Field-scale remediation of atrazine-contaminated soil using recombinant Escherichia coli expressing atrazine chlorohydrolase

We performed the first field-scale atrazine remediation study in the United States using chemically killed, recombinant organisms. This field study compared biostimulation methods for enhancing atrazine degradation with a novel bioaugmentation protocol using a killed and stabilized whole-cell suspension of recombinant Escherichia coli engineered to overproduce atrazine chlorohyrolase, AtzA. AtzA dechlorinates atrazine, producing non-toxic and non-phytotoxic hydroxyatrazine. Soil contaminated by an accidental spill of atrazine (up to 29 000 p.p.m.) supported significant populations of indigenous microorganisms capable of atrazine catabolism. Laboratory experiments indicated that supplementing soil with carbon inhibited atrazine biodegradation, but inorganic phosphate stimulated atrazine biodegradation. A subsequent field-scale study consisting of nine (0.75m3) treatment plots was designed to test four treatment protocols in triplicate. Control plots contained moistened soil; biostimulation plots received 300 p.p.m. phosphate; bioaugmentation plots received 0.5% (w/w) killed, recombinant E. coli cells encapsulating AtzA; and combination plots received phosphate plus the enzyme-containing cells. After 8 weeks, atrazine levels declined 52% in plots containing killed recombinant E. coli cells, and 77% in combination plots. In contrast, atrazine levels in control and biostimulation plots did not decline significantly. These data indicate that genetically engineered bacteria overexpressing catabolic genes significantly increased degradation in this soil heavily contaminated with atrazine

Lipopeptide surfactin produced by Bacillus amyloliquefaciens KPS46 is required for biocontrol efficacy against Xanthomonas axonopodis pv

ABSTRACT The biological control of root and foliar diseases of soybeans caused by fungi and bacteria (e.g. Xanthomonas axonopodis pv. glycines, Xag) using the plant growth promoting rhizobacterium Bacillus amyloliquefaciens KPS46 has been previously reported. Disease suppression is thought to be due, in part, to the production of secondary metabolites. While a wide variety of these active compounds has been identified, their mode of action and mechanism of disease suppression on soybeans are not fully understood. The study used HPLC to identify secondary metabolites produced by B. amyloliquefaciens KPS46 and tested these compounds for biological control activity against soybean bacterial pustule disease caused by Xag. HPLC analyses indicated that a lipopeptide surfactin was present in KPS46 cell-free culture extracts, with maximum yields of ~550 ± 20.3 mg L -1 . Exogenous application of KPS46-produced surfactin to soybean plants directly that inhibited Xag, reduced disease severity and enhanced plant growth. UV mutagenesis of KPS46 and PCR assays were carried out to assess the role of surfactin production on the biocontrol activity. An independently-generated srfAA mutant of KPS46, strain M6, was unable to produce lipopeptide surfactin. The M6 mutant also was severely affected in its ability to produce extracellular enzymes, including endoglucanase, cellulase, and protease; and had reduced motility on the surface of agar compared to the wild-type strain KPS46. In contrast, the M6 mutant had enhanced production of α-amylase, and faster growth rate in nutrient broth, than did the parental strain. Soybean plant assays using the srfAA mutant and wild-type biocontrol agents against bacterial pustule disease indicated that the mutant strain M6 had significantly less effect on disease reduction compared to the wild-type parental strain. Results of this study suggest that the ability of B. amyloliquefaciens KPS46 to reduce bacterial pustule severity on soybeans is associated with the production of a lipopeptide surfactin encoded by srfAA, and that mutations in this locus also effect extracellular enzyme production

Signal Disruption Leads to Changes in Bacterial Community Population

The disruption of bacterial signaling (quorum quenching) has been proven to be an innovative approach to influence the behavior of bacteria. In particular, lactonase enzymes that are capable of hydrolyzing the N-acyl homoserine lactone (AHL) molecules used by numerous bacteria, were reported to inhibit biofilm formation, including those of freshwater microbial communities. However, insights and tools are currently lacking to characterize, understand and explain the effects of signal disruption on complex microbial communities. Here, we produced silica capsules containing an engineered lactonase that exhibits quorum quenching activity. Capsules were used to design a filtration cartridge to selectively degrade AHLs from a recirculating bioreactor. The growth of a complex microbial community in the bioreactor, in the presence or absence of lactonase, was monitored over a 3-week period. Dynamic population analysis revealed that signal disruption using a quorum quenching lactonase can effectively reduce biofilm formation in the recirculating bioreactor system and that biofilm inhibition is concomitant to drastic changes in the composition, diversity and abundance of soil bacterial communities within these biofilms. Effects of the quorum quenching lactonase on the suspension community also affected the microbial composition, suggesting that effects of signal disruption are not limited to biofilm populations. This unexpected finding is evidence for the importance of signaling in the competition between bacteria within communities. This study provides foundational tools and data for the investigation of the importance of AHL-based signaling in the context of complex microbial communities

Proceedings of the 2014 A.S.P.E.N. Research Workshop

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141886/1/jpen0167.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141886/2/jpen0167-sup-0001.pd

Species and genus level resolution analysis of gut microbiota in Clostridium difficile patients following fecal microbiota transplantation

BACKGROUND: Clostridium difficile is an opportunistic human intestinal pathogen, and C. difficile infection (CDI) is one of the main causes of antibiotic-induced diarrhea and colitis. One successful approach to combat CDI, particularly recurrent form of CDI, is through transplantation of fecal microbiota from a healthy donor to the infected patient. In this study we investigated the distal gut microbial communities of three CDI patients before and after fecal microbiota transplantation, and we compared these communities to the composition of the donor’s fecal microbiota. We utilized phylogenetic Microbiota Array, high-throughput Illumina sequencing, and fluorescent in situ hybridization to profile microbiota composition down to the genus and species level resolution. RESULTS: The original patients’ microbiota had low diversity, was dominated by members of Gammaproteobacteria and Bacilli, and had low numbers of Clostridia and Bacteroidia. At the genus level, fecal samples of CDI patients were rich in members of the Lactobacillus, Streptococcus, and Enterobacter genera. In comparison, the donor community was dominated by Clostridia and had significantly higher diversity and evenness. The patients’ distal gut communities were completely transformed within 3 days following fecal transplantation, and these communities remained stable in each patient for at least 4 months. Despite compositional differences among recipients’ pre-treatment gut microbiota, the transplanted gut communities were highly similar among recipients post-transplantation, were indistinguishable from that of the donor, and were rich in members of Blautia, Coprococcus, and Faecalibacterium. In each case, the gut microbiota restoration led to a complete patient recovery and symptom alleviation. CONCLUSION: We conclude that C. difficile infection can be successfully treated by fecal microbiota transplantation and that this leads to stable transformation of the distal gut microbial community from the one abundant in aerotolerant species to that dominated by members of the Clostridia

Soil nitrogen transformations under elevated atmospheric CO2 and O3 during the soybean growing season

We investigated the influence of elevated CO2 and O3 on soil N cycling within the soybean growing season and across soil environments (i.e., rhizosphere and bulk soil) at the Soybean Free Air Concentration Enrichment (SoyFACE) experiment in Illinois, USA. Elevated O3 decreased soil mineral N likely through a reduction in plant material input and increased denitrification, which was evidenced by the greater abundance of the denitrifier gene nosZ. Elevated CO2 did not alter the parameters evaluated and both elevated CO2 and O3 showed no interactive effects on nitrifier and denitrifier abundance, nor on total and mineral N concentrations. These results indicate that elevated CO2 may have limited effects on N transformations in soybean agroecosystems. However, elevated O3 can lead to a decrease in soil N availability in both bulk and rhizosphere soils, and this likely also affects ecosystem productivity by reducing the mineralization rates of plant-derived residues

Climate Change Impacts on Microbiota in Beach Sand and Water: Looking Ahead

Beach sand and water have both shown relevance for human health and their microbiology have been the subjects of study for decades. Recently, the World Health Organization recommended that recreational beach sands be added to the matrices monitored for enterococci and Fungi. Global climate change is affecting beach microbial contamination, via changes to conditions like water temperature, sea level, precipitation, and waves. In addition, the world is changing, and humans travel and relocate, often carrying endemic allochthonous microbiota. Coastal areas are amongst the most frequent relocation choices, especially in regions where desertification is taking place. A warmer future will likely require looking beyond the use of traditional water quality indicators to protect human health, in order to guarantee that waterways are safe to use for bathing and recreation. Finally, since sand is a complex matrix, an alternative set of microbial standards is necessary to guarantee that the health of beach users is protected from both sand and water contaminants. We need to plan for the future safer use of beaches by adapting regulations to a climate-changing world.Financial support from CESAM (UID/AMB/50017-POCI-01-0145-FEDER-007638) and CITAB (UID/AGR/04033/2019), via FCT/MCTES, from national funds (PIDDAC), cofounded by FEDER, (PT2020 Partnership Agreement and Compete 2020).info:eu-repo/semantics/publishedVersio

Denitrifying Bacteria Active in Woodchip Bioreactors at Low-Temperature Conditions

Woodchip bioreactor technology removes nitrate from agricultural subsurface drainage by using denitrifying microorganisms. Although woodchip bioreactors have demonstrated success in many field locations, low water temperature can significantly limit bioreactor efficiency and performance. To improve bioreactor performance, it is important to identify the microbes responsible for nitrate removal at low temperature conditions. Therefore, in this study, we identified and characterized denitrifiers active at low-temperature conditions by using culture-independent and -dependent approaches. By comparative 16S rRNA (gene) analysis and culture isolation technique, Pseudomonas spp., Polaromonas spp., and Cellulomonas spp. were identified as being important bacteria responsible for denitrification in woodchip bioreactor microcosms at relatively low temperature conditions (15°C). Genome analysis of Cellulomonas sp. strain WB94 confirmed the presence of nitrite reductase gene nirK. Transcription levels of this nirK were significantly higher in the denitrifying microcosms than in the non-denitrifying microcosms. Strain WB94 was also capable of degrading cellulose and other complex polysaccharides. Taken together, our results suggest that Cellulomonas sp. denitrifiers could degrade woodchips to provide carbon source and electron donors to themselves and other denitrifiers in woodchip bioreactors at low-temperature conditions. By inoculating these denitrifiers (i.e., bioaugmentation), it might be possible to increase the nitrate removal rate of woodchip bioreactors at low-temperature conditions

Climate Change Impacts on Microbiota in Beach Sand and Water : Looking Ahead

Beach sand and water have both shown relevance for human health and their microbiology have been the subjects of study for decades. Recently, the World Health Organization recommended that recreational beach sands be added to the matrices monitored for enterococci and Fungi. Global climate change is affecting beach microbial contamination, via changes to conditions like water temperature, sea level, precipitation, and waves. In addition, the world is changing, and humans travel and relocate, often carrying endemic allochthonous microbiota. Coastal areas are amongst the most frequent relocation choices, especially in regions where desertification is taking place. A warmer future will likely require looking beyond the use of traditional water quality indicators to protect human health, in order to guarantee that waterways are safe to use for bathing and recreation. Finally, since sand is a complex matrix, an alternative set of microbial standards is necessary to guarantee that the health of beach users is protected from both sand and water contaminants. We need to plan for the future safer use of beaches by adapting regulations to a climate-changing world.Peer reviewe