Five ml of top layer with cool melted 0.7% agar-GMM or YGT that contained 10 6 conidia of the appropriate strain was added on each plate. Cultures were incubated in continuous dark or light at 37°C. A core of 12.5-mm diameter was removed from each plate at the appropriate time interval and homogenized for 1 min in 3 ml of sterile water supplemented with 0.01 % Tween 80 to release the spores. Spores were counted using a hemacytometer. The experiments were performed with four replicates. Induction of niiA promoter was performed as described previously (24). Spore data and fatty acid analysis results were statistically compared by analysis of variance (ANOVA) and Fisher's Least Significant Difference (LSD) using the Statistical Analysis System (SAS Institute, Cary, NC).  (32). The tissue fixation was performed as described previously (33). TRITC filter was used to observe the Nile Red fluorescence.

RESULTS
The A. nidulans ppoA gene encodes a protein with oxygenase and peroxidase domains.

Phenotypic characterization of the A. nidulans ∆ppoA mutant.
To assess the phenotype of a ppoA null mutant (∆ppoA), the gene was inactivated by homologous recombination with pDIT3.11 in strain pW1. PCR and Southern analysis of 138 transformants revealed the replacement of the ppoA gene with the metG gene in four transformants that showed identical phenotypes (data not shown). These transformants had no alterations in radial or vegetative growth compared to the parental strain, however both asexual and sexual development were altered as described below. One transformant, TDIT2.19, was selected for in-depth physiological and molecular analysis. Complementation of the ∆ppoA strain with a functional copy of ppoA returned wild type phenotype, thus confirming that the effects on sexual and asexual sporulation were solely due to the deletion of ppoA (data not shown). Fatty acid composition, analyzed from dark grown cultures at 37 ºC, did not significantly differ between wild type, ∆ppoA or ppoA overexpression strains (data not shown) with palmitic, oleic and linoleic acids being the most prevalent fatty acids (11,35). However, measurements of the two most abundant psi factor components, the oleic acid derived 8-hydroxy-9(Z)octadecanoic acid (8-HOE = psiBβ) and the linoleic acid derived 8-hydroxy-9(Z),12(Z)octadecadienoic acid (8-HODE = psiBα) (11)(12)(13) were statistically different in the three isogenic strains (Table III) resulting in altered 8-HOE/8-HODE ratios that were correlated with changes in the asexual to sexual spore ratios (Table IV). Deletion of the ppoA allele resulted in almost complete elimination of psiBα and a slight increase in psiBβ levels. In contrast, overexpression of ppoA led to a 15 fold and two fold increase in psiBα and psiBβ levels respectively. The presence of linoleic or oleic acid derived psiA or psiC was not detected in any samples in accordance to previous studies (11). These data demonstrated that PpoA is involved in the production of psiB components and suggested that linoleic acid can be a preferable substrate for PpoA.
Deletion of ppoA results in an increase of the asexual to sexual spore ratio; overexpression of ppoA has the opposite effect.
Spore production on GMM, media typically used to produce conidia (11), was measured three and five days after inoculation (Fig. 2). Under light conditions there was a significant increase in conidial production in the ∆ppoA strain at day 5 (p<0.01) ( Fig. 2A). Under dark conditions ∆ppoA produced significantly more conidia than the wild type at both time points (p<0.01) (Fig. 2B) but lower number of ascospores at day 5 compared to wild type (p<0.01) (Fig.  2C). The ratio of asexual spore development to sexual spore development under dark conditions increased approximately four fold in the ∆ppoA mutant after five days of cultivation (Table IV).
Sexual spore production was also assessed for cultures grown on YGT media in the dark, a condition commonly used to promote sexual spore development (9,10,31). The number of Hülle cells at day 2 and ascospores at day 4 was not affected by deletion of ppoA. However, after six days ascospore numbers decreased significantly in the ∆ppoA mutant (p<0.01) (data not shown), thus showing similarity to the ∆ppoA strain grown on GMM (Fig. 2).
Next we examined the effect of misscheduled expression of ppoA by fusing the ppoA gene to the constitutive promoter gpdA and to the inducible promoter niiA. The gpdA(p)::ppoA strain showed a significant increase in sexual spore production and a decrease in asexual sporulation under both light and dark conditions on GMM ( Fig. 3A and 3B). These results were maintained over a time period of 10 days (data not shown). In stark contrast to the ∆ppoA phenotype, the ratio of asexual to sexual spore development decreased approximately six fold in the gpdA(p)::ppoA strain after five days of cultivation (Table IV). With the exception of ascospore numbers during growth in dark, similar results were obtained with the nitrogen inducible niiA(p)::ppoA strain ( Fig. 3A and 3B). Growth of the niiA(p)::ppoA strain on the niiA repressing source ammonia yielded wild-type phenotype (data not shown) indicating that the expression level of ppoA is correlated with the increased sexual spore phenotype.

PpoA localizes in lipid bodies in Hülle cells and metulae.
The cellular localization of the PpoA protein was determined by constructing a ppoA::green fluorescent protein (GFP) fusion. Microscopic observations revealed GFP localization only to metulae (Fig. 4B), Hülle cells (Fig. 4C) and initial stages of cleistothecia formation (Fig. 4D). Metulae are mononuclear cells branching from conidiophore vesicles.
During asexual development both GFP and Nile Red dye, specific for lipid bodies (32), were localized to metulae cells (Fig. 4B). During sexual development, PpoA initially localized in Hülle cells formed at the stage of cleistothecial primordium formation (Fig. 4A) and subsequently in immature cleistothecia (12-24h old) as seen in Fig. 4C-4D. Further analyses indicated that the GFP was restricted to distinct globular organelles inside these cells. These organelles were also labeled by Nile Red. DAPI staining and a peroxisome specific GFP-fused protein showed that PpoA was not localized in either the nucleus or the peroxisome (data not shown). In contrast to the gpdA(p)::ppoA::GFP strain, the control strain the gpdA(p):GFP fusion did not show any specific localization patterns but the fluorescence was diffused in the cytoplasm of hyphae, conidiophores and conidia, Hülle cells and cleistothecia ( Fig 4E).
The co-localization of GPF-PpoA fusion protein with the Nile Red stained organelles in the Hülle cells and metulae suggested that PpoA localizes to lipid bodies. In support of this case, analysis of the amino acid sequence of PpoA indicated that it contains a conserved hydrophobic subdomain known as the "proline knot" that is characteristic for targeting plant proteins to lipid bodies ( Fig. 5) (37,38). This motif is also found in Lds and Ssp1 proteins, the latter shown also to be a spore specific lipid body protein in U. maydis teliospores (33).

ppoA expression is developmentally regulated.
Considering the phenotype of the ppoA deletion and overexpression strains as well as the localization of PpoA, we hypothesized that ppoA mRNA would be developmentally regulated.
ppoA transcript was analyzed from asexually and sexually induced wild type cultures. As depicted in Figure 6A, upon switching from mycelial growth to asexual spore inducing conditions (Panel A), ppoA was highly expressed at 6h and 12h, tapering off at 24h, the time of Considering the importance of VeA in sexual and asexual development, we speculated that veA allele would affect ppoA expression and/or PpoA activity. Therefore, we compared the expression of the ppoA gene in veA strain to veA1 and ∆veA mutants. As shown in Figure 6B, Further evidence of a PpoA/VeA interaction was observed by comparing the ∆ppoA; veA and ∆ppoA; veA1 phenotypes. Although both strains showed an increase in conidia production compared to their respective ppoA wild type (p<0.01) ( Fig. 2A, 2B and Fig. 7A, 7B), the ∆ppoA; veA1 strain also showed an increase in ascospore production in the dark in contrast to the ∆ppoA; veA strain (Fig. 2C, 7C). Ascospore production is inhibited in the veA1 genotype and this may suggest that inhibition requires PpoA activity in this genetic background. Deletion of ppoA in the ∆veA background did not alter the phenotype of a ∆veA strain (data not shown). Taken together, these data demonstrate that VeA is required for normal ppoA expression and that VeA/PpoA interactions affect sexual and asexual development.
ppoA expression is regulated by the COP9 signalosome.
Psi factor was originally isolated and chemically characterized from the psi overproducing acoC and acoB mutants, WIM145 (acoC193) and WIM146 (acoB202) respectively (8). AcoB and AcoC 3 show high amino acid sequence similarity to components of the plant and mammalian multiprotein complex COP9 signalosome (CSN) (39). The CSN complex is conserved from fission yeast to humans and functions in various physiological processes such as cell cycle, transcriptional control, light-and hormone-dependent pathways (40). Transcript analysis showed that ppoA expression is misscheduled and upregulated in both the acoC193 and acoB202 mutants (Fig. 6D, Panels A and B and data not shown), thus correlating ppoA expression with psi over production in these strains.

DISCUSSION
A striking aspect of morphogenesis in many filamentous fungi is the presence of both an asexual and a sexual spore reproductive cycle. Due to the importance of fungal spores in initiating a plant or mammalian infection, establishing a fungal colony and dissemination as well as the importance of sexual fruiting bodies as overwintering structures and aids in genus and species identification, there has been considerable interest in identifying factors that regulate fruiting body and spore formation. In our studies we are focusing on identifying endogenous signals that can integrate and balance anamorph and teleomorph development. Here we describe an A. nidulans lipid body protein, PpoA, a putative oxylipin producing oxygenase that regulates the asexual to sexual spore ratio.

PpoA is involved in the production of the linoleic acid derived oxylipin psiBα.
Chemical analysis of ∆ppoA and OE::ppoA strains demonstrated that deletion of ppoA did not alter the major fatty acid profile in A. nidulans. However, the ∆ppoA mutant was crippled in its ability to synthesize psiBα (Table III), otherwise known as 8-HODE, the same metabolite synthesized by G. graminis Lds (14). Additionally, the amount of psiBα detected in the overexpression ppoA strain was ca. 15 fold greater than that of wild type (Table IV). Slight increases in psiBβ (8-HOE) content, the oleic acid derived psi moiety, in ∆ppoA and OE::ppoA strains possibly indicated that another enzyme responsible for the oxygenation of oleic acid was upregulated in both mutants. However, we can not exclude the possibility that oleic acid can also act as a minor substrate for PpoA since 8-HOE was increased approximately two fold in the OE::ppoA strain. The fact that the other components of psi factor, psiC and psiA were not detected indicates that either these compounds are in low undetected levels, or are unstable.
PsiCβ was previously identified in a mutant strain that accumulates significant amounts of oleic acid (11). In the case of psiA, we can not exclude the possibility that it is an artifact of the extraction methodology that used for its characterization (12,13). Further biochemical analyses will clarify the actual substrates of the ppoA in vitro.

PpoA activity increases sexual sporulation and decreases asexual sporulation.
Deletion of ppoA led to a four fold increase in the asexual to sexual spore ratio, whereas overexpression of ppoA led to a six fold decrease in the asexual to sexual spore ratio (Fig. 2, 3 and Table IV). These results suggest that the PpoA product(s) act as positive regulator(s) of ascosporogenesis and/or negative regulator(s) of conidiation. Champe et al. (10) observed that exogenously applied psiBα induces premature cleistothecia formation and sexual sporulation and inhibits conidiation, which are characteristics of the OE::ppoA phenotype. This suggests an important role for psiBα in the ∆ppoA and OE::ppoA phenotypes. It is also possible that the phenotypic changes were due to the altered ratio of psiBα to psiBβ in the mutants (Table IV) or even downstream derivatives of psiB oxylipins or any combinations thereof. For instance, Calvo et al. (11) noted a correlation between increasing psiBβ content and ascospore formation, as we also observed in the ∆ppoA strain. Interestingly, exogenously applied seed linoleic acid derived oxylipins also alter the asexual to sexual spore ratio in wild type A. nidulans (31). Together these studies strongly support a role for oxylipins in maintaining a balance between sexual and asexual spore development in A. nidulans. Considering that putative homologs exist in all the filamentous fungi examined, it is possible that this phenomenon is conserved in other fungi.
Deletion of Ssp1, a protein that is predominantly expressed in mature teliospores of U. maydis led to no altered phenotype, however assessment of spore production was not reported (33).

PpoA is a lipid body protein and localizes in fruiting bodies.
Microscopic studies revealed that PpoA co-localized with organelles stained by Nile Red, a dye predictive of lipid bodies (Fig. 4). Lipid bodies are macromolecular proteolipid assemblies that contain neutral lipids important as storage fats and oils (41,42). The lipid bodies' surface proteins probably play a role in lipid-body biogenesis, trafficking, mobilization and metabolism (43,44). Lipid body proteins include prostaglandin H synthases in mammalian tissues (45), oleosin (38), caleosin and isoforms of lipoxygenases (46) in plant tissues and Ssp1 in teliospores of U. maydis (33). Previous studies have also demonstrated that the plant oleosin protein is targeted to the lipid bodies in a transformed yeast strain indicating that the lipid body localization motifs are conserved between plants and fungi (47). As shown in Figure 5

PpoA is expressed during the formation of fruiting bodies.
Our GFP studies indicated that the location of PpoA is highly correlated with its target tissues, conidiophores and cleistothecia. Figure 6A (Panels A-B) shows that ppoA expression was also associated with formation of these fruiting bodies. This correlation suggested that PpoA activity is important over time and space for normal sporulation processes in A. nidulans.
Constitutive expression of ppoA led to precocious sexual development and to delayed asexual spore formation, phenomena that were also correlated with the observed significant increase in psiB production. Examination of the putative promoter region of ppoA revealed the presence of binding domains for several known fungal (e.g. stuA) and mammalian (e.g. SREBP-1) transcription factors suggesting that the transcriptional regulation of ppoA is under the control of complex cellular molecular pathways. Huber et al. (33) analyzed the 5' region of the ssp1 gene and by deletion studies identified undefined promoter regions that are essential in maintaining the gene in a repressed state during saprophytic growth as well as an element that appears to be necessary for stage-specific regulation or induction in spores.

PpoA is regulated by veA.
The sporogenic response to linoleic acid moieties requires the presence of an intact veA gene in A. nidulans (31). The veA gene is known to have a role in activating sexual development and/ or inhibiting asexual development (5)(6)(7). Two lines of evidence (Fig. 6B, 6C) suggested that veA regulates directly or indirectly the transcription of ppoA gene: (i) in veA1 mutants ppoA was expressed at very high levels only during asexual development and (ii) deletion of veA caused inhibition of ppoA transcript accumulation. veA1 mutants are sexually defective; however disruption of ppoA in veA1 background led to wild type level ascospore production suggesting that the early high level expression of ppoA in this mutant might account for the significant increase in conidia production and decrease in ascospore development (Fig. 7). The absence of ppoA transcripts in ∆veA strains might also be correlative with the sexual defects in this strain.
Psi factor was first identified in acoB202 and acoC193 mutants, pleiotrophic strains where psi compounds are over produced (8,10). Figure 6D (Panels A-B) shows that ppoA was upregulated in the acoC193 strain, thus indicating that AcoC negatively regulates ppoA expression. Similar results were obtained examining the acoB mutant (data not shown). The amino acid sequence of AcoB (GenBank Accession # U18265) reveals that it has high similarity to the Arabidopsis CSN7 protein and AcoC 3 protein is identical to the cloned A. nidulans csnD gene (39). CSN7 and CSND are components of the COP9 signalosome, the eight-component complex found in all multicellular eukaryotes (40). Recent data indicate that the A. nidulans COP9 signalosome is a key regulator of light dependent signaling and asexual and sexual development (39). Based on our data we speculate that the Aspergillus COP9 multiprotein complex controls the transcription factors that regulate the expression and/or transcript degradation of ppoA and as a consequence the production of the corresponding oxylipin products. The elevated levels of oxylipins in acoB202 and acoC193 strains may account for some of the developmental defects in these mutants.
In conclusion we have characterized the ppoA gene, which is likely responsible for the production of the sporogenic psi factor psiBα. PpoA was required for integrating asexual and       Some of the strains are not described in the text but were used for sexual crosses to create the final prototrophic strains.