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1. Bacterial Quorum-Sensing Signal Arrests Phytoplankton Cell Division and Impacts Virus-Induced Mortality

   https://doi.org/10.1128/mSphere.00009-21  

   Pollara, Scott B. ; Becker, Jamie W. ; Nunn, Brook L. ; Boiteau, Rene ; Repeta, Daniel ; Mudge, Miranda C. ; Downing, Grayton ; Chase, Davis ; Harvey, Elizabeth L. ; Whalen, Kristen E. ( June 2021 , mSphere)

   McMahon, Katherine (Ed.)

   ABSTRACT Interactions between phytoplankton and heterotrophic bacteria fundamentally shape marine ecosystems by controlling primary production, structuring marine food webs, mediating carbon export, and influencing global climate. Phytoplankton-bacterium interactions are facilitated by secreted compounds; however, linking these chemical signals, their mechanisms of action, and their resultant ecological consequences remains a fundamental challenge. The bacterial quorum-sensing signal 2-heptyl-4-quinolone (HHQ) induces immediate, yet reversible, cellular stasis (no cell division or mortality) in the coccolithophore Emiliania huxleyi ; however, the mechanism responsible remains unknown. Using transcriptomic and proteomic approaches in combination with diagnostic biochemical and fluorescent cell-based assays, we show that HHQ exposure leads to prolonged S-phase arrest in phytoplankton coincident with the accumulation of DNA damage and a lack of repair despite the induction of the DNA damage response (DDR). While this effect is reversible, HHQ-exposed phytoplankton were also protected from viral mortality, ascribing a new role of quorum-sensing signals in regulating multitrophic interactions. Furthermore, our data demonstrate that in situ measurements of HHQ coincide with areas of enhanced micro- and nanoplankton biomass. Our results suggest bacterial communication signals as emerging players that may be one of the contributing factors that help structure complex microbial communities throughout the ocean. IMPORTANCE Bacteria and phytoplankton form close associations in the ocean that are driven by the exchange of chemical compounds. The bacterial signal 2-heptyl-4-quinolone (HHQ) slows phytoplankton growth; however, the mechanism responsible remains unknown. Here, we show that HHQ exposure leads to the accumulation of DNA damage in phytoplankton and prevents its repair. While this effect is reversible, HHQ-exposed phytoplankton are also relieved of viral mortality, elevating the ecological consequences of this complex interaction. Further results indicate that HHQ may target phytoplankton proteins involved in nucleotide biosynthesis and DNA repair, both of which are crucial targets for viral success. Our results support microbial cues as emerging players in marine ecosystems, providing a new mechanistic framework for how bacterial communication signals mediate interspecies and interkingdom behaviors. 

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2. A bacterial quorum sensing signal is a potent inhibitor of de novo pyrimidine biosynthesis in the globally abundant Emiliania huxleyi

   https://doi.org/10.3389/fmicb.2023.1266972  

   Garrett, Oscar ; Whalen, Kristen E. ( October 2023 , Frontiers in Microbiology)

   Interactions between marine phytoplankton, viruses, and bacteria drive biogeochemical cycling, shape marine trophic structures, and impact global climate. Microbially produced compounds have emerged as key players in influencing eukaryotic organismal physiology, and in turn, remodel microbial community structure. This work aimed to reveal the molecular mechanism by which the bacterial quorum sensing molecule 2-heptyl-4-quinolone (HHQ), produced by the marine gammaproteobacteriumPseudoalteromonasspp., arrests cell division and confers protection from virus-induced mortality in the bloom-forming coccolithophoreEmiliania huxleyi. Previous work has established alkylquinolones as inhibitors of dihydroorotate dehydrogenase (DHODH), a fundamental enzyme catalyzing the fourth step in pyrimidine biosynthesis and a potential antiviral drug target. An N-terminally truncated version ofE. huxleyiDHODH was heterologously expressed inE. coli, purified, and kinetically characterized. Here, we show HHQ is a potent inhibitor (K_iof 2.3 nM) ofE. huxleyiDHODH.E. huxleyicells exposed to brequinar, the canonical human DHODH inhibitor, experienced immediate, yet reversible cellular arrest, an effect which mirrors HHQ-induced cellular stasis previously observed. However, brequinar treatment lacked other notable effects observed in HHQ-exposedE. huxleyiincluding significant changes in cell size, chlorophyll fluorescence, and protection from virus-induced lysis, indicating HHQ has additional as yet undiscovered physiological targets. Together, these results suggest a novel and intricate role of bacterial quorum sensing molecules in tripartite interdomain interactions in marine ecosystems, opening new avenues for exploring the role of microbial chemical signaling in algal bloom regulation and host-pathogen dynamics.

    

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3. Quorum sensing signal disrupts viral infection dynamics in the coccolithophore Emiliania huxleyi

   https://doi.org/10.3354/ame01998  

   Harvey, EL ; Yang, H ; Castiblanco, E ; Coolahan, M ; Dallmeyer-Drennen, G ; Fukuda, N ; Greene, E ; Gonsalves, M ; Smith, S ; Whalen, KE ( June 2023 , Aquatic Microbial Ecology)

   Viruses that infect phytoplankton are abundant in all regions of the global ocean. Despite their ubiquity, little is understood regarding how biotic interactions can alter virus infection success as well as the fate of phytoplankton hosts. In previous work, the bacterially derived compound 2-heptyl-4-quinolone (HHQ) has been shown to protect the cosmopolitan coccolithophoreEmiliania huxleyifrom virus-induced mortality. The present study explores the potential mechanisms through which protection is conferred. Using a suite of transmission electron microscopy and physiological diagnostic staining techniques, we show that whenE. huxleyiis exposed to HHQ, viruses can gain entry into cells but viral replication and release is inhibited. These findings are supported by a smaller burst size, as well as lower infectious and total virus production when the host is treated with nanomolar concentrations of HHQ. Additionally, diagnostic staining results indicate that programmed cell death markers commonly associated with viral infection are not activated when infectedE. huxleyiare exposed to HHQ. Together, these results suggest that the ability of HHQ to inhibit infectious viral production protects the alga not from getting infected, but from cell lysis. This work identifies a new mechanistic role of bacterial quorum sensing molecules in mediating viral infections in marine microbial systems.

    

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4. Quorum Sensing and Antimicrobial Production Orchestrate Biofilm Dynamics in Multispecies Bacterial Communities

   https://doi.org/10.1128/spectrum.02615-22  

   Armes, April C. ; Walton, Jillian L. ; Buchan, Alison ( October 2022 , Microbiology Spectrum)

   DeAngelis, Kristen M. (Ed.)

   ABSTRACT Microbial interactions are often mediated by diffusible small molecules, including secondary metabolites, that play roles in cell-to-cell signaling and inhibition of competitors. Biofilms are often “hot spots” for high concentrations of bacteria and their secondary metabolites, which make them ideal systems for the study of small-molecule contributions to microbial interactions. Here, we use a five-member synthetic community consisting of Roseobacteraceae representatives to investigate the role of secondary metabolites on microbial biofilm dynamics. One synthetic community member, Rhodobacterales strain Y4I, possesses two acylated homoserine lactone (AHL)-based cell-to-cell signaling systems ( pgaRI and phaRI ) as well as a nonribosomal peptide synthase gene ( igi ) cluster that encodes the antimicrobial indigoidine. Through serial substitution of Y4I with mutants deficient in single signaling molecule pathways, the contribution of these small-molecule systems could be assessed. As secondary metabolite production is dependent upon central metabolites, the influence of growth substrate (i.e., complex medium versus defined medium with a single carbon substrate) on these dynamics was also considered. Depending on the Y4I mutant genotype included, community dynamics ranged from competitive to cooperative. The observed interactions were mostly competitive in nature. However, the community harboring a Y4I variant that was both impaired in quorum sensing (QS) pathways and unable to produce indigoidine ( pgaR variant) shifted toward more cooperative interactions over time. These cooperative interactions were enhanced in the defined growth medium. The results presented provide a framework for deciphering complex, small-molecule-mediated interactions that have broad application to microbial biology. IMPORTANCE Microbial biofilms play critical roles in marine ecosystems and are hot spots for microbial interactions that play a role in the development and function of these communities. Roseobacteraceae are an abundant and active family of marine heterotrophic bacteria forming close associations with phytoplankton and carrying out key transformations in biogeochemical cycles. Group members are aggressive primary colonizers of surfaces, where they set the stage for the development of multispecies biofilm communities. Few studies have examined the impact of secondary metabolites, such as cell-to-cell signaling and antimicrobial production, on marine microbial biofilm community structure. Here, we assessed the impact of secondary metabolites on microbial interactions using a synthetic, five-member Roseobacteraceae community by measuring species composition and biomass production during biofilm growth. We present evidence that secondary metabolites influence social behaviors within these multispecies microbial biofilms, thereby improving understanding of bacterial secondary metabolite production influence on social behaviors within marine microbial biofilm communities. 

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5. Phage Infection Restores PQS Signaling and Enhances Growth of a Pseudomonas aeruginosa lasI Quorum-Sensing Mutant

   https://doi.org/10.1128/jb.00557-21  

   Høyland-Kroghsbo, Nina Molin ; Bassler, Bonnie L. ( January 2022 , Journal of Bacteriology)

   Bondy-Denomy, Joseph (Ed.)

   ABSTRACT Chemical communication between bacteria and between bacteria and the bacteriophage (phage) viruses that prey on them can shape the outcomes of phage-bacterial encounters. Quorum sensing (QS) is a bacterial cell-to-cell communication process that promotes collective undertaking of group behaviors. QS relies on the production, release, accumulation, and detection of signal molecules called autoinducers. Phages can exploit QS-mediated communication to manipulate their hosts and maximize their own survival. In the opportunistic pathogen Pseudomonas aeruginosa , the LasI/R QS system induces the RhlI/R QS system, and in opposing manners, these two systems control the QS system that relies on the autoinducer called PQS. A P. aeruginosa Δ lasI mutant is impaired in PQS synthesis, leading to accumulation of the precursor molecule HHQ, and HHQ suppresses growth of the P. aeruginosa Δ lasI strain. We show that, in response to a phage infection, the P. aeruginosa Δ lasI mutant reactivates QS, which, in turn, restores pqsH expression, enabling conversion of HHQ into PQS. Moreover, downstream QS target genes encoding virulence factors are induced. Additionally, phage-infected P. aeruginosa Δ lasI cells transiently exhibit superior growth compared to uninfected cells. IMPORTANCE Clinical isolates of P. aeruginosa frequently harbor mutations in particular QS genes. Here, we show that infection by select temperate phages restores QS, a cell-to-cell communication mechanism in a P. aeruginosa QS mutant. Restoration of QS increases expression of genes encoding virulence factors. Thus, phage infection of select P. aeruginosa strains may increase bacterial pathogenicity, underscoring the importance of characterizing phage-host interactions in the context of bacterial mutants that are relevant in clinical settings. 

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