Biology subjects

Sauvageau, D.

Publications and source records attributed to Sauvageau, D..

12 recordsLinked to original sources

bioRxiv2026

Diverse phages of ammonia oxidizers with the potential to modulate nitrification

Agriculture is a major source of anthropogenic greenhouse-gas emissions and the largest source of nitrous oxide (N2O), an extremely potent greenhouse gas and ozone-depleting agent. Soil N2O emissions are largely driven by nitrification, in which ammonia-oxidizing microorganisms catalyze the oxidation of ammonia to nitrite. Nitrification not only mediates N2O fluxes but also reduces fertilization efficiency and contributes to eutrophication through nitrate leaching.\ Bacteriophage (phage)-based control of microbial communities is gathering interest; however, phages infecting ammonia-oxidizers are mostly uncharacterized, with only one lytic phage having been described, limiting the potential for developing phage-mediated nitrification control. Here, we characterized the largest set of cultivated phages infecting ammonia-oxidizing bacteria (AOB) to date: 45 dsDNA phages collected from urban wastewater, infecting four AOB species, with 16 demonstrating cross-genus host ranges and capable of eliminating nitrification activity in liquid cultures. Phylogenetic and taxonomic analyses revealed six proposed families of Caudoviricetes and several monophyletic clades, likely representing higher-level lineages. Structure-guided genome annotation revealed a diverse collection of auxiliary metabolic genes encoded by phage, ranging from a complete ABC transporter cassette to a large antimicrobial resistance gene cluster. These results unveil previously unrecognized diversity of cultivated AOB phages and their potential to alter host physiology. Our data describes a broad taxonomic and functional repertoire of cultured AOB phages, suggesting that viruses play a significant and complex role in nitrification. Moreover, we outline an effective methodological framework for isolating AOB phages from environmental samples. These results reframe our understanding of environmental nitrification and enable intensified cultivation, characterization and use of phages for its control.

microbiology↗

bioRxiv2025

The role and effects of the phage T4 Ac protein on infection

The ac gene from T4 and T4-like phages is associated with susceptibility to acriflavine, an acridine that intercalates with DNA, disrupting replication and transcription. While this interference has been shown to inhibit phage replication - resulting in reduced frequency of infection and viral titer at the population level - and mutation or knockout of the ac gene renders the phage resistant to acriflavine, the role of the ac gene product, Ac, has not yet been elucidated. This study aims to further explore the role of the ac gene in phage infection by expressing it in Escherichia coli and evaluating its effects on the host under varying growth conditions and during phage infection, both in the presence and absence of acriflavine. E. coli induced to express the ac gene from phage T4 showed an increased susceptibility to acriflavine compared to the same strain not undergoing expression (non-induced) or not carrying the ac expression plasmid (empty). Moreover, in the presence of acriflavine, the parental phage T4 was not able to infect the host variants (induced, non-induced and empty), suggesting the Ac protein is involved in a potential membrane modification leading to acriflavine hindering infection. When the ac gene was deleted from the T4 phage (T4{Delta}ac), the mutated phage was capable of infecting the three host variants (empty, non-induced and induced) in the presence of acriflavine, showing resistance to this acridine. Based on experimental results and protein structure prediction, we propose that the Ac protein integrates into the bacterial host cell membrane and interacts with the AcrAB-TolC efflux pump, either altering its conformation or blocking its function, thereby preventing the excretion of acriflavine which accumulates in the cell and impedes DNA replication

microbiology↗

bioRxiv2025

Prolific release of S-layer units and associated proteins by the methanotrophic bacterium Methylomicrobium album BG8

Some methanotrophs synthesize S-layers that overlay their outer membrane. TEM imaging revealed that Methylomicrobium album BG8 constitutively sheds abundant S-layer units, a phenotype not found in 7 other methanotrophs, even though Methylotuvimicrobium buryatense 5GB1 produces a similar structure. Release of S-layer units occurred regardless of carbon (methane or methanol) or nitrogen (ammonium or nitrate) source, with 50X trace metals, under copper deprivation, and at all growth phases. The released S-layer units were isolated from the culture medium of M. album BG8 by density gradient centrifugation for proteome analysis. The proteome revealed the S-layer protein subunits, transporters for calcium uptake including TolC and Repeats-in-Toxin (RTX) proteins, transporters for uptake for cobalamin and siderophores, cell wall biogenesis proteins, and proteins with Type I secretion system (T1SS) target domains. M. album BG8 adapted to grow at pH 4 lost its S-layer and genome analysis revealed a frameshift mutation plus reduced expression of the S-layer unit gene plus the deletion and almost no expression of an S-layer-associated porin gene. Together, the results suggest that the biogenesis and secretion of M. album BG8 S-layer is mediated by its associated T1SS, the S-layer possesses metal acquisition functions, and low pH adaptation of M. album BG8 results in loss of S-layer, likely due to reduced, or incomplete, expression of S-layer units and loss of an associated porin. The involvement of the T1SS and shedding phenotype of the S-layer in M. album BG8 could be applied towards selective secretion of proteins and other factors of bioindustrial interest. ImportanceThe methanotrophic bacterium M. album BG8 produces and sheds large quantities of S-layer units into the culture medium regardless of carbon or nitrogen source, metal availability or growth phase. Of the 8 methanotrophic bacteria screened, only M. album BG8 possessed this phenotype. Proteomics analysis of density gradient purified culture supernatant identified the S-layer protein units and proteins involved in metal uptake and S-layer biogenesis, some with secretion signals for the T1SS. M. album BG8 adapted to grow at low pH lost production of its S-layer due to mutations in the genes encoding S-layer units and an associated porin. Better understanding of M. album BG8 S-layer production and its shedding phenotype could be harnessed for exporting expressed proteins and bioproducts of industrial interest for ease of collection and downstream processing.

microbiology↗

bioRxiv2024

Comparative study on the virulence of mycobacteriophages

The global tuberculosis (TB) epidemic affected 10 million people and caused 1.3 million deaths in 2022 alone. Multidrug-resistant TB is successfully treated in less than 60% of cases by long, expensive and aggressive treatments. Mycobacteriophages, viruses that can infect bacteria such as Mycobacterium tuberculosis--the species responsible for TB, have the potential to redefine TB prevention and treatments. However, the development of phage-based products necessitates the assessment of numerous parameters, including virulence and adsorption, to ensure their performance and quality. In this work, we characterized the virulence of three different mycobacteriophages (Fionnbharth, Muddy and D29), alone and as cocktails, against a TB model host (Mycobacterium smegmatis) under planktonic and early-stage biofilm growth conditions. Phage D29 and cocktails containing D29 had the highest virulence under all conditions. Interestingly, phages Fionnbharth and Muddy and their combination showed higher virulence against early-stage biofilm than against the planktonic phenotype. Adsorption assays indicated that all three phages had lower adsorption efficiencies on the early-stage biofilm phenotype than on the planktonic one, suggesting a reduced availability of receptors in the former. Given that, despite these lower adsorption efficiencies, the virulence of the phages and phage cocktails was either unchanged or higher against the early-stage biofilm, this phenotype must display properties that are favorable to other steps of the infection process. These results inform us on the dynamics of mycobacteriophage infections, alone and in cocktail formulations, under different host growth conditions, and serve as a basis for the development of phage products targeting mycobacteria biofilms.

microbiology↗

bioRxiv2024

Diverse bacteriohemerythrin genes of Methylomonas denitrificans FJG1 provide insight into the survival and activity of proteobacterial methanotrophs under hypoxia

Proteobacterial methanotrophs are dependent on the oxidation of methane for ATP production and assimilation of carbon into biomass. Interestingly, some types of gammaproteobacterial methanotrophs thrive in oxygen-depleted zones of lakes and other aquatic ecosystems despite their reliance on oxygen to support methane oxidation. The model gammaproteobacterial methanotroph, Methylomonas denitrificans FJG1, oxidizes methane coupled to nitrate reduction under hypoxia and highly upregulates its expression of bacteriohemerythrin (Bhr). Bhr is a homolog of eukaryotic hemerythrin, which is a protein associated with oxygen binding. Ten bhr homologs were identified in the genome of M. denitrificans FJG1, requiring phylogenetic and gene expression analyses to pinpoint which homolog is likely responsible for delivering oxygen to support methane oxidation to under low oxygen conditions. This study examined the prevalence and phylogeny of the 10 bhr homologs from M. denitrificans FJG1 in the genomes of other methanotrophs and across the Bacteria. The homolog denoted "bhr-00" was specific to methanotroph genomes, was highly expressed in M. denitrificans FJG1 under hypoxia, and its predicted structure was nearly identical to a purified oxygen-scavenging hemerythrin protein from Methylococcus capsulatus Bath. Other bhr homologs upregulated from denitrifying cultures of M. denitrificans FJG1 included those with gene neighborhoods related to oxygen sensing, denitrification and chemotaxis. Together, this study uncovered potential multifunctional roles of bacteriohemerythrin genes of M. denitrificans FJG1 under low oxygen conditions and identified the bhr homolog that most likely enables and supports oxygen delivery to methane monooxygenase enzymes in anoxic ecosystems. ImportanceAerobic gammaproteobacterial methanotrophs can survive and grow in anoxic lakes, but mechanisms that provide them with oxygen to support methane oxidation remain uncharacterized. Methylomonas denitrificans FJG1 encodes 10 copies of bacteriohemerthyrin (bhr), of which 7 are expressed at the mRNA level under low oxygen conditions. Comparing the 10 bhr homologs from M. denitrificans FJG1 with those from other methanotrophs and bacterial genomes shows that two are specific to methanotrophs. Gene neighbourhoods surrounding conserved bhr genes in methanotrophs suggest a range of potential functions including oxygen respiration, oxygen sensing, chemotaxis, and nitrate reduction. The results from this study illuminate a previously undescribed diversity of structures and potential functions of bhr homologs in M. denitrificans FJG1 and related methanotrophic bacteria. The results pinpoint a methanotroph-specific homolog, bhr-00, that is likely responsible for oxygen binding and delivery to methane monooxygenase enzymes to promote methane oxidation in low oxygen ecosystems.

microbiology↗

bioRxiv2023

Targeted spectroscopy in the eye fundus

AbstractO_ST_ABSSignificanceC_ST_ABSThe assessment of biomarkers in the eye is rapidly gaining traction for the screening, diagnosis and monitoring of ocular and neurological diseases. Targeted ocular spectroscopy is a new technology that enables the user to concurrently image the eye fundus and acquire high quality spectra from a targeted region -1.5 degrees- within the imaged area. The combination of imaging and high-sensitivity spectroscopy provides structural, compositional, and functional information of selected regions of the eye fundus. This opens the door to new, non-invasive approaches to the detection of biomarkers in the eye. AimThe aim of this study was to demonstrate the multi-modal functionality and validation of the targeted ocular spectroscopy developed. This was done in vitro, using a reference target and a model eye, and in vivo. ApproachImages and spectra from different regions of a reference target and a model eye were acquired and analyzed to validate the system. The same eye model was used to obtain fluorescence images and spectra, highlighting the capability of the system to also perform targeted ocular fluorescence spectroscopy. Subsequently, in vivo imaging and diffuse reflectance spectra were acquired to assess blood oxygen saturation in the optic nerve head and the parafovea of healthy subjects. ResultsTests conducted with the reference target showed that spectral analysis could be accurately performed within specific areas of the imaging space. Moving to the model eye, distinct spectral signatures were observed for the targeted spectral analysis done in the optic disc, the retina and the macula, consistent with the variations in tissue composition and functions between these regions mimicked by the model eye. Further, it was shown that the targeted spectral analysis could also be performed in a fluorescence mode to distinguish various fluorophores present within the imaging space. Finally, in vivo ocular oximetry experiments performed in the optic nerve head and parafovea of healthy patients showed significant differences in blood oxygen saturation between these regions (p = 0.004). ConclusionsEnabling non-invasive, sensitive diffuse reflectance and fluorescence spectroscopy in specific regions of the eye fundus opens the door to a whole new range of monitoring and diagnostic capabilities, from assessment of oxygenation in glaucoma and diabetic retinopathy to photo-oxidation and photo-degradation in age-related macular degeneration.

biophysics↗

bioRxiv2022

Lysogenized phages of methanotrophic bacteria show a broad and untapped genetic diversity

Methanotrophs are a unique class of bacteria with the ability to metabolize single-carbon compounds such as methane. They play an important role in the global methane cycle and have great potential as industrial platforms for the bioconversion of methane from industrial waste streams into valuable products, such as biofuels and bioplastics. However, many aspects of methanotroph biology have yet to be elucidated, including the prevalence and impact of lysogenized bacteriophages (phages), which can greatly affect both the ecology and the industrial performance of these bacteria. The present study investigates the presence of putative prophages in three gammaproteobacterial (Methylobacter marinus A45, Methylomicrobium album BG8, Methylomonas denitrificans FJG1) and two alphaproteobacterial (Methylosinus trichosporium OB3b, Methylocystis sp. Rockwell) methanotrophs using four programs predicting putative phage sequences (PhageBoost, PHASTER, Phigaro, and Island Viewer). Mitomycin C was used to trigger induction of prophages, which was monitored through infection dynamics. Successfully induced phages from M. marinus A45 (MirA1, MirA2), M. album BG8 (MirB1), and M. trichosporium OB3b (MirO1) were isolated and characterized using transmission electron microscopy. Subsequently, bioinformatic analyses (BLAST and phylogenetics) were performed on three induced phages to obtain a profile of their respective genetic makeup. Their broad diversity and differences from previously known phages, based on whole genome and structural gene sequences, suggest they each represent a new phage family, genus and species: "Britesideviridae Inducovirus miraone", "Patronusviridae Enigmavirus miratwo", and "Kainiviridae Tripudiumvirus miroone" represented by isolates MirA1, MirA2, and MirO1, respectively.

microbiology↗

bioRxiv2021

Use of untargeted magnetic beads to capture Mycobacterium smegmatis and Mycobacterium avium paratuberculosis prior detection by mycobacteriophage D29 and Real-Time-PCR

Untargeted magnetic beads were evaluated to capture Mycobacterium smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) from spiked feces, milk, and urine. Untargeted magnetic beads recovered more M. smegmatis cells from PBS suspension than the typical centrifugation method; 96.31% of 1.68 x 104 CFU/mL viable M. smegmatis were recovered by beads and 0% by centrifugation. Likewise, the F57-qPCR detection of MAP cells was different whether they were recovered by beads or centrifugation; cycle threshold (Ct) was lower (p<0.05) for the detection of MAP cells recovered by beads than centrifugation, indicative of higher sensitivity. Magnetic separation of MAP cells from milk, urine, and feces specimens were detected by F57 and IS900 sequences. Ct values demonstrated that beads captured no less than 109 CFU/mL from feces and no less than 104 CFU/mL from milk and urine suspensions. In another detection strategy, M. smegmatis coupled to magnetic beads were infected by mycobacteriophage D29. Plaque forming units were observed after 24 h of incubation from urine samples containing 2 x 105 and 2 x 103 CFU/mL M. smegmatis. The results of this study provide a promising tool for diagnosis of Johnes disease and other mycobacterial diseases such as tuberculosis.

microbiology↗

bioRxiv2021

Transcriptomic and metabolomic responses to carbon and nitrogen sources in Methylomicrobium album BG8

Methanotrophs use methane as their sole carbon and energy source and represent an attractive platform for converting single-carbon feedstocks into value-added compounds. Optimizing these species for biotechnological applications involves choosing an optimal growth substrate based on an understanding of cellular responses to different nutrients. Although many studies of methanotrophs have examined growth rate, yield, and central carbon flux in cultures grown with different carbon and nitrogen sources, few studies have examined more global cellular responses to different media. Here, we evaluated global transcriptomic and metabolomic profiles of Methylomicrobium album BG8 when grown with methane or methanol as the carbon source and nitrate or ammonium as the nitrogen source. We identified five key physiological changes during growth on methanol: M. album BG8 cultures upregulated transcripts for the Entner-Doudoroff and pentose phosphate pathways for sugar catabolism, produced more ribosomes, remodeled its phospholipid membrane, activated various stress response systems, and upregulated glutathione-dependent formaldehyde detoxification. When using ammonium, M. album BG8 upregulated haoAB hydroxylamine dehydrogenase and the overall central metabolic activity; whereas when using nitrate, cultures upregulated genes for nitrate assimilation and conversion. Overall, we identified several nutrient source-specific responses that could provide a valuable basis for future research on the biotechnological optimization of these species. IMPORTANCEMethanotrophs are gaining increasing interest for their biotechnological potential to convert single-carbon compounds into value-added products such as industrial chemicals, fuels, and bioplastics. Optimizing these species for biotechnological applications requires a detailed understanding of how cellular activity and metabolism varies across different growth substrates. Although each of the two most commonly used carbon sources (methane or methanol) and nitrogen sources (ammonium or nitrate) in methanotroph growth media have well-described advantages and disadvantages in an industrial context, their effects on global cellular activity remain poorly characterized. Here, we comprehensively describe the transcriptomic and metabolomic changes that characterize the growth of an industrially promising methanotroph strain on multiple combinations of carbon and nitrogen sources. Our results represent a more holistic evaluation of cellular activity than previous studies of core metabolic pathways and provide a valuable basis for the future biotechnological optimization of these species.

microbiology↗

bioRxiv2020

Analysis of selection methods to develop novel phage therapy cocktails against antimicrobial resistant clinical isolates of bacteria

Antimicrobial resistance (AMR) is a major problem globally. The main bacterial organisms associated with urinary tract infection (UTI) associated sepsis are E. coli and Klebsiella along with Enterobacter species. These all have AMR strains known as ESBL (Extended Spectrum Beta-Lactamase), which are featured on the WHO priority pathogens list as critical for research. Bacteriophages (phages) as viruses that can infect and kill bacteria, could provide an effective tool to tackle these AMR strains. There is currently no gold standard for developing a phage cocktail. Here we describe a novel approach to develop an effective phage cocktail against a set of ESBL-producing E. coli and Klebsiella largely isolated from patients in UK hospitals. By comparing different measures of phage efficacy, we show which are the most robust, and suggest an efficient screening cascade that could be used to develop phage cocktails to target other AMR bacterial species. A target panel of 38 ESBL-producing clinical strains isolated from urine samples was collated and used to test phage efficacy. After an initial screening of 68 phages, six were identified and tested against these 38 strains to determine their clinical coverage and killing efficiency. To achieve this, we assessed four different methods to assess phage virulence across these bacterial isolates. These were the Direct Spot Test (DST), the Efficiency of Plating (EOP) assay, the planktonic killing assay and the biofilm assay. The final ESBL cocktail of six phages could effectively kill 23/38 strains (61%) for Klebsiella 13/19 (68%) and for E. coli 10/19 (53%) based on the planktonic killing assay data. The ESBL E. coli collection had six isolates from the prevalent UTI-associated ST131 sequence type, five of which were targeted effectively by the final cocktail. Of the four methods used to assess phage virulence, the data suggests that planktonic killing assays are as effective as the much more time-consuming EOPs and data for the two assays correlates well. This suggests that planktonic killing is a good proxy to determine which phages should be used in a cocktail. This assay when combined with the virulence index also allows phage synergy to inform cocktail design.

microbiology↗

bioRxiv2019

Streamlined production, purification, and comparison of recombinant extracellular polyhydroxybutyrate depolymerases

Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His-tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp., Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, strategies were developed to circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate-based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15 {degrees}C, and rPhaZs from Cupriavidus sp. and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs, and to perform preliminary evaluation for applications that require PHB degradation.

molecular biology↗

bioRxiv2019

The Virulence Index: A Metric for Quantitative Analysis of Phage Virulence

BackgroundOne of the main challenges in developing phage therapy and manufacturing phage products is the reliable evaluation of their efficacy, performance and quality. Since phage virulence is intrinsically difficult to fully capture, researchers have turned to rapid but partially inadequate methods for its evaluation.\n\nMaterials and MethodsThe present study demonstrates a standardized, quantitative method to assess phage virulence based on three parameters: the Virulence Index (VP) - quantifying the virulence of a phage against a host, the local virulence (vi) - assessing killing potential at given MOIs, and MV50 - the MOI at which the phage achieves 50% of its maximum theoretical virulence. This was shown through comparative analysis of the virulence of phages T4, T5 and T7.\n\nResultsUnder the conditions tested, phage T7 displayed the highest virulence, followed by phage T4 and, finally, phage T5. The impact of parameters such as temperature and medium composition on virulence was shown for each phage. The use of the method to evaluate the virulence of combinations of phages - e.g. for cocktail formulation - is also shown with phages T5 and T7.\n\nConclusionsThe method presented provides a platform for high-throughput quantitative assessment of phage virulence and quality control of phage products. It can also be applied to phage screening, evaluation of phage strains, phage mutants, infection conditions and/or the susceptibility of host strains, and the formulation of phage cocktails.

microbiology↗