Characterization of the cryptic plasmids of the Pseudomonas alcaligenes type strain

The species type strain of Pseudomonas alcaligenes contains three small cryptic plasmids (designated pECB1, 2, and 3) of 7740, 4480, and 2700 bp, respectively. Partial restriction enzyme maps have been constructed for pECB1 and 2 which on this basis do not appear to be related. pECB3 proved refractile to cutting with commonly used restriction enzymes, though it was completely rendered by those enzymes which recognize 4-bp sequences containing only G + C. This suggested that pECB3 is especially rich in these bases. Hybridization studies using labeled pECB2 as probe revealed homology with pECB3 and with regions of the bacterial chromosome, but not with pECB1. A 1214-bp region of pECB2 showed great sequence similarity to the basic replicon of pPS10, a 10-kb Pseudomonas-specific plasmid isolated from Pseudomonas syringae pv. savastonoi. The putative replicon (including the gene for a replication protein) was subcloned and both DNA strands were sequenced. Introduction of the putative replicon into the Escherichia coli plasmid pUC19 created a recombinant vector able to replicate in both E. coli and Pseudomonas spp. Minicell analyses did not reveal any peptides which could be attributed to the remaining region of pECB2 or to pECB1--a finding supported by sequencing studies. Attempts at plasmid curing were unsuccessful. A phenotypic comparison with a non-plasmid-harboring strain of P. alcaligenes, based on nutritional versatility and antibiotic susceptibility, revealed a single difference of note: the type strain alone was able to utilize benzoate for growth. Transformation of the non-harboring strain with pECB1-3, followed by selection on a minimal medium containing benzoate, gave no colonies. The advantages gained by possession of pECB1-3, if any, are at present unknown.