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Comparative proteomics of three species of ammonia-oxidizing bacteria under ammonia starved and ammonia replete conditions

ABSTRACT: In this experiment, we used advanced proteomics techniques to discern differences in energy allocation between three strains of ammonia oxidizing bacteria: Nitrosomonas europaea, Nitrosomonas ureae, and Nitrosospira multiformis, during ammonia starved and ammonia replete conditions. Replicate cultures in late log phase from the three strains were starved of ammonia for 24 hours and compared to replicate control cultures grown for the same period. All three species were grown with three biological replicates for each condition and species with the exception of two replicates from the N. ureae starved cultures due to sample processing loss. This study has, to our knowledge, produced the first complete proteomes of Nitrosospira multiformis and Nitrosomonas ureae.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Nitrosomonas Europaea Atcc 19718 Nitrosospira Multiformis Atcc 25196 Nitrosomonas Ureae

SUBMITTER: Jackie Zorz

LAB HEAD: Manuel Kleiner

PROVIDER: PXD008954 | Pride | 2018-05-16

REPOSITORIES: pride

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Project description:Investigation of the whole genome gene expression level changes relative to exponential phase growth in Nitrosomonas europaea ATCC19718 after 12 hours ammonia starvation, 144 hours ammonia starvation, and 20 minutes following ammonia addition to starved cells. The ammonia monooxygenase of chemolithotrophic ammonia oxidizing bacteria (AOB) catalyzes the first step in ammonia oxidation by converting ammonia to hydroxylamine. The monooxygenase of Nitrosomonas europaea is encoded by two nearly identical operon copies (amoCAB1,2). Several AOB, including N. europaea, also posess a divergent monocistronic copy of amoC (amoC3) of unknown function. Previous work suggested a possible functional role for amoC3 in N. europaea during recovery from extended ammonia starvation as part of the σE- stress response regulon during the recovery of N. europaea from extended ammonia starvation, thus indicating its importance during the exit of cells from starvation. We here used global transcription analysis to show that expression of amoC3 is part of a general post-starvation cellular response system in N. europaea. We also found that amoC3 is required for efficient exit from prolonged ammonia starvation, as deleting this gene impaired growth at elevated temperatures and recovery following starvation under high oxygen tensions. Deletion of the σ32 global stress response regulator demonstrated that the heat shock regulon also plays a significant role in mediating the recovery of N. europaea from starvation. These findings provide the first described phenotype associated with the divergent AmoC3 subunit which appears to function as a stress responsive subunit capable of maintaining ammonia oxidation activity under stress conditions. A twelve chip study using total RNA recovered from four timepoints for each of three biological replicates of wild-type cultures of Nitrosomonas europaea ATCC 19718. Total RNA was obtained from each biological culture replicate during exponential growth, following 12 hours ammonia starvation, 144 hours ammonia starvations, and 20 minutes following ammonia addition to starved cells.

Project description:Increasing atmospheric CO2 concentrations are causing decreased pH over vast expanses of the ocean. This decreasing pH may alter biogeochemical cycling of carbon and nitrogen via the microbial process of nitrification, a key process that couples these cycles in the ocean, but which is often sensitive to acidic conditions. Recent reports indicate a decrease in oceanic nitrification rates under experimentally lowered pH. How composition and abundance of ammonia oxidizing bacteria (AOB) and archaea (AOA) assemblages respond to decreasing oceanic pH, however, is unknown. We sampled microbes from two different acidification experiments and used a combination of qPCR and functional gene microarrays for the ammonia monooxygenase gene (amoA) to assess how acidification alters the structure of ammonia oxidizer assemblages. We show that despite widely different experimental conditions, acidification consistently altered the community composition of AOB by increasing the relative abundance of taxa related to the Nitrosomonas ureae clade. In one experiment this increase was sufficient to cause an increase in the overall abundance of AOB. There were no systematic shifts in the community structure or abundance of AOA in either experiment. These different responses to acidification underscore the important role of microbial community structure in the resiliency of marine ecosystems. amoA gene diversity from two ocean acidification experiments, Monterey Bay experiment (two time points, ambient and acidified) and Vineyard Sound experiment (ambient and acifidied, with and without nutrients) examined with 2 two-color arrays (Cy3 and Cy5): the universal standard 20-mer oligo is printed to the slide with a 70-mer oligo (an archetype). Environmental DNA sequences (fluoresced with Cy3) within 15% of the 70-mer conjugated to a 20-mer oligo (fluoresced with Cy5) complementary to the universal standard will bind to the oligo probes on the array. Signal is the ratio of Cy3 to Cy5.

			Action	DRS
	Run1_Ne1_C.msf	Msf
	Run1_Ne1_C.pep.xml	Pepxml
	Run1_Ne1_C.raw	Raw
	Run1_Ne1_S.msf	Msf
	Run1_Ne1_S.pep.xml	Pepxml

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Comparative proteomics of three species of ammonia-oxidizing bacteria under ammonia starved and ammonia replete conditions

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