bluuebunny/crossref_metadata_2025 huggingface dataset:
D-Amino-acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217
Authors: Alonso Jorge , Barredo José L. , Díez Bruno , Mellado Encarnación , Salto Francisco , García José L. , Cortés Estrella
Published: June 2009

The complete nucleotide sequence of the DAO1 gene encoding D-amino-acid oxidase (DAAO) in the yeast Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217 has been determined. The primary structure of DAAO was deduced from the nucleotide sequence of a cDNA clone that covered the entire amino acid coding sequence. Comparison of cDNA and genomic sequences of DAO1 revealed the presence of five introns. Because this is the first gene of strain ATCC 26217 that has been cloned so far, the nucleotide sequences of these introns were compared to those from other fungi. Upstream of the structural gene there was a stretch of C + T-rich DNA similar to that found in the promoter region of a number of yeast genes. The cDNA gene, which encoded a protein of 368 amino acids (molecular mass 40 kDa), was overexpressed in Escherichia coli under the control of the strong lipoprotein promoter. Interestingly, a significant fraction (13-62%) of the total DAAO activity was recovered in its apoenzyme form, the percentage depending on the culture conditions. This fact allowed a rapid purification of the recombinant DAAO by affinity chromatography. The high level of expression achieved in E. coli and the possibility of modifying its catalytic properties by protein engineering provide a new model for the study of this enzyme.
Use of resistant mutants to characterize the target of mycobacillin in Aspergillus niger membranes
Authors: Chowdhury Bhabadeb , Das Sanjoy K. , Bose Sushil K.
Published: June 2009

The mycobacillin-sensitive Aspergillus niger strain G3Br and resistant mutants of it did not show any differences in their total lipid content, although the amounts of phospholipids and sterols, particularly phosphatidylcholine and cholesterol, were lower in resistant cells. Mycobacillin resistance was accompanied by an increase in the phase-transition temperature of plasma membrane preparations. When exposed to mycobacillin, resistant and sensitive cells did not differ qualitatively with respect to most released materials (lysine, proline, Pi, Na+, K+, Ca2+); however, the release of ATP was completely inhibited in resistant cells unless they were exposed to concentrations of mycobacillin exceeding their respective MIC value. Resistant cells, under steady-state conditions, displayed greater uptake and release of the same specific materials - except ATP - as sensitive cells did under similar conditions. Thus release and uptake of those materials except ATP are not implicated in the mode of action of mycobacillin. The inhibiting action of mycobacillin (at concentrations higher than the MIC) on sensitive or resistant cells was completely antagonized by ATP (which did not form any complex with mycobacillin) but not by any of the releasable components, either alone or in combination. This observation, coupled with the authors’ recent findings on ATP release, indicates that the fungistatic action of mycobacillin is due to excessive ATP release, leading to energy starvation. Interestingly, ATP release during the first 2 h of incubation with mycobacillin was minimal, but increased to over 96% during the next 48 h. Release and uptake of ATP via liposomes, prepared with lipid and protein isolated from membranes of the mycobacillin-sensitive parent and resistant mutants, showed that mycobacillin action could be inhibited either by resistant protein or by resistant lipid. The mycobacillin target appears to be a lipid-protein site on the membrane of sensitive A. niger G3Br.
A 35.7 kb DNA fragment from the Bacillus subtilis chromosome containing a putative 12.3 kb operon involved in hexuronate catabolism and a perfectly symmetrical hypothetical catabolite-responsive element
Authors: Rivolta Carlo , Soldo Blazenka , Lazarevic Vladimir , Joris Bernard , Mauël Catherine , Karamat Dimitri
Published: June 2009

The Bacillus subtilis strain 168 chromosomal region extending from 109† to 112° has been sequenced. Among the 35 ORFs identified, cotT and rapA were the only genes that had been previously mapped and sequenced. Out of ten ORFs belonging to a single putative transcription unit, seven are probably involved in hexuronate catabolism. Their sequences are homologous to Escherichia coli genes exuT, uidB, uxaA, uxaB, uxaC, uxuA and uxuB, which are all required for the uptake of free D-glucuronate, D-galacturonate and β-glucuronide, and their transformation into glyceraldehyde 3-phosphate and pyruvate via 2-keto-3-deoxygluconate. The remaining three ORFs encode two dehydrogenases and a transcriptional regulator. The operon is preceded by a putative catabolite-responsive element (CRE), located between a hypothetical promoter and the RBS of the first gene. This element, the longest and the only so far described that is fully symmetrical, consists of a 26 bp palindrome matching the theoretical B. subtilis CRE sequence. The remaining predicted amino acid sequences that share homologies with other proteins comprise: a cytochrome P-450, a glycosyltransferase, an ATP-binding cassette transporter, a protein similar to the formate dehydrogenase α-subunit (FdhA), a protein similar to NADH dehydrogenases, and three homologues of polypeptides that have undefined functions.
Division of the genus Enterococcus into species groups using PCR-based molecular typing methods
Authors: Monstein Hans-Jürg , Quednau Mikael , Samuelsson Annika , Ahrné Siv , Isaksson Barbro , Jonasson Jon
Published: June 2009

Broad-range 16S rDNA PCR (BR-PCR) applied to DNA from 32 clinical enterococcal isolates and 12 other enterococci from a clinical reference collection followed by species-specific hybridization analysis identified 25 strains of Enterococcus faecalis and 19 Enterococcus species. Randomly amplified polymorphic DNA (RAPD)analysis using upgma clustering on the same material revealed four different clusters at a similarity level of 49%. Based on partial 16S rDNA sequence analysis of variable regions V4 and V9, it was possible to divide the 19 type strains specifying the genus Enterococcus into 12 different 16S rDNA species groups. The type strain distribution then served as a template for the analysis of the other 44 strains which were assigned to four different species groups (a-d) based on their 16S rDNA motifs. There was good agreement with the RAPD clusters. Species group a was an individual species line containing 25 strains that were identified as E. faecalis. Group b also represented an individual species line of 12 strains identified as E. faecium. The remaining seven strains that formed species groups c and d could not be fully identified to species by this analysis. It was concluded that BR-PCR of 16S rDNA followed by partial sequence analysis of the PCR products is a reliable technique for the identification and classification of enterococci. Further division of unresolved species groups should be achievable if regions other than V4 and V9 of 16S rDNA are also analysed.
Characterization of a haemolysin from Mycobacterium tuberculosis with homology to a virulence factor of Serpulina hyodysenteriae
Authors: Wren Brendan W. , Stabler Richard A. , Das Satya S. , Butcher Philip D. , Mangan Joseph A. , Clarke Jonathan D. , Casali Nicola , Parish Tanya , Stoker Neil G.
Published: June 2009

Scrutiny of sequence data from the Mycobacterium leprae genome sequencing project identified the presence of a gene encoding a 268-amino-acid polypeptide which is highly similar to a pore-forming haemolysin/cytotoxin virulence determinant, TlyA, from the swine pathogen Serpulina hyodysenteriae. Using degenerate oligonucleotide primers based on the TlyA sequences, the Mycobacterium tuberculosis homologue was amplified and this product was used to obtain the clone and sequence a 2.5 kb fragment containing the whole M. tuberculosis tlyA gene. tlyA encodes a 267-amino-acid protein with a predicted molecular mass of 28 kDa. TlyA homologues were identified by PCR in M. leprae, Mycobacterium avium and Mycobacterium bovis BCG, but appeared absent in Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium kansasii, Mycobacterium chelonae and Mycobacterium phlei. The M. tuberculosis gene appeared to be the first gene in an operon containing at least two other genes. Introduction of the M. tuberculosis tlyA gene into M. smegmatis using a mycobacterial shuttle expression plasmid converted non-haemolytic cells into those exhibiting significant haemolytic activity. Similarly, inducible haemolytic activity was observed in sonicated bacteria when tlyA was expressed as a His6-tagged fusion protein in Escherichia coli. tlyA mRNA was detected in both M. tuberculosis and M. bovis BCG using RT-PCR, confirming that this gene is expressed in organisms cultured in in vitro.
Characterization of plasmid pAW63, a second self-transmissible plasmid in Bacillus thuringiensis subsp. kurstaki HD73
Authors: Wilcks Andrea , Jayaswal Nicole , Lereclus Didier , Andrup Lars
Published: June 2009

Bacillus thuringiensis subspecies kurstaki HD73, toxic for lepidopteran larvae, contains two large self-transmissible plasmids of approximately 75 kb, pHT73 and pAW63. The conjugative plasmid pHT73 has been studied extensively and has been shown to harbour the toxin gene cry1Ac, the transposon Tn4430 and several insertion sequences. In this study it was demonstrated that the minor plasmid pAW63 is also self-transmissible and about 10-30 times more efficient in mobilizing plasmid pBC16. To facilitate direct selection for pAW63 transfer, the plasmid was tagged with the tetracycline resistance transposon Tn5401 and in intraspecies matings it was found that after 2 h, all recipients had acquired a copy of the plasmid. Mating experiments demonstrated that pAW63 could be transferred to Bacillus thuringiensis subsp. israelensis. Bacillus cereus, Bacillus licheniformis, Bacillus subtilis and Bacillus sphaericus, and that the conjugative functions were expressed in these hosts. Hybridization studies showed that the replicons of pAW63 and pHT73 were distinct from one another. Sequences homologous to transposon Tn4430 and several insertion sequences were, however, shown to reside on both plasmids.
ppc, the gene for phosphoenolpyruvate carboxylase from an extremely thermophilic bacterium, Rhodothermus obamensis: cloning, sequencing and overexpression in Escherichia coli
Authors: Takai Ken , Sako Yoshihiko , Uchida Aritsune
Published: June 2009

The ppc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) of an extremely thermophilic bacterium, Rhodothermus obamensis, was directly sequenced by the thermal asymmetric interlaced (TAIL) PCR method. An ORF for a 937 amino acid polypeptide was found in the gene. The ppc gene had a high G+C content (66.2 mol%) and the third position of the codon exhibited strong preference for G or C usage (85.0 mol%). The calculated molecular mass was 107 848 Da, which was consistent with the molecular mass of the enzyme as determined by SDS-PAGE (100 kDa). The amino acid sequence of R. obamensis PEPC was closely related to that of PEPC from another thermophile, a Thermus sp., and from a mesophile. Corynebacterium glutamicum, exhibiting 45.3% or 37.7% identity and 61.5% or 56.5% similarity, respectively. By Southern analysis, the ppc gene was found to be present in a single copy in the genomic DNA of this organism. The cloned gene was expressed in Escherichia coli using a pET expression vector system and a thermostable recombinant PEPC was obtained. Comparison of the deduced amino acid sequences of the thermophilic and mesophilic PEPCs revealed distinct or common preferences for specific amino acid composition and substitutions in the two thermophilic enzymes.
Biodegradation kinetics of toluene, m-xylene, p-xylene and their intermediates through the upper TOL pathway in Pseudomonas putida (pWWO)
Authors: Duetz Wouter A. , Wind Bea , van Andel Johan G. , Barnes Michael R. , Williams Peter A. , Rutgers Michiel
Published: June 2009

Pseudomonas putida mt-2, harbouring TOL plasmid pWWO, is capable of degrading toluene and a range of di- and tri-alkylbenzenes. In this study, chemostat-grown cells (D = 0.05 h-1, toluene or m-xylene limitation) of this strain were used to assess the kinetics of the degradation of toluene, m-xylene, p-xylene, and a number of their pathway intermediates. The conversion kinetics for the three hydrocarbons showed significant differences: the maximal conversion rates were rather similar [11-14 mmol h-1 (g dry wt)-1] but the specific affinity (the slope of the v vs s curve near the origin) of the cells for toluene [1300 I (g dry wt)-1 h-1] was only 5% and 14% of those found for m-xylene and p-xylene, respectively. Consumption kinetics of mixtures of the hydrocarbons confirmed that xylenes are strongly preferred over toluene at low substrate concentrations. The maximum flux rates of pathway intermediates through the various steps of the TOL pathway as far as ring cleavage were also determined. Supply of 0-5 mM 3-methylbenzyl alcohol or 3-methylbenzaidehyde to fully induced cells led to the transient accumulation of 3-methylbenzoate. Accumulation of the corresponding carboxylic acid (benzoate) was also observed after pulses of benzyl alcohol and benzaldehyde, which are intermediates in toluene catabolism. Analysis of consumption and accumulation rates for the various intermediates showed that the maximal rates at which the initial monooxygenation step and the conversion of the carboxylic acids by toluate 1,2-dioxygenase may occur are two- to threefold lower than those measured for the two intermediate dehydrogenation steps.
Regulation of the spvR gene of the Salmonella typhimurium virulence plasmid during exponential-phase growth in intracellular salts medium and at stationary phase in L broth
Authors: Wilson Julie A. , Gulig Paul A.
Published: June 2009

The authors previously showed that the SpvR-regulated spvABCD operon of the Salmonella typhimurium virulence plasmid is highly induced during exponential-phase growth by salmonellae intracellularly in mammalian cells and in a medium designed to mimic the intracellular environment of mammalian cells, intracellular salts medium (ISM), as well as at stationary phase in L broth (LB). The most relevant signal(s) for spv gene expression in vivo is not known. To elucidate the means by which salmonellae regulate the spv genes in response to the environment during the disease process, expression of the spvR gene, encoding the positive regulatory protein SpvR, was examined under these same growth conditions by using RNAse-protection analysis, spvR was expressed at a low, basal level during exponential growth in LB but was induced during exponential growth in ISM and during stationary phase in LB, the same conditions that increased expression of the spvABCD operon. Basal expression of spvR during exponential growth in LB was independent of both SpvR and the alternative sigma factor RpoS, whereas maximal induction of spvR was dependent on both SpvR and RpoS. In an RpoS-background, spvR message was decreased in stationary phase, whereas spvR exhibited residual RpoS-independent induction during exponential growth in ISM. Deletion of spvA from the virulence plasmid of S. typhimurium increased expression of spvR during stationary phase in LB, but not during exponential growth in ISM. These results suggest that expression of spvR is controlled by different regulatory factors, depending on the growth conditions encountered by the salmonellae.
Redox poise and oxygenation of cytochrome bd in the diazotroph Azotobacter vinelandii assessed in vivo using diode-array reflectance spectrophotometry
Authors: Kavanagh Eugene P. , Callis James B. , Edwards Sian E. , Poole Robert K. , Hill Susan
Published: June 2009

A ferrous oxygenated form of cytochrome d is characteristic of all cytochrome bd-type oxidases so far examined, but its participation in enzyme turnover is unclear. It is relatively stable, occurs in aerated cell suspensions and predominates during enzyme preparation. In this study, diode-array reflectance spectrophotometry was used to assess the redox poise and oxygenation of cytochrome bd in vivo, in the aerobic diazotroph Azotobacter vinelandii. Mutants either lacking or overproducing the cytochrome bd oxidase were used to confirm the reliability of the optical configuration. Changes in absorbance attributed to cytochromes b, c and d were followed as the O2 supply was altered either in suspensions of harvested cells or during steady-state growth. In washed cell suspensions, three states of cytochrome d, which differed in absorbance characteristics, were seen: (1) an oxygenated form that absorbs at 650 nm, (2) a form which has little absorbance at either 650 or 630 nm and (3) the reduced form that absorbs at 630 nm. The transition between states 2 and 3, but not 1 and 2, correlated with the changes in the redox states of cytochromes b
595 and b
560. The dissolved O2 concentration at which this transition occurred coincided approximately with the apparent O2 affinity for the oxidase in vivo (approx. 5 M). During steady-state growth, the cytochromes were partially reduced and the oxygenated form of cytochrome d was undetected. These in situ measurements support the view that an oxygenated form of cytochrome d (absorbing at 650 nm) in the one-electron-reduced cytochrome bd-type oxidase does not take part in enzyme turnover.
pilO, a gene required for glycosylation of Pseudomonas aeruginosa 1244 pilin
Authors: Castric Peter
Published: June 2009

Nucleotide sequencing of a region downstream from thePseudomonas aeruginosa1244 pilin structural gene,pilA, revealed an ORF potentially able to code for a protein ofMr50862. This ORF, calledpilO, was flanked by a tRNAthrgene, which was followed by a transcriptional termination sequence. The tRNAthrgene and the termination sequence were nearly identical to sequences found immediately adjacent to thepilAgene of severalP. aeruginosastrains. A 2200 base mRNA strand, which contained both thepilOandpilAtranscripts, was produced from this region, while a 650 base transcript containing onlypilAwas present in a 100-fold excess over the longer transcript. Hyperexpression of thepilAgene in a PilO-strain resulted in normal pilus-specific phage sensitivity and twitching motility. The pilin produced by this strain had a lower apparentMrand a more neutral pI compared to that produced by a strain containing a functionalpilOgene. This pilin failed to react with a sugar-specific reagent which recognized pilin produced by the strain containing a functionalpilOgene.
The effect of NaCl on the growth of a Halomonas species: accumulation and utilization of compatible solutes
Authors: Cummings Stephen P. , Gilmour D. James
Published: June 2009

The effect of NaCl on growth and compatible solute utilization was investigated in a Halomonas species. Growth of Halomonas was observed in medium of low osmolarity (high water activity) when only 01 mM Na+ was present. However, lowering the water activity, by addition of KCl or sucrose, inhibited growth in this low-Na+ medium, but growth could be restored by the addition of NaCl. The bacterium could grow on glucose as the sole carbon source in up to 355 M NaCl and was shown also to metabolize glycine betaine. However NaCl concentrations greater than 2 M inhibited growth when glycine betaine was the sole carbon source. Glycine betaine was transported into the cells by a process stimulated by NaCl irrespective of whether the carbon source was glucose or glycine betaine. Cytoplasmic levels of glycine betaine were monitored throughout the growth cycle in 2 M NaCl medium with glycine betaine as sole carbon source. As the culture aged, glycine betaine was increasingly replaced by the tetrahydropyrimidine ectoine as the major cytoplasmic solute. The increased sensitivity to high NaCl concentrations when grown on glycine betaine may be due to the glycine betaine catabolic pathway enzymes being inhibited by the increasing external solute concentration.
Rhapidosomes from Aquaspirillum itersonii: disruption and subsequent renaturation and characterization of the component proteins
Authors: Pazirandeh Mehran , Deschamps Jeffery , Baral Subash , Owlett Rachael , Aggerwal Angeli , Campbell James R.
Published: June 2009

Rhapidosomes, tubular proteinaceous microstructures found in a variety of bacteria and algae, are resistant to disruption by many denaturing agents. They have potential application as a biomaterial and may also serve as a model for the study of self-assembly. Several reagents were tested for their ability to disrupt rhapidosomes isolated from Aquaspirillum itersonii into their component proteins. Only treatments with 01 M hydrochloric acid and 6 M guanidinium hydrochloride were found to disrupt rhapidosomes. A protocol was developed to renature the disrupted component proteins using rapid dilution and subsequent dialysis of the residual guanidinium hydrochloride. The renatured proteins were shown to have secondary structure as determined by circular dichroism. Furthermore, upon renaturation the two component proteins interacted with each other to form a complex of molecular mass > 2500 kDa. This complex, which may be a precursor in the pathway to rhapidosome formation, is currently being used to study the self-assembly process of these unique structures in vitro.
Regulation of the degradative pathways from 4-toluenesulphonate and 4-toluenecarboxylate to protocatechuate in Comamonas testosteroni T-2
Authors: Schlfli Oppenberg Hans R. , Chen Gang , Leisinger Thomas , Cook Alasdair M.
Published: June 2009

Comamonas testosteroni T-2 was grown in salts medium containing intermediates of the established, inducible degradative pathway(s) for 4-toluenesulphonate/4-toluenecarboxylate. The specific activity or, if appropriate, the specific expression of pathway enzymes or their components was constant throughout growth and decreased only slowly in the stationary phase. It was found that the 4-toluenesulphonate methyl-monooxygenase system and 4-sulphobenzyl alcohol dehydrogenase (with 4-sulphobenzaldehyde dehydrogenase) were always co-induced, with similar ratios of their activities during growth with 4-toluenesulphonate, 4-toluenecarboxylate and 4-sulphobenzoate. We presume these enzymes to be co-expressed from one regulatory unit. The ratio of activities of the terephthalate 1,2-dioxygenase system to those of (1R,2S)-dihydroxy-1,4-dicarboxy-3,5-cyclohexadiene dehydrogenase was also constant, and present only during growth with 4-toluenecarboxylate or terephthalate. We presume these two enzymes to be co-expressed from a different regulatory unit. The oxygenase component of 4-sulphobenzoate 3,4-dioxygenase (PSBDOS) was expressed at high levels in most growth conditions examined, the exception being with 4-toluenecarboxylate as carbon source. However, no expression of a specific reductase activity linked to synthesis of the oxygenase of PSBDOS could be detected. The PSBDOS was thus active in vivo solely under conditions where the 4-toluenesulphonate methyl-monooxygenase system was also present, whose reductase is active with the oxygenase of the 4-sulphobenzoate 3,4-dioxygenase system in vitro, and, apparently, in vivo. The synthesis of PSBDOS is thus under the control of a third regulatory unit.
The bldA-encoded tRNA is poorly expressed in the bldi mutant of Streptomyces coelicolor A3(2)
Authors: Leskiw Brenda K. , Mah Richard
Published: June 2009

The bldA gene, encoding a leucyl tRNA recognizing the UUA codon, is expressed at significantly lower levels in the bldii mutant, Streptomyces coelicolor J703, than in the parent S. coelicolor A3(2). Expression of a TTA-containing reporter gene was reduced in the bldi mutant, as was the mature, 87 nucleotide, form of the bldA-encoded tRNA. This reduced level of the tRNA was also seen when the bldA gene was introduced on a high-copy-number plasmid into the bldi mutant, suggesting that maximal bldA expression may require a bldii-dependent promoter.
Topological analysis of the lysine-specific permease of Escherichia coli
Authors: Ellis Jeri , Carlin Arthur , Steffes Chris , Wu Jianhua , Liu Jiyang , Rosen Barry P.
Published: June 2009

Escherichia coli accumulates lysine via two systems, one specific for lysine (LysP) and a second inhibited by arginine or ornithine (LAO). The lysP gene encodes a polypeptide of 489 residues. A topological analysis of the LysP protein was performed using gene fusions. Random in-frame fusions of the lysP gene with the lacZ or blaM genes were generated. Site-directed mutagenesis was also used to generate additional blaM fusions at specific locations in the lysP gene. Two methods were used to alleviate the problem of lethal expression of some lysP::blaM fusions. First, ternary fusions were constructed in which the arsD gene was fused at the 5' end of the lysP gene and the blaM gene fused at specific sites within the lysP gene. In these plasmids lysP expression was controlled by the ars promoter. Secondly, an E. coli strain with a pcnB mutation was used with some fusions to maintain the plasmids at a reduced copy number. From analysis of 30 gene fusions, a topological model of the LysP protein is proposed in which the protein has 12 membrane-spanning regions, with the N- and C-termini in the cytosol.
Immune reactions to Bacteroides fragilis populations with three different types of capsule in a model of infection
Authors: Patrick Sheila , Lutton Deborah A. , Crockard Alistair D.
Published: June 2009

The survival and growth of populations of the obligately anaerobic pathogenic bacterium Bacteroides fragilis enriched for large capsules (LCs), small capsules (SCs) or an electron-dense layer (EDL; non-capsulate by light microscopy) were examined in a mouse model of infection over a minimum period of 20 d. Chambers which allowed the influx of leukocytes, but not the efflux of bacteria, were implanted in the mouse peritoneal cavity. The LC and EDL populations consistently attained viable cell densities of the order of 10-10 c.f.u. ml-1 within 24 h, whereas the SC population did not. However, after 3 d, all three bacterial populations maintained total viable numbers of 10-109 c.f.u. ml-1 within the chambers. LC expression was selected against within 24 h in the model, the populations becoming non-capsulate by light microscopy, whereas in the SC population expression of the SC was retained by approximately 90% of the population. The EDL population remained non-capsulate by light microscopy throughout. Lymphocytes infiltrated the chambers to an equal extent for all three B. fragilis populations and at approximately 1000 times higher concentration than chambers which contained only quarter-strength Ringer's solution. The presence of neutrophils within the chambers did not cause a decrease in the total viable bacterial count. Each population elicited antibodies specific for outer-membrane proteins and polysaccharide, as detected by immunoblotting, which cross-reacted with the other populations. Differences were observed in the immunogenicity of the outer-membrane proteins within the three populations. Neutrophils were initially the predominant cell type in the chambers, but as the total leukocyte count increased with incubation time, neutrophils were outnumbered by other leukocytes. Flow cytometric investigations indicated that by day 7 the majority of these leukocytes were B-cells. Bearing in mind the constraints of this model system, it appears that all three populations of B. fragilis have the potential for in vivo growth and that each elicits an immune reaction.
Lipopolysaccharide and porin of Roseobacter denitrificans, confirming its phylogenetic relationship to the α-3 subgroup of Proteobacteria
Authors: Neumann Uwe , Mayer Hubert , Schiltz Emile , Benz Roland , Weckesser Jürgen
Published: June 2009

Roseobacter denitrificans has rough (R)-type lipopolysaccharide, containing 2-keto-3-deoxyoctonate but no heptoses. Its lipid A has a glucosamine-containing, phosphorylated backbone. It contains the rare 3-oxotetradecanoic (3-oxomyristic) acid as the only amide-bound fatty acid and ester-bound 3-hydroxydecanoic acid, this pattern being characteristic for the a 3 subgroup of Proteobacteria. Treatment of the major outer-membrane protein (porin, apparent molecular mass 88 kDa) of Roseobacter denitrificans with EDTA (2 mM, 30°C, 20 min) resulted in the dissociation of the oligomers into monomers (apparent molecular mass 35 kDa). EDTA-sensitive dissociation has so far been observed only within the α-3 subgroup of Proteobacteria. The 12 N-terminal amino acids of the monomers exhibit sequence homology with the porins of Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas blastica. Renaming of Roseobacter denitrificans as Rhodobacter denitrificans is suggested.
Catabolite repression in the hyperthermophilic bacterium Thermotoga neapolitana is independent of cAMP
Authors: Vargas Madeline , Noll Kenneth M.
Published: June 2009

Thermotoga neapolitana is a hyperthermophilic bacterium whose phylogenetic lineage includes the most primitive of the bacterial heterotrophs. It is not known whether Thermotoga exhibits preferences for growth substrates or regulates the synthesis of degradative enzymes. We have found that T. neapolitana exhibits diauxic growth in medium containing 300 μM glucose and 1 mM lactose. We measured the activity of β-galactosidase and β-glucosidase in extracts prepared from cells grown on defined media and found that cells grown on 0.5% lactose, galactose or cellobiose contained β-galactosidase specific activities of 1.19, 1.78 and 1.34 U (mg protein)-1, respectively. Cells grown on 0.5% glucose, maltose, fructose, sucrose, xylose, ribose or starch had no measurable β-galactosidase activity. β-Glucosidase activity was found only in cells grown on cellobiose. Cells grown on the combination of 0.5% lactose or galactose and 0.05% glucose had no detectable β-galactosidase activity, whereas up to 0.5% glucose did not prevent expression of β-galactosidase or β-glucosidase activity in cells induced with 0.5% cellobiose. These activities are catalysed by separate enzymes as determined by resolution of their activities on 6% native polyacrylamide gels. Therefore, only β-galactosidase synthesis induced by lactose is subject to catabolite repression. To determine the mechanism of catabolite repression, the levels of cAMP were measured in T. neapolitana cells grown on various defined media using an enzyme-immunoassay. The cAMP levels ranged from 44 to 280 fmol (mg protein)-1 irrespective of the carbon source used. By comparison, Escherichia coli grown on lactose contained 5.1 pmol (mg protein)-1. Like Gram-positive bacteria, T. neapolitana displays a cAMP-independent mechanism for catabolite repression and this may represent the more ancient mode of regulation.
Molecular variation between the α-toxins from the type strain (NCTC 8237) and clinical isolates of Clostridium perfringens associated with disease in man and animals
Authors: Ginter A. , Williamson E. D. , Dessy F. , Coppe P. , Bullifent H. , Howells A. , Titball R. W.
Published: June 2009

The α-toxin produced by the type strain of Clostridium perfringens (NCTC 8237) was shown to differ from the α-toxins produced by most strains of C. perfringens isolated from man and from calves with respect to reactivity with a neutralizing monoclonal antibody (DY2F5D11). The difference in antibody binding correlated with three differences in the deduced amino acid sequence (Ala174 to Asp174; Thr177 to Ala177; Ser335 to Pro335) of the α-toxins. Using octapeptides synthesized on the basis of the amino acid sequences from these regions of variability, it was shown that the Ala174 to Asp174 change had the greatest effect on reducing the binding of monoclonal antibody DY2F5D11 to the α-toxin. These differences did not affect the enzymic or toxic properties of the protein. However, the phospholipase C activity of the α-toxin produced by strain NCTC 8237 was more susceptible to inactivation by chymotrypsin. The changes in amino acid sequence did not affect the ability of a C-terminal domain vaccine, derived from the α-toxin of strain NCTC 8237, to induce protection against the α-toxin from a bovine enteric strain of C. perfringens.
Physical mapping of 32 genetic markers on the Pseudomonas aeruginosa PAO1 chromosome
Authors: Liao Xiaowen , Charlebois Isabelle , Ouellet Catherine , Morency Marie-Josée , Dewar Ken , Lightfoot Jeff , Foster Jennifer , Siehnel Richard , Schweizer Herbert , Lam Joseph S. , Hancock Robert E. W. , Levesque Roger C.
Published: June 2009

The Pseudomonas aeruginosa chromosome was fractionated with the enzymes Spel and Dpnl, and genomic fragments were separated by PFGE and used for mapping a collection of 40 genes. This permitted the localization of 8 genes previously mapped and of 32 genes which had not been mapped. We showed that a careful search of databases and identification of sequences that were homologous to known genes could be used to design and synthesize DNA probes for the mapping of P. aeruginosa homologues by Southern hybridization with genomic fragments, resulting in definition of the locations of the aro-2, dapB, envA, mexA, groEL, oprH, oprM, oprP, ponA, rpoB and rpoH genetic markers. In addition, a combination of distinct DNA sources were utilized as radioactively labelled probes, including specific restriction fragments of the cloned genes (glpD, opdE, oprH, oprO, oprP, phoS), DNA fragments prepared by PCR, and single-stranded DNA prepared from phagemid libraries that had been randomly sequenced. We used a PCR approach to clone fragments of the putative yhhF, sucC, sucD, cypH, pbpB, murE, pbpC, soxR, ftsA, ftsZ and envA genes. Random sequencing of P. aeruginosa DNA from phagemid libraries and database searching permitted the cloning of sequences from the acoA, catR, hemD, pheS, proS, oprD, pyo and rpsB gene homologues. The described genomic methods permit the rapid mapping of the P. aeruginosa genome without linkage analysis.
Aeromonas trota strains, which agglutinate with Vibrio cholerae O139 Bengal antiserum, possess a serologically distinct fimbrial colonization factor
Authors: Nakasone Noboru , Iwanaga Masaaki , Yamashiro Tetsu , Nakashima Kazutoshi , Albert M. John
Published: June 2009

Summary: Pili of Aeromonas trota strain 1220, which agglutinates with Vibrio cholerae O139 Bengal antiserum, were purified and characterized. The molecular mass of the subunit protein was estimated to be 20 kDa and the pI was 5.4. The pili were immunologically unrelated to the other Aeromonas pili reported so far. However, the N-terminal amino acid sequence of the subunit pilin was similar to those of the pilins from other Aeromonas pili reported previously. Neither A. trota cells nor pili purified from strain 1220 agglutinated human and rabbit erythrocytes, but both adhered to the rabbit intestine. Bacterial cells pretreated with antipilus antibody (Fab portion) failed to adhere to the rabbit intestine. Moreover, bacteria did not adhere to the rabbit intestine pretreated with the purified pili. This pilus antigen was not detected in V. cholerae O139 Bengal and other Aeromonas spp. These findings suggest that the pilus of the A. trota strain is a novel colonization factor of Aeromonas spp.
Regulatory sequences of two flagellin genes in Bacillus thuringiensis subsp. alesti
Authors: Ankarloo Jonas , Zhang Min-Ying , Lövgren Ann
Published: June 2009

Summary: Two highly homologous flagellin genes, flaA and flaB, are expressed in Bacillus thuringiensis subsp. alesti. Both genes were found to be transcribed during vegetative growth. After the onset of sporulation, transcripts could not be detected. Both flaA and flaB were found to be transcribed from σ70-like promoters. In addition, the 3'-terminal half of flaA was cloned and sequenced, completing the sequence of flaA.
The adaptation of Rhizobium leguminosarum bv. phaseoli to oxidative stress and its overlap with other environmental stress responses
Authors: Crockford Ansley J. , Behncke Cecile , Williams Huw D.
Published: June 2009

Summary: This paper reports the adaptation of Rhizobium leguminosarum bv. phaseoli to oxidative stress and the investigation of its overlap with other environmental stress responses. Treatment of R. leguminosarum bv. phaseoli cells with low concentrations of either menadione (MD, a superoxide generating agent) or 1-chloro-2,4-dinitrobenzene (CDNB, which depletes GSH levels) induced an adaptive response which resulted in cells becoming resistant to subsequent treatment with high concentrations of these oxidative stress compounds. There was overlap between the adaptive response to MD-generated superoxide stress and the response previously demonstrated in this organism to H2O2 (A. J. Crockford, G. A. Davis & H. D. Williams, 1995, Microbiology 141, 843-851); pretreatment with H2O2 was protective against cell killing by MD and vice versa. In contrast, similar experiments indicated only a limited overlap between the responses to H2O2 and CDNB-mediated GSH depletion. It was also found that H2O2, but not MD or CDNB, adaptation protected cells against subsequent osmotic challenge and heat shock. Carbon-starved cells were more resistant to H2O2 and MD killing than exponentially growing cultures, but were more sensitive to CDNB-mediated GSH depletion. Therefore, this work shows that there is a substantial, but incomplete overlap between the responses of R. leguminosarum to different forms of oxidative and other environmental stresses.
Acid Proteinase Secreted by Candida Tropicalis: Functional Analysis of Preproregion Cleavages in C. Tropicalis and Saccharomyces Cerevisiae
Authors: Togni Giuseppe , Sanglard Dominique , Quadroni Manfredo , Foundling Stephen I. , Monod Michel
Published: June 2009

The 40 kDa secreted aspartyl proteinase (Sapt1) of Candida tropicalis is a pepsin-like enzyme encoded by the SAPT1 gene. According to the deduced amino acid sequence, Sapt1 has a putative preproregion of 60 amino acids preceding the mature enzyme. Maturation and processing of Sapt1 was analysed in C. tropicalis and Saccharomyces cerevisiae strains expressing wild-type or mutated forms of SAPT1. In S. cerevisiae the glycosylated 46 kDa proenzyme was converted to the mature 40 kDa form of Sapt1 by KEX2-dependent proteolytic cleavage following the Lys59-Arg60 sequence. The replacement of Lys59-Arg60 by Lys59-Gly60 revealed that the precursor can be processed by an autocatalytic cleavage. This alternative processing pathway to produce mature Sapt1 is less efficient than the Kex2-mediated pathway. Finally, it was shown that in C. tropicalis and S. cerevisiae the removal of the proregion was a prerequisite for the secretion of Sapt1.
The homologous regulators ANR of Pseudornonas aeruginosa and FNR of Escherichia coli have overlapping but distinct specificities for anaerobically inducible promoters
Authors: Winteler Harald V. , Haas Dieter
Published: June 2009

The anaerobic transcriptional regulator ANR induces the arginine deiminase and denitrification pathways in Pseudomonas aeruginosa during oxygen limitation. The homologous activator FNR of Escherichia coli , when introduced into an anr mutant of P. aeruginosa , could functionally replace ANR for anaerobic growth on nitrate but not for anaerobic induction of arginine deiminase. In an FNR-positive E. coli strain, the ANR-dependent promoter of the arcDABC operon, which encodes the enzymes of the arginine deiminase pathway, was not expressed. To analyse systematically these distinct induction patterns, a lacZ promoter-probe, broad-host-range plasmid containing various –40 regions (the ANR/FNR recognition sequences) and –10 promoter sequences was constructed. These constructs were tested in P. aeruginosa and in E. coli expressing either ANR or FNR. In conjunction with the consensus –10 hexamer of E. coli
σ
70 RNA polymerase (TATAAT), the consensus FNR site (TTGAT ....ATCAA) was recognized efficiently by ANR and FNR in both hosts. By contrast, when promoters contained the Arc box (TTGAC.... ATCAG), which is found in the arcDABC promoter, or a symmetrical mutant FNR site (CTGAT.... ATCAG), ANR was a more effective activator than was FNR. Conversely, an extended 22 bp, fully symmetrical FNR site allowed better activation with FNR than with ANR. Combination of the arc promoter –10 sequence (CCTAAT) with the Arc box or the consensus FNR site resulted in good ANR-dependent expression in P. aeruginosa but gave practically no expression in E. coli , suggesting that RNA polymerase of P. aeruginosa differs from the E. coli enzyme in –10 recognition specificity. In conclusion, ANR and FNR are able to activate the RNA polymerases of P. aeruginosa and E. coli when the –40 and –10 promoter elements are identical or close to the E. coli consensus sequences.
Novel pyoverdine biosynthesis gene(s) of Pseudomonas aeruginosa PAO
Authors: Stintzi Alain , Cornelis Pierre , Hohnadel Dany , Meyer Jean-Marie , Dean Charles , Poole Keith , Kourambas Sophia , Krishnapillai Viji
Published: June 2009

Conjugational mobilization of a Pseudomonas aeruginosa PAO1 cosmid bank (in pMMB33) into a pyoverdine-deficient (pvd) mutant harbouring a mutation in the 47 min region of the chromosome yielded one clone which restored yellow-green pigmentation and fluorescence when grown on iron-deficient medium. The relevant pMMB33-derivative cosmid, pPYP17, contained a 15.1 kb insert which was subcloned into pKT240 as a 10.8 kb Sacl-Clal fragment conferring the same phenotype. This derivative, pPYP180, like pPYP17, also conferred an apparent wild-type phenotype on pvd mutants previously shown to map genetically in the 23 min region of the P. aeruginosa PAO chromosome. Physical mapping indicated that the cloned DNA fragment is located at the 66-70 min region of the PAO chromosome, demonstrating that the restored apparent wild-type phenotype observed for the transconjugants was not the result of a true gene complementation. A gene interruption was obtained by replacing a 0.6 kb Bglll-Bglll region of pPYP180 necessary for the expression of the pigmentation/fluorescence phenotype, by a Hgr interposon (ΩHg). After conjugational transfer and introduction of the mutagenized fragment into the PAO1 chromosome by gene replacement, pyoverdine-deficient mutants were recovered, indicating that the fragment indeed contained at least one gene involved in pyoverdine synthesis. The yellow-green fluorescent compound produced by such cells harbouring plasmids pPYP17 or pPYP180 differed from pyoverdine in several aspects and was consequently named pseudoverdine. Although pseudoverdine was able to complex iron, it was unable to restore growth to pvd mutants in the presence of the iron chelator ethylenediamine di(o-hydroxyphenylacetic acid), or to mediate iron uptake into PAO1. Pseudoverdine lacked a peptide chain but possessed spectral properties similar to pyoverdine, suggesting that it was structurally related to the chromophore of the pyoverdine molecule. The recent structural determination of pseudoverdine as a coumarin derivative confirmed this view and sheds some light on the biosynthetic pathway of the pyoverdine chromophore.
Metabolism of glycoprotein-derived sialic acid and N-acetylglucosamine by Streptococcus oralis
Authors: Homer K. A. , Kelley S. , Hawkes J. , Beighton D. , Grootveld M. C.
Published: June 2009

Nine strains ofStreptococcus oralis, isolated from blood cultures of patients with infective endocarditis or from the oral cavity as part of the normal flora, were examined for their ability to elaborate sialidase (neuraminidase) andN-acetylglucosaminidase, enzymes which are involved in the degradation of glycoproteins. Both glycosidases were induced when bacteria were grown in a minimal medium supplemented with porcine gastric mucin, a model glycoprotein, and repressed when growth occurred in the presence of glucose. Cell-free extracts of mucin-grown cultures expressed elevated levels ofN-acetylneuraminate pyruvate-lyase (the first intracellular enzyme in the pathway ofN-acetylneuraminate catabolism),N-acetylglucosamine (GlcNAc)-6-phosphate deacetylase and glucosamine-6-phosphate deaminase (enzymes involved in the intracellular catabolism of GlcNAc 6-phosphate); activity of each of these intracellular enzymes was markedly repressed when bacteria were grown in the presence of glucose. Three strains ofS. oraliswere also grown in media supplemented with α1-acid glycoprotein, a major component of human plasma. Cells from these cultures expressed high levels of sialidase,N-acetylglucosaminidase, and the intracellular enzymes involved in the catabolism ofN-acetyl-sugars released by the action of these glycosidases. High-resolution1H-NMR spectroscopy of spent culture supernatants revealed that sialic acid and GlcNAc residues of the molecularly mobile oligosaccharide side-chains of α1-acid glycoprotein had been hydrolysed and the released sugars internalized by the bacteria. These data indicate thatS. oralishas the ability to hydrolyse constituents of oligosaccharide side-chains of host-derived glycoproteins and to utilize simultaneously these released carbohydrates. The biochemical characteristics induced by the growth ofS. oralison glycoproteins may play a role in the survival and persistence of these bacteria at the infection sitein vivo.
Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif
Authors: Pátek Miroslav , Eikmanns Bernhard J. , Pátek Jaroslav , Sahm Hermann
Published: June 2009

Relatively limited information about promoter structures in Corynebacterium glutamicum has been available until now. With the aim of isolating and characterizing such transcription initiation signals, random Sau3A fragments of C. glutamicum chromosomal DNA and of the corynebacterial phage øGA1 were cloned into the promoter probe vector pEKplCm and selected for promoter activity by chloramphenicol resistance of transformed C. glutamicum cells. The nucleotide sequence of ten chromosomal and three phage fragments was determined and the transcriptional start (TS) sites were localized by primer extension analyses. Additionally, the promoters of five previously isolated C. glutamicum genes were cloned and mapped. All of the isolated promoters were also functional in the heterologous host Escherichia coli. A comparative analysis of the newly characterized promoter sequences together with published promoters from C. glutamicum revealed conserved sequences centred about 35 bp (ttGcca) and 10 bp (TA.aaT) upstream of the TS site. The position of these motifs and the motifs themselves are comparable to the −35 and −10 promoter consensus sequences of other Gram-positive and Gram-negative bacteria, indicating that they represent transcription initiation signals in C. glutamicum. However, the C. glutamicum consensus hexamer of the −35 region is much less conserved than in E. coli, Bacillus, Lactobacillus and Streptococcus.
Determination of a 12 kb nucleotide sequence around the 76° region of the Bacillus subtilis chromosome
Authors: Yamamoto Hiroki , Uchiyama Shigeki , Fajar Aji N. , Ogasawara Naotake , Sekiguchi Junichi
Published: June 2009

The nucleotide sequence of a 12361 bp DNA segment in the 760 region of the Bacillus subtilis chromosome has been determined. Ten putative ORFs were identified. The deduced amino acid sequences of the products of two of them (glv-1 and glv-2) exhibited high similarity to those of glvG (6-phospho-β-glucosidase gene) and glvC [permease (the IIC domain) of the phosphotransferase system (PTS)], respectively, in the glv operon of Escherichia coli. The C-terminal region of Glv-2 exhibited similarity to the entire region of GlvB (the IIB domain of PTS) of E. coli, suggesting fusion of the glvC and glvB genes in B. subtilis. glv-1, yfiA and glv-2 seem to form an operon of a phosphoenolpyruvate:sugar PTS, followed by a presumed four-membered operon of an ABC transport system. Moreover, a presumed sugar symporter and its regulatory genes were located in this region.