Cattle-compost-soil: The transfer of antibiotic resistance in livestock agriculture.

Antibiotic resistance is a major global health threat. Agricultural use of antibiotics is considered to be a main contributor to the issue, influencing both animals and humans as defined by the One Health approach. The purpose of the present study was to determine the abundance of antibiotic-resistant bacterial populations and the overall bacterial diversity of cattle farm soils that have been treated with animal manure compost. Soil and manure samples were collected from different sites at Tullimba farm, NSW. Cultures were grown from these samples in the presence of 11 commonly used antibiotics and antibiotic-resistant bacteria (ARB) colonies were identified. Soil and manure bacterial diversity was also determined using 16S ribosomal RNA next-generation sequencing. Results showed that ARB abundance was greatest in fresh manure and significantly lower in composted manure. However, the application of composted manure on paddock soil led to a significant increase in soil ARB abundance. Of the antibiotics tested, the number of ARB in each sample was greatest for antibiotics that inhibited the bacterial cell wall and protein synthesis. Collectively, these results suggest that the transfer of antibiotic resistance from composted animal manure to soil may not be solely mediated through the application of live bacteria and highlight the need for further research into the mechanism of antibiotic resistance transfer.

Microbiology at Home (M@H): an Online, Self-Paced Solution To Enhance Practical Skills in Foundation Microbiology.

The online education market share is rapidly increasing, raising the demand to teach sciences outside the laboratory environment. Here, we present Microbiology at Home (M@H), a new approach that integrates hands-on microbiology experimentation with online interactive simulations using authentic scenarios in microbiology in the home environment. The M@H program includes 8 practical activities aligned to the ASM curriculum for practical skills. M@H kits are mailed to students, and each practical activity is prepacked individually with the required consumables, including microbial culture media to prepare at home using a microwave. These practicals are self-paced, and each activity is facilitated using a two-dimensional simulation package with prerecorded videos, protocols, and interactive activities. The students receive both synchronous and asynchronous support and guidance through online learning management systems fora and virtual gatherings. The M@H program was applied to an Introductory Microbiology cohort at the University of New England in 2020 and 2021. Based on student feedback, the experience not only provided real hands-on practice in microbiology but also acted to cement the engagement with the content by contextualizing it to the surrounding home environment. We anticipate that these activities will provide a way to successfully engage students with hands-on microbiology without the need for actual laboratory attendance, thus increasing accessibility to microbial protocols and applications.

The Hydroponic Rockwool Root Microbiome: Under Control or Underutilised?

Land plants have an ancient and intimate relationship with microorganisms, which influences the composition of natural ecosystems and the performance of crops. Plants shape the microbiome around their roots by releasing organic nutrients into the soil. Hydroponic horticulture aims to protect crops from damaging soil-borne pathogens by replacing soil with an artificial growing medium, such as rockwool, an inert material made from molten rock spun into fibres. Microorganisms are generally considered a problem to be managed, to keep the glasshouse clean, but the hydroponic root microbiome assembles soon after planting and flourishes with the crop. Hence, microbe-plant interactions play out in an artificial environment that is quite unlike the soil in which they evolved. Plants in a near-ideal environment have little dependency on microbial partners, but our growing appreciation of the role of microbial communities is revealing opportunities to advance practices, especially in agriculture and human health. Hydroponic systems are especially well-suited to active management of the root microbiome because they allow complete control over the root zone environment; however, they receive much less attention than other host-microbiome interactions. Novel techniques for hydroponic horticulture can be identified by extending our understanding of the microbial ecology of this unique environment.

Expression of key auxin biosynthesis genes correlates with auxin and starch content of developing wheat (Triticum aestivum) grains.

The effect of auxin on wheat (Triticum aestivum L.) grain size is contentious. Additionally, the contributions to the IAA pool from de novo synthesis versus hydrolysis of IAA-glucose are unclear. Here, we describe the first comprehensive study of tryptophan aminotransferase and indole-3-pyruvate mono-oxygenase expression from 5 to 20 days after anthesis. A comparison of expression data with measurements of endogenous IAA via combined liquid chromatography-tandem mass spectrometry using heavy isotope labelled internal standards indicates that TaTAR2-B3, TaYUC9-A1, TaYUC9-B, TaYUC9-D1, TaYUC10-A and TaYUC10-D are primarily responsible for IAA production in developing grains. Furthermore, these genes are expressed specifically in developing grains, like those found in rice (Oryza sativa L.) and maize (Zea mays L.). Our results cast doubt on the proposed role of THOUSAND-GRAIN WEIGHT gene, TaTGW6, in promoting larger grain size via negative effects on grain IAA content. Work on this gene overlooked the contribution of IAA biosynthesis from tryptophan. Although IAA synthesis occurs primarily in the endosperm, we show the TaYUC9-1 group is also strongly expressed in the embryo. Within the endosperm, TaYUC9-1 expression is highest in aleurone and transfer cells, suggesting that IAA has a key role in differentiation of these tissues as has been proposed for other cereals.

A review of the antimicrobial side of antidepressants and its putative implications on the gut microbiome.

OBJECTIVE: Serotonin reuptake inhibitors are the predominant treatment for major depressive disorder. In recent years, the diversity of the gut microbiota has emerged to play a significant role in the occurrence of major depressive disorder and other mood and anxiety disorders. Importantly, the role of the gut microbiota in the treatment of such disorders remains to be elucidated. Here, we provide a review of the literature regarding the effects of physiologically relevant concentrations of serotonin reuptake inhibitors on the gut microbiota and the implications this might have on their efficacy in the treatment of mood disorders. METHODS: First, an estimation of gut serotonin reuptake inhibitor concentrations was computed based on pharmacokinetic and gastrointestinal transit properties of serotonin reuptake inhibitors. Literature regarding the in vivo and in vitro antimicrobial properties of serotonin reuptake inhibitors was gathered, and the estimated gut concentrations were examined in the context of these data. Computer-based investigation revealed putative mechanisms for the antimicrobial effects of serotonin reuptake inhibitors. RESULTS: In vivo evidence using animal models shows an antimicrobial effect of serotonin reuptake inhibitors on the gut microbiota. Examination of the estimated physiological concentrations of serotonin reuptake inhibitors in the gastrointestinal tract collected from in vitro studies suggests that the microbial community of both the small intestine and the colon are exposed to serotonin reuptake inhibitors for at least 4 hours per day at concentrations that are likely to exert an antimicrobial effect. The potential mechanisms of the effect of serotonin reuptake inhibitors on the gut microbiota were postulated to include inhibition of efflux pumps and/or amino acid transporters. CONCLUSION: This review raises important issues regarding the role that gut microbiota play in the treatment of mood-related behaviours, which holds substantial potential clinical outcomes for patients suffering from major depressive disorder and other mood-related disorders.

Storage and handling of human faecal samples affect the gut microbiome composition: A feasibility study.

Human gut microbiome analysis through faecal sampling typically involves five stages: sample collection, storage, DNA extraction, next generation sequencing and bioinformatics analysis. Of these, the first three are considered irreversible. This feasibility study describes an assessment of methodologies used for faecal DNA extraction and sample handling, using the parameters DNA yield, purity and resultant microbial profile. Six DNA extraction techniques, including commercially available kits and manual protocols were compared on human faecal samples (n = 3). Different extraction techniques produced significant variance in DNA yield (range 2.7-164 ng/mg faeces) and microbial diversity profiles, with considerable variation in phyla dominance (Firmicutes (P < 0.001), Bacteroidetes (P = 0.003), Actinobacteria (P = 0.003), One-way ANOVA). The most effective method, with the highest DNA yield, was a simple and inexpensive extraction technique named MetaHIT. Using this method, DNA was extracted from separate faecal samples (n = 3) and had been aliquoted to seven storage conditions including three stabilizing buffers and three temperature conditions, for a period of 120-h, with storage at -80 °C as a control treatment. DNA yield and purity was not statistically different between the control and remaining treatments. 16S rDNA-based diversity profile was largely comparable across the treatments with only minor differences in genera between samples stored at room temperature in air and - 80 °C control. Overall these results suggest that the choice of DNA extraction method has a greater influence on the resultant microbial diversity profile than the short-term storage method.

Gut microbiome and depression: what we know and what we need to know.

Gut microbiome diversity has been strongly associated with mood-relating behaviours, including major depressive disorder (MDD). This association stems from the recently characterised bi-directional communication system between the gut and the brain, mediated by neuroimmune, neuroendocrine and sensory neural pathways. While the link between gut microbiome and depression is well supported by research, a major question needing to be addressed is the causality in the connection between the two, which will support the understanding of the role that the gut microbiota play in depression. In this article, we address this question by examining a theoretical 'chronology', reviewing the evidence supporting two possible sequences of events. First, we discuss that alterations in the gut microbiota populations of specific species might contribute to depression, and secondly, that depressive states might induce modification of specific gut microbiota species and eventually contribute to more severe depression. The feasibility of both sequences is supported by pre-clinical trials. For instance, research in rodents has shown an onset of depressive behaviour following faecal transplantations from patients with MDD. On the other hand, mental induction of stress and depressive behaviour in rodents resulted in reduced gut microbiota richness and diversity. Synthesis of these chronology dynamics raises important research directions to further understand the role that gut microbiota play in mood-relating behaviours, which holds substantial potential clinical outcomes for persons who experience MDD or related depressive disorders.

Quantitative analysis of aromatics for synthetic biology using liquid chromatography.

The replacement of petrochemical aromatics with bio-based molecules is a key area of current biotechnology research. To date, a small number of aromatics have been produced by recombinant bacteria in laboratory scale while industrial production still requires further strain development. While each study includes some distinct analytical methodology to quantify certain aromatics, a method that can reliably quantify a great number of aromatic products and relevant pathway intermediates is needed to accelerate strain development. In this study, we developed a robust reverse phase high performance liquid chromatography method to quantify a wide range of aromatic metabolites present in host microorganisms using the shikimate pathway, which is the major metabolic pathway for biosynthesis of aromatics. Twenty-three metabolites can be quantified precisely with the optimized method using standard HPLC equipment and UV detection, with the mobile phase used for chromatography also compatible with mass spectrometry (MS). The limit of quantification/detection is as low as 10-10 to 10-13 mol, respectively, which makes this method feasible for quantification of intracellular metabolites. This method covers most metabolic routes for aromatics biosynthesis, it is inexpensive, robust, simple, precise and sensitive, and has been demonstrated on cell extracts from S. cerevisiae genetically engineered to overproduce aromatics.

Metabolic Engineering of Pseudomonas putida KT2440 for the Production of para-Hydroxy Benzoic Acid.

para-Hydroxy benzoic acid (PHBA) is the key component for preparing parabens, a common preservatives in food, drugs, and personal care products, as well as high-performance bioplastics such as liquid crystal polymers. Pseudomonas putida KT2440 was engineered to produce PHBA from glucose via the shikimate pathway intermediate chorismate. To obtain the PHBA production strain, chorismate lyase UbiC from Escherichia coli and a feedback resistant 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase encoded by gene aroGD146N were overexpressed individually and simultaneously. In addition, genes related to product degradation (pobA) or competing for the precursor chorismate (pheA and trpE) were deleted from the genome. To further improve PHBA production, the glucose metabolism repressor hexR was knocked out in order to increase erythrose 4-phosphate and NADPH supply. The best strain achieved a maximum titer of 1.73 g L-1 and a carbon yield of 18.1% (C-mol C-mol-1) in a non-optimized fed-batch fermentation. This is to date the highest PHBA concentration produced by P. putida using a chorismate lyase.

Production of para-aminobenzoic acid from different carbon-sources in engineered Saccharomyces cerevisiae.

BACKGROUND: Biological production of the aromatic compound para-aminobenzoic acid (pABA) is of great interest to the chemical industry. Besides its application in pharmacy and as crosslinking agent for resins and dyes pABA is a potential precursor for the high-volume aromatic feedstocks terephthalic acid and para-phenylenediamine. The yeast Saccharomyces cerevisiae synthesises pABA in the shikimate pathway: Outgoing from the central shikimate pathway intermediate chorismate, pABA is formed in two enzyme-catalysed steps, encoded by the genes ABZ1 and ABZ2. In this study S. cerevisiae metabolism was genetically engineered for the overproduction of pABA. Using in silico metabolic modelling an observed impact of carbon-source on product yield was investigated and exploited to optimize production. RESULTS: A strain that incorporated the feedback resistant ARO4 (K229L) and deletions in the ARO7 and TRP3 genes, in order to channel flux to chorismate, was used to screen different ABZ1 and ABZ2 genes for pABA production. In glucose based shake-flaks fermentations the highest titer (600 µM) was reached when over-expressing the ABZ1 and ABZ2 genes from the wine yeast strains AWRI1631 and QA23, respectively. In silico metabolic modelling indicated a metabolic advantage for pABA production on glycerol and combined glycerol-ethanol carbon-sources. This was confirmed experimentally, the empirical ideal glycerol to ethanol uptake ratios of 1:2-2:1 correlated with the model. A (13)C tracer experiment determined that up to 32% of the produced pABA originated from glycerol. Finally, in fed-batch bioreactor experiments pABA titers of 1.57 mM (215 mg/L) and carbon yields of 2.64% could be achieved. CONCLUSION: In this study a combination of genetic engineering and in silico modelling has proven to be a complete and advantageous approach to increase pABA production. Especially the enzymes that catalyse the last two steps towards product formation appeared to be crucial to direct flux to pABA. A stoichiometric model for carbon-utilization proved useful to design carbon-source composition, leading to increased pABA production. The reported pABA concentrations and yields are, to date, the highest in S. cerevisiae and the second highest in a microbial production system, underlining the great potential of yeast as a cell factory for renewable aromatic feedstocks.

Formation of hydrogen sulfide from cysteine in Saccharomyces cerevisiae BY4742: genome wide screen reveals a central role of the vacuole.

Discoveries on the toxic effects of cysteine accumulation and, particularly, recent findings on the many physiological roles of one of the products of cysteine catabolism, hydrogen sulfide (H2S), are highlighting the importance of this amino acid and sulfur metabolism in a range of cellular activities. It is also highlighting how little we know about this critical part of cellular metabolism. In the work described here, a genome-wide screen using a deletion collection of Saccharomyces cerevisiae revealed a surprising set of genes associated with this process. In addition, the yeast vacuole, not previously associated with cysteine catabolism, emerged as an important compartment for cysteine degradation. Most prominent among the vacuole-related mutants were those involved in vacuole acidification; we identified each of the eight subunits of a vacuole acidification sub-complex (V1 of the yeast V-ATPase) as essential for cysteine degradation. Other functions identified included translation, RNA processing, folate-derived one-carbon metabolism, and mitochondrial iron-sulfur homeostasis. This work identified for the first time cellular factors affecting the fundamental process of cysteine catabolism. Results obtained significantly contribute to the understanding of this process and may provide insight into the underlying cause of cysteine accumulation and H2S generation in eukaryotes.

In vivo instability of chorismate causes substrate loss during fermentative production of aromatics.

Metabolic engineering of microbial strains to produce aromatic compounds deriving from the shikimate pathway is of great interest to the chemical industry as a more sustainable alternative for feedstock production. Chorismate is a significant intermediate in the shikimate pathway. In this study, the formation of phenylalanine and phenylpyruvate as by-products in strains engineered downstream of the chorismate node for increased aromatic production was explored in yeast fermentations. Tracer experiments showed that these compounds are synthesized de novo during fermentation, under conditions in which their synthesis was genetically blocked. Chorismate stability evaluation, as well as deletion mutation analysis throughout the phenylalanine biosynthesis pathway, suggested that this synthesis was a result of intracellular, non-enzymatic rearrangement of chorismate to phenylpyruvate via prephenate, which was followed by enzymatic transamination of phenylpyruvate to form phenylalanine. These results not only aid in the development of strain-engineering strategies to avoid the accumulation of by-products during fermentations aimed at increased aromatics production, but also deepen our understanding of yeast metabolism.

Fluxomics - connecting 'omics analysis and phenotypes.

In our modern 'omics era, metabolic flux analysis (fluxomics) represents the physiological counterpart of its siblings transcriptomics, proteomics and metabolomics. Fluxomics integrates in vivo measurements of metabolic fluxes with stoichiometric network models to allow the determination of absolute flux through large networks of the central carbon metabolism. There are many approaches to implement fluxomics including flux balance analysis (FBA), (13) C fluxomics and (13) C-constrained FBA as well as many experimental settings for flux measurement including dynamic, stationary and semi-stationary. Here we outline the principles of the different approaches and their relative advantages. We demonstrate the unique contribution of flux analysis for phenotype elucidation using a thoroughly studied metabolic reaction as a case study, the microbial aerobic/anaerobic shift, highlighting the importance of flux analysis as a single layer of data as well as interlaced in multi-omics studies.

In situ high throughput method for H(2)S detection during micro-scale wine fermentation.

An in situ high throughput method for the detection of H(2)S during fermentation was developed. The method utilizes a redox reaction in which sulfide ion reduces methylene blue, leading to its decolourisation. Incorporation of methylene blue into the fermentation media allows real-time detection of H(2)S during fermentation and the generation of an H(2)S production profile. Kinetic parameters extracted from the H(2)S production profile can be used to characterise genetic factors affecting H(2)S production and differentiate between environmental conditions affecting it. The method, validated here for Saccharomyces cerevisiae, is suited for high throughput screening purposes by virtue of its simplicity and the ability to detect H(2)S in micro-scale fermentations.

Effects of rehydration nutrients on H2S metabolism and formation of volatile sulfur compounds by the wine yeast VL3.

In winemaking, nutrient supplementation is a common practice for optimising fermentation and producing quality wine. Nutritionally suboptimal grape juices are often enriched with nutrients in order to manipulate the production of yeast aroma compounds. Nutrients are also added to active dry yeast (ADY) rehydration media to enhance subsequent fermentation performance. In this study we demonstrate that nutrient supplementation at rehydration also has a significant effect on the formation of volatile sulfur compounds during wine fermentations. The concentration of the 'fruity' aroma compounds, the polyfunctional thiols 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), was increased while the concentration of the 'rotten egg' aroma compound, hydrogen sulfide (H2S), was decreased. Nutrient supplementation of the rehydration media also changed the kinetics of H2S production during fermentation by advancing onset of H2S production. Microarray analysis revealed that this was not due to expression changes within the sulfate assimilation pathway, which is known to be a major contributor to H2S production. To gain insight into possible mechanisms responsible for this effect, a component of the rehydration nutrient mix, the tri-peptide glutathione (GSH) was added at rehydration and studied for its subsequent effects on H2S formation. GSH was found to be taken up during rehydration and to act as a source for H2S during the following fermentation. These findings represent a potential approach for managing sulfur aroma production through the use of rehydration nutrients.

Monitoring autophagy in yeast using FM 4-64 fluorescence.

The original observations and experiments dealing with autophagy were purely morphological in nature. Even though more and more molecular techniques have been introduced, experimenters are often asked to provide visual evidence of autophagic processes in order to back up data obtained via other means. In yeast as well, autophagosomes were initially defined morphologically and indirectly, by observing intravacuolar autophagic bodies that accumulate upon starvation. This can be achieved by electron microscopy, which affords very high resolution but is time consuming and costly, or by light microscopy, which is a relatively inaccurate method of scoring autophagy. A third alternative, which we present here, is to use the unique properties of the fluorescent dye FM 4-64 to follow the accumulation of autophagic bodies.

Caffeine induces macroautophagy and confers a cytocidal effect on food spoilage yeast in combination with benzoic acid.

Weak organic acids are an important class of food preservatives that are particularly efficacious towards yeast and fungal spoilage. While acids with small aliphatic chains appear to function by acidification of the cytosol and are required at high concentrations to inhibit growth, more hydrophobic organic acids such as sorbic and benzoic acid have been suggested to function by perturbing membrane dynamics and are growth-inhibitory at much lower concentrations. We previously demonstrated that benzoic acid has selective effects on membrane trafficking in Saccharomyces cerevisiae. Benzoic acid selectively blocks macroautophagy in S. cerevisiae while acetic acid does not, and sorbic acid does so to a lesser extent. Indeed, while both benzoic acid and nitrogen starvation are cytostatic when assayed separately, the combination of these treatments is cytocidal, because macroautophagy is essential for survival during nitrogen starvation. In this report, we demonstrate that Zygosaccharomyces bailii, a food spoilage yeast with relatively high resistance to weak acid stress, also exhibits a cytocidal response to the combination of benzoic acid and nitrogen starvation. In addition, we show that nitrogen starvation can be replaced by caffeine supplementation. Caffeine induces a starvation response that includes the induction of macroautophagy, and the combination of caffeine and benzoic acid is cytocidal, as predicted from the nitrogen starvation data.

Aup1p, a yeast mitochondrial protein phosphatase homolog, is required for efficient stationary phase mitophagy and cell survival.

Autophagy is a catabolic membrane-trafficking process that occurs in all eukaryotic cells and leads to the hydrolytic degradation of cytosolic material in the vacuolar or lysosomal lumen. Mitophagy, a selective form of autophagy targeting mitochondria, is poorly understood at present. Several recent reports suggest that mitophagy is a selective process that targets damaged mitochondria, whereas other studies imply a role for mitophagy in cell death processes. In a screen for protein phosphatase homologs that functionally interact with the autophagy-dedicated protein kinase Atg1p in yeast, we have identified Aup1p, encoded by Saccharomyces cerevisiae reading frame YCR079w. Aup1p is highly similar to a family of protein phosphatase homologs in animal cells that are predicted to localize to mitochondria based on sequence analysis. Interestingly, we found that Aup1p localizes to the mitochondrial intermembrane space and is required for efficient mitophagy in stationary phase cells. Viability studies demonstrate that Aup1p is required for efficient survival of cells in prolonged stationary phase cultures, implying a pro-survival role for mitophagy under our working conditions. Our data suggest that Aup1p may be part of a signal transduction mechanism that marks mitochondria for sequestration into autophagosomes.