Triclosan inhibition of mycobacterial InhA in Saccharomyces cerevisiae: yeast mitochondria as a novel platform for in vivo antimycolate assays.

AIMS: To demonstrate the suitability of yeast to act as a novel biotechnological platform for conducting in vivo inhibition assays using drugs with low efficacies towards their mycobacterial targets, such as occurs in the situation with triclosan and InhA. METHODS AND RESULTS: A surrogate yeast host represented by Saccharomyces cerevisiae etr1Delta cells lacking Etr1p, the 2-trans-enoyl-thioester reductase of mitochondrial type 2 fatty acid synthase (FASII), was designed to rely on the Mycobacterium tuberculosis FASII enzyme InhA. Although InhA is 10,000 times less sensitive to the antimicrobial drug triclosan than is bacterial FabI, the respiratory growth of yeast cells depending on InhA was severely affected on glycerol medium containing triclosan. CONCLUSIONS: The yeast system could detect enzyme inhibition despite the use of a drug with only low efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: Tuberculosis affects a third of the human population, and InhA is a major drug target for combating this disease. InhA is inhibited by isoniazid, but triclosan-derived compounds are presently being developed as antimycolates. A demonstration of triclosan inhibition of InhA in yeast represents a meaningful variation in studying this effect in mycobacteria, because it occurred without the potentially confusing aspects of perturbing protein-protein interactions which are presumed vital to mycobacterial FASII, inactivating other important enzymes or eliciting a dedicated transcriptional response in Myco. tuberculosis.

Degradation of conjugated linoleic acid isomers in the yeast Saccharomyces cerevisiae.

Propagation of Saccharomyces cerevisiae cells in conjugated linoleic acid (CLA) medium resulted in activation of the transcriptional machinery that responds to fatty acids. Cells utilized efficiently trans-10,cis-12 CLA, but not the corresponding cis-9,trans-11 isomer, probably due to the formation of cis-3,trans-5-dienoyl-CoA intermediates that are recalcitrant to beta-oxidation.

Candida tropicalis Etr1p and Saccharomyces cerevisiae Ybr026p (Mrf1'p), 2-enoyl thioester reductases essential for mitochondrial respiratory competence.

We report here on the identification and characterization of novel 2-enoyl thioester reductases of fatty acid metabolism, Etr1p from Candida tropicalis and its homolog Ybr026p (Mrf1'p) from Saccharomyces cerevisiae. Overexpression of these proteins in S. cerevisiae led to the development of significantly enlarged mitochondria, whereas deletion of the S. cerevisiae YBR026c gene resulted in rudimentary mitochondria with decreased contents of cytochromes and a respiration-deficient phenotype. Immunolocalization and in vivo targeting experiments showed these proteins to be predominantly mitochondrial. Mitochondrial targeting was essential for complementation of the mutant phenotype, since targeting of the reductases to other subcellular locations failed to reestablish respiratory growth. The mutant phenotype was also complemented by a mitochondrially targeted FabI protein from Escherichia coli. FabI represents a nonhomologous 2-enoyl-acyl carrier protein reductase that participates in the last step of the type II fatty acid synthesis. This indicated that 2-enoyl thioester reductase activity was critical for the mitochondrial function. We conclude that Etr1p and Ybr026p are novel 2-enoyl thioester reductases required for respiration and the maintenance of the mitochondrial compartment, putatively acting in mitochondrial synthesis of fatty acids.

Saccharomyces cerevisiae Adr1p governs fatty acid beta-oxidation and peroxisome proliferation by regulating POX1 and PEX11.

Saccharomyces cerevisiae Adr1p is essential for fatty acid degradation and peroxisome proliferation. Here, the role of Adr1p was examined with respect to the transcriptional regulation of the Pip2p-Oaf1p dependent genes POX1 and PEX11. POX1 encodes the rate-limiting enzyme of peroxisomal beta-oxidation, acyl-CoA oxidase. The POX1 promoter was shown to contain a canonical Adr1p element (UAS1), within which the oleate response element (ORE) was nested. PEX11 codes for a peroxin that is critical for normal peroxisome proliferation, and its promoter was shown similarly to contain a UAS1-like element overlapping the ORE. Northern analysis demonstrated that transcriptional up-regulation of both POX1 and PEX11 was abolished in adr1 Delta mutant cells, and immunoblotting confirmed that the abundance of their gene products was dramatically reduced. Studies of an overlapping ORE/UAS1 arrangement in the CTA1 promoter revealed synergy between these elements. We conclude that overlapping ORE and UAS1 elements in conjunction with their binding factors Pip2p-Oaf1p and Adr1p coordinate the carbon flux through beta-oxidation with peroxisome proliferation.

The tetratricopeptide repeat domains of human, tobacco, and nematode PEX5 proteins are functionally interchangeable with the analogous native domain for peroxisomal import of PTS1-terminated proteins in yeast.

In the yeast Saccharomyces cerevisiae, beta-oxidation of fatty acids is compartmentalised in peroxisomes. Most yeast peroxisomal matrix proteins contain a type 1C-terminal peroxisomal targeting signal (PTS1) consisting of the tripeptide SKL or a conservative variant thereof. PTS1-terminated proteins are imported by Pex5p, which interacts with the targeting signal via a tetratricopeptide repeat (TPR) domain. Yeast cells devoid of Pex5p are unable to import PTS1-containing proteins and cannot degrade fatty acids. Here, the PEX5-TPR domains from human, tobacco, and nematode were inserted into a TPR-less yeast Pex5p construct to generate Pex5p chimaeras. These hybrid proteins were examined for functional complementation of the pex5delta mutant phenotype. Expression of the Pex5p chimaeras in pex5delta mutant cells restored peroxisomal import of PTS1-terminated proteins. Chimaera expression also re-established degradation of oleic acid, allowing growth on this fatty acid as a sole carbon source. We conclude that, in the context of Pex5p chimaeras, the human, tobacco, and nematode Pex5p-TPR domains are functionally interchangeable with the native domain for the peroxisomal import of yeast proteins terminating with canonical PTS1s. Non-conserved yeast PTS1s, such as HRL and HKL, did not interact with the tobacco PEX5-TPR domain in the two-hybrid system. HRL occurs at the C-terminus of the peroxisomal protein Eci1p, which is required for growth on unsaturated fatty acids. Although mutant pex5delta cells expressing a yeast/tobacco Pex5p chimaera failed to import a GFP-Eci1p reporter protein, they were able to grow on oleic acid. We reason that this is due to a cryptic PTS in native Eci1p that can function in a redundant system with the C-terminal HRL.

Peroxisomal degradation of trans-unsaturated fatty acids in the yeast Saccharomyces cerevisiae.

Degradation of trans-unsaturated fatty acids was studied in the yeast Saccharomyces cerevisiae. Propagation of yeast cells on trans-9 elaidic acid medium resulted in transcriptional up-regulation of the SPS19 gene, whose promoter contains an oleate response element. This up-regulation depended on the Pip2p-Oaf1p transcription factor and was accompanied by induction of import-competent peroxisomes. Utilization of trans fatty acids as a single carbon and energy source was evaluated by monitoring the formation of clear zones around cell growth on turbid media containing fatty acids dispersed with Tween 80. For metabolizing odd-numbered trans double bonds, cells required the beta-oxidation auxiliary enzyme Delta(3)-Delta(2)-enoyl-CoA isomerase Eci1p. Metabolism of the corresponding even-numbered double bonds proceeded in the absence of Sps19p (2,4-dienoyl-CoA reductase) and Dci1p (Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase). trans-2,trans-4-Dienoyl-CoAs could enter beta-oxidation directly via Fox2p (2-enoyl-CoA hydratase 2 and d-specific 3-hydroxyacyl-CoA dehydrogenase) without the involvement of Sps19p, whereas trans-2,cis-4-dienoyl-CoAs could not. This reductase-independent metabolism of trans-2,trans-4-dienoyl-CoAs resembled the situation postulated for mammalian mitochondria in which oleic acid is degraded through a di-isomerase-dependent pathway. In this hypothetical process, trans-2,trans-4-dienoyl-CoA metabolites are generated by Delta(3)-Delta(2)-enoyl-CoA isomerase and Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase and are degraded by 2-enoyl-CoA hydratase 1 in the absence of 2,4-dienoyl-CoA reductase. Growth of a yeast fox2sps19Delta mutant in which Fox2p was exchanged with rat peroxisomal multifunctional enzyme type 1 on trans-9,trans-12 linolelaidic acid medium gave credence to this theory. We propose an amendment to the current scheme of the carbon flux through beta-oxidation taking into account the dispensability of beta-oxidation auxiliary enzymes for metabolizing trans double bonds at even-numbered positions.

Predicting the function and subcellular location of Caenorhabditis elegans proteins similar to Saccharomyces cerevisiae beta-oxidation enzymes.

The role of peroxisomal processes in the maintenance of neurons has not been thoroughly investigated. We propose using Caenorhabditis elegans as a model organism for studying the molecular basis underlying neurodegeneration in certain human peroxisomal disorders, e.g. Zellweger syndrome, since the nematode neural network is well characterized and relatively simple in function. Here we have identified C. elegans PEX-5 (C34C6.6) representing the receptor for peroxisomal targeting signal type 1 (PTS1), defective in patients with such disorders. PEX-5 interacted strongly in a two-hybrid assay with Gal4p-SKL, and a screen using PEX-5 identified interaction partners that were predominantly terminated with PTS1 or its variants. A list of C. elegans proteins with similarities to well-characterized yeast beta-oxidation enzymes was compiled by homology probing. The possible subcellular localization of these orthologues was predicted using an algorithm based on trafficking signals. Examining the C termini of selected nematode proteins for PTS1 function substantiated predictions made regarding the proteins' peroxisomal location. It is concluded that the eukaryotic PEX5-dependent route for importing PTS1-containing proteins into peroxisomes is conserved in nematodes. C. elegans might emerge as an attractive model system for studying the importance of peroxisomes and affiliated processes in neurodegeneration, and also for studying a beta-oxidation process that is potentially compartmentalized in both mitochondria and peroxisomes.

Adr1p-dependent regulation of the oleic acid-inducible yeast gene SPS19 encoding the peroxisomal beta-oxidation auxiliary enzyme 2,4-dienoyl-CoA reductase.

The role of Saccharomyces cerevisiae Adr1p was examined with respect to the transcriptional regulation of the SPS19 gene encoding the peroxisomal beta-oxidation auxiliary enzyme 2,4-dienoyl-CoA reductase. The SPS19 promoter contains both an oleate response element that binds the Pip2p-Oaf1p transcription factor as well as a canonical Adr1p-binding element, termed UAS1(SPS19). Northern analysis demonstrated that transcriptional up-regulation of SPS19 was abolished in cells devoid of Adr1p. Expression of an SPS19-lacZ reporter gene was shown to be quiescent in the adr1Delta mutant and abnormally elevated in cells containing multiple ADR1 copies. UAS1(SPS19) was able to compete for formation of a specific complex between recombinant Adr1p-LacZ and UAS1(CTA1) representing the corresponding Adr1p-binding element in the promoter of the catalase A gene, and to interact directly with this fusion protein. We conclude that in the presence of fatty acids in the medium transcription of SPS19 is directly regulated by both Pip2p-Oaf1p and Adr1p.

A novel element in the promoter of the Saccharomyces cerevisiae gene SPS19 enhances ORE-dependent up-regulation in oleic acid and is essential for de-repression.

In Saccharomyces cerevisiae cells grown on oleic acid, genes encoding enzymes of beta-oxidation are induced by the interaction of a transcription factor composed of Pip2p and Oaflp with an oleate response element (ORE) in their promoters. The SPS19 gene, which encodes peroxisomal 2,4-dienoyl-CoA reductase, an auxiliary beta-oxidation enzyme, has been shown previously to be up-regulated by a canonical ORE. To determine whether additional elements contribute to this transcriptional upregulation, deletion analysis of the SPS19 promoter was conducted using SPS19-lacZ reporter genes. In a reporter construct containing a deletion adjacent to the ORE, transcriptional activation of SPS19 in oleic acid medium was impaired. Together with an additional segment that overlaps a portion of the canonical ORE, this region forms a continuous element (termed UAS(SPS19)) that is essential for de-repression of SPS19 when glucose levels are low. The potentially bi-partite UAS(SPS19) element was able to initiate bi-directional transcription from a promoterless CYC1-lacZ reporter construct under de-repression conditions, whereas the canonical ORE was not. In oleic acid-containing medium, UAS(SPS19) stimulated transcription of the reporter gene 2.4-fold compared to the intact SPS19 ORE, but did so only in the presence of Pip2p and Oaf1p. UAS(SPS19), which is similar to a transcriptional enhancer in the promoter of the sporulation-specific gene SPS4, was shown specifically to bind several proteins, including Pip2p and Oaflp. We propose that UAS(SPS19) and other sequences like it are required to enhance the transcriptional effects mediated by more specific response elements.

Function of human mitochondrial 2,4-dienoyl-CoA reductase and rat monofunctional Delta3-Delta2-enoyl-CoA isomerase in beta-oxidation of unsaturated fatty acids.

Human 2,4-dienoyl-CoA reductase (2,4-reductase; DECR) and rat monofunctional Delta(3)-Delta(2)-enoyl-CoA isomerase (rat 3, 2-isomerase; ECI) are thought to be mitochondrial auxiliary enzymes involved in the beta-oxidation of unsaturated fatty acids. However, their function during this process has not been demonstrated. Although they lack obvious peroxisomal targeting signals (PTSs), both proteins have been suggested previously to also occur in the mammalian peroxisomal compartment. The putative function and peroxisomal location of the two mammalian proteins can be examined in yeast, since beta-oxidation of unsaturated fatty acids is a compartmentalized process in Saccharomyces cerevisiae requiring peroxisomal 2,4-dienoyl-CoA reductase (Sps19p) and peroxisomal 3, 2-isomerase (Eci1p). A yeast sps19Delta mutant expressing human 2, 4-reductase ending with the native C-terminus could not grow on petroselinic acid [cis-C(18:1(6))] medium but could grow when the protein was extended with a PTS tripeptide, SKL (Ser-Lys-Leu). We therefore reason that the human protein is a physiological 2, 4-reductase but that it is probably not peroxisomal. Rat 3, 2-isomerase expressed in a yeast eci1Delta strain was able to re-establish growth on oleic acid [cis-C(18:1(9))] medium irrespective of an SKL extension. Since we had shown that Delta(2,4) double bonds could not be metabolized extra-peroxisomally to restore growth of the sps19Delta strain, we postulate that rat 3,2-isomerase acted on the Delta(3) unsaturated metabolite of oleic acid by replacing the mutant's missing activity from within the peroxisomes. Immunoblotting of fractionated yeast cells expressing rat 3, 2-isomerase in combination with electron microscopy supported our proposal that the protein functioned in peroxisomes. The results presented here shed new light on the function and location of human mitochondrial 2,4-reductase and rat monofunctional 3,2-isomerase.

Alternatives to the isomerase-dependent pathway for the beta-oxidation of oleic acid are dispensable in Saccharomyces cerevisiae. Identification of YOR180c/DCI1 encoding peroxisomal delta(3,5)-delta(2,4)-dienoyl-CoA isomerase.

Fatty acids with double bonds at odd-numbered positions such as oleic acid can enter beta-oxidation via a pathway relying solely on the auxiliary enzyme Delta(3)-Delta(2)-enoyl-CoA isomerase, termed the isomerase-dependent pathway. Two novel alternative pathways have recently been postulated to exist in mammals, and these additionally depend on Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase (di-isomerase-dependent) or on Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase and 2,4-dienoyl-CoA reductase (reductase-dependent). We report the identification of the Saccharomyces cerevisiae oleic acid-inducible DCI1 (YOR180c) gene encoding peroxisomal di-isomerase. Enzyme assays conducted on soluble extracts derived from yeast cells overproducing Dci1p using 3,5,8,11,14-eicosapentenoyl-CoA as substrate demonstrated a specific di-isomerase activity of 6 nmol x min(-1) per mg of protein. Similarly enriched extracts from eci1Delta cells lacking peroxisomal 3,2-isomerase additionally contained an intrinsic 3,2-isomerase activity that could generate 3, 5,8,11,14-eicosapentenoyl-CoA from 2,5,8,11,14-eicosapentenoyl-CoA but not metabolize trans-3-hexenoyl-CoA. Amplification of this intrinsic activity replaced Eci1p since it restored growth of the eci1Delta strain on petroselinic acid for which di-isomerase is not required whereas Eci1p is. Heterologous expression in yeast of rat di-isomerase resulted in a peroxisomal protein that was enzymatically active but did not re-establish growth of the eci1Delta mutant on oleic acid. A strain devoid of Dci1p grew on oleic acid to wild-type levels, whereas one lacking both Eci1p and Dci1p grew as poorly as the eci1Delta mutant. Hence, we reasoned that yeast di-isomerase does not additionally represent a physiological 3,2-isomerase and that Dci1p and the postulated alternative pathways in which it is entrained are dispensable for degrading oleic acid.

Peroxisomal Delta3-cis-Delta2-trans-enoyl-CoA isomerase encoded by ECI1 is required for growth of the yeast Saccharomyces cerevisiae on unsaturated fatty acids.

We have identified the Saccharomyces cerevisiae gene ECI1 encoding Delta3-cis-Delta2-trans-enoyl-CoA isomerase that acts as an auxiliary enzyme in the beta-oxidation of (poly)unsaturated fatty acids. A mutant devoid of Eci1p was unable to grow on media containing unsaturated fatty acids such as oleic acid but was proficient for growth when a saturated fatty acid such as palmitic acid was the sole carbon source. Levels of ECI1 transcript were elevated in cells grown on oleic acid medium due to the presence in the ECI1 promoter of an oleate response element that bound the transcription factors Pip2p and Oaf1p. Eci1p was heterologously expressed in Escherichia coli and purified to homogeneity. It was found to be a hexameric protein with a subunit of molecular mass 32, 000 Da that converted 3-hexenoyl-CoA to trans-2-hexenoyl-CoA. Eci1p is the only known member of the hydratase/isomerase protein family with isomerase and/or 2-enoyl-CoA hydratase 1 activities that does not contain a conserved glutamate at its active site. Using a green fluorescent protein fusion, Eci1p was shown to be located in peroxisomes of wild-type yeast cells. Rat peroxisomal multifunctional enzyme type I containing Delta3-cis-Delta2-trans-enoyl-CoA isomerase activity was expressed in ECI1-deleted yeast cells, and this restored growth on oleic acid.

Fate and role of peroxisomes during the life cycle of the yeast Saccharomyces cerevisiae: inheritance of peroxisomes during meiosis.

Sporulation in the yeast Saccharormyces cerevisiae is a meiotic developmental process that occurs in MATa/MATalpha heterozygotes in response to nutrient deprivation. Here, the fate and role of peroxisomes during sporulation and germination has been examined by a combination of immunoelectron microscopy and the use of pex mutants defective in peroxisomal functions. Using a green fluorescent protein probe targeted to peroxisomes we show that peroxisomes are inherited through meiosis and that they do not increase in number either during sporulation or spore germination. In addition, there is no requirement for peroxisome degradation prior to spore packaging. Unlike the situation in filamentous fungi, peroxisomes do not proliferate during the yeast life cycle. Functional peroxisomes are dispensable for efficient meiotic development on acetate medium since homozygous delta pex6 diploids sporulated well and produced mature spores that were resistant to diethyl ether. Like haploids, diploid cells can proliferate their peroxisomes in response to oleate as sole carbon source in liquid medium, but under these conditions they do not sporulate. On solid oleate medium, homozygous pex5, delta pex6, and pex7 cells were unable to sporulate efficiently, whereas the wild type was. The results presented here are discussed in terms of the transmission of organelles to progeny cells.

The Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA reductase is encoded by the oleate-inducible gene SPS19.

beta-Oxidation is compartmentalized in mammals into both mitochondria and peroxisomes. Fatty acids with double bonds at even-numbered positions require for their degradation the auxiliary enzyme 2,4-dienoyl-CoA reductase, and at least three isoforms, two mitochondrial and one peroxisomal, exist in the rat. The Saccharomyces cerevisiae Sps19p is 34% similar to the human and rat mitochondrial reductases, and an SPS19 deleted strain was unable to utilize petroselineate (cis-C18:1(6)) as the sole carbon source, but remained viable on oleate (cis-C18:1(9)). Sps19p was purified to homogeneity from oleate-induced cells and the homodimeric enzyme (native molecular weight 69,000) converted 2,4-hexadienoyl-CoA into 3-hexenoyl-CoA in an NADPH-dependent manner and therefore contained 2,4-dienoyl-CoA reductase activity. Antibodies raised against Sps19p decorated the peroxisomal matrix of oleate-induced cells. SPS19 shares with the sporulation-specific SPS18 a common promoter region that contains an oleate response element. This element unidirectionally regulates transcription of the reductase and is sufficient for oleate induction of a promoterless CYC1-lacZ reporter gene. SPS19 is dispensable for growth and sporulation on solid acetate and oleate media, but is essential for these processes to occur on petroselineate.

Regulation of the yeast SPS19 gene encoding peroxisomal 2,4-dienoyl-CoA reductase by the transcription factors Pip2p and Oaf1p: beta-oxidation is dispensable for Saccharomyces cerevisiae sporulation in acetate medium.

The yeast SPS19 gene encoding the peroxisomally targeted 2,4-dienoyl-CoA reductase shares its promoter region (291 bp) with the sporulation-specific gene SPS18. SPS19 is induced during sporulation in diploids but to a lesser extent than SPS18; under oleate induction conditions, SPS19, but not SPS18, is transcribed via an oleate response element (ORE) independently of ploidy or sporulation. The SPS19 ORE is the binding target of the Pip2p and Oaf1p transcription factors, and an SPS19-lacZ reporter gene, which is highly expressed in oleate-induced cells, is not activated in haploids devoid of either protein. We examined the expression of CYC1-lacZ reporter constructs carrying the SPS19 and CTA1 OREs in diploids propagated under sporulation conditions and have shown that OREs are not sufficient for heterologous expression during yeast development. In addition, diploids deleted at either PIP2 or OAF1 demonstrated abundant ascosporogenesis, indicating that these genes are not essential for sporulation. A deltapex6 strain lacking peroxisomal structures and one devoid of fatty acyl-CoA oxidase (deltapox1), the first step in fungal beta-oxidation, were both proficient for sporulation and, hence, beta-oxidation and the peroxisomal compartment containing it are dispensable for meiotic development.

Carrier detection in X-linked retinitis pigmentosa.

X-linked retinitis pigmentosa (XLRP) is manifested in affected males in their first decade and results in blindness by the third or fourth decade. Carrier detection is difficult since most carrier females show no or only equivocal signs well into or beyond their reproductive years. The genes, or the mutations causing RP have not been identified but at least two have been localised to the short arm of the X chromosome provisionally named RP2 and RP3. Identifying inheritance of one or other of these genes must be done by linkage in families using close, informative DNA markers. Here we report the localisation of a highly informative polymerase chain reaction (PCR) detectable microsatellite marker DXS538 using a previously studied family with X-linked RP3 in which recombination had occurred in the region of importance. The DXS538 dinucleotide repeat locus was previously localised to Xp21.1-p11.21 to study RP3 in one XLRP family. Using published RFLP data we narrowed the localisation of DXS538 to the region Xp21.1-p11.23. Thus DXS538 is now a convenient diagnostic tool, aiding carrier detection of XLRP in females, as shown in the family presented here.

Exploiting biological materials in forensic science.

One of the important problems in forensic science is the limited availability of biological samples left behind at the scene of the crime. Research in the area of obtaining DNA data from such limited biological samples has resulted in successful court convictions. The ability to carry out DNA fingerprinting from such minute sources relies both on the successful extraction of DNA as well as its subsequent characterisation and analysis. Improved techniques designed to obtain DNA from such samples combined with a small-volume polymerase chain reaction (PCR) and highly informative genetic markers will mean that law enforcement agencies will increasingly be better equipped to deal with violent crime. This review describes some of the sources from which DNA is obtained as well as the techniques used to derive from them valuable genetic information.

Use of reporter genes for the isolation and characterisation of different classes of sporulation mutants in the yeast Saccharomyces cerevisiae.

Reporter genes consisting of sporulation-specific promoters fused to lacZ were used as markers to monitor the sporulation pathway of the yeast Saccharomyces cerevisiae. Strains transformed with these lacZ gene fusions expressed beta-galactosidase (assayable on plates using the substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, X-gal) in a sporulation-dependent manner. Mutagenesis experiments performed on transformed strains resulted in the recovery of a number of novel sporulation mutants. Three classes of mutants were obtained: those which overexpressed the reporter gene under sporulation conditions, those which did not express the gene under any conditions, and those which expressed the gene in vegetative cells not undergoing sporulation. On the basis of the blue colony-colour produced in the presence of X-gal these have been described as superblue, white, and blue vegetative mutants, respectively. These were further characterised using earlier reporter genes and other marker systems. This study established that the multicopy reporter plasmids chosen do not interfere with sporulation; they are valid tools for monitoring the pathway and they provide a way to isolate mutations not readily selected by other markers.