Functional redundancy of genes for sulphate activation enzymes in Rhizobium sp. BR816

Abstract

The broad-host-range, heat-tolerant Rhizobium strain BR816 produces sulphated Nod metabolites. Two ORFs highly homologous to the Sinorhizo&ium meliloti nodPQ genes were isolated and sequenced. It was found that Rhizo&ium sp. BR816 contained two copies of these genes; one copy was localized on the symbiotic plasmid, the other on the megaplasmid. Both nodP genes were interrupted by insertion of antibiotic resistance cassettes, thus constructing a double nodPlP2 mutant strain. However, no detectable differences in Nod factor TLC profile from this mutant were observed as compared to the wild-type strain. Additionally, plant inoculation experiments did not reveal differences between the mutant strain and the wild-type. It is proposed that a third, functionally homologous locus complements mutations in the Nod factor sulphation genes. Southern blot analysis suggested that this locus contains genes necessary for the sulphation of amino acids.

Identification of a Third Sulfate Activation System inSinorhizobiumsp. Strain BR816: the CysDN Sulfate Activation Complex

“…DNA fragments were recovered from agarose gels by using the Nucleotrap kit (MachereyNagel). Southern blotting and hybridizations were carried out as previously described (25). Sequencing of DNA fragments cloned in the pUC18-pUC19 vectors was performed on an automated ALF sequencer with fluorescein-labeled universal and synthetic oligonucleotide primers (Amersham Pharmacia Biotech, Uppsala, Sweden).…”

“…strain BR816 possesses two nodPQ copies. Although both copies are functional, as demonstrated by genetic complementation of an R. tropici nodP mutant, the double mutants did not show any detectable changes in the amount of sulfated Nod factors produced by this strain (25). It was suggested that in Sinorhizobium sp.…”

“…For example, Rhizobium-legume symbiotic interactions are mediated by a host-specific bacterial signaling molecule (the Nod factor), which can be sulfated. In general, rhizobial species that produce sulfated Nod factors possess at least two sulfate activation systems (6,12,24,25,40). The three genes that are indispensable for Nod factor sulfation, nodP, nodQ, and nodH, were first isolated from Sinorhizobium meliloti.…”

“…The sulfate decoration on the Nod factors secreted by S. meliloti is essential for nodulation of alfalfa (40). Except for S. meliloti, it is still unclear whether rhizobia producing sulfated Nod factors use only the nodPQdependent PAPS pool as a source of activated sulfate for Nod factor sulfation, the housekeeping PAPS pool, or both (25). Previously, Laeremans et al (25) demonstrated that Sinorhizobium sp.…”

Signals in Soybean's Inoculants

“…Quantitative analysis of GUS A activity was then carried out with p-nitrophenyl-ß-D-glucuronide (pNPG) as the substrate in microtiter plates and GUS A activity was examined in VERSA max microplate reader (Molecular Devices). To determination of Nod factors profile, the nodulation factors were radioactively labelled and they were isolated by following a slightly modified protocol of (Laeremans et al, 1998). 100μL from Bradyrhizobium cultures, growth for two nights, were inoculated in 900μL of each fresh culture medium and the concentration was adjusted to 5x10 8 CFU per medium milliliter.…”