Browsing by Author "Mathieu, Jacques"
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Item 1,4-Dioxane-degrading consortia can be enriched from uncontaminated soils: prevalence of Mycobacterium and soluble di-iron monooxygenase genes(Wiley, 2017) He, Ya; Mathieu, Jacques; da Silva, Marcio L.B.; Li, Mengyan; Alvarez, Pedro J.J.Two bacterial consortia were enriched from uncontaminated soil by virtue of their ability to grow on 1,4-dioxane (dioxane) as a sole carbon and energy source. Their specific dioxane degradation rates at 30°C, pH = 7 (i.e. 5.7 to 7.1 g-dioxane per g-protein per day) were comparable to those of two dioxane-metabolizing archetypes: Pseudonocardia dioxanivoransCB1190 and Mycobacterium dioxanotrophicusPH-06. Based on 16S rRNA sequencing, Mycobacterium was the dominant genus. Acetylene inhibition tests suggest that dioxane degradation was mediated by monooxygenases. However, qPCR analyses targeting the tetrahydrofuran/dioxane monooxygenase gene (thmA/dxmA) (which is, to date, the only sequenced dioxane monooxygenase gene) were negative, indicating that other (as yet unknown) catabolic gene(s) were responsible. DNA sequence analyses also showed threefold to sevenfold enrichment of group 5 and group 6 soluble di-iron monooxygenase (SDIMO) genes relative to the original soil samples. Whereas biodegradation of trace levels of dioxane is a common challenge at contaminated sites, both consortia degraded dioxane at low initial concentrations (300 μg l−1) below detectable levels (5 μg l−1) in bioaugmented microcosms prepared with impacted groundwater. Overall, this work shows that dioxane-degrading bacteria (and the associated natural attenuation potential) exist even in some uncontaminated soils, and may be enriched to broaden bioaugmentation options for sites experiencing insufficient dioxane catabolic capacity.Item Bacterial Endospores as Phage Genome Carriers and Protective Shells(American Society for Microbiology, 2018) Gabiatti, Naiana; Yu, Pingfeng; Mathieu, Jacques; Lu, Grant W.; Wang, Xifan; Zhang, Hangjun; Soares, Hugo M.; Alvarez, Pedro J.J.Bacterial endospores can serve as phage genome protection shells against various environmental stresses to enhance microbial control applications. The genomes of polyvalent lytic Bacillus phages PBSC1 and PBSC2, which infect both B. subtilis subsp. subtilis and B. cereusNRS 248, were incorporated into B. subtilis endospores (without integration into the host chromosome). When PBSC1 and PBSC2 were released from germinating endospores, they significantly inhibited the growth of the targeted opportunistic pathogen B. cereus Optimal endospore entrapment was achieved when phages were introduced to the fast-sporulating prespores at a multiplicity of infection of 1. Longer endospore maturation (48 h versus 24 h) increased both spore yield and efficiency of entrapment. Compared with free phages, spore-protected phage genomes showed significantly higher resistance toward high temperatures (60 to 80°C), extreme pH (pH 2 or pH 12), and copper ions (0.1 to 10 mg/liter). Endospore germination is inducible by low concentrations of l-alanine or by a germinant mixture (l-asparagine, d-glucose, d-fructose, and K+) to trigger the expression, assembly, and consequent release of phage particles within 60 to 90 min. Overall, the superior resiliency of polyvalent phages protected by endospores might enable nonrefrigerated phage storage and enhance phage applications after exposure to adverse environmental conditions.IMPORTANCE: Bacteriophages are being considered for the control of multidrug-resistant and other problematic bacteria in environmental systems. However, the efficacy of phage-based microbial control is limited by infectivity loss during phage delivery and/or storage. Here, we exploit the pseudolysogenic state of phages, which involves incorporation of their genome into bacterial endospores (without integration into the host chromosome), to enhance survival in unfavorable environments. We isolated polyvalent (broad-host-range) phages that efficiently infect both benign and opportunistically pathogenic Bacillusstrains and encapsulated the phage genomes in B. subtilis endospores to significantly improve resistance to various environmental stressors. Encapsulation by spores also significantly enhanced phage genome viability during storage. We also show that endospore germination can be induced on demand with nutrient germinants that trigger the release of active phages. Overall, we demonstrate that encapsulation of polyvalent phage genomes into benign endospores holds great promise for broadening the scope and efficacy of phage biocontrol.Item Phage biocontrol in water treatment and reuse systems: a nascent field with significant innovation opportunities(Elsevier, 2025) Hong, Pei-Ying; Mathieu, Jacques; Cheng, Hong; Narayanasamy, Shaman; Castillo, Darwin A; Goel, Ramesh; Alvarez, Pedro JJ; Rice Water InstituteWhile the use of phages in the food and biomedical sectors occurs commercially, their application in the water sector is less common and is typically demonstrated at a lower technological readiness level. This is so despite the potential that phages have to enhance the control of problematic bacteria (including pathogens) and protect infrastructure within the water sector. Fulfilling the great potential of this nascent field requires more research and development. Here, we highlight innovation opportunities and discern critical knowledge gaps and research needs to facilitate the use of phages as precise biocontrol agents in the water sector. First, while the advent of sequencing technologies made it easier to identify bacterial communities and understand their functional roles, identifying and cultivating the appropriate phages that can be effective against the bacterial target requires more research. The large volumes of water to be spiked with phages also require optimizing the phage biocontrol strategy, minimizing the associated costs and enhancing scaling up. In addition, bacterial hosts may gain phage resistance after long-term exposure, which is common in most water-engineered systems, and strategies to minimize or delay resistance must be considered. In this opinion, we provide an overview of pertinent literature and bioinformatic tools that help identify appropriate bacterial hosts and phages for water systems applications. We then discuss strategies that can aid in prolonging the efficacy and enhancing the feasibility of phage biocontrol approaches.Item Rapid Metabolism of 1,4-Dioxane to below Health Advisory Levels by Thiamine-Amended Rhodococcus ruber Strain 219(American Chemical Society, 2021) Simmer, Reid A.; Richards, Patrick M.; Ewald, Jessica M.; Schwarz, Cory; da Silva, Marcio L.B.; Mathieu, Jacques; Alvarez, Pedro J.J.; Schnoor, Jerald L.Bioremediation is a promising treatment technology for 1,4-dioxane-contaminated groundwater. However, metabolic dioxane-degrading bacteria identified to date are limited by their slow kinetics and inability to sustain growth at low dioxane concentrations (<100 μg/L). Furthermore, strains may underperform because of missing growth factors, such as amino acids or vitamins. In this work, we reevaluate Rhodococcus ruber strain 219 as a dioxane-degrading strain with bioaugmentation potential. We report rapid growth and metabolic dioxane degradation by R. ruber 219 when supplemented with thiamine (vitamin B1). We also discern that the strain lacks a complete de novo thiamine synthesis pathway, indicating that R. ruber 219 is a probable thiamine auxotroph. However, when supplemented with thiamine, the strain’s Monod kinetics (Ks = 0.015 ± 0.03 μg/L) and exceedingly low Smin (0.49 ± 1.16 μg/L) suggest this strain can maintain growth at very low dioxane concentrations (<100 μg/L). Accordingly, we demonstrate that thiamine-grown R. ruber 219 sustains degradation of dilute dioxane (<100 μg/L) to below health advisory levels. This is the first study to report sustained metabolic dioxane biodegradation to below health advisory levels of 0.35 μg/L. Overall, our findings solidify R. ruber 219 as a promising candidate for bioremediation of dioxane-contaminated groundwater.Item Recombination-assisted megaprimer (RAM) cloning(Elsevier, 2014) Mathieu, Jacques; Alvarez, Emilia; Alvarez, Pedro J.J.No molecular cloning technique is considered universally reliable, and many suffer from being too laborious, complex, or expensive. Restriction-free cloning is among the simplest, most rapid, and cost-effective methods, but does not always provide successful results. We modified this method to enhance its success rate through the use of exponential amplification coupled with homologous end-joining. This new method, recombination-assisted megaprimer (RAM) cloning, significantly extends the application of restriction-free cloning, and allows efficient vector construction with much less time and effort when restriction-free cloning fails to provide satisfactory results. The following modifications were made to the protocol: 1) Limited number of PCR cycles for both megaprimer synthesis and the cloning reaction to reduce error propagation; 2) Elimination of phosphorylation and ligation steps previously reported for cloning methods that used exponential amplification, through the inclusion of a reverse primer in the cloning reaction with a 20 base pair region of homology to the forward primer; 3) The inclusion of 1 M betaine to enhance both reaction specificity and yield.Item Whole-Genome Sequence of the 1,4-Dioxane-Degrading Bacterium Mycobacterium dioxanotrophicus PH-06(American Society for Microbiology, 2017) He, Ya; Wei, Kangfei; Si, Kaiwei; Mathieu, Jacques; Li, Mengyan; Alvarez, Pedro J.J.We report here the complete genome sequence of Mycobacterium dioxanotrophicus PH-06, which is capable of using 1,4-dioxane as a sole source of carbon and energy. The reported sequence will enable the elucidation of this novel metabolic pathway and the development of molecular biomarkers to assess bioremediation potential at contaminated sites.