Transcription of All amoC Copies Is Associated with Recovery of Nitrosomonas europaea from Ammonia Starvation

Abstract

The chemolithotrophic ammonia-oxidizing bacterium Nitrosomonas europaea is known to be highly resistant to starvation conditions. The transcriptional response of N. europaea to ammonia addition following short-and long-term starvation was examined by primer extension and S1 nuclease protection analyses of genes encoding enzymes for ammonia oxidation (amoCAB operons) and CO 2 fixation (cbbLS), a third, lone copy of amoC (amoC 3 ), and two representative housekeeping genes (glyA and rpsJ). Primer extension analysis of RNA isolated from growing, starved, and recovering cells revealed two differentially regulated promoters upstream of the two amoCAB operons. The distal 70 type amoCAB promoter was constitutively active in the presence of ammonia, but the proximal promoter was only active when cells were recovering from ammonia starvation. The lone, divergent copy of amoC (amoC 3 ) was expressed only during recovery. Both the proximal amoC 1,2 promoter and the amoC 3 promoter are similar to gram-negative E promoters, thus implicating E in the regulation of the recovery response. Although modeling of subunit interactions suggested that a nonconservative proline substitution in AmoC 3 may modify the activity of the holoenzyme, characterization of a ⌬amoC 3 strain showed no significant difference in starvation recovery under conditions evaluated. In contrast to the amo transcripts, a delayed appearance of transcripts for a gene required for CO 2 fixation (cbbL) suggested that its transcription is retarded until sufficient energy is available. Overall, these data revealed a programmed exit from starvation likely involving regulation by E and the coordinated regulation of catabolic and anabolic genes.Ammonia-oxidizing bacteria fulfill an important biological role because they carry out the first reaction in the oxidative pathway of the nitrogen cycle. These bacteria obtain energy for growth from the oxidation of ammonia and acquire the majority of their carbon through the fixation of CO 2 via the Calvin cycle. Since these microorganisms must compete with plants and other microorganisms that assimilate ammonia for biosynthesis (47, 48), it is likely that they have developed adaptive strategies to cope with periods of ammonia starvation. Both Nitrosomonas cryotolerans and Nitrosomonas europaea have been shown to be particularly resilient to energy source deprivation. Following 10 weeks of starvation, N. cryotolerans retained 85% of initial viability, showed no apparent degradation of DNA, protein, or RNA, and maintained an active electron transport system (21). Studies of N. europaea demonstrated persistence of the AmoB protein and immediate oxidation of added ammonia following 1 year of starvation for ammonia (38,51). This remarkable resistance to starvation likely reflects unique physiological facets of these highly specialized microorganisms. In this study, we examined transcription of genes for key energy-generating (ammonia oxidation) and energyconsuming (CO 2 fixation) reactions during recovery of N. europaea from ammo...

Ammonia availability shapes the seasonal distribution and activity of archaeal and bacterial ammonia oxidizers in the Puget Sound Estuary

“…Thus, AOB (varying between 10 3 and10 5 cells mL 21 in the water column) may employ an opportunistic strategy to take advantage of transient spikes in ammonia concentration. This strategy is supported by our prior studies of the response of N. europaea to recovery from extended ammonia starvation (Berube et al 2007). The average number of amoA transcripts per cell for AOA was similar to values reported by Church and colleagues (2010) and a laboratory study of N. maritimus (0.01 to 2.6 per cell; Nakagawa and Stahl 2013) but was much higher than that reported from the Black Sea (Lam et al 2007).…”

“…In the present study, although AOA amoA gene copy numbers outnumbered those of AOB, higher cellular transcript numbers were observed for AOB (Table 4). This may reflect the larger cell size of AOB (Lam et al 2007) or, as suggested by our earlier studies of ammonia-starved N. europaea, the greater stability of AOB transcripts in the absence of cell growth (Berube et al 2007). Thus, a direct association between transcript abundance and activity for different groups of microorganisms in nature must be viewed with caution (Lam et al 2007;Prosser and Nicole 2008).…”

Integrated metatranscriptomic and metagenomic analyses of stratified microbial assemblages in the open ocean

“…The normalized expression levels of Crenarchaea-related amt and amoABC genes (especially amoC gene) was among the highest in our data sets (orders of magnitude higher than most other protein-coding genes) (Figure 3b). Interestingly, the anomalously high amoC gene expression seems to be common, as it is also observed in bacterial nitrifiers (Berube et al, 2007), for as-yet unknown reasons. Consistent with a quantitative PCR-based study (Church et al, 2010), the amoABC transcripts were detected in high abundance at 125 m depth despite the small planktonic crenarchaeal population size (Supplementary Figure S4B, right panel).…”

Mutagenesis of the hydrocarbon monooxygenase indicates a metal centre in subunit-C, and not subunit-B, is essential for copper-containing membrane monooxygenase activity

“…Methanotrophs and nitrifiers commonly contain multiple alleles of CuMMO genes and additional copies of the gene encoding subunit-C appear to be essential in some cases (Berube & Stahl, 2012;Berube et al, 2007;Stolyar et al, 1999). The extent of sequence divergence between operons varies, but in at least one case this allelic variation represents kinetically distinct forms.…”