SummaryWhile it is generally accepted that anaerobic metabolism is required during infection, supporting experimental data have only been described in a limited number of studies. To provide additional evidence on the role of anaerobic metabolism in bacterial pathogens while invading mammalian hosts, we analysed the effect of the inactivation of FNR, the major regulatory protein involved in the adaptation to oxygen restrictive conditions, and of two of the FNRregulated genes on the survival of Neisseria meningitidis serogroup B ( MenB ) in vivo . We found that fnr deletion resulted in more than 1 log reduction in the meningococcal capacity to proliferate both in infant rats and in mice. To identify which of the FNRregulated genes were responsible for this attenuated phenotype, we defined the FNR regulon by combining DNA microarray analysis and FNR-DNA binding studies. Under oxygen-restricted conditions, FNR positively controlled the transcription of nine transcriptional units, the most upregulated of which were the two operons NMB0388-galM and mapA-pgm b implicated in sugar metabolism and fermentation. When galM and mapA were knocked out, the mutants were attenuated by 2 and 3 logs respectively. As the operons are controlled by FNR, from these data we conclude that MenB survival in the host anatomical sites where oxygen is limiting is supported by sugar fermentation.

Our whole-genome microarray studies of Neisseria meningitidis MC58 previously identified a set of 153 genes whose transcription was activated during growth in iron. In this study, Fur-mediated regulation of the iron-activated nspA gene was confirmed, whereas iron-activated regulation of the secY gene was demonstrated to be Fur independent. Analysis of the Fur binding sequences in the nspA gene and an additional iron-activated and Fur-regulated gene identified a hexameric (G/T)ATAAT unit in the operator regions of these genes similar to that observed in Fur-and iron-repressed genes. These studies indicate that the expression of the ironactivated nspA and secY genes in N. meningitidis occur by Fur-dependent and -independent mechanisms, respectively.It is well established that the iron-responsive regulatory protein Fur functions as a repressor of gene transcription in several microorganisms. In its most basic state, Fur forms a dimer together with divalent cations, such as ferrous iron, and binds to a consensus sequence (the Fur box) that overlaps the promoters of iron-regulated genes to prevent their transcription. Recent studies indicate that in some organisms, Fur may also function as a positive regulator of gene transcription, together with iron (8,(9)(10)(11)(12)24), although the mechanism of iron activation by Fur is not well elucidated. Our recent studies of N. meningitidis group B, using a combination of microarray technology, computational analysis, and in vitro binding studies, revealed that a large number of genes are activated during growth in the presence of iron and that a number of these iron-activated genes have putative Fur-binding sequences to which Fur was demonstrated to bind (17). However, the biological significance of Fur binding to the operator regions of these genes has not been defined, as an N. meningitidis fur mutant had not been constructed at the time of that study.Of interest within the group of iron-activated genes under the potential control of Fur were candidate genes involved in the virulence potential of N. meningitidis, including the nspA (NMB0663) (1, 21) and secY (NMB0162) (18, 28) genes. Neisserial surface protein A (NspA) is an 18.6-kDa membrane protein of unknown function that was first described to confer protection against meningococcal infection in animal models of infection (1,21,22). NspA is highly conserved and expressed by all N. meningitidis strains tested (22,25). Recent studies indicate that conserved epitopes of the NspA protein confer protection against N. meningitidis serogroup B challenge in a mouse model of meningococcal infection (26). The N. meningitidis secY gene encodes a putative preprotein translocase (SecY) whose homolog in Escherichia coli has been studied extensively; in E. coli, it functions as an essential component of the protein translocation machinery of the cytoplasmic membrane. Sec-dependent protein secretion in the pathogenic Neisseria has been reported (18, 28); however, the function of the N. meningitidis SecY protein is not known, nor has ...

Thermophilic microorganisms which can colonize at extreme ecological niches are known as extremophiles. Because of their capacity to withstand high temperatures, enzymes from these organisms are relatively heat stable. The versatile enzyme properties of these organisms make them excellent candidates in biotechnology. In general, fungi have been widely used for the production of proteins and enzymes, since they can grow rapidly in a low cost media and they secrete proteins into the extra-cellular medium. Recently, these organisms have also been used in large scale fermentation as host for the expression of heterologous proteins in industrial applications. However, little is known about the regulation and genetic manipulations of these fungi. We have previously shown the regulation of gene expression in a thermophilic fungus, Thermyces lanuginosus, using an inducible invertase system. The aim of this review is to elucidate the recent advances of thermophilic fungi, and their implications in industrial applications are discussed.