Modulation of host immune responses stimulated by Salmonella vaccine carrier strains by using different promoters to drive the expression of the recombinant antigen.

We evaluated whether immune responses stimulated by Salmonella vaccine carriers can be modulated by using different promoters to drive antigen expression. Mice were orally immunized with strains transfected with plasmids carrying beta-galactosidase (beta-gal) under the control of either a constitutive or an in vivo-activated promoter. While alpha-gal-reactive IgG1, IgG2a, IgG2b and IgG3 were detected in sera of mice immunized with Salmonella expressing constitutively beta-gal, higher titers dominated by IgG2a and IgG2b were detected in sera when the in vivo-activated promoter was used.

Vectors to achieve selective expression of vaccine antigens within eukaryotic cells using Salmonella spp. as carrier strains.

A set of expression vectors was constructed which allows the expression of recombinant antigens under the control of Salmonella typhi promoters that are selectively activated after infection of eukaryotic cells. The pUC18Not derivatives contain a promoter downstream of the early transcriptional terminator from phage T7 and followed by a multiple cloning site, whereas the pBluescript II S/K derivatives contain the ribosomal RNA T(1) transcriptional terminator and also the strong translation signals of the Escherichia coli atpE gene. The expression cassettes are flanked by NotI or PacI sites to simplify their subcloning where required.

An Escherichia coli-Enterococcus faecalis shuttle vector as a tool for the construction of a group B Streptococcus heterologous mutant expressing the beta antigen (Bac) of the C protein complex.

Group B streptococci (GBS) represent a very important group of human pathogens. So far little is known about the mechanisms by which these bacteria can cause disease and the bacterial factors involved. One putative virulence factor is the beta antigen of the C protein complex (Bac), which can bind to the Fc region of human IgA.

The EspD protein of enterohemorrhagic Escherichia coli is required for the formation of bacterial surface appendages and is incorporated in the cytoplasmic membranes of target cells.

The formation of EspA-containing surface appendages in pathogenic Escherichia coli strains, both enteropathogenic E. coli (EPEC) and Shiga toxin-producing E. coli strains, is essential for critical events in the infective process, e.

Seasonal dynamics of bacterioplankton community structure in a eutrophic lake as determined by 5S rRNA analysis.

Community structure of bacterioplankton was studied during the major growth season for phytoplankton (April to October) in the epilimnion of a temperate eutrophic lake (Lake Plusssee, northern Germany) by using comparative 5S rRNA analysis. Estimates of the relative abundances of single taxonomic groups were made on the basis of the amounts of single 5S rRNA bands obtained after high-resolution electrophoresis of RNA directly from the bacterioplankton. Full-sequence analysis of single environmental 5S rRNAs enabled the identification of single taxonomic groups of bacteria.

A functional 4-hydroxysalicylate/hydroxyquinol degradative pathway gene cluster is linked to the initial dibenzo-p-dioxin pathway genes in Sphingomonas sp. strain RW1.

The bacterium Sphingomonas sp. strain RW1 is able to use dibenzo-p-dioxin, dibenzofuran, and several hydroxylated derivatives as sole sources of carbon and energy. We have determined and analyzed the nucleic acid sequence of a 9,997-bp HindIII fragment downstream of cistrons dxnA1A2, which encode the dioxygenase component of the initial dioxygenase system of the corresponding catabolic pathways.

Transcriptional regulation of the esp genes of enterohemorrhagic Escherichia coli.

We have determined that the genes encoding the secreted proteins EspA, EspD, and EspB of enterohemorrhagic Escherichia coli (EHEC) are organized in a single operon. The esp operon is controlled by a promoter located 94 bp upstream from the ATG start codon of the espA gene. The promoter is activated in the early logarithmic growth phase, upon bacterial contact with eukaryotic cells and in response to Ca2+, Mn2+, and HEPES.

Degradation of chlorobenzenes at nanomolar concentrations by Burkholderia sp. strain PS14 in liquid cultures and in soil.

The utilization of 1,2,4,5-tetrachloro-, 1,2,4-trichloro-, the three isomeric dichlorobenzenes and fructose as the sole carbon and energy sources at nanomolar concentrations was studied in batch experiments with Burkholderia sp. strain PS14. In liquid culture, all chlorobenzenes were metabolized within 1 h from their initial concentration of 500 nM to below their detection limits of 0.

Bacteria designed for bioremediation.

Although many environmental pollutants are efficiently degraded by microorganisms, others persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.

Initial reactions in the biodegradation of 1-chloro-4-nitrobenzene by a newly isolated bacterium, strain LW1.

Bacterial strain LW1, which belongs to the family Comamonadaceae, utilizes 1-chloro-4-nitrobenzene (1C4NB) as a sole source of carbon, nitrogen, and energy. Suspensions of 1C4NB-grown cells removed 1C4NB from culture fluids, and there was a concomitant release of ammonia and chloride. Under anaerobic conditions LW1 transformed 1C4NB into a product which was identified as 2-amino-5-chlorophenol by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry.

Identification of pneumococcal surface protein A as a lactoferrin-binding protein of Streptococcus pneumoniae.

Lactoferrin (Lf), an iron-sequestering glycoprotein, predominates in mucosal secretions, where the level of free extracellular iron (10(-18) M) is not sufficient for bacterial growth. This represents a mechanism of resistance to bacterial infections by prevention of colonization of the host by pathogens. In this study we were able to show that Streptococcus pneumoniae specifically recognizes and binds the iron carrier protein human Lf (hLf).

Protective immune response against Streptococcus pyogenes in mice after intranasal vaccination with the fibronectin-binding protein SfbI.

Despite the significant impact on human health of Streptococcus pyogenes, an efficacious vaccine has not yet been developed. Here, the potential as a vaccine candidate of a major streptococcal adhesin, the fibronectin-binding protein SfbI, was evaluated. Intranasal immunization of mice with either SfbI alone or coupled to cholera toxin B subunit (CTB) triggered efficient SfbI-specific humoral (mainly IgG) and lung mucosal (14% of total IgA) responses.

Salmonella vaccine carrier strains: effective delivery system to trigger anti-tumor immunity by oral route.

Recombinant Salmonella strains expressing heterologous antigens can be delivered by oral route triggering the elicitation of efficient antigen-specific humoral, T helper and cytotoxic responses. The potential of attenuated Salmonella spp. to trigger anti-tumor immunity was evaluated for the first time by using beta-galactosidase (beta-gal) as a model tumor-associated antigen (TAA).

Streptococcal invasion.

The genus Streptococcus consists of large number of species many of which are pathogenic to humans and animals. Although streptococci have long been considered as extracellular pathogens, they are capable of causing serious invasive infections such as necrotizing fasciitis and meningitis. Streptococcal invasion, therefore, has been a focus of many studies in recent years.

Pathogenicity island 2 mutants of Salmonella typhimurium are efficient carriers for heterologous antigens and enable modulation of immune responses.

The potential use as vaccine delivery system of Salmonella typhimurium strains harboring defined mutations in the sseC (HH104) and sseD (MvP101) genes, which encode putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2, was evaluated and compared with that of the well-characterized aroA mutant strain SL7207 by using beta-galactosidase (beta-Gal) as a model antigen. When orally administered to immune-competent or gamma interferon-deficient (IFN-gamma-/-) BALB/c mice, both mutants were found to be highly attenuated (50% lethal dose, >10(9) bacteria). Both strains were also able to efficiently colonize and persist in Peyer's patches.

Genetic and biochemical analyses of the tec operon suggest a route for evolution of chlorobenzene degradation genes.

The TecA broad-spectrum chlorobenzene dioxygenase of Burkholderia sp. strain PS12 catalyzes the first step in the mineralization of 1,2,4, 5-tetrachlorobenzene. The catabolic genes were localized on a small plasmid that belongs to the IncPbeta incompatibility group.

Fibronectin-binding protein I of Streptococcus pyogenes promotes T cell-independent proliferation of murine B lymphocytes and enhances the expression of MHC class II molecules on antigen-presenting cells.

We have previously shown that fibronectin-binding protein I (SfbI) of Streptococcus pyogenes can act as an adjuvant for mucosal-delivered antigens (Medina, E., Talay, S. R.

Identification of chlorobenzene dioxygenase sequence elements involved in dechlorination of 1,2,4,5-tetrachlorobenzene.

The TecA chlorobenzene dioxygenase and the TodCBA toluene dioxygenase exhibit substantial sequence similarity yet have different substrate specificities. Escherichia coli cells producing recombinant TecA enzyme dioxygenate and simultaneously eliminate a halogen substituent from 1,2,4,5-tetrachlorobenzene but show no activity toward benzene, whereas those producing TodCBA dioxygenate benzene but not tetrachlorobenzene. A hybrid TecA dioxygenase variant containing the large alpha-subunit of the TodCBA dioxygenase exhibited a TodCBA dioxygenase specificity.

A putative lichenysin A synthetase operon in Bacillus licheniformis: initial characterization.

Certain Bacillus licheniformis strains isolated from oil wells have been shown to produce a very effective biosurfactant, lichenysin A, which is structurally similar to another less active lipopeptide, surfactin. Surfactin, like many small peptides in prokaryotes and lower eukaryotes, is synthesized non-ribosomally by multi-enzyme peptide synthetase complex. Analysis of several peptide synthetases of bacterial and fungal origin has revealed a high degree of sequence conservation.

A second two-component regulatory system of Bordetella bronchiseptica required for bacterial resistance to oxidative stress, production of acid phosphatase, and in vivo persistence.

Random minitransposon mutagenesis was used to identify genes involved in the survival of Bordetella bronchiseptica within eukaryotic cells. One of the mutants which exhibited a reduced ability to survive intracellularly harbored a minitransposon insertion in a locus (ris) which displays a high degree of homology to two-component regulatory systems. This system exhibited less than 25% amino acid sequence homology to the only other two-component regulatory system described in Bordetella spp.

Alcanivorax borkumensis gen. nov., sp. nov., a new, hydrocarbon-degrading and surfactant-producing marine bacterium.

During screening for biosurfactant-producing, n-alkane-degrading marine bacteria, six heterotrophic bacterial strains were isolated from enriched mixed cultures, obtained from sea water/sediment samples collected near the isle of Borkum (North Sea), using Mihagol-S (C14,15-n-alkanes) as principal carbon source. These Gram-negative, aerobic, rod-shaped bacteria use a limited number of organic compounds, including aliphatic hydrocarbons, volatile fatty acids, and pyruvate and its methyl ether. During cultivation on n-alkanes as sole source of carbon and energy, all strains produced both extracellular and cell-bound surface-active glucose lipids which reduced the surface tension of water from 72 to 29 mN m-1 (16).

Pas, a novel protein required for protein secretion and attaching and effacing activities of enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological phenotype collectively known as the attaching and effacing lesion. The genes encoding the products responsible for this phenotype are located on a 35-kb pathogenicity island designated the locus of enterocyte effacement, which is also shared by enteropathogenic E. coli.

Biochemical and genetic characterization of a gentisate 1, 2-dioxygenase from Sphingomonas sp. strain RW5.

A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.

Genetic analysis of dioxin dioxygenase of Sphingomonas sp. Strain RW1: catabolic genes dispersed on the genome.

The dioxin dioxygenase of Sphingomonas sp. strain RW1 activates dibenzo-p-dioxin and dibenzofuran for further metabolism by introducing two atoms of oxygen at a pair of vicinal carbon atoms, one of which is involved in one of the bridges between the two aromatic rings, i.e.

Multifunctional g3p-peptide tag for current phage display systems.

We have previously described a monoclonal antibody (mAb), 10C3, directed against the gene-3 protein (g3p) of filamentous phage M13, which was produced to study g3p fusion protein expression in Escherichia coli and its incorporation in the phage capsid [Tesar, M., Beckmann, C., Röttgen, P.