Deletion of the Caenorhabditis elegans homologues of the CLN3 gene, involved in human juvenile neuronal ceroid lipofuscinosis, causes a mild progeric phenotype.

The CLN3 gene is involved in juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten-Spielmeyer-Vogt disease, a severe hereditary neurodegenerative lysosomal storage disorder characterized by progressive disease pathology, with loss of vision as the first symptom. Another characteristic of JNCL is the lysosomal accumulation of autofluorescent lipopigments, forming fingerprint storage patterns visible by electron microscopy. The function of the CLN3 protein is still unknown, although the evolutionarily conserved CLN3 protein is being functionally analysed using different experimental models. We have explored the potential of the nematode Caenorhabditis elegans as a model for Batten disease in order to bridge the gap between the unicellular yeast and very complex mouse JNCL models. C. elegans has three genes homologous to CLN3, for each of which deletion mutants were isolated. Cln-3.1 deletion mutants have a decreased lifespan, and cln-3.2 deletion mutants a decreased brood size. However, the neuronal or movement defects and aberrant lipopigment distribution or accumulation observed in JNCL were not found in the worms. To detect possible redundancy, single deletion mutants were crossed to obtain double and triple mutants, which were viable but showed no JNCL-specific defects. The cln-3 triple mutants show a more prominent decrease in lifespan and brood size, the latter most conspicuously at the end of the egg-laying period, suggesting premature ageing. To focus our functional analysis we examined the C. elegans cln-3 expression patterns, using promoter-GFP (green fluorescent protein) gene fusions. Fluorescence patterns suggest cln-3.1 expression in the intestine, cln-3.2 expression in the hypoderm, and cln-3.3 expression in intestinal muscle, male-specific posterior muscle and hypoderm. Further life stage- and tissue-specific analysis of the processes causing the phenotype of the cln-3 triple mutants may provide more information about the function of the cln-3 protein and contribute to a better understanding of the basic processes affected in Batten disease patients.

Caenorhabditis elegans homologues of the CLN3 gene, mutated in juvenile neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCLs) are the most common hereditary neurodegenerative disorders of childhood. The first symptom of this heterogeneous group of devastating lysosomal storage diseases is progressive visual failure. The different forms of NCL can be distinguished by age of onset, clinical features and the characteristics of the accumulated materials. The juvenile form, Batten-Spielmeyer-Vogt disease which is caused by mutations in the CLN3 gene, is the most frequent form of the disease in which loss of vision becomes apparent around the age of 5-8 years. The gene was found to encode a novel integral membrane protein localizing to the lysosomes, confirming that the primary defect in NCL is in lysosomal function. The CLN3 protein function is still unknown, and is examined in several model organisms. We are studying the nematode Caenorhabditis elegans, and have identified three CLN3 homologues. In order to investigate the role of the CLN3 protein in C. elegans, Cecln-3 deletion mutants are being isolated from an ethyl methanesulphonate (EMS)-induced deletion mutant library. Examination of these mutants may provide us with information that will help in dissecting the processes in which the CLN3 protein is involved. In this library two mutated C. elegans Cln-3 loci have been identified, of which one mutant, NL748, was isolated. This mutant contains a deletion of the whole gene. The deletion mutant was characterized with regard to life expectancy, and showed no significant differences when compared with wild-type.

Phenazine-1-carboxamide production in the biocontrol strain Pseudomonas chlororaphis PCL1391 is regulated by multiple factors secreted into the growth medium.

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. The production of phenazine-1-carboxamide (PCN) is crucial for this biocontrol activity. In vitro production of PCN is observed only at high-population densities, suggesting that production is under the regulation of quorum sensing. The main autoinducer molecule produced by PCL1391 was identified structurally as N-hexanoyl-L-homoserine lactone (C6-HSL). The two other autoinducers that were produced comigrate with N-butanoyl-L-homoserine lactone (C4-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL). Two PCL1391 mutants lacking production of PCN were defective in the genes phzI and phzR, respectively, the nucleotide sequences of which were determined completely. Production of PCN by the phzI mutant could be complemented by the addition of exogenous synthetic C6-HSL, but not by C4-HSL, C8-HSL, or any other HSL tested. Expression analyses of Tn5luxAB reporter strains of phzI, phzR, and the phz biosynthetic operon clearly showed that phzI expression and PCN production is regulated by C6-HSL in a population density-dependent manner. The introduction of multiple copies of the regulatory genes phzI and phzR on various plasmids resulted in an increase of the production of HSLs, expression of the PCN biosynthetic operon, and consequently, PCN production, up to a sixfold increase in a copy-dependent manner. Surprisingly, our expression studies show that an additional, yet unidentified factor(s), which are neither PCN nor C4-HSL or C8-HSL, secreted into the growth medium of the overnight cultures, is involved in the positive regulation of phzI, and is able to induce PCN biosynthesis at low cell densities in a growing culture, resulting in an increase of PCN production.