Marine Metagenomics: New Tools for the Study and Exploitation of Marine Microbial Metabolism

The marine environment is extremely diverse, with huge variations in pressure and temperature. Nevertheless, life, especially microbial life, thrives throughout the marine biosphere and microbes have adapted to all the divergent environments present. Large scale DNA sequence based approaches have recently been used to investigate the marine environment and these studies have revealed that the oceans harbor unprecedented microbial diversity. Novel gene families with representatives only within such metagenomic datasets represent a large proportion of the ocean metagenome. The presence of so many new gene families from these uncultured and highly diverse microbial populations represents a challenge for the understanding of and exploitation of the biology and biochemistry of the ocean environment. The application of new metagenomic and single cell genomics tools offers new ways to explore the complete metabolic diversity of the marine biome

Structural modification of the Pseudomonas aeruginosa alkylquinoline cell–cell communication signal, HHQ, leads to benzofuranoquinolines with anti-virulence behaviour in ESKAPE pathogens

Microbial populations have evolved intricate networks of negotiation and communication through which they can coexist in natural and host ecosystems. The nature of these systems can be complex and they are, for the most part, poorly understood at the polymicrobial level. The Pseudomonas Quinolone Signal (PQS) and its precursor 4- hydroxy- 2-heptylquinoline (HHQ) are signal molecules produced by the important nosocomial pathogen Pseudomonas aeruginosa. They are known to modulate the behaviour of co-colonizing bacterial and fungal pathogens such as Bacillus atropheaus, Candida albicans and Aspergillus fumigatus. While the structural basis for alkyl-quinolone signalling within P. aeruginosa has been studied extensively, less is known about how structural derivatives of these molecules can influ-ence multicellular behaviour and population- level decision-making in other co-colonizing organisms. In this study, we investigated a suite of small molecules derived initially from the HHQ framework, for anti-virulence activity against ESKAPE pathogens, at the species and strain levels. Somewhat surprisingly, with appropriate substitution, loss of the alkyl chain (present in HHQ and PQS) did not result in a loss of activity, presenting a more easily accessible synthetic framework for investigation. Virulence profiling uncovered significant levels of inter-strain variation among the responses of clinical and environmental isolates to small-molecule challenge. While several lead compounds were identified in this study, further work is needed to appreciate the extent of strain- level tolerance to small-molecule anti-infectives among pathogenic organisms.National Forum for the Enhancement of Teaching and Learning in Higher Education SFI/12/IP/1315, US Cystic Fibrosis Foundation SFI/12/RC/2275, National Health and Medical Research Council (NHMRC) of Australia SFI/12/RC/2275_P2, UCC Strategic Research Fund and Science Foundation Ireland (SFI) SSPC-3 12/RC/2275_2, Synthesis and Solid State Pharmaceutical Centre (SSPC) HRB-ILP-POR-2019-004, MRCG-2018-16, Universidade do Algarve TL19UCC1481/02, OGARA1710, APP1183640 2020-5,info:eu-repo/semantics/publishedVersio

Transcriptome profiling defines a novel regulon modulated by the LysR-type transcriptional regulator MexT in Pseudomonas aeruginosa

The LysR-family regulator MexT modulates the expression of the MexEF-OprN efflux system in the human pathogen Pseudomonas aeruginosa. Recently, we demonstrated that MexT regulates certain virulence phenotypes, including the type-three secretion system and early attachment independent of its role in regulating MexEF-OprN. In this study, transcriptome profiling was utilized to investigate the global nature of MexT regulation in P. aeruginosa PAO1 and an isogenic mexEF mutant. Twelve genes of unknown function were highly induced by overexpressing MexT independent of MexEF-OprN. A well-conserved DNA motif was identified in the upstream regulatory region of nine of these genes and upstream of mexE. Reporter fusion analysis demonstrated that the expression of the genes was significantly induced by MexT in P. aeruginosa and a heterogenous Escherichia coli strain and that the conserved sequence was required for this induction. The conserved DNA motif was further characterized as the MexT binding site by site-directed mutagenesis and electrophoretic mobility shift assays. Genes containing this conserved regulatory sequence were identified across other Pseudomonas species, and their expression was activated by MexT. Thus, a novel regulon directly modulated by MexT, that includes but is independent of mexEF-oprN, has been identified

CpxR activates expression of target promoters.

<p>CpxR activates expression of target promoters.</p

Inhibition of co-colonizing cystic fibrosis-associated pathogens by Pseudomonas aeruginosa and Burkholderia multivorans

Cystic fibrosis (CF) is a recessive genetic disease characterized by chronic respiratory infections and inflammation causing permanent lung damage. Recurrent infections are caused by Gram-negative antibiotic-resistant bacterial pathogens such as Pseudomonas aeruginosa, Burkholderia cepacia complex (Bcc) and the emerging pathogen genus Pandoraea. In this study, the interactions between co-colonizing CF pathogens were investigated. Both Pandoraea and Bcc elicited potent pro-inflammatory responses that were significantly greater than Ps. aeruginosa. The original aim was to examine whether combinations of pro-inflammatory pathogens would further exacerbate inflammation. In contrast, when these pathogens were colonized in the presence of Ps. aeruginosa the pro-inflammatory response was significantly decreased. Real-time PCR quantification of bacterial DNA from mixed cultures indicated that Ps. aeruginosa significantly inhibited the growth of Burkholderia multivorans, Burkholderia cenocepacia, Pandoraea pulmonicola and Pandoraea apista, which may be a factor in its dominance as a colonizer of CF patients. Ps. aeruginosa cell-free supernatant also suppressed growth of these pathogens, indicating that inhibition was innate rather than a response to the presence of a competitor. Screening of a Ps. aeruginosa mutant library highlighted a role for quorum sensing and pyoverdine biosynthesis genes in the inhibition of B. cenocepacia. Pyoverdine was confirmed to contribute to the inhibition of B. cenocepacia strain J2315. B. multivorans was the only species that could significantly inhibit Ps. aeruginosa growth. B. multivorans also inhibited B. cenocepacia and Pa. apista. In conclusion, both Ps. aeruginosa and B. multivorans are capable of suppressing growth and virulence of co-colonizing CF pathogens

Direct binding of CpxR to the promoter region of <i>mexA in vitro</i>.

<p>DNase I footprinting assays of the <i>mexA</i> promoter DNA fragment were performed in the absence (A) and presence (B) of purified CpxR. The FAM-labelled 322-bp DNA fragments (50 nM) pre-incubated in the absence or presence of 1.5 μM phosphorylated CpxR were subjected to DNase I digestion and fragment length analysis. The fluorescence signal of the labelled DNA fragments is plotted against the sequence of the fragment. The protected region bound by CpxR is shown with the conserved binding motif in red.</p

CpxR mediates enhancement of antibiotic resistance in <i>mexR</i>-deleted <i>P</i>. <i>aeruginosa</i> laboratory standard strains.

<p>CpxR mediates enhancement of antibiotic resistance in <i>mexR</i>-deleted <i>P</i>. <i>aeruginosa</i> laboratory standard strains.</p

CpxR activates expression of target promoters.

<p>CpxR activates expression of target promoters.</p