TheSinorhizobium meliloti furGene Regulates, with Dependence on Mn(II), Transcription of thesitABCDOperon, Encoding a Metal-Type Transporter

Abstract

Sinorhizobium meliloti is an alpha-proteobacterium able to induce nitrogen-fixing nodules on roots of specific legumes. In order to propagate in the soil and for successful symbiotic interaction the bacterium needs to sequester metals like iron and manganese from its environment. The metal uptake has to be in turn tightly regulated to avoid toxic effects. In this report we describe the characterization of a chromosomal region of S. meliloti encoding the sitABCD operon and the putative regulatory fur gene. It is generally assumed that the sitABCD operon encodes a metal-type transporter and that the fur gene is involved in iron ion uptake regulation. A constructed S. meliloti sitA deletion mutant was found to be growth dependent on Mn(II) and to a lesser degree on Fe(II). The sitA promoter was strongly repressed by Mn(II), with dependence on Fur, and moderately by Fe(II). Applying a genome-wide S. meliloti microarray it was shown that in the fur deletion mutant 23 genes were up-regulated and 10 genes were down-regulated when compared to the wild-type strain. Among the up-regulated genes only the sitABCD operon could be associated with metal uptake. On the other hand, the complete rhbABCDEF operon, which is involved in siderophore synthesis, was identified among the down-regulated genes. Thus, in S. meliloti Fur is not a global repressor of iron uptake. Under symbiotic conditions the sitA promoter was strongly expressed and the S. meliloti sitA mutant exhibited an attenuated nitrogen fixation activity resulting in a decreased fresh weight of the host plant Medicago sativa.The root nodule bacteria of the genera Rhizobium, Bradyrhizobium, Mesorhizobium, Azorhizobium, and Sinorhizobium, collectively known as rhizobia, are able to establish a nitrogenfixing symbiosis with their respective leguminous host plants. During this symbiotic interaction the rhizobia first induce the formation of root nodules on their respective hosts, colonize the nodules, and finally, after differentiation into bacteroids, reduce atmospheric nitrogen to ammonia. The fixed nitrogen is then delivered to the host plants in the form of alanine or ammonia. The plant in turn supplies the bacteria with various nutrients. This symbiotic interaction has been described in detail recently (44,57,61).One interesting aspect of the rhizobium-legume symbiosis is the influence of transition metals on nodule formation and nitrogen fixation. A number of these micronutrients are known to be essential for bacterial metabolism (36), yet the mechanisms of metal import and their regulation in rhizobia are largely unknown. One of the most important transition metals is iron, which due to its importance in metabolic electron transport chains is essential for almost all known organisms. Despite the fact that iron is the fourth most abundant element on earth its availability to microorganisms is restricted because of its low solubility under aerobic conditions and physiological pH values. Thus, bacteria have evolved a number of strategies to overcome iron deficiencies ...

Disruption of sitA Compromises Sinorhizobium meliloti for Manganese Uptake Required for Protection against Oxidative Stress

“…These characterizations were consistent with the decrease in healthy pink nodules observed on sitA::mTn5 mutant-inoculated plants. While we were characterizing the sitA::mTn5 mutant, a report describing a sitA deletion was published (10). In agreement with our observations, that report showed that acetylene reduction was decreased for alfalfa inoculated with the sitA deletion strain, but the physiology of the symbiotic defect was not further characterized.…”

“…Our findings are consistent with studies of SitABCD homologs in S. enterica serovar Typhimurium, which showed that SitABCD can transport both Fe 2ϩ and Mn 2ϩ but that transport of Mn 2ϩ is favored 100 times over that of Fe 2ϩ (30). Previous work suggested that Mn 2ϩ alone could not rescue the growth defect of a sitA deletion strain (10). The authors of that work graciously sent us their sitA deletion strain for comparison with our sitA::mTn5 strain.…”

Bacterial outer membrane channel for divalent metal ion acquisition

“…Association of Mn 2+ with Mur confers DNA binding activity on the protein, resulting in occupancy of the mntH promoter by Mur and transcriptional repression (13,15). The responsiveness of the Fur-like regulator Mur to Mn 2+ in B. japonicum and other α-Proteobacteria rather than to Fe 2+ (13,(15)(16)(17)(18)(19) may reflect an adaptation by this group to a more manganese-dependent metabolism. In support of this, an mntH mutant of B. japonicum has a severe growth phenotype (13), whereas other bacterial mntH mutants do not unless they are exposed to oxidative stress (20).…”

Evidence that the Rhizobium regulatory protein RirA binds to cis-acting iron-responsive operators (IROs) at promoters of some Fe-regulated genes

“…Consistent with this, Rhizobium has a gene whose product resembles a 'classical' Fur, but mutations in the fur-like gene do not affect Feresponsive regulation of the fhu, vbs, rpoI, hmu and tonB genes alluded to above (Wexler et al, 2003). Indeed, this Fur-like protein is actually a manganese-sensing regulator, termed 'Mur', which represses the operon that specifies a Mn 2+ ABC transporter, SitABCD, in Mn 2+ -replete cells (Diaz-Mireles et al, 2004;Chao et al, 2004). …”