Phosphate assimilation in Rhizobium (Sinorhizobium) meliloti: identification of a pit-like gene

Abstract

Rhizobium meliloti mutants defective in the phoCDET-encoded phosphate transport system form root nodules on alfalfa plants that fail to fix nitrogen (Fix-). We have previously reported that two classes of second-site mutations can suppress the Fix- phenotype of phoCDET mutants to Fix+. Here we show that one of these suppressor loci (sfx1) contains two genes, orfA and pit, which appear to form an operon transcribed in the order orfA-pit. The Pit protein is homologous to various phosphate transporters, and we present evidence that three suppressor mutations arose from a single thymidine deletion in a hepta-thymidine sequence centered 54 nucleotides upstream of the orfA transcription start site. This mutation increased the level of orfA-pit transcription. These data, together with previous biochemical evidence, show that the orfA-pit genes encode a Pi transport system that is expressed in wild-type cells grown with excess Pi but repressed in cells under conditions of Pi limitation. In phoCDET mutant cells, orfA-pit expression is repressed, but this repression is alleviated by the second-site suppressor mutations. Suppression increases orfA-pit expression compensating for the deficiencies in phosphate assimilation and symbiosis of the phoCDET mutants.

FIG. 1

Growth of Rm1021 (wild type) (■), RmG490 (phoCΩ490) (•), RmG762 (phoCΩ490 sfx1) (★), RmG822 (phoCΩ490 sfx1 orfA23::Tn5) (⧫), RmG830 (phoCΩ490 sfx1 pit10::Tn5) (▾), and RmH842 (phoCΩ490 sfx1 recF12::TnphoA) (▿) in MOPS-buffered minimal medium supplemented with 2 mM P_i. Each time point represent the average of triplicate values. The growth curve for strain RmG821 [phoCΩ490 sfx1 (orf2)2::Tn5] was similar to that for strain Rm1021, and the growth curves for phoCΩ490 sfx1::Tn5 recombinants Ω16, -3, -J, -5, and -10A (Fig. 3) were similar to those for RmG822 and RmG830. In order to simplify the figure, these results are not shown.

FIG. 2

P_i uptake in different R. meliloti mutants. [³³P]orthophosphoric acid was added to a final concentration of 4 μM. Results for R. meliloti Rm1021 (wild type) (⧫), RmG490 (phoCΩ490) (▴), RmG762 (phoCΩ490 sfx1) (■), and RmG830 (phoCΩ490 pit10::Tn5) (•) are shown. The symbols represent the means of triplicate assays, and each line gives the linear regression for all data points for one strain. Background values for all strains were adjusted so that the results for the extrapolated zero time showed no P_i uptake.

FIG. 3

Restriction maps of pTH90 and related plasmids. The upper diagram shows the regions which remained following deletion from the BamHI site within Tn5 transposons (Ω2.15, -1.4, -1.5, -E, -2.3, -3.10, and -2.2) to the BamHI site in the polylinker of pTH90. Below the map for pTH276 are diagrammed the deduced orfA-pit genes and two partial recF and orf2 open reading frames together with the Tn5 insertions that disrupted the sfx1 locus (Ω16, -3, -J, -5, -10, -23, and -10A) and the Ω2 and Ω12 insertions that did not. The orientation of fragments subcloned into pRK7813 are indicated relative to the position of the plac promoter. The subclones are pTH276 (4.8-kb HindIII/SacI), pTH305 (2.6-kb HindIII/EcoRV₁), pTH304 (2.5-kb HindIII/SmaI₁), and pTH347 and pTH348 (2.6-kb partial EcoRI in orientations I and II). The ability of the plasmids to suppress the Fix⁻ and/or slow-growth phenotype of RmG490 (phoCΩ490) in MOPS P2 is indicated in parentheses (+, suppression; −, no suppression). Restriction site abbreviations: B, BamHI; Bg, BglII; C, ClaI; H, HindIII; R, EcoRI; Sc, SacI; Sm, SmaI; Sp, SphI; V, EcoRV; X, XhoI. The subscripts following the R and V are used to specify particular sites.

FIG. 4

Determination of the orfA transcriptional start sites. The autoradiograph shows the products of the sequencing reaction by using primer 55 and plasmid pTH191 (sfx1) as a template. The results for primers 81 and 82 yielded a start site identical to that with primer 55 (data not shown). The relevant sequence is shown on the left, and the arrow indicates the position of the extension product. Lane 1, RmG591 (sfx1) carrying pTH347 (sfx1 orfA-pit in orientation I); lane 2, RmG591; lane 3, Rm1021 (wild type); lane 4, Rm1021 carrying pTH391 (orfA-pit); lane 5, RmG591 carrying pTH348 (sfx1 orfA-pit in orientation II). The sequence on the right of the figure is the wild-type recF-orfA intergenic region. The deduced orfA transcriptional start site is in boldface type and underlined. The translational start sites for orfA and recF are in boldface type with arrows above indicating the direction of transcription. The three different primers used are indicated by thin underlines. The hepta-thymidine repeat is underlined, and a consensus PHO box sequence is shown below the putative PhoB-binding site. Numbers to the right of the sequence are nucleotide positions relative to the orfA transcriptional start site.

FIG. 5

Histogram showing AP (a) and β-galactosidase (b) activities from the R. meliloti wild type (wt), RmG212, and the RmG212 phoC mutant, RmH667. The strains represented in data sets 2 to 5 carried plasmid-borne lacZ gene fusions to the pit or orfA genes from the wild-type and sfx1 loci, i.e., plasmids pTH376, pTH378, pTH365, and pTH367, respectively. Cells were assayed following 38 h of growth in MOPS-buffered minimal medium with no phosphate added (■) or supplemented with 2 mM phosphate (□). Each datum point represents the average of triplicate values ± standard error (error bar).