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1. Membrane-Binding Biomolecules Influence the Rate of Vesicle Exchange between Bacteria

   https://doi.org/10.1128/aem.01346-22  

   Tran, Frances; Gangan, Manasi S.; Weaver, Brian P.; Boedicker, James Q. (December 2022, Applied and Environmental Microbiology)

   Nikel, Pablo Ivan (Ed.)

   ABSTRACT The exchange of bacterial extracellular vesicles facilitates molecular exchange between cells, including the horizontal transfer of genetic material. Given the implications of such transfer events on cell physiology and adaptation, some bacterial cells have likely evolved mechanisms to regulate vesicle exchange. Past work has identified mechanisms that influence the formation of extracellular vesicles, including the production of small molecules that modulate membrane structure; however, whether these mechanisms also modulate vesicle uptake and have an overall impact on the rate of vesicle exchange is unknown. Here, we show that membrane-binding molecules produced by microbes influence both the formation and uptake of extracellular vesicles and have the overall impact of increasing the vesicle exchange rate within a bacterial coculture. In effect, production of compounds that increase vesicle exchange rates encourage gene exchange between neighboring cells. The ability of several membrane-binding compounds to increase vesicle exchange was demonstrated. Three of these compounds, nisin, colistin, and polymyxin B, are antimicrobial peptides added at sub-inhibitory concentrations. These results suggest that a potential function of exogenous compounds that bind to membranes may be the regulation of vesicle exchange between cells. IMPORTANCE The exchange of bacterial extracellular vesicles is one route of gene transfer between bacteria, although it was unclear if bacteria developed strategies to modulate the rate of gene transfer within vesicles. In eukaryotes, there are many examples of specialized molecules that have evolved to facilitate the production, loading, and uptake of vesicles. Recent work with bacteria has shown that some small molecules influence membrane curvature and induce vesicle formation. Here, we show that similar compounds facilitate vesicle uptake, thereby increasing the overall rate of vesicle exchange within bacterial populations. The addition of membrane-binding compounds, several of them antibiotics at subinhibitory concentrations, to a bacterial coculture increased the rate of horizontal gene transfer via vesicle exchange. 

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2. Plasmid Characteristics Modulate the Propensity of Gene Exchange in Bacterial Vesicles

   https://doi.org/10.1128/JB.00430-18  

   Tran, Frances; Boedicker, James Q.; Becker, Anke (April 2019, Journal of Bacteriology)

   ABSTRACT Horizontal gene transfer is responsible for the exchange of many types of genetic elements, including plasmids. Properties of the exchanged genetic element are known to influence the efficiency of transfer via the mechanisms of conjugation, transduction, and transformation. Recently, an alternative general pathway of horizontal gene transfer has been identified, namely, gene exchange by extracellular vesicles. Although extracellular vesicles have been shown to facilitate the exchange of several types of plasmids, the influence of plasmid characteristics on genetic exchange within vesicles is unclear. Here, a set of different plasmids was constructed to systematically test the impact of plasmid properties, specifically, plasmid copy number, size, and origin of replication, on gene transfer in vesicles. The influence of each property on the production, packaging, and uptake of vesicles containing bacterial plasmids was quantified, revealing how plasmid properties modulate vesicle-mediated horizontal gene transfer. The loading of plasmids into vesicles correlates with the plasmid copy number and is influenced by characteristics that help set the number of plasmids within a cell, including size and origin of replication. Plasmid origin also has a separate impact on both vesicle loading and uptake, demonstrating that the origin of replication is a major determinant of the propensity of specific plasmids to transfer within extracellular vesicles. IMPORTANCE Extracellular vesicle formation and exchange are common within bacterial populations. Vesicles package multiple types of biomolecules, including genetic material. The exchange of extracellular vesicles containing genetic material facilitates interspecies DNA transfer and may be a promiscuous mechanism of horizontal gene transfer. Unlike other mechanisms of horizontal gene transfer, it is unclear whether characteristics of the exchanged DNA impact the likelihood of transfer in vesicles. Here, we systematically examine the influence of plasmid copy number, size, and origin of replication on the loading of DNA into vesicles and the uptake of DNA containing vesicles by recipient cells. These results reveal how each plasmid characteristic impacts gene transfer in vesicles and contribute to a greater understanding of the importance of vesicle-mediated gene exchange in the landscape of horizontal gene transfer. 

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3. Exometabolite Dynamics over Stationary Phase Reveal Strain-Specific Responses

   https://doi.org/10.1128/mSystems.00493-20  

   Chodkowski, John L.; Shade, Ashley (December 2020, mSystems)

   Gibbons, Sean M. (Ed.)

   ABSTRACT Microbial exponential growth is expected to occur infrequently in environments that have long periods of nutrient starvation punctuated by short periods of high nutrient flux. These conditions likely impose nongrowth states for microbes. However, nongrowth states are uncharacterized for the majority of environmental bacteria, especially in regard to exometabolite production. We compared exometabolites produced over stationary phase across three environmental bacteria: Burkholderia thailandensis E264 (ATCC 700388), Chromobacterium violaceum ATCC 31532, and Pseudomonas syringae pv. tomato DC3000 (ATCC BAA-871). We grew each strain in monoculture and investigated exometabolite dynamics from mid-exponential to stationary phases. We focused on exometabolites that were released into the medium and accumulated over 45 h, including approximately 20 h of stationary phase. We also analyzed transcripts (transcriptome sequencing [RNA-seq]) to interpret exometabolite output. We found that the majority of exometabolites released were strain specific, with a subset of identified exometabolites involved in both central and secondary metabolism. Transcript analysis supported that exometabolites were released from intact cells, as various transporters had either increased or consistent transcripts through time. Interestingly, we found that succinate was one of the most abundant identifiable exometabolites for all strains and that each strain rerouted their metabolic pathways involved in succinate production during stationary phase. These results show that nongrowth states can be metabolically dynamic and that environmental bacteria can enrich a minimal environment with diverse chemical compounds as a consequence of growth and postgrowth maintenance in stationary phase. This work provides insights into microbial community interactions via exometabolites under conditions of growth cessation or limitation. IMPORTANCE Nongrowth states are common for bacteria that live in environments that are densely populated and predominantly nutrient exhausted, and yet these states remain largely uncharacterized in cellular metabolism and metabolite output. Here, we investigated and compared stationary-phase exometabolites and RNA transcripts for each of three environmental bacterial strains. We observed that diverse exometabolites were produced and provide evidence that these exometabolites accumulate over time through release by intact cells. Additionally, each bacterial strain had a characteristic exometabolite profile and exhibited dynamics in exometabolite composition. This work affirms that stationary phase is metabolically dynamic, with each strain tested creating a unique chemical signature in the extracellular space and altering metabolism in stationary phase. These findings set the stage for understanding how bacterial populations can support surrounding neighbors in environments with prolonged nutrient exhaustion through exometabolite-mediated interspecies interactions. 

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4. Dynamics of a methanol-fed marine denitrifying biofilm: 2—impact of environmental changes on the microbial community

   https://doi.org/10.7717/peerj.7467  

   Villemur, Richard; Payette, Geneviève; Geoffroy, Valérie; Mauffrey, Florian; Martineau, Christine (January 2019, PeerJ)

   null (Ed.)

   Background The biofilm of a methanol-fed, marine denitrification system is composed of a multi-species microbial community, among which Hyphomicrobium nitrativorans and Methylophaga nitratireducenticrescens are the principal bacteria involved in the denitrifying activities. To assess its resilience to environmental changes, the biofilm was cultivated in artificial seawater (ASW) under anoxic conditions and exposed to a range of specific environmental conditions. We previously reported the impact of these changes on the denitrifying activities and the co-occurrence of H. nitrativorans strain NL23 and M. nitratireducenticrescens in the biofilm cultures. Here, we report the impact of these changes on the dynamics of the overall microbial community of the denitrifying biofilm. Methods The original biofilm (OB) taken from the denitrification system was cultivated in ASW under anoxic conditions with a range of NaCl concentrations, and with four combinations of nitrate/methanol concentrations and temperatures. The OB was also cultivated in the commercial Instant Ocean seawater (IO). The bacterial diversity of the biofilm cultures and the OB was determined by 16S ribosomal RNA gene sequences. Culture approach was used to isolate other denitrifying bacteria from the biofilm cultures. The metatranscriptomes of selected biofilm cultures were derived, along with the transcriptomes of planktonic pure cultures of H. nitrativorans strain NL23 and M. nitratireducenticrescens strain GP59. Results High proportions of M. nitratireducenticrescens occurred in the biofilm cultures. H. nitrativorans strain NL23 was found in high proportion in the OB, but was absent in the biofilm cultures cultivated in the ASW medium at 2.75% NaCl. It was found however in low proportions in the biofilm cultures cultivated in the ASW medium at 0–1% NaCl and in the IO biofilm cultures. Denitrifying bacterial isolates affiliated to Marinobacter spp. and Paracoccus spp. were isolated. Up regulation of the denitrification genes of strains GP59 and NL23 occurred in the biofilm cultures compared to the planktonic pure cultures. Denitrifying bacteria affiliated to the Stappia spp. were metabolically active in the biofilm cultures. Conclusions These results illustrate the dynamics of the microbial community in the denitrifying biofilm cultures in adapting to different environmental conditions. The NaCl concentration is an important factor affecting the microbial community in the biofilm cultures. Up regulation of the denitrification genes of M. nitratireducenticrescens strain GP59 and H. nitrativorans strain NL23 in the biofilm cultures suggests different mechanisms of regulation of the denitrification pathway in the biofilm. Other denitrifying heterotrophic bacteria are present in low proportions, suggesting that the biofilm has the potential to adapt to heterotrophic, non-methylotrophic environments. 

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5. Heterotrophic Bacteria Dominate Catalase Expression during Microcystis Blooms

   https://doi.org/10.1128/aem.02544-21  

   Smith, Derek J.; Berry, Michelle A.; Cory, Rose M.; Johengen, Thomas H.; Kling, George W.; Davis, Timothy W.; Dick, Gregory J. (July 2022, Applied and Environmental Microbiology)

   Nojiri, Hideaki (Ed.)

   ABSTRACT In the oligotrophic oceans, key autotrophs depend on “helper” bacteria to reduce oxidative stress from hydrogen peroxide (H 2 O 2 ) in the extracellular environment. H 2 O 2 is also a ubiquitous stressor in freshwaters, but the effects of H 2 O 2 on autotrophs and their interactions with bacteria are less well understood in freshwaters. Naturally occurring H 2 O 2 in freshwater systems is proposed to impact the proportion of microcystin-producing (toxic) and non-microcystin-producing (nontoxic) Microcystis in blooms, which influences toxin concentrations and human health impacts. However, how different strains of Microcystis respond to naturally occurring H 2 O 2 concentrations and the microbes responsible for H 2 O 2 decomposition in freshwater cyanobacterial blooms are unknown. To address these knowledge gaps, we used metagenomics and metatranscriptomics to track the presence and expression of genes for H 2 O 2 decomposition by microbes during a cyanobacterial bloom in western Lake Erie in the summer of 2014. katG encodes the key enzyme for decomposing extracellular H 2 O 2 but was absent in most Microcystis cells. katG transcript relative abundance was dominated by heterotrophic bacteria. In axenic Microcystis cultures, an H 2 O 2 scavenger (pyruvate) significantly improved growth rates of one toxic strain while other toxic and nontoxic strains were unaffected. These results indicate that heterotrophic bacteria play a key role in H 2 O 2 decomposition in Microcystis blooms and suggest that their activity may affect the fitness of some Microcystis strains and thus the strain composition of Microcystis blooms but not along a toxic versus nontoxic dichotomy. IMPORTANCE Cyanobacterial harmful algal blooms (CHABs) threaten freshwater ecosystems globally through the production of toxins. Toxin production by cyanobacterial species and strains during CHABs varies widely over time and space, but the ecological drivers of the succession of toxin-producing species remain unclear. Hydrogen peroxide (H 2 O 2 ) is ubiquitous in natural waters, inhibits microbial growth, and may determine the relative proportions of Microcystis strains during blooms. However, the mechanisms and organismal interactions involved in H 2 O 2 decomposition are unexplored in CHABs. This study shows that some strains of bloom-forming freshwater cyanobacteria benefit from detoxification of H 2 O 2 by associated heterotrophic bacteria, which may impact bloom development. 

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