This idea is supported by the increased expression in response to additional phosphate that occurs even when PhoB is absent. the PSR. Our results show that phosphate availability affects glutamine synthetase activity and expression, which are often used as indicators of NSR activity, but that nitrogen availability did not affect alkaline phosphatase activity and expression, which are indicators of PSR activity. We conclude that the NSR is co-regulated by nitrogen and phosphate, whereas the PSR does not appear to be co-regulated by nitrogen in addition to its known phosphate regulation. Keywords: nitrogen stress, phosphate stress, glutamine synthetase, alkaline phosphatase, PIInitrogen regulatory proteins, Phob == Introduction == Sinorhizobium melilotiare important bacteria due to their ability to fix atmospheric nitrogen during symbiosis with alfalfa and other legumes. Nitrogen availability is a common problem that farmers need to address in order to approach the yield potential of many crops. The application of nitrogen fertilizers to the soil is a common solution (Ladha et al., 2005; Hirsch and Mauchline, 2015). Using fertilizers is expensive and their use is also associated with significant environmental costs (Ladha et al., 2005; Spiertz, 2010; Hirsch and Mauchline, 2015). A significant fraction of the fertilizer typically runs off farmland and contaminates groundwater and river ecosystems. Microbial transformation of nitrogen fertilizers can lower soil pH, and affect crops that do not have the ability to compensate for the increased acidity. One way to reduce the need for nitrogen fertilizer applications is to grow legumes that can form symbiotic relationships with rhizobia, allowing biological nitrogen fixation (BNF) to PXS-5153A occurin situand on demand. In the process of establishing a symbiosis with a legume like alfalfa, nodules are formed on the roots after the bacteria infect emerging root hairs. In these nodules, bacteria invade plant cells and differentiate into an organelle-like state called a bacteroid. The bacteroids produce nitrogenase and associated proteins, which reduce atmospheric dinitrogen into ammonia, a process termed BNF. The PXS-5153A plant can use the ammonia for incorporation into amino acids, nucleic acids, and other compounds necessary for plant survival (Patriarca et al., 2004; Oldroyd and Downie, 2008). In order to support nitrogen fixation, plants supply the bacteroids with carbon compounds and the nodule maintains a microaerobic environment that is compatible with bacterial respiration and nitrogen fixation. Establishing a symbiotic relationship is facultative for both the plant and bacteria and nodules are formedde novoin response to the plants need for nitrogen. For BNF to occur and provide useable nitrogen to legumes, free-living rhizobia must be present in the soil and the bacteria therefore need to be able to survive in various soil conditions, adapting to nutrient limitations, soil acidity and moisture content. Soil nutrient availability can be dynamic, with the availability of nutrients shifting as the season and presence of other microorganisms changes (Vitousek et al., 2010; Fanin et al., 2016). Rhizobia have stress response pathways to deal with numerous environmental limitations and help them persist in the rhizosphere (Al-Niemi et al., 1997; Krol and Becker, 2004; Yurgel et al., 2012). Nutrient stress responses alter gene expression and protein activity to improve uptake and catabolism of substrates that contain phosphorus or nitrogen, more efficiently incorporate the compounds into metabolism and, in some situations, use biosynthetic pathways that are not as dependent on these nutrients (Battesti et al., 2011). The nitrogen and phosphate stress responses (PSRs) inS. melilotihave each been characterized. While certain aspects may not be entirely understood yet, a fair amount of detail has been elucidated for each pathway. During nitrogen limitation, the nitrogen stress response (NSR) inS. melilotiis active (Figure1A). The NSR increases uptake and mobilization of nitrogen while increasing the efficiency with which available nitrogen is used (Merrick and Edwards, 1995; Arcondeguy et al., 2001; Leigh and Dodsworth, 2007). Regulation of the core NSR inS. melilotiinvolves a sensor protein, GlnD, two PIIproteins, GlnB and GlnK, and the NtrB/NtrC two component regulatory system. GlnD, a uridylyltransferase/uridylyl-cleavage enzyme, senses the ratio between glutamine and alpha-ketoglutarate, an index of nitrogen availability (Merrick and Edwards, 1995; Ikeda et al., 1996; Jiang et al., 2012). When alpha-ketoglutarate levels are high, indicating nitrogen deficiency, GlnD PXS-5153A uridylylates GlnB and GlnK. GlnB-UMP and GlnK-UMP act on GlnE which regulates the adenylylation state of glutamine synthetase I (GSI) (Atkinson and HDAC7 Ninfa, 1998). GSI-AMP is inactive and when the adenylyl group is removed, GSI becomes activated allowing it to add ammonium to glutamate to form glutamine, a molecule which is used to donate.
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