Variability of outer membrane protein P1 and its evaluation as a vaccine candidate against experimental otitis media due to nontypeable Haemophilus influenzae: an unambiguous, multifaceted approach.

Candidate vaccine antigens for preventing otitis media caused by nontypeable Haemophilus influenzae (NTHI) should possess one or more conserved epitopes. We sought to evaluate the candidacy of P1, a surface-expressed outer membrane protein knowing that this antigen is subject to diversifying selection. Therefore, we selected NTHI strains from among >500 phylogenically variant isolates representative of the diversity found in natural populations of H. influenzae. Twenty-three variants of P1 (

An epidemic of burkholderia cepacia transmitted between patients with and without cystic fibrosis.

Burkholderia cepacia is an important pathogen in cystic fibrosis (CF) and an infrequent cause of nosocomial infection in non-CF patients. This report describes a large hospital outbreak that appeared to involve both patient groups, a previously unrecognized phenomenon. Ribotype restriction fragment length polymorphism (RFLP) profiles and pulsed-field gel electrophoresis-resolved macrochromosomal RFLPs were analyzed, a ribotype-based phylogenic tree was constructed, and case-control and cohort studies were performed. A single dominant clone was found in both CF and non-CF groups. Phylogenic analysis suggests that it has evolved independently and that such highly transmissible strains can emerge rapidly and randomly. Acquisition risk in the CF patients was linked to hospitalization (odds ratio=5.47, P=.0158, confidence interval=1. 28-26.86) and was associated with significantly increased mortality rates. Infection control policies must now consider this threat of transmission between non-CF and CF patients.

[Integration via Tn10 and expression of Bti cryIVA gene in the chromosome DNA of flourescent Pseudomonas bacterial strains].

cryIVA gene of the Bacillus thuringiensis subsp. israelensis was subcloned into the Tn10 of a suicide transposon plasmid vector and a transposon plasmid pLF97A carrying cryIVA was constructed. By electroporation and transposition, cryIVA gene integrated into chromosome DNA of F.P.DE2. Thus the genetic engineered bacterial strain, F.P.DE202, was constructed. Southern blotting revealed that this integration was happened in different loci of the chromosome. Western blotting demonstrated that cryIVA was expressed in the engineered strain, and that the expressed product possesed the pesticidal ability against the third-instar larva of Bradysia odoriphaga.

Characterization and cytokine stimulating activities of heteroglycans from Tremella fuciformis.

Three heteroglycans, T1a, T1b, and T1c, have been isolated from the body of Tremella fuciformis Berk. They are composed of mannose (Man), xylose (Xyl), glucose (Glc), fucose (Fuc), and glucuronic acid (GlcA). According to methylation analysis and partial acidic hydrolysis the main chains of T1a, T1b, and T1c consisted of (1-->3)-linked Man, which was branched at the 2, 4, or 6 positions. The branching points were linked with nonreducing terminal GIcA-residues or (1-->6)-linked glucan-chains. Molecular weights of the three heteroglycans are 53,000, 18,000, and 12,000 D respectively, but they undergo self-aggregation in water. T1a-T1c induce human monocytes to produce interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) in vitro. Acidic hydrolysate fractions of T1a (T1a-1, 2, 3, 4, 5) with molecular weight from 53,000 to 1,000 D, also induce human monocytes to produce IL-6 as efficient as T1a.

The emergence of a highly transmissible lineage of cbl+ Pseudomonas (Burkholderia) cepacia causing CF centre epidemics in North America and Britain.

The rapid increase in Pseudomonas (Burkholderia) cepacia infection in cystic fibrosis (CF) patients suggests epidemic transmission, but the degree of transmissibility remains controversial as conflicting conclusions have been drawn from studies at different CF centres. This report provides the first DNA sequence-based documentation of a divergent evolutionary lineage of P. cepacia associated with CF centre epidemics in North America (Toronto) and Europe (Edinburgh). The involved epidemic clone encoded and expressed novel cable (Cbl) pili that bind to CF mucin. The sequence of the cblA pilin subunit gene carried by the epidemic isolates proved to be invariant. Although it remains to be determined how many distinct, highly transmissible lineages exist, our results provide both a DNA sequence and chromosomal fingerprint that can be used to screen for one such particularly infectious, transatlantic clone.

Structurally variant classes of pilus appendage fibers coexpressed from Burkholderia (Pseudomonas) cepacia.

One or more of five morphologically distinct classes of appendage pili were determined to be peritrichously expressed by Burkholderia (formerly Pseudomonas) cepacia isolated from disparate sources. B. cepacia-encoded cblA pilin gene hybridization-based analysis revealed that one associated class, cable (Cbl) adhesin type IIB. cepacia pili, correlates with epidemically transmitted strains from a single cystic fibrosis (CF) center. When only phenotypic assays were available, correlations between the source and the pilus type were nonetheless observed: filamentous (Fil) type IIIB. cepacia pili correlated with CF-associated nonepidemic isolates, spine (Spn) type IVB. cepacia pili correlated with clinical (non-CF) isolates, and spike (Spk) type VB. cepacia pili correlated with environmental isolates. Further, Cbl, Fil, or Spk pili typically appear as an internal framework for constitutively coexpressed, peritrichously arranged dense mats of fine, curly mesh (Msh) type IB. cepacia pili. Constitutive coexpression of dense mats of Msh type IB. cepacia pili in association with a labyrinth of either Cbl, Fil, or Spk pili suggests possible cooperative pilus interactions mediating adhesion-based colonization in the differing environments from which the strains were isolated. Despite such correlations, phylogenetic analyses indicate that with the exception of the epidemically transmitted clusters of isolates, the remaining B. cepacia strains from the other three sources exhibited an equal degree of genetic relatedness independent of origin. As previously found for Escherichia coli, this discrepancy could be accounted for by selection-driven, in vivo horizontal transfer events between distantly related members of the species B. cepacia, leading to the genetic acquisition of environmentally appropriate adhesion-based colonization pilus operons.

Transmissibility of Pseudomonas cepacia infection in clinic patients and lung-transplant recipients with cystic fibrosis.

BACKGROUND: In patients with cystic fibrosis, infection with Pseudomonas cepacia is associated with poor outcomes. However, the extent of person-to-person transmission and the source of P. cepacia infection after lung transplantation are not well defined. Using DNA-based typing systems, we sought to determine the genetic relatedness of P. cepacia infection at one cystic fibrosis center. METHODS: We analyzed 65 P. cepacia isolates gathered over a period of eight years at a single cystic fibrosis center from 17 clinic patients and from 5 patients who underwent double-lung transplantation. The isolates were analyzed by ribotyping and chromosomal fingerprinting based on pulsed-field gel electrophoresis. RESULTS: Analyses of serial isolates revealed that each clinic patient and transplant recipient harbored a different P. cepacia clone that was persistent. In the transplant recipients, the preoperative and postoperative isolates were identical. In the two patients with disseminated infection after lung transplantation, isolates from multiple sites were identical and indicated clonal expansion of the previous respiratory P. cepacia strain. Pulsed-field gel electrophoresis proved both more discriminative and more practical than ribotyping as a means of defining the genetic relatedness of the P. cepacia isolates. CONCLUSIONS: Our serial analyses in patients with cystic fibrosis at one center found distinct strains of P. cepacia persistently infecting each patient and no evidence of person-to-person transmission of this organism. P. cepacia infection after lung transplantation was due to the persistence of the strain present before transplantation.

Recognition sequence specificity of signal peptidase I and the role of signal peptide in secretion of protein in Bacillus subtilis.

By recombinant DNA technology, the N-terminal of the beta-protein encoding region of plasmid pUB110 is fused with the structure gene of alpha-amylase from Bacillus licheniformis. This gene fusion is called beta Amy. It is able to transcribe and translate in phase. Protein fusion can be secreted into the medium mediated by beta-signal peptide. The efficiency of secretion is about 10% of the synthesized pre-alpha-amylase. By comparing the secretion capacities and analysing the restriction sites on beta-Amy genes and the molecular weights of the mature alpha-amylase secreted by B. subtilis harbouring different plasmids, it is indicated in vivo that the recognition and cleavage sequence for signal peptidase I of B. subtilis is Ala-Ala-Ala Ala. The results also indicate that the secretion of the alpha-amylase in B. subtilis is in accordance with the post-translational transportation mode.

[Molecular cloning of alpha-amylase gene from Bacillus megaterium and its expression in Bacillus subtilis].

Using Bacteriophage lambda and plasmid pAT153 and pNQ122 as vectors, alpha-Amylase gene from B. megaterium has been cloned into both hosts of E. coli and B. subtilis. Expression level of the gene is 250 times higher than B. megaterium when it resides in B. subtilis. The enzyme produced by B. subtilis harboring the hybrid plasmid can digest amylase into maltose and maltotriose at first, then turn them to maltose and glucose, as incubation time extended. It also can digest maltotriose to maltose and glucose. As a control, the extracts from the broth of recipient strain have no detectable amylase activities. Therefore the enzyme coded by this gene is defined as saccharifying type alpha-amylase. Its molecular weight is about 58,000 daltons.

[Study on Bacillus pumilus as a recipient strain for genetic engineering of Bacillus].

Bacillus pumilus 289 can be transformed easily by plasmid pUB110 through protoplast transformation with the frequency of 10(-5)--10(-3), similar to B. subtilis AS 1.1176, a derivative strain of B. subtilis 168. The regeneration frequency of its protoplast is only slightly lower than B. subtilis AS1.1176 (0.3-12.0% compare to 1.53--24.16%). Plasmid pUB110 can be maintained in both bacterial strains stably. The frequency of loss of the plasmid in both strains is lower than 3% after 45 generations in LB medium. But it is quite different that the hybrid plasmid (pUB110 with 3.9 kb foreign DNA fragment) can be maintained much more stably in B. pumilus 289 than in B. subtilis AS1.1176. The frequency of loss of the plasmid is lower than 5% in B. pumilus 289 and 24% in B. subtilis AS1.1176 after 25 generations when they grown in SH medium. The expression level of foreign gene in B. pumilus 289 is also much higher than that in B. subtilis AS1.1176. Therefore B. pumilus 289 is valuable to be exploited as recipient strain for genetic engineering of Bacillus in the future.

Rho-dependent transcription termination of a bacterial operon is antagonized by an extrachromosomal gene product.

The psu gene product of "phasmid" (phage-plasmid) P4 acts as a transcription antitermination factor in trans and in cis, respectively, within the morphogenic operons of its P2 phage helper during lytic viral development and on P4 itself during the establishment stage of its alternative mode of propagation as a plasmid. Here we show that psu also antagonizes activity of the Escherichia coli transcription termination factor rho at the terminator of the trp operon. Such a finding provides to our knowledge the first direct evidence for antitermination activity at a known rho-dependent site by the psu gene product. It also reveals an example of an extrachromosomal gene product that acts on specific sites of three different genomes to regulate expression of unlinked families of genes.

Mutants of Streptococcus pneumoniae that contain a temperature-sensitive autolysin.

Two mutants of Streptococcus pneumoniae deficient in autolysin activity produced a protein that showed immunological identity with the N-acetyl-muramyl-L-alanyl-amidase present in the wild-type strain, when tested with antiserum obtained against this enzyme. The protein was produced by the mutant cultures grown either at 37 degrees C or at 30 degrees C, although only the cell extracts obtained at 30 degrees C showed significant cell wall hydrolysing activity. In contrast to the lysis resistance of these bacteria grown at 37 degrees C, mutant cultures grown at 30 degrees C exhibited significant degrees of autolysis when treated with detergent or cell wall inhibitors. Extracts of the mutant cultures contained a cell wall hydrolysing activity that was rapidly inactivated during incubation at 37 degrees C.