Transcriptional and posttranscriptional events control copper-responsive expression of a Rhodobacter capsulatus multicopper oxidase

Abstract

The copper-regulated Rhodobacter capsulatus cutO (multicopper oxidase) gene confers copper tolerance and is carried in the tricistronic orf635-cutO-cutR operon. Transcription of cutO strictly depends on the promoter upstream of orf635, as demonstrated by lacZ reporter fusions to nested promoter fragments. Remarkably, orf635 expression was not affected by copper availability, whereas cutO and cutR were expressed only in the presence of copper. Differential regulation was abolished by site-directed mutations within the orf635-cutO intergenic region, suggesting that this region encodes a copper-responsive mRNA element. Bioinformatic predictions and RNA structure probing experiments revealed an intergenic stem-loop structure as the candidate mRNA element. This is the first posttranscriptional copper response mechanism reported in bacteria.

Fig 1

Identification of copper tolerance genes by mutational analysis. (A) Genetic and physical map of the R. capsulatus cutO (multicopper oxidase gene; RCAP_rcc02110) region. The localizations and sizes of genes and open reading frames (orf) are given by arrows. Only restriction sites relevant for cloning of kanamycin resistance (Km) cassettes are shown. Km cassettes are not drawn to scale. Arrowheads indicate the direction of Km transcription. Designations of R. capsulatus mutants and copper susceptibilities (Cu^T, copper tolerant; Cu^S, copper sensitive) are shown next to the Km cassettes. (B) Copper tolerance of wild-type and mutant strains. Liquid cultures were plated on RCV minimal medium plates, before filter discs soaked with 2.5 mM CuSO₄ were placed on top. Inhibition zones around the discs were documented after 2 days of growth.

Fig 2

Copper-dependent cutO expression requires two cis-regulatory elements. (A) Transcriptional lacZ reporter fusions. Plasmids carrying lacZ fusions to orf635, cutO, and cutR based on narrow-host-range plasmid pSUP202 were used to construct chromosomal R. capsulatus reporter strains. Episomal lacZ fusions are based on broad-host-range plasmid pBBR1MCS-5. The lacZ gene is not drawn to scale. (B) Expression studies. R. capsulatus reporter strains were grown in RCV minimal medium with 2 μM CuSO₄ (+Cu) or without copper addition (−Cu) prior to determination of β-galactosidase activities as described earlier (53). β-Galactosidase activities, given in Miller units (30), and standard deviations were calculated from five independent experiments for each strain.

Fig 3

Expression of in-frame lacZ fusions to orf635, cutO, and cutR. (A) Translational (in-frame) lacZ reporter fusions. Plasmids carrying lacZ fusions to orf635, cutO, and cutR based on narrow-host-range plasmid pSUP202 were used to construct chromosomal R. capsulatus reporter strains. The lacZ gene is not drawn to scale. (B) Expression studies. R. capsulatus reporter strains were grown in RCV minimal medium with 2 μM CuSO₄ (+Cu) or without copper addition (−Cu) prior to determination of β-galactosidase activities as described earlier (53). β-Galactosidase activities, given in Miller units (30), and standard deviations were calculated from five independent experiments for each strain.

Fig 4

Site-directed mutations abolishing copper control of cutO expression. (A) Structure prediction of orf635-cutO intergenic mRNA. Intergenic mRNA was predicted to form two stems (S-I and S-II) and two loops (L-I and L-II) using the program mfold (57). (B) Site-directed mutagenesis of the orf635-cutO intergenic region. To examine the role of the orf635-cutO intergenic region in copper regulation, appropriate site-directed mutations were introduced into plasmid pJL5 (cutO-lacZ). (C) Transcriptional analysis. R. capsulatus strains carrying pJL5 derivatives were grown in RCV minimal medium with 2 μM CuSO₄ (+Cu) or without copper addition (−Cu). β-Galactosidase activities, given in Miller units (30), and standard deviations were calculated from four independent experiments for each strain.

Fig 5

Identification of paired and unpaired regions in orf635-cutO intergenic mRNA. (A) In vitro transcription of the orf635-cutO intergenic region. Plasmid pAM39 served as a template for in vitro transcription of the orf635-cutO intergenic region starting at a T7 promoter (P_T7) recognized by T7 RNA polymerase. (B) RNA structure probing experiments. In vitro transcripts were incubated with nuclease S1, RNase V, or RNase T1 prior to separation of cleavage products by polyacrylamide gel electrophoresis. RNase/nuclease digestions were carried out without copper addition or in the presence of 2 μM CuCl as indicated. All samples contained 2 μM dithiothreitol (DTT). Assays without RNase/nuclease addition served as controls (co). Lane L, alkaline ladder.

Fig 6

Model of copper-dependent cutO expression. Copper-dependent cutO expression requires two cis-regulatory elements, the constitutive orf635 promoter (P_const), and the orf635-cutO intergenic region. RT-PCR products corresponding to orf635-cutO and cutO-cutR borders were previously identified in the presence but not in the absence of copper (53). For further details, see text.