bluuebunny/crossref_metadata_2025 huggingface dataset:
Site-directed mutagenesis of streptococcal plasmin receptor protein (Plr) identifies the C-terminal Lys334 as essential for plasmin binding, but mutation of the plr gene does not reduce plasmin binding to group A streptococci
Authors: Winram Scott B. , Lottenberg Richard
Published: June 2009

Plasmin(ogen) binding is a common property of many pathogenic bacteria including group A streptococci. Previous analysis of a putative plasmin receptor protein, Plr, from the group A streptococcal strain 64/14 revealed that it is a glyceraldehyde-3-phosphate dehydrogenase and that the plr gene is present on the chromosome as a single copy. This study continues the functional characterization of Plr as a plasmin receptor. Attempts at insertional inactivation of the plr gene suggested that this single-copy gene may be essential for cell viability. Therefore, an alternative strategy was applied to manipulate this gene in vivo. Site-directed mutagenesis of Plr revealed that a C-terminal lysyl residue is required for wild-type levels of plasmin binding. Mutated Plr proteins expressed in Escherichia coli demonstrated reduced plasmin-binding activity yet retained glyceraldehyde-3-phosphate dehydrogenase activity. A novel integration vector was constructed to precisely replace the wild-type copy of the plr gene with these mutations. Isogenic streptococcal strains expressing altered Plr bound equivalent amounts of plasmin as wild-type streptococci. These data suggest that Plr does not function as a unique plasmin receptor, and underscore the need to identify other plasmin-binding structures on group A streptococci and to assess the importance of the plasminogen system in pathogenesis by inactivation of plasminogen activators and the use of appropriate animal models.
Physical map of the chromosome of Aeromonas salmonicida and genomic comparisons between Aeromonas strains
Authors: Umelo Elizabeth , Trust Trevor J.
Published: June 2009

I-Ceul and Pmel physical maps of the Aeromonas salmonicida A449 chromosome were constructed using PFGE. The circular chromosome of A. salmonicida A449 was estimated to be 4658.30 kb. The approximate location of several genes, including those encoding proteins implicated in virulence, were identified. The map showed that the known virulence-factor-encoding genes were not clustered. The I-Ceul genomic digestion fingerprints of several typical and atypical strains of A. salmonicida were compared. The results confirmed the homogeneity of typical strains, which provided further support for the clonality of the population structure of this group. Extensive diversity was observed in the I-Ceul digestion fingerprint of atypical strains, although a clonality was observed in the strains isolated from diseased goldfish. The results suggest that comparison of I-Ceul digestion fingerprints could be used as a powerful taxonomic tool to subdivide the atypical strains and also help clarify some of the current confusion associated with the taxonomy of the genus Aeromonas.
Excess production of phage λ delayed early proteins under conditions supporting high Escherichia coli growth rates
Authors: Gabig Magdalena , Obuchowski Michal , WeLgrzyn Alicja , Szalewska-Palasz Agnieszka , Thomas Mark S. , WeLgrzyn Grzegorz
Published: June 2009

Bacteriophage λ is unable to lysogenize Escherichia coli hosts harbouring the rpoA341 mutation due to a drastic reduction in transcription from CII-activated lysogenic promoters (p
E, p
1 and p
aQ). In addition, the level of early transcripts involved in the lytic pathway of λ development is also decreased in this genetic background due to impaired N-dependent antitermination. Here, it is demonstrated that despite the reduced level of early lytic p
L- and p
R-derived transcripts, lytic growth of bacteriophage λ is not affected in rich media. The level of the late lytic, p
R-derived transcripts also remains unaffected by the rpoA341 mutation under these conditions. However, it was found that whilst there is no significant difference in the phage burst size in rpoA
+ and rpoA341 hosts growing in rich media, phage λ is not able to produce progeny in the rpoA341 mutant growing in minimal medium, in contrast to otherwise isogenic rpoA
+ bacteria. Provision of an excess of the phage replication proteins O and P in trans or overproduction of the antitermination protein N restore the ability of phage λ to produce progeny in the rpoA341 mutant under the latter conditions. These results suggest that in rich media phage λ produces some early proteins in excess of that needed for its effective propagation and indicate that replication proteins may be limiting factors for phage lytic growth in poor media.
Temporal changes in the frequency of colicinogeny in Escherichia coli from house mice
Authors: Gordon David M. , Riley Margaret A. , Pinou Theodora
Published: June 2009

Escherichia coli was isolated from feral house mice (Mus domesticus) during the course of a mouse plague in the state of Victoria, Australia. The isolates were characterized for the production of colicins and their resistance to the co-occurring colicins. Of the 447 isolates examined, 59% were found to be colicinogenic. Phenotypic and PCR-based genotypic methods were used to determine the types of colicins being produced. Colicin E2 was the most common, representing 27% of the colicin-producing isolates. Colicin la was produced by 3% of the colicinogenic isolates. The remaining colicins could not be identified, but phenotypic and PCR data argue that at least nine different colicin types are present in this collection of E. coli. The frequency of colicinogenic isolates declined from 71% to 43% over the 7 months of the study. All colicin types appeared to decline in frequency. Concurrently, the resistance of isolates to colicin E2 increased from about 50% to 70%. Two hypotheses are proposed to explain the decline in the frequency of colicinogeny in this population of E. coli. The first relates to the within-host interactions occurring among colicinogenic, colicin-susceptible and colicin-resistant populations within a host. The second relates to the among-host interactions between susceptible and colicinogenic populations and the effect of host population densities on these interactions.
Bacterial chemotactic motility is important for the initiation of wheat root colonization by Azospirillum brasilense
Authors: Van de Broek Ann , Lambrecht Mark , Vanderleyden Jos
Published: June 2009

Bacteria of the genusAzospirillumare able to colonize plant roots. Using the glucuronidase (GUS) reporter system, variousAzospirillummutants, including mutants affected in chemotactic motility or extracellular polysaccharide biosynthesis, were investigated for their capacity to initiate wheat root colonization at the root hair zones. Only non-flagellated mutants and a generally non-chemotactic mutant exhibited a strongly reduced colonization ability as compared to the wild-type. No role of theAzospirillumcalcofluor-binding polysaccharide in primary wheat root colonization could be observed. This is the first report demonstrating directly, by using different motility mutants, the requirement of bacterial motility in the establishment of theAzospirillum-plant root association.
Shigella and enteroinvasive Escherichia coli strains are derived from distinct ancestral strains of E. coli
Authors: Rolland Karine , Lambert-Zechovsky Nicole , Picard Bertrand , Denamur Erick
Published: June 2009

The differentiation between Shigella subspecies, and the phylogenetic position of Shigella clones within Escherichia coli clones was determined by analysis of restriction fragment length polymorphisms of rDNA (ribotyping). Seventy-five Shigella strains belonging to the four subspecies and 13 enteroinvasive E. coli (EIEC) strains were compared with the 72 E. coli strains of the ECOR collection, which have been classified into four phylogenetic groups (A, B1, B2 and D). Seventeen Shigella dysenteriae ribotypes, 12 Shigella flexneri ribotypes, 23 Shigella boydii ribotypes, 12 Shigella sonnei ribotypes and 13 EIEC ribotypes were identified following digestion with HindIII and EcoRI. Correspondence analysis of the data showed that S. boydii serotype 13 strains were distantly related to the other Shigella strains, and that S. sonnei and S. flexneri were distinct from S. boydii and S. dysenteriae. The ribotypes of Shigella and ECOR strains were indistinguishable, and S. sonnei, S. flexneri and most S. dysenteriae strains were closely related to phylogenetic group D, whereas S. dysenteriae serotype 1 strains belonged to phylogenetic group B1, and S. boydii strains were evenly distributed between the two groups. The Shigella strains were distantly related to group B2, which contains E. coli strains frequently implicated in extra-intestinal infections in humans. In contrast, the 13 EIEC strains were more widely distributed between phylogenetic groups B1, A and B2. Thus, there was no primordial Shigella species and Shigella and EIEC strains are derived from different ancestral strains.
Crystalline surface protein of Peptostreptococcus anaerobius
Authors: Kotiranta Anja , Haapasalo Markus , Lounatmaa Kari , Kari Kirsti
Published: June 2009

The surface ultrastructure of three anaerobic Gram-positive cocci frequently encountered in oral infections, Peptostreptococcus micros, P. magnus and P. anaerobius, was studied. The type strains of P. micros (DSM 20468) and P. anaerobius (ATCC 27337), several clinical isolates of both species and the type strain of P. magnus (DSM 20470) were included. Thin-sectioned cells studied by electron microscopy revealed a homogeneous layer outside the peptidoglycan layer in P. anaerobius. In P. micros and P. magnus a more amorphous layer was present. No periodic structures were seen in negatively stained whole cells of these three species. However, in freeze-etched cells of P. anaerobius a crystalline surface protein layer (S-layer) was detected. No periodicity was seen in any of the P. micros strains or the magnus type strain by the methods used, but a periodic pattern was observed in negatively stained specimens of cell wall fragments of sonicated P. anaerobius cells. No capsular material was visible outside the S-layer in P. anaerobius. The cells of the Peptostreptococcus spp. were extracted for 30 min with detergents and urea. One er cent SDS and 6 M urea both extracted a major 78 kDa protein from all strains of P. anaerobius. Extraction of P. micros and P. magnus cells did not reveal any major protein bands comparable to that of P. anaerobius. Surface biotinylation of cells followed by Western blotting and detection by alkaline-phosphatase-conjugated extravidin showed strong staining of the 78 kDa band in P. anaerobius, further indicating that this molecule is located on the surface of the cell and is the S-protein. Another protein, of 127 kDa, was also detected by surface biotinylation in all P. anaerobius strains. No differences were detected in colony morphology or cell surface structures between the four strains. Isoelectric focusing of the proteins of P. anaerobius revealed that the S-proteins of individual strains have different pl values, ranging from 5.3 to 6.9.
The signal transducer encoded by ampG is essential for induction of chromosomal AmpC -lactamase in Escherichia coli by -lactam antibiotics and unspecific inducers
Authors: Schmidt Herbert , Korfmann Gisela , Barth Holger , Martin Hans H.
Published: June 2009

Chemical mutagenesis of the AmpC
-lactamase-hyperinducible Escherichia coli strain SN0301/pNu305 carrying the cloned ampC and ampR genes from Citrobacter freundii OS60 gave four independent mutants in which
-lactamase was no longer inducible, or was inducible only to a low level, by
-lactam antibiotics. The genes ampC, ampR, ampD and ampE, which were essential for
-lactamase induction, were functional in these mutants. In all four mutants, the sites of mutation were mapped to 9.9 min on the E. coli chromosome. Complementation with wild-type ampG restored inducibility of
-lactamase to wild-type levels. The nucleotide sequence of all four mutant ampG alleles (ampG1, ampG3, ampG4 and ampG5) was determined. In three of the mutants, a single base exchange led to an amino acid change from glycine to aspartate at different sites in the deduced amino acid sequence. In the fourth mutant (ampG4), with low-level inducibility, the nucleotide sequence was identical to wild-type ampG. Spontaneous back-mutation of the chromosomal ampG1 mutant resulted in restoration of wild-type inducibility and a return to the wild-type ampG sequence. Unspecific induction by components of the growth medium was also dependent on intact ampG function.
Organization of carbamoyl-phosphate synthase genes in Neisseria gonorrhoeae includes a large, variable intergenic sequence which is also present in other Neisseria species
Authors: Lawson Fiona S. , Billowes Finola M. , Dillon Jo-Anne R.
Published: June 2009

The carbamoyl-phosphate synthase (CPS) enzyme in prokaryotes is a heterodimer, encoded by genes commonly called carA and carB. In most prokaryotes examined, these genes are separated by up to 24 bp and are cotranscribed. In Pseudomonas aeruginosa, carA and carB are also co-transcribed, but are separated by 682 bp. We have determined the complete DNA sequence of the carA and carB genes of Neisseria gonorrhoeae strain CH811. carA (1125 bp) and carB (3237 bp) are similar in size and sequence to other prokaryotic CPS genes, however they are separated by an intervening sequence of 3290 bp which has no similarity to the intervening sequence between other CPS genes; furthermore, putative transcription terminators are found downstream of both carA and carB. Several neisserial repetitive sequences were identified within the 9 kb sequenced, as well as novel 120 and 150 bp repeats (designated RS6 and RS7, respectively) which were found within the intervening sequence between carA and carB. To determine whether the intervening sequence observed in N. gonorrhoeae CH811 was not unusual, the sequence between carA and carB was amplified by PCR from 30 isolates of N. gonorrhoeae. The intervening sequence was found to vary in size, from approximately 2·2 to 3·7 kb, although the carA and carB genes themselves did not vary in size in isolates with functional CPS enzyme. A similar large, variably sized intervening sequence was also found between the carA and carB genes of 12 isolates of N. meningitidis and 18 commensal Neisseria isolates comprising nine species. This unexpected organization of the CPS genes in N. gonorrhoeae is therefore widespread throughout the genus Neisseria.
The immunogenicity of recombinant Mycobacterium smegmatis bearing BCG genes
Authors: Falcone Valeria , Bassey Effiong , Jacobs William , Collins Frank
Published: June 2009

Specific pathogen-free C57BL/6 mice infected with recombinantMycobacterium smegmatis(rM. smegmatis) bearing BCG genes showed increased splenic survival compared to those receiving the vector control (plasmid DNA only). The mouse-passaged rM. smegmatis(J3R) survived in peritoneal macrophages better than the vector control, regardless of whether the macrophages were infectedin vivo or in vitro. When rM. smegmatisJ3R was cultured in synthetic Proskauer-Beck-Tween medium, protein bands characteristic of BCG culture filtrates and not present in the vector control preparation were observed. Mice immunized with two doses of heat-killed J3R suspended in Freund's adjuvant were able to limit the growth of virulentMycobacterium tuberculosiswithin the lung and spleen compared to that observed in control mice receiving adjuvanted vector control or Freund's adjuvant alone.
Actin cortical cytoskeleton and cell wall synthesis in regenerating protoplasts of the Saccharomyces cerevisiae actin mutant DBY 1693
Authors: Kopecká Marie , Gabriel Miroslav
Published: June 2009

The relationship between the actin cytoskeleton and cell wall synthesis was studied by light and electron microscopy in protoplasts ofSaccharomyces cerevisiaeDBY 1693 containing theact1-1allele. Since protoplasting also disturbs the actin cytoskeleton, these mutant protoplasts had a double error in their actin cytoskeletons. In the period between the onset of wall synthesis and completion of the wall, protoplasts grown at the permissive temperature showed an even distribution of actin patches all over the surface on which a new cell wall was being synthesized. After wall completion, actin patches partially disappeared, but then re-appeared, accumulated in growth regions at the start of polarized growth. This was compared with the pattern of actin patches observed in intact temperature-sensitive actin mutant cells cultivated at the permissive temperature. Electron microscopy of freeze-etched replicas revealed finger-like invaginations of the plasma membrane in both the actin mutant cells and their protoplasts. These structures showed a very similar distribution to the actin patches detected by rhodamine phalloidin staining in the fluorescence microscope. A hypothesis is presented, explaining the role of actin patches/finger-like invaginations of the plasma membrane in the synthesis of β-(13)-D-glucan wall microfibrils in yeast cells.
Hirsutellin A, a toxic protein produced in vitro by Hirsutella thompsonii
Authors: Mazet Isabelle , Vey Alain
Published: June 2009

A toxic protein, hirsutellin A, has been purified from the mite fungal pathogen, Hirsutella thompsonii, using ammonium sulphate precipitation, ion exchange chromatography and gel filtration on Bio-Gel P-10. The protein has been characterized as a monomer with a molecular mass of 15 kDa and an isoelectric point of 105. The amino acid composition and the N-terminal sequence of hirsutellin A (34 amino acids) have been determined. From these results, the toxin appears to be distinct from other known proteins. It is not glycosylated, and does not show proteolytic activity. The toxin is also antigenic, thermostable and not inactivated by treatments with proteolytic enzymes. Toxicity bioassays showed that injection of larvae of the waxmoth, Galleria mellonella, with hirsutellin A at low dosages [1 g toxin (g body wt)−1] caused a high mortality rate. Hirsutellin A was also toxic per os to neonatal larvae of the mosquito Aedes aegypti.
Four glucosyltransferases, GtfJ, GtfK, GtfL and GtfM, from Streptococcus salivarius ATCC 25975
Authors: Simpson Christine L. , Cheetham Norman W. H. , Jacques Nicholas A.
Published: June 2009

The four recombinant glucosyltransferases (GTFs), GtfJ, GtfK, GtfL and GtfM, that had previously been cloned from Streptococcus salivarius ATCC 25975, were individually expressed in Escherichia coli and their glucan products and kinetic properties were analysed. GtfJ was a primer-dependent GTF which synthesized an insoluble glucan composed mainly of α-(13)-linked glucosyl residues in the presence of dextran T-10. GtfK was primer-stimulated, and produced a linear soluble dextran without any detectable branch points both in the absence and in the presence of dextran T-10. GtfL was primer-independent and produced a mixed-linkage insoluble glucan composed of approximately equal proportions of α-(13)-and α-(16)-linked glucosyl residues. GtfL was inhibited by dextran T-10. GtfM was primer-independent and produced a soluble dextran with approximately 5% α-(13)-linked glucosyl residues. GtfM was essentially unaffected by the presence of dextran T-10. The results confirmed that each enzyme represented one of the four possible combinations of primer-dependency and product solubility and that each possessed unique biosynthetic properties. The soluble dextrans formed by GtfK and GtfM, as well as the mixed-linkage insoluble glucan formed by GtfL, were also capable of acting as primers for the primer-dependent GtfJ and the primer-stimulated GtfK. Unexpectedly, the linear dextran produced by GtfK was by far the least effective either at priming itself or at activating and priming the primer-dependent GtfJ.
A convenient and reproducible method to genetically transform bacteria of the genus Bifidobacterium
Authors: Argnani Alessandra , Leer Rob J. , van Luijk Nicole , Pouwels Peter H.
Published: June 2009

A protocol was developed for the introduction of foreign plasmid DNA into variousBifidobacteriumstrains. The method, which is applicable to allBifidobacteriumspecies tested so far, is based on electroporation of bacteria made competent by preincubation in electroporation buffer for several hours at 4 °C. Transformation ofBifidobacteriumcould be achieved with a plasmid vector originating fromBifidobacteriumand with plasmid vectors fromCorynebacterium,but not with vectors carrying replicons fromLactococcusorLactobacillus.
Metabolism of methanesulfonic acid involves a multicomponent monooxygenase enzyme
Authors: Higgins Timothy P. , Davey Margaret , Trickett Jim , Kelly Don P. , Murrell J. Colin
Published: June 2009

Summary: A novel methylotroph, strain M2, capable of utilizing methanesulfonic acid (MSA) as a sole source of carbon and energy was the subject of these investigations. The initial step in the biodegradative pathway of MSA in strain M2 involved an inducible NADH-specific monooxygenase enzyme (MSAMO). Partial purification of MSAMO from cell-free extracts by ion-exchange chromatography led to the loss of MSAMO activity. Activity was restored by the mixing of three distinct protein fractions designated A, B and C. The reconstituted enzyme had a narrow substrate specificity relative to crude cell-free extracts. Addition of FAD and ferrous ions to the reconstituted enzyme complex resulted in a fivefold increase in enzyme activity, suggesting the loss of FAD and ferrous ion from the multicomponent enzyme on purification. Analysis of mutants of strain M2 defective in the metabolism of C1 compounds indicated that methanol was not an intermediate in the degradative pathway of MSA and also confirmed the involvement of a multicomponent enzyme in the degradation of MSA by methylotroph strain M2.
The influence of A-band and B-band lipopolysaccharide on the surface characteristics and adhesion of Pseudomonas aeruginosa to surfaces
Authors: Makin Stephen A. , Beveridge Terrance J.
Published: June 2009

Summary: Pseudomonas aeruginosa PAO1 possesses two distinct lipopolysaccharide (LPS) O-polysaccharide species, A- and B-band LPS, the relative expression of which appears to be under environmental control. In an attempt to identify the influence these LPS types have on surface characteristics and adhesion, we examined the surface hydrophobicity and surface charge of P. aeruginosa PAO1 (O5 serotype) and its isogenic LPS derivatives which possessed A+B−, A+B− and A−B− LPS. The surface characteristics of the strains affected their ability to adhere to hydrophilic (glass) and hydrophobic (polystyrene) surfaces. Cells possessing only A-band LPS demonstrated the highest surface hydrophobicity, followed by the strain lacking both A- and B-band LPS. The presence of B-band LPS resulted in a more hydrophilic surface. Strains lacking B-band LPS (A+B− and A−B−) had more electronegative surfaces than those possessing B-band LPS (A+B+ and A−B−), with cells lacking both A- and B-band LPS showing the highest surface electronegativity. These data suggest that the main surface-charge-determining groups reside in the core region of the LPS molecule. Cells with the lowest surface hydrophobicity and lowest surface charge (A−B−, A−B+) adhered to glass the most efficiently, implying a role for electrostatic interaction, whereas adhesion to polystyrene mirrored the relative hydrophobicities of the strains (A+B−>A+B−>A−B−>A+B+). It is postulated that phenotypic variation in the relative expression of A- and B-band LPS may be a mechanism by which P. aeruginosa can alter its overall surface characteristics in such a way as to influence adhesion and favour survival.
The fructokinase from Rhizobium leguminosarum biovar trifolii belongs to group I fructokinase enzymes and is encoded separately from other carbohydrate metabolism enzymes
Authors: Fennington George J. , Hughes Thomas A.
Published: June 2009

Summary: The Rhizobium leguminosarum bv. trifolii BAL fructokinase (frk) gene was isolated on a 2.4 kb BamHI fragment from the cosmid pLA72 by complementation analysis of the Tn5-induced frk mutant BAL79, and confirmed by hybridization analysis. The nucleotide sequence of the frk gene was found to contain an open reading frame consisting of 978 bp encoding 326 amino acids, which was then compared to known fructokinase sequences. The fructokinase gene was not contained in an operon and is encoded separately from other enzymes of carbohydrate metabolism. Its product is therefore assigned to the group I fructokinases. A putative promoter (TTGACA-N16-GTTGAT), ribosome-binding site and termination sequence were identified. The Frk protein contained several motifs conserved in other known fructokinase sequences, including an ATP-binding and a substrate-binding motif. The hydropathy plot derived from the frk gene sequence data revealed the fructokinase as a hydrophilic protein. The fructokinase protein was purified to electrophoretic homogeneity by a three-step method using chromatofocusing, affinity chromatography and gel filtration. Its purity was confirmed by SDSPAGE and it was visualized as a single band by silver staining. The N-terminal amino acid sequence of the purified fructokinase confirmed the proposed open reading frame of the frk gene. The purified fructokinase had a molecular mass of 36.5 kDa, pl of 4.65, pH activity range of 6.0-9.0 (maximum activity at pH 8.0) and a Mg2+ requirement. It had a K
m of 0.31 mM and a V
max of 31 μmol fructose 6-phosphate (mg protein)−1 min with fructose as substrate. The R. leguminosarum bv. trifolii BAL fructokinase was biochemically and molecularly similar to other bacterial fructokinases.
Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana
Authors: Strobel Gary , Yang Xianshu , Sears Joe , Kramer Robert , Sidhu Rajinder S. , Hess W. M.
Published: June 2009

Summary: Pestalotiopsis microspora was isolated from the inner bark of a small limb of Himalayan yew, Taxus wallachiana, and was shown to produce taxol in mycelial culture. Taxol was identified by spectroscopic and chromatographic comparisons with authentic taxol. Optimal taxol production occurred after 2-3 weeks in still culture at 23°C. [14C]Acetate and [14C]phenylalanine served as precursors for fungal [14C]taxol. These observations on P. microspora are discussed in relation to the biological importance of taxol production by fungi in general.
SYR2, A Gene Necessary for Syringomycin Growth Inhibition of Saccharomyces Cerevisiae
Authors: Cliften Paul , Wang Yeelan , Mochizuki Daisuke , Miyakawa Tokichi , Wangspa Rungrach , Hughes Joanne , Takemoto Jon Y.
Published: June 2009

The Pseudomonas syringae cyclic lipodepsipeptide syringomycin inhibits the growth of Saccharomyces cerevisiae. A novel yeast gene, SYR2 was found to complement two syringomycin-resistant S. cerevisiae mutants. SYR2 was cloned, sequenced, and shown to encode a 349 amino acid protein located in the endoplasmic reticulum. SYR2 was identical to SUR2 which is involved in survival during nutritional starvation. Gene disruption or overexpression of SYR2 did not affect cell viability or ergosterol levels, but did influence cellular phospholipid levels. The findings suggest that phospholipids are important for the growth inhibitory action of syringomycin.
Reclassification of the Penicillium Roqueforti Group into Three Species on the Basis of Molecular Genetic and Biochemical Profiles
Authors: Boysen Marianne , Skouboe Pernille , Frisvad Jens , Rossen Lone
Published: June 2009

Penicillium roqueforti is currently divided into two varieties, one used for cheese starter cultures, P. roqueforti var. roqueforti and one ubiquitous patulin-producing variety, P. roqueforti var. carneum. The ribosomal regions comprising the 5.8S gene and the internal transcribed spacers, ITS I and ITS II, have been analysed from 10 isolates belonging to each variety. The 10 P. roqueforti var. carneum isolates were separated into two groups of five on the basis of 12 base-pair differences in the ITS regions. One of the groups of P. roqueforti var. carneum in the following designated P. carneum differed from P. roqueforti var. roqueforti here designated P. roqueforti in just two positions, while the other group, here called P. paneum differed from P. roqueforti in 12 positions. Random Amplified Polymorphic DNA (RAPD) analysis substantiated these findings, and a comparison of secondary metabolites produced by the three groups showed that the P. roqueforti isolates all produce Penicillium Roqueforti (PR) toxin, marcfortines and fumigaclavine A, while the P. carneum isolates produce patulin, penitrem A and mycophenolic acid, as well as unidentified metabolites. P. paneum produces secondary metabolites in five chromophore families including the known mycotoxins patulin and botryodiploidin. On the basis of these findings it is proposed that P. roqueforti is reclassified into three species named P. roqueforti, P. carneum and P. paneum.
Regulation of Escherichia Coli Adenylate Cyclase Activity during Hexose Phosphate Transport
Authors: Dumay Valérie , Danchin Antoine , Crasnier Martine
Published: June 2009

In Escherichia coli, cAMP levels vary with the carbon source used in the culture medium. These levels are dependent on the cellular concentration of phosphorylated EnzymeIIAglc, a component of the glucose-phosphotransferase system, which activates adenylate cyclase (AC). When cells are grown on glucose 6-phosphate (Glc6P), the cAMP level is particularly low. In this study, we investigated the mechanism leading to the low cAMP level when Glc6P is used as the carbon source, i.e. the mechanism preventing the activation of AC by phosphorylated EnzymellAglc. Glc6P is transported via the Uhp system which is inducible by extracellular Glc6P. The Uhp system comprises a permease UhpT and three proteins UhpA, UhpB and UhpC which are necessary for uhpT gene transcription. Controlled expression of UhpT in the absence of the regulatory proteins (UhpA, UhpB and UhpC) allowed us to demonstrate that (i) the Uhp regulatory proteins do not prevent the activation of AC by direct interaction with EnzymellAglc and (ii) an increase in the amount of UhpT synthesized (corresponding to an increase in the amount of Glc6P transported) correlates with a decrease in the cAMP level. We present data indicating that Glc6P per se or its degradation is unlikely to be responsible for the low cAMP level. It is concluded that the level of cAMP in the cell is determined by the flux of Glc6P through UhpT.
Effect of oxygen, inoculum composition and flow rate on development of mixed-culture oral biof ilms
Authors: Bradshaw David J. , Marsh Philip D. , Allison Clive , Schilling Kurt M.
Published: June 2009

The effect of aeration on the development of a defined biofilm consortium of oral bacteria was investigated in a two-stage chemostat system. An inoculum comprising 10 species, including both facultatively anaerobic and obligately anaerobic bacteria, and species associated with oral health and disease, was inoculated into an anaerobic first-stage chemostat vessel. The effluent from this chemostat was linked to an aerated [200 ml CO2/air (5:95, v/v) min–1] second-stage vessel, in which removable hydroxyapatite discs were inserted to allow biofilm formation. Comparisons were made of planktonic and biofilm communities in the aerated second-stage vessel by means of viable counts. Both planktonic and early biofilm communities were dominated by Neisseria subflava, comprising > 40% of total c>f.u. in the fluid phase, and > 80% of c.f.u. in 2 h biofilms. Obligate anaerobes persisted in this mixed culture, and succession in biofilms led them to predominate only after 7 d. Despite the continuous addition of air, the dissolved oxygen tension (dO2) within the culture remained low (< 5% of air saturation), and the redox potential (E
h) was –275 mV. In order to assess the significance of the presence of N. subflava in community development, a subsequent experiment omitted this aerobe from the inoculum, to produce a nine-species culture. The planktonic phase was predominated by three streptococcal species, Prevotella nigrescens and Fusobacterium nucleatum. Biofilms again underwent successional changes, with anaerobes increasing in proportion with time. In contrast to the culture including N. subflava, dO2 was 50–60% of air saturation, and the Eh was + 50 mV. In the final experiment, the rate of addition of first-stage culture was reduced to 1/10 of that in the previous experiment, in order to determine whether anaerobes were growing, rather than merely persisting in the aerated culture. The data for the planktonic phase indicated that the anaerobes were growing in aerated (dO2 40–50%, E
h +100 mV) conditions. Once again, anaerobes increased in proportion in older biofilms. The study indicates that mixed cultures can protect obligate anaerobes from the toxic effects of oxygen, both in the biofilm and planktonic modes of growth.
Cloning, sequencing and disruption of a bromoperoxidase-catalase gene in Streptomyces venezuelae: evidence that it is not required for chlorination in chloramphenicol biosynthesis
Authors: Facey Sandra J. , Groß Frank , Vining Leo C. , Yang Keqian , van Pé Karl-Heinz
Published: June 2009

Genomic DNA libraries of Streptomyces venezuelae ISP5230 and of a mutant blocked at the chlorination step of chloramphenicol biosynthesis were probed by hybridization with a synthetic oligonucleotide corresponding to the N-terminal amino acid sequence of a bromoperoxidase-catalase purified from the wild-type strain. Hybridizing fragments obtained from the two strains were cloned and sequenced. Analysis of the nucleotide sequences demonstrated that the fragments contained the same 1449 bp open reading frame with no differences in nucleotide sequence. The deduced polypeptide encoded 483 amino acids with a calculated M
r of 54200; the N-terminal sequence was identical to that of the bromoperoxidase-catalase purified from wild-type S. venezuelae. Comparison of the amino acid sequence predicted for the cloned bromoperoxidase-catalase gene (bca) with database protein sequences showed a significant similarity to a group of prokaryotic and eukaryotic catalases, but none to other peroxidases or haloperoxidases. Replacement of the bca gene in the wild-type strain of S. venezuelae with a copy disrupted by insertion of a DNA fragment encoding apramycin resistance did not prevent chloramphenicol production. The results suggest that the role of the enzyme in S. venezuelae is related to its activity as a catalase rather than as a halogenating agent.
Saccharomyces cerevisiae expressing bacterial polyhydroxybutyrate synthase produces poly-3-hydroxybutyrate
Authors: Leaf Timothy A. , Peterson Marvin S. , Stoup Sandra K. , Somers David , Srienc Friedrich
Published: June 2009

The polyhydroxybutyrate (PHB) synthase gene of the bacterium Alcaligenes eutrophus was used to construct a yeast plasmid which enabled expression of the functional synthase enzyme in Saccharomyces cerevisiae. Cells transformed with the synthase plasmid accumulated up to 0.5% of cell dry weight as PHB, with accumulation occurring in the stationary phase of batch growth. The identity of PHB in recombinant yeast cells was confirmed with 1HNMR spectra of chloroform-extracted cell material. In addition, freeze-fracture electron microscopy revealed cytoplasmic granules exhibiting plastic deformations characteristic for PHB. GC results indicated a low background level of PHB in the wild-type strain, but intact polymer could not be detected by 1H-NMR. Formation of PHB in the recombinant strain implies the participation of native yeast enzymes in the synthesis of D-3-hydroxybutyryl-CoA (3-HB-CoA). Inhibition studies with cerulenin indicated that the fatty acid synthesis pathway is not involved in PHB precursor formation. Wild-type cell-free extracts showed D-3-HB-CoA dehydrogenase activity [150-200 nmol min-1 (mg protein)-1] and acetoacetyl-CoA thiolase activity [10-20 nmol min-1 (mg protein)-1], which together could synthesize monomer from acetyl-CoA. PHB accumulation was simultaneous with ethanol production, suggesting that PHB can act as an alternate electron sink in fermentative metabolism. We propose that PHB synthesis in recombinant yeast is catalysed by native cytoplasmic acetoacetyl-CoA thiolase, a native β-oxidation protein possessing D-3-HB-CoA dehydrogenase activity and heterologous PHB synthase.
Fuse1 alcohols induce hyphal-like extensions and pseudohyphal formationin yeasts
Authors: Dickinson J. Richard
Published: June 2009

At a concentration of 0*5% (vlv), isoamyl alcohol induced the formationof hyphal-like extensions in haploid and diploid strains of Saccharomyces cerewisiae in liquid complex medium. These extensions, which develop via budinitiation and elongation, undergo DNA replication and nuclear division andappear similar in many respects to an aberrant form of the cell division cycle. However, in 025 % (vh) isoamyl alcohol, 5. cerewisiae formed pseudohyphae. Other ‘fusel’ alcohols (which are the products of amino acid catabolism) also induced hyphal-like extensions in this yeast, with n-amyl alcohol being as equally effective as isoamyl alcohol. lsoamyl alcohol induced the formation of pseudohyphae in two species of Candida and both hyphal-like extensions and pseudohyphae in Brettanomyces anomalus, suggesting a close relationship or a common basis to the development of the two morphologies.
The consensus coding sequence (CCDS) project: Identifying a common protein-coding gene set for the human and mouse genomes
Authors: Pruitt Kim D. , Harrow Jennifer , Harte Rachel A. , Wallin Craig , Diekhans Mark , Maglott Donna R. , Searle Steve , Farrell Catherine M. , Loveland Jane E. , Ruef Barbara J. , Hart Elizabeth , Suner Marie-Marthe , Landrum Melissa J. , Aken Bronwen , Ayling Sarah , Baertsch Robert , Fernandez-Banet Julio , Cherry Joshua L. , Curwen Val , DiCuccio Michael , Kellis Manolis , Lee Jennifer , Lin Michael F. , Schuster Michael , Shkeda Andrew , Amid Clara , Brown Garth , Dukhanina Oksana , Frankish Adam , Hart Jennifer , Maidak Bonnie L. , Mudge Jonathan , Murphy Michael R. , Murphy Terence , Rajan Jeena , Rajput Bhanu , Riddick Lillian D. , Snow Catherine , Steward Charles , Webb David , Weber Janet A. , Wilming Laurens , Wu Wenyu , Birney Ewan , Haussler David , Hubbard Tim , Ostell James , Durbin Richard , Lipman David
Published: June 2009

Effective use of the human and mouse genomes requires reliable identification of genes and their products. Although multiple public resources provide annotation, different methods are used that can result in similar but not identical representation of genes, transcripts, and proteins. The collaborative consensus coding sequence (CCDS) project tracks identical protein annotations on the reference mouse and human genomes with a stable identifier (CCDS ID), and ensures that they are consistently represented on the NCBI, Ensembl, and UCSC Genome Browsers. Importantly, the project coordinates on manually reviewing inconsistent protein annotations between sites, as well as annotations for which new evidence suggests a revision is needed, to progressively converge on a complete protein-coding set for the human and mouse reference genomes, while maintaining a high standard of reliability and biological accuracy. To date, the project has identified 20,159 human and 17,707 mouse consensus coding regions from 17,052 human and 16,893 mouse genes. Three evaluation methods indicate that the entries in the CCDS set are highly likely to represent real proteins, more so than annotations from contributing groups not included in CCDS. The CCDS database thus centralizes the function of identifying well-supported, identically-annotated, protein-coding regions.
Outcome Research and Interagency Work with Children: What Does it Tell us About What the CAMHS Contribution Should Look Like?
Authors: Worrall‐Davies Anne , Cottrell David
Published: June 2009

Child and adolescent mental health (CAMH) services in the UK have a long history of multiagency working. In this article, we explore the difficulties in agreeing an evidence‐base for interagency work, and describe some of the challenges this poses for practitioners and service planners. We use current literature to outline the barriers and facilitators to good multi‐agency practice and explore the outcomes of integrated professional work with children and young people, the development of ‘comprehensive CAMHS’ positions, and CAMH work as an exemplar of a multi‐agency service.
Cardiac air transit following venous air embolism and right ventricular air aspiration*
Authors: Schäfer S. T. , Lindemann J. , Neumann A. , Brendt P. , Kaiser G. M. , Peters J.
Published: June 2009

Using intra‐cardiac echocardiography in anaesthetised swine we tested the hypotheses that embolised air (i) passes immediately through the right atrium into the ventricle; (ii) persists in the right ventricle for a long time; (iii) is detectable for longer within the right ventricle or main pulmonary artery than the right atrium, and (iv) right ventricular aspiration recovers more air than right atrial aspiration. Following intravenous injection of different air volumes the air appeared in the right atrium in a mean (95% CI) of 3 s (2.5–3.5 s) and almost simultaneously in the right ventricle after 5 s (3.9–6.0 s), but air persisted for longer in the right ventricle (420 s; (367–473 s)) and pulmonary artery (541 s; (475–606 s)) than in the right atrium (404 s (353–457 s)), particularly with larger air volumes and in the semi‐upright position. More air was recovered via a right ventricular catheter than an atrial catheter (52% vs 25%, p < 0.01).
Non‐infant adoption from care: lessons for safeguarding children
Authors: Rees C. A. , Selwyn J.
Published: June 2009

Aim  To explore use of adoption in remedying abuse and neglect, to inform child protection practice and to identify professional responsibilities to adoptive families.

Method  A cohort of 130 children was identified for whom adoption was recommended in 1991–1996 at a mean age of 5.7 years (range 3–11). All were in local authority care for child protection reasons. Background information was gathered from social work records. The children were traced between 6 and 11 years later and their adopters interviewed. The Strengths and Difficulties Questionnaire and the Parent–Child Communication Scale were completed.

Results  All but three birth parents had traumatic childhoods involving abuse, neglect and/or time in care. Eighty‐six per cent had violent adult relationships. Mental illness, learning difficulties and substance abuse were prominent. Sixty‐seven per cent of families were known to social services when children were born, but 98% experienced abuse or neglect. Most adopters found the first year challenging, though rewarding. Depression, anxiety and marital problems were common. Children's learning difficulties, conduct problems, emotional ‘phoniness’ and rejection affected closeness. At follow‐up 28% described rewarding, happy placements, and 62% described continuing difficulties tempered by rewards. However, 10% reported no rewards. Hyperactivity and inattention frequently persisted despite stable adoption and were associated with conduct and attachment difficulties. Use of professional services was substantial. Thirty‐eight per cent of children failed to achieve stable adoption. Later entry to care correlated with poorer outcome.

Conclusions  This study highlights the importance in safeguarding children of considering the implications of parental childhood experiences, and indicates the risk of delay. The high prevalence of domestic violence in birth families indicates the need for better resources for managing emotional dysregulation. Adoption is a valuable therapeutic tool, but professional responsibilities in supporting it need to be acknowledged and adequately resourced. Dysfunctional hypothalamus‐pituitary‐adrenal axis programming may contribute to persisting difficulties. Supporting substitute care should be considered integral to safeguarding children.
Brood development into sexual females depends on the presence of a queen but not on temperature in an ant dispersing by colony fission, Aphaenogaster senilis
Authors: BOULAY RAPHAËL , CERDÁ XIM , FERTIN ARNOLD , ICHINOSE KATSYUA , LENOIR ALAIN
Published: June 2009

Abstract. 1. In eusocial insects, colony fission is a mode of dispersal by which a young queen leaves her nest with some workers to found a new colony. In these species, adult females (workers and the queen) should allocate most resources to increasing their colony size, which constrains the possibility of fission. In contrast, developing diploid larvae should have a preference for becoming a queen and having their own offspring, rather than becoming workers and rearing the offspring of other females.

2. In the ant Aphaenogaster senilis, queens are produced in very small numbers, suggesting that adult females control larval development. We used a 6‐year series of data on more than 300 nests to determine the annual cycle of worker and queen production. Although both overlapped, the latter mostly occurred in the second half of the summer, after a major peak of worker emergence. Young queens were also often produced in nests whose reproductive queen had died, thus allowing her replacement. Overall, we estimate that only 0.07% of diploid larvae actually develop into gynes.

3. Laboratory experiments indicated that brood is bipotent until the second larval instar. Diploid larval development into queen was favoured by the removal of the mother queen, but was not affected by rearing temperature.

4. Our data suggest that most diploid broods are forced by the adults to develop into workers rather than into gynes. However, when the queen is not present due to death or after a fission event, a few larvae are allowed to develop into gynes. One way for workers to limit the development of larvae might be by controlling the amount of food they receive.
Non‐invasive foetal RHD genotyping via real‐time PCR of foetal DNA from Chinese RhD‐negative maternal plasma
Authors: Wang X. D. , Wang B. L. , Ye S. L. , Liao Y. Q. , Wang L. F. , He Z. M.
Published: June 2009

Background  A majority of studies predicting the foetal RhD blood group in free foetal DNA from RhD‐negative maternal plasma have been conducted in Caucasian populations, whereas limited data have been accumulated for Asian populations. In this study, we assessed the feasibility of prenatal genotyping of RHD in RhD‐negative Chinese pregnant women.

Materials and methods  Cell‐free plasma DNA was extracted from 78 RhD‐negative Chinese women carrying a singleton foetus (gestation between 14 and 40 weeks). Foetal DNA was confirmed by testing SRY or nine different polymorphic STR loci in the maternal plasma and buffy coat. Foetal RHD exons 5, 7 and 10 and intron 4 were successfully amplified with RQ‐PCR. The RHD1227A allele was examined in all RhD‐positive individuals. The foetal RHD genotyping results were compared with the infant cord blood serological analysis.

Results  Among the 78 specimens, RHD genotyping results of 70 cases were in complete concordance with serological results from foetal umbilical cord blood. Sixty of these cases were identified as RhD‐positive, and 10 cases were typed as RhD‐negative. In addition, five cases were ‘false‐positives’, while three cases were considered inconclusive. The detection rate was 89·7% (70/78). In four of the five ‘false‐positive’ cases, the RhDel phenotype was assessed by detecting the RHD1227A allele. Thus, this method yielded a 94·9% (74/78) accuracy rate.

Conclusions  The correct foetal RhD phenotype may be accurately predicted from RhD‐negative maternal plasma in Chinese subjects. The RHD1227A allele proved to be an important genetic marker in the RhDel Chinese population.