Immunogenicity of recombinant protective antigen and efficacy against aerosol challenge with anthrax.

Immunization with a recombinant form of the protective antigen (rPA) from Bacillus anthracis has been carried out with rhesus macaques. Rhesus macaques immunized with 25 mug or more of B. subtilis-expressed rPA bound to alhydrogel had a significantly increased immunoglobulin G (IgG) response to rPA compared with macaques receiving the existing licensed vaccine from the United Kingdom (anthrax vaccine precipitated [AVP]), although the isotype profile was unchanged, with bias towards the IgG1 and IgG2 subclasses. Immune macaque sera from all immunized groups contained toxin-neutralizing antibody and recognized all the domains of PA. While the recognition of the N terminus of PA (domains 1 to 3) was predominant in macaques immunized with the existing vaccines (AVP and the U.S. vaccine anthrax vaccine adsorbed), macaques immunized with rPA recognized the N- and C-terminal domains of PA. Antiserum derived from immunized macaques protected macrophages in vitro against the cytotoxic effects of lethal toxin. Passive transfer of IgG purified from immune macaque serum into naive A/J mice conferred protection against challenge with B. anthracis in a dose-related manner. The protection conferred by passive transfer of 500 mug macaque IgG correlated significantly (P = 0.003; r = 0.4) with the titers of neutralizing antibody in donor macaques. Subsequently, a separate group of rhesus macaques immunized with 50 mug of Escherichia coli-derived rPA adsorbed to alhydrogel was fully protected against a target dose of 200 50% lethal doses of aerosolized B. anthracis. These data provide some preliminary evidence for the existence of immune correlates of protection against anthrax infection in rhesus macaques immunized with rPA.

The nucleotide sequence of a transposable haloalkanoic acid dehalogenase regulatory gene (dehRI) from Pseudomonas putida strain PP3 and its relationship with sigma 54-dependent activators.

The mobile genetic element, DEH found in Pseudomonas putida PP3 carries a 2-haloalkanoic acid dehalogenase structural gene, dehI, and its associated regulatory gene, dehRI. The nucleotide sequence of dehRI was determined. The gene had an open reading frame putatively encoding for a 64 kDa protein containing 571 amino acid residues. The protein was similar to previously published sequences of several other sigma 54-dependent activator proteins. Amino acid sequence analysis showed that the deduced DehRI protein clustered with the NifA nitrogenase regulatory activator family, and was most closely related, with 47.7% similarity, to a 'NifA-like' deduced partial sequence from a plasmid-encoded ORF in Pseudomonas sp. strain NS671, associated with L-amino acid production. The domain structure of DehRI was analysed by alignment with other NifA-like and NtrC-like sequences and showed a highly conserved central region of approximately 230 amino acids, and a potential DNA-binding domain. No homology was detected between the deduced DehRI and other sigma 54-dependent activator sequences at the N-terminus, a result which was consistent with that region being the domain which recognised inducer.

Localization and functional analysis of structural and regulatory dehalogenase genes carried on DEH from Pseudomonas putida PP3.

Pseudomonas putida PP3 expressed two dehalogenases, DehI and DehII. The DehI gene (dehI) was located on a mobile DNA element (DEH) which inserted at high frequencies into target plasmids from its chromosomal location. From a recombinant TOL plasmid (pWW0) containing a 6.0-kb DEH element inserted into the plasmid's 5.6-kb EcoRI-G restriction endonuclease fragment, an 11.6-kb EcoRI fragment was cloned. Subcloning analysis and insertion mutagenesis produced a structural map of the DEH element and located the dehalogenase functions. The gene dehI was transcribed from a regulated promoter on DEH which was expressed in P. putida and Escherichia coli. The direction of transcription of dehI was determined, and it was also found to be under positive control, activated by an adjacent regulatory gene (dehRI). Expression of dehI in clones containing the intact DEH supported good growth on 2-monochloropropionate (2MCPA). Subclones lacking dehRI expressed dehI at levels which allowed only slow growth on 2MCPA, even when dehI expression was initiated from vector promoters. Expression of dehI in P. putida containing the intact DEH element required rpoN, suggesting that it was omega 54 dependent. The intact DEH element transferred to P. putida on a suicide plasmid donor pAWT34 (pBR325 replicon), and dehI was stably inherited, without vector DNA sequences, in transformants selected on 2MCPA. This indicated that the cloned DEH element contained functions associated with recombination.

Environmentally directed mutations in the dehalogenase system of Pseudomonas putida strain PP3.

Favourable mutations involving the two dehalogenases (DehI and DehII) of Pseudomonas putida PP3 and derivative strains containing the cloned gene for DehI (dehI) occurred in response to specific environmental conditions, namely: starvation conditions; the presence of dehalogenase substrates (halogenated alkanoic acids--HAAs) which were toxic to P. putida; and/or the presence of a potential growth substrate. Fluctuation tests showed that these mutations were environmentally directed by the presence of HAAs. The mutations were associated with complex DNA rearrangements involving the movement of dehI located on a transposon DEH. Some mutations resulted in switching off the expression of either one or both of the dehalogenases, events which were effective in protecting P. putida from toxic compounds in its growth environment. Other mutations partially restored P. putida's dehalogenating capability under conditions where toxic substrates were absent. Restoration of the capability to untilize HAAs was favoured when normal growth substrates were present in the environment.