Project description:Investigation of whole genome gene expression level changes in a Nitrosomonas europaea (ATCC 19718) wildtype and pFur::Kan mutant [kanamycin resistance cassette insertion in the promoter region of the fur gene (NE0616)] strains grown in Fe-replete and Fe-limited media. The Nitrosomonas europaea (ATCC 19718) wiltype cells grown in Fe-limited media were compared to cells grown in Fe-replete media to gain a better understanding of the metabolic changes occurring in response to iron stress. The Nitrosomonas europaea (ATCC 19718) pFur::Kan mutant strain grown in Fe-replete & Fe-limited media were compared to wildtype cells grown in Fe=replete & Fe-limited media to gain a better understanding of the role Fur (NE0616) plays in iron homeostasis control.
Project description:Investigation of whole genome gene expression level changes in a Nitrosomonas europaea (ATCC 19718) wildtype and pFur::Kan mutant [kanamycin resistance cassette insertion in the promoter region of the fur gene (NE0616)] strains grown in Fe-replete and Fe-limited media. The Nitrosomonas europaea (ATCC 19718) wiltype cells grown in Fe-limited media were compared to cells grown in Fe-replete media to gain a better understanding of the metabolic changes occurring in response to iron stress. The Nitrosomonas europaea (ATCC 19718) pFur::Kan mutant strain grown in Fe-replete & Fe-limited media were compared to wildtype cells grown in Fe=replete & Fe-limited media to gain a better understanding of the role Fur (NE0616) plays in iron homeostasis control. A 4-plex 3 chip study using total RNA recovered from three separate wild-type cultures each of N. europaea grown in Fe-replete media and Fe-limited media and three seperate cultures each of N. europaea pFur::Kan mutant strain grown in Fe-replete and Fe-limited media. Each chip measures the expression level of 2368 genes from Nitrosomonas europaea (ATCC19718) with 4 X 72,000 60-mer 14 probe pairs per gene, with two-fold technical redundancy.
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.
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:Nitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH3) to obtain energy for growth on carbon dioxide (CO2), and can also produce nitrous oxide (N2O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses of N. europaea to replete (> 5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na2CO3) supplemented with atmospheric CO2. IC-limited cultures oxidized 25 to 58% of available NH3 to nitrite, depending on dilution rate and Na2CO3 concentration. IC limitation resulted in a 1.5-fold increase in cellular maintenance energy requirements compared to NH3-limited cultures. Rates of N2O production increased 2- and 6.3 fold under the two IC-limited conditions increasing the percentage of oxidized NH3-N being transformed to N2O-N from 0.5% (replete) to 4.4% (0.25 mM Na2CO3). Transcriptome analysis showed differential expression (p ≤ 0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits, and increased expression of genes involved in C1 metabolism including RuBisCO (cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C1 moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924-0927) correlated with increased production of N2O. Together, these data suggest that N. europaea adapts physiologically during IC-limited steady state growth, which leads to uncoupling of NH3 oxidation from growth and increased N2O production.
Project description:Nitrosomonas europaeais a chemolithoautotrophic bacterium that oxidizes ammonia (NH3) to obtain energy for growth on carbon dioxide (CO2), and can also produce nitrous oxide (N2O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses ofN. europaeato replete (> 5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na2CO3) supplemented with atmospheric CO2 IC-limited cultures oxidized 25 to 58% of available NH3to nitrite, depending on dilution rate and Na2CO3concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to NH3-limited cultures. Rates of N2O production increased 2.5- and 6.3-fold under the two IC-limited conditions increasing the percentage of oxidized NH3-N being transformed to N2O-N from 0.5% (replete) up to 4.4% (0.2 mM Na2CO3). Transcriptome analysis showed differential expression (p⤠0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits, and increased expression of genes involved in C1 metabolism including RuBisCO (cbbgene cluster), carbonic anhydrase, folate-linked metabolism of C1 moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924-0927) correlated with increased production of N2O. Together, these data suggest thatN. europaeaadapts physiologically during IC-limited steady state growth, which leads to uncoupling of NH3oxidation from growth and increased N2O production. Transcriptome responses of N. europaea in 60 mM NH4+ to suboptimum levels of carbonate (1.0 mM and 0.2 mM) in continuous steady-state culture in a bioreactor.
Project description:Pure cultures of ammonia oxidizing bacterium, Nitrosomonas europaea, are exposed to cyanide in pseudo-steady state batch reactor in presence of ammonia. Nitrosomonas europaea are generally regarded as the most sensitive organism to various inhibitors commonly encountered in the wastewater treatment plants (WWTP). To find stress genes of Nitrosomonas europaea to cyanide known as inhibitor of respiratory process, whole-genome transcript response to cyanide was determined in this research using microarray and qRT-PCR. When 1 uM NaCN inhibits nitrification about 50 %, transcript levels of 35 genes were increased while transcript levels of 29 genes were decreased, showing more than 2-fold in total 2460 genes. moeZ (NE2353), homologue with rhodanases related to detoxification of CN-, showed 7-fold up regulation and gene cluster including moeZ also showed significant up regulation. Keywords: cyanide, stress response, moeZ