The ubiquitous opportunistic pathogen Pseudomonas aeruginosa has five aerobic terminal oxidases: bo 3 -type quinol oxidase (Cyo), cyanide-insensitive oxidase (CIO), aa 3 -type cytochrome c oxidase (aa 3 ), and two cbb 3 -type cytochrome c oxidases (cbb 3 -1 and cbb 3 -2). These terminal oxidases are differentially regulated under various growth conditions and are thought to contribute to the survival of this microorganism in a wide variety of environmental niches. Here, we constructed multiple mutant strains of P. aeruginosa that express only one aerobic terminal oxidase to investigate the enzymatic characteristics and in vivo function of each enzyme. The K m values of Cyo, CIO, and aa 3 for oxygen were similar and were 1 order of magnitude higher than those of cbb 3 -1 and cbb 3 -2, indicating that Cyo, CIO, and aa 3 are low-affinity enzymes and that cbb 3 -1 and cbb 3 -2 are high-affinity enzymes. Although cbb 3 -1 and cbb 3 -2 exhibited different expression patterns in response to oxygen concentration, they had similar K m values for oxygen. Both cbb 3 -1 and cbb 3 -2 utilized cytochrome c 4 as the main electron donor under normal growth conditions. The electron transport chains terminated by cbb 3 -1 and cbb 3 -2 generate a proton gradient across the cell membrane with similar efficiencies. The electron transport chain of aa 3 had the highest proton translocation efficiency, whereas that of CIO had the lowest efficiency. The enzymatic properties of the terminal oxidases reported here are partially in agreement with their regulatory patterns and may explain the environmental adaptability and versatility of P. aeruginosa.

The ubiquitous opportunistic human pathogen Pseudomonas aeruginosa has five terminal oxidases for aerobic respiration and uses them under different growth conditions. Two of them are cbb 3 -type cytochrome c oxidases encoded by the gene clusters ccoN1O1Q1P1 and ccoN2O2Q2P2, which are the main terminal oxidases under high-and low-oxygen conditions, respectively. P. aeruginosa also has two orphan gene clusters, ccoN3Q3 and ccoN4Q4, encoding the core catalytic CcoN isosubunits, but the roles of these genes have not been clarified. We found that 16 active cbb 3 isoforms could be produced by combinations of four CcoN, two CcoO, and two CcoP isosubunits. The CcoN3-or CcoN4-containing isoforms were produced in the WT cell membrane in response to nitrite and cyanide, respectively. The strains carrying these isoforms were more resistant to nitrite or cyanide under low-oxygen conditions. These results indicate that P. aeruginosa gains resistance to respiratory inhibitors using multiple cbb 3 isoforms with different features, which are produced through exchanges of multiple core catalytic isosubunits.

Pseudomonas aeruginosa has one A-type (caa3) and multiple C-type (cbb3) cytochrome c oxidases as well as two quinol oxidases for aerobic respiration. The caa3 oxidase is highly efficient in creating a proton gradient across the cell membrane, but it is not expressed under normal growth conditions and its physiological role has not been investigated. In the present study, a mutant strain deficient in the coxBA-PA0107-coxC genes encoding caa3 exhibited normal growth under any test conditions, but it had low relative fitness under carbon starvation conditions, indicating that the expression of caa3 is advantageous under starvation conditions. A mutant that lacked four terminal oxidase gene clusters except for the cox genes was unable to grow aerobically because of low expression level of caa3. However, suppressor mutants that grew aerobically using caa3 as the only terminal oxidase emerged after aerobic subculturing. Analyses of the suppressor mutants revealed that a mutation of roxS encoding a sensor kinase of a two-component regulator RoxSR was necessary for the aerobic growth in synthetic medium. Two additional mutations in the 5′-flanking region of coxB were necessary for the aerobic growth in LB medium. Although the expression level of caa3 was higher in the suppressor mutants, their growth rates were lower than when the other terminal oxidases were utilized, suggesting that caa3 was not suited for utilization as the only terminal oxidase. Overexpression of the cox genes also inhibited the aerobic growth of the wild-type strain. These results indicate that caa3 is tightly regulated to be expressed only under starvation conditions at low level and it functions in cooperation with other terminal oxidases to facilitate survival in nutrient starvation conditions.

In the present study, we attempted to control the pH profile of the catalytic activity of the industrially relevant alkaline protease KP-43, by incorporating 3-nitro-l-tyrosine and 3-chloro-l-tyrosine at and near the catalytic site. Thirty KP-43 variants containing these non-natural amino acids at the specific positions were synthesized in Escherichia coli host cells with expanded genetic codes. The variant with 3-nitrotyrosine at position 205, near the substrate binding site, retained its catalytic activity at the neutral pH and showed a 60% activity reduction at pH 10.5. This reduction in the alkaline domain is desirable for enhancing the stability of the enzyme in the liquid laundary detergent, whereas the wild-type molecule showed a 20% increase in response to the same pH shift. The engineered pH dependency of the activity of the variant was ascribed partly to a lowered substrate affinity under the alkaline conditions, in which the incorporated 3-nitrotyrosine was probably charged negatively due to the phenolic pKa lower than that of tyrosine.

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