bluuebunny/crossref_metadata_2025 huggingface dataset:
A chromosomal ars operon homologue of Pseudomonas aeruginosa confers increased resistance to arsenic and antimony in Escherichia coli
Authors: Cai Jie , Salmon Kirsty , DuBow Michael S.
Published: June 2009

SUMMARY: Operons encoding homologous arsenic-resistance determinants (ars) have been discovered in bacterial plasmids from Gram-positive and Gram-negative, organisms, as well as in the Escherichia coli chromosome. However, evidence for this arsenic-resistance determinant in the medically and environmentally important bacterial species Pseudomonas aeruginosa is conflicting. Here the identification of a P. aeruginosa chromosomal ars operon homologue via cloning and complementation of an E. coli ars mutant is reported. The P. aeruginosa chromosomal ars operon contains three potential ORFs encoding proteins with significant sequence similarity to those encoded by the arsR, arsB and arsC genes of the plasmid-based and E. coli chromosomal ars operons. The cloned P. aeruginosa chromosomal ars operon confers augmented resistance to arsenic and antimony oxyanions in an E. coli arsB mutant and in wild-type P. aeruginosa. Expression of the operon was induced by arsenite at the mRNA level. DNA sequences homologous with this operon were detected in some, but not all, species of the genus Pseudomonas, suggesting that its conservation follows their taxonomic-based evolution.
Selection and characterization of mercury- independent activation mutants of the Tn501 transcriptional regulator, MerR
Authors: Parkhill Julian , Lawley Blair , Hobman Jonathan L. , Brown Nigel L.
Published: June 2009

SUMMARY: MerR is the transcriptional regulator of the mercury-resistance (mer) operon of transposon TnSO1, acting at the mer promoter as both an activator in the presence of mercuric salts and a repressor in their absence. This paper reports a method for selection of constitutive activator mutants, which activate transcription in the absence of Hg", and the characterization of these MerRAC proteins. At least two mutations in the MerR protein were found necessary for strong mercury-independent activation, and these mutations lie in the C- terminal two-thirds of the MerR protein near the Hg"-binding cysteines. Anm triple mutation was shown t o increase activation over the corresponding double mutations. All mutant proteins caused further activation in the presence of Hg". The data support a mechanism in which a conformational change of one or both MerR subunits in the homodimer drives a distortion of DNA bound t o a helix-turn-helix structure in the N-terminal region. A mutation in this putative helix-turn-helix region severely reduced both the repressor and activator functions of MerR.
Porin alteration and active efflux: two in vivo drug resistance strategies used by Enterobacter aerogenes
Authors: Mallea Monique , Chevalier Jacqueline , Bornet Charleric , Eyraud Annie , Davin-Regli Anne , Bollet Claude , Pages Jean-Marie
Published: June 2009

SUMMARY: Entembacter aemgenes is among the five most frequently isolated nosocomial pathogens in France, and this bacterium also shows increasing multidrug resistance. In this study, various E. aerogenes strains isolated from hospital units were characterized for their outer-membrane proteins, antibiotic susceptibilities (inhibition diameters and MICs) and resistance mechanisms associated with modification of envelope permeability (porin alteration and active efflux). Diminished outer-membrane permeability due to porin alterations was found in conjunction with the expression of an enzymic barrier in resistant isolates. Interestingly, changes in the functional expression of porins appeared to play a special role in susceptibility to cefepime. An active efflux to quinolones was also identified. Simultaneous changes in envelope permeability, i.e. a porin deficiency (in) and an efflux mechanism (out), were clearly evident in two clinical strains.
Biofilm susceptibility to bacteriophage attack: the role of phage-borne polysaccharide depolymerase
Authors: Hughes Kevin A. , Sutherland Ian W. , Jones Martin V.
Published: June 2009

SUMMARY: Biof ilm bacteria Enterobecter agglomerans 53b and Serratia marcescens Serr were isolated from a food processing factory. A bacteriophage (SF153b), which could infect and lyse strain 53b, was isolated from sewage. This has been shown to possess a polysaccharide depolymerase enzyme specific for the exopolysaccharide (EPS) of strain 53b. Using batch culture and chemostat-linked Modified Robbins Device systems it was observed that SF1 53b could degrade the EPS of a mono-species biofilm (strain 53b) and infect the cells. The disruption of the biofilm by phage was a combination of EPS degradation by the depolymerase and infection and subsequent cell lysis by the phage. Strain Serr biofilms were not susceptible to the phage and the biofilm EPS was not degraded by the phage glycanase, with the result that the biofilm was unaffected by the addition of SF153b phage. Scanning electron microscopy confirmed that specific phage could extensively degrade susceptible biofilms and continue to infect biofilm bacteria whilst EPS degradation was occurring.
A Mycobacterium tuberculosis operon encoding ESAT=6 and a novel low-molecular-mass culture filtrate protein (CFP-10)
Authors: Berthet Fransois-Xavier , Rasmussen Peter Birk , Rosenkrands Ida , Andersen Peter , Gicquel Brigitte
Published: June 2009

SUMMARY: The early secreted antigenic target 6 kDa protein (ESAT-6) is a potent T-cell protein antigen synthesized by Mycobacterium tuberculosis. Its corresponding gene (esat-6) is located in RD1, a 10 kb DNA region deleted in the attenuated tuberculosis vaccine strain Mycobacterium bowis BCG. The promoter region of M. tuberculosis esat-6 was cloned and characterized. A new gene, designated lhp and cotranscribed with esat-6, was identified. Moreover, computer searches in the M. tuberculosis genome identified 13 genes related to the lhplesat-6 operon, defining a novel gene family. The transcription initiation sites of the lhplesat-6 operon were mapped using M. tuberculosis RNA. The corresponding promoter signals were not recognized in Mycobacterium smegmatis, in whichtranscription of lhplesat-6 is initiated at different locations. The M. tuberculosis lhp gene product was identified as CFP-10, a low- molecular-mass protein found in the short-term culture filtrate. These results show that the genes encoding CFP-10 and ESAT-6 are transcribed together in M. tuberculosis and that both code for small exported proteins.
Functional characterization of the Erwinia chrysanthemi Outs protein, an element of a type II secretion system
Authors: Shevchik Vladimir E. , Condemine Guy
Published: June 2009

SUMMARY: Secretion of pectate lyases and a cellulase occurs in winis chrysanthemi through a type II secretion machinery, the Out system. Proper insertion of the secretin OutD in the outer membrane requires the presence of Outs. Outs is an outer-membrane lipoprotein that interacts directly with OutD. Using ligand-blotting experiments, it has been shown that this interaction requires at least the 62 C-terminal amino acids of OutD. When this domain was added to the C-terminal extremity of the secreted pectate lyase PelD, the construct was stabilized by Outs but not inserted into the outer membrane. Thus, this
domain is sufficient to interact with Outs but it is unable to confer the ability to be inserted into the outer membrane in the presence of Outs. A screen for outs mutants unable to secrete pectate lyases gave only mutants unable to properly localize OutD in the outer membrane and no mutant in the protection function. Thus, the interaction between Outs and OutD can probably not be abolished by the mutation of a single amino acid, and the insertion of OutD in the outer membrane may require additional proteins.
The transcriptional regulator gene phrR in Sinorhizobium meliloti WSM419 is regulated by low pH and other stresses
Authors: Reeve Wayne G. , Tiwari Ravi P. , Wong Cheryl M. , Dilworth Michael J. , Glenn Andrew R.
Published: June 2009

Summary: The phrR gene in Sinorhizobium meliloti (previously known as Rhizobium meliloti) WSM419, directly downstream from actA, is induced by low pH or certain stresses (e.g. high concentrations of Zn2+, Cu2+, H2O2 or ethanol), but not in stationary phase or by other stresses (e.g. phosphate limitation, elevated temperature, high concentrations of sucrose or iron). A DNA fragment containing the wild-type phrR gene could not be cloned and inverse PCR was therefore used to amplify a 3�5 kb BamHI fragment containing phrR from the mutant S. meliloti TG2-6 (actA::Tn5). DNA fragments from a BamHI/SalI digest of the amplified product were cloned into pUK21 and sequenced. The phrR open reading frame contiguous to actA appears to code for a 15�2 kDa protein showing significant identity with the proteins encoded by y4wC and y4aM in Rhizobium sp. NGR234. All three proteins resemble transcriptional regulators in containing a DNA-binding helix-turn-helix motif similar to that reported for URF4 in Rhodospirillum rubrum and repressors in coliphage.
Ultrastructural analysis of the sporozoite of Cryptosporidium parvum
Authors: Tetley Laurence , Brown Samantha M. A. , McDonald Vincent , Coombs Graham H.
Published: June 2009

Summary: Cryopreparation of live sporozoites and oocysts of the apicomplexan parasite Cryptosporidium parvum, followed by transmission electron microscopy, was undertaken to show the 3D arrangement of organelles, their number and distribution. Profiles of parasites obtained from energy-filtering transmission electron microscopy of serial sections provided 3D reconstructions from which morphometric data and stereo images were derived. The results suggest that sporozoites have a single rhoptry containing an organized lamellar body, no mitochondria or conventional Golgi apparatus, and one or two crystalline bodies. Micronemes were shown to be spherical, numerous and apically located, and to account for 0·8% of the total cell volume. Dense granules were less numerous, larger, accounted for 5·8% of the cell volume, and were located more posteriorly than micronemes. A structure juxtaposed to the nucleus with similarities to the plastid-like organelle reported for other members of the Apicomplexa was observed. The detailed analysis illustrates the advantages of cryopreparation in retaining ultrastructural fidelity of labile or difficult to preserve structures such as the sporozoite of Cryptosporidium.
A low G+C content genetic island in Mycobacterium avium subsp. paratuberculosis and M. avium subsp. silvaticum with homologous genes in Mycobacterium tuberculosis
Authors: Tizard Mark , Bull Tim , Millar Douglas , Doran Tim , Martin Helene , Sumar Nazira , Ford Jon , Hermon-Taylor John
Published: June 2009

Summary: The technique of representation difference analysis PCR has been applied to find genes specific to Mycobacterium avium subsp. paratuberculosis. This generated a 671 bp fragment which was used to isolate a larger genetic element found in the enteric pathogens M. avium subsp. paratuberculosis and M. avium subsp. silvaticum but which was absent from the very closely related and relatively benign M. avium subsp. avium. This element, designated GS, is greater than 6·5 kbp in length and has a G+C content 9 mol% lower than other genes from this species. There is a previously uncharacterized insertion sequence associated with one end. The GS element encodes five ORFs in M. avium subsp. paratuberculosis and M. avium subsp. silvaticum, all of which have counterparts encoded in Mycobacterium tuberculosis. Database searches revealed homologues for these ORFs in a number of bacterial species, predominantly Gram-negative organisms, including a number of enteric pathogens. These homologous genes encode functions related to LPS or extracellular polysaccharide biosynthesis. This element has a number of features in common with pathogenicity islands such as its low G+C content, an association with a putative insertion sequence and a grouping of genes of related function with a possible link to virulence. No direct link to pathogenicity has been shown but GS may belong to a group of related ‘genetic islands’ and represents the first such element to be identified in mycobacteria.
Cell integrity and morphogenesis in a budding yeast septin mutant
Authors: Cid Victor J. , Adamíková Lubica , Cenamor Rosa , Molina María , Sánchez Miguel , Nombela César
Published: June 2009

Summary: The non-sporulating diploid strain V327 of Saccharomyces cerevisiae was previously isolated in a search for thermosensitive autolytic mutants. This strain is very efficient at releasing intracellular proteins into the medium when incubated at high temperatures. The expression of this lytic phenotype depends on a morphogenetic defect, consisting of the appearance of elongated chains of cells. Transmission electron microscopy revealed a mislocalization of septa at semi-permissive temperatures and a total lack of septation together with abnormal cell wall architecture at a non-permissive temperature. The septin-encoding CDC10 gene was cloned by complementation of the pleiotropic phenotype of the V327 mutant. Rescue and sequencing of CDC10 alleles from V327 revealed a point mutation that created a single amino acid change in a region which is well conserved among septins. This new allele was named cdc10-11. The construction of a cdc10-11 haploid strain by substituting the CDC10 gene with the rescued allele permitted further genetic analyses of the mutation and allowed the construction of new homozygous cdc10-11 diploid strains that showed a reduced ability to sporulate. Fusing both the wild-type and the cdc10-11 alleles to green fluorescent protein (GFP) demonstrated that the mutation does not affect the localization of this septin to the bud neck at the standard growth temperature of 24 °C, although the morphogenetic phenotype at 37 °C parallels the disappearance of Cdc10-GFP at the ring encircling the septum area.
Regulation of photosynthetic gene expression in purple bacteria
Authors: Pemberton John M. , Horne Irene M. , McEwan Alastair G.
Published: June 2009

Purple phototrophic bacteria have the ability to capture and use sunlight efficiently as an energy source. In these organisms, photosynthesis is carried out under anaerobic conditions. The introduction of oxygen into a culture growing phototrophically results in a rapid decrease in the synthesis of components of the photosynthetic apparatus and a change to an alternative source of energy, usually derived from the degradation of organic compounds under aerobic conditions (chemoheterotrophy). Switching back and forth between anaerobic (photosynthetic) and aerobic growth requires tight regulation of photosynthetic gene expression at the molecular level. Initial experiments by Cohen-Bazire et al. (1957) showed quite clearly that the regulation of photosynthetic gene expression was in response to two environmental stimuli. The most potent stimulus was oxygen; its presence shut down production of photosynthetic pigments very rapidly. To a lesser extent photosynthetic gene expression responded to light intensity. Low light intensity produced high levels of photosynthetic pigments; high light intensities caused a decrease, but the effect was less dramatic than that observed for oxygen. Since these initial observations were made in Rhodobacter sphaeroides some forty years ago, a great deal has been revealed as to the nature of the genes that encode the various components of the photosynthetic apparatus. Recent progress in the understanding of the regulation of expression of these genes in R. sphaeroides and Rhodobacter capsulatus is the subject of this review.
Anionogenic groups and surface sialoglycoconjugate structures of yeast forms of the human pathogen Paracoccidioides brasiliensis
Authors: Soares Regina M. A. , Silva-Filho Fernando Costa , Rozental Sonia , Angluster Jayme , de Souza Wanderley , Alviano Celuta S. , Travassos Luiz R.
Published: June 2009

The surface anionogenic groups and sialoglycoconjugate structures of Paracoccidioides brasiliensis yeast forms were analysed by cell microelectrophoresis, binding assays with lectins and viral particles, ultrastructural cytochemistry, enzymic digestion and flow cytofluorimetry. P. brasiliensis yeast forms, particularly the budding primordia, reacted strongly with cationized ferritin. Binding assays showed that the reaction with sialic-acid-specific Limax flavus lectin (LFA) was distributed over the entire P. brasiliensis cell wall. Treatment of yeast forms with neuraminidase significantly reduced their negative surface charge and LFA labelling, which suggests that sialic acid residues are major anionogenic groups exposed on the P. brasiliensis surface. Furthermore, after neuraminidase treatment, labelling with Arachis hypogaea (peanut) agglutinin increased due to unmasking of subterminal βD-galactopyranosyl residues. The sialic acid linkages to galactose are α2,6 and α2,3 as assessed, respectively, by fungal attachment to M1/5 and M1/5 HS8 strains of influenza A virus and binding of Sambucus niger and Maackia amurensis agglutinins. The α2,6 linkage clearly predominated in both experiments. Flow cytofluorimetry analysis revealed the heterogenicity of P. brasiliensis yeast cell populations, which comprised young and mature budding yeasts. Both express binding sites to LFA and Limulus polyphemus agglutinin.
Appearance of a stress-response protein, phage-shock protein A, in Escherichia coli exposed to hydrophobic organic solvents
Authors: Kobayashi Hideki , Yamamoto Mami , Aono Rikizo
Published: June 2009

A 28 kDa protein associated with the inner membrane was induced strongly in Escherichia coli K-12 cells grown in the presence of a hydrophobic organic solvent, n-hexane or cyclooctane. These organic solvents suppressed the growth (growth rate and yield) of E. coli. A partial amino acid sequence showed that this protein was the phage-shock protein PspA. PspA is known to be induced in E. coli cells under extreme stress conditions. The results suggest that E. coli cells are subject to strong stress in the presence of organic solvents. Introduction of a multi-copy plasmid vector carrying the psp operon into E. coli improved the survival frequency of cells exposed suddenly to n-hexane but not the growth rate of cells growing in the presence of n-hexane.
An improved physical and genetic map of Campylobacter jejuni NCTC 11168 (UA580)
Authors: Karlyshev Andrey V. , Henderson John , Ketley Julian M. , Wren Brendan W.
Published: June 2009

Campylobacter jejuni is recognized as the major cause of food-borne gastrointestinal disease in the developed world. To facilitate the molecular genetic analysis of this pathogen, an approximately 18-fold redundant Tropist3 cosmid library was constructed from C. jejuni NCTC 11168 genomic DNA. The cosmid library was partially ordered by hybridization to 15 pulsed-field electrophoresis (PFGE) restriction fragments. This analysis confirmed the estimated size of the genome to be 1730 kb, but suggested discrepancies in some regions of the published physical map. The precise locations of two of the three rRNA gene clusters were mapped using a combination of restriction fingerprinting, sample sequencing and riboprobing. Additionally, 15 further genes were located on the rev-recd map. A more detailed physical and genetic map of C. jejuni NCTC 11168 is presented.
Kluyveromyces marxianus flocculence and growth at high temperature is dependent on the presence of the protein p37
Authors: Falcão Moreira R. , Fernandes P. A. , Moradas-Ferreira P.
Published: June 2009

A Kluyveromyces marxianus mutant deficient in p37, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-like protein, was obtained and characterized with respect to flocculation behaviour, resistance to temperatures above the optimum for growth, morphology, growth, calcofluor white sensitivity and GAPDH activity. In YPD media, the mutant cells were unable to flocculate and were thermosensitive. However, this thermosensitivity could be overcome by the presence of calcium. Calcofluor white was toxic to the mutant, indicating that the mutation affects cell wall structure. The contribution of p37 to total GAPDH activity was 25% when cells were using glucose as carbon source and 50% when cells were growing in 3% ethanol. These results indicate that p37 is likely to be involved in thermotolerance and flocculation, which can be related to its contribution to cell wall integrity.
A response-regulator-like activator of antibiotic synthesis from Streptomyces coelicolor A3(2) with an amino-terminal domain that lacks a phosphorylation pocket
Authors: Guthrie Ellen P. , Flaxman Christine S. , White Janet , Hodgson David A. , Bibb Mervyn J. , Chater Keith F.
Published: June 2009

InStreptomyces coelicolorA3(2),bldAmutants that lack the tRNA for the rare leucine codon UUA fail to make the red undecylprodigiosin antibiotic complex. To find out why, red-pigmented while bald (Pwb) derivatives of abldAmutant were isolated. Using a cloning strategy that allowed for (and demonstrated) dominance of the mutations, they were localized to theredgene cluster. By using insert-mediated integration of a ?C31 phage-based vector, one of the Pwb mutations was more precisely located betweenredstructural genes to a segment of approximately 1 kb about 4 kb from the known pathway-specific regulatory generedD.The segment contained most of an ORF (redZ) encoding a protein (RedZ) with end-to-end similarity to response regulators of diverse function from a variety of bacteria. Remarkably, in RedZ hydrophobic residues replace nearly all of the charged residues that usually make up the phosphorylation pocket present in typical response regulators, including the aspartic acid residue that is normally phosphorylated by a cognate sensory protein kinase. A single TTA codon inredZprovided a potential explanation for thebldA-dependence of undecylprodigiosin synthesis. This codon was unchanged in three Pwb mutants, but further analysis of one of the mutants revealed a potential up-promoter mutation. It seems possible that a combination of low-level natural translation of the UUA codon by a charged non-cognate tRNA, coupled with increased transcription ofredZin the Pwb mutant, allows the accumulation of a threshold level of the RedD protein.
Rhizobium leguminosarum bv. viciae produces a novel cyclic trihydroxamate siderophore, vicibactin
Authors: Dilworth Michael J. , Carson Kerry C. , Giles Robin G. F. , Byrne Lindsay T. , Glenn Andrew R.
Published: June 2009

Trihydroxamate siderophores were isolated from iron-deficient cultures of three strains of Rhizobium leguminosarum biovar viciae, two from Japan (WSM709, WSM710) and one from the Mediterranean (WU235), and from a Tn5-induced mutant of WSM710 (MNF7101). The first three all produced the same compound (vicibactin), which was uncharged and could be purified by solvent extraction into benzyl alcohol. The gallium and ferric complexes of vicibactin were extractable into benzyl alcohol at pH 5.0, while metal-free vicibactin could be extracted with good yield at pH 8.0. The trihydroxamate from MNF7101 (vicibactin 7101) could not be extracted into benzyl alcohol, but its cationic nature permitted purification by chromatography on Sephadex CM-25 (NH+
4 form). Relative molecular masses and empirical formulae were obtained from fast-atom-bombardment MS. The structures were derived from one- and two-dimensional 1H and 13C NMR spectroscopy, using DQF-COSY, NOESY, HMQC and HMBC techniques on the compounds dissolved in methanol-d
4 and DMSO-d
6. Vicibactin proves to be a cyclic molecule containing three residues each of (R)-2,5-diamino-N
2-acetyl-N
5-hydroxypentanoic acid (N
2-acetyl-N
5-hydroxy-D-ornithine) and (R)-3-hydroxybutanoic acid, arranged alternately, with alternating ester and peptide bonds. Vicibactin 7101 differed only in lacking the acetyl substitution on the N2 of the N
5-hydroxyornithine, resulting in net positive charge; it was still functional as a siderophore and promoted 55Fe uptake by iron-starved cells of WSM710 in the presence of an excess of phosphate. The rate of vicibactin biosynthesis by iron-deficient cells of WSM710 was essentially constant between pH 5.5 and 7.0, but much decreased at pH 5.0. When iron-starved cultures were supplemented with potential precursors for vicibactin, the rates of its synthesis were consistent with both β-hydroxybutyrate and ornithine being precursors. At least three genes seem likely to be involved in synthesis of vicibactin from ornithine and β-hydroxybutyrate: a hydroxylase adding the -OH group to the N5 of ornithine, an acetylase adding the acetyl group to the N2 of ornithine, and a peptide synthetase system.
A Candida albicans 37 kDa polypeptide with homology to the laminin receptor is a component of the translational machinery
Authors: Montero Mercedes , Marcilla Antonio , Sentandreu Rafael , Valentin Eulogio
Published: June 2009

A cDNA encoding a 37 kDa protein was isolated from an expression library using antibodies raised against mycelial cell walls fromCandida albicans.The 37 kDa protein has over 60% sequence identity with the 37 kDa laminin-binding protein (LBP) from humans and over 80% identity with the Yst proteins ofSaccharomyces cerevisiae. TheC. albicansprotein was named CaYst1. It was found in membrane and ribosome fractions but surprisingly, was not found in cell walls. Unlike the human LBP, CaYst1p does not bind laminin. These data indicate that CaYst1p is not a cell-surface receptor for laminin as has been proposed for the human LBP. Instead, like theS. cerevisiaeYst proteins, it appears to be a ribosomal protein. This conclusion is supported by the finding thatCaYST1-cDNA complements the lethal phenotype linked to the disruption of bothYSTgenes inS. cerevisiae.
Effects on Bacillus subtilis of conditional expression of the accBC operon encoding subunits of acetyl coenzyme A carboxylase, the first enzyme of fatty acid synthesis
Authors: Perez Carlos A. , Marini Patricia , de Mendoza Diego
Published: June 2009

A Bacillus subtilis strain was constructed in which the operon accBC, encoding the biotin carboxyl carrier protein (BCCP) and biotin carboxylase (BC) subunits of acetyl-CoA carboxylase (ACC), was placed under the control of the IPTG-inducible promoter spac. A reduction in the levels of BCCP resulted in a decrease of de novo fatty acid synthesis and in the total content of membrane fatty acids. This strain was dependent upon the presence of IPTG for a normal growth phenotype. Growth was specifically restored by supplying exogenous long branched-chain fatty acids in the medium, indicating that the inducer-dependent phenotype was specifically related to a conditional block in fatty acid biosynthesis. The strain showed a strong decrease in sporulation frequency when it was induced to sporulate in an IPTG-free medium. Germination and outgrowth were both delayed in spores of the mutant obtained in the absence of IPTG.
Citrate synthase and 2-methylcitrate synthase: structural, functional and evolutionary relationships
Authors: Gerike Ursula , Hough David W. , Russell Nicholas J. , Dyall-Smith Michael L. , Danson Michael J.
Published: June 2009

Following the complete sequencing of the Escherichia coli genome, it has been shown that the proposed second citrate synthase of this organism, recently described by the authors, is in fact a 2-methylcitrate synthase that possesses citrate synthase activity as a minor component. Whereas the hexameric citrate synthase is constitutively produced, the 2-methylcitrate synthase is induced during growth on propionate, and the catabolism of propionate to succinate and pyruvate via 2-methylcitrate is proposed. The citrate synthases of the psychrotolerant eubacterium DS2-3R, and of the thermophilic archaea Thermoplasma acidophilum and Pyrococcus furiosus, are approximately 40% identical in sequence to the Escherichia coli 2-methylcitrate synthase andalso possess 2-methylcitrate synthase activity. The data are discussed with respect tothe structure, function and evolution of citrate synthase and 2-methylcitrate synthase.
The terminal structures of linear plasmids from Rhodococcus opacus
Authors: Kalkus Jutta , Menne Renate , Reh Michael , Schlegel Hans G.
Published: June 2009

The telomers of several linear plasmids of Rhodococcus opacus (formerly Nocardia opaca) were studied. The plasmids pHG201, pHG204 and pHG205 carry proteins bound to their ends, as shown by gel retardation experiments. A sequence hybridizing with the terminal sequence of pHG207, a recombinant linear plasmid consisting of the left part of pHG204 and the right part of pHG205, which was analysed in a previous study by the authors, could be detected in all linear plasmids of the wild-type R. opacus strains MR11 and MR22. However, only pHG204 and pHG206 carry terminal inverted repeats (TIRs) like pHG207. Cloning and sequencing of the terminal fragment of pHG204 revealed a nearly perfect TIR of 1016 bp. In contrast, the termini of pHG201 and pHG205 share little homology. Sequence analysis of the two end fragments of pHG201 revealed a similarity of only 65% within the terminal 34/32 bp and a perfect TIR of only 3 bp. The results support the assumption that long TIRs are not absolutely necessary for replication and maintenance of linear plasmids.
Sequence and structural relationships of leucocins A-, B- and C-TA33a from Leuconostoc mesenteroides TA33a
Authors: Papathanasopoulos Maria A. , Dykes Gary A. , Revol-Junelles Anne-Marie , Delfour Antoine , von Holy Alexander , Hastings John W.
Published: June 2009

Amino acid sequences of two of the three bacteriocins from Leuconostoc mesenteroides TA33a were determined and their sequence-structure relationships investigated. Leucocin B-TA33a consists of 31 amino acid residues, with a molecular mass of 3466 Da. Leucocin B-TA33a does not belong to the pediocin family of bacteriocins, but shares 62% homology with mesenterocin 52B. A partial sequence of 36 amino acids of leucocin C-TA33a (4598 Da) was determined. Leucocin C-TA33a belongs to the class II bacteriocins having the consensus YGNGV motif. The third bacteriocin, leucocin A-TA33a, is identical to leucocin A-UAL 187. Circular dichroism spectra of the leucocins in aqueous solution and micellar SDS indicated that they undergo a structural transition when in a membrane-mimicking environment. Theoretical predictions from circular dichroism data suggest that leucocins A-, B- and C-TA33a adopt a β-structure (48%) in membrane-mimicking environments. Sequence alignments and secondary structure predictions for the N-terminus of leucocins A- and C-TA33a predicted that Cys-9 and Cys-14 are connected by a disulfide bridge and form two β-strands.
Antibacterial action of dipeptides containing an inhibitor of glucosamine-6-phosphate isomerase
Authors: Chmara Henryk , Milewski Slawomir , Andruszkiewicz Ryszard , Mignini Fiorenzo , Borowski Edward
Published: June 2009

Several dipeptides, containing the N
3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) moiety linked to protein and non-protein amino acids, exhibited a strong growth-inhibitory and bactericidal effect against Bacillus subtilis. FMDP-dipeptides were efficiently transported into bacterial cells by a di-tripeptide permease and subsequently cleaved by intracellular Mn2+/Co2+-dependent peptidases. Cleavage rates [0.1-5.6 μmol min-1 (mg protein)-1] were about two orders of magnitude lower than transport rates [40-200 μmol min-1 (mg dry wt)-1]. The released FMDP inactivated glucosamine-6-phosphate (GlcN-6-P) isomerase, an enzyme catalysing the first committed step in a biosynthetic pathway leading to amino sugar-nucleotide precursors of bacterial peptidoglycan. Inhibition of GlcN-6-P isomerase precluded peptidoglycan biosynthesis and resulted in a strong bacteriolytic effect. Results of the studies on consequences of GlcN-6-P isomerase inhibition upon the action of FMDP-dipeptides provided evidence demonstrating that the lack of endogenous GlcN-6-P could be a reason for the triggering of bacterial autolysis. Peptides containing the inhibitors of GlcN-6-P isomerase are one of the very few antimicrobial agents known that exhibit both bactericidal and fungicidal effects.
Repression of nitrogen catabolic genes by ammonia and glutamine in nitrogen-limited continuous cultures of Saccharomyces cerevisiae
Authors: ter Schure Eelko G. , Silljé Herman H. W. , Vermeulen Edgar E. , Kalhorn Jan-Willem , Verkleij Arie J. , Boonstra Johannes , Verrips C. Theo
Published: June 2009

Growth of Saccharomyces cerevisiae on ammonia and glutamine decreases the expression of many nitrogen catabolic genes to low levels. To discriminate between ammonia- and glutamine-driven repression of GAP1, PUT4, GDH1 and GLN1, a gln1-37 mutant was used. This mutant is not able to convert ammonia into glutamine. Glutamine-limited continuous cultures were used to completely derepress the expression of GAP1, PUT4, GDH1 and GLN1. Following an ammonia pulse, the expression of GAP1, PUT4 and GDH1 decreased while the intracellular glutamine concentration remained constant, both in the cytoplasm and in the vacuole. Therefore, it was concluded that ammonia causes gene repression independent of the intracellular glutamine concentration. The expression of GLN1 was not decreased by an ammonia pulse but solely by a glutamine pulse. Analysis of the mRNA levels of ILV5 and HIS4 showed that the response of the two biosynthetic genes, GDH1 and GLN1, to ammonia and glutamine in the wild-type and gln1-37 was not due to changes in general transcription of biosynthetic genes. Ure2p has been shown to be an essential element for nitrogen-regulated gene expression. Deletion of URE2 in the gln1-37 background prevented repression of gene expression by ammonia, showing that the ammonia-induced repression is not caused by a general stress response but represents a specific signal for nitrogen catabolite regulation.
Evidence for a role for the gumB and gumC gene products in the formation of xanthan from its pentasaccharide repeating unit by Xanthomonas campestris
Authors: Vojnov Adrián A. , Zorreguieta Angeles , Dow J. Maxwell , Daniels Michael J. , Dankert Marcelo A.
Published: June 2009

The biosynthesis of the extracellular polysaccharide xanthan in Xanthomonas campestris pv. campestris is directed by a cluster of 12 genes, gumB-gumM. Several xanthan-deficient mutants of the wild-type strain 8004 have previously been described which carry Tn5 insertions in this region of the chromosome. Here it is shown that the transposon insertion in one of these mutants, strain 8397, is located 15 bp upstream of the translational start site of the gumB gene. EDTA-treated cells of strain 8397 were able to synthesize the lipid-linked pentasaccharide repeating unit of xanthan from the three nucleotide sugar donors (UDP-glucose, GDP-mannose and UDP-glucuronic acid) but were unable to polymerize the pentasaccharide into mature xanthan. A subclone of the gum gene cluster carrying gumB and gumC restored xanthan production to strain 8397 to levels approximately 28% of the wild-type. In contrast, subclones carrying gumB or gumC alone were not effective. These results are discussed with reference to previous speculations, based on computer analysis, that gumB and gumC are both involved in the translocation of xanthan across the bacterial membranes.
Yersinia pseudotuberculosis and Yersinia pestis show increased outer membrane permeability to hydrophobic agents which correlates with lipopolysaccharide acyl-chain fluidity
Authors: Bengoechea Jose-Antonio , Brandenburg Klaus , Seydel Ulrich , Díaz Ramón , Moriyón Ignacio
Published: June 2009

The hydrophobic probe N-phenyl-1-naphthylamine accumulated less in nonpathogenic Yersinia spp. and non-pathogenic and pathogenic Yersinia enterocolitica than in Yersinia pseudotuberculosis or Yersinia pestis. This was largely due to differences in the activity of efflux systems, but also to differences in outer membrane permeability because uptake of the probe in KCN/arsenate-poisoned cells was slower in the former group than in Y. pseudotuberculosis and Y. pestis. The probe accumulation rate was higher in Y. pseudotuberculosis and Y. pestis grown at 37 °C than at 26 °C and was always highest in Y. pestis. These yersiniae had LPSs with shorter polysaccharides than Y. enterocolitica, particularly when grown at 37 °C. Gel liquid-crystalline phase transitions (Tc 28–31 °C) were observed in LPS aggregates of Y. enterocolitica grown at 26 and 37 °C, with no differences between nonpathogenic and pathogenic strains. Y. pseudotuberculosis and Y. pestis LPSs showed no phase transitions and, although the fluidity of LPSs of Y. pseudotuberculosis and Y. enterocolitica grown at 26 °C were close below the Tc of the latter, they were always in a more fluid state than Y. enterocolitica LPS. Comparison with previous studies of Salmonella choleraesuis subsp. choleraesuis serotype minnesota rough LPS showed that the increased fluidity and absence of transition of Y. pseudotuberculosis and Y. pestis LPSs cannot be explained by their shorter polysaccharides and suggested differences at the lipid A/core level. It is proposed that differences in LPS-LPS interactions and efflux activity explain the above observations and reflect the adaptation of Yersinia spp. to different habitats.
Inducible chitinolytic system of Aspergillus fumigatus
Authors: Escott Gemma M. , Hearn Veronica M. , Adams David J.
Published: June 2009

Incubation of Aspergillus fumigatus NCPF 2140 in growth medium containing 1 % chitin as sole carbon source led to induction of specific extracellular chitinolytic activity of 1.5 μmol GlcNAc released min-1 (mg protein)-1. The effect was repressed by the inclusion of GlcNAc in the medium, indicating regulation by a negative feedback mechanism. Extracellular chitinase activity was inhibited by allosamidin (IC50 0.12 μM). Multiple chitinolytic enzymes were detected on zymograms of extracellular preparations; levels of individual enzymes induced were dependent upon whether cells were incubated with purified colloidal chitin or a crude preparation of crystalline chitin. A major, inducible, 45 kDa chitinase was purified using ammonium sulphate precipitation, chitin affinity chromatography and a novel procedure involving the electroelution of the enzyme from a substrate gel containing glycol chitin. The enzyme is a glycoprotein with endochitinase activity.
The yvsA-yvqA (293°-289°) region of the Bacillus subtilis chromosome containing genes involved in metal ion uptake and a putative sigma factor
Authors: Wipat Anil , Brignell S. Caroline , Guy B. Jane , Rose Matthias , Emmerson Peter T. , Harwood Colin R.
Published: June 2009

The region between yvsA (293°) and yvqA (289°) of the Bacillus subtilis chromosome has been sequenced within the framework of the B. subtilis 168 international sequencing programme. A primary analysis of the 42 ORFs identified in this 43 kb region is presented. The region included a high proportion of genes that did not show homology with genes in other bacteria. The identified ORFs showed homology to proteins involved in the transport of metal ions, two-component signal transducers, ATP-binding-cassette-type transporters and a sigma factor.
The catecholate siderophores of Azotobacter vinelandii: their affinity for iron and role in oxygen stress management
Authors: Cornish Anthony S. , Page William J.
Published: June 2009

In iron-limited medium, Azotobacter vinelandii strain UW produces three catecholate siderophores: the tricatecholate protochelin, the dicatecholate azotochelin and the monocatecholate aminochelin. Each siderophore was found to bind Fe3+ preferentially to Fe2+, in a ligand:Fe ratio of 1:1, 3:2 and 3:1, respectively. Protochelin had the highest affinity for Fe3+, with a calculated proton-independent solubility coefficient of 10439, comparable to ferrioxamine B. Iron-limited wild-type strain UW grown under N2-fixing or nitrogen-sufficient conditions hyper-produced catecholate siderophores in response to oxidative stress caused by high aeration. In addition, superoxide dismutase activity was greatly diminished in iron-limited cells, whereas catalase activity was maintained. The ferredoxin I (Fdl)-negative A. vinelandii strain LM100 also hyper-produced catecholates, especially protochelin, under oxidative stress conditions, but had decreased activities of both superoxide dismutase and catalase, and was about 10 times more sensitive to paraquat than strain UW. Protochelin and azotochelin held Fe3+ firmly enough to prevent its reduction by.O-
2 and did not promote the generation of hydroxyl radical by the Fenton reaction. Ferric-aminochelin was unable to resist reduction by O-
2 and was a Fenton catalyst. These data suggest that under iron-limited conditions, A. vinelandii suffers oxidative stress caused by.O-
2. The catecholate siderophores azotochelin, and especially protochelin, are hyper-produced to offer chemical protection from oxidative damage catalysed by.O-
2 and Fe3+. The results are also consistent with Fdl being required for oxidative stress management in A. vinelandii.
Phenol hydroxylase cloned from Ralstonia eutropha strain E2 exhibits novel kinetic properties
Authors: Hino Sanae , Watanabe Kazuya , Takahashi Nobuhiro
Published: June 2009

Ralstonia eutropha strain E2 (previously Alcaligenes sp.) is a phenol-degrading bacterium expressing phenol-oxygenating activity with a low Ks (the apparent half-saturation constant in Haldane's equation) and an extremely high KSI (the apparent inhibition constant). To identify the molecular basis for these novel cellular kinetic properties, a 9.5 kb DNA fragment that allowed Pseudomonas aeruginosa PAO1c (Phl- Cat+) to grow on phenol as the sole carbon source was cloned from strain E2 into plasmid pRO1614. PAO1c harbouring this plasmid (designated pROE217) transformed phenol to catechol, indicating that this fragment contains gene(s) for phenol hydroxylase. The cloned genes consist of eight complete ORFs, designated poxRABCDEFG. The products are homologous to those of dmpRKLMNOPQ of Pseudomonas sp. CF600, sharing 30--65% identity: this suggests that the phenol hydroxylase is a multicomponent enzyme. The kinetic constants for phenol-oxygenating activity of PA01c(pROE217) were determined, and these were compared with those of strain E2. The kinetic constants of PAO1c derivatives expressing different phenol hydroxylases were also determined. A comparison of these kinetic data suggests that phenol hydroxylase, the first enzyme in the phenol-degradative pathway, determines Ks and KSI values for the cellular phenol-oxygenating activity. It is thus suggested that the phenol hydroxylase cloned from strain E2 exhibits the novel kinetic properties that were observed with intact cells of strain E2.
The aberrant positioning of nuclei and the microtubular cytoskeleton in Saccharomyces cerevisiae due to improper actin function
Authors: Kopecká Marie , Gabriel Miroslav
Published: June 2009

An excentric position of the nuclei, random orientation of mitoses, and multinuclear budding cells were identified in part of a population of temperature-sensitive (ts) Saccharomyces cerevisiae actin mutants at the permissive temperature of 23 ° by fluorescence and electron microscopy. The phenotype resembled that of mutants in -tubulin, dynein, JNM1, NUM1, ACT3, ACT5, myosins, profilin, tropomyosin 1, SLA2 and other genes. The question was addressed whether the cause was (i) defects in cell polarity in some ts actin mutants, manifested by lack of asymmetry of actin cortical patches, or (ii) lack of cytoplasmic or astral microtubules. The results indicated that in the cells with the nuclear defects, actin cortical patches showed the normal asymmetric distribution typical of undisturbed polarity. Cytoplasmic, astral and spindle microtubules were also preserved. The principal difference found between the wild-type and actin mutant cells was in actin cables, which in the actin mutants were developed insufficiently. It is suggested that actin cables serve as a ‘suspensory apparatus’ and/or ‘intracellular corridor’, predetermining: the location of the nucleus in the central position in interphase; the axis of nuclear movement to the bud neck before mitosis; the direction of the elongating nucleus during mitosis; and the motion of each nucleus from an excentric to a central position during cytokinesis, in cooperation with the above-mentioned and other gene products.
Evidence of past recombination events among the genes encoding the Erp antigens of Borrelia burgdorferi
Authors: Stevenson Brian , Casjens Sherwood , Rosa Patricia
Published: June 2009

A single Borrelia burgdorferi bacterium may contain six or more different 32 kb circular plasmids (cp32s). Although these plasmids are homologous throughout much of their sequences, two loci have been identified at which they can vary significantly. The cp32 plasmids and their relatives each contain two adjacent genes, orfC and orf3, that vary in sequence between plasmids found within clones of individual bacteria. The orfC gene product is homologous to proteins involved in partitioning of bacterial plasmids, and the differences at this locus between plasmids may account for their compatibility. The orfC-orf3 loci are located approximately 5 kb from another variable locus called erp. The orfC-orf3 loci were used as physically linked markers to assess genetic rearrangements in the erp loci; this revealed examples of recombination involving both individual genes and entire erp loci. Recombination of the genes encoding the Erp antigens might contribute to the evasion of the mammalian immune response and could play roles in the establishment and persistence of B. burgdorferi infections in mammalian hosts.
Degradation pathway of CopA, the antisense RNA that controls replication of plasmid R1
Authors: Söderbom Fredrik , Wagner E. Gerhart H.
Published: June 2009

RNA decay in bacteria is carried out by a number of enzymes that participate in the coordinated degradation of their substrates. Endo- and exonucleolytic cleavages as well as polyadenylation are generally involved in determining the half-life of RNAs. Small, untranslated antisense RNAs are suitable model systems to study decay. A study of the pathway of degradation of CopA, the copy number regulator RNA of plasmid R1, is reported here. Strains carrying mutations in the genes encoding RNase E, polynucleotide phosphorylase (PNPase), RNase II and poly(A) polymerase I (PcnB/PAP I) -- alone or in combination -- were used to investigate degradation patterns and relative half-lives of CopA. The results obtained suggest that RNase E initiates CopA decay. Both PNPase and RNase II can degrade the major 3-cleavage product generated by RNase E. This exonucleolytic degradation is aided by PcnB, which may imply a requirement for A-tailing. RNase II can partially protect CopA's 3′-end from PNPase-dependent degradation. Other RNases are probably involved in decay, since in rnblpnp double mutants, decay still occurs, albeit at a reduced rate. Experiments using purified RNase E identified cleavage sites in CopA in the vicinity of, but not identical to, those mapped in vivo, suggesting that the cleavage site specificity of this RNase is modulated by additional proteins in the cell. A model of CopA decay is presented and discussed.
Transposon mutagenesis with IS6100 in the avermectin-producer Streptomyces avermitilis
Authors: Weaden Jenny , Dyson Paul
Published: June 2009

The insertion sequence IS6100 was shown to undergo intermolecular transposition from a temperature-sensitive delivery plasmid to the genome of the avermectin-producer Streptomyces avermitilis, creating cointegrates. Evidence from both Southern hybridization and the range of auxotrophic mutations present in a transposon library was consistent with random transposition. It was not possible to increase transposase expression by readthrough transcription from a copy of the tipA promoter located adjacent to the insertion sequence. This was in part due to the absence of a homologue of the Streptomyces lividans transcriptional activator TipALin S. avermitilis. However, recombinant S. avermitilis strains carrying the S. lividans tip operon were also deficient for induction of the promoter. The frequency of reversion of different auxotrophic mutations by precise excision, involving recombination across 8 bp direct repeats, was shown to vary by at least five orders of magnitude. This dependence of recombination frequency on chromosomal location may contribute to the stability of repetitive modular type I polyketide biosynthetic genes.