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Microorganisms Mar 2022Extracellular factors and growth conditions can affect the formation and development of bacterial biofilms. The biofilm of has been studied for decades, but so far,...
Extracellular factors and growth conditions can affect the formation and development of bacterial biofilms. The biofilm of has been studied for decades, but so far, little attention has been paid to the components of the medium that may affect the biofilm development in a closed system. It is known that Fis strongly enhances biofilm in complete LB medium. However, this is not the case in the defined M9 medium, which led us to question why the bacterium behaves differently in these two media. Detailed analysis of the individual medium components revealed that tryptone as the LB proteinaceous component maintains biofilm in its older stages. Although the growth parameters of planktonic cells were similar in the media containing tryptone or an equivalent concentration of amino acids, only the tryptone had a positive effect on the mature biofilm of the wild type strain of . Thus, the peptides in the environment may influence mature biofilm as a structural factor and not only as an energy source. Testing the effect of other biopolymers on biofilm formation showed variable results even for polymers with a similar charge, indicating that biopolymers can affect biofilm through a number of bacterial factors.
PubMed: 35336191
DOI: 10.3390/microorganisms10030618 -
Scientific Reports Dec 2023Pseudomonads are ubiquitous bacteria with importance in medicine, soil, agriculture, and biomanufacturing. We report a novel Pseudomonas putida phage, MiCath, which is...
Pseudomonads are ubiquitous bacteria with importance in medicine, soil, agriculture, and biomanufacturing. We report a novel Pseudomonas putida phage, MiCath, which is the first known phage infecting P. putida S12, a strain increasingly used as a synthetic biology chassis. MiCath was isolated from garden soil under a tomato plant using P. putida S12 as a host and was also found to infect four other P. putida strains. MiCath has a ~ 61 kbp double-stranded DNA genome which encodes 97 predicted open reading frames (ORFs); functions could only be predicted for 48 ORFs using comparative genomics. Functions include structural phage proteins, other common phage proteins (e.g., terminase), a queuosine gene cassette, a cas4 exonuclease, and an endosialidase. Restriction digestion analysis suggests the queuosine gene cassette encodes a pathway capable of modification of guanine residues. When compared to other phage genomes, MiCath shares at most 74% nucleotide identity over 2% of the genome with any sequenced phage. Overall, MiCath is a novel phage with no close relatives, encoding many unique gene products.
Topics: Bacteriophages; Genome, Viral; Pseudomonas putida; DNA, Viral; Nucleoside Q; Sequence Analysis, DNA; Soil; Open Reading Frames; Phylogeny
PubMed: 38071193
DOI: 10.1038/s41598-023-48634-z -
Frontiers in Microbiology 2022The stringent response is a ubiquitous bacterial reaction triggered by nutrient deprivation and mediated by the intracellular concentrations of ppGpp and pppGpp. These...
The stringent response is a ubiquitous bacterial reaction triggered by nutrient deprivation and mediated by the intracellular concentrations of ppGpp and pppGpp. These alarmones, jointly referred to as (p)ppGpp, control gene transcription, mRNA translation and protein activity to adjust the metabolism and growth rate to environmental changes. While the ability of (p)ppGpp to mediate cell growth slowdown and metabolism adaptation has been demonstrated in , it's role in remains unclear. The aims of this study were therefore to determine which forms of (p)ppGpp are synthetized in response to severe growth inhibition in , and to decipher the mechanisms of (p)ppGpp-mediated metabolic regulation in this bacterium. We exposed exponentially growing cells of to serine hydroxamate (SHX), a serine analog known to trigger the stringent response, and tracked the dynamics of intra- and extracellular metabolites using untargeted quantitative MS and NMR-based metabolomics, respectively. We found that SHX promotes ppGpp and pppGpp accumulation few minutes after exposure and arrests bacterial growth. Meanwhile, central carbon metabolites increase in concentration while purine pathway intermediates drop sharply. Importantly, in a Δ mutant and a ppGpp strain in which (p)ppGpp synthesis genes were deleted, SHX exposure inhibited cell growth but led to an accumulation of purine pathway metabolites instead of a decrease, suggesting that as observed in other bacteria, (p)ppGpp downregulates the purine pathway in . Extracellular accumulations of pyruvate and acetate were observed as a specific metabolic consequence of the stringent response. Overall, our results show that (p)ppGpp rapidly remodels the central carbon metabolism and the purine biosynthesis pathway in . These data represent a hypothesis-generating resource for future studies on the stringent response.
PubMed: 35495732
DOI: 10.3389/fmicb.2022.872749 -
Applied and Environmental Microbiology Oct 2020With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, KT2440 is emerging as an important chassis organism for metabolic...
With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of the organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. The gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes coexist, making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged random barcode transposon sequencing (RB-Tn-Seq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on fatty acids of varying chain lengths indicated specific enzyme substrate preferences and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data, we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with coenzyme A (CoA). Because fatty acids and alcohols may serve as both feedstocks and final products of metabolic-engineering efforts, the fitness data presented here will help guide future genomic modifications toward higher titers, rates, and yields. To engineer novel metabolic pathways into , a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here, we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of the bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and to channel the flux of specific pathway intermediates as desired.
Topics: Alcohols; DNA Transposable Elements; DNA, Bacterial; Fatty Acids; Metabolic Networks and Pathways; Pseudomonas putida; Sequence Analysis, DNA
PubMed: 32826213
DOI: 10.1128/AEM.01665-20 -
Current Research in Food Science 2021is widely recognized as a spoiler of fresh foods under cold storage, and recently associated also with infections in clinical settings. The presence of antibiotic...
is widely recognized as a spoiler of fresh foods under cold storage, and recently associated also with infections in clinical settings. The presence of antibiotic resistance genes (ARGs) could be acquired and transmitted by horizontal genetic transfer and further increase the risk associated with its persistence in food and the need to be deeper investigated. Thus, in this work we presented a genomic and phenotypic analysis of the psychrotrophic ITEM 17297 to provide new insight into AR mechanisms by this species until now widely studied only for its spoilage traits. ITEM 17297 displayed resistance to several classes of antibiotics and it also formed huge amounts of biofilm; this latter registered increases at 15 °C in comparison to the optimum growth condition (30 °C). After ITEM 17297 biofilms exposure to antibiotic concentrations higher than 10-fold their MIC values no eradication occurred; interestingly, biomasses of biofilm cultivated at 15 °C increased their amount in a dose-dependent manner. Genomic analyses revealed determinants (RND-systems, ABC-transporters, and MFS-efflux pumps) for multi-drugs resistance (β-lactams, macrolides, nalidixic acid, tetracycline, fusidic acid and bacitracin) and a novel C allele. Biofilm and motility related pathways were depicted underlying their contribution to AR. Based on these results, underestimated psychrotrophic pseudomonas, such as the herein studied ITEM 17297 strain, might assume relevance in relation to the risk associated with the transfer of antimicrobial resistance genes to humans through cold stored contaminated foods. biofilm and AR related molecular targets herein identified will provide a basis to clarify the interaction between AR and biofilm formation and to develop novel strategies to counteract the persistence of multidrug resistant in the food chain.
PubMed: 33718885
DOI: 10.1016/j.crfs.2021.02.001 -
Journal of Global Antimicrobial... Mar 2024Wild birds are vectors of antimicrobial resistance. Birds living in close contact with humans or other animals, like feral pigeons (Columba livia), might be especially...
OBJECTIVES
Wild birds are vectors of antimicrobial resistance. Birds living in close contact with humans or other animals, like feral pigeons (Columba livia), might be especially prone to acquire resistance genes such as those encoding extended-spectrum beta-lactamases (ESBLs) and carbapenemases.
METHODS
Cloacal samples (n = 206) of free-living feral pigeons (C. livia) were collected in Sousse and Monastir, Tunisia. Antimicrobial susceptibility profiles were determined by disc-diffusion, and resistant isolates were short- and long-read whole-genome sequenced. Sequence analysis was performed using tools of the Centre for Genomic Epidemiology, and Phylogenetic analysis was performed based on the core-genome MLST.
RESULTS
Fourteen (14/206, 6.8%) pigeons harboured Enterobacterales resistant to last-generations cephalosporins, of which 10 were CTX-M-15- or CTX-M-27-producers, while two (1.0%) carried a VIM-2-producing Pseudomonas putida. Positive pigeons lived on four different livestock farms. Three STs (ST206, ST5584, ST8149) were identified among E. coli, of which ST5584 and ST8149 were found in two different farms. Genetic diversity was also observed in Enterobacter cloacae and P. putida isolates. The bla genes were chromosomally encoded, while the bla genes were carried on highly similar IncF/F-:A-:B53 plasmids. The bla gene was located on a class 1 integron co-harbouring several resistance genes.
CONCLUSION
Pigeons living on livestock farms carried clinically important resistance genes encoding ESBLs and carbapenemases. Our results evidenced that both clonal (ST8149 and ST5584) and plasmidic (IncF/F-:A-:B53) transfers played a role in the spread of resistance genes among pigeons. Further studies are needed to identify factors favouring the transfer and persistence of resistance genes within the pigeon communities.
Topics: Animals; Humans; Columbidae; Escherichia coli; Pseudomonas putida; Multilocus Sequence Typing; Tunisia; Phylogeny; beta-Lactamases; Anti-Infective Agents
PubMed: 38145799
DOI: 10.1016/j.jgar.2023.12.013 -
Environmental Microbiology Feb 2023The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently...
The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.
Topics: Pentoses; Xylose; Arabinose; Pseudomonas putida; Oxidative Stress
PubMed: 36465038
DOI: 10.1111/1462-2920.16296 -
BMC Microbiology Jan 2021Pseudomonas putida KT2440 is a metabolically versatile, HV1-certified, genetically accessible, and thus interesting microbial chassis for biotechnological applications....
BACKGROUND
Pseudomonas putida KT2440 is a metabolically versatile, HV1-certified, genetically accessible, and thus interesting microbial chassis for biotechnological applications. However, its obligate aerobic nature hampers production of oxygen sensitive products and drives up costs in large scale fermentation. The inability to perform anaerobic fermentation has been attributed to insufficient ATP production and an inability to produce pyrimidines under these conditions. Addressing these bottlenecks enabled growth under micro-oxic conditions but does not lead to growth or survival under anoxic conditions.
RESULTS
Here, a data-driven approach was used to develop a rational design for a P. putida KT2440 derivative strain capable of anaerobic respiration. To come to the design, data derived from a genome comparison of 1628 Pseudomonas strains was combined with genome-scale metabolic modelling simulations and a transcriptome dataset of 47 samples representing 14 environmental conditions from the facultative anaerobe Pseudomonas aeruginosa.
CONCLUSIONS
The results indicate that the implementation of anaerobic respiration in P. putida KT2440 would require at least 49 additional genes of known function, at least 8 genes encoding proteins of unknown function, and 3 externally added vitamins.
Topics: Anaerobiosis; Bacterial Proteins; Computer Simulation; Databases, Genetic; Fermentation; Gene Expression Profiling; Metabolic Engineering; Microbial Viability; Pseudomonas putida; Pyrimidines
PubMed: 33407113
DOI: 10.1186/s12866-020-02058-1 -
In Vivo (Athens, Greece) Jun 2020The Covid-19 pandemic is a world-wide crisis without an effective therapy. While most approaches to therapy are using repurposed drugs that were developed for other... (Review)
Review
The Covid-19 pandemic is a world-wide crisis without an effective therapy. While most approaches to therapy are using repurposed drugs that were developed for other diseases, it is thought that targeting the biology of the SARS-CoV-2 virus, which causes Covid-19, can result in an effective therapeutic treatment. The coronavirus RNA cap structure is methylated by two viral methyltransferases that transfer methyl groups from S-adenosylmethionine (SAM). The proper methylation of the virus depends on the level of methionine in the host to form SAM. Herein, we propose to restrict methionine availability by treating the patient with oral recombinant methioninase, aiming to treat Covid-19. By restricting methionine we not only interdict viral replication, which depends on the viral RNA cap methyaltion, but also inhibit the proliferation of the infected cells, which have an increased requirement for methionine. Most importantly, the virally-induced T-cell- and macrophage-mediated cytokine storm, which seems to be a significant cause for Covid-19 deaths, can also be inhibited by restricting methionine, since T-cell and macrophrage activation greatly increases the methionine requirement for these cells. The evidence reviewed here suggests that oral recombinant methioninase could be a promising treatment for coronavirus patients.
Topics: Administration, Oral; Antiviral Agents; Bacterial Proteins; Betacoronavirus; COVID-19; Carbon-Sulfur Lyases; Clinical Trials as Topic; Coronavirus Infections; Cytokine Release Syndrome; Humans; Lymphocyte Activation; Macrophage Activation; Meta-Analysis as Topic; Methionine; Methylation; Pandemics; Pneumonia, Viral; Pseudomonas putida; RNA Caps; RNA Processing, Post-Transcriptional; RNA, Viral; Recombinant Proteins; S-Adenosylmethionine; SARS-CoV-2; T-Lymphocyte Subsets; Virus Replication
PubMed: 32503816
DOI: 10.21873/invivo.11948 -
Heliyon Nov 2023In this work, it is presented a first approach of a mathematical and kinetic analysis for improving the decoloration and further degradation process of an azo dye named...
In this work, it is presented a first approach of a mathematical and kinetic analysis for improving the decoloration and further degradation process of an azo dye named acid red 27 (AR27), by means of a novel microbial consortium formed by the fungus and the bacterium . A multivariate analysis was carried out by simulating scenarios with different operating conditions and developing a specific mathematical model based on kinetic equations describing all stages of the biological process, from microbial growth and substrate consuming to decoloration and degradation of intermediate compounds. Additionally, a sensitivity analysis was performed by using a factorial design and the Response Surface Method (RSM), for determining individual and interactive effects of variables like, initial glucose concentration, initial dye concentration and the moment in time for bacterial inoculation, on response variables assessed in terms of the minimum time for: full decoloration of AR27 (R = 2.375 days); maximum production of aromatic metabolites (R = 1.575 days); and full depletion of aromatic metabolites (R = 12.9 days). Using RSM the following conditions improved the biological process, being: an initial glucose concentration of 20 g l, an initial AR27 concentration of 0.2 g l and an inoculation moment in time of at day 1. The mathematical model is a feasible tool for describing AR27 decoloration and its further degradation by the microbial consortium of and , this model will also work as a mathematical basis for designing novel bio-reaction systems than can operate with the same principle of the described consortium.
PubMed: 38027625
DOI: 10.1016/j.heliyon.2023.e21793