A new small regulatory protein, HmuP, modulates haemin acquisition in Sinorhizobium meliloti

Abstract

Sinorhizobium meliloti has multiple systems for iron acquisition, including the use of haem as an iron source. Haem internalization involves the ShmR haem outer membrane receptor and the hmuTUV locus, which participates in haem transport across the cytoplasmic membrane. Previous studies have demonstrated that expression of the shmR gene is negatively regulated by iron through RirA. Here, we identify hmuP in a genetic screen for mutants that displayed aberrant control of shmR. The normal induction of shmR in response to iron limitation was lost in the hmuP mutant, showing that this gene positively affects shmR expression. Moreover, the HmuP protein is not part of the haemin transporter system. Analysis of gene expression and siderophore production indicates that disruption of hmuP does not affect other genes related to the iron-restriction response. Our results strongly indicate that the main function of HmuP is the transcriptional regulation of shmR. Sequence alignment of HmuP homologues and comparison with the NMR structure of Rhodopseudomonas palustris CGA009 HmuP protein revealed that certain amino acids localized within predicted beta-sheets are well conserved. Our data indicate that at least one of the beta-sheets is important for HmuP activity.

Fig. 1.

β-Galactosidase activity in the SM1021 shmR : : lacZ mutant (SHMR) and in its derivative mutant (B20). Cells were grown to early stationary phase in TY broth. One hundred-fold dilutions were made in TY supplemented with either 150 μM EDDHA or 37 μM FeCl₃ and grown for 48 h. β-Galactosidase activity is expressed as nmol o-nitrophenol min⁻¹ (mg protein)⁻¹. The data shown are the mean of two independent experiments done in triplicate. Error bars, 1 sd.

Fig. 2.

Genetic organization of the hmuP gene in S. meliloti 1021. A black oval indicates a rho-independent transcriptional terminator. The Tn5-1063a insertion site and orientation are illustrated with a white arrow not to scale. The small numbered arrows indicate the positions of primers used for RT-PCR: HmuPF (1), GSP1 (2), HmuSF (3), HmuTR (4). The transcription start site of hmuP was determined by 5′-RACE. The bent arrow indicates the transcription start site and the numbers show the distance (bp) from it to the initiation codon (GTG) and to the putative RirA box.

Fig. 3.

In vivo effect of hmuP mutation on shmR : : gusA activity. (a) The pRG1SMc02726 construction integrated into the SM1021 or HMUP genome. Small numbered arrows indicate the position of primers shmRforward (1) and 2030F (2). (b) Strains SM1021 : : pRG1SMc02726 and HMUP : : pRG1SMc02726 containing the indicated pOT2 derivatives were grown until early stationary phase in M9 broth. One hundred-fold dilutions were made in M9 supplemented with 300 μM EDDHA and grown for 48 h. β-Glucuronidase activity is expressed as β-glucuronidase arbitrary units. The data shown are the mean of two independent experiments done in triplicate. Error bars, 1 sd.

Fig. 4.

Growth assays. Cells were grown in TY broth until early stationary phase. Appropriate dilutions were made in TY broth and then plated in TY solid media supplemented with 300 μM EDDHA and 10 μM haemin. Dilutions were also spotted in TY media, and all the strains displayed a similar growth pattern (data not shown). Cells were grown for 5 days and colony sizes were determined. The experiments were done in triplicate with similar results. (a) Effect of HmuP on the ability of S. meliloti 1021 to use haemin as an iron source. (b) Effect of hmuP mutations on HmuP activity.

Fig. 5.

Effect of HmuP on the expression of iron-regulated genes. mRNAs from wild-type (black bars) and hmuP mutant (white bars) cells grown in M9 minimal media supplemented with 300 μM EDDHA were analysed by qPCR. The genes assessed are indicated below the bars. The data are expressed as the relative starting quantity (SQ) of mRNA normalized to the housekeeping gene gapA. The data are expressed as the mean of three replicates; error bars, 1 sd.