ABSTRACT Heliobacteria are the only phototrophic members of the gram-positive bacteria. Heliomicrobium modesticaldum can grow chemotrophically in the dark using pyruvate as a carbon source. It can grow phototrophically only on acetate (plus CO 2 ). We found that dihydrogen production occurred only when fixing N 2 and increased ~5-fold upon replacing N 2 with argon. Introduction of genes encoding a [FeFe] hydrogenase and maturases ( hydAEFG ) from Acetivibrio thermocellus conferred the ability to produce H 2 at much higher levels. The modified cells made the most H 2 when grown chemotrophically, less when grown mixotrophically, and none when grown phototrophically, suggesting that only excess electrons were available for H 2 production. We tested several molecules as potential reduced fermentation products by this species. Only propanoate was observed, and its extracellular concentration could reach several millimolar. There was an inverse correlation between light intensity and propanoate production when the cells were grown on pyruvate, and phototrophic cultures produced no detectable propanoate. We conclude that propanoate is the major reduced fermentation product made by Hmb. modesticaldum, and we propose a pathway to produce it that is essentially a mini-cycle within the reductive TCA (rTCA) cycle, combining the first four reactions of the rTCA cycle with methylmalonyl-CoA mutase and epimerase, and methylmalonyl-CoA:pyruvate carboxytransferase. This pathway would, in effect, dispose of four reducing equivalents while driving the conversion of pyruvate to propanoate. The genes for the last six enzymes of this pathway seem to be part of an operon; the genes and potential operon are conserved in other heliobacteria, suggesting that this pathway exists in all or most heliobacteria. IMPORTANCE This study establishes that heliobacteria, the only phototrophic members of the Bacillota , produce propanoic acid as the reduced fermentation product when growing chemotrophically. The pathway used to make it resembles that of the Propionibacteria , except in the context of a mini-cycle within the reductive TCA (rTCA) cycle. This work confirms that the Heliomicrobium genus lacks a functional hydrogenase to make hydrogen as a way to dispose of excess electrons. Finally, it is shown that these organisms can be engineered to make dihydrogen as a reduced fermentation product if the genes for a clostridial hydrogenase and assembly proteins are expressed in Heliomicrobium modesticaldum .
ABSTRACT Co-cultures of filamentous microorganisms are a tool to unlock the biosynthetic machinery encoded in silent gene clusters. However, if bioactive compounds are to be produced biotechnologically, it is essential to understand the dynamics of microbial partnership. In this context, we have previously introduced a co-cultivation platform based on low-cost crystalline cellulose as a substrate. Here, the fungus Trichoderma reesei RUT-C30 degrades α-cellulose into soluble sugars to support the growth of the natural product-producing bacterium Streptomyces coelicolor A3(2), which is unable to utilize cellulose. This co-cultivation approach mimics a carbon-limited interaction scenario of natural habitats. We investigated how the population dynamics of both strains affect metabolite production by combining high-throughput online fluorescence monitoring with high-resolution liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based metabolomics. We then determined the metabolic profile of the cultures and identified metabolites specifically induced during co-cultivation. The coupled analysis of population ratios and metabolomic data indicated that the highest number of induced metabolites compared to axenic cultures is observed in co-cultures with a balanced ratio of both strains. This analysis further led to deciphering a biotransformation of dimerumic acid, a hydroxamate siderophore produced by T. reesei , into the dehydroxylated derivatives eleutherazine B and talarazine A by S. coelicolor . Additionally, a small cluster of unique metabolites strongly induced in co-cultures was found to be structurally similar to steroidal lactones of the withanolide class, thus highlighting the potential for the discovery of natural products. Overall, the platform described combines natural product prospecting with a biotechnological production strategy that can be extended also to other co-cultures. IMPORTANCE This work provides a comprehensive analysis of population dynamics and metabolite production in a complex microbial co-culture. This approach integrates three key aspects. First, high-throughput online monitoring enables biomass tracking through fluorescence labeling in a system containing two filamentous microorganisms with distinct macro- and micromorphologies and a non-soluble fibrous substrate. Second, concomitant metabolomics analysis during co-cultivation settings helps to resolve metabolic interaction patterns. Interestingly, a metal-acquisition compound produced by the first microbe is chemically modified by the second, suggesting a competition strategy for an essential micronutrient. Third, from a biotechnological perspective, substrate solubilization resembles a fed-batch strategy that minimizes carbon catabolite repression during secondary metabolite production. Overall, this platform advances our understanding of microbial interactions and culture conditions that can be transferred to other culture platforms of relevance in bioprocess intensification.
ABSTRACT Microplastics (MPs), resulting from plastic objects and waste degradation, are increasingly abundant, particularly in marine environments. They exhibit a hydrophobic surface on which biofilms form easily. Metagenomic analyses of these biofilms have revealed that they often contain bacterial species potentially pathogenic to humans or animals. For this reason, MPs are suspected to present a risk to public health by acting as a vector for pathogenic bacterial species. To better understand this hazard, we studied different factors potentially affecting the bacterial attachment rate to MPs. Focusing on the fish pathogen Vibrio anguillarum , a collection of 16 GFP-labeled strains was assembled, including isolates from infected fish. Their attachment rates were measured using fluorescence microscopy on three types of MPs (milled polypropylene and polyethylene terephthalate particles, and commercially available polystyrene beads). A strong effect of the particle type was found, with higher attachment rates on milled particles, whose surface appeared qualitatively rougher based on visual examination of scanning electron microscopy images. Our results also revealed an intraspecific variation in attachment rate, highlighting the need for testing several strains of the same species to assess attachment rate and related hazards. Finally, it was observed that when a biofilm had formed by field incubation of the MPs, the differences in attachment rates between particle types were erased. The attachment of V. anguillarum depends on a combination of strain-related factors, particle characteristics, and environmental conditions, highlighting the need to combine microbiology, materials science, and environmental sciences to achieve a comprehensive risk assessment associated with MPs. IMPORTANCE Microplastics (MPs) are increasingly abundant, particularly in marine environments. They are suspected to present a risk to public health by acting as a vector for pathogenic bacterial species. Focusing on the fish pathogen Vibrio anguillarum , we measured the attachment rates of 16 strains on three types of microplastics (milled polypropylene and polyethylene terephthalate particles, and commercially available polystyrene beads). This study highlights a strong effect of the particle type, with higher attachment rates on irregular particles, as well as a strong intraspecific variation. In a second experiment, it was observed that when a natural marine biofilm had already formed on the microplastic particles, differences in attachment rates between particle types were erased. Our findings emphasize the importance of accounting for multiple factors when assessing the risks of MPs as carriers of pathogenic bacteria, with our study addressing the first step of the conceptual pathway linking attachment, persistence, transport, exposure, and ultimately infection.
ABSTRACT Heterologous production of metabolites and proteins requires the transfer of foreign DNA into a host. One common barrier to the genetic engineering of bacteria is their plethora of phage defense systems. Targeted deletion of defense system components can improve DNA transfer for downstream applications. We sought to improve the transformation efficiency of Burkholderia sp. FERM BP-3421, a β-proteobacterial host that has great potential for the high-titer production of bioactive small molecules. We investigated restriction-modification systems and the recently described Kiwa phage defense system. Through deletion of three restriction enzyme genes, transformation efficiency was improved nearly 7-fold. The electroporation protocol was also optimized with respect to temperature, cell density, and buffer composition, which resulted in an additional 40-fold improvement in transformation efficiency, reaching 1 × 10 6 CFU/µg plasmid DNA. Interestingly, a mimicry by methylation approach, where DNA is pre-methylated using two host DNA methyltransferases, outperformed the transformation efficiency obtained with restriction enzyme deletion mutants nearly 3-fold. Moreover, contrary to previous reports in Escherichia coli , deletion of the Kiwa system did not improve transformation efficiency into Burkholderia sp. FERM BP-3421. Understanding the impact of bacterial innate immune systems on genetic tractability is vital to improving non-model host strains to produce high-value molecules of interest. IMPORTANCE The ability to transfer DNA into bacteria is key to genetic engineering. However, bacteria have evolved immune defenses to recognize and protect themselves from foreign DNA, thwarting genetic manipulation. Here, we identified putative defense systems in a β-proteobacterial strain of interest and characterized their impact on DNA transfer. The defense systems studied belong to the well-known class of restriction-modification systems and to the recently described Kiwa system. Intriguingly, we observed higher DNA transfer efficiency by avoiding restriction-modification systems via pre-methylation rather than by restriction gene deletion. Future studies should investigate the underlying mechanism for this difference. Moreover, contrary to what was reported for γ-proteobacteria Escherichia coli , we showed that the Kiwa system does not impair DNA transfer into the β-proteobacteria Burkholderia sp. strain. There has been significant interest in the discovery of novel defense systems. Learning more about their complexity is important to enable genetic manipulation of previously intractable strains.
ABSTRACT Methanogens are responsible for producing approximately one billion metric tons of methane each year within Earth’s anaerobic environments as a byproduct of their energy metabolism. They play a vital role in the global carbon cycle. There are three main pathways for methanogenesis: hydrogenotrophic, which uses H 2 and CO 2 to produce methane; methylotrophic, which utilizes methylated compounds like methanol and amines; and aceticlastic, which converts acetate into methane and CO 2 . Instead of substrate-level ATP production, methanogens conserve energy through a membrane-bound electron transport chain and flavin-based electron bifurcation (FBEB). Beyond the canonical modes of growth in methanogens, Methanosarcina acetivorans , a model acetotrophic methanogen, can conserve energy via direct interspecies electron transfer (DIET). Here, we review the metabolic pathways and electron transport processes involved in energy conservation during fermentative, respiratory, and DIET modes. Additionally, we discuss the role of FBEB in energy conservation in gut-associated Methanomassiliicoccus luminyensis . Finally, we discuss the potential roles of heterodisulfide reductase (Hdr) and the process of electron transfer during the reaction mechanism of electron-bifurcating HdrA2B2C2 complex in cytochrome-containing methanogens. Understanding the complex redox control mechanisms in these organisms has broader implications for sustainable energy and environmental management.
