Isolation, molecular & physiological characterization of sulfate-reducing, heterotrophic, diazotrophs

Nitrogen (N2) fixation is the process by which N2 gas is converted to biologically reactive ammonia, and is a cellular capability widely distributed amongst prokaryotes. This process is essential for the input of new, reactive N in a variety of environments. Heterotrophic bacterial N fixers residing in estuarine sediments have only recently been acknowledged as important contributors to the overall N budget of these ecosystems and many specifics about their role in estuarine N cycling remain unknown, partly due to a lack of knowledge about their autecology and a lack of cultivated representatives. Nitrogenase reductase (nifH) gene composition and prevalence in Narragansett Bay sediments has revealed that two distinct phylogenetic groups dominate N-fixation. Analysis of nifH transcripts has revealed one active group to be the Desulfovibrionaceae , belonging to the Deltaproteobacteria. We see nifH expression from this group across sampling sites and times, despite the fact that Narragansett Bay sediments are replete with combined N, which is thought to inhibit N fixation in the environment. Here were present the genomic and physiological data in relation to N2 fixation by two heterotrophic members of the Desulfovibrionaceae, isolated from sediments of the Narragansett Bay estuary in 2010 and 2011, respectively: Desulfovibrio sp. NAR1 and Desulfovibrio spNAR2.To elucidate how nitrogenase activity in these organisms responds to the presence of different sources of combined N, and to link observed physiology with genomic potential (i.e. gene content), we performed a two-part study that coupled high-throughput genome sequencing and analysis with physiological investigations of growth on different N sources with N fixation rate measurements. The genomes of the two diazotrophic Narragansett Bay Desulfovibrio isolates (NAR1 and NAR2) were sequenced using a high throughput platform, subsequently assembled, annotated, and investigated for genes related to N fixation and overall N metabolism which were then compared across 34 additional Desulfovibrio genomes which were publicly available. To link findings at the molecular level with observations at the physiological level, N fixation rates were measured using the acetylene reduction assay (ARA) under conditions free of reactive N, and under the following combined N conditions: 12 mM urea, 12 mM NO3-, and 12 mM NH3. Both isolates can sustain growth by N2 fixation in the absence of biologically available N and our data indicate that nitrogenase activity is completely inhibited by the presence of ammonia, yet uninhibited by nitrate and urea, which are other forms of combined N found in Narragansett Bay. This agrees with observations made at the genome level, as neither our isolates nor the other Desulfovibrio examined in comparison appeared to have the genetic capability to use NO3- or urea catabolically to meet cellular N demands. This study indicates that the Desulfovibrionaceae are restricted in terms of the N sources they are capable of using, and that this may be a factor contributing to the observed N fixation by this group in sediments that are not limited for sources of combined N. Genome sequencing also reveals both isolates to be metabolically versatile and unique. The NAR1 isolate possesses genes involved in bacterial mercury methylation, and displays near obligate biofilm formation. Genes were also found in the NAR1 isolate which suggest the involvement of c-di-GMP in cell-to-cell communication and biofilm formation. This is particularly interesting since biofilm formation and quorum sensing is not well characterized among the Desulfovibrio , despite biofilm formation being displayed by many members of this genus. While investigating the role of these organisms as important contributors of fixed N in Narragansett Bay, it was critical that we examine these additional aspects of their metabolism in order to gain a better understanding of controls on growth that may also impact biomass and the ability of these organisms to achieve significant rates of N fixation in the environment