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PubMed1995

Characterisation of a haemolysin related to Vp-TDH produced by a Kanagawa phenomenon-negative clinical isolate of Vibrio parahaemolyticus.

The production of a family of haemolysins--thermostable direct haemolysin (Vp-TDH), Vp-TDH-related haemolysin (Vp-TRH) and Vp-TDH/I--has been reported in clinical isolates of Vibrio parahaemolyticus. This paper describes a fourth type of haemolysin--Vp-TDH/II--produced by a Kanagawa phenomenon-negative clinical isolate of V. parahaemolyticus (O13:K, untypable). Vp-TDH/II was purified by ammonium sulphate precipitation and successive filtrations on DEAE-cellulose, hydroxyapatite, Sepharose 4B and Mono Q columns. Vp-TDH/II was biophysicochemically and immunologically similar to, but not identical to Vp-TDH, Vp-TRH and Vp-TDH/I. Vp-TDH/II stimulated vascular permeability in rabbit skin and was lethal to mice. Purified Vp-TDH/II and viable cells of the Vp-TDH/II-producing strain both induced fluid accumulation in ligated rabbit intestine. The plasmid-determined structural gene for Vp-TDH/II was cloned and the nucleotide sequence determined. The deduced amino acid sequence of Vp-TDH/II differed from those of Vp-TDH, Vp-TRH and Vp-TDH/I.

Amino Acid Sequence↗
PubMed1996

Functional analysis of the beta and epsilon lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation.

Carotenoids with cyclic end groups are essential components of the photosynthetic membranes in all plants, algae, and cyanobacteria. These lipid-soluble compounds protect against photooxidation, harvest light for photosynthesis, and dissipate excess light energy absorbed by the antenna pigments. The cyclization of lycopene (psi, psi-carotene) is a key branch point in the pathway of carotenoid biosynthesis. Two types of cyclic end groups are found in higher plant carotenoids: the beta and epsilon rings. Carotenoids with two beta rings are ubiquitous, and those with one beta and one epsilon ring are common; however, carotenoids with two epsilon rings are rare. We have identified and sequenced cDNAs that encode the enzymes catalyzing the formation of these two rings in Arabidopsis. These beta and epsilon cyclases are encoded by related, single-copy genes, and both enzymes use the linear, symmetrical lycopene as a substrate. However, the epsilon cyclase adds only one ring, forming the monocyclic delta-carotene (epsilon, psi-carotene), whereas the beta cyclase introduces a ring at both ends of lycopene to form the bicyclic beta-carotene (beta, beta-carotene). When combined, the beta and epsilon cyclases convert lycopene to alpha-carotene (beta, epsilon-carotene), a carotenoid with one beta and one epsilon ring. The inability of the epsilon cyclase to catalyze the introduction of a second epsilon ring reveals the mechanism by which production and proportions of beta,beta- and beta, epsilon-carotenoids may be controlled and adjusted in plants and algae, while avoiding the formation of the inappropriate epsilon,epsilon-carotenoids.

Amino Acid Sequence↗
PubMed1992

Broad-host-range Rhizobium species strain NGR234 secretes a family of carbamoylated, and fucosylated, nodulation signals that are O-acetylated or sulphated.

Rhizobium species strain NGR234 is the most promiscuous known rhizobium. In addition to the non-legume Parasponia andersonii, it nodulates at least 70 genera of legumes. Here we show that the nodulation genes of this bacterium determine the production of a large family of Nod-factors which are N-acylated chitin pentamers carrying a variety of substituents. The terminal non-reducing glucosamine is N-acylated with vaccenic or palmitic acids, is N-methylated, and carries varying numbers of carbamoyl groups. The reducing N-acetyl-glucosamine residue is substituted on position 6 with 2-O-methyl-L-fucose which may be acetylated or sulphated or non-substituted. All three internal residues are N-acetylated. At pico- to nanomolar concentrations, these signal molecules exhibit biological activities on the tropical legumes Macroptilium and Vigna (Phaseoleae), as well as on both the temperate genera Medicago (Trifoliae) and Vicia (Viciae). These data strongly suggest that the uniquely broad host range of NGR234 is mediated by the synthesis of a family of varied sulphated and non-sulphated lipo-oligosaccharide signals.

Acetylation↗
PubMed1995

The role of chromatin-associated protein Hbsu in beta-mediated DNA recombination is to facilitate the joining of distant recombination sites.

The beta recombinase is unable to mediate in vitro DNA recombination between two directly oriented recombination sites unless a bacterial chromatin-associated protein (Bacillus subtilis Hbsu or Escherichia [correction of Eschrichia] coli HU] is provided. By electron microscopy, we show that the role of Hbsu is to help in joining the recombination sites to form a stable synaptic complex. Some evidence supports the fact that Hbsu works by recognizing and stabilizing a DNA structure at the recombination site, rather than by serving as a bridge between beta recombinase dimers through a protein-protein interaction. We show that the mammalian HMG1 protein, which shares neither sequence nor structural homology with Hbsu, can also stimulate beta-mediated recombination. These chromatin-associated proteins share the property of binding to DNA in a relatively non-specific fashion, bending it, and having a marked preference for altered DNA structures. Hbsu, HU or HMG1 proteins probably bind specifically at the crossing-over region, since at limiting protein-DNA molar ratios they could not be outcompeted by an excess of a DNA lacking the crossing over site. Distamycin, a minor groove binder that induces local distortions in DNA, did not affect the binding of beta protein to DNA, but inhibited the formation of the synaptic complex.

Bacterial Proteins↗
PubMed1989

Photofootprinting of drug-binding sites on DNA using diazo- and azido-9-aminoacridine derivatives.

It is demonstrated that DNA photofootprinting analysis of the intercalating depsipeptide echinomycin, and the minor groove-binders distamicyn, 4',6-diamidino-2-phenylindole (DAPI) and Hoechst 33258 can be performed using 9-[6-(2-diazocyclopentadienylcarbonyloxy)hexylamino]acridine (DHA) [Nielsen et al. (1988) Nucleic Acids Res. 16, 3877-3888] or 2-methoxy-6-azido-9-aminoacridine (MAA) [Jeppesen et al. (1988) Nucleic Acids Res. 16, 5755-5770]. Both the extent of the drug-binding sites and their relative strength can be determined with either reagent. DNA has the advantage of giving virtually sequence-uniform DNA photocleavage. On the other hand, structural changes in the DNA are detected by MAA. Using the 232-base-pair EcoRI-PvuII pUC19 restriction fragment, it is found that cleavage protection by distamycin, DAPI and Hoechst 33258 all require an (A.T)4 sequence, whereas protection by echinomycin was confined to a G + C-rich 8-base-pair region.

Aminoacridines↗
PubMed1996

Divergicin 750, a novel bacteriocin produced by Carnobacterium divergens 750.

Divergicin 750, a bacteriocin produced by Carnobacterium divergens 750, preferentially inhibited the growth of strains of Carnobacterium and Enterococcus. Selected strains of Listeria monocytogenes and Clostridium perfringens were also inhibited. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential S-Sepharose, hydrophobic interaction and reversed phase chromatography. The complete amino acid sequence was determined by Edman degradation. The peptide consisted of 34 amino acid residues. The calculated M(r) from the peptide sequence, 3447.7, agreed well with that obtained by mass spectrometry. Divergicin 750 did not show any sequence similarities to other known bacteriocins. The plasmid-located structural gene encoding divergicin 750 (dvn750) was cloned and sequenced. The gene encoded a primary translation product of 63 amino acids with a deduced M(r) = 6789.4 which is cleaved between amino acid residues 29 and 30 to yield the mature bacteriocin.

Amino Acid Sequence↗
PubMed1993

Analysis of pristinamycin-resistant Staphylococcus epidermidis isolates responsible for an outbreak in a Parisian hospital.

In 1990, over a 6-month period, an increase from 1 to 10% in the incidence of pristinamycin resistance among coagulase-negative staphylococci was observed in four intensive care units of a Parisian hospital. Twenty-three such isolates, as well as 25 pristinamycin-susceptible Staphylococcus epidermidis isolates, were collected and typed by analyzing various bacterial constituents. Two structurally related plasmids of 7.3 and 14.3 kb, carrying the gene vga encoding resistance to pristinamycin, were detected in the 23 pristinamycin-resistant coagulase-negative staphylococci which were identified as S. epidermidis. Although related by numerous common characteristics, 20 of these 23 isolates could be divided into two types, A (17 isolates) and B (three isolates). These types were characterized on the basis of their plasmid contents and hybridization patterns obtained when the EcoRI-digested DNA was probed with plasmid pIP1551 containing an internal fragment of the insertion sequence IS256. These findings suggest that the dissemination of type A epidemic strains was, in large part, responsible for the outbreak.

Base Sequence↗
PubMed1995

Sequencing of the gene encoding the major pilin of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli and evidence that CFA/III is related to type IV pili.

The plasmid-encoded structural gene cofA necessary for the production of the major pilin subunit of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli was identified, and the nucleotide sequence of the gene was determined. cofA consists of 714 nucleotides encoding a 238-amino-acid protein (molecular weight of 25,309). CofA seems to be a precursor of CFA/III pilin, because the first 23 residues of the N-terminal amino acid sequence of the purified CFA/III pili coincided with the deduced amino acid sequence for residues 32 to 54 of CofA. Western blot (immunoblot) analysis of CofA also indicated its processing to form mature pilin in the presence of the downstream region of cofA. These results suggest that the major pilin of CFA/III pili is produced as a precursor form which is posttranslationally modified to the mature pilin and forms morphological pili after cleavage of the Gly-30-Met-31 junction, probably by a protease encoded by an as-yet-unknown gene located downstream of cofA. Interestingly, the N-terminal 30-amino-acid sequence of mature CFA/III shows the highest identity (76.7%) to TcpA pilin of Vibrio cholerae, which is a type IV class B pilin.

Antigens, Bacterial↗
PubMed1980

Transcription of bacteriocinogenic plasmid CloDF13 in vivo and in vitro: structure of the cloacin immunity operon.

Escherichia coli minicells harboring plasmid CloDF13 synthesized at least 25 messenger ribonucleic acid (RNA) species; three of these RNAs, a 2,400-, a 2,200-, and a 100-nucleotide RNA, were synthesized in relatively large amounts. Using insertion and deletion mutants of CloDF13 as well as an RNA blotting technique, we could demonstrate that these three RNAs are transcripts from the CloDF13 DNA region from 0 to 40%. This region contains the cloacin and immunity genes and the genetic information involved in plasmid DNA replication. A transcription map of this region is presented and discussed. The data indicate that the cloacin and immunity genes were coordinately transcribed into messenger RNAs of about 2,400 and 2,200 nucleotides, which differ in length at their 3' terminus. RNA polymerase binding studies and in vitro transcription assays indicated that transcription of these genes initiates at a promoter located around 32% on the CloDF13 map. Furthermore, it is shown that a 100-nucleotide RNA is encoded by the CloDF13 DNA region between 7.7 and 8.8% on the plasmid genome; the synthesis of this RNA proceeds in a direction opposite to the transcription of the cloacin and immunity genes.

Bacteriocins↗
PubMed1991

Fine-structure analysis of the P1 plasmid partition site.

P1 plasmid partition requires two plasmid-encoded Par proteins and a cis-acting site. The site, parS, lies in a region consisting of a 13-bp palindrome and an adjacent AT-rich sequence. A series of point mutations were analyzed for their effects on partition site activity. The results indicated that only the left arm of the palindrome and some adjacent bases were needed. The limits of the functional site were further refined to a maximum of 22 bp, which includes binding sites for the P1 ParB protein. Mutations in the 22-bp site cause concomitant defects in partition and the ability to exert partition-mediated incompatibility. Like the region immediately to the left of the 22-bp region, the right arm of the palindrome is not essential for partition but does contain information that affects the specificity of incompatibility.

Bacterial Proteins↗
PubMed1993

Fine-structure analysis of the P7 plasmid partition site.

The par region of bacteriophage P7 is responsible for active partition of the P7 plasmid prophage into daughter cells. The cis-acting partition site was defined precisely as a 75-bp sequence that was necessary and sufficient to promote correct segregation of an unstable vector plasmid when the two P7 partition proteins, ParA and ParB, were supplied in trans. Roughly the same region was necessary to exert partition-mediated incompatibility. The minimal site contains an integration host factor (IHF) protein binding site bracketed by regions containing heptamer repeat sequences that individually bind ParB. An additional sequence forms the left boundary of the site. Site-directed mutations in the latter sequence, as well as the IHF motif and the rightmost ParB box, blocked site function. Although the P7 site shares 55% sequence identity with its counterpart in bacteriophage P1, functional interactions between the partition sites and the Par proteins of the two plasmids were entirely species specific in vivo. The P1 sequence has similar IHF and ParB binding motifs, but the left boundary sequence differs radically and may define a point of species-specific contact with the Par proteins. No evidence was found for the existence of a functional P7 analog of the P1 parS core, a small subregion of the P1 site that, in isolation, acts as an enfeebled partition site with modified incompatibility properties.

Bacterial Proteins↗
PubMed1993

Dynamic structure of Agrobacterium tumefaciens Ti plasmids.

Agrobacterium tumefaciens C58F is a variant of strain C58 which generates a high proportion of avirulent mutants in the presence of the virulence (vir) gene inducer acetosyringone. These mutants are altered in the Ti plasmid and do not respond to the acetosyringone signal (C. Fortin, E. W. Nester, and P. Dion, J. Bacteriol. 174:5676-5685, 1992). The physical organization of the Ti plasmid was compared in strain C58 and its variant. One feature distinguishing pTiC58F from its parent plasmid was the presence of the insertion element IS426. Three copies of this element were detected in the strain C58 chromosome, whereas two additional copies were found in strain C58F, including one copy in the Ti plasmid. This particular copy of IS426 was associated with the region of arginine and nopaline catabolism of pTiC58F. Most of the avirulent mutants recovered following growth of strain C58F in the presence of acetosyringone were complemented by clones carrying either virA or virG. Element IS426 was no longer found in the arginine and nopaline catabolism region of the Ti plasmids from the virA and virG mutants, but it resided in the particular KpnI fragment containing the modified vir locus. Behavior of a strain C58F derivative, which was inactivated in a chromosomal component required for the response to acetosyringone, was consistent with the possibility that vir gene induction is essential to the massive production of avirulent mutants.

Acetophenones↗
PubMed1993

Biosynthesis of the corrin macrocycle of coenzyme B12 in Pseudomonas denitrificans.

Studies with cell-free protein preparations from a series of recombinant strains of Pseudomonas denitrificans demonstrated that precorrin-3 is converted into a further trimethylated intermediate, named precorrin-3B, along the pathway to coenzyme B12. It was then shown that the part of the pathway from precorrin-3 (called precorrin-3A hereafter) to precorrin-6x involves three intermediates, precorrin-3B, precorrin-4, and precorrin-5. Precorrin-3B was isolated in its native (reduced) as well as its oxidized (factor-IIIB) states, and precorrin-4 was isolated in its oxidized form only (factor-IV). Both factors were in vitro precursors of precorrin-6x. The synthesis of precorrin-6x from precorrin-3A was shown to be catalyzed by four enzymes, CobG, CobJ, CobM, and CobF, intervening in this order. They were purified to homogeneity. CobG, which converts precorrin-3A to precorrin-3B, was found to be an iron-sulfur protein responsible for the oxidation known to occur between precorrin-3A and precorrin-6x, and CobJ, CobM, and CobF are the C-17, C-11, and C-1 methylases, respectively. The acetate fragment is extruded after precorrin-4 formation. This study combined with our recent structural studies on factor-IV (D. Thibaut, L. Debussche, D. Fréchet, F. Herman, M. Vuilhorgne, and F. Blanche, J. Chem. Soc. Chem. Commun. 1993:513-515, 1993) and precorrin-3B (L. Debussche, D. Thibaut, M. Danzer, F. Debu, D. Fréchet, F. Herman, F. Blanche, and M. Vuilhorgne, J. Chem. Soc. Chem. Commun. 1993:1100-1103, 1993) provides a first step-by-step picture of the sequence of the enzymatic reactions leading to the corrin ring in P. denitrificans.

Carbon Isotopes↗
PubMed1994

Substratum-induced morphological changes in a marine bacterium and their relevance to biofilm structure.

The effects of surfaces on the physiology of bacteria adhering to surfaces or immobilized within biofilms are receiving more interest. A study of the effects of hydrophobic and hydrophilic substrata on the colonization behavior of a marine bacterium, SW5, revealed major differences in the morphology of SW5 on these surfaces. Using epifluorescence, scanning confocal laser, and on-line visualization (time-lapse video) microscopy, the organisms at hydrophobic surfaces were characterized by the formation of tightly packed biofilms, consisting of single and paired cells, whereas those at hydrophilic surfaces exhibited sparse colonization and the formation of chains more than 100 microns long, anchored at the surface by the terminal (colonizing) cell. The results are discussed in terms of the possible factors inducing the observed morphological differences and the significance of these differences in terms of biofilm structure and plasmid transfer when SW5 is the recipient organism.

Bacteria↗
PubMed1990

Identification of a site on herpes simplex virus type 1 glycoprotein D that is essential for infectivity.

Herpes simplex virus glycoprotein D (gD) plays an essential role during penetration of the virus into cells. There is evidence that it recognizes a specific receptor after initial attachment of virions to cell surface heparan sulfate and also that gD-1, gD-2, and gp50 (the pseudorabies virus gD homolog) bind to the same receptor. Although the antigenic structure of gD has been studied intensively, little is known about functional regions of the protein. Antigenic site I is a major target for neutralizing antibodies and has been partially mapped by using deletion mutants and neutralization-resistant viruses. Working on the assumption that such a site may overlap with a functional region of gD, we showed previously that combining two or more amino acid substitutions within site I prevents gD-1 from functioning and is therefore lethal. We have now used a complementation assay to measure the functional activity of a panel of deletion mutants and compared the results with an antigenic analysis. Several mutations cause gross changes in protein folding and destroy functional activity, whereas deletions at the N and C termini have little or no effect on either. In contrast, deletion of residues 234 to 244 has only localized effects on antigenicity but completely abolishes functional activity. This region, which is part of antigenic site Ib, is therefore essential for gD-1 function. The complementation assay was also used to show that a gD-negative type 1 virus can be rescued by gD-2 and by two gD-1-gD-2 hybrids but not by gp50, providing some support for the existence of a common receptor for herpes simplex virus types 1 and 2 but not pseudorabies virus. Alternatively, gp50 may lack a signal for incorporation into herpes simplex virions.

Animals↗
PubMed1992

Intracellular modifications induced by poliovirus reduce the requirement for structural motifs in the 5' noncoding region of the genome involved in internal initiation of protein synthesis.

A series of genetic deletions based partly on two RNA secondary structure models (M. A. Skinner, V. R. Racaniello, G. Dunn, J. Cooper, P. D. Minor, and J. W. Almond, J. Mol. Biol. 207:379-392, 1989; E. V. Pilipenko, V. M. Blinov, L. I. Romanova, A. N. Sinyakov, S. V. Maslova, and V. I. Agol, Virology 168:201-209, 1989) was made in the cDNA encoding the 5' noncoding region (5' NCR) of the poliovirus genome in order to study the sequences that direct the internal entry of ribosomes. The modified cDNAs were placed between two open reading frames in a single transcriptional unit and used to transfect cells in culture. Internal entry of ribosomes was detected by measuring translation from the second open reading frame in the bicistronic mRNA. When assayed alone, a large proportion of the poliovirus 5' NCR superstructure including several well-defined stem-loops was required for ribosome entry and efficient translation. However, in cells cotransfected with a complete infectious poliovirus cDNA, the requirement for the stem-loops in this large superstructure was reduced. The results suggest that virus infection modifies the cellular translational machinery, so that shortened forms of the 5' NCR are sufficient for cap-independent translation, and that the internal entry of ribosomes occurs by two distinct modes during the virus replication cycle.

DNA Mutational Analysis↗
PubMed1993

Cooperative binding at a distance by even-skipped protein correlates with repression and suggests a mechanism of silencing.

In this study, we examined how the Drosophila developmental control gene even-skipped (eve) represses transcription. Tissue culture cells were used to show that eve contains domains which inhibit transcriptional activators present at the Ultrabithorax (Ubx) proximal promoter when bound up to 1.5 kb away from these activators. Different portions of eve were fused to a heterologous DNA binding domain to show that three adjacent regions of eve contribute to silencing. There appear to be two mechanisms by which eve protein represses transcription. In this study, we used in vitro transcription and DNA binding experiments to provide evidence for one of these mechanisms. Repression in vitro correlates with binding of eve protein to two low-affinity sites in the Ubx proximal promoter. Occupancy of these low-affinity sites is dependent upon cooperative binding of other eve molecules to a separate high-affinity site. Some of these sites are separated by over 150 bp of DNA, and the data suggest that this intervening DNA is bent to form a looped structure similar to those caused by prokaryotic repressors. One of the low-affinity sites overlaps an activator element bound by the zeste transcription factor. Binding of eve protein is shown to exclude binding by zeste protein. These data suggest a mechanism for silencing whereby a repressor protein would be targeted to DNA by a high-affinity element, which itself does not overlap activator elements. Cooperative binding of further repressor molecules to distant low-affinity sites, and competition with activators bound at these sites lead to repression at a distance.

Animals↗