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See detailPlasmid-associated bacteriocin production by Lactobacillus LMG21688 Listeria monocytogenes growth rebound in a food system.
Kouakou, P.; Dortu, C.; Dubois Dauphin, Robin ULg et al

in FEMS Microbiology Letters (2010), 306(1), 37-44

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See detailCharacterization of the proteins encoded by the Bacillus subtilis yoxA-dacC operon.
Duez, Colette ULg; Zervosen, Astrid ULg; Teller, Nathalie et al

in FEMS Microbiology Letters (2009), 300

Abstract In Bacillus subtilis, the yoxA and dacC genes were proposed to form an operon. The yoxA gene was overexpressed in Escherichia coli and its product fused to a polyhistidine tag was purified. An ... [more ▼]

Abstract In Bacillus subtilis, the yoxA and dacC genes were proposed to form an operon. The yoxA gene was overexpressed in Escherichia coli and its product fused to a polyhistidine tag was purified. An aldose-1-epimerase or mutarotase activity was measured with the YoxA protein that we propose to rename as GalM by analogy with its counterpart in E. coli. The peptide d-Glu-delta-m-A(2)pm-d-Ala-m-A(2)pm-d-Ala mimicking the B. subtilis and E. coli interpeptide bridge was synthesized and incubated with the purified dacC product, the PBP4a. A clear dd-endopeptidase activity was obtained with this penicillin-binding protein, or PBP. The possible role of this class of PBP, present in almost all bacteria, is discussed. [less ▲]

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See detailGenome comparison of Bifidobacterium longum strains NCC2705 and CRC-002 using suppression subtractive hybridization.
Delcenserie, Véronique ULg; Lessard, Marie*-Helene; LaPointe, Gisele et al

in FEMS Microbiology Letters (2008), 280(1), 50-6

Because probiotic effects are strain dependent, genomic explanations of these differences will contribute to understanding their mechanisms of action. The genomic sequence of the Bifidobacterium longum ... [more ▼]

Because probiotic effects are strain dependent, genomic explanations of these differences will contribute to understanding their mechanisms of action. The genomic sequence of the Bifidobacterium longum probiotic strain NCC2705 was determined, but little is known about the genetic diversity between strains of this species. Suppression subtractive hybridization (SSH) is a powerful method for generating a set of DNA fragments differing between two closely related bacterial strains. The purpose of this study was to identify genetic differences between genomes of B. longum strains NCC2705 and CRC-002 using PCR-based SSH. Strain CRC-002 produces exopolysaccharides whereas NCC2705 is not known for reliable exopolysaccharide production. Thirty-five and 30 different sequences were obtained from the SSH libraries of strains CRC-002 and NCC2705, respectively. Specific CRC-002 genes found were predicted to be involved in the biosynthesis of exopolysaccharides and metabolism of other carbohydrates, and these genes were not present in the genome of strain NCC2705. The identification of an endo-1,4-beta-xylanase gene in the CRC-002 SSH library is an important difference because xylanase genes have previously been proposed as a defining characteristic of the NCC2705 strain. The results demonstrate that the SSH technique was useful to highlight potential genes involved in complex sugar metabolism that differ between the two probiotic strains. [less ▲]

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See detailRNA silencing in the dermatophyte Microsporum canis
Vermout, S.; Tabart, J.; Baldo, Aline ULg et al

in FEMS Microbiology Letters (2007), 275(1), 38-45

Dermatomycoses caused by Microsporum canis are frequent in domestic animals and easily transmissible to humans. Several proteases secreted by this fungus were identified as potential virulence factors ... [more ▼]

Dermatomycoses caused by Microsporum canis are frequent in domestic animals and easily transmissible to humans. Several proteases secreted by this fungus were identified as potential virulence factors, but the construction of deficient strains is required to investigate their role in the pathogenesis of the disease. Using target genes encoding two of these proteases, a first evaluation of the utility of RNA-mediated silencing as a reverse genetic tool in dermatophytes was carried out. SUB3 and DPPIV, respectively coding for a subtilisin and a dipeptidyl peptidase, were both down-regulated, by means of two plasmid constructs designed to express an RNA hairpin that corresponds to part of their respective sequence. The degree of attenuation was evaluated by enzymatic assay of the transformants culture supernatants, and by real-time reverse transcriptase-polymerase chain reaction. Enzymatic activities and expression levels varied from less than 5% to 100% of that of control transformants obtained with plasmid without hairpin inserts. Inhibition was globally more efficient for SUB3 than for DPPIV. These results show that RNA silencing can be used for functional genomics in M. canis, and particularly to circumvent the limits and technical difficulties of conventional disruption methods. [less ▲]

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See detailMycoplasma bovis shares insertion sequences with Mycoplasma agalactiae and Mycoplasma mycoides subsp mycoides SC: Evolutionary and developmental aspects
Thomas, Anne; Linden, Annick ULg; Mainil, Jacques ULg et al

in FEMS Microbiology Letters (2005), 245(2), 249-255

Three new insertion elements, IS Mbov1, ISMbov2 and ISMbov3, which are closely related to ISMag1 (Mycoplasma agalactiae), ISMmy1 and IS1634 (both Mycoplasma in mycoides subsp. in mycoides SC ... [more ▼]

Three new insertion elements, IS Mbov1, ISMbov2 and ISMbov3, which are closely related to ISMag1 (Mycoplasma agalactiae), ISMmy1 and IS1634 (both Mycoplasma in mycoides subsp. in mycoides SC), respectively, have been discovered in Mycoplasma bovis, an important pathogen of cattle. Southern blotting showed that the genome of M. bovis harbours 6-12 copies of ISMbov1, 11-15 copies of ISMbov2 and 4-10 copies of ISMbov3, depending on the strain. A fourth insertion element, the IS30-like element, is present in 4-8 copies. This high number of IS elements in AI bovis, which represent a substantial part of its genome, and their relatedness with IS elements of both M. agalactiae and M. mycoides subsp. mycoides SC suggest the occurrence of two evolutionary events: (i) a divergent evolution into ill. agalactiae and M. bovis upon infection of different hosts; (ii) a horizontal transfer of IS elements during co-infection with M. mycoides subsp. mycoides SC and Ad. bovis of a same bovine host. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. [less ▲]

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See detailThe Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues.
Ghysels, Bart ULg; Ochsner, Urs; Mollman, Ute et al

in FEMS microbiology letters (2005), 246(2), 167-74

Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes ... [more ▼]

Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes a high affinity siderophore, pyoverdine, and the low affinity siderophore, pyochelin. Uptake of the iron-siderophore complexes is an active process that requires specific outer membrane located receptors, which are dependent of the inner membrane-associated protein TonB and two other inner membrane proteins, ExbB and ExbC. P. aeruginosa is also capable of using a remarkable variety of heterologous siderophores as sources of iron, apparently by expressing their cognate receptors. Illustrative of this feature are the 32 (of which 28 putative) siderophore receptor genes observed in the P. aeruginosa PAO1 genome. However, except for a few (pyoverdine, pyochelin, enterobactin), the vast majority of P. aeruginosa siderophore receptor genes still remain to be characterized. Ten synthetic iron chelators of catecholate type stimulated growth of a pyoverdine/pyochelin deficient P. aeruginosa PAO1 mutant under condition of severe iron limitation. Null mutants of the 32 putative TonB-dependent siderophore receptor encoding genes engineered in the same genetic background were screened for obvious deficiencies in uptake of the synthetic siderophores, but none showed decreased growth stimulation in the presence of the different siderophores. However, a double knock-out mutant of ferrienterobactin receptor encoding gene pfeA (PA 2688) and pirA (PA0931) failed to be stimulated by 4 of the tested synthetic catecholate siderophores whose chemical structures resemble enterobactin. Ferric-enterobactin also failed to stimulate growth of the double pfeA-pirA mutant although, like its synthetic analogues, it stimulated growth of the corresponding single mutants. Hence, we confirmed that pirA represents a second P. aeruginosa ferric-enterobactin receptor. The example of these two enterobactin receptors probably illustrates a more general phenomenon of siderophore receptor redundancy in P. aeruginosa. [less ▲]

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See detailImmunocytochemical detection of DNA and RNA in endosymbiont-bearing trypanosomatids.
Motta, Maria Cristina M; de Souza, Wanderley; Thiry, Marc ULg

in FEMS Microbiology Letters (2003), 221(1), 17-23

Research about the kinetoplast of trypanosomatids has yielded valuable information about the organization of extranuclear structure. However, the ultrastructural localization of nucleic acids within these ... [more ▼]

Research about the kinetoplast of trypanosomatids has yielded valuable information about the organization of extranuclear structure. However, the ultrastructural localization of nucleic acids within these protozoa remains uncertain. We have applied cytochemical and immunocytochemical approaches to precisely identify DNA and RNA in lower endosymbiont-bearing trypanosomatids. Using the Terminal deoxynucleotidyl Transferase (TdT) immunogold technique, we showed that nuclear DNA is seen associated with the nuclear envelope during the trypanosomatid cell cycle. By combining the TdT technique with the acetylation method, which improves the contrast between structures containing fibrils and granules, we have demonstrated that the nucleolus of endosymbiont-bearing trypanosomatids is composed of two constituents: a granular component and a DNA-positive fibrillar zone. Moreover, we revealed that DNA of endosymbiotic bacteria consisted of electron-dense filaments which are usually in close contact with the prokaryote envelope. Using a Lowicryl post-embedding immunogold labeling procedure with anti-RNA antibodies, we showed the presence of RNA not only over the cytoplasm, the interchromatin spaces and the nucleolus, but also over the kinetoplast and virus-like particles present in Crithidia desouzai. [less ▲]

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See detailPleiotropic Mutants Hypersensitive to Heavy Metals and to Oxidative Stress in Chlamydomonas Reinhardtii
Hanikenne, Marc ULg; Matagne, René F.; Loppes, Roland ULg

in FEMS Microbiology Letters (2001), 196(2),

Insertional mutagenesis was used in Chlamydomonas reinhardtii to isolate original mutants hypersensitive to multiple drugs and physical agents. Out of 5200 transformants analyzed, 13 mutants belonging to ... [more ▼]

Insertional mutagenesis was used in Chlamydomonas reinhardtii to isolate original mutants hypersensitive to multiple drugs and physical agents. Out of 5200 transformants analyzed, 13 mutants belonging to seven phenotypic classes were isolated. Five were exclusively sensitive to cadmium and represented two loci. The other mutants were pleiotropic and presented a cross sensitivity to several (2--6) of the following agents: cadmium, copper, lead, paraquat, hydrogen peroxide, UVC and light. In all mutants analyzed, the hypersensitive phenotype was most probably due to a single mutational event. The sensitivity of several pleiotropic mutants to a broad range of physical and chemical agents suggests that the disrupted genes are involved in multiple stress responses. [less ▲]

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See detailIntracellular pH-dependent efflux of the fluorescent probe pyranine in the yeast Yarrowia lipolytica
Aguedo, Mario ULg; Waché, Y.; Belin, J.-M.

in FEMS Microbiology Letters (2001), 200(2), 185-189

8-Hydroxypyrene-1,3,6-trisulfonic acid (pyranine) can be used as a vital intracellular pH (pHi) indicator. In the yeast Yarrowia lipolytica, a partial efflux of the probe was detected by using the pH ... [more ▼]

8-Hydroxypyrene-1,3,6-trisulfonic acid (pyranine) can be used as a vital intracellular pH (pHi) indicator. In the yeast Yarrowia lipolytica, a partial efflux of the probe was detected by using the pH-independent wavelength of 415 nm. A simplified correction of the fluorescent signals was applied, enabling to show for this species a good near-neutral pHi maintenance capacity in a pH 3.9 medium. Octanoic acid, which is known to have toxic effects on yeast, decreased the pHi and increased the 260-nm-absorbing compounds leakage. However, this acid inhibited the fluorescent probe efflux linearly with its concentration suggesting a pHi-dependent efflux of pyranine from cells. © 2001 Federation of European Microbiological Societies. [less ▲]

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See detailComparison of Eae, Tir, Esp a and Esp b Genes of Bovine and Human Attaching and Effacing Escherichia Coli by Multiplex Polymerase Chain Reaction
China, B.; Goffaux, F.; Pirson, V. et al

in FEMS Microbiology Letters (1999), 178(1), 177-82

Attaching and effacing Escherichia coli (AEEC) virulence genes include the eae, the tir, the espA and the espB genes. These genes have been sequenced from several AEEC strains. The sequences alignments ... [more ▼]

Attaching and effacing Escherichia coli (AEEC) virulence genes include the eae, the tir, the espA and the espB genes. These genes have been sequenced from several AEEC strains. The sequences alignments revealed the presence of constant and variable regions. Multiplex polymerase chain reactions were developed, in order to determine the subtype of each gene present in a particular isolate. AEEC strains isolated from calves dead of diarrhea, from healthy calves and from infected humans were compared. The same pathotypes were found in sick and healthy calves but in inverted proportion. These pathotypes were also found in human AEEC. Although, the human EHEC strains from serotype O157 possessed their own pathotype. [less ▲]

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See detailArthrospira ('Spirulina') strains from four continents are resolved into only two clusters, based on amplified ribosomal DNA restriction analysis of the internally transcribed spacer
Scheldeman, Patsy; Baurain, Denis ULg; Bouhy, Rachel et al

in FEMS Microbiology Letters (1999), 172(2), 213-22

We present the results of a phylogenetic study, based on amplified ribosomal DNA restriction analysis of the rDNA operon, of 37 Arthrospira ('Spirulina') cultivated clonal strains from four continents. In ... [more ▼]

We present the results of a phylogenetic study, based on amplified ribosomal DNA restriction analysis of the rDNA operon, of 37 Arthrospira ('Spirulina') cultivated clonal strains from four continents. In addition, duplicates from different culture collections or markedly different morphotypes of particular strains established as clonal cultures were treated as separate entries, resulting in a total of 51 tested cultures. The strain Spirulina laxissima SAG 256.80 was included as outgroup. The 16S rRNA genes appeared too conserved for discrimination of the strains by amplified ribosomal DNA restriction analysis, and thus the internally transcribed spacer was selected as molecular taxonomic marker. The internally transcribed spacer sequences situated between the 16S and the 23S rRNA were amplified by polymerase chain reaction and yielded amplicons of about 540 bp. Direct use of cells for polymerase chain reaction seemed to inhibit the amplification reaction. This was overcome by the design of a crude lysis protocol and addition of bovine serum albumin in the polymerase chain reaction mix. The amplicons were digested with four restriction enzymes (EcoRV, Hhal, Hinfl, Msel) and the banding patterns obtained were analyzed. Cluster analysis showed the separation of all the strains into two main clusters. No clear relationships could be observed between this division into two clusters and the geographic origin of the strains, or their designation in the culture collections, or their morphology. [less ▲]

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See detailInducible class C beta-lactamases produced by psychrophilic bacteria
Pierrard, Annick ULg; Ledent, P.; Docquier, J. D. et al

in FEMS Microbiology Letters (1998), 161

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See detailBovine Attaching and Effacing Escherichia Coli Possess a Pathogenesis Island Related to the LEE of the Human Enteropathogenic Escherichia Coli Strain E2348/69
Goffaux, F.; Mainil, Jacques ULg; Pirson, V. et al

in FEMS Microbiology Letters (1997), 154(2), 415-421

Attaching and effacing Escherichia coli (AEEC) has been described as a cause of diarrhea in calves. The molecular pathogenesis of AEEC was mainly studied in human enteropathogenic E. coli strain E2348/69 ... [more ▼]

Attaching and effacing Escherichia coli (AEEC) has been described as a cause of diarrhea in calves. The molecular pathogenesis of AEEC was mainly studied in human enteropathogenic E. coli strain E2348/69 in which the virulence correlated with the presence of a 35.4 kb pathogenesis island called LEE. We showed that several strains isolated from calves with diarrhea were able to produce attaching and effacing lesions in a rabbit ileal loop model and that they possess a pathogenesis island related to the LEE. Moreover, we showed that the LEE from bovine strains was inserted mainly at a different position in the chromosome compared to the human enteropathogenic E. coli strain E2348/69. [less ▲]

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See detailUse of an Automatic DNA Sequencer for S1 Mapping: Transcriptional Analysis of the Streptomyces Coelicolor A3(2) Dnak Operon
Brans, Alain ULg; Loriaux, Axelle; Thamm, Iris ULg et al

in FEMS Microbiology Letters (1997), 149(2), 189-94

The transcription start point of the dnaK operon of Streptomyces coelicolor A3(2) has been determined by S1 mapping, using the EMBL automated fluorescent DNA sequencer. The -35 and -10 hexamers correspond ... [more ▼]

The transcription start point of the dnaK operon of Streptomyces coelicolor A3(2) has been determined by S1 mapping, using the EMBL automated fluorescent DNA sequencer. The -35 and -10 hexamers correspond to a sigma 70-type promoter. This promoter responds to heat shock and involves an inverted repeat different from the CIRCE sequence characteristic of the Gram-positive heat-shock promoters. [less ▲]

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See detailA Sequence-Specific DNA-Binding Protein Interacts with the Xlnc Upstream Region of Streptomyces Sp. Strain Ec3
Giannotta, F.; Georis, J.; Moreau, A. et al

in FEMS Microbiology Letters (1996), 142(1), 91-7

The alignment of the promoter region of several Streptomyces xylanases shows three conserved sequences which could be involved in gene regulation. By electromobility shift assays these specific sequences ... [more ▼]

The alignment of the promoter region of several Streptomyces xylanases shows three conserved sequences which could be involved in gene regulation. By electromobility shift assays these specific sequences, present only in Streptomyces xylanolytic strains, were identified as protein-binding sites. The sequence required for efficient recognition by the retarding protein appeared to be a 4-bp inverted repeat: 5'-CTTT-Nx-AAAG-3'. The DNA-protein affinity was influenced by the culture conditions. [less ▲]

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See detailOverproduction and properties of the mannuronate alginate lyase AlxMB
Malissard, Martine; Chavagnat, Frédéric; Duez, Colette ULg et al

in FEMS Microbiology Letters (1995), 126(2), 105-111

In previous studies (Malissard et al., FEMS Microbiol. Lett. (1993) 110, 101-106), the alginate lyase AlxM of the marine bacterium ATCC 433367 was produced in Escherichia coli TC4/pAL-A3 with a yield of ... [more ▼]

In previous studies (Malissard et al., FEMS Microbiol. Lett. (1993) 110, 101-106), the alginate lyase AlxM of the marine bacterium ATCC 433367 was produced in Escherichia coli TC4/pAL-A3 with a yield of 50 mu g per litre of culture. The polypeptide chain was cleaved between two cysteine residues, C169 and C183, themselves linked by a disulphide bridge. AlxM has now been overproduced in E. coli BL21(DE3)/pAL-Sur/pLysS. Under conditions in which formation of inclusion bodies can be avoided, the enzyme is synthesized as a catalytically active, water-soluble, unnicked polypeptide with a yield of 32 mg per litre of culture. It has been purified to protein homogeneity using a one-step procedure. The nicked ALxM(A) and unnicked ALxM(B) alginate lyases have identical alginate-degrading activities at high salt concentrations. [less ▲]

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See detailSite-directed mutagenesis of penicillin-binding protein 3 of Escherichia coli: role of Val-545
Ayala, Juan; Goffin, Colette; Nguyen-Distèche, Martine ULg et al

in FEMS Microbiology Letters (1994), 121(2), 251-256

Val545 of the Escherichia coli penicillin-binding protein 3 is essential to the acyl transfer mechanism through which the active-site serine 307 is acylated by benzylpenicillin and cephalexin and to the ... [more ▼]

Val545 of the Escherichia coli penicillin-binding protein 3 is essential to the acyl transfer mechanism through which the active-site serine 307 is acylated by benzylpenicillin and cephalexin and to the mechanism through which the protein allows rapidly growing cells to divide. [less ▲]

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See detailSite-directed mutagenesis of dicarboxylic acid residues of the penicillin-binding module of the Escherichia coli penicillin-binding protein 3
Goffin, Colette ULg; Ayala, J. A.; Nguyen-Distèche, Martine ULg et al

in FEMS Microbiology Letters (1993), 113(3), 247-251

The glutamic acid E396, aspartic acid D409 and glutamic acid E411 residues of the Escherichia coli penicillin-binding protein 3 were each converted into an alanine residue. As deduced from penicillin ... [more ▼]

The glutamic acid E396, aspartic acid D409 and glutamic acid E411 residues of the Escherichia coli penicillin-binding protein 3 were each converted into an alanine residue. As deduced from penicillin-binding and complementation experiments, none of these dicarboxylic acid residues is involved in the mechanism of acylation by penicillin and none of them is essential for the in vivo functioning of the PBP. The mutation E396, however, causes an increased thermolability of the protein. [less ▲]

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