bluuebunny/crossref_metadata_2025 huggingface dataset:
A mutational analysis of Dictyostelium discoideum multicellular development
Authors: Carrin Ines , Murgia Irene , McLachlan Andrew , Kay Robert R.
Published: June 2009

We have collectedDictyosteliummutants that arrest in development after aggregation, but before first finger formation. A total of 118 mutant strains were isolated and are referred to as mound(mnd)mutants. Nine complementation groups(mndA-mndl), containing 46 of the mutant strains, were defined by parasexual methods. A statistical analysis suggested that there are about 118 genes which, when mutated, confer the mound phenotype. Of these genes, about 60 are predicted to be mutated in our collection: the 9 assigned to complementation groups and another 51 unassigned mutants.mndA, G, HandIwere assigned to linkage groups VII, IV, II and VI, respectively. Development of the mutant strains was characterized by terminal morphology, neutral red staining and expression of marker mRNAs for prespore and prestalk cells. Three broad classes were recognized. (1) Postaggregative mutants - those blocked early in multicellular development. They did not express any of the prestalk or prespore marker mRNAs and generally arrested as low mounds or ridges. (2) Pathway mutants - those blocked specifically in either prestalk or prespore differentiation. They expressed either prestalk or prespore marker mRNAs, but not both, and generally proceeded further morphologically than post-aggregative mutants. (3) Morphogenesis mutants - those apparently blocked in morphogenesis rather than cell differentiation. They expressed all the cell-type marker mRNAs tested. Most arrested as tight mounds lacking a tip and of defined upper size, but some mutants produced aberrant tips. The majority of mutants tested synergized with wild-type: 24/28 strains which cannot make spores when developed alone, were able do so when allowed to develop with an equal number of wild-type cells. We suggest that some of the morphogenesis mutants have a cytoskeletal defect which prevents first finger formation and that these mutants can be physically carried through development by the wildtype (synergy by ‘piggy-backing’).
Flux Distributions in Anaerobic, Glucose-Limited Continuous Cultures of Saccharomyces Cerevisiae
Authors: Nissen Torben L. , Schulze Ulrik , Nielsen Jens , Villadsen John
Published: June 2009

A stoichiometric model describing the anaerobic metabolism of Saccharomyces cerevisiae during growth on a defined medium was derived. The model was used to calculate intracellular fluxes based on measurements of the uptake of substrates from the medium, the secretion of products from the cells, and of the rate of biomass formation. Furthermore, measurements of the biomass composition and of the activity of key enzymes were used in the calculations. The stoichiometric network consists of 37 pathway reactions involving 43 compounds of which 13 were measured (acetate, CO2, ethanol, glucose, glycerol, NH+
4, pyruvate, succinate, carbohydrates, DNA, lipids, proteins and RNA). The model was used to calculate the production rates of malate and fumarate and the ethanol measurement was used to validate the model. All rate measurements were performed on glucose-limited continuous cultures in a high-performance bioreactor. Carbon balances closed within 98%. The calculations comprised flux distributions at specific growth rates of 0.10 and 0.30 h−1. The fluxes through reactions located around important branch points of the metabolism were compared, i.e. the split between the pentose phosphate and the Embden-Meyerhoff-Parnas pathways. Also the model was used to show the probable existence of a redox shunt across the inner mitochondrial membrane consisting of the reactions catalysed by the mitochondrial and the cytosolic alcohol dehydrogenase. Finally it was concluded that cytosolic isocitrate dehydrogenase is probably not present during growth on glucose. The importance of basing the flux analysis on accurate measurements was demonstrated through a sensitivity analysis. It was found that the accuracy of the measurements of CO2, ethanol, glucose, glycerol and protein was critical for the correct calculation of the flux distribution.
A novel 1,3-β-glucan synthase from the oomycete Saprolegnia monoica
Authors: Billon-Grand Geneviève , Marais Marie-France , Joseleau Jean-Paul , Girard Vincent , Gay Lucien , Fãvre Michel
Published: June 2009

An apparently novel 1,3-β-glucan synthase from the oomycete Saprolegnia monoica has been characterized. The enzyme exhibits properties that differ markedly from those of the enzyme previously described [Fèvre, M. & Dumas, C. (1977). J Gen Microbiol 103, 297-306] as it is active at alkaline pH, stimulated by the divalent cations Ca2+, Mg2+ and Mn2+, and appears to be located mainly in the apical part of the hypha. Taking into consideration the differences in pH optimum and effect of divalent ions, each enzyme activity could be assayed in the presence of the other. The insoluble polymeric product of the enzyme with alkaline pH optimum was characterized as a linear 1,3-β-glucan. Comparisons of the general properties of 1,3-β-glucan synthases suggest that enzymes from the oomycetes are more closely related to enzymes from higher plants than to those of true fungi, reflecting the fact that the oomycetes are highly divergent from chitinous fungi.
Genetic identification of chemotactic transducers for amino acids in Pseudomonas aeruginosa
Authors: Taguchi Kazunori , Fukutomi Hiroyuki , Kuroda Akio , Kato Junichi , Ohtake Hisao
Published: June 2009

Two chemotactic transducer genes (termed pctB and pctC and an open reading frame (orf1) were found in the pctA-flanking region which was previously identified as a chemotactic transducer gene in Pseudomonas aeruginosa. The pctB and pctC genes encode predicted polypeptides of 629 and 632 amino acids, respectively. Overall, PctB and PctC had 81 and 75% amino acid identities with PctA, respectively. A null mutant strain PCT2, which contained a deletion in the entire pcfC, orf1, pctA and pctB genes, did not show chemotaxis towards all 20 commonly occurring L-amino acids. This mutant strain also failed to respond to amino acid catabolites (cadaverine, 4-aminobutyrate and putrescine) that are strong attractants for the wild-type strain PAO1. To study the role of each gene product in L-amino acid taxis, plasmids harbouring the pctC, orf1, pctA, or pctB genes were constructed and introduced into strain PCT2 by transformation. The orf1 gene did not complement the defect in chemotaxis of strain PCT2. The pctA gene restored the ability of strain PCT2 to respond to 18 L-amino acids, suggesting that PctA plays a major role in detecting L-amino acids in P. aeruginosa. The pctB and pctC genes complemented the defect in chemotaxis to only seven (Ala, Arg, Glu, Lys, Met, Tyr, Gin) and two (His, Pro) L-amino acids, respectively.
Sequence completion, identification and definition of the fengycin operon in Bacillus subtilis 168
Authors: Tosato Valentina , Albertini Alessandra M. , Zotti Michela , Sonda Sabrina , Bruschi Carlo V.
Published: June 2009

A 15 kb DNA fragment from the Bacillus subtilis chromosome between citB and ppsC has been sequenced, and new ORFs encoding putative enzymes involved in lipopolypeptide synthesis, which complete a partial operon previously reported, and a new set of enzymes responsible for lipid metabolism have been identified. From the analysis of DNA sequence homology of the fragment it was deduced that these new peptide synthetase genes are part of an operon for the biosynthesis of the fungicide fengycin.
Lactobacilli carry cryptic genes encoding peptidase-related proteins: characterization of a prolidase gene (pepQ) and a related cryptic gene (orfZ) from Lactobacillus delbrueckii subsp. bulgarkus
Authors: Rantanen Terhi , Palva Airi
Published: June 2009

Two genes, pepQ and orfZ, encoding a prolidase and a prolidase-like protein, respectively, were cloned and characterized from Lactobacillus delbrueckii subsp. bulgaricus. The identity of the pepQ and orfZ genes with the Lactobacillus delbrueckii subsp. lactis prolidase gene (pepQ) was shown to be 98% and 60%, respectively. Both pepQ and orfZ were preceded by a putative promoter region. Northern analysis of pepQ mRNA revealed a 1.1 kb transcript indicating that pepQ forms a monocistronic transcriptional unit. Under the growth conditions used, no evidence was obtained that orfZ was expressed, either by mRNA size determination in Northern analysis or by primer extension analysis. With reverse transcription-PCR, however, the presence of monocistronic orfZ transcripts was established. The orfZ gene could also be overexpressed in E. coli using the vector pKK223-3. The size of the protein synthesized, 41 kDa, confirmed the molecular mass of OrfZ calculated according to DNA sequence analysis. In contrast to PepQ, which showed a substrate specificity characteristic of prolidase enzymes, no enzymic activity for the orfZ-encoded protein was found with the peptide substrates tested. These results indicate that orfZ is a cryptic gene, which is expressed at a very low level under the growth conditions used. It is noteworthy that homologues of the Lb. delbrueckii subsp. bulgaricus orfZ and pepQ genes appeared to be present in both Lb. delbrueckii subsp. lactis and Lactobacillus helveticus.
Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene
Authors: Sanglard Dominique , Ischer Françoise , Monod Michel , Bille Jacques
Published: June 2009

Resistance to azole antifungal agents in Candida albicans can be mediated by multidrug efflux transporters. In a previous study, we identified at least two such transporters, Cdr1p and Benp, which belong to the class of ATP-binding cassette (ABC) transporters and of major facilitators, respectively. To isolate additional factors potentially responsible for resistance to azole antifungal agents in C. albicans, the hypersusceptibility of a Saccharomyces cerevisiae multidrug transporter mutant, δpdr5, to these agents was complemented with a C. albicans genomic library. Several new genes were isolated, one of which was a new ABC transporter gene called CDR2 (
Candida
drug resistance). The protein Cdr2p encoded by this gene exhibited 84% identity with Cdr1p and could confer resistance to azole antifungal agents, to other antifungals (terbinafine, amorolfine) and to a variety of metabolic inhibitors. The disruption of CDR2 in the C. albicans strain CAF4-2 did not render cells more susceptible to these substances. When the disruption of CDR2 was performed in the background of a mutant in which CDR1 was deleted, the resulting double δcdr1 δcdr2 mutant was more susceptible to these agents than the single δcdr1 mutant. The absence of hypersusceptibility of the single δcdr2 mutant could be explained by the absence of CDR2 mRNA in azole-susceptible C albicans strains. CDR2 was overexpressed, however, in clinical C. albicans isolates resistant to azole antifungal agents as described previously for CDR1, but to levels exceeding or equal to those reached by CDR1. Interestingly, CDR2 expression was restored in δcdr1 mutants reverting spontaneously to wild-type levels of susceptibility to azole antifungal agents. These data demonstrate that CDR2 plays an important role in mediating the resistance of C. albicans to azole antifungal agents.
Electrotransformation of Streptococcus pneumoniae: evidence for restriction of DNA on entry
Authors: Lefrançois Jacques , Sicard A. Michel
Published: June 2009

Electrotransformation is a method generally used in biotechnology to introduce recombinant DNA into a wide range of bacteria. However, the mechanism of DNA entry is poorly understood. We report that inStreptococcus pneumoniae,a naturally transformable species, electrotransformation efficiently introduces a plasmid replicon. DNA is strongly restricted by the restriction-modification systemsDpnlandDpnllwhich degrade methylated and non-methylated DNA, respectively, at GATC sequences. This suggests that in electrotransformation double-stranded DNA penetrates into these bacteria without a single-stranded DNA step in contrast to natural transformation. Single-stranded DNA by Itself is able to electrotransform very weakly and linearized double-stranded plasmid DNA yields barely detectable levels of transformants.
Activation of the Erwinia carotovora subsp. carotovora pectin lyase structural gene pnIA: a role for RdgB
Authors: Liu Yang , Cui Yaya , Mukherjee Asita , Chatterjee Arun K.
Published: June 2009

The activation of pectin lyase (Pnl) production in Erwinia carotovora subsp. carotovora strain 71 occurs upon DNA damage via a unique regulatory circuit involving recA, rdgA and rdgB. In a similar Pnl-inducible system reconstituted in Escherichia coli, the rdgB product was found to activate the expression of pnIA, the structural gene for pectin lyase. The kinetic data presented here also show that transcription of pnIA followed that of rdgB in Er. carotovora subsp. carotovora, indicating a temporal order of gene expression. By deletion analysis we have localized the promoter/regulatory region within a 66 bp DNA segment upstream of the pnIA transcriptional start site. This region contains the -10 consensus sequence but not the sequences corresponding to the E. coli -35 region. For DNA-binding studies, rdgB was overexpressed in E. coli and a 14 kDa polypeptide was identified as the gene product. RdgB from crude extracts or a purified preparation caused an identical gel mobility shift of a 164 bp DNA segment containing the pnIA promoter/regulatory region. Utilizing DNase I protection assay the RdgB-binding site was localized between nucleotides -29 and -56, i.e. overlapping the position of the putative -35 box. The findings reported here, taken along with our previous observation that the rdgB product is required for pnIA expression, establishes that rdgB encodes a transcriptional factor which specifically interacts with the pnIA promoter/regulatory region.
Structure of the Clostridium stercorarium gene celY encoding the exo-1,4-�-glucanase Avicelase II
Authors: Bronnenmeier Karin , Kundt Kerstin , Riedel Kathrin , Schwarz Wolfgang H. , Staudenbauer Walter L.
Published: June 2009

The nucleotide sequence of the celY gene coding for the thermostable exo-1,4-�-glucanase Avicelase II of Clostridium stercorarium was determined. The gene consists of an ORF of 2742 bp which encodes a preprotein of 914 amino acids with a molecular mass of 103 kDa. The signal-peptide cleavage site was identified by comparison with the N-terminal amino acid sequence of Avicelase II purified from C. stercorarium. The celY gene is located in close vicinity to the celZ gene coding for the endo-1,4-�-glucanase Avicelase I. The CelY-encoding sequence was isolated from genomic DNA of C. stercorarium with the PCR technique. The recombinant enzyme produced in Escherichia coli as a LacZ'-CelY fusion protein could be purified using a simple two-step procedure. The properties of CelY proved to be consistent with those of Avicelase II purified from C. stercorarium. Sequence comparison revealed that CelY consists of an N-terminal catalytic domain flanked by a domain of 95 amino acids with unknown function joined to a type III cellulose-binding domain. The catalytic domain belongs to the recently proposed family L of cellulases (family 48 of glycosyl hydrolases).
Identification of Mycobacterium paratuberculosis gene expression signals
Authors: Bannantine John P. , Barletta Raúl G. , Thoen Charles O. , Andrews Robert E.
Published: June 2009

Mycobacterium paratuberculosis promoter-containing clones were isolated from a genomic DNA library constructed in the transcriptional-translational fusion vector pYUB76. The promoter-containing DNA fragments were identified in the surrogate host Mycobacterium smegmatis by expression of the promoterless lacZ reporter gene of pYUB76. The expression signals exhibited a wide range of strengths, as indicated by their corresponding β-galactosidase activities. Eight clones were sequenced and characterized further. Predicted open reading frames and codon usage were identified by computer analysis. Database searching for related sequences using the BLAST method revealed no homologies. Transcriptional activity was measured by slot-blot hybridization with steady-state RNA isolated from lacZ
+
M. smegmatis clones. Primer extension analysis identified the transcription start sites within the cloned fragments. The promoter regions characterized in this study were used to establish a consensus promoter sequence for M. paratuberculosis. M. paratuberculosis consensus hexanucleotide sequences of TGMCGT and CGGCCS centred approximately 35 and 10 bp upstream from the transcription startpoints do not correspond to the consensus hexanucleotides of Escherichia coli promoters.
Identification of vegetative proteins for a two-dimensional protein index of Bacillus subtilis
Authors: Schmid Roland , Bernhardt Jörg , Antelmann Haike , Völker Andrea , Mach Hiltraut , Völker Uwe , Hecker Michael
Published: June 2009

Twenty-three of the most prominent spots which are visible on two-dimensional (2-D) protein gels of Bacillus subtilis crude extracts were selected as marker spots for the construction of a 2-D protein index. N-terminal sequencing of the corresponding proteins resulted in the identification of enzymes involved in glycolysis, TCA cycle, pentose phosphate cycle, amino acid metabolism, nucleotide biosynthesis and translation. Using computer analysis of the 2-D protein gels, most of these metabolic enzymes were found to be synthesized at a reduced rate after different stresses and glucose starvation. Such an approach permits a rapid and global evaluation of the regulation of different branches of metabolism in response to various physiological conditions.
Photoreactivation in an archaeon from geothermal environments
Authors: Grogan Dennis W.
Published: June 2009

UV-inactivated cells of Sulfolobus acidocaldarius rapidly regained viability when exposed to white light This recovery was strictly dependent upon illumination with visible light and was not attenuated by prior dark-incubation. The kinetics of photoreactivation were determined at several temperatures and at several wavelengths of light. The results obtained in vivo were consistent with a DNA photolyase having a broad action spectrum. Photoreactivation of S. acidocaldarius apparently represents the first DNA repair process to be measured in an archaeon which grows optimally near 80°C.
A 12 kb nucleotide sequence containing the alanine dehydrogenase gene at 279° on the Bacillus subtilis chromosome
Authors: Oudega Bauke , Vandenbol Micheline , Koningstein Gregory
Published: June 2009

In the framework of the European project aimed at the sequencing of the Bacillus subtilis genome, a DNA fragment of 12315 bp was cloned and sequenced. The DNA fragment is located between rrnB (275°) and pai (284°). Twelve ORFs were predicted to encode putative proteins. Two of these (ald and yukl) coincided with known B. subtilis genes. The products of two other genes (yukK and yukL) showed significant similarity to known proteins present in databases, e.g. pyoverdine synthase of Pseudomonas aeruginosa and pristinamycine synthase D of Streptomyces pristinaespiralis.
Mitochondrial superoxide dismutase is essential for ethanol tolerance of Saccharomyces cerevisiae in the post-diauxic phase
Authors: Costa V. , Amorim M. A. , Reis E. , Quintanilha A. , Moradas-Ferreira P.
Published: June 2009

This work reports the role of both superoxide dismutases - CuZnSOD (encoded by SOD1) and MnSOD (encoded by SOD2) - in the build-up of tolerance to ethanol during growth of Saccharomyces cerevisiae from exponential to post-diauxic phase. Both enzyme activities increase from the exponential phase to the diauxic shift and from the diauxic shift to the post-diauxic phase. The levels of mRNA-SOD1 and mRNA-SOD2 increase from the exponential phase to the diauxic shift; however, during the post-diauxic phase mRNA-SOD1 levels decrease while mRNA-SOD2 levels remain unchanged. These data indicate the existence of two regulatory mechanisms involved in the induction of SOD activity during growth: synthesis de novo of the proteins (until the diauxic shift), and post-transcriptional or post-translational regulation (during the post-diauxic phase). Ethanol does not alter the activities of either enzyme in cells from the diauxic shift or post-diauxic phases, although the respective mRNA levels decrease in post-diauxic-phase cells treated with ethanol (14% or 20%). Results of experiments with sod1 and sod2 mutants show that MnSOD, but not CuZnSOD, is essential for ethanol tolerance of diauxic-shift and post-diauxic-phase cells. Evidence that ethanol toxicity is correlated with the production of reactive oxygen species in the mitochondria is obtained from results with respiration-deficient mutants. In these cells, the induction of superoxide dismutase activity by ethanol is low; also, the respiratory deficiency restores the capacity of sod2 cells to acquire ethanol tolerance.
Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae: effects of insert position and host background
Authors: Stentebjerg-Olesen Bodil , Pallesen Lars , Jensen Lars Bogø , Christiansen Gunna , Klemm Per
Published: June 2009

The potential of the major structural protein of type 1 fimbriae as a display system for heterologous sequences was tested. As a reporter-epitope, a heterologous sequence mimicking a neutralizing epitope of the cholera toxin B chain was inserted, in one or two copies, into four different positions in thefimgene. This was carried out by introduction of new restriction sites by PCR-mediated site-directed mutagenesis offimin positions predicted to correspond to optimally surface-located regions of the subunit protein. Subsequently, the synthetic cholera-toxin-encoding DNA segment was inserted. Several of the chosen positions seemed amenable even for large foreign inserts; the chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Display of chimeric fimbriae was tested with respect to host background in three differentEscherichia colistrains, i.e. an isogenic set of K-12 strains, differing in the presence of an indigenousfimgene cluster, as well as a wild-type isolate. Immunization of rabbits with purified chimeric fimbriae resulted in serum which specifically recognized cholera toxin B chain, confirming the utility of the employed strategy.
Restoration of wild-type virulence to Tri5 disruption mutants of Gibberella zeae via gene reversion and mutant complementation
Authors: Proctor Robert H. , Hohn Thomas M. , McCormick Susan P.
Published: June 2009

Summary: Gibberella zeae is a pathogen of small grain crops and produces trichothecene mycotoxins in infected host tissue. The role of trichothecenes in the virulence of G. zeae was previously investigated using trichothecene-non-producing mutants that were generated via transformation-mediated disruption of a gene (Tri5) that encodes the first enzyme in the trichothecene biosynthetic pathway. The mutants were less virulent on some hosts than the wild-type strain from which they were derived. Here, we used two approaches to determine whether the reduced virulence of mutants was due specifically to Tri5 disruption or to non-target effects caused by the transformation process. First, we generated a revertant from a Tri5 disruption mutant by allowing the mutant to pass through the sexual phase of its life cycle. In approximately 2% of the resulting progeny the disrupted Tri5 had reverted to wild-type; however, only one of three revertant progeny also regained the ability to produce trichothecenes. In the second approach, we complemented the Tri5 mutation in a disruption mutant by transforming the mutant with a plasmid carrying a functional copy of Tri5. In all transformants examined, the ability to produce trichothecenes was restored. The restoration of trichothecene production in the revertant progeny and in the complemented mutant was accompanied by restoration of wild-type or near wild-type levels of virulence on wheat seedlings (cultivar Wheaton). The results indicate that the reduced virulence of the mutants was caused by disruption of Tri5 rather than non-target effects resulting from the transformation process. The results also provide further evidence that trichothecenes contribute to the virulence of plant-pathogenic fungi.
Expression of the eicosapentaenoic acid synthesis gene cluster from Shewanella sp. in a transgenic marine cyanobacterium, Synechococcus sp.
Authors: Takeyama Haruko , Takeda Daisuke , Yazawa Kazunaga , Yamada Akiko , Matsunaga Tadashi
Published: June 2009

Summary: The eicosapentaenoic acid (EPA) synthesis gene cluster isolated from a marine bacterium, Shewanella putrefaciens strain SCRC-2738, was cloned and expressed in the marine cyanobacterium Synechococcus sp. A broad-host-range cosmid vector, pJRD215 (10.2 kb, Smr Kmr), was used to clone a 38 kb insert, pEPA, containing the EPA synthesis gene cluster, creating plasmid pJRDEPA (approx. 48 kb). This plasmid was transferred to the cyanobacterial host at a frequency of 2.2 x 10−7. Cyanobacterial transconjugants grown at 29 °C produced 0.12 mg EPA (g dry weight)−1, whereas those grown at 23 °C produced 0.56 mg EPA (g dry weight)−1. The yield was further improved to 0.64 mg (g dry weight)−1 by incubation for 1 d at 17 °C. This is believed to be the first successful cloning and expression of such a large heterologous gene cluster in a marine cyanobacterium.
Analysis of the Bacillus subtilis genome: cloning and nucleotide sequence of a 62 kb region between 275° (rrnB) and 284° (pai)
Authors: Oudega Bauke , Koningstein Gregory , Rodrigues Luísa , de Sales Ramon Maria , Hilbert Helmut , Düsterhöft Andreas , Pohl Thomas M. , Weitzenegger Thomas
Published: June 2009

Summary: In the framework of the international project aimed at the sequencing of the Bacillus subtilis genome, five DNA fragments in the region between rrnB (275° and pai (284°) were cloned by inverse and combinatorial long-range PCR and their nucleotide sequences were determined and analysed. Together these sequences constituted a contig of 62229 bp. On the basis of the position of NotI and SfiI restriction sites, the orientation and order of known genetic markers was determined to be pai (284°) - degQ comQ comP comAA comAB - pbpD - kapB kinB patB - mcpB tlpA mcpA tlpB - rrnB (275°). Fifty-four ORFs were detected. Thirteen of these coincided with known B. subtilis genes, and 41 new ORFs were found. Of the predicted new gene products, 12 showed no significant similarity to other known proteins, whereas ten showed strong similarity to proteins of other organisms with unknown function. Nineteen predicted proteins showed strong similarity to known proteins of other organisms, for instance a Na+/H+ antiporter system of Bacillus alcalophilus, a sugar transport system found in Mycoplasma genitalium, NADH-dependent butanol dehydrogenase of Clostridium acetobutylicum, glucose-6-phosphate isomerase A of B. subtilis, exo-1,4-α-glucosidase activity of Bacillus stearothermophilus and L-rhamnose isomerase of Escherichia coli.
Sequence, localization and characteristics of the replicator region of the symbiotic plasmid of Rhizobium etli
Authors: Ramírez-Romero Miguel A. , Bustos Patricia , Girard Lourdes , Rodríguez Oscar , Cevallos Miguel A. , Dávila Guillermo
Published: June 2009

Summary: The replicator region of the symbiotic plasmid of Rhizobium etli CFN42 was cloned and sequenced. A plasmid derivative (pH3) harbouring a 5.6 kb HindIII fragment from the symbiotic plasmid was found to be capable of independent replication and eliminated the symbiotic plasmid when introduced into a R. etli CFNX101 strain (a recA derivative). The stability and the copy number of pH3 were the same as that of the symbiotic plasmid, indicating that the information required for stable replication and incompatibility resides in the 5.6 kb HindIII fragment. The sequence analysis of this fragment showed the presence of three ORFs similar in sequence and organization to repA, repB and repC described for the replicator regions of the Agrobacterium plasmids pTiB6S3 and pRiA4b and for the R. leguminosarum cryptic plasmid pRL8JI. Hybridization studies showed that p42d-like replicator sequences are found in the symbiotic plasmids of other R. etli strains and in a ‘cryptic’ plasmid of R. tropici.
The gshB gene in the cyanobacterium Synechococcus sp. PCC 7942 encodes a functional glutathione synthetase
Authors: Okumura Noriko , Masamoto Kazumori , Wada Hajime
Published: June 2009

Summary: The gene homologous to glutathione synthetase of Escherichia coli was inactivated in the cyanobacterium Synechococcus sp. PCC 7942. The region of genomic DNA including the mutation site was isolated from the mutant by plasmid rescue and the native gene of the wild-type was cloned from a genomic DNA library of the wild-type using the flanking DNA as a probe. The wild-type gene, designated gshB, encodes a polypeptide of 323 amino acids with a molecular mass of 35 kDa. The deduced amino acid sequence resembles glutathione synthetases of bacteria, but not those of higher organisms. When gshB was overexpressed in E. coli, glutathione synthetase activity was increased markedly in the E. coli extract. In addition, the Synechococcus sp. PCC 7942 gshB mutants had lost their ability to synthesize glutathione. These findings demonstrate that the gshB gene of Synechococcus sp. PCC 7942 is a structural gene for glutathione synthetase and is involved in the biosynthesis of glutathione.
Ribosome analysis reveals prominent activity of an uncultured member of the class Actinobacteria in grassland soils
Authors: Felske Andreas , Rheims Holger , Wolterink Arthur , Stackebrandt Erko , Akkermans Antoon D. L.
Published: June 2009

Summary: A 16S rRNA-based molecular ecological study was performed to search for dominant bacterial sequences in Drentse A grassland soils (The Netherlands). In the first step, a library of 165 clones was generated from PCR-amplified 16S rDNA. By sequence comparison, clone DA079 and two other identical clones could be affiliated to a group of recently described uncultured Actinobacteria. This group contained 16S rDNA clone sequences obtained from different environments across the world. To determine whether such uncultured organisms were part of the physiologically active population in the soil, ribosomes were isolated from the environment and 16S rRNA was partially amplified via RT-PCR using conserved primers for members of the domain Bacteria. Subsequent sequence-specific separation by temperature-gradient gel electrophoresis (TGGE) generated fingerprints of the amplicons. Such community fingerprints were compared with the TGGE pattern of PCR-amplified rDNA of clone DA079 which was generated with the same set of primers. One of the dominant fingerprint bands matched with the band obtained from the actinobacterial clone. Southern blot hybridization with a probe made from clone DA079 confirmed sequence identity of clone and fingerprint band. This is the first report that a member of the novel actinobacterial group may play a physiologically active role in a native microbial community.
Alterations in cellular lipids may be responsible for the transient nature of the yeast heat shock response
Authors: Chatterjee Mahua T. , Khalawan Seunath A. , Curran Brendan P. G.
Published: June 2009

Summary: The stress-sensing systems leading to the cellular heat shock response (HSR) and the mechanism responsible for the desensitizing of this response in stress-acclimated cells are largely unknown. Here it is demonstrated that there is a close correlation between a 3 ° increase in the temperature required for maximal activation of a heat-shock (HS)-inducible gene in Saccharomyces cerevisiae and an increase in the percentage of cellular unsaturated fatty acids when cells are subjected to extended periods of growth at 37 °. The latter occurs with the same kinetics as HS gene down-regulation during a prolonged HS and is reversed by reacclimation to growth at 25 °. The transient nature of the HS may therefore be due to a lipid-mediated decrease in cellular heat sensitivity. Further evidence that unsaturated fatty acids desensitize cells to heat, with a resultant down-regulation of the HSR, is provided by demonstrating a 9 ° increase in the temperature required for maximal induction of this HS-inducible gene in cells containing high levels of unsaturated fatty acids assimilated during anaerobic growth at 25 °.
Seroreactive species-specific lipooligosaccharides of Mycobacterium mucogenicum sp. nov. (formerly Mycobacterium chelonae-like organisms): identification and chemical characterization
Authors: Muñoz Manuel , Raynaud Catherine , Lanéelle Marie-Antoinette , Julián Esther , Marín Luz María López , Silve Gaby , Ausina Vicente , Daffé Mamadou , Luquin Marina
Published: June 2009

Summary: Strains of the new species Mycobacterium mucogenicum exhibit physiological and biochemical features very similar to those of the other species of the Mycobacterium fortuitum complex. To define taxonomic criteria for easy identification of M. mucogenicum, the glycolipid patterns of the reference strains and of 32 environmental and clinical isolates were examined by TLC. It was concluded that all M. mucogenicum strains of smooth colony morphology contained species-specific alkali-labile glycoconjugates. Three different patterns were observed among the strains of the smooth colony type. Fractionation followed by conventional chemical analyses of the purified glycolipids showed the specific glycolipids to be lipooligosaccharides (LOS). The three LOS showed a similar fatty acid composition consisting of straight chain (dodeca-, tetradeca-, hexadecanoyl and hexadecenoyl) and methyl-branched (2,4-dimethyleicosanoyl and 2,4-dimethyleicosenoyl) fatty acyl substituents. The most commonly encountered LOS (present in 76% of the smooth strains) contained a tetraacylated pentasaccharide composed of four moles of glucose and one mole of a 2,4-di-O-methylhexose. A LOS composed of arabinose, glucose and mannose was present in 20% of the smooth strains, whereas the newly proposed type strain of M. mucogenicum (ATCC 49650) was the only strain that contained a LOS composed of glucose and galactose. Serological studies clearly differentiated most of the strains of M. mucogenicum from those of the other members of the M. fortuitum complex, and demonstrated the existence of serovars within the former species. Altogether, these data confirm the validity of the new species but show ATCC 49651 to be the most representative strain.
In situ identification of nocardioform actinomycetes in activated sludge using fluorescent rRNA-targeted oligonucleotide probes
Authors: Schuppler Markus , Wagner Michael , Schön Georg , Göbel Ulf B.
Published: June 2009

Summary: Hitherto, few environmental samples have been investigated by a ‘full cycle rRNA analysis’. Here the results of in situ hybridization experiments with specific rRNA-targeted oligonucleotide probes developed on the basis of new sequences derived from a previously described comparative 16S rRNA analysis of nocardioform actinomycetes in activated sludge are reported. Application of the specific probes enabled identification and discrimination of the distinct populations of nocardioform actinomycetes in activated sludge. One of the specific probes (DLP) detected rod-shaped bacteria which were found in 13 of the 16 investigated sludge samples from various wastewater treatment plants, suggesting their importance in the wastewater treatment process. Another probe (GLP2) hybridized with typically branched filaments of nocardioforms mainly found in samples from enhanced biological phosphorus removal plants, suggesting that these bacteria are involved in sludge foaming. The combination of in situ hybridization with fluorescently labelled rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy improved the detection of nocardioform actinomycetes, which often showed only weak signals inside the activated-sludge flocs.
Unconventional organization of the division and cell wall gene cluster of Streptococcus pneumoniae
Authors: Massidda Orietta , Anderluzzi Daniela , Friedli Laurence , Feger Georg
Published: June 2009

SUMMARY: The genes responsible for cell wall biosynthesis and cell division (dcw genes) were identified and sequenced in Streptococcus pneumoniae. The genetic organization of the dcw cluster in Streptococcus pneumoniae differed significantly from the clusters of other bacteria reported to date. In particular, the genes corresponding to the 2 min region of the Escherichia colichromosome were found distributed in three genetically separate regions of the Streptococcus pneumoniae chromosome. The first region contained the expected ftsA and ftsZ cell division genes at one end and pbp2b, ddl and murF at the o her end. The murD, murG and diw/B genes, always found located upstream of ftsA, were found in a second region separated from the first. A third region contained the yllC, yllD, pbp2x and mraY genes. The chromosomal region downstream of ftsZ was also sequenced and characterized. In Streptococcus pneumoniae this region contains four ORFs, all of unknown function, and an ORF encoding the Bacillus subtilis DivlVA homologue. The gene order and the organization of this region was found to be conserved in Staphylococcus aureus, Streptococcus pyogenes and Bacillus subtilis, raising the possibility that previously unidentified loci may also be involved in division.
Molecular constituents of the replication apparatus in the plasmodium of Physarum polycephalum: identification by photoaffinity labelling
Authors: Doerhoefer Sabine , Khodyreva Svetlana , Safronov lgor V. , WIasoff Wjatschesslaw A. , Anarbaev Rushid , Lavrik Olga I. , Holler Eggehard
Published: June 2009

SUMMARY: The plasmodium of Physarum polycephalum has long been considered a model system for syncytically growing cells, but important details of the DNA replication apparatus, such as the DNA polymerase E and other replication factors, have not been detected. In this study, a new variation of photoaffinity labelling and immunoblotting was used to detect DNA polymerases and other factors in nuclear extracts of P. polycaphalum. Proteins were specifically cross-l inked with photoreactive arylazido-dCMP residues incorporated during extension of template-primer DNA. The DNA synthesized in situ was labelled. After nucleolytic removal of protruding DNA, the proteins were separated by SDS-gel electrophoresis, electroblotted on membranes and subjected to autoradiography. The a,s,eand -like DNA polymerases were labelled, as were histones and replication-factor-like proteins. Cytoplasmic extracts were devoid of these species. Abundant proliferating-cell nuclear antigen and replication protein A large subunit were labelled and found to be of unusual mass. A number of subunits of purified DNA polymerase holoenzymes were labelled. In contrast, only the DNA-polymerizing subunits could be labelled in nuclear extracts. Higher-order complexes in the nuclear extract may make subunits inaccessible to photo-cross-linking. Complex formation is promoted by -poly(~-malate), a plasmodium-specific putative storage and carrier molecule that supports DNA replication in the synchronized nuclei. Percoll, a polyvinylpyrrolidone-coated colloidal silica, partially disrupted these complexes. A 200 kDa fragment of DNA polymerase E and a 135 kDa -like DNA polymerase did not participate in the complexes, suggesting functions unlike those of the other polymerases. DNA polymerase molecules were intact during proliferation of plasmodia, but were nicked before their clearance from the nuclei at growth arrest.
A factor produced by Escherichia coli K-12 inhibits the growth of E. coli mutants defective in the cytochrome bd quinol oxidase complex: enterochelin rediscovered
Authors: Cook Gregory M. , Loder Caroline , Søballe Britta , Stafford Graham P. , Membrillo-Hernández Jorge , Poole Robert K.
Published: June 2009

Summary: Escherichia coli produces an extracellular factor that inhibits the aerobic growth of Cyd- mutants, defective in the synthesis or assembly of the cytochrome bd-type quinol oxidase. This paper shows that such a factor is the iron-chelating siderophore enterochelin. Mutants in entA or aroB, defective in the production of enterochelin, did not produce the factor that inhibits the growth of cydAB and cydDC mutants; purified enterochelin inhibited the growth of Cyd- mutants, but not that of wild-type cells. Other iron-chelating agents, particularly ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA), whose complex with Fe(III) has a large stability constant (log K = 33·9), also inhibited the growth of Cyd- mutants at micromolar concentrations, but not that of wild-type cells. Supplementation of agar plates with Fe(III) or boiled catalase prevented the inhibition of Cyd- mutants by the extracellular factor. Spontaneous mutants isolated by being able to grow in the presence of the extracellular factor on plates also showed increased resistance to iron chelators. The reducing agent ascorbate, ascorbate plus In(III), ascorbate plus Ga(III), or Ga(III) alone, also alleviated inhibition by the extracellular factor, presumably by reducing iron to Fe(II) and complexing of the siderophore with alternative trivalent metal cations. The preferential inhibition of Cyd- mutants by the extracellular factor and other iron chelators is not due to decrease in expression, activity or assembly of cytochrome bo', the major alternative oxidase mediating quinol oxidation. Cyd- mutants overproduce siderophores, presumably reflecting intracellular iron deprivation.
Three new members of the serine-aspartate repeat protein multigene family of Staphylococcus aureus
Authors: Josefsson Elisabet , McCrea Kirk W. , Eidhin Deirdre Ni , O'Connell David , Cox Jos , Hook Magnus , Foster Timothy J.
Published: June 2009

Summary: Three new genes encoding the serine-aspartate (SD) repeat-containing proteins SdrC, SdrD and SdrE were found in Staphylococcus aureus strain Newman. The SD repeats had earlier been found in the S. aureus fibrinogen-binding clumping factors ClfA and ClfB. The clfA and clfB genes encode high-molecular-mass fibrinogen-binding proteins that are anchored to the cell surface of S. aureus. The sdr genes now reported are closely linked and tandemly arrayed. The putative Sdr proteins have both organizational and sequence similarity to ClfA and ClfB. At the N-terminus, putative secretory signal sequences precede approximately 500 residue A regions. The A regions of the Sdr and Clf proteins exhibit only 20–30% residue identity when aligned with any other member of the family. The only conserved sequence is the consensus motif TYTFTDYVD. The Sdr proteins differ from ClfA and ClfB by having two to five additional 110–113 residue repeated sequences (b-motifs) located between region A and the r-region. Each b-motif contains a consensus Ca2+-binding ef-hand loop normally found in eukaryotic proteins. The structural integrity of recombinant Sdrd(B1-B5) protein comprising the five b-repeats of SdrD was shown by bisANS fluorescence analysis to be Ca2+-dependent, suggesting that the ef-hands are functional. When Ca2+was removed the structure collapsed to an unfolded conformation. The original structure was restored by addition of Ca2+. The C-terminal r-domains of the Sdr proteins contain 132–170 sd residues. These are followed by conserved wall-anchoring regions characteristic of many surface proteins of Gram-positive bacteria. The Sdr locus was present in all 31 S. aureus strains from human and bovine sources tested by Southern hybridization, although in a few strains it contained two rather than three genes.
The site-specific recombination locus of mycobacteriophage Ms6 determines DNA integration at the tRNAAlagene of Mycobacterium spp.
Authors: Freitas-Vieira Acilino , Anes Elsa , Moniz-Pereira J.
Published: June 2009

Summary: Genetic determinants of the temperate mycobacteriophage Ms6 required for chromosomal integration were identified. DNA sequence analysis of an attP-containing fragment revealed an ORF encoding a protein of 372 amino acid residues with a C-terminus similar to other conserved C-terminal regions typical of the phage integrase family. Comparison of the sequences of attP, attB and bacteria-prophage junctions attL and attR showed a 26 bp common core sequence, where recombination takes place, near the 5′ end of the integrase gene. Nucleotide sequence analysis of the attB chromosomal region showed that the core site overlaps the 3′ end of the tRNAAlagene. An integration-proficient plasmid vector was constructed and efficiently inserted at the tRNAAlagene of Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra. It was demonstrated that Ms6 and D29 integrative systems can be used in conjunction for inserting genes at multiple loci. The site-specific integration system of mycobacteriophage Ms6 is a new tool for mycobacterial genetic analysis and is poorly related to those of the L5 bacteriophage family.
Inactivation of the Kluyveromyces lactis KIPDA1 gene leads to loss of pyruvate dehydrogenase activity, impairs growth on glucose and triggers aerobic alcoholic fermentation
Authors: Zeeman Anne-Marie , Luttik Marijke A. H. , Thiele Claudia , van Dijken Johannes P. , Pronk Jack T. , Steensma H. Yde
Published: June 2009

Summary: The KIPDA1 gene, encoding the E1α subunit of the mitochondrial pyruvate-dehydrogenase (PDH) complex was isolated from a Kluyveromyces lactis genomic library by screening with a 1·1 kb internal fragment of the Saccharomyces cerevisiae PDA1 gene. The predicted amino acid sequence encoded by KIPDA1 showed 87% similarity and 79% identity to its S. cerevisiae counterpart. Disruption of KIPDA1 resulted in complete absence of PDH activity in cell extracts. The maximum specific growth rate on glucose of null mutants was 3·5-fold lower than that of the wild-type, whereas growth on ethanol was unaffected. Wild-type K. lactis CBS 2359 exhibits a Crabtree-negative phenotype, i.e. no ethanol was produced in aerobic batch cultures grown on glucose. In contrast, substantial amounts of ethanol and acetaldehyde were produced in aerobic cultures of an isogenic Klpda1 null mutant. A wild-type specific growth rate was restored after introduction of an intact KIPDA1 gene but not, as previously found for S. cerevisiae pda1 mutants, by cultivation in the presence of leucine. The occurrence of aerobic fermentation and slow growth of the Klpda1 null mutant indicate that, although present, the enzymes of the PDH bypass (pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase) could not efficiently replace the PDH complex during batch cultivation on glucose. Only at relatively low growth rates (D = 0·10 h-1) in aerobic, glucose-limited chemostat cultures, could the PDH bypass completely replace the PDH complex, thus allowing fully respiratory growth. This resulted in a lower biomass yield [g biomass (g glucose)-1] than in the wild-type due to a higher consumption of ATP in the PDH bypass compared to the formation of acetyl-CoA via the PDH complex.