Jane C. Schneider

Protein secretion systems of Pseudomonas aeruginosa and P. fluorescens

Gram-negative bacteria have evolved numerous systems for the export of proteins across their dual-membrane envelopes. Three of these systems (types I, III and IV) secrete proteins across both membranes in a single energy-coupled step. Four systems (Sec, Tat, MscL and Holins) secrete only across the inner membrane, and four systems [the main terminal branch (MTB), fimbrial usher porin (FUP), autotransporter (AT) and two-partner secretion families (TPS)] secrete only across the outer membrane. We have examined the genome sequences of Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens Pf0-1 for these systems. All systems except type IV were found in P. aeruginosa, and all except types III and IV were found in P. fluorescens. The numbers of each such system were variable depending on the system and species examined. Biochemical and physiological functions were assigned to these systems when possible, and the structural constituents were analyzed. Available information regarding the mechanisms of transport and energy coupling as well as physiological functions is summarized. This report serves to identify and characterize protein secretion systems in two divergent pseudomonads, one an opportunistic human pathogen, the other a plant symbiont.

Auxotrophic markers pyrF and proC can replace antibiotic markers on protein production plasmids in high-cell-density Pseudomonas fluorescens fermentation

The use of antibiotic‐resistance genes as selectable markers in transgenic organisms is coming under increased scrutiny, for fear that they may spread to human pathogens, thereby reducing the effectiveness of antibiotic therapy. A current Pseudomonas fluorescens protein expression system uses a tetracycline resistance gene (tetR/tetA) to maintain an expression plasmid under control of a repressible promoter and a kanamycin resistance gene (kanR) to maintain a plasmid carrying a repressor gene. We investigated using auxotrophic markers to replace these two antibiotic resistance genes: pyrF (encoding orotidine‐5′‐phosphate decarboxylase) in place of tetR/tetA and proC (encoding pyrroline‐5‐carboxylate reductase) in place of kanR, complementing their respective precise chromosomal deletions created by allele exchange using a suicide vector carrying pyrF as a counterselectable marker. The resulting strains, devoid of antibiotic‐resistance genes, were shown to achieve high productivity of nitrilase and thermostable α‐amylase equal to that of the former antibiotic‐resistant production host. The production plasmids were stable. The pyrF (uracil‐dependent) background of the production host strain also allows us to sequentially alter the genome to incorporate other desired genomic changes, deletions, or insertions using 5′‐fluoroorotic acid counterselection, restoring the selectable marker after each step.