ActP controls copper homeostasis in Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti preventing low pH‐induced copper toxicity

Abstract

Summary Two ‘calcium‐irreparable’ acid‐sensitive mutants were identified after mutagenizing Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti with Tn5. Each mutant contains a single copy of the transposon which, inserted within the actP gene, prevents expression of a P‐type ATPase that belongs to the CPx heavy metal‐transporting subfamily. Here, we show that both actP‐knockout mutants show sensitivity to copper; omission of this heavy metal from low pH‐buffered media restores acid tolerance to these strains. Furthermore, complementation of the mutant phenotype requires only the actP gene. An actP–gusA fusion in R. leguminosarum was transcriptionally regulated by copper in a pH‐dependent manner. Downstream to actP in both organisms is the hmrR gene that encodes a heavy metal‐responsive regulator (HmrR) that belongs to the merR class of regulatory genes. Insertional inactivation of hmrR abolished transcriptional activation of actP by copper ions and increased the basal level of its expression in their absence. These observations suggest that HmrR can regulate actP transcription positively and negatively. We show that copper homeostasis is an essential mechanism for the acid tolerance of these root nodule bacteria since it prevents this heavy metal from becoming overtly toxic in acidic conditions.

The Escherichia coli Copper-responsivecopA Promoter Is Activated by Gold

“…1) has no effect on CueR response to Hg(II), as the spacing between Cys 112 and Cys 120 in CueR is different to that between Cys 117 and Cys 126 in MerR, and more subtle positioning and charge effects will undoubtedly be invovled in metal recognition. The recently described copper-dependent regulator, HmrR, from rhizobia (27) contains cysteines equivalent to Cys 112 and Cys 120 , but lacks a sequence equivalent to the CCHH motif at positions 129 -132, providing indirect confirmation of our analysis. The detail of metal binding will require structural information on the CueR protein.…”

PAS domain containing chemoreceptor couples dynamic changes in metabolism with chemotaxis

“…At least three independent experiments were performed, and at least five different fields of view from each experiment were analyzed. was cloned into the pFUS1 vector (33) to generate a transcriptional fusion with the promoterless gusA, yielding PaerC-gusA (pFUSPaerC; Table S2). The nifH promoter activity was assessed using a PnifH-gusA fusion from the pFAJ318 vector (Table S2).…”

The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to enhance survival in lethal acid conditions

“…Escherichia coli strains were cultured at 37 uC using LB or Antibiotic Medium 3 (Oxoid) when using gentamicin (Reeve et al, 1999). Strains of Sinorhizobium were grown at 28 uC using TY, JMM or MJMM (O'Hara et al, 1989;Reeve et al, 2002) media. Minimal medium contained glutamate (3 mM) as nitrogen source.…”

“…Construction of mutants from the acid-tolerant strain Sinorhizobium medicae WSM419 has allowed the identification of some of the genes required in acidic conditions (Dilworth et al, 2001). The protein products of these genes include ActA (an apolipoprotein acyl transferase; Tiwari et al, 1996a), ActS (a histidine kinase 'sensor'; Tiwari et al, 1996b), ActR (a response regulator; Tiwari et al, 1996b) and ActP (a CPx heavy metaltransporting ATPase; Reeve et al, 2002). The acid-sensitive mutants with lesions in actA, actS and actR can be restored to wild-type levels of acid-tolerance by addition of high concentrations of calcium (50 mM), but those with lesions in actP cannot be repaired by calcium (Reeve et al, 2002).…”