Microbiome yarns : microbiomology of curly and straight hair

Non peer reviewe

Broad-host range expression vectors containing manipulated meta-cleavage pathway regulatory elements of the TOL plasmid

AbstractThe construction of pERD20 and pERD21, two broad-host range expression vectors, is described. The vectors contain the Pm promoter of the meta-cleavage pathway operon of the TOL plasmid pWWO; this promoter is present within a polylinker which provides a number of downstream cloning sites close to the transcription initiation site. Transcription from the Pm promoter in these vectors is controlled not by the natural positive regulator of Pm, the Xy1S protein, but by an Xy1S mutant analogue, Xy1S2tr6, which ??hibits an altered effector specificity and can mediate a 3–8-fold higher level of transcription than can Xy1S in a wide range of temperatures. Controlled expression of cloned genes can be achieved in a broad spectrum of Gram negative bacteria grown at a wide range of temperatures

Construction of Minitransposons for Constitutive and Inducible Expression of Pertussis Toxin in Bvg-Negative Bordetella-Bronchiseptica

Appropriately detoxified pertussis toxin (PT) of Bordetella pertussis is considered to be an essential component of new-generation whooping cough vaccines, but the development of a procedure to obtain high levels of purified toxin has been and continues to be a major difficulty. To produce a system enabling the biological separation of PT from other virulence determinants of B. pertussis and the attainment of high yields of the toxin, minitransposons containing the PT operon were constructed and stably integrated into the chromosome of Bordetella virulence regulatory gene (bvg)-negative Bordetella bronchiseptica ATCC 10580. Since the minitransposons introduced into Bordetella spp. lack the cognate transposase function, they are unable to undergo further transposition events or mediate gene deletions and rearrangements that lead to strain instability. The TnPtacPT minitransposon contains the PT operon under the control of the tac promoter and directs IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible expression of PT in B. bronchiseptica ATCC 10580. The level of IPTG-induced PT expression was, however, lower than that found for the wild-type B. pertussis Tohama I strain. The TnfusPT minitransposon contains a promoterless PT operon which is only expressed after insertion of the transposon downstream of an appropriately oriented indigenous promoter. After "promoter probing" of B. bronchiseptica with the transposon, clones were screened for PT production by immunoblotting with specific monoclonal antibodies. One clone, designated B. bronchiseptica 10580:: TnfusPT1, expresses significantly higher levels of PT than does B. pertussis Tohama I. The recombinant toxin produced was biologically active in the Chinese hamster ovary cell-clustering assay. High-level expression of PT from a B. bronchiseptica host promoter should provide better yields of the toxin from bacteria not producing other bvg-regulated pathogenesis factors that may play a role in the undesired side effects of current pertussis vaccine preparations

Specific Lung Mucosal and Systemic Immune-Responses After Oral Immunization of Mice with Salmonella-Typhimurium-Aroa, Salmonella-Typhi Ty21a, and Invasive Escherichia-Coli Expressing Recombinant Pertussis Toxin S1 Subunit

Pertussis toxin (PT) is considered an essential protective component for incorporation into new generation vaccines against Bordetella pertussis, the causative agent of whooping cough. Traditionally, antipertussis vaccination has employed an intramuscular route. An alternative to this approach is to stimulate mucosal and systemic immune responses by oral immunization with live vaccine carrier strains of Salmonella spp. or Escherichia coli. Recombinant S1 subunit of pertussis toxin was expressed in the attenuated aroA mutant of Salmonella typhimurium, SL3261, in the human typhoid vaccine strain Salmonella typhi Ty21a, and in E. coli CAG629 containing the Shigella flexneri plasmid pWR110, which encodes bacterial invasiveness of epithelial cells. Expression of recombinant PT S1 subunit (rPT-S1) did not affect in vitro invasiveness of the tested strains, which retained the ability to adhere to and invade the embryonic human intestinal cell line HI-407. Following oral immunization of mice with the live vaccine strains expressing rPT-S1, immunoglobulin G (IgG), IgA, and IgM responses were monitored. IgG specific to PT was detected in serum samples of mice, while IgG and IgA specific to PT were detected in lung washes after oral immunization with living Salmonella spp. or E. coli (pWR110) expressing rPT-S1. Utilization of live oral vaccines expressing B. pertussis antigens, which stimulate both a systemic and lung mucosal response, may provide an attractive alternative to purified component vaccines against whooping cough

Biological activities specified by antibiotic resistance plasmids

Bacteria can display resistance to a wide spectrum of noxious agents and environmental conditions, and these properties are often mediated by genes located on extrachromosomal DNA elements called plasmids. Replication, vertical and horizontal transmission and evolution of these elements are discussed, and examples of the genes responsible for the resistance phenotypes are given. Selective forces that drive the evolution of new combinations of bacterial properties of particular importance in clinical situations are analyse

Biological activities specified by antibiotic resistance plasmids

Bacteria can display resistance to a wide spectrum of noxious agents and environmental conditions, and these properties are often mediated by genes located on extrachromosomal DNA elements called plasmids. Replication, vertical and horizontal transmission and evolution of these elements are discussed, and examples of the genes responsible for the resistance phenotypes are given. Selective forces that drive the evolution of new combinations of bacterial properties of particular importance in clinical situations are analyse