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Glycosylphosphatidyl inositol (GPI)-anchored proteins in Candida albicans are responsible for a vast range of functions, and deletions in certain GPI-anchored proteins severely reduce adhesion and virulence of this organism. In addition, completely modified GPIs are necessary for virulence. GPI anchor biosynthesis is essential for viability and starts with the transfer of N-acetylglucosamine to phosphatidylinositol. This step is catalysed by a multi-subunit complex, GPI--N-acetylglucosarninyltransferase (GPI-GnT). In this, the first report to our knowledge on a subunit of the Candida GPI--GnT complex, we show that CaGpil 9p is the functional equivalent of the Saccharomyces cerevisiae Gpi19p. An N-terminal truncation mutant of CaGpi19p functionally complements a conditionally lethal S. cerevisiae gpi19 mutant. Further, we constructed a conditional null mutant of CaGPI19 by disrupting one allele and placing the remaining copy under the control of the MET3 promoter. Repression leads to growth defects, cell wall biogenesis aberrations, azole sensitivity and hyperfilamention. In addition, there is a noticeable gene dosage effect, with the heterozygote also displaying intermediate degrees of most phenotypes. The mutants also displayed a reduced susceptibility to the antifungal agent amphotericin B. Collectively, the results suggest that CaGPI19 is required for normal morphology and cell wall architecture. DOI 10.1099/mic.0.039628-0

scyllo-lnositol (SI) is a stereoisomer of inositol whose catabolism has not been characterized in bacteria. We found that Bacillus subtilis 168 was able to grow using SI as its sole carbon source and that this growth was dependent on a functional iol operon for catabolism of myo-inositol (MI; another inositol isomer, which is abundant in nature). Previous studies elucidated the MI catabolic pathway in B. subtilis as comprising multiple stepwise reactions catalysed by a series of lol enzymes. The first step of the pathway converts MI to scyllo-inosose (SIS) and involves the MI dehydrogenase IolG. Since IolG does not act on SI, we suspected that there could be another enzyme converting SI into SIS, namely an SI dehydrogenase. Within the whole genome, seven genes paralogous to iolG have been identified and two of these, iolX and iolW (formerly known as yisS and yvaA, respectively), were selected as candidate genes for the putative SI dehydrogenase since they were both prominently expressed when B. subtilis was grown on medium containing SI. iolX and iolW were cloned in Escherichia coil and both were shown to encode a functional enzyme, revealing the two distinct SI dehydrogenases in B. subtilis. Since inactivation of ioIX impaired growth with SI as the carbon source, IoIX was identified as a catabolic enzyme required for SI catabolism and it was shown to be [NAD.sup.+] dependent. The physiological role of IoIW remains unclear, but it may be capable of producing SI from SIS with NADPH oxidation. DOI 10.1099/mic.0.037499-0

Inositol is essential in eukaryotes, and must be imported or synthesized. Inositol biosynthesis in Saccharomyces cerevisiae is controlled by three non-essential genes that make up the inositol regulon: SclNO2 and SclNO4, which together encode a heterodimeric transcriptional activator, and ScOPI1, which encodes a transcriptional repressor. ScOpi1p inhibits the Sclno2-Sclno4p activator in response to extracellular inositol levels. An important gene controlled by the inositol regulon is SclNO1, which encodes inositol-3-phosphate synthase, a key enzyme in inositol biosynthesis. In the pathogenic yeast Candida albicans, homologues of the S. cerevisiae inositol regulon genes are 'transcriptionally rewired'. Instead of regulating the CAINO1 gene, CAINO2 and CAINO4 regulate ribosomal genes. Another Candida species that is a prevalent cause of infections is Candida glabrata; however, C. glabrata is phylogenetically more closely related to S. cerevisiae than C. albicans. Experiments were designed to determine if C. glabrata homologues of the inositol regulon genes function similarly to S. cerevisiae or are transcriptionally rewired. CglNO2, CglNO4 and CgOPI1 regulate CglNO1 in a manner similar to that observed in S. cerevisiae. However, unlike in S. cerevisiae, CgOPI1 is essential. Genetic data indicate that CgOPI1 is a repressor that affects viability by regulating activation of a target of the inositol regulon. DOI 10.1099/mic.0.030072-0

In Salmonella enterica serovar Typhimurium, the genomic island GEI4417/4436 has recently been identified to be responsible for myo-inositol (MI) utilization. Here, two of the four island-encoded permeases are identified as the MI transporters of this pathogen. In-frame deletion of iolT1 (STM4418) led to a severe growth defect, and deletion of iolT1 (STM4419) to a slight growth defect in the presence of MI. These phenotypes could be complemented by providing the putative transporter genes in trans. Bioluminescence-based reporter assays demonstrated a strong induction of their promoters [P.sub.iolT1] and [P.sub.iolT2] in the presence of MI but not of glucose. Deletion of iolR, which encodes the negative regulator of most genes involved in MI degradation, resulted in upregulation of PioiT1 and PioIT2, indicating that the expression of IolT1 and IolT2 is repressed by IolR. This finding was supported by bandshift assays using purified IolR. Both transporters are located in the membrane when expressed in Escherichia coli. Heterologously expressed IolT1 had its optimal activity at pH 5.5. Together with the strongly reduced MI uptake in the presence of protonophores, this indicates that IolT1 operates as a proton symporter. Using myo-[1,2-[[sup.3]H](N)]inositol, a saturable uptake activity of IolT1 with a [K.sub.m] value between 0.49 and 0.79 mM was determined in DH5[alpha] expressing IolT1, in S. enterica serovar Typhimurium strain 14028, and in mutant 14028 [DELTA]iolT2. Phylogenetic analysis of IolT1 identified putative MI transporters in Gram-negative bacteria also able to utilize MI. DOI 10.1099/mic.0.032250-0

myo-Inositol (inositol) is an essential nutrient that is used for building phosphatidylinositol and its derivatives in eukaryotes and even in some eubacteria such as the mycobacteria. As a consequence, fungal, protozoan and mycobacterial pathogens must be able to acquire inositol in order to proliferate and cause infection in their hosts. There are two primary mechanisms for acquiring inositol. One is to synthesize inositol from glucose 6-phosphate using two sequentially acting enzymes: inositol-3-phosphate synthase (Ino1p) converts glucose 6-phosphate to inositol 3-phosphate, and then inositol monophosphatase (IMPase) dephosphorylates inositol 3-phosphate to generate inositol. The other mechanism is to import inositol from the environment via inositol transporters. Inositol is readily abundant in the bloodstream of mammalian hosts, providing a source from which many pathogens could potentially import inositol. However, despite this abundance of inositol in the host, some pathogens such as the bacterium Mycobacterium tuberculosis and the protist parasite Trypanosoma brucei must be able to make inositol de novo in order to cause disease (M. tuberculosis) or even grow (T. brucei). Other pathogens such as the fungus Candida albicans are equally adept at causing disease by importing inositol or by making it de novo. The role of inositol acquisition in the biology and pathogenesis of the parasite Leishmania and the fungus Cryptococcus are being explored as well. The specific strategies used by these pathogens to acquire inositol while in the host are discussed in relation to each pathogen's unique metabolic requirements.

Inositol is considered a growth factor in yeast cells and it plays an important role in Candida as an essential precursor for phospholipomannan, a glycophosphatidylinositol (GPI)-anchored glycolipid on the cell surface of Candida which is involved in the pathogenicity of this opportunistic fungus and which binds to and stimulates human macrophages. In addition, inositol plays an essential role in the phosphatidylinositol signal transduction pathway, which controls many cell cycle events. Here, high-affinity myo-inositol uptake in Candida albicans has been characterized, with an apparent [K.sub.m] value of 240 [+ or -] 15 [mu]M, which appears to be active and energy-dependent as revealed by inhibition with azide and protonophores (FCCP, dinitrophenol). Candida myo-inositol transport was sodium-independent but proton-coupled with an apparent [K.sub.m] value of 11.04 [+ or -] 1.1 nM for [H.sup.+], equal pH 7.96 [+ or -] 0.05, suggesting that the C. albicans myo-inositoI-[H.sup.+] transporter is fully activated at physiological pH. C. albicans inositol transport was not affected by cytochalasin B, phloretin or phlorizin, an inhibitor of mammalian sodium-dependent inositol transport. Furthermore, myo-inositol transport showed high substrate specificity for inositol and was not significantly affected by hexose or pentose sugars as competitors, despite their structural similarity. Transport kinetics in the presence of eight different inositol isomers as competitors revealed that proton bonds between the C-2, C-3 and C-4 hydroxyl groups of myo-inositol and the transporter protein play a critical role for substrate recognition and binding. It is concluded that C. albicans myo-inositoI-[H.sup.+] transport differs kinetically and pharmacologically from the human sodium-dependent myo-inositol transport system and constitutes an attractive target for delivery of cytotoxic inositol analogues in this pathogenic fungus.

Disruption of sblA in Streptomyces lividans is discussed. This disruption makes expression of a heterologous alpha-amylase gene in the presence of glucose possible. In a transposition mutant of S. lividans TK24, the usually glucose-repressible expression of a heterologous alpha amylase gene was rendered resistant to glucose repression.

Enzyme assays suggest the involvement of 2-keto-myo-inositol and D-2,3-diketo-4-deoxy epi-inositol in the myo-inositol catabolism of Rhizobium leguminosarum bv. viciae. Growth of R. leguminosarum on myo-inositol increases the enzyme's myo-inositol dehydrogenase and 2-keto-myo-inositol dehydratase. Examination of the growth in glucose as carbon source and resuspension studies in myo-inositol show that O2 consumption is an inducible route. Mutation studies reveal the necessity of intra-cellular dicarboxylation for the repression of these enzymes. Isolation and studies of three transposon mutants that do not induce both enzymes suggest that myo-inositol is not vital as an energy source in bacteriods.