Microorganisms can accelerate the rate of Mn (super 2+) oxidation by up to five orders of magnitude compared to abiotic Mn (super 2+) oxidation. Mn (super 2+) oxidation in Pseudomonas putida strain GB-1 involves an enzyme incorporated in the outer membrane that oxidizes Mn (super 2+) extracellularly. This Mn (super 2+) -oxidizing factor has to be synthesized inside the cell and transported across the outer membrane. We used a method known as transposon mutagenesis to generate two mutants that are incapable of Mn (super 2+) oxidation because they are unable to transport the Mn (super 2+) -oxidizing factor across the outer membrane. However, when cells were lysed, Mn (super 2+) oxidation occurred, verifying that transport and not synthesis of the Mn (super 2+) -oxidizing factor was affected. Transport of the Mn (super 2+) -oxidizing factor was restored when normal sequences obtained from a genomic library of Pseudomonas putida strain GB-1 were introduced into the mutant strains. By sequencing the DNA of the disrupted genes of these two mutants it was determined that the affected genes are very similar to the xpc gene family of the related species, Pseudomonas putida WCS358 and Pseudomonas aeruginosa. This gene family is known to be involved in the two-step protein secretion process in Gram-negative bacteria. In one of the two mutants, the disruption occurs in the gene that encodes a subunit of a complex that spans the membrane. In the other mutant the disruption occurs in the pilD/xcpA gene, which encodes an enzyme (peptidase) that modifies the subunits that are assembled into this membrane-spanning complex. These results indicate the involvement of a two-step protein secretion pathway in the transport of the Mn (super 2+) -oxidizing factor of Pseudomonas putida strain GB-1.