Producer immunity towards the lantibiotic Pep5: identification of the immunity gene pepI and localization and functional analysis of its gene product

The lantibiotic Pep5 is produced by Staphylococcus epidermidis 5. Pep5 production and producer immunity are associated with the 20-kb plasmid pED503. A 1.3-kb KpnI fragment of pED503, containing the Pep5 structural gene pepA, was subcloned into the Escherichia coli-Staphylococcus shuttle vector pCU1, and the recombinant plasmid pMR2 was transferred to the Pep5- and immunity-negative mutant S. epidermidis 5 Pep5- (devoid of pED503). This clone did not produce active Pep5 but showed the same degree of insensitivity towards Pep5 as did the wild-type strain. Sequencing of the 1.3-kb KpnI-fragment and analysis of mutants demonstrated the involvement of two genes in Pep5 immunity, the structural gene pepA itself and pepI, a short open reading frame upstream of pepA. To identify the 69-amino-acid pepI gene product, we constructed an E. coli maltose-binding protein-PepI fusion clone. The immunity peptide PepI was detected in the soluble and membrane fractions of the wild-type strain and the immune mutants (harboring the plasmids pMR2 and pMR11) by immunoblotting with anti-maltose-binding protein-PepI antiserum. Strains harboring either pepI without pepA or pepI with incomplete pepA were not immune and did not produce PepI. Washing the membrane with salts and EDTA reduced the amount of PepI in this fraction, and treatment with Triton X-100 almost completely removed the peptide. Furthermore, PepI was hydrolyzed by proteases added to osmotically stabilized protoplasts. This suggests that PepI is loosely attached to the outside of the cytoplasmic membrane. Proline uptake and efflux experiments with immune and nonimmune strains also indicated that PepI may act at the membrane site.     

Isolation, characterization, and heterologous expression of the novel lantibiotic epicidin 280 and analysis of its biosynthetic gene cluster

Epicidin 280 is a novel type A lantibiotic produced by Staphylococcus epidermidis BN 280. During C18 reverse-phase high-performance liquid chromatography two epicidin 280 peaks were obtained; the two compounds had molecular masses of 3,133 +/- 1.5 and 3,136 +/- 1.5 Da, comparable antibiotic activities, and identical amino acid compositions. Amino acid sequence analysis revealed that epicidin 280 exhibits 75% similarity to Pep5. The strains that produce epicidin 280 and Pep5 exhibit cross-immunity, indicating that the immunity peptides cross-function in antagonization of both lantibiotics. The complete epicidin 280 gene cluster was cloned and was found to comprise at least five open reading frames (eciI, eciA, eciP, eciB, and eciC, in that order). The proteins encoded by these open reading frames exhibit significant sequence similarity to the biosynthetic proteins of the Pep5 operon of Staphylococcus epidermidis 5. A gene for an ABC transporter, which is present in the Pep5 gene cluster but is necessary only for high yields (G. Bierbaum, M. Reis, C. Szekat, and H.-G. Sahl, Appl. Environ. Microbiol. 60:4332-4338, 1994), was not detected. Instead, upstream of the immunity gene eciI we found an open reading frame, eciO, which could code for a novel lantibiotic modification enzyme involved in reduction of an N-terminally located oxopropionyl residue. Epicidin 280 produced by the heterologous host Staphylococcus carnosus TM 300 after introduction of eciIAPBC (i.e., no eciO was present) behaved homogeneously during reverse-phase chromatography.     

Prolactin induction of the alpha 2-Macroglobulin gene in rat ovarian granulosa cells: stat 5 activation and binding to the interleukin-6 response element

Lantibiotics form a group of modified peptides with unique structures, containing post-translationally modified amino acids such as dehydrated and lanthionine residues. In the gram-positive bacteria that secrete these lantibiotics, the gene clusters flanking the structural genes for various linear (type A) lantibiotics have recently been characterized. The best studied representatives are those of nisin (nis), subtilin (spa), epidermin (epi), Pep5 (pep), cytolysin (cyl), lactocin S (las) and lacticin 481 (lct). Comparison of the lantibiotic gene clusters shows that they contain conserved genes that probably encode similar functions. The nis, spa, epi and pep clusters contain lanB and lanC genes that are presumed to code for two types of enzymes that have been implicated in the modification reactions characteristic of all lantibiotics, i.e. dehydration and thio-ether ring formation. The cyl, las and lct gene clusters have no homologue of the lanB gene, but they do contain a much larger lanM gene that is the lanC gene homologue. Most lantibiotic gene clusters contain a lanP gene encoding a serine protease that is presumably involved in the proteolytic processing of the prelantibiotics. All clusters contain a lanT gene encoding an ABC transporter likely to be involved in the export of (precursors of) the lantibiotics. The lanE, lanF and lanG genes in the nis, spa and epi clusters encode another transport system that is possibly involved in self-protection. In the nisin and subtilin gene clusters two tandem genes, lanR and lanK, have been located that code for a two-component regulatory system. Finally, non-homologous genes are found in some lantibiotic gene clusters. The nisI and spaI genes encode lipoproteins that are involved in immunity, the pepI gene encodes a membrane-located immunity protein, and epiD encodes an enzyme involved in a post-translational modification found only in the C-terminus of epidermin. Several genes of unknown function are also found in the las gene cluster. A database has been assembled for all putative gene products of type A lantibiotic gene clusters. Database searches, multiple sequence alignment and secondary structure prediction have been used to identify conserved sequence segments in the LanB, LanC, LanE, LanF, LanG, LanK, LanM, LanP, LanR and LanT gene products that may be essential for structure and function. This database allows for a rapid screening of newly determined sequences in lantibiotic gene clusters.     

Escherichia coli DNA topoisomerase I and suppression of killing by Tn5 transposase overproduction: topoisomerase I modulates Tn5 transposition

Tn5 transposase (Tnp) overproduction is lethal to Escherichia coli. The overproduction causes cell filamentation and abnormal chromosome segregation. Here we present three lines of evidence strongly suggesting that Tnp overproduction killing is due to titration of topoisomerase I. First, a suppressor mutation of transposase overproduction killing, stkD10, is localized in topA (the gene for topoisomerase I). The stkD10 mutant has the following characteristics: first, it has an increased abundance of topoisomerase I protein, the topoisomerase I is defective for the DNA relaxation activity, and DNA gyrase activity is reduced; second, the suppressor phenotype of a second mutation localized in rpoH, stkA14 (H. Yigit and W. S. Reznikoff, J. Bacteriol. 179:1704-1713, 1997), can be explained by an increase in topA expression; and third, overexpression of wild-type topA partially suppresses the killing. Finally, topoisomerase I was found to enhance Tn5 transposition up to 30-fold in vivo.     

hyp gene products in Alcaligenes eutrophus are part of a hydrogenase-maturation system

In Alcaligenes eutrophus H16 the hyp gene complex consists of six open reading frames hypA1, B1, F1, C, D and E whose products are involved in maturation of the two NiFe hydrogenases: an NAD-reducing cytoplasmic enzyme (SH) and a membrane-bound electron-transport-coupled protein (MBH). hypB1 and hypF1 were originally considered to form a single open reading frame designated hypB [Dernedde, J., Eitinger, M. & Friedrich, B. (1993) Arch. Microbiol. 159, 545-553]. Re-examination of the relevant sequence identified hypB1 and hypF1 as two distinct genes. Non-polar in-frame deletions in the individual hyp genes were constructed in vitro and transferred via gene replacement to the wild-type strain. The resulting mutants fall into two classes. Deletions in hypC, D and E (class I) gave a clear negative phenotype, while hypA1, B1 and F1 deletion mutants (class II) were not impaired in hydrogen metabolism. Class I mutants were unable to grow on hydrogen under autotrophic conditions. The enzymatic activities of SH and MBH were disrupted in all three class I mutants. Immunoblot analysis showed the presence of the H2-activating SH subunit (HoxH) at levels comparable to those observed in the wild-type strain whereas the other three subunits (HoxF, U and Y) were only detectable in trace amounts, probably due to proteolytic degradation. Likewise, MBH was less stable in hypC, D and E deletion mutants and was not attached to the cytoplasmic membrane. In the wild-type strain, HoxH and the MBH large subunit (HoxG) undergo C-terminal proteolytic processing before attaining enzymatic activity. In class I mutants this maturation was blocked. 63Ni-incorporation experiments identified both hydrogenases as nickel-free apoproteins in these mutants. Although class II mutants bearing deletions in hypA1, B1 and F1 showed no alteration of the wild-type phenotype, a role for these genes in the incorporation of nickel and hence hydrogenase maturation cannot be excluded, since there is experimental evidence that this set of genes is duplicated in A. eutrophus.     

pMH2, a small plasmid bearing the nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette

A small plasmid containing the entire nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette has been constructed, using pACYC177 as a vector. Two cosmid clones taken from a gene library of E. agglomerans plasmid pEA3 were used as a source of nif genes. A SmaI fragment of peaMS2-2, containing the H,D,K,Y,E,N,X,U,S,V,W,Z,M,L,A and B genes and an ApaI fragment of peaMS2-16 containing nif A,B,Q,F and J were selected to construct pMH2. The resulting plasmid of 33 kb carries the complete nif gene cluster as a nif cassette on a single XbaI fragment. The nif construct pMH2 in Escherichia coli strains has significant nitrogenase activity compared to wild-type E. agglomerans 333. The nif gene cluster construct was found to be very stable.     

Conformation and lipid binding properties of four peptides derived from the membrane-binding domain of CTP:phosphocholine cytidylyltransferase

We are probing the mechanism of the lipid selective membrane interactions of CTP:phosphocholine cytidylyltransferase (CT). We have proposed that the membrane binding domain of CT (domain M) consists of a continuous amphipathic alpha-helix between residues approximately 240-295 [Dunne, S. J., et al. (1996) Biochemistry 35, 11975-11984]. This study examined the secondary structure and membrane binding properties of synthetic peptides derived from domain M: a 62mer peptide encompassing the entire domain (Pep62), a 33mer corresponding to the N-terminal portion (PepNH1), and two 33mers corresponding to the three C-terminal 11mer repeats, one with the wild-type sequence (Pep33Ser), and one with the three serines in the nonpolar face substituted with alanine (Pep33Ala). Peptide secondary structure was analyzed by circular dichroism, and lipid interactions were analyzed by a direct vesicle binding assay, by effects of lipid vesicles on peptide tryptophan fluorescence, and by monolayer surface pressure changes. All peptides bound to vesicles as alpha-helices with selectivity for anionic lipids. Binding involved intercalation of the peptide tryptophan into the hydrophobic membrane core. PepNH1, the peptide with the highest positive charge density, showed strong selectivity for anionic lipids. PepNH1 and Pep33Ser did not bind to PC vesicles; however, the more hydrophobic peptides, Pep33Ala and Pep62, did bind to PC vesicles, with apparent partition coefficients for PC that were only approximately 1 order of magnitude lower than those for PC/PG (1/1). Our results suggest that the polar serines interrupting the nonpolar face of the amphipathic helix serve to lower the lipid affinity and thereby enhance selectivity for anionic lipids. Although diacylglycerol is an activator of the enzyme, none of the peptides responded differentially to PC/diacylglycerol vesicles versus pure PC vesicles, suggesting that domain M alone is not sufficient for the enzyme's response to diacylglycerol. Increases in surface pressure at an air-water interface indicated that the domain M peptides had strong surface-seeking tendencies. This supports a binding orientation for domain M parallel to the membrane surface. Binding of CT peptides to spread lipid monolayers caused surface pressure reductions, suggesting condensation of lipids in the formation of lipid-peptide complexes. At low monolayer surface pressures, Pep62 interacted equally with anionic and zwitterionic phospholipids. This suggests that one determinant of the selectivity for anionic lipids is the lipid packing density (area per molecule).     

The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice

In this work we identified and characterized a gene cluster containing three internalin genes of Listeria monocytogenes EGD. These genes, termed inlG, inlH and inlE, encode proteins of 490, 548 and 499 amino acids, respectively, which belong to the family of large, cell wall-bound internalins. The inlGHE gene cluster is flanked by two listerial house-keeping genes encoding proteins homologous to the 6-phospho-beta-glucosidase and the succinyl-diaminopimelate desuccinylase of E. coli. A similar internalin gene cluster, inlC2DE, localised to the same position on the L. monocytogenes EGD chromosome was recently described in a different isolate (Dramsi S, Dehoux P, Lebrun M, Goossens PL, Cossart P (1997) Infect Immun 65: 1615-1625). Sequence comparison of the two inl gene clusters indicates that inlG is a new internalin gene, while inlH was generated by a site-specific recombination, leading to an in-frame deletion which removed the 3'-terminal end of inlC2 and the 5'-terminal part of inlD. The third gene of the inlGHE cluster, inlE, is almost identical to the previously reported inlE gene. Our data show that the inlGHE gene cluster is probably transcribed from a major PrfA-independent promoter located upstream of inlG. PCR analysis revealed the presence of the newly identified inl genes inlG and inlH in most L. monocytogenes isolates tested. A mutant which has lost inlG, inlH and inlE by an in-frame deletion exhibited, after oral infection of mice, a significant loss in virulence and shows drastically reduced numbers of viable bacteria in both liver and spleen when compared to the wild-type strain.     

Mhc class I and non-class I gene organization in the proximal H2-M region of the mouse

A bacterial artificial chromosome (BAC) contig was constructed across the proximal part of the H2-M region from the major histocompatibility complex (Mhc) of mouse strain 129 (H2bc). The contig is composed of 28 clones that span approximately 1 megabasepair (Mb), from H2-T1 to Mog, and contains three H2-T genes and 18 H2-M genes. We report the fine mapping of the H2-M class I gene cluster, which includes the previously reported M4-M6, the M1 family, the M10 family, and four additional class I genes. All but two of the H2-M class I genes are conserved among haplotypes H2k, H2b, and H2bc, and only two genes are found in polymorphic HindIII fragments. Six evolutionarily conserved non-class I genes were mapped to a 180 kilobase interval in the distal part of the class I region in mouse, and their order Znf173-Rfb30-Tctex5-Tctex6- Tctex4-Mog was found conserved between human and mouse. In this Znf173-Mog interval, three mouse class I genes, M6, M4, and M5, which are conserved among haplotypes, occupy the same map position as the human HLA-A class I cluster, which varies among haplotypes and is diverged in sequence from the mouse genes. These results further support the view that class I gene diverge and evolve independently between species.     

The Mof2/Sui1 protein is a general monitor of translational accuracy

Although it is essential for protein synthesis to be highly accurate, a number of cases of directed ribosomal frameshifting have been reported in RNA viruses, as well as in procaryotic and eucaryotic genes. Changes in the efficiency of ribosomal frameshifting can have major effects on the ability of cells to propagate viruses which use this mechanism. Furthermore, studies of this process can illuminate the mechanisms involved in the maintenance of the normal translation reading frame. The yeast Saccharomyces cerevisiae killer virus system uses programmed -1 ribosomal frameshifting to synthesize its gene products. Strains harboring the mof2-1 allele demonstrated a fivefold increase in frameshifting and prevented killer virus propagation. In this report, we present the results of the cloning and characterization of the wild-type MOF2 gene. mof2-1 is a novel allele of SUI1, a gene previously shown to play a role in translation initiation start site selection. Strains harboring the mof2-1 allele demonstrated a mutant start site selection phenotype and increased efficiency of programmed -1 ribosomal frameshifting and conferred paromomycin sensitivity. The increased frameshifting observed in vivo was reproduced in extracts prepared from mof2-1 cells. Addition of purified wild-type Mof2p/Sui1p reduced frameshifting efficiencies to wild-type levels. Expression of the human SUI1 homolog in yeast corrects all of the mof2-1 phenotypes, demonstrating that the function of this protein is conserved throughout evolution. Taken together, these results suggest that Mof2p/Sui1p functions as a general modulator of accuracy at both the initiation and elongation phases of translation.     

Structure-function relationships in Anabaena ferredoxin: correlations between X-ray crystal structures, reduction potentials, and rate constants of electron transfer to ferredoxin:NADP+ reductase for site-specific ferredoxin mutants

A combination of structural, thermodynamic, and transient kinetic data on wild-type and mutant Anabaena vegetative cell ferredoxins has been used to investigate the nature of the protein-protein interactions leading to electron transfer from reduced ferredoxin to oxidized ferredoxin:NADP+ reductase (FNR). We have determined the reduction potentials of wild-type vegetative ferredoxin, heterocyst ferredoxin, and 12 site-specific mutants at seven surface residues of vegetative ferredoxin, as well as the one- and two-electron reduction potentials of FNR, both alone and in complexes with wild-type and three mutant ferredoxins. X-ray crystallographic structure determinations have been carried out for six of the ferredoxin mutants. None of the mutants showed significant structural changes in the immediate vicinity of the [2Fe-2S] cluster, despite large decreases in electron-transfer reactivity (for E94K and S47A) and sizable increases in reduction potential (80 mV for E94K and 47 mV for S47A). Furthermore, the relatively small changes in Calpha backbone atom positions which were observed in these mutants do not correlate with the kinetic and thermodynamic properties. In sharp contrast to the S47A mutant, S47T retains electron-transfer activity, and its reduction potential is 100 mV more negative than that of the S47A mutant, implicating the importance of the hydrogen bond which exists between the side chain hydroxyl group of S47 and the side chain carboxyl oxygen of E94. Other ferredoxin mutations that alter both reduction potential and electron-transfer reactivity are E94Q, F65A, and F65I, whereas D62K, D68K, Q70K, E94D, and F65Y have reduction potentials and electron-transfer reactivity that are similar to those of wild-type ferredoxin. In electrostatic complexes with recombinant FNR, three of the kinetically impaired ferredoxin mutants, as did wild-type ferredoxin, induced large (approximately 40 mV) positive shifts in the reduction potential of the flavoprotein, thereby making electron transfer thermodynamically feasible. On the basis of these observations, we conclude that nonconservative mutations of three critical residues (S47, F65, and E94) on the surface of ferredoxin have large parallel effects on both the reduction potential and the electron-transfer reactivity of the [2Fe-2S] cluster and that the reduction potential changes are not the principal factor governing electron-transfer reactivity. Rather, the kinetic properties are most likely controlled by the specific orientations of the proteins within the transient electron-transfer complex.     

Novel temperature-sensitive mutants of Escherichia coli that are unable to grow in the absence of wild-type tRNA6Leu

Escherichia coli has only a single copy of a gene for tRNA6Leu (Y. Komine et al., J. Mol. Biol. 212:579-598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O2-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA4Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O2-methyluridine) as its anticodon, tRNA6Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA6Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su+6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA6Leu. These tRNA6Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaA gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA4Leu restored the growth of group I mutants at 42 degrees C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA4Leu poorly recognizes the UUG codon at 42 degreesC and that the wild-type tRNA6Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA6Leu remains to be investigated.     

Enzyme stability in systems with organic solvents

In this paper, we describe the cloning of the MS5 gene, a gene essential for male fertility in Arabidopsis. We previously defined the MS5 locus by characterizing an EMS-induced allele, ms5-1. We identified a new allele of MS5 (ms5-2) that was T-DNA-generated and used the T-DNA tag to clone the gene. Sequencing of mutant and wild-type alleles together with complementation of the ms5-1 mutant phenotype with a wild-type genomic clone confirmed the identity of the gene. Differences between the phenotypes of the two mutant alleles could be attributed to differences in mutant gene structure. The semi-dominant and dominant negative phenotypes of the ms5-2 mutant probably result from production of a truncated polypeptide. An unknown locus in Landsberg erecta can counteract the dominant negative phenotype of ms5-2. Mutations in MS5 cause the formation 'polyads'--tetrads with more than four pools of chromosomes after male meiosis. Similarities between the MS5 sequence and that of a number of proteins were found; two that may be significant were with a synaptonemal complex protein and with a regulatory subunit of a cyclin-dependent kinase. The MS5 gene is a member of a small gene family highly conserved amongst plant species.     

Modified ligands to FA and FB in photosystem I. Proposed chemical rescue of a [4Fe-4S] cluster with an external thiolate in alanine, glycine, and serine mutants of PsaC

The FB and FA electron acceptors in Photosystem I (PS I) are [4Fe-4S] clusters ligated by cysteines provided by PsaC. In a previous study (Mehari, T., Qiao, F., Scott, M. P., Nellis, D., Zhao, J., Bryant, D., and Golbeck, J. H. (1995) J. Biol. Chem. 270, 28108-28117), we showed that when cysteines 14 and 51 were replaced with serine or alanine, the free proteins contained a S = 1/2, [4Fe-4S] cluster at the unmodified site and a mixed population of S = 1/2, [3Fe-4S] and S = 3/2, [4Fe-4S] clusters at the modified site. We show here that these mutant PsaC proteins can be rebound to P700-FX cores, resulting in fully functional PS I complexes. The low temperature EPR spectra of the C14XPsaC.PS I complexes (where X = S, A, or G) show the photoreduction of a wild-type FA cluster and a modified FB' cluster, the latter with g values of 2.115, 1.899, and 1.852 and linewidths of 110, 70, and 85 MHz. Since neither alanine nor glycine contains a suitable side group, an external thiolate provided by beta-mercaptoethanol has likely been recruited to supply the requisite ligand to the [4Fe-4S] cluster. The EPR spectrum of the C51SPsaC.PS I complex differs from that of the C51APsaC.PS I or C51GPsaC.PS I complexes by the presence of an additional set of resonances, which may be derived from the serine oxygen-ligated cluster. In all other mutant PS I complexes, a wild-type spin-coupled interaction spectrum appears when FA and FB are simultaneously reduced. Single turnover flash studies indicate approximately 50% efficient electron transfer to FA/FB in the C14SPsaC.PS I, C51SPsaC.PS I, C14GPsaC.PS I, and C51GPsaC.PS I mutants and less than 40% in the C14APsaC.PS I and C51APsaC.PS I mutants, compared with approximately 76% in the PS I core reconstructed with wild-type PsaC. These data are consistent with the measurements of the rates of cytochrome c6-NADP+ reductase activity, indicating lower rates in the alanine mutants. It is proposed that the chemical rescue of a [4Fe-4S] cluster with a recruited external thiolate at the modified site allows the mutant PsaC proteins to rebind to PS I and to function in forward electron transfer.     

Pet127p, a membrane-associated protein involved in stability and processing of Saccharomyces cerevisiae mitochondrial RNAs

Nuclear mutations that inactivate the Saccharomyces cerevisiae gene PET127 dramatically increased the levels of mutant COX3 and COX2 mitochondrial mRNAs that were destabilized by mutations in their 5' untranslated leaders. The stabilizing effect of pet127 delta mutations occurred both in the presence and in the absence of translation. In addition, pet127 delta mutations had pleiotropic effects on the stability and 5' end processing of some wild-type mRNAs and the 15S rRNA but produced only a leaky nonrespiratory phenotype at 37 degrees C. Overexpression of PET127 completely blocked respiratory growth and caused cells to lose wild-type mitochondrial DNA, suggesting that too much Pet127p prevents mitochondrial gene expression. Epitope-tagged Pet127p was specifically located in mitochondria and associated with membranes. These findings suggest that Pet127p plays a role in RNA surveillance and/or RNA processing and that these functions may be membrane bound in yeast mitochondria.     

Mapping of a protective epitope of the CopB outer membrane protein of Moraxella catarrhalis

A monoclonal antibody (MAb) (MAb 10F3) directed against the CopB outer membrane protein of Moraxella catarrhalis previously was found to enhance pulmonary clearance of M. catarrhalis in an animal model (M. Helminen, I. Maciver, J. L. Latimer, L. D. Cope, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 61:2003-2010, 1993). In the present study, this same MAb was shown to exert complement-dependent bactericidal activity against this pathogen in vitro. Nucleotide sequence analysis of the copB gene from two MAb 10F3-reactive and two MAb 10F3-unreactive strains of M. catarrhalis revealed that the deduced amino acid sequences of these four CopB proteins were at least 90% identical. Comparison of the amino acid sequences of these proteins allowed localization of possible MAb 10F3 binding sites to five relatively small regions of the CopB protein from M. catarrhalis O35E. When five synthetic peptides representing these regions were tested for their ability to bind MAb 10F3 in a direct enzyme-linked immunosorbent assay system, an oligopeptide containing 26 amino acids was shown to bind this MAb. The actual binding region for MAb 10F3 was localized further through the use of overlapping decapeptides that spanned this 26-mer. A fusion protein containing the same 26-mer readily bound MAb 10F3 and was used to immunize mice. The resultant antiserum contained antibodies that reacted with the CopB protein of the homologous M. catarrhalis strain in Western blot analysis and bound to the surface of both homologous and heterologous strains of M. catarrhalis.     

Reduction of CDP-diacylglycerol synthase activity results in the excretion of inositol by Saccharomyces cerevisiae

A yeast mutant, cdg1, was isolated on the basis of an inositol excretion phenotype. This mutant exhibited pleiotropic deficiencies in phospholipid biosynthesis, including reduced levels of CDP-diacylglycerol (DAG) synthase activity (Klig, L. S., Homann, M. J., Kohlwein, S. D., Kelley, M. J., Henry, S. A., and Carman, G. M. (1988) J. Bacteriol. 170, 1878-1886). In this study we present evidence that the molecular basis for the inositol excretion phenotype is a G305/A305 point mutation (Cys102 --> Tyr substitution) within the CDS1 gene (encodes CDP-DAG synthase) of this mutant. Expression of CDP-DAG synthase activity from a plasmid-borne copy of the CDS1 gene in the cdg1 mutant was not down-regulated, and this expression also corrected the inositol excretion phenotype. Introduction of the above mutated gene (CDS1*) controlled by its endogenous promoter on a single copy plasmid into a cds1-null background reconstituted a transformant with the cdg1 phenotype, including reduced CDP-DAG synthase activity, elevated phosphatidylserine synthase activity, and inositol excretion into the growth medium. Expression of CDS1* in a single copy in the cdg1 mutant raised CDP-DAG synthase activity from 15 to 30% of derepressed wild-type yeast levels but still did not correct the inositol excretion phenotype. CDP-DAG synthase activity was not regulated in response to precursors of phospholipid biosynthesis in the cdg1 mutant either with or without a trans copy of the CDS1* gene. An open reading frame was identified 5' to the CDS1 locus, YBR0314, which also resulted in inositol excretion when present in trans in multiple copies.