Phase-variable expression of an operon encoding extracellular alkaline protease, a serine protease homolog, and lipase in Pseudomonas brassicacearum

Abstract

The rhizobacterium Pseudomonas brassicacearum forms phenotypic variants which do not show extracellular protease and lipase activity. The operon encoding these enzymes, a serine protease homolog, and a type I secretion machinery was characterized. Transcriptional lacZ gene fusions revealed that the expression of the operon is under the control of phase variation.

FIG. 1

Phenotypic variation of P. brassicacearum strain NFM421. (a) Seven-day-old colony grown on PAF medium (Difco Laboratories, Detroit, Mich.) with a sector corresponding to phase II cells. (b) Colony morphology of the two phases. (c and d) Extracellular protease and lipase activity in phase I but not phase II cells was revealed on 10-fold-diluted tryptic soy broth medium (Difco Laboratories) agar plates containing 1% skim milk (c) or 1% tributyrin and 0.8% gum arabic (d). Bacteria were grown at 30°C.

FIG. 2

Genetic organization and restriction map of the sequenced 12-kb fragment. Restriction sites indicated are BamHI (B), EcoRI (E), HindIII (H), KpnI (K), PstI (P), SacI (Sa), SmaI (Sm), and XhoI (X). Thin arrows indicate the open reading frames. The two putative terminators are indicated by stem-loop structure. Thick arrows represent fragments subcloned in pEMBL19, pPC7, and pPC8 or pUR6500HE (pAG525), in the orientation indicated relative to the lac or tac promoter, or in pMP220, in the orientation indicated relative to the promoterless lacZ gene.

FIG. 3

Confocal microscopy observation of E. coli DH5α without plasmid (a) and with plasmid pAG525 (b) after growth on LB plates supplemented with 0.5 mM isopropyl-β-d-thiogalactoside. Bars: (a) 5.0 μm; (b) 20.0 μm.