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Madhushani, Anjana
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Publications (5 of 5) Show all publications
Wirebrand, L., Madhushani, A. W. K., Irie, Y. & Shingler, V. (2018). Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF600. Environmental Microbiology, 20(1), 186-199
Open this publication in new window or tab >>Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF600
2018 (English)In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 20, no 1, p. 186-199Article in journal (Refereed) Published
Abstract [en]

The dmp-system encoded on the IncP-2 pVI150 plasmid of Pseudomonas putida CF600 confers the ability to assimilate (methyl)phenols. Regulation of the dmp-genes is subject to sophisticated control, which includes global regulatory input to subvert expression of the pathway in the presence of preferred carbon sources. Previously we have shown that in P. putida, translational inhibition exerted by the carbon repression control protein Crc operates hand-in-hand with the RNA chaperon protein Hfq to reduce translation of the DmpR regulator of the Dmp-pathway. Here we show that Crc and Hfq co-target four additional sites to form riboprotein complexes within the proximity of the translational initiation sites of genes encoding the first two steps of the Dmp-pathway to mediate two-layered control in the face of selection of preferred substrates. Furthermore, we present evidence that Crc plays a hitherto unsuspected role in maintaining the pVI150 plasmid within a bacterial population, which has implications for (methyl)phenol degradation and a wide variety of other physiological processes encoded by the IncP-2 group of Pseudomonas-specific mega-plasmids.

Keywords
catabolite repression, translational regulation, Crc, CrcZ, CrcY, Hfq, Phenol catabolism, IncP-2 plasmids
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-141902 (URN)10.1111/1462-2920.13966 (DOI)000419784100015 ()29076626 (PubMedID)
Available from: 2017-11-15 Created: 2017-11-15 Last updated: 2018-06-09Bibliographically approved
Madhushani, W. K. (2015). Multiple regulatory inputs for hierarchical control of phenol catabolism by Pseudomonas putida. (Doctoral dissertation). Umeå: Umeå University
Open this publication in new window or tab >>Multiple regulatory inputs for hierarchical control of phenol catabolism by Pseudomonas putida
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Metabolically versatile bacteria have evolved diverse strategies to adapt to different environmental niches and respond to fluctuating physico-chemical parameters. In order to survive in soil and water habitats, they employ specific and global regulatory circuits to integrate external and internal signals to counteract stress and optimise their energy status. One strategic endurance mechanism is the ability to choose the most energetically favourable carbon source amongst a number on offer.

Pseudomonas putida strains possess large genomes that underlie much of their ability to use diverse carbon sources as growth substrates. Their metabolic potential is frequently expanded by possession of catabolic plasmids to include the ability to grow at the expense of seemingly obnoxious carbon sources such as phenols. However, this ability comes with a metabolic price tag. Carbon source repression is one of the main regulatory networks employed to subvert use of these expensive pathways in favour of alternative sources that provide a higher metabolic gain. This thesis identifies some of the key regulatory elements and factors used by P. putida to supress expression of plasmid-encoded enzymes for degradation of phenols until they are beneficial.

I first present evidence for a newly identified DNA and RNA motif within the regulatory region of the gene encoding the master regulator of phenol catabolism – DmpR. The former of these motifs functions to decrease the number of transcripts originating from the dmpR promoter, while the latter mediates a regulatory checkpoint for translational repression by Crc – the carbon repression control protein of P. putida. The ability of Crc to form repressive riboprotein complexes with RNA is shown to be dependent on the RNA chaperone protein Hfq – a co-partnership demonstrated to be required for many previously identified Crc-targets implicated in hierarchical assimilation of different carbon sources in P. putida. Finally, I present evidence for a model in which Crc and Hfq co-target multiple RNA motifs to bring about a two-tiered regulation to subvert catabolism of phenols in the face of preferred substrates – one at the level of the regulator DmpR and another at the level of translation of the catabolic enzymes.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2015. p. 46
Keywords
P. putida, Phenol catabolism, dmpR, dmp-System, 5’-LR, Carbon Catabolite Repression, Transcriptional & Translational regulation, Crc, CrcZ, CrcY, Hfq
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-106878 (URN)978-91-7601-313-7 (ISBN)
Public defence
2015-09-07, E04, Unod R1, Norrlands universitetssjukhus, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2015-08-17 Created: 2015-08-11 Last updated: 2018-06-07Bibliographically approved
Moreno, R., Hernandez-Arranz, S., La Rosa, R., Yuste, L., Madhushani, A., Shingler, V. & Rojo, F. (2015). The Crc and Hfq proteins of Pseudomonas putida cooperate in catabolite repression and formation of ribonucleic acid complexes with specific target motifs. Environmental Microbiology, 17(1), 105-118
Open this publication in new window or tab >>The Crc and Hfq proteins of Pseudomonas putida cooperate in catabolite repression and formation of ribonucleic acid complexes with specific target motifs
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2015 (English)In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 17, no 1, p. 105-118Article in journal (Refereed) Published
Abstract [en]

The Crc protein is a global regulator that has a key role in catabolite repression and optimization of metabolism in Pseudomonads. Crc inhibits gene expression post-transcriptionally, preventing translation of mRNAs bearing an AAnAAnAA motif [the catabolite activity (CA) motif] close to the translation start site. Although Crc was initially believed to bind RNA by itself, this idea was recently challenged by results suggesting that a protein co-purifying with Crc, presumably the Hfq protein, could account for the detected RNA-binding activity. Hfq is an abundant protein that has a central role in post-transcriptional gene regulation. Herein, we show that the Pseudomonas putidaHfq protein can recognize the CA motifs of RNAs through its distal face and that Crc facilitates formation of a more stable complex at these targets. Crc was unable to bind RNA in the absence of Hfq. However, pull-down assays showed that Crc and Hfq can form a co-complex with RNA containing a CA motif in vitro. Inactivation of the hfq or the crc gene impaired catabolite repression to a similar extent. We propose that Crc and Hfq cooperate in catabolite repression, probably through forming a stable co-complex with RNAs containing CA motifs to result in inhibition of translation initiation.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-100780 (URN)10.1111/1462-2920.12499 (DOI)000349152800010 ()24803210 (PubMedID)
Available from: 2015-04-01 Created: 2015-03-09 Last updated: 2018-06-07Bibliographically approved
Madhushani, A., del Peso-Santos, T., Moreno, R., Rojo, F. & Shingler, V. (2015). Transcriptional and translational control through the 5 '-leader region of the dmpR master regulatory gene of phenol metabolism. Environmental Microbiology, 17(1), 119-133
Open this publication in new window or tab >>Transcriptional and translational control through the 5 '-leader region of the dmpR master regulatory gene of phenol metabolism
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2015 (English)In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 17, no 1, p. 119-133Article in journal (Refereed) Published
Abstract [en]

Expression of pathways for dissimilation of toxic aromatic compounds such as (methyl)phenols interfaces both stress-response and carbon catabolite repression control cascades. In Pseudomonas putida, carbon catabolite repression is mediated by the protein Crc - a translational repressor that counteracts utilization of less-preferred carbon sources as growth substrates until they are needed. In this work we dissect the regulatory role of the 5-leader region (5-LR) of the dmpR gene that encodes the master regulator of (methyl)phenol catabolism. Using deletion and substitution mutants combined with artificial manipulations of Crc availability in P.putida, we present evidence that a DNA motif within the 5-leader region is critical for inhibition of the output from the Pr promoter that drives transcription of dmpR, while the RNA chaperone Hfq facilitates Crc-mediated translation repression through the 5-leader region of the dmpR mRNA. The results are discussed in the light of a model in which Hfq assists Crc to target a sequence within a loop formed by secondary structure of the 5-LR mRNA. Our results support the idea that Crc functions as a global translational inhibitor to co-ordinate hierarchical carbon utilization in Pseudomonads.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-100785 (URN)10.1111/1462-2920.12511 (DOI)000349152800011 ()24889314 (PubMedID)
Available from: 2015-03-11 Created: 2015-03-09 Last updated: 2018-06-07Bibliographically approved
Madhushani, A., Wirebrand, L. & Shingler, V.Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF600.
Open this publication in new window or tab >>Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF600
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-106886 (URN)
Available from: 2015-08-12 Created: 2015-08-12 Last updated: 2018-06-07Bibliographically approved
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