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RpoS differentially affects the general stress response and biofilm formation in the endophytic Serratia plymuthica G3

Liu X, Wu Y, Chen Y, Xu F, Halliday N, Gao K, et al.

Res. Microbiol., 2016 Apr;167(3):168-77.
PMID: 26671319 DOI: 10.1016/j.resmic.2015.11.003

The σ(S) subunit RpoS of RNA polymerase functions as a master regulator of the general stress response in Escherichia coli and related bacteria. RpoS has been reported to modulate biocontrol properties in the rhizobacterium Serratia plymuthica IC1270. However, the role of RpoS in the stress response and biofilm formation in S. plymuthica remains largely unknown. Here we studied the role of RpoS from an endophytic S. plymuthica G3 in regulating these phenotypes. Mutational analysis demonstrated that RpoS positively regulates the global stress response to acid or alkaline stresses, oxidative stress, hyperosmolarity, heat shock and carbon starvation, in addition to proteolytic and chitinolytic activities. Interestingly, rpoS mutations resulted in significantly enhanced swimming motility, biofilm formation and production of the plant auxin indole-3-acetic acid (IAA), which may contribute to competitive colonization and environmental fitness for survival. These findings provide further insight into the strain-specific role of RpoS in the endophytic strain G3 of S. plymuthica, where it confers resistance to general stresses encountered within the plant environment. The heterogeneous functionality of RpoS in rhizosphere and endophytic S. plymuthica populations may provide a selective advantage for better adaptation to various physiological and environmental stresses.
Functional identification of the prnABCD operon and its regulation in Serratia plymuthica

Liu X, Yu X, Yang Y, Heeb S, Gao S, Chan KG, et al.

Appl Microbiol Biotechnol, 2018 Apr;102(8):3711-3721.
PMID: 29511844 DOI: 10.1007/s00253-018-8857-0

The antibiotic pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite that plays an important role in the biocontrol of plant diseases due to its broad-spectrum of antimicrobial activities. The PRN biosynthetic gene cluster remains to be characterised in Serratia plymuthica, though it is highly conserved in PRN-producing bacteria. To better understand PRN biosynthesis and its regulation in Serratia, the prnABCD operon from S. plymuthica G3 was cloned, sequenced and expressed in Escherichia coli DH5α. Furthermore, an engineered strain prnind which is a conditional mutant of G3 prnABCD under the control of the Ptac promoter was constructed. This mutant was able to overproduce PRN with isopropylthiogalactoside (IPTG) induction by overexpressing prnABCD, whilst behaving as a conditional mutant of G3 prnABCD in the absence of IPTG. These results confirmed that prnABCD is responsible for PRN biosynthesis in strain G3. Further experiments involving lux-/dsRed-based promoter fusions, combined with site-directed mutagenesis of the putative σS extended -10 region in the prnA promoter, and liquid chromatography-mass spectrometry (LC-MS) analysis extended our previous knowledge about G3, revealing that quorum sensing (QS) regulates PRN biosynthesis through cross talk with RpoS, which may directly activated prnABCD transcription. These findings suggest that PRN in S. plymuthica G3 is produced in a tightly controlled manner, and has diverse functions, such as modulation of cell motility, in addition to antimicrobial activities. Meanwhile, the construction of inducible mutants could be a powerful tool to improve PRN production, beyond its potential use for the investigation of the biological function of PRN.
Fulltext Characterization of N-acylhomoserine lactone-degrading bacteria associated with the Zingiber officinale (ginger) rhizosphere: co-existence of quorum quenching and quorum sensing in Acinetobacter and Burkholderia

Chan KG, Atkinson S, Mathee K, Sam CK, Chhabra SR, Cámara M, et al.

BMC Microbiol, 2011 Mar 08;11:51.
PMID: 21385437 DOI: 10.1186/1471-2180-11-51

BACKGROUND: Cell-to-cell communication (quorum sensing (QS)) co-ordinates bacterial behaviour at a population level. Consequently the behaviour of a natural multi-species community is likely to depend at least in part on co-existing QS and quorum quenching (QQ) activities. Here we sought to discover novel N-acylhomoserine lactone (AHL)-dependent QS and QQ strains by investigating a bacterial community associated with the rhizosphere of ginger (Zingiber officinale) growing in the Malaysian rainforest.
RESULTS: By using a basal growth medium containing N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) as the sole source of carbon and nitrogen, the ginger rhizosphere associated bacteria were enriched for strains with AHL-degrading capabilities. Three isolates belonging to the genera Acinetobacter (GG2), Burkholderia (GG4) and Klebsiella (Se14) were identified and selected for further study. Strains GG2 and Se14 exhibited the broadest spectrum of AHL-degrading activities via lactonolysis while GG4 reduced 3-oxo-AHLs to the corresponding 3-hydroxy compounds. In GG2 and GG4, QQ was found to co-exist with AHL-dependent QS and GG2 was shown to inactivate both self-generated and exogenously supplied AHLs. GG2, GG4 and Se14 were each able to attenuate virulence factor production in both human and plant pathogens.
CONCLUSIONS: Collectively our data show that ginger rhizosphere bacteria which make and degrade a wide range of AHLs are likely to play a collective role in determining the QS-dependent phenotype of a polymicrobial community.
Biotic inactivation of the Pseudomonas aeruginosa quinolone signal molecule

Soh EY, Chhabra SR, Halliday N, Heeb S, Müller C, Birmes FS, et al.

Environ Microbiol, 2015 Nov;17(11):4352-65.
PMID: 25809238 DOI: 10.1111/1462-2920.12857

In Pseudomonas aeruginosa, quorum sensing (QS) regulates the production of secondary metabolites, many of which are antimicrobials that impact on polymicrobial community composition. Consequently, quenching QS modulates the environmental impact of P. aeruginosa. To identify bacteria capable of inactivating the QS signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), a minimal medium containing PQS as the sole carbon source was used to enrich a Malaysian rainforest soil sample. This yielded an Achromobacter xylosoxidans strain (Q19) that inactivated PQS, yielding a new fluorescent compound (I-PQS) confirmed as PQS-derived using deuterated PQS. The I-PQS structure was elucidated using mass spectrometry and nuclear magnetic resonance spectroscopy as 2-heptyl-2-hydroxy-1,2-dihydroquinoline-3,4-dione (HHQD). Achromobacter xylosoxidans Q19 oxidized PQS congeners with alkyl chains ranging from C1 to C5 and also N-methyl PQS, yielding the corresponding 2-hydroxy-1,2-dihydroquinoline-3,4-diones, but was unable to inactivate the PQS precursor HHQ. This indicates that the hydroxyl group at position 3 in PQS is essential and that A. xylosoxidans inactivates PQS via a pathway involving the incorporation of oxygen at C2 of the heterocyclic ring. The conversion of PQS to HHQD also occurred on incubation with 12/17 A. xylosoxidans strains recovered from cystic fibrosis patients, with P. aeruginosa and with Arthrobacter, suggesting that formation of hydroxylated PQS may be a common mechanism of inactivation.
Fulltext Genome-wide mapping of the RNA targets of the Pseudomonas aeruginosa riboregulatory protein RsmN

Romero M, Silistre H, Lovelock L, Wright VJ, Chan KG, Hong KW, et al.

Nucleic Acids Res, 2018 Jul 27;46(13):6823-6840.
PMID: 29718466 DOI: 10.1093/nar/gky324

Pseudomonads typically carry multiple non-identical alleles of the post-transcriptional regulator rsmA. In Pseudomonas aeruginosa, RsmN is notable in that its structural rearrangement confers distinct and overlapping functions with RsmA. However, little is known about the specificities of RsmN for its target RNAs and overall impact on the biology of this pathogen. We purified and mapped 503 transcripts directly bound by RsmN in P. aeruginosa. About 200 of the mRNAs identified encode proteins of demonstrated function including some determining acute and chronic virulence traits. For example, RsmN reduces biofilm development both directly and indirectly via multiple pathways, involving control of Pel exopolysaccharide biosynthesis and c-di-GMP levels. The RsmN targets identified are also shared with RsmA, although deletion of rsmN generally results in less pronounced phenotypes than those observed for ΔrsmA or ΔrsmArsmNind mutants, probably as a consequence of different binding affinities. Targets newly identified for the Rsm system include the small non-coding RNA CrcZ involved in carbon catabolite repression, for which differential binding of RsmN and RsmA to specific CrcZ regions is demonstrated. The results presented here provide new insights into the intricacy of riboregulatory networks involving multiple but distinct RsmA homologues.
Fulltext Genome-Wide Analysis of Targets for Post-Transcriptional Regulation by Rsm Proteins in Pseudomonas putida

Huertas-Rosales Ó, Romero M, Chan KG, Hong KW, Cámara M, Heeb S, et al.

Front Mol Biosci, 2021;8:624061.
PMID: 33693029 DOI: 10.3389/fmolb.2021.624061

Post-transcriptional regulation is an important step in the control of bacterial gene expression in response to environmental and cellular signals. Pseudomonas putida KT2440 harbors three known members of the CsrA/RsmA family of post-transcriptional regulators: RsmA, RsmE and RsmI. We have carried out a global analysis to identify RNA sequences bound in vivo by each of these proteins. Affinity purification and sequencing of RNA molecules associated with Rsm proteins were used to discover direct binding targets, corresponding to 437 unique RNA molecules, 75 of them being common to the three proteins. Relevant targets include genes encoding proteins involved in signal transduction and regulation, metabolism, transport and secretion, stress responses, and the turnover of the intracellular second messenger c-di-GMP. To our knowledge, this is the first combined global analysis in a bacterium harboring three Rsm homologs. It offers a broad overview of the network of processes subjected to this type of regulation and opens the way to define what are the sequence and structure determinants that define common or differential recognition of specific RNA molecules by these proteins.