Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation

Abstract

bRhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent K m and k cat values for PQS of ϳ27 M and 21 s ؊1 , respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases.A wide variety of bacteria employ quorum sensing (QS) systems to communicate and coordinate their behavior according to their cell density by sensing self-generated small signal molecules (1). QS systems allow bacteria to act cooperatively in processes such as biofilm formation or pathogenesis. The opportunistic pathogen Pseudomonas aeruginosa uses a complex QS network comprising several interconnected signaling circuits to regulate group motility, biofilm maturation, and a battery of virulence factors (for a recent review, see reference 2). The Las and Rhl circuits use the signal molecules N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL) and N-butanoyl homoserine lactone (C4-HSL), respectively, whereas the Pqs circuit employs the alkylquinolone (AQ) signals 2-heptyl-3-hydroxy-4(1H)-quinolone (Pseudomonas quinolone signal [PQS]) and 2-heptyl-4(1H)-quinolone (HHQ), which act as coinducers of the transcriptional regulator PqsR (3-6). The PqsR-AQ complex mainly activates the transcription of the pqsABCDE operon coding for the enzymes of AQ biosynthesis (7, 8); moreover, PqsE, via unknown mechanisms, modulates the expression of large arrays of target genes (9-11). PqsE was termed the "PQS response protein," because its disruption negatively affected the production of PQS-mediated exoproducts (3,9,12...

Interference with Pseudomonas aeruginosa Quorum Sensing and Virulence by the Mycobacterial Pseudomonas Quinolone Signal Dioxygenase AqdC in Combination with the N -Acylhomoserine Lactone Lactonase QsdA

“…The enzymes were purified to electrophoretic homogeneity by Ni-nitrilotriacetic acid (NTA) affinity chromatography and stored in 20 mM Tris buffer (pH 8) with 10% (vol/vol) glycerol at Ϫ80°C. Recombinant His-tagged HodC and AqdC1 were purified as described previously (15,30).…”

“…The catalytic activity of AqdC, AqdC1, and HodC was measured in a continuous spectrophotometric assay at 30°C, following consumption of PQS or other 2-alkyl-3-hydroxy-4(1H)quinolones at 337 nm (16). The assay buffer used in this study (50 mM Tris, 2 mM EDTA, 10% polyethylene glycol 1500 [PEG 1500], 4% dimethyl sulfoxide [DMSO] [pH 8]) differs from the one reported by Müller et al (15) and further improved PQS solubility. The extinction coefficient of PQS and its congeners in the assay buffer is 10,169 M Ϫ1 cm Ϫ1 .…”

“…Rue61a, which shows the highest activity toward the C 2 and C 1 congeners (Fig. 3A) (15). A major drawback of HodC for use as a quorum quenching enzyme, besides its low activity for PQS, is its degradation by extracellular proteases produced by P. aeruginosa (13,30).…”

“…3B). Nevertheless, among the PQS-cleaving enzymes described so far (13,15), AqdC is the most promising enzyme due to its high activity combined with a comparatively good persistence in P. aeruginosa cultures.…”

“…The soil isolate Rhodococcus erythropolis BG43 was the first bacterium identified to be capable of specifically degrading HHQ and PQS (14). Two gene clusters, termed aqdA1B1C1 and aqdRA2B2C2, are involved in HHQ and PQS degradation, and PQS dioxygenase activity was shown for the purified AqdC1 enzyme (15). Remarkably, a gene cluster homologous to aqdRA2B2C2 is also present in the genome of Mycobacteroides (formerly Mycobacterium) abscessus subsp.…”