Regulation of phenylacetic acid uptake is σ54 dependent in Pseudomonas putidaCA-3
© O' Leary et al; licensee BioMed Central Ltd. 2011
Received: 27 April 2011
Accepted: 13 October 2011
Published: 13 October 2011
Styrene is a toxic and potentially carcinogenic alkenylbenzene used extensively in the polymer processing industry. Significant quantities of contaminated liquid waste are generated annually as a consequence. However, styrene is not a true xenobiotic and microbial pathways for its aerobic assimilation, via an intermediate, phenylacetic acid, have been identified in a diverse range of environmental isolates. The potential for microbial bioremediation of styrene waste has received considerable research attention over the last number of years. As a result the structure, organisation and encoded function of the genes responsible for styrene and phenylacetic acid sensing, uptake and catabolism have been elucidated. However, a limited understanding persists in relation to host specific regulatory molecules which may impart additional control over these pathways. In this study the styrene degrader Pseudomonas putida CA-3 was subjected to random mini-Tn5 mutagenesis and mutants screened for altered styrene/phenylacetic acid utilisation profiles potentially linked to non-catabolon encoded regulatory influences.
One mutant, D7, capable of growth on styrene, but not on phenylacetic acid, harboured a Tn5 insertion in the rpoN gene encoding σ54. Complementation of the D7 mutant with the wild type rpoN gene restored the ability of this strain to utilise phenylacetic acid as a sole carbon source. Subsequent RT-PCR analyses revealed that a phenylacetate permease, PaaL, was expressed in wild type P. putida CA-3 cells utilising styrene or phenylacetic acid, but could not be detected in the disrupted D7 mutant. Expression of plasmid borne paaL in mutant D7 was found to fully restore the phenylacetic acid utilisation capacity of the strain to wild type levels. Bioinformatic analysis of the paaL promoter from P. putida CA-3 revealed two σ54 consensus binding sites in a non-archetypal configuration, with the transcriptional start site being resolved by primer extension analysis. Comparative analyses of genomes encoding phenylacetyl CoA, (PACoA), catabolic operons identified a common association among styrene degradation linked PACoA catabolons in Pseudomonas species studied to date.
In summary, this is the first study to report RpoN dependent transcriptional activation of the PACoA catabolon paaL gene, encoding a transport protein essential for phenylacetic acid utilisation in P. putida CA-3. Bioinformatic analysis is provided to suggest this regulatory link may be common among styrene degrading Pseudomonads.
Results and Discussion
Random Tn5mutant library screening and characterisation
Identification and complementation of the rpoNgene disruption
The insertion site of the mini-Tn5 transposon was mapped using two consecutive rounds of arbitrary PCR and the resulting amplicons sequenced and analysed using the GenBank, BLASTn algorithm. The chromosomal region immediately downstream of the Tn5 insertion displayed over 98% sequence similarity to rpoN genes from other P. putida strains, suggesting the gene was disrupted in mutant D7. The nucleotide sequence of the full gene was subsequently generated and submitted to Genbank under the accession number HM756586. In P. putida KT2440 the rpoN gene forms part of an operon with 4 putative downstream genes encoding members of the phosphotransferase system, including ptsN and ptsO . While such an operonic structure has not been demonstrated for P. putida CA-3, the possibility existed that the observed phenylacetic acid negative phenotype of the D7 mutant may in fact have been as a result of downstream pleiotropic effects of the Tn5 insertion in rpoN. However, complementation of the disrupted rpoN with the cloned, full length wild type gene, (D7-RpoN+), was found to completely restore the strain's ability to grow on styrene and phenylacetic acid, respectively, Figure 2(a) and 2(b). Thus a σ54 deficiency in D7 appeared to be the primary factor responsible for the loss of phenylacetic acid utilisation in this strain. Control experiments with P. putida CA-3 wild type and D7 strains carrying the pBBR1MCS-5 expression vector without insert, revealed that the growth profiles presented in Figure 2 were not affected by plasmid maintenance demands or antibiotic presence in the respective media, (results not shown).
RT-PCR of PACoA catabolon genes in wild type P. putida CA-3 and rpoNdisrupted D7 mutant strains
Over-expression of PaaL in wild type P. putida CA-3 and rpoNdisrupted D7 mutant strains
Cloning and bioinformatic analysis of the paaL promoter from P. putidaCA-3
Relative sequence identities of paaL genes and promoters from diverse Pseudomonasspecies
Clustal W alignment of microbial paaL genes and promoters.
Percentage Sequence Identity
To our knowledge this is the first study to report σ54 dependent regulation of PaaL expression in phenylacetic acid utilisation by a Pseudomonas species. Since other groups have previously suggested σ70 dependent regulation of the transport system, [5, 10, 12, 20] we questioned whether such regulation might be unique to P. putida CA-3, or have a potentially broader significance in the field of styrene/phenylacetic acid microbial catabolism. Our analyses of the genetic diversity of paaL genes and promoters suggest that a relatively recent recombination event involving de novo clustering of paa genes  with the sty operon may have occurred. In this scenario, incorporation of the σ54 dependent regulation of paaL may have been an arbitrary event, following the "black cat/white cat" random promoter association model proposed by Cases and de Lorenzo in relation to novel catabolic pathways . However, irrespective of the origins of σ54 regulation of paaL, the identical promoter structures suggest that biotechnological applications targeting this pathway should consider the potential for a functional role of σ54 dependent regulation in phenylacetic acid assimilation by these strains.
Bacterial strains, plasmids and growth conditions
P. putida CA-3 is a styrene degrading, bioreactor isolate previously characterised by our group . Cultures were maintained on LB agar for use in overnight inoculations into cultivation media. P. putida CA-3 was routinely grown in 100 ml of liquid minimal salt media in 1 L flasks at 30°C, shaking at 120 rpm. The basal salts media contained 7.0 g K2HPO4, 3.0 g KH2PO4, 1.0 g (NH4)2SO4 per litre distilled water, and 2 ml of 1 M MgSO4 added post autoclaving. Carbon sources were added to the following concentrations; 15 mM phenylacetic acid and 10 mM citrate. Growth on styrene required substrate provision in the gaseous phase via addition of 70 μl of liquid styrene to a test tube fixed centrally to the bottom of a baffled 1 L Erlenmeyer flask . Cell growth was monitored by measuring optical density at 540 nm. E. coli CC118λpir hosted the mini-Tn5 derivative pUTKm1 . The suicide plasmid has the R6K origin of replication and encodes resistance to kanamycin and ampicillin. HB101 (pRK600) was used as a helper in triparental mating experiments, providing both resistance to chloramphenicol and the tra function for pUTKm1 mobilization . PCR2.1-TOPO vector was used to clone polymerase chain reaction (pcr) amplification products and transformations performed with One shot® Top10F' competent E. coli cells, (Invitrogen, California). E. coli strains were grown on Luria Burtani medium at 37°C. Host/plasmid associations were maintained during growth via the incorporation of appropriate antibiotics to media at the following concentrations; 100 μg/ml ampicillin, 25 μg/ml chloramphenicol, 50 μg/ml kanamycin and 20 μg/ml gentamycin.
Nucleic acid manipulations
Primers for PCR amplifications.
Styrene monooxygenase activity was assessed colorimetrically using whole cell transformations of indole to indigo as previously described . PACoA ligase activity was measured via the method of Martinez-Blanco et al . Activities are expressed as nmol product formed min-1 (mg cell dry weight)-1 for both assays. Cells were harvested at mid-exponential phase unless otherwise stated.
A triparental mating approach was used to introduce pUTKm1 into P. putida CA-3, as previously described . The mating reaction was plated out on minimal salts media containing 10 mM citrate and 50 μg/ml kanamycin to select for P. putida CA-3 transconjugants harbouring successful, mini-Tn5 genomic insertions. 12, 500 transconjugants were screened for transposition events that disrupted phenylacetic acid metabolism on solid minimal media containing 15 mM phenylacetic acid and kanamycin 50. Transconjugants which failed to grow on phenylacetic acid were subsequently screened for an ability to utilise styrene as a sole carbon source.
Mapping of transposon insertion sites
Arbitrarily primed PCR was employed to map the gene disruption sites utilising previously published oligonucleotide sequences and appropriate thermal cycling parameters . Products were visualised on 1% agarose gels, purified using a QIAEX II Gel extraction kit and sequenced using the mini-Tn5 internal primer, TNInt2 (Table 2).
RNA was isolated from P. putida CA-3 using a Qiagen RNeasy® Mini Kit, as per the manufacturer's instructions. The purified RNA was treated with TURBO DNA-free™ DNase kit, (Ambion), to ensure complete removal of DNA. All RNA samples were routinely subjected to 16S rRNA gene PCR to confirm the absence of DNA contamination. Reverse transcription was performed with 1 μg of total RNA using random hexamer priming, 1 mM dNTPs, 10 U Transcriptor reverse transcriptase with 1× reaction buffer, (Roche), and SUPERNaseIn (Ambion) in a 20 μl reaction volume. Reactions were incubated at 25°C for 10 minutes, followed by 30 minutes at 55°C. 2 μl of the respective RT reactions were employed as template in subsequent PCR reactions. Amplification of the 16S rRNA gene acted as positive control for RT-PCR analyses (universal primers 27f, 1429r), while the following pathway operon specific targets were selected for transcriptional profiling; paaF encoding PACoA ligase, paaG encoding a member of the ring hydroxylation complex, and the paaL encoding phenylacetate permease. Oligonucleotide sequences for the respective gene targets are provided in Table 2.
Complementation of the RpoN disrupted mutant
Available nucleotide sequences of rpoN genes from P. putida species were retrieved from the GenBank database and used to construct degenerate primers for the amplification of rpoN from P. putida CA-3. Restriction sites were mis-primed into the oligonucleotides, (Sig54f-Hind and Sig54r-Xba, respectively), to allow directional cloning into the pBBR1MCS-5 expression vector enabling lac promoter expression . Amplification of the desired rpoN target was confirmed by sequencing, prior to enzymatic restriction and ligation using standard conditions (GenBank accession no. HM756586). Transformations were carried out with Top 10F' competent E. coli cells, (Invitrogen, California), in accordance with the manufacturer's instructions. The pBBR1MCS-5 vector facilitated blue/white colony screening on LB-IPTG-β-gal-Gent20 medium to identify successful cloning events, which were confirmed by culturing, plasmid isolation and restriction with HindIII and XbaI. Conjugal transfer of this RpoN expression vector into P. putida CA-3 D7 (carrying a Tn5::rpoN gene disruption), was performed by tri-parental mating with the Top 10F' E. coli host and the HB101(pRK600) helper, as previously described. P. putida CA-3 D7 transconjugants were isolated from the mating mix by spread plating 50 μl aliquots onto minimal salts media containing10 mM citrate and 20 μg/ml gentamycin. The pBBR1MCS-5 vector, (lacking any insert), was also transferred into P. putida CA-3 wild type and D7 mutant strains to provide controls for subsequent growth studies. All growth curves were conducted in triplicate.
Cloning and over expression of the phenylacetate permease, PaaL
Degenerate paaL primers, harbouring similar mis-primed restriction enzyme sites as before (paaLf-Hind & paaLr-Xba, Table 2), were designed based on sequence data from P. fluorescens ST and Pseudomonas sp. Y2, [20, 22]. Cloning, screening and vector/insert confirmation in the Top 10F' E. coli host was conducted as described previously. Tri-parental mating to achieve conjugal transfer of the vector into rpoN disrupted P. putida CA-3 cells was also performed as before. Transconjugants were subsequently screened for any restoration of the ability to grow in minimal salts media with phenylacetic acid as the sole carbon source. To determine whether strict regulation of PaaL expression represented a rate limiting feature of extracellular phenylacetic acid utilisation in wild type P. putida CA-3, the PaaL expression vector was also conjugally transferred into the parent strain. RT-PCR analysis was employed to confirm constitutive expression of PaaL from the vector under non inducing growth on minimal salts citrate. Over expression strains were subsequently grown in minimal salts media with phenylacetic acid to facilitate growth profiling and PACoA ligase activity determination. All growth curves were conducted in triplicate. It should be noted that a degenerate pcr strategy was employed to screen the P. putida CA-3 genome for a paaM permease gene homologue, but none was detected.
Isolation and analysis of the paaLpromoter
Primers were designed to amplify the promoter region of the paaL gene based on the sequence data of the PACoA catabolon of Pseudomonas sp. strain Y2. The primer set (paaLproF and paaLproR, Table 2), amplified a 964 base pair region spanning the 3' end of the paaG gene, the intergenic region and the 5' end of paaL. The complete paaL gene and promoter region have been submitted to GenBank, (Accession number HM638062). A number of putative σ54 dependent promoters of transport proteins from the P. putida KT2440 genome, , were comparatively analysed with the CA-3 paaL promoter using the MEME software suite and the TOMTOM motif comparison tool to identify any highly conserved motifs, [29, 30]. 5'RACE primer extension analysis (Ambion) was also carried out to map the paaL transcriptional start site, as per the manufacturer's instructions. In brief, this approach involved the generation of 5' adapter ligated RNA, reverse transcription with random decamers and PCR amplification from cDNA using 5' adapter specific and 3' gene specific primers, OP2-55 and GS-441 (Table 2). The PCR thermal cycling conditions included a 5 min hot start at 94°C, followed by 45 cycles of 94°C × 60 s, 55°C × 45 s and 72°C × 30 s.
This work was funded by the Science, Technology, Research and Innovation for the Environment 2007-2013 (STRIVE) Fellowship programme of the Irish Environmental Protection Agency. (Grant No: 2007-FS-ET-9-M5).
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