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PubMed1975

[Blood microfiltration. Scanning electron microscopic study of the aggregates retained by a transfusion filter made of polyurethane foam].

Preliminary studies of the transfusion filter Bentley PF 127, a polyfilter type with a graded serie of diameters of microfenestration are reported. Dog blood has been used in all instances of the trial phase. Variations of the hematological factors as well as biochemical disparities have been examined and all deposits were assessed by means of scanning electron microscope. Amounts of deposits increased with the blood age. As far as banked dog blood develops less microaggregates during storage than human blood, the SEM pictures reported are a plea for banked blood microfiltration in any transfusion to human beings. The deposits which were trapped in the polyurethane foam, had previously passed through a screen filter with pore size slightly wider than the standard one (250 microns instead of 170 microns). Unfortunately the possibility of thrombus formation is serious as far as banked blood is rather fragile, and due to a slow flow rate, the time of blood contact with the filter is enough to allow thrombus development. However, the amounts of clots greatly increased with the age of the blood. The importance of filtration by adsorption was not very visible. The future of such a depth filter is questionable: should we prefer a transfusion screen filter with small pore size, the efficiently of which is determined by its pore size, and which traps the microaggregates by mechanical retention, or a depth filter which is supposed to retain the microaggregates regard less of the size but which could be very easily thrombus invaded and does not allow a suffisant blood flow rate for patients needing large amounts of blood in period of initial resuscitation? The debate is open but we should recognize that a screen filter with small pore size is widely used in the hospitals.

Animals↗
PubMed2003

Transferrin-dependent expression of TbpA by Histophilus ovis involves a poly G tract within tbpA.

A poly G tract in tbpA of Histophilus ovis strain 3384Y was suspected of being responsible for the transferrin (Tf)-dependent expression of TbpA. The region encompassing the poly G tract was amplified using DNA from H. ovis strains 9L and 3384Y grown under iron-replete conditions and under iron-restricted conditions in the presence of bovine Tf. Sequence analysis of the amplification products revealed that regardless of the growth conditions, the poly G tract in strain 9L contained eight Gs, a situation that maintains the correct reading frame of the gene. Similarly, the poly G tract in strain 3384Y contained eight Gs when the organisms were grown under iron-restricted conditions in the presence of bovine Tf but when grown under iron-replete conditions, the poly G tract contained nine Gs resulting in a frame shift and the introduction of a premature stop codon. It is concluded that the Tf-dependent expression of TbpA in H. ovis strain 3384Y is due to a form of phase variation.

Amino Acid Sequence↗
PubMed1997

Insertionally inactivated and inducible recA alleles for use in Neisseria.

Two classes of recA mutations have been constructed for use in Neisseria gonorrhoeae: three insertionally inactivated ('knockout') mutations and three LacI-regulatable constructs that can be shifted between Rec- and Rec+ by the removal or addition of IPTG. The effects of regulating recA expression on the processes of DNA transformation, DNA repair and pilin-phase variation are described. These regulatable cassettes can also be used to control the expression of any chromosomal gene.

Alleles↗
PubMed2001

Genetic analysis of virulence factors of Mannheimia (Pasteurella) haemolytica A1.

Using a molecular genetic approach, the genes that code for the various virulence factors of Mannheimia haemolytica A1 have been cloned for detailed characterizations. These included analysis of the encoded proteins, their biological activities, secretion of the molecules from the bacterium as well as their use in a vaccine component. Two newly characterized antigens of M. haemolytica A1 have been identified. The first one is a TonB-dependent iron regulated outer-membrane receptor that is distinct from the transferrin binding proteins. The 84kDa Irp protein exhibits features including a TonB box and a 50 amino acid region that can adopt occluded beta-barrel structures similar to the "plug" domain of the Escherichia coli FhuA and FepA crystal structures. Homologues of Irp were identified by analysis of the genome sequences of a number of Gram negative mucosal pathogens, including Neisseria meningitidis and N. gonorrhoeae. The Neisserial irp genes were cloned by PCR and expressed the 84kDa protein as expected, demonstrating that they are functional genes. In addition to being regulated by iron and Fur, irp(Mh) undergoes phase variation by a slipped-strand mispairing mechanism and may represent a contingency locus for iron acquisition during an infection. Another locus that codes for a putative adhesin molecule has also been partially characterized. This putative adhesin protein is highly homologous with the high-molecular-weight adhesin proteins of non-piliated non-typable strains of Haemophilus influenzae (NTHi) including Hia, Hsf, HMW1, HMW2. Currently, we have cloned the DNA that codes for 2223 amino acids (225kDa) and is still missing the stop codon. It is anticipated that when complete, the protein could be close to 240kDa, similar to the molecular mass of Hsf. Though incomplete, analysis of the adhesin showed that it exhibits characteristics of autotransporter (AT) proteins. The role of this high-molecular-weight adhesin in infection is being investigated.

Adhesins, Bacterial↗
PubMed1997

Convergent flow phenomenon mimics the appearance of venous thrombosis in gradient-echo images with or without the presence of a contrast agent.

We have observed signal voids at the junction of the renal vein and the inferior vena cava in the Spoiled Grass images. They mimicked the magnetic resonance appearance of an intraluminal thrombus with and without the presence of a contrast agent. The patency of the vessels was unveiled by fast Spoiled Grass sequence with reduced echo time as well as by Doppler ultrasound. Phantom studies revealed patterns of counterrotating vortices at the confluence. The cause of this image artifact was subsequently deduced as the intravoxel spin phase dispersion arising from the impinging flows of the renal vein and inferior vena cava. It is concluded that in regions where complex flow patterns reside, fast imaging sequences that reduce spin phase variations should always be conducted in addition to other routine sequences to exclude uncertainties in image interpretation.

Aged↗
PubMed2002

Detection of the icaADBC gene cluster and biofilm formation in Staphylococcus epidermidis isolates from catheter-related urinary tract infections.

Biofilm production in Staphylococcus epidermidis is an important virulence factor that is mediated by the expression of the icaADBC operon. In this study 41 S. epidermidis isolates obtained from catheter-related urinary tract infections were analyzed for the presence of the icaADBC operon and biofilm formation. Eighteen of 41 isolates (44%) were shown to carry ica-specific DNA, but only 11 isolates (27%) produced biofilms spontaneously under normal growth conditions. Upon induction by external stress or antibiotics, biofilm formation could be stimulated in five of seven ica-positive, biofilm-negative isolates, indicating that the icaADBC expression was down-regulated in these strains. Genetic analyses of the ica gene clusters of the remaining two ica-positive, biofilm-negative strains revealed a spontaneous ICAC::IS256 insertion in one strain. Insertion of the element caused a target site duplication of seven base pairs and a biofilm-negative phenotype. After repeated passages the insertion mutant was able to revert to a biofilm-forming phenotype which was due to the precise excision of IS256 from the icaC gene. The data show that icaC::IS256 integrations occur during S. epidermidis polymer-related infections and the results highlight the biological relevance of the IS256-mediated phase variation of biofilm production in S. epidermidis during an infection.

Biofilms↗
PubMed2001

Deciphering Campylobacter jejuni cell surface interactions from the genome sequence.

The completion of the Campylobacter jejuni genome sequence is a landmark in Campylobacter research. Discoveries directly arising from these data include the identification of a capsular polysaccharide, extensive capacity for phase variable gene expression and lipo-oligosaccharide structural phase variation. The recent identification of a unique system of general protein glycosylation in C. jejuni, a C. jejuni protein that is translocated into eukaryotic cells, and plasmid-encoded components of a putative type IV secretion system are likely to be significant in terms of the host-pathogen interaction.

Antigenic Variation↗
PubMed2007

Following photoinduced dynamics in bacteriorhodopsin with 7-fs impulsive vibrational spectroscopy.

Sub-10-fs laser pulses are used to impulsively photoexcite bacteriorhodopsin (BR) suspensions and probe the evolution of the resulting vibrational wave packets. Fourier analysis of the spectral modulations induced by transform-limited as well as linearly chirped excitation pulses allows the delineation of excited- and ground-state contributions to the data. On the basis of amplitude and phase variations of the modulations as a function of the dispersed probe wavelength, periodic modulations in absorption above 540 nm are assigned to ground-state vibrational coherences induced by resonance impulsive Raman spectral activity (RISRS). Probing at wavelengths below 540 nm-the red edge of the intense excited-state absorption band-uncovers new vibrational features which are accordingly assigned to wave packet motions along bound coordinates on the short-lived reactive electronic surface. They consist of high- and low-frequency shoulders adjacent to the strong C=C stretching and methyl rock modes, respectively, which have ground-state frequencies of 1008 and 1530 cm-1. Brief activity centered at approximately 900 cm-1, which is characteristic of ground-state HOOP modes, and strong modulations in the torsional frequency range appear as well. Possible assignments of the bands and their implication to photoinduced reaction dynamics in BR are discussed. Reasons for the absence of similar signatures in the pump-probe spectral modulations at longer probing wavelengths are considered as well.

Absorption↗
PubMed2005

Channel specific rate constants relevant to the thermal decomposition of disilane.

Rate constants for the thermal dissociation of Si2H6 are predicted with a novel transition state model. The saddle points for dissociation on the Si2H6 potential energy surface are lower in energy than the corresponding separated products, as confirmed by high level ab initio quantum mechanical calculations. Thus, the dissociations of Si2H6 to produce SiH2 + SiH4 (R1) and H3SiSiH + H2 (R2) both proceed through tight inner transition states followed by loose outer transition states. The present "dual" transition state model couples variational phase space theory treatments of the outer transition states with ab initio based fixed harmonic vibrator treatments of the inner transition states to obtain effective numbers of states for the two transition states acting in series. It is found that, at least near room temperature, such a dual transition state model is generally required for the proper description of each of the dissociations. Only at quite high temperatures, i.e., above 2000 K for (R1) and 600 K for (R2), does a single fixed inner transition state provide an adequate description. Similarly, only at quite low temperatures (below 100 and 10 K for (R1) and (R2), respectively) does a single outer transition state provide an adequate description. Pressure dependent rate constants are obtained from solutions to the multichannel master equation. These calculations confirm that dissociation channel (R2) is negligible under conditions relevant to the thermal chemical vapor deposition (CVD) processes. Rate constants for the chemical activation reactions, SiH2 + SiH4 --> Si2H6 (R-1) and SiH2 + SiH4 --> H3SiSiH + H2 (R3), are also evaluated within the dual transition state model. It is found that reaction R3 is the dominant channel for low pressures and high temperatures, i.e., below 100 Torr for temperatures above 1100 K.

Journal Article↗
PubMed2002

Microbe-plant interactions: principles and mechanisms.

The present status of research on the molecular basis of microbe-plant interactions is discussed. Principles and mechanisms which play a role in the interactions of microbial pathogens, biofertilizers, phytostimulators, rhizoremediators and biocontrol agents with the plants are treated. Special emphasis is given to colonization, phase variation, two-component systems, quorum sensing, complex regulation of the syntheses of extracellular enzymes and secondary metabolites, Type 4 pili and Type III and Type IV secretion systems.

Bacteria↗
PubMed2001

Cloning and expression of Helicobacter pylori GDP-l-fucose synthesizing enzymes (GMD and GMER) in Saccharomyces cerevisiae.

Helicobacter pylori is a Gram-negative gastric pathogen causing diseases from mild gastric infections to gastric cancer. The difference in clinical outcome has been suggested to be due to strain differences. H. pylori undergoes phase variation by changing its lipopolysaccharide structure according to the environmental conditions. The O-antigen of H. pylori contains fucosylated glycans, similar to Lewis structures found in human gastric epithelium. These Lewis glycans of H. pylori have been suggested to play a role in pathogenesis in the adhesion of the bacterium to gastric epithelium. In the synthesis of fucosylated structures, GDP-l-fucose is needed as a fucose donor. Here, we cloned the two key enzymes of GDP-l-fucose synthesis, H. pylori gmd coding for GDP-d-mannose dehydratase (GMD), and gmer coding for GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase/4-reductase (GMER) and expressed them in an enzymatically active form in Saccharomyces cerevisiae. The end product of these enzymes, GDP-l-fucose was used as a fucose donor in a fucosyltransferase assay converting sialyl-N-acetyllactosamine to sialyl Lewis X.

Base Sequence↗
PubMed1998

Leucine alters the interaction of the leucine-responsive regulatory protein (Lrp) with the fim switch to stimulate site-specific recombination in Escherichia coli.

The leucine-responsive regulatory protein (Lrp) is a global regulator that controls the expression of numerous operons in Escherichia coli. Lrp can act as a repressor or as an activator of transcription with its effects being potentiated, repressed or unaffected by the presence of exogenous leucine. The phase variation of type 1 fimbria in E. coli provides a unique system in which to investigate the effects of leucine on Lrp, as it is the only known example in which Lrp is a positive regulator and leucine potentiates this effect. Previous studies determined that Lrp binds with high affinity to two sites within the fim switch (fim sites 1 and 2), and binding to these sites stimulates recombination. Here, it is shown that, even though leucine stimulates the fim switch in vivo, it nevertheless causes a slight decrease in Lrp binding to the fim switch in vitro. These contradictory results are explicable by the finding that Lrp binding to a third region adjacent to fim sites 1 and 2 inhibits recombination. According to this model, leucine stimulates recombination by selectively disrupting Lrp binding to this newly characterized region, while having little or no effect on Lrp binding to fim sites 1 and 2.

Bacterial Proteins↗
PubMed2001

Control of FimY translation and type 1 fimbrial production by the arginine tRNA encoded by fimU in Salmonella enterica serovar Typhimurium.

Expression of type 1 fimbriae in Salmonella enterica serovar Typhimurium undergoes phase variation or alteration between a fimbriate and a non-fimbriate phenotype. This variation is known to be dependent upon environmental conditions in vitro and is thought to be a complex process involving regulation by a number of proteins. The regulatory genes located within the fim cluster include fimZ, fimY and fimW. A fourth gene of the cluster, fimU, encodes a tRNA molecule specific for rare arginine codons. We have shown previously that fimU affects the expression of S. typhimurium type 1 fimbriae, and that fimU is functionally related to the Escherichia coli gene argU. A high frequency of rare arginine codons was found within the three fim regulatory genes, and five of these codons were clustered within fimY alone. To investigate the affects of fimU on FimY production, a FimY fusion with the E. coli maltose-binding protein was constructed and expressed in an E. coli argU background. Western blots of extracts from the argU mutant and parental strain indicated that production of FimY was significantly reduced in the absence of a functional tRNAArg(UCU). FimY production in this mutant could be restored to high levels when fimU was introduced on a plasmid, and also when three rare arginine codons, located within the first 14 positions within fimY, were exchanged for major arginine codons. A Tn10 insertion from a Salmonella enteritidis fimU mutant was transduced into S. typhimurium, and this strain was analysed for the expression of type 1 fimbriae. The resulting S. typhimurium fimU mutant was found to be non-fimbriate under all conditions tested and could be complemented by the introduction of fimU alone on a plasmid. In addition, this mutant could be complemented by transformation with fimY altered in the first three rare arginine codons. Reverse transcriptase-polymerase chain reaction confirmed that the fimY transcript was present at similar levels in the fimU mutant and parental strain. These results indicated that the observed inhibition of protein expression was not occurring at the transcriptional level. Analysis of expression of the malEfimY fusion in the S. typhimurium fimU mutant and parental strain confirmed the data observed in E. coli. In contrast, a FimW fusion was found to be produced at similar levels in both the fimU mutant and the parental strain. Together, these data indicate that the absence of a functional fimU results in the inhibition of efficient FimY translation, and thus type 1 fimbrial production in S. typhimurium.

Bacterial Proteins↗
PubMed2001

A phase-variable capsule is involved in virulence of Campylobacter jejuni 81-176.

Campylobacter jejuni strain 81-176 (HS36, 23) synthesizes two distinct glycan structures, as visualized by immunoblotting of proteinase K-digested whole-cell preparations. A site-specific insertional mutant in the kpsM gene results in loss of expression of a high-molecular-weight (HMW) glycan (apparent Mr 26 kDa to > 85 kDa) and increased resolution of a second ladder-like glycan (apparent Mr 26-50 kDa). The kpsM mutant of 81-176 is no longer typeable in either HS23 or HS36 antisera, indicating that the HMW glycan structure is the serodeterminant of HS23 and HS36. Both the kpsM-dependent HMW glycan and the kpsM-independent ladder-like structure appear to be capsular in nature, as both are attached to phospholipid rather than lipid A. Additionally, the 81-176 kpsM gene can complement a deletion in Escherichia coli kpsM, allowing the expression of an alpha2,8 polysialic acid capsule in E. coli. Loss of the HMW glycan in 81-176 kpsM also increases the surface hydrophobicity and serum sensitivity of the bacterium. The kpsM mutant is also significantly reduced in invasion of INT407 cells and reduced in virulence in a ferret diarrhoeal disease model. The expression of the kpsM-dependent capsule undergoes phase variation at a high frequency.

ATP-Binding Cassette Transporters↗
PubMed2002

Identification of a gene (lpt-3) required for the addition of phosphoethanolamine to the lipopolysaccharide inner core of Neisseria meningitidis and its role in mediating susceptibility to bactericidal killing and opsonophagocytosis.

We have identified a gene, lpt-3, that is required for the addition of phosphoethanolamine to the 3-position (PEtn-3) on the beta-chain heptose (HepII) of the inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm). The presence of this PEtn-3 substituent is characteristic of the LPS of a majority ( approximately 70%) of hypervirulent Nm strains, irrespective of capsular serogroup, and is required for the binding of a previously described monoclonal antibody (mAb B5) to a surface-accessible epitope. All strains of Nm that have PEtn-3 possess the lpt-3 gene. In some lpt-3-containing strains, the 3-position on HepII is preferentially substituted by glucose instead of PEtn, the result of lgtG phase variation mediated by slippage of a homopolymeric tract of cytidines. Inactivation of lpt-3 resulted in loss of PEtn-3, lack of reactivity with mAb B5 and conferred relative resistance to bactericidal killing and opsonophagocytosis by mAb B5 in vitro. Thus, the identification of lpt-3 has facilitated rigorous genetic, structural and immunobiological definition of an immunodominant epitope that is a candidate immunogen for inclusion in an LPS-based vaccine to protect against invasive meningococcal disease.

Antibodies, Monoclonal↗
PubMed2002

Genetic and functional analysis of the phosphorylcholine moiety of commensal Neisseria lipopolysaccharide.

Phosphorylcholine (ChoP) is a common surface feature of many mucosal organisms, including Neisseria spp., in which it is present exclusively on pili of pathogenic Neisseria and on the lipopolysaccharide (LPS) of commensal Neisseria (Cn). Its presence in Cn has been confirmed by nuclear magnetic resonance. It appears that choline is the main source for the production of ChoP by Cn. We have sequenced a locus, containing four genes (licA-D) with 47-73% identity to the lic1 locus of Haemophilus influenzae (Hi) and 21-40% identity to lic genes in Streptococcus pneumoniae, involved in the production and incorporation of ChoP. The arrangement of the Cn genes and the presence of CAAT repeats, responsible for phase variation of ChoP expression, resemble Hi and differ from S. pneumoniae. Cn DNA flanking the lic locus contains genes ilvE and NMA2149 with >85% identity to the pathogenic Neisseria genes. However, there are no lic genes in the corresponding location or elsewhere in pathogenic Neisseria. This suggests either the loss of the locus from pathogenic Neisseria or a horizontal transfer of genes to Cn, perhaps from H. influenzae spp. As in Hi, ChoP enhances adherence to and invasion of human epithelial cells via the receptor for platelet-activating factor. However, ChoP expression also increases susceptibility to serum killing mediated by complement and C-reactive protein. Taken together, these observations support the hypothesis that the ability of many organisms to switch off ChoP expression rapidly represents an important adaptation to different environments encountered during the colonization/infection process and that the ChoP moiety apparently synthesized by distinct means in pathogenic and commensal Neisseria represents an advantage in the colonization properties of these bacteria.

Amino Acid Sequence↗
PubMed2002

Genetics of the phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2).

The phage growth limitation (Pgl) system, encoded by Streptomyces coelicolor A3(2), confers protection against the temperate bacteriophage phiC31 and its homoimmune relatives. The Pgl phenotype is characterized by the ability of Pgl+ hosts to support a phage burst on initial infection but subsequent cycles are severely attenuated. Previously, two adjacent genes pglY and pglZ were shown to be required for Pgl. It had been shown by Southern blotting that Streptomyces lividans, a close relative of S. coelicolor and naturally Pgl-, does not contain homologues of pglYZ and that introduction of pglYZ into S. lividans is not sufficient to confer a Pgl+ phenotype. Moreover, the mechanism of the Pgl+<--> Pgl- phase variation associated with this phenotype is also not understood. Here we describe two novel genes, pglW and pglX, that were shown to be part of this system by complementation of Pgl- mutants and by insertional mutagenesis. pglW encodes a 169 kDa protein that includes putative motifs for both serine/threonine protein kinase activity and DNA binding. pglX encodes a 136 kDa protein with putative adenine-specific DNA methyltransferase activity. pglW and pglX have overlapping stop-start codons suggesting transcriptional and translational coupling. S1 mapping of transcripts initiating up-stream of pglW indicated that, like pglYZ, pglWX is expressed in uninfected cultures. A homologue of pglX with 76% amino acid identity was identified in S. coelicolor, and insertional mutagenesis indicated that this gene was not required for the Pgl+ phenotype. Southern blots indicated that S. lividans does not contain homologues of pglW or pglX. A plasmid encoding pglWXYZ was able to confer the Pgl+ phenotype to S. lividans implying that these four genes constitute the whole system.

Bacteriophages↗
PubMed2003

Distant cis-active sequences and sialic acid control the expression of fimB in Escherichia coli K-12.

The phase variation of type 1 fimbriation in Escherichia coli is controlled by the inversion of a 314 bp element of DNA, determined by FimB (switching in both directions) or FimE (switching from the ON-to-OFF orientation predominantly), and influenced by auxiliary factors IHF, Lrp and H-NS. The fimB gene is separated from the divergently transcribed yjhATS operon by a large (1.4 kbp) intergenic region of unknown function. Here, we show that fimB expression is regulated by multiple cis-active sequences that lie far upstream (>600 bp) of the transcription start sites for the recombinase gene. Two regions characterized further (regions 1 and 2) show sequence identity, and each coincides with a methylation-protected Dam (5'-GATC) site. Regions 1 and 2 apparently control fimB expression by an antirepression mechanism that involves additional sequences proximal to yjhA. Region 1 encompasses a 27 bp DNA sequence conserved upstream of genes known (nanAT ) or suspected (yjhBC) to be involved in sialic acid metabolism, and we show that FimB expression and recombination are suppressed by N-acetylneuraminic acid. We propose that E. coli recognizes the amino sugars as a harbinger of potential host defence activation, and suppresses the expression of type 1 fimbriae in response.

DNA-Binding Proteins↗