Elucidating novel pathways and regulation of nitrogen assimilation in alpha proteobacteria exemplified by the soil organism Paracoccus denitrificans

Nitrogen is one of the critical elements that is required for all forms of biological life on Earth because it is a building block for DNA and proteins. All biological life forms must therefore have mechanisms by which they can capture nitrogen from the environment and incorporate it into important cellular components. This process is called NITROGEN ASSIMILATION. Life forms at the top of the food chain, such as Humans, assimilate nitrogen from organic nitrogen sources extracted from the plants and animals that they ingest and digest. However, some life forms lower down in the food chain, such as bacteria, can assimilate nitrogen from simple inorganic forms such as nitrogen gas, nitrate or ammonium as well as from simple organic forms such as amino acids. Many agricultural soils are rich in nitrates as they are applied by farmers to encourage high yielding plant crop production and water run-off from soils is in turn enriching rivers, lakes and oceans in nitrate. This then provides a source of nitrogen that can lead to proliferation of the bacteria that can utilise nitrate as a nitrogen-source for assimilation. In agricultural fields the diversion of nitrate towards a 'food source' for soil microbes, rather than the crops for which it was intended, is economically wasteful. However, some estimates suggest that this may be happening on a large scale. It is then timely to seek an academic knowledge base from which it might be possible to develop strategies to lessen these losses. This requires an understanding of the molecular machinery with which soil bacteria incorporate nitrogen from nitrate into cellular nitrogen in the process of NITRATE ASSIMILATION and also the means by which this process is regulated when other sources of nitrogen are present. However, under certain conditions the metabolism of nitrate in soils by bacteria takes on an added complication. If a soil becomes anaerobic, for example through water logging, some species of soil bacteria can begin to 'breathe' nitrate. Essentially, this NITRATE RESPIRATION process is an alternative to oxygen-respiration enabling the bacteria to sustain energy generation for growth. It also consumes the costly fertilisers added to soils. What then if the bacterium can do both NITRATE ASSIMILATION and NITRATE RESPIRATION? Many species of soil denitrifying bacterium can indeed do just this and so provide what amounts to a double whammy for the availability of nitrate to crops. What is not yet clear many species of soil denitrifying bacteria is how the NITRATE ASSIMILATION process is regulated and.to what extent the molecular systems involved in NITRATE ASSIMILATION overlap with those involved in NITRATE RESPIRATION. In this research programme we will address these questions using a soil bacterium called Paracoccus denitrificans. For many years this species of bacterium has been a model organism for the study of the nitrate respiration as part of a process called denitrification in which the soluble soil nitrate is converted to nitrogen gas and so lost to the atmosphere. The importance of this species as a model organism led to the United States Department of Energy providing the funds to enable the sequencing of the DNA of it's genome. This has allowed us to identify the genes that encode a nitrate assimilation system, which we term the Nas system. The study of this Nas system provides the focus for this research programme.

The importance of inorganic nitrate for the nutrition and growth of marine and freshwater autotrophic phytoplankton has long been recognised, in particularly because of the 'blooms' formed in nitrate and phosphate polluted water and their possible roles as carbon dioxide sinks. By contrast, the utilisation of nitrate by heterotrophic bacteria has historically received less attention. Up until now, the primary role of heterotrophic bacteria has classically been considered to be the decomposition and mineralization of dissolved and particulate organic nitrogen. Consequently bacterial nitrate assimilation has not been a pathway currently widely considered in text-book models of the carbon and nitrogen cycles. However, recent data now suggests that in many environments a large number of bacterial species will utilise nitrate extensively for growth and so may be significant consumers of this form inorganic nitrogen in soils. Significant heterotrophic bacterial utilisation of nitrates will have a great impact on the fluxes of nitrogen, carbon and other minerals, such as phosphorous in soils, sediments and marine environments. In this proposal the the mechanism and regulation of nitrogen assimilation from nitrate in the model soil denitrifyign bacterium Paracoccus denitrificans will be assessed. The focus will be the mechanism of nitrate and nitrite signal sensing and transduction; the integration of the regulation of nitrate assimilation into global cellular regulation of nitrogen assimilation; and a mechanism of nitrate reduction to nitrate that reveals overlapping pathways of assimilatory and respiratory functions.