bluuebunny/crossref_metadata_2025 huggingface dataset:
A geographically widespread plasmid from Thiobacillus ferrooxidans has genes for ferredoxin-, FNR-, prismane- and NADH-oxidoreductase-like proteins which are also located on the chromosome
Authors: Dominy Clifford N. , Deane Shelly M. , Rawlings Douglas E.
Published: June 2009

During a search for genes encoding electron transport proteins from a Thiobacillus ferrooxidans ATCC 33020 gene bank, a 19.8 kb plasmid, pTF5, which conferred increased sensitivity to the antimicrobial agent metronidazole upon an Escherichia coli mutant, was isolated and cloned in E. coli. The plasmid had an identical restriction enzyme map to a plasmid which has been found in T. ferrooxidans strains isolated from many different parts of the world. The plasmid was present at between two and four copies per genome and contained a region of approximately 5.6 kb which was also found on the chromosome. This region was sequenced and found to have four complete ORFs, which when translated had high percentage amino acid similarity to [3Fe-4S,4Fe-4S] ferredoxins, proteins of the FNR regulator family, prismane-like proteins and the NADH oxidoreductase subunit of a methane monooxygenase. In vitro protein analysis using an E. coli-derived transcription-translation system indicated that three of the four products (FdxA, PsmA and RedA) were expressed in the heterologous system. Ferredoxins, prismane-like proteins and NADH oxidoreductases are redox-active proteins and it is likely that the proteins on pTF5 represent an electron transport system of as yet unknown function. Surprisingly, although genes for redox-active proteins have been isolated from other bacteria by screening gene banks for increased sensitivity to metronidazole, the region of pTF5 containing the genes for these proteins was not responsible for the increase in metronidazole sensitivity conferred by the plasmid. The region of pTF5 which did confer increased metronidazole sensitivity to an E. coli metronidazole-resistant mutant was a 319 bp region of DNA close to the origin of plasmid replication. This region contained no ORFs and was identical to that previously reported for the replicon of a 9.8 kb T. ferrooxidans plasmid, pTFI91.
Identification of new regulatory genes controlling synthesis of folate-dependent enzymes in Aspergillus nidulans
Authors: Lewandowska Irmina , Balińska Malgorzata , Paszewski Andrzej
Published: June 2009

Prototrophic revertants of a metH2 strain of Aspergillus nidulans which is impaired in the regulation of synthesis of folate-dependent enzymes were isolated and six of them analysed. In three of the isolates reversion was the result of an intragenic suppressor mutation in the metH locus. In the remaining strains suppressor mutations occurred in independent genes. These genes, designated folA, folB and folC, are linked and located in chromosome VI. Mutations in these genes render synthesis of some folate enzymes, particularly folylpolyglutamate synthetase, insensitive to methionine-mediated repression.
Molecular analysis of a Clostridium butyricum NCIMB 7423 gene encoding 4-α-glucanotransferase and characterization of the recombinant enzyme produced in Escherichia coli
Authors: Goda Sayed K. , Eissa Omima , Akhtar Muhammad , Minton Nigel P.
Published: June 2009

An Escherichia coli clone was detected in a Clostridium butyricum NCIMB 7423 plasmid library capable of degrading soluble amylose. Deletion subcloning of its recombinant plasmid indicated that the gene(s) responsible for amylose degradation was localized on a 1·8 kb NspHI-Scal fragment. This region was sequenced in its entirety and shown to encompass a large ORF capable of encoding a protein with a calculated molecular mass of 57184 Da. Although the deduced amino acid sequence showed only weak similarity with known amylases, significant sequence identity was apparent with the 4-α-glucanotransferase enzymes of Streptococcus pneumoniae (46.9%), potato (42.9%) and E. coli (16.2%). The clostridial gene (designated malQ) was followed by a second ORF which, through its homology to the equivalent enzymes of E. coli and S. pneumoniae, was deduced to encode maltodextrin phosphorylase (MalP). The translation stop codon of malQ overlapped the translation start codon of the putative malP gene, suggesting that the two genes may be both transcriptionally and translationally coupled. 4-α-Glucanotransferase catalyses a disproportionation reaction in which single or multiple glucose units from oligosaccharides are transferred to the 4-hydroxyl group of acceptor sugars. Characterization of the recombinant C. butyricum enzyme demonstrated that glucose, maltose and maltotriose could act as acceptor, whereas of the three only maltotriose could act as donor. The enzyme therefore shares properties with the E. coli MalQ protein, but differs significantly from the glucanotransferase of Thermotoga maritima, which is unable to use maltotriose as donor or glucose as acceptor. Physiologically, the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase provides C. butyricum with a mechanism of utilizing amylose/maltodextrins with little drain on cellular ATP reserves.
Evidence for Serpulina hyodysenteriae being recombinant, with an epidemic population structure
Authors: Trott Darren J. , Oxberry Sophy L. , Hampson David J.
Published: June 2009

The population structure of Serpulina hyodysenteriae was investigated using multilocus enzyme electrophoresis. A total of 231 isolates were divided into 50 electrophoretic types (ETs), with a mean genetic diversity of 0·29 for the number of ETs and 0·23 for the number of isolates. Subsets of isolates from two Australian states (71 isolates from Victoria and 68 isolates from Queensland) exhibited as much genetic variation as the entire collection. The calculated index of association (I
A) for the number of ETs (0·29±0·17) was not significantly different from zero, and hence provided evidence for the occurrence of significant genetic recombination accounting for the observed variation between strains. In contrast, the I
A for the number of isolates (3·93.0·03) was significantly different from zero, with seven of the 50 ETs (ETs 4, 6, 13, 14, 20, 33 and 35) containing 51% of all the isolates. Even when multiple isolates from the same farm were removed from the analysis, the I
A value for the number of isolates remained significantly greater than zero (I
A 9·87±0·04), indicating that it was not biased by their inclusion. The results suggest that S. hyodysenteriae has an epidemic population structure.
The lag phase rather than the exponential-growth phase on glucose is associated with a higher cAMP level in wild-type and cAPK-attenuated strains of the yeast Saccharomyces cerevisiae
Authors: Ma Pingsheng , Gon�alves Teresa , Maretzek Ant�nio , Dias Maria C. Loureiro , Thevelein Johan M.
Published: June 2009

In the yeast Saccharomyces cerevisiae several phenotypic properties controlled by cAMP-dependent protein kinase (cAPK) are indicative of high cAPK activity during growth on glucose and low activity during growth on non-fermentable carbon sources and in stationary phase. It has been a matter of debate whether the apparently higher activity of cAPK in cells growing on glucose is due to a higher cAMP level or to an alternative mechanism activating cAPK. The cAMP level during diauxic growth of yeast cells in cultures with different initial glucose levels and different initial cell densities has been reinvestigated and the previously reported twofold increase in cAMP during growth initiation has been confirmed. However, this increase was transient and entirely associated with the lag phase of growth. The initiation of exponential growth on glucose was associated with a decrease in the cAMP level and there was no correlation between this decrease in cAMP and the depletion of glucose in the medium. In mutants defective in feedback inhibition of cAMP synthesis, resuspension of exponential-phase glucose-grown cells in glucose medium caused an extended lag phase during which a huge, transient accumulation of cAMP occurred. The latter required the presence of glucose and nitrogen, but not phosphate or sulfate, and was not due to intracellular acidification, as shown by in vivo
31P-NMR spectroscopy. The initiation of exponential growth on glucose was also associated in this case with a decrease in cAMP rather than an increase. This behaviour was also observed in strains with attenuated catalytic subunit activity and lacking the regulatory subunit and even in strains without catalytic subunits of cAPK. This might indicate that other mechanisms are able to cause down-regulation of cAMP synthesis in a way mimicking feedback inhibition. Transfer of glucose-growing cells of wild-type or cAPK-attenuated strains to a nitrogen starvation medium resulted in an increase in the cAMP level rather than a decrease. The results indicate that the apparent changes in cAPK activity in vivo during diauxic growth on glucose and during nitrogen starvation cannot be explained on the basis of changes in the cAMP level.
Flagellar flhA, flhB and flhE genes, organized in an operon, cluster upstream from the inv locus in Yersinia enterocolitica
Authors: Fauconnier Alan , Allaoui Abdelmounaaïm , Campos Andrés , Van Elsen Ary , Cornelis Guy R. , Bollen Alex
Published: June 2009

Theinvgene ofYersinia enterocoliticacodes for invasin, a member of the invasin/intimin-like protein family, which mediates the internalization of the bacterium into cultured epithelial cells. The putative inclusion of inv into a pathogenicity island was tested by investigating its flanking sequences. Indeed, the enteropathogenicEscherichia coli(EPEC) intimin, a member of the same family of proteins, is encoded byeaeA,a gene which belongs to a pathogenicity island. An ORF located upstream frominvwas of particular interest since it appeared homologous both to the flagellarflhAgene and tosepA,an EPEC gene lying inside the same pathogenicity island as eaeA. A mutant in this ORF was non-motile and non-flagellated while its invasion phenotype remained unaffected. These data indicated that the ORF corresponded to theflhAgene ofY. enterocolitica.Subsequently, theflhBandflhEgenes, located respectively upstream and downstream fromflhA,were identified. The threeflhgenes appear to be transcribed from a single operon calledflhB,according to the nomenclature used forSalmonella typhimurium.Intergenic sequence betweenflhEandinvincludes a grey hole, with no recognizable function. Downstream frominv,we have detected the flagellarflgMoperon as already reported. Finally, the incongruous localization ofinvamidst the flagellar cluster is discussed; while transposition could explain this phenomenon, no trace of such an event was detected.
Expression of Campylobacter hyoilei lipo-oligosaccharide (LOS) antigens in Escherichia coli
Authors: Korolik Victoria , Fry Ben N. , Alderton Malcolm R. , van der Zeijst Bernard A. M. , Coloe Peter J.
Published: June 2009

Campylobacter spp. are well recognized as primary pathogens in animals and in people. To isolate and define the genetic regions encoding major surface antigens of Campylobacter hyoilei, genomic DNA of the type strain of the species, RMIT-32A, was cloned into a cosmid vector, pLA2917, in Escherichia coli and the resulting genomic library was screened using antiserum raised to the parent C. hyoilei strain. Six cosmid clones were found to express a series of immunoreactive bands in the 15-25 kDa range. These bands were proteinase K-resistant and were found in the LPS fraction of the cells, suggesting that the recombinant cosmids expressed C. hyoilei lipo-oligosaccharide (LOS) antigen(s). The minimum DNA insert size required for expression of C. hyoilei LOS antigen(s) in E. coli was 11-8 kb. This region was subcloned into the plasmid vector pBR322. The partial sequencing of the 11.8 kb region showed that it contains two ORFs, designated rfbF and rfbP, showing homology with the rfbF gene from Serratia marcescens and the rfbP gene from Salmonella typhimurium. Both genes are involved in LPS synthesis. The region also contained a sequence homologous to the rfaC gene of E. coli and Sal. typhimurium which is involved in core oligosaccharide synthesis.
Transcription-frequency-dependent modulation of an attenuator in a ribosomal protein-RNA polymerase operon requires an upstream site
Authors: Steward Keith L. , Pierre Rebecca St , Linn Thomas
Published: June 2009

Although the attenuator located between the ribosomal protein and RNA polymerase gene domains of theEscherichia coli rplKAJLrpoBCoperon has a maximum termination efficiency of 80%, the level of termination is diminished with decreasing transcription frequency. In this report, the use of transcriptional fusions to further investigate the mechanism of transcription-frequency-dependent regulation is described. The termination efficiency of two other weak terminators was assayed over a wide range of transcription frequencies programmed by different strength promoters. The results indicated that a decrease in termination efficiency with decreasing transcription frequency is not an inherent property of weak terminators. Deletion of the 165 bp segment located 439-274 bp upstream of the attenuator abrogated the difference in termination efficiency normally seen between high and low levels of transcription. This suggests that acis-acting site located in this upstream region is necessary for transcription-frequency-dependent modulation of the attenuator's function. However, this site apparently works only in combination with the attenuator, since it did not cause transcription-frequency-dependent modulation when placed upstream of two other weak terminators. Analysis of the readthrough frequencies of single or tandem copies of the attenuator indicated that the transcription complexes which pass through the attenuator have not been converted to termination-resistant complexes in a manner analogous to the N-mediated antitermination system of lambda. Finally, an examination of termination efficiency in threenusAmutants suggested that although NusA increases readthrough at the attenuator it is not directly involved in transcription-frequency-dependent modulation.
Iron uptake in Ustilago maydis: studies with fluorescent ferrichrome analogues
Authors: Ardon Orly , Weizman Haim , Libman Jacqueline , Shanzer Abraham , Chen Yona , Hadar Yitzhak
Published: June 2009

Iron uptake by the phytopathogenic fungus Ustilago maydis was studied using synthetic biomimetic ferrichrome analogues and their fluorescently labelled derivatives as structural and dynamic probes, respectively. The use of structurally distinct analogues enabled determination of the structural requirements for recognition by the fungal iron-uptake system. The application of fluorescently labelled derivatives which convert from a non-fluorescent to a fluorescent state upon iron (III) release enabled monitoring of iron uptake in real time both fluorimetrically and microscopically. Different rates of 55Fe uptake were found for two structurally distinct synthetic analogues, B9 and B5, which differ in their amino acid building blocks. B9 mediated uptake of 55Fe at a higher rate than B5. The behaviour of the fluorescent derivatives B9-Ant (anthracene-labelled B9) and B5-Ant (anthracene-labelled B5) paralleled that of their non-labelled precursors. Exposure of fungal cells to B9-Ant led to a higher increase of fluorescence in the medium than exposure to B5-Ant, indicating a more effective iron uptake from B9-Ant. By using fluorescence microscopy it was possible to trace the label of B9-Ant. Fluorescence was localized in regularly shaped vesicles in the treated cells. The rate of fluorescence appearance within the cells lagged behind the rate of iron uptake, suggesting use of the siderophores for iron storage.
A mutation that affects fibril protein, development, cohesion and gene expression in Myxococcus xanthus
Authors: Smith Daniel R. , Dworkin Martin
Published: June 2009

Extracellular matrix fibrils are involved in the cell-cell interactions of the social prokaryote, Myxococcus xanthus. The fibrils are composed of a carbohydrate backbone and a set of five integral fibrillar proteins (IFPs) ranging from 14 to 66 kDa. As part of an attempt to understand the function(s) of the IFPs, a mutant (ifp-1:20) was generated that lacks IFP-1:20, one of the fibril proteins, as shown by Western blot analysis of both whole cells and isolated fibrils. Unlike those of the parent strain, the fibrils of the mutant were removed easily from the cells by shear forces. Development in ifp-1:20 was aberrant - aggregation and early mound formation were delayed by 6-10 h and mature fruiting bodies never formed. Myxospore production was also greatly reduced. Additionally, fibril-mediated cohesion in ifp-1:20 was changed. Cohesion resulted in chains of cells rather than the characteristic clumps of cells seen for the parent strain. Isolated ifp-1:20 fibrils, unlike wild-type fibrils, could not rescue cohesion of non-cohesive, fibril-negative dsp cells, supporting the notion that the fibrils were functionally altered. The mutation also reduced developmental gene expression by three- to fourfold in Ω4521, a transposon insertion mutant expressed early in development. Expression of a later developmental gene fusion was not affected, suggesting that the fibrils may not be required for later developmental gene expression. These data suggest that intact fibrils may function early in development to facilitate close cell proximity for signal exchange.
Transcriptional regulation of the aconitase genes (acnA and acnB) of Escherichia coli
Authors: Cunningham Louise , Gruer Megan J. , Guest John R.
Published: June 2009

Escherichia colicontains two differentially regulated aconitase genes, acnA and acnB. Two acnA promoters transcribing from start points located 407 bp (P1
acnA) and 50 bp (P2
acnA) upstream of the acnA coding region, and one acnB promoter (P
acnB) with a start point 95 bp upstream of the acnB coding region, were identified by primer extension analysis. A 2.8 kb acnA monocistronic transcript was detected by Northern blot hybridization, but only in redox-stressed (methyl-viologen-treated) cultures, and a 2.5 kb acnB monocistronic transcript was detected in exponential- but not stationary-phase cultures. These findings are consistent with previous observations that acnA is specifically subject to SoxRS-mediated activation, whereas acnB encodes the major aconitase that is synthesized earlier in the growth cycle than AcnA. Further studies with acn-lacZ gene fusions and a wider range of transcription regulators indicated that acnA expression is initiated by σ38 from P1
acnA’ and from P2
acnA it is activated directly or indirectly by CRP, FruR, Fur and SoxRS, anc repressed by ArcA and FNR. In contrast, acnB expression is activated by CRP and repressed by ArcA, FruR and Fis from P
acnB Comparable studies with fum-lacZ fusions indicated that transcription of fumC, but not of fumA or fumB, is initiated by RNA polymerase containing σ38. It is concluded that AcnB is the major citric acid cycle enzyme, whereas AcnA is an aerobic stationary-phase enzyme that is specifically induced by iron and redox-stress.
Adhesion of coagulase-negative staphylococci grouped according to Physico-chemical surface properties
Authors: van der Mei Henny C. , van de Belt-Gritter Betsy , Reid Gregor , Bialkowska-Hobrzanska Hanna , Busscher Henk J.
Published: June 2009

Physico-chemical cell surface properties of 23 coagulase-negative staphylococcal strains, including contact angles, zeta potentials and elemental cell surface composition were measured, together with the adhesion of all strains to hexadecane. The data were employed in a hierarchical cluster analysis, revealing that the 23 strains comprised essentially four different groups. Groups I-III were somewhat similar to each other, but group IV was markedly distinguished from the other strains, predominantly through an elevated acidity of the cell surface. These group distinctions were not related to the presence of a capsule or slime on the strains. Adhesion of the strains to hexadecane depended critically on electrostatic interactions between the hexadecane and the staphylococci, and adhesion only occurred when the electrostatic repulsion between hexadecane and the micro-organisms was less than 500 kT at closest approach. Adhesion of six representative strains from all four groups in a parallel plate flow chamber to silicone rubber, an implant material with similar hydrophobicity to hexadecane, did not show such a critical dependence, nor did it relate with the group distinction Possibly microbial adhesion to substratum surfaces like silicone rubber is more complicated than adhesion to an ideally smooth and homogeneous hexadecane surface in an aqueous solution. Adhesion of all six strains to silicone rubber with an adsorbed conditioning film of plasma proteins was less than that to bare silicone rubber: initial deposition rates dropped from 2000-3000 cm-2 s-1 to 100-300 cm-2 s-1 after adsorption of plasma proteins, while the stationary end-point adhesion decreased from 10 x 106-15 x 106 cm-2 to 1 x 106-5 x 106 cm-2. The adhering staphylococci poorly withstood the passage of an air-bubble through the parallel plate flow chamber regard! of the presence of a conditioning film, indicating a low affinity of these relatively hydrophilic strains for hydrophobic substratum surfaces.
2-Phenylethylamine catabolism by Escherichia coli K-12: gene organization and expression
Authors: Hanlon Steven P. , Hill Terena K. , Flavell Margaret A. , Stringfellow Joseph M. , Cooper Ronald A.
Published: June 2009

A gene encoding phenylacetaldehyde dehydrogenase (PAD), the enzyme involved together with a copper-topaquinone-containing amine oxidase in the initial steps of 2-phenylethylamine catabolism, was located at 31.1 min on the Escherichia coli K-12 genetic map. It was immediately adjacent to the gene encoding the amine oxidase but transcribed in the opposite direction. The purified PAD acted almost equally well on phenylacetaldehyde, 4-hydroxyphenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde. It had a subunit size of 54 kDa and its deduced amino acid sequence was approximately 40% identical to various eukaryotic and prokaryotic aldehyde dehydrogenases. A third gene encoding a positive regulatory protein required for expression of the amine oxidase and PAD genes was located next to the PAD gene. A gene previously located in this position was reported to encode a second amine oxidase but this was not confirmed. The nucleotide sequence from 1447 to 1450 kb on the E. coli K-12 physical map has been determined.
Lactobacillus delbrueckii subsp. lactis DSM7290 pepG gene encodes a novel cysteine aminopeptidase
Authors: Klein Jurgen R. , Schick Joachim , Henrich Bemhard , Plapp Roland
Published: June 2009

A number of Escherichia coli clones were isolated from a Lactobacillus delbrueckii subsp. lactis gene library capable of hydrolysing the chromogenic substrate Gly-Ala-β-naphthylamide (Gly-Ala-βNA). Some of the recombinant plasmids carried by these clones have been shown to encode the cysteine aminopeptidase gene pepC. Nucleotide sequence analyses of the plasmid inserts of the remaining clones resulted in the identification of two adjacent ORFs encoding proteins exhibiting a high degree of similarity between themselves (72.6%) and with PepC. One gene, designated pepG, was overexpressed in E. coli and the crude extracts obtained were shown to be peptidolytically active both against chromogenic substrates and peptides, and in a Salmonella typhimurium growth test. PepC and PepG activities were compared using chromogenic βNA and p-nitroanilide substrates and leucine or proline-containing peptides were applied in growth experiments of recombinant Sal. typhimurium. The results indicate that the enzymes, although structurally related, have different substrate preferences. No enzyme activity could be ascribed to the second ORF (orfW), despite the production of a visible protein using a T7 RNA polymerase system. Primer extension analysis, using mRNA isolated from Lb. delbrueckii subsp. lactis DSM7290 did establish that orfW was transcribed.
Characterization of the ftsH gene of Bacillus subtilis
Authors: Lysenko Elena , Ogura Teru , Cutting Simon M.
Published: June 2009

Members of the AAA-protein family are found in both prokaryotes and eukaryotes. These ATPases are involved in a number of diverse activities ranging from protein secretion to cell cycle control. This paper reports the functional analysis of the Bacillus subtilis ftsH gene, which encodes a member of this protein family. In cells containing reduced levels of a truncated FtsH protein cell growth was impaired under certain nutritional conditions. In a hypersaline environment FtsH was required in increased amounts for the cells' recovery from osmotic stress. In the absence of FtsH the abundance of several of the major penicillin-binding proteins (PBP2A and 2B) in the cytoplasmic membrane was affected. Lastly, it has been established that FtsH is required for entry into the developmental life cycle.
An interfacial uptake mechanism for the degradation of pyrene by a Rhodococcus strain
Authors: Bouchez Murielle , Blanchet Denis , Vandecasteele Jean-Paul
Published: June 2009

The mechanism of uptake of polycyclic aromatic hydrocarbons (PAHs) was studied using a kinetic approach by electrolytic respirometry. In the case of the degradation of pyrene dissolved in a non-water-soluble non-degradable solvent (2,2,4,4,6,8,8-heptamethylnonane), by a Rhodococcus sp., two successive phases of exponential growth, during which over 80% of substrate degradation took place, were clearly characterized. During the second phase of biodegradation, rates of pyrene uptake were higher than those determined in abiotic conditions for the physicochemical transfer of pyrene from the solvent to the aqueous phase and no evidence for the presence of glycolipidic biosurfactants was obtained. The value of the specific growth rate for the first phase (𝜇max) was independent of the volume of the solvent phase and of the concentration of pyrene and was, in all cases, higher than that for the second phase (𝜇i). The 𝜇i values increased with the volume of the solvent phase but were independent of pyrene concentration, a clear indication of an interfacial uptake mechanism. The experimental kinetic data fitted well with a mathematical model incorporating PAH uptake both from the interface and from the aqueous medium by a population consisting of adsorbed cells in dynamic equilibrium with the cells in the aqueous medium, interfacial uptake being predominant in these experiments. Similar results were obtained for the degradation of fluoranthene. This newly demonstrated mechanism of PAH uptake is of great significance for the degradation of higher PAHs.
Purification of two Bacillus subtilis proteins which cross-react with antibodies directed against eukaryotic protein kinase C, the His HPr kinase and trigger factor
Authors: Zouari Naïla , Roche Benoît , Seegers Jos F. M. L. , Séror Simone J.
Published: June 2009

As in eukaryotes, phosphorylation of Ser and Thr residues in proteins appears to be a common phenomenon in bacteria. Surprisingly, however, very few Ser/Thr protein kinases have been identified and in this study antibodies directed against mammalian protein kinase C (PKC) have been used in attempts to isolate conserved Ser/Thr protein kinases. Using the mAb M7 against rat brain PKC, a single 70 kDa band was identified in total cell extracts of Bacillus subtilis by Western blotting after SDS-PAGE, whilst using polyclonal antibody α-PKC1p against Saccharomyces cerevisiae PKC a single 67 kDa band was identified by the same procedure. The two proteins were purified independently on the basis of antibody recognition employing two-dimensional gel electrophoresis as a final step, which allowed subsequent microsequencing. The 70 kDa band was thus identified as the phosphoenolpyruvate-dependent His HPr kinase, Enzyme I of the phosphotransferase system. This identity was confirmed using a mutant deleted for ptsl, encoding Enzyme I. The 67 kDa protein was identified as a previously unknown B. subtilis ‘trigger factor’, homologous to an Escherichia coli protein-folding enzyme, peptidylprolyl cis-trans-isomerase implicated in cell division.
The TBP gene from Aspergillus nidulans -structure and expression in Saccharomyces cerevisiae
Authors: Kucharski Robert , Bartnik Ewa
Published: June 2009

The genomic and cDNA copy of the TATA-binding protein (TBP) gene from the filamentous fungus Aspergillus nidulans have been cloned. The gene is interrupted by four introns, one of which is in the long 5' untranslated region of 615 bp. The transcription initiation site was established and the levels of mRNA were analysed under diverse growth conditions and found to vary severalfold. The gene encodes a protein of 268 amino acids composed of an N-terminal domain of 88 amino acids with no significant homology to other TBPs and a C-terminal domain of 180 amino acids with about 95% homology to other fungal TBPs. A cDNA clone under the yeast ADH1 promoter was able to substitute for the yeast TBP gene in vivo; however, the transformants obtained grew poorly at 35°C and on galactose and glycerol at 30°C, though they could grow in the presence of copper ions or aminotriazole at this temperature. This phenotype may be the result of altered function of A. nidulans TBP in certain yeast transcription activation pathways.
The signal peptidase II (lsp) gene of Bacillus subtilis
Authors: Prágai Zoltán , Tjalsma Harold , Bolhuis Albert , van Dijl Jan Maarten , Venema Gerard , Bron Sierd
Published: June 2009

The gene encoding the type II signal peptidase (SPase II) ofBacillus subtiliswas isolated by screening a genomic DNA library of this bacterium for the ability to increase the levels of globomycin resistance inEscherichia coli,and to complement the growth deficiency at the non-permissive temperature ofE. colistrain Y815 carrying a temperature-sensitive mutation in itslspgene for SPase II. The deduced amino acid sequence of theB. subtilisSPase II showed significant similarity with those of other known SPase II enzymes. Activity of theB. subtilisSPase II was demonstrated by a pulse-labelling experiment inE. coli.InB. subtilis,thelspgene is flanked by the isoleucyl-tRNA synthetase (ileS) gene and the pyrimicline biosynthetic (pyr) gene cluster, which is known to map at 139° of the chromosome. In the Gram-positive bacteria studied thus far,lspappears to be the first gene in an operon. The promoter-distal gene (orf4) of this operon specifies a hypothetical protein in bacteria and yeast.
Directed introduction of DNA cleavage sites to produce a high-resolution genetic and physical map of the Acinetobacter sp. strain ADP1 (BD413UE) chromosome
Authors: Gralton Elizabeth M. , Campbell Alan L. , Neidle Ellen L.
Published: June 2009

The natural transformability of the soil bacterium Acinetobacter sp. ADP1 (BD413UE), formerly classified as A. calcoaceticus, has facilitated previous physiological and biochemical investigations. In the present studies, the natural transformation system was exploited to generate a physical and genetic map of this strain's 3780·191 kbp circular chromosome. Previously isolated Acinetobacter genes were modified in vitro to incorporate a recognition sequence for the restriction endonuclease Not l. Following transformation of the wild-type strain by the modified DNA, homologous recombination placed each engineered Not l cleavage site at the chromosomal location of the corresponding gene. This allowed precise gene localization and orientation of more than 40 genes relative to a physical map which was constructed with transverse alternating field electrophoresis (TAFE) and Southern hybridization methods. The positions of Not l, Asc l and l-Ceu l recognition sites were determined, and the latter enzyme identified the presence of seven ribosomal RNA operons. Multiple chromosomal copies of insertion sequence IS 1236 were indicated by hybridization. Several of these copies were concentrated in one region of the chromosome in which a spontaneous deletion of approximately 100 kbp occurred. Moreover, contrary to previous reports, ColE1-based plasmids appeared to replicate autonomously in Acinetobacter sp. ADP1.
A secreted aspartic proteinase from Glomerella cingulata: purification of the enzyme and molecular cloning of the cDNA
Authors: Clark Sarah J. , Templeton Matthew D. , Sullivan Patrick A.
Published: June 2009

A secreted aspartic proteinase from Glomerella cingulata (GcSAP) as purified to homogeneity by ion exchange chromatography. The enzyme has an M
r of 36000 as estimated by SDS-PAGE, optimal activity from pH 3∙5 to pH 4∙0 and is inhibited by pepstatin. The N-terminal sequence, 23 residues long, was used to design a gene-specific primer. This was used in 3ʹ RACE (rapid amplification of cDNA ends) PCR to amplify a 1∙2 kb fragment of the gcsap DNA. A second gene-specific primer was designed and used in 5ʹ RACE PCR to clone the 5՛ region. This yielded a 600 bp DNA fragment and completed the open reading frame. The gcsap open reading frame encodes a protein with a 78 residue prepro-sequence typical of other fungal secreted aspartic proteinases. Based on the deduced sequence, the mature enzyme contains 329 amino acids and shows approximately 40% identity to other fungal aspartic proteinases. Subsequent cloning and sequencing of gcsap fragments obtained from PCR with genomic DNA revealed a 73 bp intron beginning at nt 728. Southern nalyses at medium and high stringency indicated that G. cingulata possesses ne gene for the secreted aspartic proteinase, and Northern blots indicated that gene expression was induced by exogenous protein and repressed by ammonium salts. GcSAP s a putative pathogenicity factor of G. cingulata, and it will now be possible to create SAP- mutants and assess the role GcSAP lays in pathogenicity.
Differences in genetic diversity of nonecapsulated Haemophilus influenzae from various diseases
Authors: van Alphen Loek , Caugant Dominique A. , Duim Birgitta , O'Rourke Maria , Bowler Lucas D.
Published: June 2009

Genetic relationships among 80 isolates of nonencapsulated Haemophilus influenzae recovered from different disease types were determined by multilocus enzyme electrophoresis (MEE) at 13 enzyme loci in an attempt to assess the association between multilocus genotype and disease. The isolates were obtained from 15 patients with meningitis, 10 with otitis media, 19 with chronic bronchitis, 20 with cystic fibrosis, and 16 were obtained from healthy carriers. The 80 isolates were assigned to 69 electrophoretic types (ETs) falling into 5 groups. Isolates from each disease entity were represented by a variety of genotypes; however, cluster analysis from a matrix of genetic distances between ETs revealed that the ETs of the otitis media and meningitis isolates were all clustered within a genetic distance of 0∙55 (group 1). In addition, no genotypes were shared between H. influenzae carrier isolates and isolates from cases of disease. H. influenzae isolates from healthy individuals were distributed significantly differently from those from chronic bronchitis meningitis and otitis media patients. The genetic diversity (H) of carrier strains was greatest, although not statistically different from that of isolates from patients with disease. It was concluded that the genetic distribution of acute disease isolates is not random over the five ET groups, although the genetic diversity within the groups is not different. The effect of bacterial persistence in the host on the genetic diversity of H. influenzae is discussed.
Physical mapping shows that the unstable oxytetracycline gene cluster of Streptomyces rimosus lies close to one end of the linear chromosome
Authors: Pandza Kenan , Pfalzer Guido , Cullum John , Hranueli Daslav
Published: June 2009

A restriction map of the 8 Mb linear chromosome of Streptomyces rimosus R6-501 was constructed for the enzymes AseI (13 fragments) and DraI (7 fragments). Linking clones for all 12 AseI sites and 5 of the 6 DraI sites were isolated. The chromosome has terminal inverted repeats of 550 kb, which are the longest yet reported for a Streptomyces species. The oxytetracycline gene cluster lies about 600 kb from one end, which might account for its frequent spontaneous amplification and deletion. Several other markers were localized on the chromosome (dnaA and recA, the rrn operons, the attachment site for pSAM2 and prophages RP2 and RP3). Comparison of the conserved markers with the map of Streptomyces coelicolor A3(2) suggested there are differences in genome organization between the two species.
Resuscitation of ‘non-culturable’ cells from aged cultures of Campylobacter jejuni
Authors: Bovill R. A. , Mackey B. M.
Published: June 2009

When stationary phase batch cultures of Campylobacter jejuni were stored in sealed flasks under static conditions, viable numbers declined from 2 x 109c.f.u. ml-1to around 103-106c.f.u. ml-1within 4-6 weeks. When the aged cultures were sparged with a microaerobic gas mixture, there was a rapid increase in viable numbers accompanied by a change from predominantly coccoid to vibrioid morphology. The most probable number (MPN) technique was used to distinguish resuscitation of injured or dormant cells from multiplication of residual viable cells. MPN estimates using fresh Brucella broth containing 0.2% mucin revealed that plate counts underestimated the true viable count by up to 23-fold. The experiments clearly demonstrated that a proportion of surviving cells in aged cultures were in an injured or latent state that prevented growth on agar plates. It is possible that the size of this fraction is greater than was demonstrated and that much higher recoveries would be obtained under other recovery conditions. Nevertheless, from presently available evidence, it must be concluded that the size of the latent fraction is quite small and that most of the increase in count that occurs on regassing a spent culture comes from multiplication of residual viable cells.
A 16 kDa protein family overexpressed by Streptococcus thermophilus PB18 in acid environments
Authors: González-Márquez Humberto , Perrin Clarisse , Bracquart Patrice , Guimont Christiane , Linden Guy
Published: June 2009

The one- and two-dimensional protein patterns of Streptococcus thermophilus PB18 in the exponential and stationary phases of growth were analysed. Onedimensional SDS-PAGE showed that a 16 kDa protein was overexpressed in stationary phase as well as 2 h after an acid shock, and that it was not expressed when the bacteria reached the stationary phase in medium with limiting lactose concentrations (5 or 10 g l-1), in which the pH (5∙5) was not as acid as in control cultures (pH 4∙7, lactose 20 g l-1). The results support the idea that this protein is expressed in response to the acidic environment and not in response to the growth phase. Two-dimensional PAGE showed that nine proteins were expressed only during the exponential phase and ten others only during the stationary phase. The 16 kDa band seen in one-dimensional SDS-PAGE corresponded to a 16 kDa protein family observed on two-dimensional SDS-PAGE/IEF gels, whose expression was increased 8∙5-fold when the extracellular pH reached a critical value below 5∙0. The N-terminal sequences of proteins from two spots on the two-dimensional gels (members of the 16 kDa family) were determined and found to be identical. The physiological role of this protein family has not yet been elucidated.
Respiration of Pseudomonas fluorescens as a function of intracellular substrate concentration
Authors: Dorofeyev Alexander G. , Bazin Michael J. , Lynch James M. , Panikov Nicolai S.
Published: June 2009

A kinetic method to measure the intracellular concentration of respiratory substrates in short-term starvation-enrichment experiments is proposed. Samples of bacterial suspension from steady-state chemostat cultures were subjected to 25 min starvation, followed by pulse addition of [14C]glucose. Residual substrate utilization rates and respiration rates (uptake of dissolved O2) before and after amendment were recorded. Increases in pool sizes (δL) during transients were calculated on the basis of C balance. The dependence of respiration rate qresp on δL was found to obey modified Michaelis-Menten kinetics: q
resp = Q
resp (LC+δL)/(K
L+LC+δL) [Q
resp is maximal respiration rate (29.1 mmol O2 h-1per g biomass C), K
L = 12.14 mg C per g biomass C], where LC is the absolute value of the pool size before amendment. Direct chemical determination of LC in cold TCA extracts revealed two fractions. The first fraction was mobile and showed a close correlation with both respiration and L. The second, ‘stable’, fraction did not correlate with respiration dynamics and was interpreted as material formed artifactually by acid degradation of polymeric cell components.
Molecular and immunological characterization of OprL, the 18 kDa outer-membrane peptidoglycan-associated lipoprotein (PAL) of Pseudomonas aeruginosa
Authors: Lim Antonio , De Vos Daniel , Brauns Mathieu , Mossialos Dimitris , Gaballa Ahmed , Qing Ding , Cornells Pierre
Published: June 2009

Immunological screening of aPseudomonas aeruginosacosmid library led to the identification of clones producing an 18 kDa outer-membrane protein. This protein reacted in Western blots with a polyclonal antiserum against outer-membrane proteins ofP. aeruginosaand with a monoclonal antibody (MA1-6) specific for OprL, the peptidoglycan-associated outer-membrane lipoprotein (PAL). Sequencing of pOML7, a subclone expressingoprL,revealed an ORF of 504 bp encoding a polypeptide with a typical lipoprotein signal recognition sequence. Another ORF was found upstream ofoprL,with homology to the ToIB protein ofEscherichia coliandHaemophilus influenzae.Downstream ofoprL,a second ORF, of 321 bp, was found (orf2), encoding a protein with a signal peptide and with no homology with proteins of known biological function. After the stop codon oforf2,a rho-independent terminator sequence was detected which is part of theP. aeruginosaPA01 insertion element IS222. OprL showed homologies with all known PALs from Gram-negative bacteria, especially in the C-terminal part. mAb MA1-6 reacted withP. aeruginosacells in immunofluorescence, and withE. colicells expressingoprL,which had an abnormal, elongated morphology, an indication that production of the protein perturbed the division process.
>Filamentous growth of the budding yeast Saccharomyces cerevisiae induced by overexpression of the WH12 gene
Authors: Radcliffe P. A. , Binley K. M. , Trevethick J. , Hall M. , Sudbery P. E.
Published: June 2009

The WH12 gene of the budding yeast Saccharomyces cerevisiae is required for the arrest of cell proliferation upon nutrient exhaustion: whi2 mutants carry on dividing and in the absence of growth become abnormally SmaIl. It is reported here that overexpression of Whi2 from the GAL1 promoter results in filamentous growth - cells fail to complete cytokinesis, the budding pattern changes from axial to polar, cells become elongated and cell size increases threefold. In many ways, these filaments resemble the pseudohyphae which result from nitrogen-limited growth and the filaments seen during the invasive growth of haploids. However, Whi2-induced filament formation is reduced, but not blocked, by mutations in STE7, STE12 or STE20 which do block pseudohypha formation. Furthermore, pseudohypha formation can still occur in a diploid in which both copies of the WH12 gene have been deleted. Thus Whi2-induced filament formation and pseudohypha formation must come about through the action of different pathways. Despite this, a mutation in the STE11 gene, which is required for pseudohypha formation, does block Whi2-induced filament formation. Concanavalin A pulse-chase experiments show that new cell wall material is incorporated only into the tips of the apical cells. An extragenic suppressor of the whi2 allele also results in filamentous growth. These results suggest that Whi2 negatively regulates a function required for the budding mode of cell proliferation including cytokinesis. This function is defined wholly or in part by the fswl allele.
Anaerobic taurine oxidation: a novel reaction by a nitrate-reducing Alcaligenes sp.
Authors: Denger Karin , Laue Heike , Cook Alasdair M.
Published: June 2009

Enrichment cultures were prepared under strictly anoxic conditions in medium representing fresh water and containing an organosulfonate as electron donor and carbon source, and nitrate as electron acceptor. The inoculum was from the anaerobic digestor of two communal sewage works. The natural organosulfonates 2-aminoethanesulfonate (taurine), dl-2-amino-3-sulfopropionate (cysteate) and 2-hydroxyethanesulfonate (isethionate) all gave positive enrichments, whereas unsubstituted alkanesulfonates, such as methanesulfonate and arenesulfonates, gave no enrichment. Two representative enrichments were used to obtain pure cultures, and strains NKNTAU (utilizing taurine) and NKNIS (utilizing isethionate) were isolated. Strain NKNTAU was examined in detail. Out of 18 tested organosulfonates, it utilized only one, taurine, and was identified as a novel Alcaligenes sp., a facultatively anaerobic bacterium. Carbon from taurine was converted to cell material and carbon dioxide. The amino group was released as ammonium ion and the sulfonate moiety was recovered as sulfate. Nitrate was reduced to nitrogen gas.
The symbiotic phenotypes of exopolysaccharide-defective mutants of Rhizobium sp. strain TAL1145 do not differ on determinate- and indeterminate-nodulating tree legumes
Authors: Parveen Nikhat , Webb David T. , Borthakur Dulal
Published: June 2009

Three classes of exopolysaccharide (EPS) defective mutants were isolated by Tn3Hogus -insertion mutagenesis of Rhizobium sp. strain TAL1145, which nodulates tree legumes. The class I and class III mutants produced 10-22% of the EPS produced by TAL1145 and appeared partially mucoid while the class II mutants formed small, opaque and non-mucoid colonies. Size-fractionation of the soluble EPSs made by these mutants in the culture supernatant indicated that the class I and the class III mutants produced reduced levels of both highland low-molecular-mass EPSs while the class II mutants lacked both these EPSs but produced a small amount of a medium-molecular-mass anthrone-reactive EPS. The succinyl and acetyl substituents observed in the TAL1145 EPS were absent in the EPS of the class II mutants. When examined under UV, the class I and class III mutants grown on Calcofluor-containing YEM agar showed dim blue fluorescence, compared to the bright blue fluorescence of the wild-type strain, whereas the class II mutants did not fluoresce. While the dim blue fluorescence of the class III mutants changed to yellow-green after 10 d, the fluorescence of the class I mutants did not change after prolonged incubation. Unlike the EPS-defective mutants of other rhizobia, these mutants did not show different symbiotic phenotypes on determinate- and indeterminate-nodulating tree legumes. The class I and the class III mutants formed small ineffective nodules on both types of legumes whereas the class II mutants formed normal nitrogen-fixing nodules on both types. The genes disrupted in the class I and class III mutants form a single complementation group while those disrupted in the class II mutants constitute another. All the three classes of EPS-defective mutants were located within a 10.8 kb region and complemented by two overlapping cosmids.