Two-pronged survival strategy for the major cystic fibrosis pathogen, Pseudomonas aeruginosa, lacking the capacity to degrade nitric oxide during anaerobic respiration

Abstract

Protection from NO gas, a toxic byproduct of anaerobic respiration in Pseudomonas aeruginosa, is mediated by nitric oxide (NO) reductase (NOR), the norCB gene product. Nevertheless, a norCB mutant that accumulated approximately 13.6 microM NO paradoxically survived anaerobic growth. Transcription of genes encoding nitrate and nitrite reductases, the enzymes responsible for NO production, was reduced >50- and 2.5-fold in the norCB mutant. This was due, in part, to a predicted compromise of the [4Fe-4S](2+) cluster in the anaerobic regulator ANR by physiological NO levels, resulting in an inability to bind to its cognate promoter DNA sequences. Remarkably, two O(2)-dependent dioxygenases, homogentisate-1,2-dioxygenase (HmgA) and 4-hydroxyphenylpyruvate dioxygenase (Hpd), were derepressed in the norCB mutant. Electron paramagnetic resonance studies showed that HmgA and Hpd bound NO avidly, and helped protect the norCB mutant in anaerobic biofilms. These data suggest that protection of a P. aeruginosa norCB mutant against anaerobic NO toxicity occurs by both control of NO supply and reassignment of metabolic enzymes to the task of NO sequestration.

Proteomic, Microarray, and Signature-Tagged Mutagenesis Analyses of AnaerobicPseudomonas aeruginosaat pH 6.5, Likely Representing Chronic, Late-Stage Cystic Fibrosis Airway Conditions

“…Of the top 100 upregulated genes, the most induced class of anaerobic respiratory genes are norCBD, encoding subunits of the protective NOR. NOR functions to detoxify potentially harmful NO during anaerobic respiration in P. aeruginosa (63). Not surprisingly, these genes are conveniently localized in a predicted operon on the P. aeruginosa genome (Fig.…”

“…However, none of these specific PseudoCyc pathway categories reached statistical significance. Among the most logically repressed genes were those encoding two dioxygenases, homogentisate-1,2-dioxygenase (PA2009) and 4-hydroxyphenylpyruvate dioxygenase (PA0865), both of which are members of the tyrosine degradation pathway and dependent upon the presence of oxygen (63). Two other gene members of the tyrosine degradation pathway, maleylacetoacetate isomerase (PA2007) and fumarylacetoacetase (PA2008), were also downregulated.…”

“…Predictably, the norCBD genes, encoding the protective enzyme NOR, were induced under both conditions. We have recently shown that a P. aeruginosa mutant lacking NOR (e.g., norCB) grows abysmally slowly during anaerobic growth but does not die (63). This is due to an elegant two-pronged mechanism.…”

“…typically the most repressed proteins, namely, homogentisate-1,2-dioxygenase (HmgA) and 4-hydroxyphenylpyruvate dioxygenase (Hpd), both of which possess inherent NO-scavenging activity (63). Despite the fact that only NO 3 Ϫ and NO 2 Ϫ were used as alternative electron acceptors in these studies, genes/ proteins involved in the arginine deiminase pathway, including arcA/arcB, were also activated, especially transcriptionally, by NO 2 Ϫ (see Table S2 in the supplemental material), and proteomically using LC-MS (Table 2), confirming previous results indicating that some of the master anaerobic regulators, including ANR, DNR, and ArgR, are fully operative as regulators of the arginine deiminase pathway (20,31).…”

Anaerobiosis-Induced Loss of Cytotoxicity Is Due to Inactivation of Quorum Sensing in Pseudomonas aeruginosa

“…To enhance anaerobic growth, bacteria were inoculated in a flask with a stirrer bar in it and the flask was placed on top of the stirrer plate to allow homogeneous mixing. Anaerobic growth was supported by the addition of 0.4% KNO 3 to the culture medium (60).…”