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The production of most factors involved in Bordetella pertussisvirulence is controlled by a two-component regulatory system termed BvgA/S. In the Bvg + phase virulence-activated genes ( vags) are expressed, and virulence-repressed genes ( vrgs) are down-regulated. The expression of these genes can also be modulated by MgSO 4 or nicotinic acid. In this study we used microarrays to analyse the influence of BvgA/S or modulation on the expression of nearly 200 selected genes. With the exception of one vrg, all previously known vags and vrgs were correctly assigned as such, and the microarray analyses identified several new vags and vrgs, including genes coding for putative autotransporters, two-component systems, extracellular sigma factors, the adenylate cyclase accessory genes cyaBDE, and two genes coding for components of a type III secretion system. For most of the new vrgs and vags the results of the microarray analyses were confirmed by RT-PCR analysis and/or lacZfusions. The degree of regulation and modulation varied between genes, and showed a continuum from strongly BvgA/S-activated genes to strongly BvgA/S-repressed genes. The microarray analyses also led to the identification of a subset of vags and vrgs that are differentially regulated and modulated by MgSO 4 or nicotinic acid, indicating that these genes may be targets for multiple regulatory circuits. For example, the expression of bilA, a gene predicted to encode an intimin-like protein, was found to be activated by BvgA/S and up-modulated by nicotinic acid. Furthermore, surprisingly, in the strain analysed here, which produces only type 2 fimbriae, the fim3 gene was identified as a vrg, while fim2 was confirmed to be a vag.

We describe in this paper the preparation and characterization of semicarbazide glass slides and their use for the fabrication of microarrays using site-specific α-oxo semicarbazone ligation. The functional density and homogeneity of the semicarbazide glass slides were optimized by analyzing the reactivity of the layer toward a synthetic glyoxylyl fluorescent probe. Oligonucleotide microarrays were prepared by site-specific immobilization of glyoxylyl oligodeoxynucleotides. The slides were directly used in the hybridization assays using fluorescence detection and displayed a significant gain in sensibility as compared to the aldehyde glass slide/amino oligodeoxynucleotide chemistry. Semicarbazide slides were also used for the immobilization of a biotinylated peptide α-oxo aldehyde. The peptide microarrays allowed model interaction studies with streptavidin or an anti-biotin antibody.

Hirudin synthesized by the medicinal leech Hirudo medicinalis is the most potent antithrombic factor found in nature. We have used Saccharomyces cerevisiae for the efficient production of recombinant hirudin variant 2 (rHV2). The mature hirudin coding sequence was placed in frame with the prepro sequence of the yeast MFα1 gene. Multicopy plasmids carrying different fused genes transcribed from the MFα1 promoter were used to transform a yeast strain selecting for uracil prototrophy. Analysis of proteins from culture supernatants revealed efficient secretion of hirudin in all cases. However, only precursors in which the HV2 mature sequence directly followed the cleavage site for yscF (a protease involved in the maturation of yeast α factor) were processed correctly to yield biologically active hirudin.

Multiple or pleiotropic drug resistance in the yeast Saccharomyces cerevisiae can arise from overexpression of the Pdr5 and Snq2 ATP binding cassette multidrug transporters. Expression of Pdr5 and Snq2 is regulated by the two transcription factors Pdr1 and Pdr3, as multidrug-resistant pdr1 and pdr3 gain-of-function mutants overexpress both drug efflux pumps. One such pdr1 mutant allele was previously cloned in a genetic screen by its ability to suppress the squelching toxicity mediated by an estradiol-inducible chimeric VP16-human estrogen receptor (VEO) expressed in yeast (Gilbert, D. M. , Heery, D. M., Losson, R., Chambon, P., and Lemoine, Y. (1993) Mol. Cell. Biol. 13, 462-472). In this study, we demonstrate that relief of estradiol toxicity in yeast cells expressing VEO requires functional PDR5 and SNQ2 genes, since a Deltapdr5 Deltasnq2 double deletion leads to an increased estradiol toxicity. Furthermore, using URA3 as an estradiol-inducible reporter gene, we show that Pdr5 and Snq2, when overexpressed from high-copy plasmids, can reduce the intracellular concentration of estradiol. In contrast, a Deltapdr5 Deltasnq2 double deletion mutant accumulates almost 30-fold more intracellular estradiol than the isogenic wild type. Indirect immunofluorescence showed that a pdr1-3 mutant massively overexpresses Pdr5 at the plasma membrane, suggesting that estradiol efflux from the cells occurs across the plasma membrane. Our data demonstrate that Pdr5 and Snq2 can transport steroid substrates in vivo and suggest that steroids and/or related membrane lipids could represent physiological substrates for certain yeast ABC transporters, which are otherwise involved in the development of pleiotropic drug resistance.

Highly purified preparations of cytochrome b6 f complex from the unicellar freshwater alga Chlamydomonas reinhardtii contain about 1 molecule of chlorophyll a/cytochrome f. Several lines of evidence indicate that the chlorophyll is an authentic component of the complex rather than a contaminant. In particular, (i) the stoichiometry is constant; (ii) the chlorophyll is associated with the complex at a specific binding site, as evidenced by resonance Raman spectroscopy; (iii) it does not originate from free chlorophyll released from thylakoid membranes upon solubilization; and (iv) its rate of exchange with free, radioactive chlorophyll a is extremely slow (weeks). Some of the putative functional roles for a chlorophyll in the b6f complex are experimentally ruled out, and its possible evolutionary origin is briefly discussed.

Using 8.8 kb of genetic information from Bacillus sphaericus, it was possible to confer to Escherichia coli bio−strains, including ΔbioA-D, bioC−, bioH−, the ability to convert exogenous pimelate into biotin. The biogenes were borne on two recombinant plasmids with inserts of 4.3 kb and 4.5 kb, which had been isolated from a genomic bank of HindIII-digested B. sphaericusDNA, by phenotypic complementation of various E. coli biomutants. The B. sphaericus bioDand bioAgenes were unambiguously identified within the 4.3-kb insert and shown to be closely linked to bioY(coding for a protein with a presently unknown function) and to bioB[Ohsawa et al., Gene 80 (1989) 39–48]. These genes are clustered in the order bioDAYB. The 4.5-kb fragment contains genetic information for three different proteins, the products of bioX, bioWand bioF. Complementation studies using an E. coli bioFmutant and a B. subtilis bio112TG3 strain, revealed that the third ORF of this cluster encodes 7-keto-8-aminopelargonic acid synthetase. A combination of bioWand bioFallows an efficient complementation of E. coli bioCand bioHmutants, provided that pimelate is added to the biotin-depleted growth medium. No function could be identified for the product of bioX. The gene order of this cluster is bioXWF. By sequence analysis, the two cloned DNA fragments were shown to bear overlapping open reading frames and secondary structures at their 3′ ends, typical of transcription terminators. The presence, upstream from the coding sequences in both inserts, of an identical 15-bp sequence, located within sequences exhibiting a center of imperfect symmetry, strongly suggests the existence in B. sphaericusof a common regulatory mechanism acting on these regions.