Culture and molecular identification of fungal contaminants in edible bird nests.

Widespread food poisoning due to microbial contamination has been a major concern for the food industry, consumers and governing authorities. This study is designed to determine the levels of fungal contamination in edible bird nests (EBNs) using culture and molecular techniques. Raw EBNs were collected from five house farms, and commercial EBNs were purchased from five Chinese traditional medicine shops (companies A-E) in Peninsular Malaysia. The fungal contents in the raw and commercial EBNs, and boiled and unboiled EBNs were determined. Culturable fungi were isolated and identified. In this study, the use of these methods revealed that all EBNs had fungal colony-forming units (CFUs) that exceeded the limit set by Standards and Industrial Research Institute of Malaysia (SIRIM) for yeast and moulds in EBNs. There was a significant difference (p < 0.05) in the number of types of fungi isolated from raw and commercial EBNs, but no significant difference in the reduction of the number of types of fungi after boiling the EBNs (p > 0.05). The types of fungi isolated from the unboiled raw EBNs were mainly soil, plant and environmental fungi, while the types of fungi isolated from the boiled raw EBNs, unboiled and boiled commercial EBNs were mainly environmental fungi. Aspergillus sp., Candida sp., Cladosporium sp., Neurospora sp. and Penicillum sp. were the most common fungi isolated from the unboiled and boiled raw and commercial EBNs. Some of these fungi are mycotoxin producers and cause opportunistic infections in humans. Further studies to determine the mycotoxin levels and methods to prevent or remove these contaminations from EBNs for safe consumption are necessary. The establishment and implementation of stringent regulations for the standards of EBNs should be regularly updated and monitored to improve the quality of the EBNs and consumer safety.

The structural gene for a phosphorus-repressible phosphate permease in Neurospora crassa can complement a mutation in positive regulatory gene nuc-1.

van+, a gene encoding a phosphorus-repressible phosphate permease, was isolated by its ability to complement nuc-1, a positive regulatory locus that normally regulates van+ expression. This was unexpected because the nuc-1 host already contained a resident van+ gene. Plasmids carrying van+ complemented a nuc-2 mutation as well. Probing of RNA from untransformed wild-type (nuc-1+) and constitutive (nuc-1c) strains by van+ probes indicated that levels of the van+ transcript were subject to control by nuc-1+. Probing of the same RNAs with a cosmid clone, containing approximately 15 kilobases of upstream and downstream DNA, revealed no other detectable phosphorus-regulated transcripts within this 40-kilobase region of the chromosome.

The Neurospora crassa metallothionein gene. Regulation of expression and chromosomal location.

The promoter region of the Neurospora crassa metallothionein gene contains no sequences which are similar to the mammalian or the yeast metal responsive elements (Münger, K., Germann, U. A., and Lerch, K. (1985) EMBO J. 4, 2665-2668). We therefore studied the regulation of expression of the N. crassa metallothionein gene in response to different metal ions (Cu2+, Cd2+, Zn2+, Co2+, and Ni2+) by Northern analysis. Only copper led to the induction of metallothionein mRNA. In N. crassa cultures inoculated and grown in copper-supplemented media, metallothionein mRNA appeared during the late logarithmic growth period (about 30 h after inoculation) and was detectable for a time period of more than 30 h. In response to copper shock, however, rapidly increasing amounts of metallothionein mRNA were detected within minutes after copper administration at any time in vegetatively growing mycelia of N. crassa. Maximum levels were detected about 1 h after addition of copper to the medium. The half-life time of the mRNA was estimated as 2.5 h. The amounts of copper metallothionein reach a maximum level at 3 h after induction and thereafter remain constant. The rapid induction by copper ions of metallothionein mRNA and metallothionein together with the remarkable stability of the native protein intracellularly suggest that this protein serves an important homeostatic role in the copper metabolism in this fungus. The structural gene of N. crassa metallothionein has been located on chromosome VI using restriction fragment-length polymorphisms as genetic markers.

Cloning of the ARO cluster gene of Neurospora crassa and its expression in Escherichia coli.

We have constructed a phage, lambda Ncl, which comprises a 4.0 kb HindIII insert of Neurospora DNA into the immunity region of the vector lambda 598. lambda Ncl complements the aroD6 mutation of E. coli, permitting the formation of galaxy plaques on medium lacking aromatic supplements, and transforms an aro-9 qa-2 Neurospora mutant to prototrophy at a low frequency. Low levels of 5-dehydroquinate hydrolyase (E.C.4.2.1.10.), with properties unlike those of the catabolic isoenzyme that is coded by qa-2, are present in E. coli aroD6 cell lysates following infection with lambda Ncl. lambda Ncl does not hybridize with qa-2 DNA and it is concluded that it contains at least the aro-9 region of the pentafunctional aro cluster gene.

General control of arginine biosynthetic enzymes in Neurospora crassa.

The response of six amino acid synthetic enzymes, including four concerned with arginine synthesis, one with histidine synthesis and one with lysine synthesis, to conditions of histidine and arginine limitation and to exogenously provided amino acids is described in Neurospora crassa. The activities of all these enzymes increased in response to lowered levels of histidine or arginine, but showed little or no repression in wild-type cultures supplemented with casein hydrolysate. The activity of glucose-6-phosphate dehydrogenase (an enzyme not concerned with amino acid synthesis) remained unaffected by any of these conditions. In the case of the four arginine synthetic enzymes evidence is presented that suggests the "general' system implicated in the 'cross-pathway' response to histidine is also entirely responsible for the derepression occurring under conditions of arginine limitation. Further investigations into the control of one enzyme, ornithine carbamoyltransferase, are also reported. These show that the enzyme is stable, and suggest that its derepression in response to histidine limitation entails new protein synthesis and involves control at a stage prior to translation.

Isolation and characterization of Neurospora crassa mutants impaired in feedback control of ornithine synthesis.

Thirty-two independent mutants were isolated which overcame the proline requirement of pro-3 mutations in Neurospora crassa. The mutations were not revertants, appeared to be allelic, were closely linked or allelic to arg-6, and in strains unable to degrade ornithine no longer suppressed the proline requirement. The suppressor mutations did not alter the levels of biosynthetic or catabolic enzymes, yet allowed accumulation of ornithine. Suppressed strains unable to degrade arginine still produced ornithine (as detected by growth) in arginine-supplemented medium. The results suggest that the suppressor mutants were impaired in the feedback inhibition of ornithine synthesis by arginine. The activity of the appropriate biosynthetic enzyme was less sensitive to inhibition by arginine. The potential usefulness of such mutations is discussed.

Genetic control of phosphorus assimilation in Neurospora crassa: dose-dependent dominance and recessiveness in constitutive mutants.

Mutants called nuc-1c, constitutive for alkaline phosphatase synthesis, were isolated and mapped very close to nuc-1 mutants in which this enzyme is not expressed. nuc-1 is epistatic to nuc-1c. nuc-1c acts only if it is cis to normal nuc-1 function. The preparation of partial diploids heterozygous for various nuc-1 alleles is described; nuc-1c is dominant to nuc-1+, which in turn is dominant to nuc-1. In heterocaryons with nuc-1+, nuc-1c is dominant when it is present in high proportion, but essentially recessive if it is present in low proportions. In heterocaryons with nuc-1, nuc-1c is again dominant when present in high proportions, but in low proportions it "complements" to give essentially normal repressibility. A model of regulation consistent with these findings is presented.

A comparative study of DNA-induced transformants and spontaneous revertants of inositolless Neurospora crassa.

Inositolless (inl-) Neurospora crassa strains were treated with DNA (allo-DNA) of wild type N. Crassa. Hyphal fragments of a mycelial suspension of the N. Crassa ragged inl- strain used as recipient in our transformation experiments were found to consist of units containing 100--1000 nuclei. In this strain the inositol-independent (inl+) nuclei appearing after DNA treatment or by spontaneous reversion are present in the cytoplasm together with a large number of inl- nuclei. Thus, both transformation and reversion initially must result in heterokaryosis. Under appropriate conditions the inl- nuclei can be detected in the transformed and spontaneous inl+ phenotype revertant strains. Spontaneous revertants are usually characterized by the loss of their inl+ nuclei after transfers on inositol-supplemented medium. On minimal medium, the growth rate of transformed strains is significantly lower than that of spontaneous revertants. The inl+ gene appearing after DNA treatment or by spontaneous reversion is inherited as a trait bound to chromosomes. In crosses with the transformed strains, there is a significant increase in the number of non-Mendelian (6:2 and 5:3) tetrads in the inl locus.