The alternative oxidase (AOX) gene in Vibrio fischeri is controlled by NsrR and upregulated in response to nitric oxide

Abstract

Alternative oxidase (AOX) is a respiratory oxidase found in certain eukaryotes and bacteria; however, its role in bacterial physiology is unclear. Exploiting the genetic tractability of the bacterium Vibrio fischeri, we explore the regulation of aox expression and AOX function. Using quantitative PCR and reporter assays, we demonstrate that aox expression is induced in the presence of nitric oxide (NO), and that the NO-responsive regulatory protein NsrR mediates the response. We have identified key amino acid residues important for NsrR function and experimentally confirmed a bioinformatically predicted NsrR binding site upstream of aox. Microrespirometry demonstrated that oxygen consumption by V. fischeri CydAB quinol oxidase is inhibited by NO treatment, whereas oxygen consumption by AOX is less sensitive to NO. NADH oxidation assays using inverted membrane vesicles confirmed that NO directly inhibits CydAB, and that AOX is resistant to NO inhibition. These results indicate a role for V. fischeri AOX in aerobic respiration during NO stress.

Figure 1

A. DNA sequence upstream of aox in V. fischeri ES114 (ORF VF_0578). The sequence begins with the start codon for the oppositely transcribed gene (VF_0577) and ends with the start codon for VF_0578, which are marked with bold text. The putative NsrR binding site is underlined, with arrows indicating the inverted repeats. The mapped transcriptional start site is indicated in bold lower case. B. The putative NsrR binding site in wild type is compared to base pair changes introduced via site-directed mutagenesis (indicated by underlined lower case bold font) and insertions found in suppressor mutants (underlined bold font). These modified sequences were used in the in vivo LacZ-based binding assays (Figure 2).

Figure 2

In vivo assay for NsrR binding to the aox promoter region. β-galactosidase assays were performed on AKD712 (chromosomal aox-lacZ fusion strain) containing either an empty control plasmid (pAKD700), pAKD700 containing approximately 200 bp of the DNA sequence upstream of the start codon encoded by aox (WT; pAKD750), pAKD700 containing a PCR-modified aox promoter region (modified; pAKD751), and pAKD700 containing the aox promoter region PCR-amplified from either suppressor mutant OG4 or OG1-10. Sequences are shown in Fig. 1. Plasmids are maintained at ∼10 copies per chromosome, and when they contain an NsrR binding site will effectively titrate out NsrR, relieving repression of aox transcription and resulting in production of LacZ. Cells were grown to mid-log phase in mineral-salts medium containing GlcNAc prior to harvesting for the assay. Data are the average of three independent cultures from one representative experiment. Error bars indicate standard error of the mean. The experiment was repeated three times. Asterisks indicate significant differences in mean activity compared to the pAKD700 control as determined using a student’s t-test. ** p < 0.01; * p<0.05

Figure 3

In vivo assay for NsrR activity. β-galactosidase assays were performed on AKD785 (nsrR mutant, chromosomal aox-lacZ fusion strain) in which NsrR variants were overexpressed. nsrR genes from wild type and ΔcydAB suppressor mutants were cloned into pAKD601B, placing expression under the control of an IPTG-inducible promoter. Cells were grown in LBS medium overnight in the presence of kanamycin and IPTG before harvesting for the assay. High levels of β-galactosidase activity indicate a non-functional version of NsrR that can no longer bind to the aox promoter region and repress expression of lacZ. Bars are labeled with the name of the suppressor mutant from which the variant nsrR was isolated, along with the wild-type (WT) and empty plasmid controls. Data are the average of three independent cultures from one representative experiment. Error bars indicate standard error of the mean. Asterisks indicate significant differences in mean activity compared to the wild-type control as determined using a student’s t-test (p < 0.01). The experiment was repeated three times with similar results.

Figure 4

Oxygen consumption profiles for V. fischeri strains demonstrating the response to treatment with 80 µM DEA NONOate. Cells were grown in mineral salts medium containing GlcNAc to mid-log phase and placed in the respirometer chamber. DEA NONOate was injected into the chamber at an oxygen concentration of 190 µmol/L (indicated by the arrow). A. Profiles for wild type (solid line; OD₆₀₀ of 0.310, total protein content of 1.29 mg) and the ΔnsrR strain AKD711 (dashed line; OD₆₀₀ of 0.304, total protein content of 1.28 mg). The oxygen consumption profiles for AKD780 (aox mutant) and AKD786 (aox nsrR double mutant) were not consistently different from those of the wild type and nsrR mutant, respectively (data not shown). B. Profiles for AKD788 (expressing only CydAB; solid line; OD₆₀₀ of 0.299, total protein content of 1.25 mg) and AKD789 (expressing only AOX; dashed line; OD₆₀₀ of 0.311, total protein content of 1.27 mg). Graphs demonstrate oxygen consumption profiles for one set of cultures and are representative of three independent experiments.

Figure 5

NADH oxidation assays using inverted membrane vesicles from strains AKD788 (only expressed terminal oxidase CydAB; white bars) and AKD789 (only expressed terminal oxidase AOX; gray bars). Vesicles were prepared from aerobically grown log-phase cells and assayed at 23 °C. NADH oxidation rate is reported as µmol NADH oxidized per minute, per mg of protein. Samples were either not treated with exogenous chemicals (untreated), treated with 80 µM DEA NONOate (NO), or 3 mM potassium cyanide (KCN). Data presented are the average rates from three independent sets of cultures from two separate experiments performed on different days. Error bars represent standard error of the mean. Asterisks indicate statistically significant differences (p < 0.002) from the corresponding untreated samples as determined using a student’s t-test