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Microbial Cell Factories Dec 2022Extremolytes enable microbes to withstand even the most extreme conditions in nature. Due to their unique protective properties, the small organic molecules, more and...
BACKGROUND
Extremolytes enable microbes to withstand even the most extreme conditions in nature. Due to their unique protective properties, the small organic molecules, more and more, become high-value active ingredients for the cosmetics and the pharmaceutical industries. While ectoine, the industrial extremolyte flagship, has been successfully commercialized before, an economically viable route to its highly interesting derivative 5-hydroxyectoine (hydroxyectoine) is not existing.
RESULTS
Here, we demonstrate high-level hydroxyectoine production, using metabolically engineered strains of C. glutamicum that express a codon-optimized, heterologous ectD gene, encoding for ectoine hydroxylase, to convert supplemented ectoine in the presence of sucrose as growth substrate into the desired derivative. Fourteen out of sixteen codon-optimized ectD variants from phylogenetically diverse bacterial and archaeal donors enabled hydroxyectoine production, showing the strategy to work almost regardless of the origin of the gene. The genes from Pseudomonas stutzeri (PST) and Mycobacterium smegmatis (MSM) worked best and enabled hydroxyectoine production up to 97% yield. Metabolic analyses revealed high enrichment of the ectoines inside the cells, which, inter alia, reduced the synthesis of other compatible solutes, including proline and trehalose. After further optimization, C. glutamicum Ptuf ectD achieved a titre of 74 g L hydroxyectoine at 70% selectivity within 12 h, using a simple batch process. In a two-step procedure, hydroxyectoine production from ectoine, previously synthesized fermentatively with C. glutamicum ectABC, was successfully achieved without intermediate purification.
CONCLUSIONS
C. glutamicum is a well-known and industrially proven host, allowing the synthesis of commercial products with granted GRAS status, a great benefit for a safe production of hydroxyectoine as active ingredient for cosmetic and pharmaceutical applications. Because ectoine is already available at commercial scale, its use as precursor appears straightforward. In the future, two-step processes might provide hydroxyectoine de novo from sugar.
Topics: Corynebacterium glutamicum; Amino Acids, Diamino; Bacteria
PubMed: 36578077
DOI: 10.1186/s12934-022-02003-z -
Enrichment of native plastic-associated biofilm communities to enhance polyester degrading activity.Environmental Microbiology Dec 2023Plastic pollution is an increasing worldwide problem urgently requiring a solution. While recycling rates are increasing globally, only 9% of all plastic waste has been...
Plastic pollution is an increasing worldwide problem urgently requiring a solution. While recycling rates are increasing globally, only 9% of all plastic waste has been recycled, and with the cost and limited downstream uses of recycled plastic, an alternative is needed. Here, we found that expanded polystyrene (EPS) promoted high levels of bacterial biofilm formation and sought out environmental EPS waste to characterize these native communities. We demonstrated that the EPS attached communities had limited plastic degrading activity. We then performed a long-term enrichment experiment where we placed a robust selection pressure on these communities by limiting carbon availability such that the waste plastic was the only carbon source. Seven of the resulting enriched bacterial communities had increased plastic degrading activity compared to the starting bacterial communities. Pseudomonas stutzeri was predominantly identified in six of the seven enriched communities as the strongest polyester degrader. Sequencing of one isolate of P. stutzeri revealed two putative polyesterases and one putative MHETase. This indicates that waste plastic-associated biofilms are a source for bacteria that have plastic-degrading potential, and that this potential can be unlocked through selective pressure and further in vitro enrichment experiments, resulting in biodegradative communities that are better than nature.
Topics: Polyesters; Bacteria; Polystyrenes; Biofilms; Carbon
PubMed: 37515381
DOI: 10.1111/1462-2920.16466 -
Applied and Environmental Microbiology Apr 2022Antibiotic-resistant bacteria and the spread of antibiotic resistance genes (ARGs) pose a serious risk to human and veterinary health. While many studies focus on the...
Antibiotic-resistant bacteria and the spread of antibiotic resistance genes (ARGs) pose a serious risk to human and veterinary health. While many studies focus on the movement of live antibiotic-resistant bacteria to the environment, it is unclear whether extracellular ARGs (eARGs) from dead cells can transfer to live bacteria to facilitate the evolution of antibiotic resistance in nature. Here, we use eARGs from dead, antibiotic-resistant Pseudomonas stutzeri cells to track the movement of eARGs to live P. stutzeri cells via natural transformation, a mechanism of horizontal gene transfer involving the genomic integration of eARGs. In sterile, antibiotic-free agricultural soil, we manipulated the eARG concentration, soil moisture, and proximity to eARGs. We found that transformation occurred in soils inoculated with just 0.25 μg of eDNA g soil, indicating that even low concentrations of soil eDNA can facilitate transformation (previous estimates suggested ∼2 to 40 μg eDNA g soil). When eDNA was increased to 5 μg g soil, there was a 5-fold increase in the number of antibiotic-resistant P. stutzeri cells. We found that eARGs were transformed under soil moistures typical of terrestrial systems (5 to 30% gravimetric water content) but inhibited at very high soil moistures (>30%). Overall, this work demonstrates that dead bacteria and their eARGs are an overlooked path to antibiotic resistance. More generally, the spread of eARGs in antibiotic-free soil suggests that transformation allows genetic variants to establish in the absence of antibiotic selection and that the soil environment plays a critical role in regulating transformation. Bacterial death can release eARGs into the environment. Agricultural soils can contain upwards of 10 ARGs g soil, which may facilitate the movement of eARGs from dead to live bacteria through a mechanism of horizontal gene transfer called natural transformation. Here, we track the spread of eARGs from dead, antibiotic-resistant Pseudomonas stutzeri cells to live antibiotic-susceptible P. stutzeri cells in sterile agricultural soil. Transformation increased with the abundance of eARGs and occurred in soils ranging from 5 to 40% gravimetric soil moisture but was lowest in wet soils (>30%). Transformants appeared in soil after 24 h and persisted for up to 15 days even when eDNA concentrations were only a fraction of those found in field soils. Overall, our results show that natural transformation allows eARGs to spread and persist in antibiotic-free soils and that the biological activity of eDNA after bacterial death makes environmental eARGs a public health concern.
Topics: Anti-Bacterial Agents; Bacteria; DNA; Drug Resistance, Microbial; Genes, Bacterial; Humans; Soil; Soil Microbiology; Wastewater
PubMed: 35323025
DOI: 10.1128/aem.02280-21 -
Antibiotics (Basel, Switzerland) May 2023Carbapenems are considered a last resort for the treatment of multi-drug-resistant bacterial infections in humans. In this study, we investigated the occurrence of...
Carbapenems are considered a last resort for the treatment of multi-drug-resistant bacterial infections in humans. In this study, we investigated the occurrence of carbapenem-resistant bacteria in feedlots in Alberta, Canada. The presumptive carbapenem-resistant isolates ( = 116) recovered after ertapenem enrichment were subjected to antimicrobial susceptibility testing against 12 different antibiotics, including four carbapenems. Of these, 72% of the isolates ( = 84) showed resistance to ertapenem, while 27% of the isolates ( = 31) were resistant to at least one other carbapenem, with all except one isolate being resistant to at least two other drug classes. Of these 31 isolates, 90% were carbapenemase positive, while a subset of 36 ertapenem-only resistant isolates were carbapenemase negative. The positive isolates belonged to three genera; , , and , with the majority being ( = 20) as identified by 16S rRNA gene sequencing. Whole genome sequencing identified intrinsic carbapenem resistance genes, including and its variants (), (), (), (), and and (). The acquired carbapenem resistance gene () was identified in and . In a comparative genomic analysis, clinical clustered separately from those recovered from bovine feces. In conclusion, despite the use of selective enrichment methods, finding carbapenem-resistant bacteria within a feedlot environment was a rarity.
PubMed: 37370279
DOI: 10.3390/antibiotics12060960 -
BMC Genomics Apr 2015Phylogenetic heterogeneity across Pseudomonas genus is complemented by its diverse genome architecture enriched by accessory genetic elements (plasmids, transposons, and...
BACKGROUND
Phylogenetic heterogeneity across Pseudomonas genus is complemented by its diverse genome architecture enriched by accessory genetic elements (plasmids, transposons, and integrons) conferring resistance across this genus. Here, we sequenced a stress tolerant genotype i.e. Pseudomonas sp. strain RL isolated from a hexachlorocyclohexane (HCH) contaminated pond (45 mg of total HCH g(-1) sediment) and further compared its gene repertoire with 17 reference ecotypes belonging to P. stutzeri, P. mendocina, P. aeruginosa, P. psychrotolerans and P. denitrificans, representing metabolically diverse ecosystems (i.e. marine, clinical, and soil/sludge). Metagenomic data from HCH contaminated pond sediment and similar HCH contaminated sites were further used to analyze the pan-genome dynamics of Pseudomonas genotypes enriched across increasing HCH gradient.
RESULTS
Although strain RL demonstrated clear species demarcation (ANI ≤ 80.03%) from the rest of its phylogenetic relatives, it was found to be closest to P. stutzeri clade which was further complemented functionally. Comparative functional analysis elucidated strain specific enrichment of metabolic pathways like α-linoleic acid degradation and carbazole degradation in Pseudomonas sp. strain RL and P. stutzeri XLDN-R, respectively. Composition based methods (%codon bias and %G + C difference) further highlighted the significance of horizontal gene transfer (HGT) in evolution of nitrogen metabolism, two-component system (TCS) and methionine metabolism across the Pseudomonas genomes used in this study. An intact mobile class-I integron (3,552 bp) with a captured gene cassette encoding for dihydrofolate reductase (dhfra1) was detected in strain RL, distinctly demarcated from other integron harboring species (i.e. P. aeruginosa, P. stutzeri, and P. putida). Mobility of this integron was confirmed by its association with Tnp21-like transposon (95% identity) suggesting stress specific mobilization across HCH contaminated sites. Metagenomics data from pond sediment and recently surveyed HCH adulterated soils revealed the in situ enrichment of integron associated transposase gene (TnpA6100) across increasing HCH contamination (0.7 to 450 mg HCH g(-1) of soil).
CONCLUSIONS
Unlocking the potential of comparative genomics supplemented with metagenomics, we have attempted to resolve the environment and strain specific demarcations across 18 Pseudomonas gene complements. Pan-genome analyses of these strains indicate at astoundingly diverse metabolic strategies and provide genetic basis for the cosmopolitan existence of this taxon.
Topics: Base Sequence; Gene Transfer, Horizontal; Genome, Bacterial; Genotype; Hexachlorocyclohexane; Integrons; Metagenomics; Phylogeny; Pseudomonas; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Soil Microbiology; Water Microbiology; Water Pollutants, Chemical
PubMed: 25898829
DOI: 10.1186/s12864-015-1488-2 -
International Journal of Molecular... Feb 2023In marine environments, biofilm can cause negative impacts, including the biofouling process. In the search for new non-toxic formulations that inhibit biofilm,...
In marine environments, biofilm can cause negative impacts, including the biofouling process. In the search for new non-toxic formulations that inhibit biofilm, biosurfactants (BS) produced by the genus have demonstrated considerable potential. To elucidate the changes that BS from promote in growth inhibition and biofilm formation, this research performed a nuclear magnetic resonance (NMR) metabolomic profile analysis to compare the metabolic differences between planktonic cells and biofilms of , a pioneer fouling bacteria. The multivariate analysis showed a clear separation between groups with a higher concentration of metabolites in the biofilm than in planktonic cells of . When planktonic and biofilm stages were treated with BS, some differences were found among them. In planktonic cells, the addition of BS had a minor effect on growth inhibition, but at a metabolic level, NADP+, trehalose, acetone, glucose, and betaine were up-regulated in response to osmotic stress. When the biofilm was treated with the BS, a clear inhibition was observed and metabolites such as glucose, acetic acid, histidine, lactic acid, phenylalanine, uracil, and NADP+ were also up-regulated, while trehalose and histamine were down-regulated in response to the antibacterial effect of the BS.
Topics: Biofouling; Pseudomonas stutzeri; Plankton; NADP; Trehalose; Biofilms; Bacillus
PubMed: 36835662
DOI: 10.3390/ijms24044249 -
Molecular Microbiology Mar 2018Bacterial response to metals can require complex regulation. We report an overlapping regulation for copper and zinc resistance genes in the denitrifying bacterium,...
Bacterial response to metals can require complex regulation. We report an overlapping regulation for copper and zinc resistance genes in the denitrifying bacterium, Pseudomonas stutzeri RCH2, by three two-component regulatory proteins CopR1, CopR2 and CzcR. We conducted genome-wide evaluations to identify gene targets of two paralogous regulators, CopR1 and CopR2, annotated for copper signaling, and compared the results with the gene targets for CzcR, implicated in zinc signaling. We discovered that the CopRs and CzcR have largely common targets, and crossregulate a core set of P. stutzeri copper and zinc responsive genes. We established that this crossregulation is enabled by a conserved binding motif in the upstream regulatory regions of the target genes. The crossregulation is physiologically relevant as these regulators synergistically and antagonistically target multicopper oxidases, metal efflux and sequestration systems. CopR1 and CopR2 upregulate two cop operons encoding copper tolerance genes, while all three regulators downregulate a putative copper chaperone, Psest_1595. CzcR also upregulated the oprD gene and the CzcIABC Zn efflux system, while CopR1 and CopR2 downregulated these genes. Our study suggests that crossregulation of copper and zinc homeostasis can be advantageous, and in P. stutzeri this is enabled by shared binding motifs for multiple response regulators.
Topics: Bacterial Proteins; Copper; DNA, Bacterial; Gene Expression Regulation, Bacterial; Homeostasis; Molecular Chaperones; Operon; Protein Binding; Pseudomonas stutzeri; Signal Transduction; Zinc
PubMed: 29341298
DOI: 10.1111/mmi.13909 -
Archives of Razi Institute Oct 2022Psoriasis is an autoimmune, persistent, inflammatory skin illness that is influenced by a variety of circumstances. Psoriasis etiology is strongly linked to bacteria,...
Psoriasis is an autoimmune, persistent, inflammatory skin illness that is influenced by a variety of circumstances. Psoriasis etiology is strongly linked to bacteria, particularly those in the pharynx and skin. This research intended to deepen our understanding by elucidating the connection between the skin microbiome and psoriasis to improve therapeutic balance using probiotics, antimicrobials, and even topical microbiota transplantation. In this work, the culture-dependent approach is utilized to compare the skin microbiomes of psoriatic and healthy individuals. On brane-heart infusion agar, swabs from 60 psoriasis patients in the flare-up stag and 40 healthy controls were cultured and grown for 48 hours. The resultant colonies were then subcultured and purified to produce a single pure colony. Using Macconkey agar for preliminary colony identification, Vitek then characterized the purified colonies. The results revealed substantial bacterial species and phyla variations between psoriatic patients and healthy controls. In addition, increased rates of opportunistic infections and were detected in psoriatic patients' normal skin and lesions.
Topics: Adult; Female; Humans; Male; Agar; Bacteria; Culture Media; Psoriasis; Skin
PubMed: 37123117
DOI: 10.22092/ARI.2022.359398.2414 -
Ecotoxicology and Environmental Safety Feb 2023Crude oil pollution is environmentally ubiquitous and has become a global public concern about its impact on human health. Asphaltenes are the key components of heavy...
Biodegradation of asphaltenes by an indigenous bioemulsifier-producing Pseudomonas stutzeri YWX-1 from shale oil in the Ordos Basin: Biochemical characterization and complete genome analysis.
Crude oil pollution is environmentally ubiquitous and has become a global public concern about its impact on human health. Asphaltenes are the key components of heavy crude oil (HCO) that are underutilized due to their high viscosity and density, and yet, the associated information about biodegradation is extremely limited in the literature. In the present study, an indigenous bacterium with effective asphaltene-degrading activity was isolated from oil shale and identified as Pseudomonas stutzeri by a polyphasic taxonomic approach, named YWX-1. Supplemented with 75 g L heavy crude oil as the sole carbon source for growth in basic mineral salts liquid medium (MSM), strain YWX-1 was able to remove 49% of asphaletene fractions within 14 days, when it was cultivated with an initial inoculation size of 1%. During the degradation process, the bioemulsifier produced by strain YWX-1 could emulsify HCO obviously into particles, as well as it had the ability to solubilize asphaletenes. The bioemulsifier was identified to be a mixture of polysaccharide and protein through Fourier transform infrared spectroscopy (FT-IR). The genome of strain YWX-1 contains one circular chromosome of 4488441 bp with 63.98% GC content and 4145 protein coding genes without any plasmid. Further genome annotation indicated that strain YWX-1 possesses a serial of genes involved in bio-emulsification and asphaltenes biodegradation. This work suggested that P. stutzeri YWX-1 could be a promising species for bioremediation of HCO and its genome analysis provided insight into the molecular basis of asphaltene biodegradation and bioemulsifier production.
Topics: Humans; Biodegradation, Environmental; Pseudomonas stutzeri; Spectroscopy, Fourier Transform Infrared; Petroleum; Minerals
PubMed: 36669280
DOI: 10.1016/j.ecoenv.2023.114551 -
BMC Microbiology Apr 2019Pythium insidiosum is a member of the oomycetes class of aquatic fungus-like microorganisms. It can infect humans and animals through skin wounds and the eyes, causing...
BACKGROUND
Pythium insidiosum is a member of the oomycetes class of aquatic fungus-like microorganisms. It can infect humans and animals through skin wounds and the eyes, causing pythiosis, an infectious disease with high morbidity and mortality rates. Antifungal agents are ineffective as pythiosis treatments because ergosterol, the target site of most antifungal agents, is not found in the P. insidiosum cytoplasmic membrane. The best choice for treatment is surgical removal of the infected organ. While natural plant products or secretory substances from bacterial flora have exhibited in vitro anti-P. insidiosum activity, their mechanism of action remains unknown. Therefore, this study hypothesized that the mechanism of action could be related to changes in P. insidiosum biochemical composition (such as lipid, carbohydrate, protein or nucleic acid) following exposure to the inhibitory substances. The biochemical composition of P. insidiosum was investigated by Synchrotron radiation-based Fourier-transform infrared (FTIR) microspectroscopy.
RESULTS
Fraction No.6 from the crude extract of P. stutzeri ST1302, fraction No.1 from the crude extract of K. pneumoniae ST2501 and xanthyletin were used as anti-P. insidiosum substances, with MFCs at 3.125, 1.57-1.91, 0.003 mg/ml, respectively. The synchrotron FTIR results show that the deconvoluted peak distributions in the amide I, amide II, and mixed regions were significantly different between the treatment and control groups.
CONCLUSIONS
Xanthyletin and the secondary metabolites from P. stutzeri ST1302 and K. pneumoniae ST2501 exerted anti-P. insidiosum activity that clearly changed the proteins in P. insidiosum. Further study, including proteomics analysis and in vivo susceptibility testing, should be undertaken to develop a better understanding of the mechanism of anti-P. insidiosum activity.
Topics: Antifungal Agents; Coumarins; Klebsiella pneumoniae; Microbial Sensitivity Tests; Pseudomonas stutzeri; Pythium; Secondary Metabolism; Spectroscopy, Fourier Transform Infrared
PubMed: 30991991
DOI: 10.1186/s12866-019-1452-4