[Search for destruction factors of bacterial biofilms: comparison of phage properties in a group of Pseudomonas putida bacteriophages and specificity of their halo-formation products].

Comparison of Pseudomonas putida group of phages attributed to five species (af, phi15, phi27, phi2F, and pf16) with their common property of halo-formation (formation of lightening zones) around phage plaques was conducted. The halo around phage plaques appears as a result of reduction or disappearance of bacterial polysaccharide capsules. The concentration of viable bacteria remains unchanged within the halo. A comparison of specificities of halo-formation products from various phages was conducted by a simple method. These products were shown to be highly specific and inactive on other species of pseudomonads. Phage-resistant P. putida mutants scored with respect to various phages, which lost phage adsorption ability, were tolerant to the effect of halo-formation products in most cases. Apparently, the capsular polysaccharides, which serve as a substrate for depolymerases and are the primary phage receptors, may be often lost. Results of partial sequencing of the af phage genome revealed an open reading frame that encodes the enzyme transglycosylase similar rather to transglycosylases of oligotrophic bacteria belonging to different species than to lysozymes of other phages. Possibly, it is a polyfunctional enzyme combining functions of lysozyme and an enzyme that executes the penetration of phage particle across extracellular slime and capsule.

Homotypic interactions among bacteriophage phiKMV early proteins.

Little is known about the bacteriophage proteins expressed immediately after infection of the host cell. Most of these early proteins are probably involved in bacteriophage-host interactions redirecting the bacterial metabolism to phage production. Interaction analysis of the first 16 phiKMV gene products (gp) identified homotypic interactions of gp7, gp9 and gp15. Two related yeast two-hybrid procedures, a matrix and a minilibrary approach, were applied to detect protein-protein interactions. A two-step selection procedure enabled drastic reduction of the background. Interactions were confirmed by drop tests. Multimerization of gp15 is consistent with its putative function as a DNA helicase involved in DNA replication. Homotypic interaction of gp7 and gp9 suggests they function as dimers or multimers. The absence of heterotypic interactions among early phiKMV proteins hints at their functional independence from other early phage proteins and their involvement in phage-host interactions that are important for creating optimal conditions for phage propagation. Besides, these results demonstrate the compatibility of phiKMV early gene products with the yeast two-hybrid system. Therefore, they are promising candidates to screen for interactions with host proteins.

Stability analysis of the bacteriophage phiKMV lysin gp36C and its putative role during infection.

The kinetic, thermodynamic and structural stability of gp36C, the virion-associated peptidoglycan hydrolase domain of bacteriophage phiKMV, is analyzed. Recombinant gp36C is highly thermoresistant (k = 0.595 h(-1) at 95 degrees C), but not thermostable (T(m) = 50.2 degrees C, DeltaH(cal) = 6.86 x 10(4) cal mol(-1)). However, aggregation influences kinetic stability in an unusual manner since aggregation is more pronounced at 55 degrees C than at higher temperatures. Furthermore, gp36C reversibly unfolds in a two-state endothermic transition, and circular dichroism analysis shows that gp36C almost completely refolds after a 3-h heat treatment at 85 degrees C. These properties are in agreement with gp36C being part of the extensible tail which is ejected in an unfolded state during phage infection.

Functional display of family 11 endoxylanases on the surface of phage M13.

Two family 11 endoxylanases (EC 3.2.1.8) were functionally displayed on the surface of bacteriophage M13. The genes encoding endo-1,4-xylanase I from Aspergillus niger (ExlA) and endo-1,4-xylanase A from Bacillus subtilis (XynA) were fused to the gene encoding the minor coat protein g3p in phagemid vector pHOS31. Phage rescue resulted in functional monovalent display of the enzymes as was demonstrated by three independent tests. Firstly, purified recombinant phage particles showed a clear hydrolytic activity in an activity assay based on insoluble, chromagenic arabinoxylan substrate. Secondly, specific binding of endoxylanase displaying phages to immobilized endoxylanase inhibitors was demonstrated by interaction ELISA. Finally, two rounds of selection and amplification in a biopanning procedure against immobilized endoxylanase inhibitor were performed. Phages displaying endoxylanases were strongly enriched from background phages displaying unrelated proteins. These results open perspectives to use phage display for analysing protein-protein interactions at the interface between endoxylanases and their inhibitors. In addition, this technology should enable engineering of endoxylanases into novel variants with altered binding properties towards endoxylanase inhibitors.

Characterization of the bacteriophage PhiKMV DNA ligase.

Gene 17 product (gp17) of the Pseudomonas aeruginosa-infecting bacteriophage phiKMV shows in silico similarity to T7 DNA ligase. In a semi-quantitative activity assay, it is shown that gp17 is a functional, ATP-dependent DNA ligase, in spite of some structural differences related to DNA-binding properties). Enzymatic activity of His6-based purified expression product was optimised (4 degrees C at 24h for sticky end double-stranded DNA fragments) and estimated at 0.5 Weiss U/microg.

Identification and characterization of a highly thermostable bacteriophage lysozyme.

Pseudomonas aeruginosa bacteriophage phiKMV is a T7-like lytic phage. Liquid chromatography-mass spectrometry of the structural proteins revealed gene product 36 (gp36) as part of the phiKMV phage particle. The presence of a lysozyme domain in the C terminal of this protein (gp36C) was verified by turbidimetric assays on chloroform-treated P. aeruginosa PAO1 and Escherichia coli WK6 cells. The molecular mass (20,884 Da) and pI (6.4) of recombinant gp36C were determined, as were the optimal enzymatic conditions (pH 6.0 in 16.7 mM phosphate buffer) and activity (4800 U/mg). Recombinant gp36C is a highly thermostable lysozyme, retaining 26% of its activity after 2 h at 100 degrees C and 21% after autoclaving. This thermostability could prove an interesting characteristic for food conservation technology.

Influence of maternal antibodies on Chlamydophila psittaci-specific immune responses in turkeys elicited by naked DNA.

Plasmid DNA (pcDNA1::MOMP D) expressing the major outer membrane protein (MOMP) of an avian Chlamydophila psittaci serovar D strain was tested for its ability to induce protective immunity against C. psittaci challenge in the presence of maternal antibodies. A combined parenteral (intramuscular injection) and mucosal route (DNA drops administered to the nares) of DNA inoculation was used. Following pcDNA1::MOMP vaccination, both T helper and B cell memory were primed. However, high maternal antibodies titres affected the induction of vaccine-specific antibody responses as assessed by MOMP-specific antibody levels in enzyme-linked immunosorbent assay (ELISA). Cell-mediated immunity was unaltered as demonstrated by the significantly heightened proliferative responses of peripheral blood lymphocytes (PBL) following vaccination. DNA vaccination could significantly reduce clinical symptoms, pharyngeal and cloacal excretion as well as Chlamydophila replication, even in the presence of maternal antibodies.

PHIRE, a deterministic approach to reveal regulatory elements in bacteriophage genomes.

MOTIVATION: In silico genome analysis of bacteriophage genomes focuses mainly on gene discovery and functional assignment. The search for regulatory elements contained within these genome sequences is often based on prior knowledge of other genomic elements or on learning algorithms of experimentally determined data, potentially leading to a biased prediction output. The PHage In silico Regulatory Elements (PHIRE) program is a standalone program in Visual Basic. It performs an algorithmic string-based search on bacteriophage genome sequences to uncover and extract subsequence alignments hinting at regulatory elements contained within these genomes, in a deterministic manner without any prior experimental or predictive knowledge. RESULTS: The PHIRE program was tested on known phage genomes with experimentally verified regulatory elements. PHIRE was able to extract phage regulatory sequences correctly for bacteriophages T7, T3, YeO3-12 and lambda, based solely on the genome sequence. For 11 bacteriophages, new predictions of conserved phage-specific putative regulatory elements were made, further corroborating this approach. AVAILABILITY: http://www.agr.kuleuven.ac.be/logt/PHIRE.htm. Freely available for academic use. Commercial users should contact the corresponding author.

Molecular identification and chromosomal localization of genes encoding Triticum aestivum xylanase inhibitor I-like proteins in cereals.

TAXI ( Triticum aestivum xylanase inhibitor) proteins are present in wheat flour and are known to inhibit glycosyl hydrolase family 11 endoxylanases, enzymes which are commonly applied in grain processing. Here, we describe the PCR-based molecular identification of genes encoding endoxylanase inhibitors HVXI and SCXI, the TAXI-like proteins from barley ( Hordeum vulgare) and rye ( Secale cereale) respectively. The HVXI coding sequence encodes a mature protein of 384 amino acids preceded by a 19 amino acid long signal sequence. SCXI-II/III has an open reading frame encoding a signal peptide of 21 amino acids and a mature protein of 375 amino acids. As for TAXI-I, no introns were detected in the untranslated regions and coding sequences identified. These newly identified sequences allowed us to perform a multiple sequence alignment with TAXI-I and similar proteins. Rice TAXI-type proteins clustered together with the cereal endoxylanase inhibitors. Dicotyledonous proteins with sequence similarity to TAXI-I, including the tomato xyloglucan-specific endoglucanase inhibiting protein, formed a different clade. The TAXI-type proteins may hence be part of a superfamily of proteins all involved in plant responses to biotic or abiotic stress and for which a function as glycosyl hydrolase inhibitors can be suggested. The chromosomal localization of the TAXI-I gene identified on wheat chromosome 3B, of the SCXI-II/III gene identified on rye chromosome 6R, and the presence of a cluster of TAXI-like genes on rice chromosome 1, allowed us to assign the location of TAXI-like genes to the wheat-rye translocation area 3BL/6RL characterized by RFLP markers XGlb33 and Xpsr454 and isozyme Est-5. In rice, RFLP marker C1310S corresponds to a TAXI-like protein encoding sequence.

Gene content and density in banana ( Musa acuminata) as revealed by genomic sequencing of BAC clones.

The complete sequence of Musa acuminata bacterial artificial chromosome (BAC) clones is presented and, consequently, the first analysis of the banana genome organization. One clone (MuH9) is 82,723 bp long with an overall G+C content of 38.2%. Twelve putative protein-coding sequences were identified, representing a gene density of one per 6.9 kb, which is slightly less than that previously reported for Arabidopsis but similar to rice. One coding sequence was identified as a partial M. acuminata malate synthase, while the remaining sequences showed a similarity to predicted or hypothetical proteins identified in genome sequence data. A second BAC clone (MuG9) is 73,268 bp long with an overall G+C content of 38.5%. Only seven putative coding regions were discovered, representing a gene density of only one gene per 10.5 kb, which is strikingly lower than that of the first BAC. One coding sequence showed significant homology to the soybean ribonucleotide reductase (large subunit). A transition point between coding regions and repeated sequences was found at approximately 45 kb, separating the coding upstream BAC end from its downstream end that mainly contained transposon-like sequences and regions similar to known repetitive sequences of M. acuminata. This gene organization resembles Gramineae genome sequences, where genes are clustered in gene-rich regions separated by gene-poor DNA containing abundant transposons.

Molecular characterization and phylogenetic utility of the rDNA external transcribed spacer region in Stylosanthes (Fabaceae).

Supplementary Material The nucleotide sequence of the ribosomal external transcribed spacer (ETS) region of Stylosanthes mexicana was determined and used to evaluate its potential for examination of intra- and inter-specific relationships in Stylosanthes, as compared to the use of the internal transcribed spacer (ITS) region. The entire ETS region comprises 1,145 bp and is composed of a region of non-repetitive sequences consisting of three subregions with organizational and nucleotide-sequence conservation, and a triplicated segment of about 100 bp. A primer designed in the second conserved subregion allowed us to amplify and sequence directly the 3' part (423-431 bp) of the ETS from 22 genotypes of 12 representative Stylosanthes species that were previously used in phylogenetic analysis of the genus. The study revealed that the right-hand part of Stylosanthes ETS contains approximately twice as much variable and informative characters than the ITS. Moreover, pairwise sequence-divergence values are twice as high, on average, when compared to the ITS. The ITS and ETS datasets are consistent in phylogenetic reconstruction of Stylosanthes, and combined parsimony analysis resulted in a strict consensus tree that is better resolved and generally better supported than trees obtained from separate analysis of the spacer regions.

Missing links in the divergence of Chlamydophila abortus from Chlamydophila psittaci.

Pathological and serological evidence and DNA-DNA reassociation data indicate that Chlamydophila psittaci and Chlamydophila abortus are separate species. C. psittaci causes avian systemic disease and C. abortus causes abortion. Both previously belonged to Chlamydia psittaci are associated with zoonotic and enzootic outbreaks. Genetic studies suggest that they are closely related and because of the recent availability of diverse C. psittaci strains and comparative data for several genes, it was possible to explore this relationship. The parrot C. psittaci strain 84/2334 was found to have DNA sequences that were identical to an extrachromosomal plasmid in duck C. psittaci strain N352, to rnpB in strain R54 from a brown skua and to the rrn intergenic spacer in parakeet strain Prk/Daruma (from Germany, Antarctica and Japan, respectively). Analysis of ompA and the rrn spacer revealed progressive diversification of the strains, with 84/2334 resembling what might have been a recent ancestor of C. abortus. Another C. psittaci strain (VS225) showed evidence of having undergone convergent evolution towards the C. abortus-like genotype, whereas strain R54 diverged independently. For the first time, these studies link C. abortus in an evolutionary context to the C. psittaci lineage. It has been concluded that C. abortus diverged from C. psittaci, and so strain R54 was designated a C. psittaci strain. It is recommended that characterization of C. psittaci and C. abortus strains should utilize more than a single method and more than a single gene.

Regulation of inhibin alpha- and beta(A)-subunit messenger ribonucleic acid levels by gonadotropins and IGF-I in cultured chicken granulosa cells.

A quantitative competitive reverse transcription polymerase chain reaction assay (QC RT-PCR) for quantifying the absolute levels of the expression of inhibin alpha- and beta(A)-subunits in chicken granulosa cells showed that these subunits are expressed in different amounts depending on follicular maturation. The present study determined the regulation of the expression of these subunits. The individual effect of different doses of IGF-I, LH or FSH (1-100 ng/ml) or the combination of IGF-I with either LH or FSH at different concentrations, on the expression of inhibin alpha- and beta(A)-subunit was determined on cultured granulosa cells of F(1) and the combined F(4)+F(5) follicle. Cells were cultured for 48 h in 6-well plates with or without added hormones. Culture medium was discarded, cells were washed and total RNA was extracted from the cells. Five hundred nanograms of total RNA was reverse transcribed using specific primers and coamplified with an internal standard, as described previously, to determine expression level in the cells. IGF-I, LH, and FSH enhanced the inhibin alpha-subunit mRNA levels in a dose dependent manner in both F(1) and the combined F(4)+F(5) whereas inhibin beta(A)-subunit was not affected. The effects of FSH, LH were more expressed in F(1) follicles compared to F(4)+F(5) on the alpha-subunit. The addition of IGF-I and either LH or FSH during the culture period significantly increased the stimulatory effects of both LH and FSH on the expression of inhibin alpha-subunit in F(1) follicles but had no significant effect on the inhibin beta(A)-subunit. The results suggest that the changing expression levels of inhibin alpha-subunit during follicular development are the result of the regulatory effect of the interaction between IGF-I and the gonadotropins and that the regulation of this subunit may be the main factor for the regulation of the protein inhibin levels. Other factors may be also implicated in the changing expression levels of the beta(A)-subunit.

Chlamydophila psittaci DNA vaccination in turkeys in the presence of maternal antibodies.

Plasmid DNA (pcDNA1::MOMP D) expressing the major outer membrane protein of an avian Chlamydophila psittaci serovar D strain was tested for its ability to induce protective immunity against Chlamydophila psittaci challenge in the presence of maternal antibodies. A combined parenteral (intramuscular injection) and mucosal route (DNA drops administered to the nares) of DNA inoculation was used. Only placebo-vaccinated turkeys showed a primary response following challenge, although DNA vaccination didn't generate high antibody titres. Following pcDNA::MOMP vaccination, both T-helper and B-cell memory were primed. However, high maternal antibodies titres affected the induction of vaccine-specific antibody responses as assessed by MOMP-specific antibody levels in ELISA. Cell-mediated immunity was unaltered as demonstrated by the significantly heightened proliferative responses of peripheral blood lymphocytes following vaccination. DNA vaccination could significantly reduce clinical symptoms, pharyngeal and cloacal excretion as well as chlamydophila replication, even in the presence of maternal antibodies.

The specific isolation of complete 5S rDNA units from chromosome 1A of hexaploid, tetraploid, and diploid wheat species using PCR with head-to-head oriented primers.

The presence of 5S rDNA units on chromosome 1A of Triticum aestivum was shown by the development of a specific PCR test, using head-to-head oriented primers. This primer set allowed the amplification of complete 5S DNA units and was used to isolate SS-Rrna-A1 sequences from polyploid and diploid wheat species. Multiple-alignment and parsimony analyses of the 132 sequences divided the sequences into four types. The isolates from T. aestivum and the tetraploid species (T. dicoccoides, T. dicoccum, T durum, T. araraticum, and T timopheevi) were all of one type, which was shown to be closely related to the type mainly characteristic for T. urartu. The other two types were isolated exclusively from the diploid species T. monococcum, T aegilopoides, T. thaoudar, and T. sinskajae and the hexaploid species T. zhukovski. Triticum monococcum was the only species for which representatives of each of the four sequence types were found to be present. Further, we discuss the possible multicluster arrangement of the 5S-Rrna-A1 array.

Genetic immunization for Chlamydia psittaci.

Plasmid DNA expressing the major outer membrane protein (MOMP) of an avian Chlamydia psittaci serovar A strain, has been tested for its ability to raise an immune response and induce protection against challenge with the same serovar. A combined parenteral (intramuscular injection) and mucosal route (DNA drops administered to the nares) of DNA inoculation was compared to gene gun-based immunization. The gene gun delivery of pcDNA1/MOMP as well as the intramuscular-intranasal DNA delivery primed both T-helper and B-cell memory although rMOMP-expressing cells did not induce high antibody responses. Evidence for the priming of the memory was provided by the fact that the pcDNA1/MOMP inoculations raised antibodies belonging to the IgG and not IgM isotype. However, in response to challenge only 5 out of 15 vaccinated turkeys showed four fold increases in serum IgG after challenge. By contrast, evidence for the priming of T-cell memory in response to challenge was found in all vaccinated turkeys as shown by the significantly heightened proliferative responses of peripheral blood lymphocytes following vaccination. Both immunization methods produced similar serological and lymphocyte proliferative responses. Notwithstanding the immunization method, a significant level of protection was observed in all pcDNA1/MOMP immunized turkeys. The efficacy of MOMP-based DNA vaccination as a means of preventing severe clinical signs, lesions and chlamydia excretion in a turkey model of Chlamydia psittaci infection was demonstrated.