Paul Casaz

The Pseudomonas fluorescens transcription activator AdnA is required for adhesion and motility.

The locations of two mutations that prevent adhesion of Pseudomonas fluorescens Pf0-1 to sand columns and seeds (adn, adhesion) were identified. Both lie in a single gene showing homology to the NtrC/NifA family of transcription activators. The predicted 55 kDa protein encoded by adnA is most closely related to activators involved in expression of flagellar proteins, consistent with the lack of flagella in adnA strains. Constitutive adnA expression restored motility and adhesion to an adnA strain, demonstrating that the observed phenotypes are due to lack of AdnA and not a consequence of other mutations or polar effects of mutations in adnA on other genes.

The adnA transcriptional factor affects persistence and spread of Pseudomonas fluorescens under natural field conditions.

ABSTRACT A soil plot was inoculated with a mixture of Pseudomonas fluorescens Pf0-2, the wild type, and Pf0-5a, a Tn5insertion mutant in adnA, at 7.84 log CFU/g of soil. Over a period of 231 days, culturable populations of both strains were measured at selected times below and away from the point of inoculation. Pf0-5a did not spread as fast and attained significantly lower populations than Pf0-2. At sample depths below the inoculation site, the adnA mutant showed a significant decrease in CFU/g of soil as compared to Pf0-2. Pf0-2 was first detected at the 1.5-cm annular site at 3 days after inoculation, whereas Pf0-5a required 7 days to travel the same distance. At this distance, the wild-type strain could be detected at a 21.5- to 25-cm depth, whereas Pf0-5a could be detected only as deep as 15.5 to 18 cm. At 4.5 cm from the site of inoculation and in soil fractions corresponding to 13 to 18 cm, Pf0-2 was the only strain detected. These results suggest that the transcription factor AdnA provides a fitness advantage in P. fluorescens, allowing it to spread and survive in soil under field conditions.

Analytical characterization of a monoclonal antibody therapeutic reveals a three-light chain species that is efficiently removed using hydrophobic interaction chromatography.

Size exclusion high performance liquid chromatography analysis of a human monoclonal antibody (mAb) showed the presence of a new species that eluted with a retention time between the dimeric and monomeric species of the antibody. Extensive characterization of this species, referred to as “shoulder,” indicated that it was a mAb containing an extra light chain and had a molecular weight of approximately 175 kDa. The extra light chain was found to be non-covalently associated with the Fab portion of the protein. The relative amount of shoulder (typically 1−3% of the total mAb present) varied with the Chinese hamster ovary cell line producing the mAb and was not influenced by the growth conditions. Our three-step mAb purification platform using protein A, anion exchange, and cation exchange process steps was successful at removing dimer and higher and lower molecular weight species, but not the shoulder impurity. It was found that hydrophobic interaction chromatography could be used in place of cation exchange to exploit the subtle differences in hydrophobicity between monomer and shoulder. We developed an antibody polishing process using Butyl Sepharose HP resin that is capable of removing the majority of high and low molecular weight impurities yielding 99% pure mAb monomer, virtually devoid of the shoulder species, with a step recovery of about 80%.