Post-transcriptional regulation by copper with a new upstream Open Reading Frame

Copper is essential to most living beings but also toxic. Bacteria have thus developed homeostatic mechanisms to tightly control its intracellular concentration. The 3-gene operon bp2923-bfrG-bp2921 is down-regulated by copper and notably encodes a TonB-dependent transporter in Bordetella pertussis. We show that the protein encoded by bp2923, which is a member of the DUF2946 family, represents a new type of upstream Open Reading Frame (uORF) involved in post-transcriptional regulation of the downstream genes. In the absence of copper, the entire operon is transcribed and translated. Perception of copper by the nascent bp2923-coded protein via its conserved CXXC motif triggers Rho-dependent transcription termination between the first and second genes by relieving translation arrest on a conserved C-terminal RAPP motif. Homologues of bp2923 are widespread in bacterial genomes, where they head operons predicted to participate in copper homeostasis. This work has unveiled an original mode of genetic regulation by a transition metal and identified a regulatory function for a member of an uncharacterized family of bacterial proteins that we have named CruR, for copper-responsive upstream regulator.


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Bacteria have evolved complex mechanisms to respond to changes of their environment, and 37 notably to strictly regulate the availability of necessary but harmful transition metals. Copper 38 is such a metal both essential and harmful to living beings (Solioz, 2018). Its properties as a 39 redox cycling metal have been put to use in electron transfer chains as a cofactor of heme- systems against copper include export of Cu 1+ from the cytoplasm or its passivation by 53 sequestration, the detoxification of Cu 1+ into Cu 2+ in the periplasm and its extrusion to the 54 extracellular medium (Solioz, 2018). 55 Bacteria also need to acquire copper from their environment (Stewart et al., 2019), and the Pfam family, predicted to be exported. Those proteins are characterized by two conserved 89 sequence motifs, CXXC (where X represent non-conserved residues) and RAPP (Fig. 1b).   BfrG was detected by immunoblot analyses of cellular extracts of B. pertussis grown in the 107 absence but not in the presence of copper, whereas Fe and Zn had no effect on its expression, 108 indicating that the regulation of the operon is copper-specific (Expanded Fig. 1). Those results 109 were confirmed by qRT-PCR experiments showing a dramatic reduction of bfrG mRNA 110 abundance in the presence of Cu (Expanded Fig. 1). In contrast, Fe had no effect and Zn 111 moderately affected bfrG mRNA levels, suggesting limited cross-regulation. 112 We performed qRT-PCR experiments on each of the three genes of the operon and 113 normalized the results against a housekeeping gene. We also generated chromosomal reporter 114 fusions by inserting lacZ in frame with the first codons of each gene to assess the effect of 115 copper on their expression. bfrG and bp2921 were expressed at moderate levels in copper-116 restricted medium (Fig. 1c,d). Addition of copper to the medium abolished their transcription 117 and translation, consistent with RNAseq data (Rivera-Millot et al., 2021). Interestingly, bp2923 118 was hardly translated despite being the first gene of the operon, and it did not appear to be 119 regulated by copper (Fig. 1c,d). qRT-PCR analyses on bfrG at various times after Cu addition 120 showed a fast decrease in transcript abundance ( Supplementary Fig. S3).

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Importance of bp2923 for post-transcriptional regulation of bfrG and bp2921 123 We investigated a potential regulatory role of the 5' region of the operon by introducing a large, 124 in-frame deletion of bp2923 to avoid polar effects, yielding the recombinant BP∆2923 strain 125 (Fig. 2a). We then tested the effect of copper on the expression of bfrG using both the bfrG-126 lacZ fusion and qRT-PCR. Copper regulation was abolished in BP∆2923, and bfrG was 127 expressed at lower levels than in the parental strain, BPSM (Fig. 2b). The introduction of 128 bp2923 at another chromosomal locus in BP∆2923 did not complement the deletion (Fig. 2b). 129 Thus, its first position in the operon is required to control both the expression levels and the 130 regulation by copper of the downstream gene. To determine if the Bp2923 protein or the bp2923 131 mRNA was involved in this regulation, we tested the bfrG-lacZ fusion in BP2923-OCU, in 132 which the protein sequence is intact but the mRNA sequence is modified (Fig. 2a). bfrG was 133 expressed, although at a slightly lower level than in BPSM, and its expression responded poorly 134 to Cu, suggesting a role of the mRNA sequence in regulation (Fig. 2b).  lacZ fusion and abolished its regulation by copper, unlike the FS117-123 mutations (Fig. 2a,b).

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Thus, the amino acid sequence encoded in this region appears to be unimportant, whereas 149 mutations that generate secondary structures in the mRNA affect Cu regulation (Fig. 2b). The

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A Rho-dependent termination mechanism accounts for the effects of the mutations in the 177 unstructured region (Fig. 2b). Thus, the frameshift did not affect regulation by copper because 178 the rut site was preserved. In contrast, mutations that disrupt the rut site abolished regulation.

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The observation that they also increased the expression level of bfrG in the absence of copper 180 suggests a background level of termination in the wt operon without added copper.

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Importance of Bp2923 protein for bfrG expression and regulation 183 We next investigated a potential role of the bp2923-encoded protein for regulation. We Role of conserved features of the Bp2923 protein for regulation 193 The Bp2923 protein belongs to the DUF2946 family, which is characterized by two highly 194 conserved sequence motifs. The RAPP motif, which is encoded by frequent B. pertussis codons, 195 is located at positions 139-142 three residues before the C terminus (Fig. 1b). It is reminiscent 216 The CXXC and the RAPP motifs are separated from each other by 45 intervening residues.

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Genome mining identified more than 2000 DUF2946 protein sequences in databases, mostly in 218 Proteobacteria (Suppl . Table S1), and their analysis showed that the spacing between the two 219 motifs is conserved to within one or two residues in the family (Fig. 4c). We therefore probed 220 its importance for expression and regulation of bfrG by shortening or lengthening the spacing 221 by one, two or five residues in the Bp2923 protein (mutants bp2923-1, bp2923-2, bp2923+1, 222 bp2923+2 and bp2923+5, respectively; Fig. 4a). We designed those mutations to minimize 223 their effects on the mRNA structure ( Supplementary Fig. S4). Deletion or addition of one or 224 two residues moderately affected the bfrG expression levels, but not its regulation by copper.

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In contrast, addition of five residues abolished the effect of copper, showing that the two motifs 226 must be adequately spaced for proper regulation, within a limited degree of variation ( Fig. 4b). 227 Bp2923 is therefore a new regulatory protein that we propose to name CruR for Copper- In silico analyses revealed that CruR homologues are widespread among b, g and a 265 Proteobacteria (Suppplementary Table S1). A few were also found in Planctomycetes, 266 Firmicutes and Deinococcus. In most cases, potential operonic structures were identified with 267 these genes in first position (Expanded Fig. 2). bp2923 homologues frequently precede TBDT-