Bacterial metabolism of 2-methylquinoline and naturally occurring 2-alkyl-4(1H) quinolones: (I) Transcriptional regulation of 2-methylquinoline degradation, (II) Bacterial strains and enzymes for the inactivation of 2-alkyl-4(1H)quinolones
Final Report Abstract
Arthrobacter sp. strain Rue61a is able to utilize 2-methylquinoline as source of carbon and energy. Previous work had suggested that 2-methylquinoline degradation is encoded by three catabolic operons located on the linear plasmid pAL1. Two PaaX-type regulators were proposed to be involved in control of these operons, which in view of the notion that PaaX proteins are restricted to the phenylacetate catabolon was unexpected. The first part of this project aimed at characterizing the transcriptional regulation of the degradation pathway and to fully characterize the pathway. The second part aimed at addressing the question of whether bacteria inactivate or degrade naturally occurring alkylquinolones (AQs), especially 2-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)- quinolone (PQS, the “Pseudomonas quinolone signal”), which are quorum sensing signalling molecules of Pseudomonas aeruginosa involved in the regulation of virulence factor expression. Our studies on the transcriptional regulation of 2-methylquinoline degradation showed that two pathway-specific repressors MeqR1 and MeqR2, which both respond to anthraniloyl-CoA, fine-tune the expression of genes coding for enzymes of quinaldine conversion to anthranilate (upper pathway) and anthranilate degradation via a CoA thioester pathway (lower pathway). MeqR1 appears to be the major repressor of the lower pathway operon. The upper pathway operon which comprises genes for 1H-4- oxoquinaldine conversion to anthranilate is mainly affected by MeqR2, while both MeqR proteins appear to have a similar impact on the operon coding for the first enzyme of the upper pathway. The expression of meqR2 is autoregulated by its own gene product and also by MeqR1, but meqR1 transcription is not affected by the MeqR proteins. The pathway-specific regulatory network appears to be embedded in a more complex system that also integrates information from other nutritional signals. In order to fully characterize the metabolic potential of strain Rue61a and to get access to candidate genes possibly involved in global control of gene expression, its complete genome was sequenced in cooperation with R. Daniel, and we performed physiological tests as well as an analysis of the CoA-metabolome (with J. Vorholt). The genome consists of a circular 4.7 Mbp chromosome, a circular 231 kbp plasmid which contributes to heavy metal resistance, and the linear plasmid pAL1. The genome reflects the saprophytic lifestyle and nutritional versatility of the organism and a strong adaptive potential to environmental stress. Strain Rue61a has a limited set of aromatic degradation pathways, enabling the utilization of aromatic carboxylic acids which are products of lignin depolymerization. Anthranilate utilization proceeds exclusively via a CoA thioester pathway. We observed that the dioxygenase Hod (1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase) from strain Rue61a catalyzes the cleavage of the P. aeruginosa signalling molecule PQS. Quorum quenching experiments indicated that Hod exogenously added to P. aeruginosa cultures downregulated the expression of key virulence factors, highlighting the potential of quenching AQ-dependent quorum sensing and hence virulence through enzymatic signal degradation (cooperation with P. Williams). However, as a cytoplasmic enzyme involved in 2-methylquinoline degradation, Hod is not a “natural quorum quenching enzyme”. To produce HHQ and other AQs for functional studies, we constructed a recombinant P. putida strain which expresses the pqsABCD genes for AQ biosynthesis. HHQ can be isolated by preparative HPLC of culture extracts. To generate tools for the functional screening of genomic libraries for genes encoding novel enzymes active towards AQ signalling molecules, we have constructed bioluminescent E. coli and P. putida reporter strains that respond to HHQ and PQS in the nM to µM range. These bioreporters can also be used in screenings for organic antagonists of PqsR, the key transcriptional regulator of PQS biosynthesis.
Publications
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(2009) Dioxygenase-mediated quenching of quinolone-dependent quorum sensing in Pseudomonas aeruginosa. Chem. & Biol. 16:1259−1267
Pustelny C, Albers A, Büldt-Karentzopoulos K, Parschat K, Chhabra SR, Camara M, Williams P, Fetzner S
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(2011) Synthesis and biotransformation of 2-alkyl-4(1H)-quinolones by recombinant Pseudomonas putida KT2440. Appl. Microbiol. Biotechnol. 91:1399−1408
Niewerth H, Bergander K, Chhabra SR, Williams P, Fetzner S
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(2012) Complete genome sequence and metabolic potential of the quinaldine-degrading bacterium Arthrobacter sp. Rue61a. BMC Genomics 13: 534
Niewerth H, Schuldes J, Parschat K, Kiefer P, Vorholt JA, Daniel R, Fetzner S
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(2013) A Pseudomonas putida bioreporter for the detection of enzymes active on 2-alkyl-4(1H)-quinolone signalling molecules. Appl. Microbiol. Biotechnol. 97:751−760
Müller C, Fetzner S
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(2013) The PaaX-type repressor MeqR2 of Arthrobacter sp. Rue61a involved in regulation of quinaldine catabolism binds to its own and to catabolic promoters and specifically responds to anthraniloyl-CoA. J. Bacteriol. 195:1068−1080
Niewerth H, Parschat K, Rauschenberg M, Ravoo BJ, Fetzner S
