Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites

Abstract

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA'-'lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Figure 1

Posttranscriptional, gacA-dependent regulation of the Pseudomonas hcnA and aprA genes in P. fluorescens. All constructs were made in the vector pME6010. *, β-galactosidase expression (Miller units) of the lacZ fusions shown was tested in the P. fluorescens strains CHA0 and CHA89 when cells reached an OD₆₀₀ of 2.0–2.5. Activities are mean values of triplicate experiments ± standard deviation.

Figure 2

Cell density-dependent expression of a translational hcnA′-′lacZ fusion from the hcn promoter on pME3219 (circles) or from the tac promoter on pME6530 (squares) in CHA0 (open symbols) and CHA89 (closed symbols). Bacterial growth reached a plateau at OD₆₀₀ 4–5.

Figure 3

Alignment of the regions containing the RBS of the hcnA genes of P. fluorescens CHA0 and P. aeruginosa PAO1, the aprA gene of P. fluorescens CHA0, and the E. coli lacZ gene. In each case, translation is initiated at the ATG codon shown at the 3′ end.

Figure 4

Posttranscriptional, GacA-dependent regulation of a translational hcnA′-′lacZ fusion (derived from the P. fluorescens hcn operon): influence of mutations in the 5′ leader sequence. *, Nucleotides of importance are designated as follows: doubly underlined, RBS; underlined, artificial restriction sites; shaded boxes, E. coli lacZ sequence; open box, ATG initiation codon of hcnA; encircled, base substitutions; ●, deletion. ^†, β-galactosidase expression (Miller units) was determined in strains CHA0 and CHA89, when cells reached an OD₆₀₀ of 2.0–2.5. Activities are mean values from triplicate experiments ± standard deviation.

Figure 5

(A) Cloning of the rsmA gene of P. fluorescens CHA0. The P. aeruginosa rsmA gene was isolated by PCR and was used as a probe to identify the homolog in P. fluorescens by Southern hybridization. A 1.25-kilobase genomic PstI fragment containing rsmA of P. fluorescens was cloned. In pME6073, rsmA on a 0.55-kilobase PstI/PvuII fragment is expressed from the lac promoter of the vector pME6001; in pME6083, rsmA is deleted. The serV gene, which encodes a serine tRNA, appears to belong to a separate transcription unit. (B) Alignment of RsmA and CsrA amino acid sequences. A, CsrA of E. coli (GenBank accession no. L07596); B, RsmA of E. carotovora subsp. carotovora 71 (L40173); C, RsmA of P. aeruginosa (AF061757); D, RsmA of P. fluorescens. Amino acid residues that are conserved in at least three of the four proteins are shown in black boxes.