A Periplasmic Complex of the Nitrite Reductase NirS, the Chaperone DnaK, and the Flagellum Protein FliC Is Essential for Flagellum Assembly and Motility in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a ubiquitously occurring environmental bacterium and opportunistic pathogen responsible for various acute and chronic infections. Obviously, anaerobic energy generation via denitrification contributes to its ecological success. To investigate the structural basis for the interconnection of the denitrification machinery to other essential cellular processes, we have sought to identify the protein interaction partners of the denitrification enzyme nitrite reductase NirS in the periplasm. We employed NirS as an affinity-purifiable bait to identify interacting proteins in vivo. Results obtained revealed that both the flagellar structural protein FliC and the protein chaperone DnaK form a complex with NirS in the periplasm. The interacting domains of NirS and FliC were tentatively identified. The NirS-interacting stretch of amino acids lies within its cytochrome c domain. Motility assays and ultrastructure analyses revealed that a nirS mutant was defective in the formation of flagella and correspondingly in swimming motility. In contrast, the fliC mutant revealed an intact denitrification pathway. However, deletion of the nirF gene, coding for a heme d 1 biosynthetic enzyme, which leads to catalytically inactive NirS, did not abolish swimming ability. This pointed to a structural function for the NirS protein. FliC and NirS were found colocalized with DnaK at the cell surface of P. aeruginosa. A function of the detected periplasmic NirS-DnaK-FliC complex in flagellum formation and motility was concluded and discussed. IMPORTANCEPhysiological functions in Gram-negative bacteria are connected with the cellular compartment of the periplasm and its membranes. Central enzymatic steps of anaerobic energy generation and the motility mediated by flagellar activity use these cellular structures in addition to multiple other processes. Almost nothing is known about the protein network functionally connecting these processes in the periplasm. Here, we demonstrate the existence of a ternary complex consisting of the denitrifying enzyme NirS, the chaperone DnaK, and the flagellar protein FliC in the periplasm of the pathogenic bacterium P. aeruginosa. The dependence of flagellum formation and motility on the presence of an intact NirS was shown, structurally connecting both cellular processes, which are important for biofilm formation and pathogenicity of the bacterium. P seudomonas aeruginosa is a metabolically versatile bacterium inhabiting multiple environmental niches (1). It is known for its highly efficient growth in the absence of oxygen. Fast anaerobic growth is mediated via the utilization of different N-oxides as electron acceptors in the respiratory chains of denitrification (2). Anaerobic respiratory growth via denitrification also sustains biofilm formation on environmental surfaces, in the mucus in the lung of cystic fibrosis patients, on the epithelium of the urinary tract infected individuals, and in burn wounds (3).The periplasm, the cellular compartment bounded by the cytoplasmi...

Origin and Impact of Nitric Oxide in Pseudomonas aeruginosa Biofilms

“…Very recently, NIR was also shown to control flagellum production and swimming motility under anaerobic conditions, independently from its ability to produce NO, by serving as a scaffold to form a ternary complex with the chaperone DnaK and the flagellar protein FliC in the periplasm (66). In the same study, the nirS mutant showed a partial restoration of swimming ability under aerobic conditions, whereas swarming mobility was previously shown to be affected under aerobiosis (63).…”

“…An intriguing possibility suggested by the latter study, which will require further investigation, is that NIR promotes motility in different ways, depending on oxygen availability, either by producing NO to increase rhamnolipid synthesis or activate other signaling pathways, or by directly controlling flagellum formation by protein-protein interactions. As underlined by Borrero-de Acuna et al (66), to fully understand these phenomena, the assembly of flagella under aerobic and anaerobic conditions will have to be analyzed in further detail. Since it is known that NO transcriptionally activates nirS expression, the production of a catalytically inactive version Ϫ ) is reduced to molecular nitrogen (N 2 ) in four reductive steps, each catalyzed by a specific reductase, namely, nitrate reductase (NAR), nitrite reductase (NIR), NO reductase (NOR), and nitrous oxide reductase (N 2 OR).…”

Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

“…Afterward, the bacteria were dehydrated and subsequently embedded in Lowicryl K4M resin. After polymerization by ultraviolet light treatment, ultrathin sections were cut with a diamond knife, collected onto Butvar-coated nickel grids, and incubated with specific antibodies (44). First, sections were incubated with the first round of purified IgG antibodies overnight at 5°C.…”

“…The P. aeruginosa strains were grown to the exponential growth phase (optical density at 578 nm [OD 578 ] of 0.8). The protein complexes were cross-linked in vivo as previously described (44). Formaldehyde, used as the cross-linking agent, was injected into the anaerobic denitrifying culture in the late stationary phase to a final concentration of 0.125% (vol/vol).…”

“…1) (40-43). Recently, we described the existence of a stable NirS-DnaK-FliC complex in the periplasm of P. aeruginosa that was involved in flagellum assembly, using a combined membrane and periplasmic proteomics and electron microscopy approach (44). Here, we employed this experimental approach (45)(46)(47) to determine the nature of the protein-protein interactions of the denitrification machinery.…”