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 sigma(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 sigma(E) promoters, thus implicating sigma(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 DeltaamoC(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 sigma(E) and the coordinated regulation of catabolic and anabolic genes.

FIG. 1.

Primer extension analysis of amoC_1,2, amoC₃, and cbbL expression during exponential growth and recovery of N. europaea ATCC 19718 from 1, 6, and 21 days of starvation. (A) Nitrite accumulation and ratios of amoCp₂ transcripts to amoCp₁ transcripts (determined by densitometry) during recovery. (B) amoC_1,2 expression levels, as determined by densitometry, showing the contribution of each promoter to total amoC_1,2 abundance. (C, D, E) Primer extension analysis of amoC_1,2, amoC₃, and cbbL, respectively. Transcripts derived from each amoC_1,2 promoter are indicated at left.

FIG. 2.

(A) Primer extension analysis of amoC_1,2 following a 3-h recovery of a 1-month stationary-phase culture of N. europaea NCIMB 11850. Transcription start sites associated with each promoter are indicated at left. (B) amoC_1,2 expression levels, as determined by densitometry.

FIG. 3.

Transcription start sites, promoters, and predicted 5′ untranslated region secondary structures associated with amoC_1,2, amoC₃, glyA, rpsJ, and cbbL. Transcription start sites determined by primer extension and S1 analysis are indicated by open circles and diamonds, respectively. Putative σ⁷⁰ promoters are underlined and putative σ^ECF promoters are boxed. ΔG values for each structure were determined by mfold version 3.1 at a fixed temperature of 37°C. Start codons (AUG) and stop codons (UAA) are indicated as needed. The positions of the transcription start sites relative to the start codons are indicated next to each start site. Bacterial promoter consensus sequences were obtained from the review by Wösten (53).

FIG. 4.

Verification of the amoC₃ deletion. (A) PCR analysis of the recombination site. (B) Primers used (Table 1) and PCR products corresponding to each lane. (C) Primer extension analysis of amoC₃ expression in wild-type (WT) and ΔamoC₃ cells following 20 min of recovery in 25 mM NH₃ after 24 h of starvation.

FIG. 5.

ClustalW alignment of AmoC and PmoC amino acid sequences. Identities are highlighted in black, and similarities are highlighted in gray. Conserved residues involved in coordination of the tetrahedral metal binding site are indicated by asterisks. The proline substitutions in the AmoC₃ proteins from N. europaea ATCC 19718 and Nitrosomonas eutropha C71 are boxed.