Mutation of GOGAT prevents pea bacteroid formation and N₂ fixation by globally downregulating transport of organic nitrogen sources

Abstract

SummaryMutation of gltB (encoding glutamate oxoglutarate amidotransferase or GOGAT) in RU2307 increased the intracellular Gln : Glu ratio and inhibited amino acid transport via Aap and Bra. The mechanism probably involves global post-translational inhibition independent of Ntr. Transport was separately restored by increased gene expression of Aap or heterologous transporters. Likewise, second site suppressor mutations in the RNA chaperone Hfq elevated transport by Aap and Bra by increasing mRNA levels. Microarrays showed Hfq regulates 34 ABC transporter genes, including aap, bra and opp. The genes coding for integral membrane proteins and ABC subunits aapQMP braDEFGC were more strongly elevated in the hfq mutants than solute-binding proteins (aapJ braC). aapQMP and braDEFG are immediately downstream of stem-loops, indicating Hfq attenuates downstream translation and stability of mRNA, explaining differential expression of ABC genes. RU2307 nodulated peas and bacteria grew down infection threads, but bacteroid development was arrested and N 2 was not fixed. This probably results from an inability to synthesize or transport amino acids. However, GOGAT and GOGAT/AldA double mutants carrying suppressor mutations that increased amino acid uptake fixed N2 on pea plants. Thus de novo ammonium assimilation into amino acids is unnecessary in bacteroids demonstrating sufficient amino acids are supplied by plants.

Quantitative Proteomic Analysis of the Hfq-Regulon in Sinorhizobium meliloti 2011

“…As for Bialaphos, this sensitivity could be a direct effect of the accumulation of several proteins involved in the transport of L-amino acids, like the amino acid permease (Aap) transport system or other putative amino acid transporters (SMc02259, SMb20706). It is worth pointing out that the tight control of amino acid transporter expression by Hfq has also been recognized in the pea symbiont Rhizobium leguminosarum [63]. In this related α-proteobacterium, a number of spontaneous second site suppressor mutants were found to arise from a gltB mutant, which is unable to carry out de novo amino acids synthesis and shows reduced amino acid transport via Aap and Bra systems.…”

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids

“…However, glutamate levels were 20-fold lower in bacteroids relative to free-living cells (Fig. 2), consistent with GS/GOGAT activity being both low and not essential in mature bacteroids (51). Levels of metabolites derived from glutamate, including glutathione and N-acetylglutamate, were also reduced, while levels of many other amino acids were either altered only slightly or unchanged in bacteroids (Fig.…”

Riboregulation in plant-associated α-proteobacteria

“…46,59,64 Furthermore, R. leguminosarum Hfq negatively regulates the broad range amino acid transport systems Aap and Bra which have a key role in nitrogen exchange between the bacteroid and the infected plant cell and suppressor mutations in hfq rescued R. leguminosarum GOGAT mutants that were deficient in nitrogen fixation. 65 α-proteobacterial hfq mutants impaired in virulence and symbiosis, as observed for several symbiotic α-rhizobia, phytopathogenic A. tumefaciens, and animal-pathogenic Brucella species, imply that Hfq is not only important for free-living states but also for establishment and maintenance of chronic microbial infections of eukaryotic hosts. 66,67 Co-immunoprecipitation (CoIP) of an epitope-tagged Hfq and its associated RNA in S. meliloti followed by PCR identified a few selected trans-sRNAs.…”