AntR-mediated bidirectional activation of antA and antR, anthranilate degradative genes in Pseudomonas aeruginosa
Highlights
► AntR activates two divergent genes, antA and antR bidirectionally. ► Two AntR-responsive elements (AREs) exist between antA and antR. ► AntR binds to AREs with different affinity and transcribes two genes asymmetrically. ► Both AREs are important for the transcriptional activation in both directions.
Introduction
While transcription has been long considered to be promoted in one direction from a promoter, a recent study showed the interesting result that many transcriptions are bidirectionally promoted by transcription machinery (Xu et al., 2009). In bacteria, it has been reported that some transcriptions are bidirectionally regulated by transcription factors, such as LasR and QscR, quorum sensing regulators of Pseudomonas aeruginosa, which activate two divergent genes bidirectionally on a single binding site (Ha et al., 2012, Schuster et al., 2004). In this study, we report that AntR, a transcriptional activator involved in anthranilate metabolism regulates genes bidirectionally.
Members of the genus Pseudomonas have remarkable metabolic versatility and ubiquitously colonize a wide variety of terrestrial and aquatic habitats (Silby et al., 2011). They are of great interest because of their metabolic potentials to produce or degrade diverse molecules, and their importance in the pathogenic infection. One of them, P. aeruginosa is an opportunistic pathogen that causes serious infections in nematodes, insects, plants, animals, and humans, especially immunocompromised individuals, such as burn victims, and in cystic fibrosis patients (Silby et al., 2011, Van Delden and Iglewski, 1998). This organism produces various metabolites, such as phenazines, pyocyanin, quinolones, acyl-homoserine lactones, anthranilate, and so on, and most of them are secreted and accumulated during growth, to which medicinal and industrial attentions are paid for clinical considerations and biotechnological applications.
Among the metabolites, anthranilate is an important intermediate for the synthesis of tryptophan and Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxyl-4-quinolone), and degradation of aromatic compounds, such as tryptophan toward the tri-carboxylic acid (TCA) cycle in Pseudomonas spp. (Choi et al., 2011, Pesci et al., 1999, Phillips, 2011). Since tryptophan is an essential amino acid for growth, PQS is a quorum sensing (QS) system that importantly works for P. aeruginosa physiology (Diggle et al., 2007), and the degradation of an aromatic compound is a prerequisite for the metabolic versatility of Pseudomonas spp., the regulation of anthranilate metabolism is very significant and finely tuned for the Pseudomonas physiology. In P. aeruginosa, anthranilate is produced by biosynthesis from chorismate by anthranilate synthases, or alternatively supplied by the degradation of tryptophan through kynurenine pathway (Farrow and Pesci, 2007).
The degradation of anthranilate in Pseudomonas spp. is achieved by anthranilate dioxygenase complex which is involved in the conversions of anthranilate to catechol (Chang et al., 2003, Urata et al., 2004). antABC operon encodes the anthranilate dioxygenase complex and it has been reported that the expression of antA is induced by anthranilate in Acinetobacter (Bundy et al., 1998). The transcriptional activator responsible for the antA regulation is AntR in P. aeruginosa and a carbazole-degradative plasmid pCAR1 of Pseudomonas resinovorans (Oglesby et al., 2008, Urata et al., 2004). AntR encoded by antR is a LysR-type regulator that uses anthranilate as a ligand to be activated (Oglesby et al., 2008, Urata et al., 2004). The activated AntR with anthranilate binds to the antA promoter (antAp) and activates the transcription of antABC (Oglesby et al., 2008). This means that the anthranilate degradation can be triggered by anthranilate itself in a feed-back regulation.
As an important intermediate at a metabolic branch point, the regulation of anthranilate metabolism is complexly intertwined with QS response in P. aeruginosa. In a previous study, we showed that antA is repressed by LasR, a QS regulator during log phase and activated by RhlR, another QS regulator in a late stationary phase, whereas pqs operon for PQS synthesis is activated by LasR in the log phase and repressed by RhlR (Choi et al., 2011). We also showed that QscR, a third QS regulator represses both antA and pqs operons in a different growth phase (Choi et al., 2011). This complex growth phase differential regulation of antABC by QS regulators is mediated by AntR, as a direct regulator of antAp (Choi et al., 2011, Oglesby et al., 2008).
In P. aeruginosa, antABC operon and antR gene are divergently located on a chromosome (Fig. 1B). In this study, to better understand the transcriptional regulation of antABC by AntR, we mapped a transcriptional start site and the AntR-regulatory element of antAp. We found that AntR activates two divergent genes, antA and antR bidirectionally, and there are two AntR-responsive elements (AREs) in the intergenic region between antA and antR. We also suggest here that two AREs have different affinities to AntR and both are important in the asymmetric and bidirectional activation of antA and antR.
Section snippets
Plasmids and bacterial strains and growth conditions
Bacterial strains and plasmids used in this study were listed in Table 1. Bacteria were grown in Luria–Bertani (LB) broth at 37 °C with vigorous shaking and the growth was monitored by optical density at 600 nm (OD600). Antibiotics were used at the following concentrations: ampicillin, 50 μg/ml; carbenicillin, 150 μg/ml; and gentamicin, 12.5 μg/ml (for Escherichia coli) or 60 μg/ml (P. aeruginosa). To prepare the Pseudomonas spent medium, PAO1 cells were grown up to OD600 of 3, cells were removed by
Structure of antA promoter and location of AntR-responsive element
A previous study showed that AntR directly binds to and activates the antAp in the presence of anthranilate (Choi et al., 2011, Oglesby et al., 2008). To precisely know the locations of the promoter and AntR-responsive elements (AREs) of antAp, we determined the transcriptional start site of antAp by primer extension analysis. As shown in Fig. 1A, a transcriptional start site (+ 1) that was highly inducible at a late stationary phase was detected and well-conserved RpoD (σ70)-type − 10 and − 35
Discussion
antABC and antR are divergently located in P. aeruginosa and involved in the conversions of anthranilate to catechol and its regulation, respectively. This study provides a report about the bidirectional transcription mechanism of antABC and antR by AntR in P. aeruginosa. The intergenic region between antR and antA is 316 bp long and two genes could possibly share the upstream regulatory element. The data presented in this study demonstrate that the AntR-control of two genes is mediated by two
Acknowledgment
This work was supported by the Pusan National University (PNU, Bio-Scientific Research Grant) (PNU-2010-101-239).
References (25)
The Pseudomonas aeruginosa 4-quinolone signal molecules HHQ and PQS play multifunctional roles in quorum sensing and iron entrapment
Chem. Biol.
(2007)- et al.
Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains
Plasmid
(2000) - et al.
Broad-host-range expression vectors that carry the l-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator
Gene
(1999) The influence of iron on Pseudomonas aeruginosa physiology: a regulatory link between iron and quorum sensing
J. Biol. Chem.
(2008)Structure, mechanism, and substrate specificity of kynureninase
Biochim. Biophys. Acta
(2011)- et al.
Similarities between the antABC-encoded anthranilate dioxygenase and the benABC-encoded benzoate dioxygenase of Acinetobacter sp. strain ADP1
J. Bacteriol.
(1998) - et al.
Characterization and regulation of the genes for a novel anthranilate 1,2-dioxygenase from Burkholderia cepacia DBO1
J. Bacteriol.
(2003) Growth phase-differential quorum sensing regulation of anthranilate metabolism in Pseudomonas aeruginosa
Mol. Cells
(2011)- et al.
QscR, a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa
Proc. Natl. Acad. Sci. U. S. A.
(2001) - et al.
Construction of broad-host-range plasmid vectors for easy visible selection and analysis of promoters
J. Bacteriol.
(1990)
Two distinct pathways supply anthranilate as a precursor of the Pseudomonas quinolone signal
J Bacteriol.
Expression of the TOL plasmid xylS gene in Pseudomonas putida occurs from a alpha 70-dependent promoter or from alpha 70- and alpha 54-dependent tandem promoters according to the compound used for growth
J. Bacteriol.
Cited by (16)
Identification of a repressor and an activator of azoreductase gene expression in Pseudomonas putida and Xanthomonas oryzae
2018, Biochemical and Biophysical Research CommunicationsCitation Excerpt :LTTRs can act as either activators or repressors [15]. Recent examples of a LTTRs that function as activators are AntR, which positively regulates antA and antR, anthranilate degradative genes in P. aeruginosa [19], and HsdR controlling steroid degradation via 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) in Comamonas testosterone [20]. LTTRs as repressors are also reported as RovM in Yersinia pestis which involved in invasion, motility, and virulence [21], and CatR which used cis, cis-muconate as co-factor to regulate catechol catabolism in P. putida [22].
Modulation of QscR, a quorum sensing receptor of Pseudomonas aeruginosa, by truncation of a signal binding domain
2013, Research in MicrobiologyCitation Excerpt :Two parental plasmids, pJN105 and pJL42, were used to construct the plasmids for in vivo expression of wild type QscR (QscRwt) and its N-terminal truncated version, QscR160–237 (Table 1). Protein expression using pJN105 and pJL42 systems has been previously published elsewhere (Elsen et al., 2011; Kim et al., 2012; Lee et al., 2006; Lequette et al., 2006; Petrova and Sauer, 2011; Rampioni et al., 2007). Plasmids overexpressing the differently truncated QscRs, pJN105Q150–237, pJN105Q160–237 and pJN105Q170–237 were constructed in the same way as pJN105Q; the QscRwt-overexpressing plasmid had been constructed previously (Lee et al., 2006).
ReComBat: Batch-effect removal in large-scale multi-source gene-expression data integration
2022, Bioinformatics AdvancesThe arac-type transcriptional regulator glir (Pa3027) activates genes of glycerolipid metabolism in pseudomonas aeruginosa
2021, International Journal of Molecular Sciences
- 1
These authors equally contributed to this work.