-
Microbial Genomics Jan 2023High-risk clone types in are problematic global multidrug-resistant clones. However, apart from their ability to resist antimicrobial treatment, not much is known about...
High-risk clone types in are problematic global multidrug-resistant clones. However, apart from their ability to resist antimicrobial treatment, not much is known about what sets these clones apart from the multitude of other clones. In high-risk clone ST111, it has previously been shown that replacement of the native serotype biosynthetic gene cluster (O4) by a different gene cluster (O12) by horizontal gene transfer and recombination may have contributed to the global success of this clone. However, the extent to which isolates undergo this type of serotype switching has not been adequately explored in . In the present study, a bioinformatics tool has been developed and utilized to provide a first estimate of serotype switching in groups of multidrug resistant (MDR) clinical isolates. The tool detects serotype switching by analysis of core-genome phylogeny and serotype. Analysis of a national survey of MDR isolates found a prevalence of 3.9 % of serotype-switched isolates in high-risk clone types ST111, ST244 and ST253. A global survey of MDR isolates was additionally analysed, and it was found that 2.3 % of isolates had undergone a serotype switch. To further understand this process, we determined the exact boundaries of the horizontally transferred serotype O12 island. We found that the size of the serotype island correlates with the clone type of the receiving isolate and additionally we found intra-clone type variations in size and boundaries. This suggests multiple serotype switch events. Moreover, we found that the housekeeping gene is co-transferred with the O12 serotype island, which prompted us to analyse this allele for all serotype O12 isolates. We found that 95 % of ST111 O12 isolates had a resistant allele and 86 % of all O12 isolates had a resistant allele. The rates of resistant alleles in isolates with other prevalent serotypes are all lower. Together, these results show that the transfer and acquisition of serotype O12 in high-risk clone ST111 has happened multiple times and may be facilitated by multiple donors, which clearly suggests a strong selection pressure for this process. However, gyrA-mediated antibiotic resistance may not be the only evolutionary driver.
Topics: Serogroup; Pseudomonas aeruginosa; Drug Resistance, Microbial; Gene Transfer, Horizontal
PubMed: 36748704
DOI: 10.1099/mgen.0.000919 -
Applied and Environmental Microbiology Nov 2021Silicate mineral weathering (dissolution) plays important roles in soil formation and global biogeochemical cycling. In this study, a combination of genomics,...
Silicate mineral weathering (dissolution) plays important roles in soil formation and global biogeochemical cycling. In this study, a combination of genomics, transcriptomics, and genetics was used to identify the molecular basis of mineral weathering activity and acid tolerance in Pseudomonas azotoformans F77. Biotite was chosen as a silicate mineral to investigate mineral weathering. The genome of strain F77 was sequenced, and the genes significantly upregulated when grown in the presence of biotite included mineral weathering-related genes associated with gluconic acid metabolism, flagellar assembly, and pilus biosynthesis and acid tolerance-related genes associated with neutralizing component production, reducing power, and proton efflux. The biotite-weathering behaviors of strain F77 and its mutants that were created by deleting the , , and genes, which are involved in gluconic acid metabolism, and the , , and genes, which are involved in acid tolerance, were determined. The Fe and Al concentrations in the strain F77-inoculated medium increased 2.2- to 13.7-fold compared to the controls. The cell numbers of strain F77 increased over time, while the pH values in the medium ranged from 3.75 to 3.90 between 20 and 36 h of incubation. The release of Al and Fe was significantly reduced in the F77 Δ, F77 Δ, F77 Δ, and F77 Δ mutants. Bacterial growth was significantly reduced in the presence of biotite in the F77 Δ and F77 Δ mutants. Our results demonstrated the acid tolerance of strain F77 and suggested that multiple genes and metabolic pathways in strain F77 are involved in biotite weathering and acid tolerance during the mineral weathering process. Acid production and tolerance play important roles in effective and persistent mineral weathering in bacteria, although the molecular mechanisms governing acid production and acid tolerance in bacteria have not been fully elucidated. In this study, the molecular mechanisms underlying biotite (as a silicate mineral) weathering (dissolution) and acid tolerance of F77 were characterized using genomics, transcriptomics, and genetics analyses. Our results showed that the genes and metabolic pathways for gluconic acid metabolism, flagellar assembly, and pilus biosynthesis may play important roles in mineral weathering by strain F77. Notably, the genes associated with neutralizing component production, reducing power, and proton efflux may be related to acid tolerance in strain F77. The expression of these acid production- and acid tolerance-related genes was observed to be increased by biotite in strain F77. Our findings may help to elucidate the molecular mechanisms governing mineral weathering and, especially, acid tolerance in mineral-weathering bacteria.
Topics: Genomics; Minerals; Phenotype; Protons; Pseudomonas; Silicates; Transcriptome
PubMed: 34586903
DOI: 10.1128/AEM.01552-21 -
Journal of Bacteriology Sep 2022The Aer2 chemoreceptor from Pseudomonas aeruginosa is an O sensor involved in stress responses, virulence, and tuning the behavior of the chemotaxis (Che) system. Aer2...
The Aer2 chemoreceptor from Pseudomonas aeruginosa is an O sensor involved in stress responses, virulence, and tuning the behavior of the chemotaxis (Che) system. Aer2 is the sole receptor of the Che2 system. It is soluble, but membrane associated, and forms complexes at the cell pole during stationary phase. The domain arrangement of Aer2 is unusual, with a PAS sensing domain sandwiched between five HAMP domains, followed by a C-terminal kinase-control output domain. The first three HAMP domains form a poly-HAMP chain N-terminal to the PAS sensing domain. HAMP domains are often located between signal input and output domains, where they transduce signals. Given that HAMP1 to 3 reside N-terminal to the input-output pathway, we undertook a systematic examination of their function in Aer2. We found that HAMP1 to 3 influence PAS signaling over a considerable distance, as the majority of HAMP1, 2 and 3 mutations, and deletions of helical phase stutters, led to nonresponsive signal-off or off-biased receptors. PAS signal-on lesions that mimic activated Aer2 also failed to override N-terminal HAMP signal-off replacements. This indicates that HAMP1 to 3 are critical coupling partners for PAS signaling and likely function as a cohesive unit and moveable scaffold to correctly orient and poise PAS dimers for O-mediated signaling in Aer2. HAMP1 additionally controlled the clustering and polar localization of Aer2 in P. aeruginosa. Localization was not driven by HAMP1 charge, and HAMP1 signal-off mutants still localized. Employing HAMP as a clustering and localization determinant, as well as a facilitator of PAS signaling, are newly recognized roles for HAMP domains. P. aeruginosa is an opportunistic pathogen that interprets environmental stimuli via 26 chemoreceptors that signal through 4 distinct chemosensory systems. The second chemosensory system, Che2, contains a receptor named Aer2 that senses O and mediates stress responses and virulence and tunes chemotactic behavior. Aer2 is membrane associated, but soluble, and has three N-terminal HAMP domains (HAMP1 to 3) that reside outside the signal input-output pathway of Aer2. In this study, we determined that HAMP1 to 3 facilitate O-dependent signaling from the PAS sensing domain and that HAMP1 controls the formation of Aer2-containing polar foci in P. aeruginosa. Both of these are newly recognized roles for HAMP domains that may be applicable to other non-signal-transducing HAMP domains and poly-HAMP chains.
Topics: Carrier Proteins; Chemotaxis; Pseudomonas aeruginosa; Signal Transduction
PubMed: 35916529
DOI: 10.1128/jb.00225-22 -
Essays in Biochemistry Jul 2021Biotechnological production in bacteria enables access to numerous valuable chemical compounds. Nowadays, advanced molecular genetic toolsets, enzyme engineering as well...
Biotechnological production in bacteria enables access to numerous valuable chemical compounds. Nowadays, advanced molecular genetic toolsets, enzyme engineering as well as the combinatorial use of biocatalysts, pathways, and circuits even bring new-to-nature compounds within reach. However, the associated substrates and biosynthetic products often cause severe chemical stress to the bacterial hosts. Species of the Pseudomonas clade thus represent especially valuable chassis as they are endowed with multiple stress response mechanisms, which allow them to cope with a variety of harmful chemicals. A built-in cell envelope stress response enables fast adaptations that sustain membrane integrity under adverse conditions. Further, effective export machineries can prevent intracellular accumulation of diverse harmful compounds. Finally, toxic chemicals such as reactive aldehydes can be eliminated by oxidation and stress-induced damage can be recovered. Exploiting and engineering these features will be essential to support an effective production of natural compounds and new chemicals. In this article, we therefore discuss major resistance strategies of Pseudomonads along with approaches pursued for their targeted exploitation and engineering in a biotechnological context. We further highlight strategies for the identification of yet unknown tolerance-associated genes and their utilisation for engineering next-generation chassis and finally discuss effective measures for pathway fine-tuning to establish stable cell factories for the effective production of natural compounds and novel biochemicals.
Topics: Biosynthetic Pathways; Biotechnology; Oxidation-Reduction; Pseudomonas; Pseudomonas putida
PubMed: 34223620
DOI: 10.1042/EBC20200173 -
Annual Review of Microbiology Sep 2019Cooperation has fascinated biologists since Darwin. How did cooperative behaviors evolve despite the fitness cost to the cooperator? Bacteria have cooperative behaviors... (Review)
Review
Cooperation has fascinated biologists since Darwin. How did cooperative behaviors evolve despite the fitness cost to the cooperator? Bacteria have cooperative behaviors that make excellent models to take on this age-old problem from both proximate (molecular) and ultimate (evolutionary) angles. We delve into swarming, a phenomenon where billions of bacteria move cooperatively across distances of centimeters in a matter of a few hours. Experiments with swarming have unveiled a strategy called metabolic prudence that stabilizes cooperation, have showed the importance of spatial structure, and have revealed a regulatory network that integrates environmental stimuli and direct cooperative behavior, similar to a machine learning algorithm. The study of swarming elucidates more than proximate mechanisms: It exposes ultimate mechanisms valid to all scales, from cells in cancerous tumors to animals in large communities.
Topics: Adaptation, Physiological; Gene Expression Regulation, Bacterial; Gene Regulatory Networks; Locomotion; Microbial Interactions; Models, Theoretical; Pseudomonas aeruginosa
PubMed: 31180806
DOI: 10.1146/annurev-micro-020518-120033 -
Applied and Environmental Microbiology Apr 2020The quinolone ring is a common core structure of natural products exhibiting antimicrobial, cytotoxic, and signaling activities. A prominent example is the quinolone...
The quinolone ring is a common core structure of natural products exhibiting antimicrobial, cytotoxic, and signaling activities. A prominent example is the quinolone signal (PQS), a quorum-sensing signal molecule involved in the regulation of virulence of The key reaction to quinolone inactivation and biodegradation is the cleavage of the 3-hydroxy-4(1)-quinolone ring, catalyzed by dioxygenases (HQDs), which are members of the α/β-hydrolase fold superfamily. The α/β-hydrolase fold core domain consists of a β-sheet surrounded by α-helices, with an active site usually containing a catalytic triad comprising a nucleophilic residue, an acidic residue, and a histidine. The nucleophile is located at the tip of a sharp turn, called the "nucleophilic elbow." In this work, we developed a search workflow for the identification of HQD proteins from databases. Search and validation criteria include an [H-x(2)-W] motif at the nucleophilic elbow, an [HFP-x(4)-P] motif comprising the catalytic histidine, the presence of a helical cap domain, the positioning of the triad's acidic residue at the end of β-strand 6, and a set of conserved hydrophobic residues contributing to the substrate cavity. The 161 candidate proteins identified from the UniProtKB database originate from environmental and plant-associated microorganisms from all domains of life. Verification and characterization of HQD activity of 9 new candidate proteins confirmed the reliability of the search strategy and suggested residues correlating with distinct substrate preferences. Among the new HQDs, PQS dioxygenases from , , and likely are part of a catabolic pathway for alkylquinolone utilization. Functional annotation of protein sequences is a major requirement for the investigation of metabolic pathways and the identification of sought-after biocatalysts. To identify heterocyclic ring-cleaving dioxygenases within the huge superfamily of α/β-hydrolase fold proteins, we defined search and validation criteria for the primarily motif-based identification of 3-hydroxy-4(1)-quinolone 2,4-dioxygenases (HQD). HQDs are key enzymes for the inactivation of metabolites, which can have signaling, antimicrobial, or cytotoxic functions. The HQD candidates detected in this study occur particularly in environmental and plant-associated microorganisms. Because HQDs active toward the quinolone signal (PQS) likely contribute to interactions within microbial communities and modulate the virulence of , we analyzed the catalytic properties of a PQS-cleaving subset of HQDs and specified characteristics to identify PQS-cleaving dioxygenases within the HQD family.
Topics: Amino Acid Sequence; Bacterial Proteins; Hydrolases; Pseudomonas aeruginosa; Quinolones; Quorum Sensing; Sequence Alignment
PubMed: 32086305
DOI: 10.1128/AEM.00279-20 -
Frontiers in Cellular and Infection... 2021is a common opportunistic pathogen that causes acute nosocomial necrotizing pneumonia and is the predominant source of chronic lung infections in patients with the...
is a common opportunistic pathogen that causes acute nosocomial necrotizing pneumonia and is the predominant source of chronic lung infections in patients with the genetic disorder cystic fibrosis. Early diagnosis in infected patients and monitoring contamination is therefore of great importance in controlling disease spread and development with timely drugs intervention treatment and cut off infection source. Traditional culture-biochemical methods are time consuming and highly dependent on technicians and expensive instruments. To address these challenges, the present study aimed to develop a rapid, sensitive, and specific, on-site detection method for based on recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) technology. The experimental process included screening and modification of primer and probe sets targeting the unique virulence gene (); specificity detection in 29 strains of and 23 closely-related pathogenic bacteria; sensitivity measurements with gradient-diluted genomic DNA and probit regression analysis; and clinical application evaluation using 574 patients samples and calculating coincidence rate and kappa index value in comparison with the culture-biochemical method. The RPA-LFS assay could complete the amplification process at 37°C constant temperature within 30 min and results could be visualized by the naked eye within 10 min on LFS. The assay displayed high sensitivity with a limit of detection of 3.05 CFU/reaction. It also demonstrated high specificity by showing no cross reaction with other pathogenic bacteria, and rapidness by being completed in less than an hour. Furthermore, when used with clinical samples, the assay had a coincidence rate of 98.26% with the culture-biochemical method and a kappa index value of 0.9433. These data indicate that the RPA-LFS assay represents a major improvement for detection, especially in resource-limited areas.
Topics: Humans; Nucleic Acid Amplification Techniques; Pseudomonas aeruginosa; Recombinases; Sensitivity and Specificity; Technology
PubMed: 34595129
DOI: 10.3389/fcimb.2021.698929 -
Sovremennye Tekhnologii V Meditsine 2021was to develop a new software tool for identifying gene mutations that determine the porin-mediated resistance to antibiotics in gram-negative bacteria and to...
UNLABELLED
was to develop a new software tool for identifying gene mutations that determine the porin-mediated resistance to antibiotics in gram-negative bacteria and to demonstrate the functionality of this program by detecting porin-mediated resistance to carbapenems in clinical isolates of .
MATERIALS AND METHODS
The proposed algorithm is based on searching for a correspondence between the reference and the studied genes. When the sought nucleotide sequence is found in the analyzed genome, it is compared with the reference one and analyzed. The genomic analysis is then verified by comparing between the amino acid sequences encoded by the reference and studied genes. The genes of the susceptible ATCC 27853 strain were used as the reference nucleotide sequences encoding for porins (OprD, OpdD, and OpdP) involved in the transport of carbapenems into the bacterial cell. The complete genomes of clinical isolates from the PATRIC database 3.6.9 and our own collection were used to test the functionality of the proposed program. The analyzed isolates were phenotypically characterized according to the CLSI standard. The search for carbapenemase genes in the studied genomes of was carried out using the ResFinder 4.1.
RESULTS
The developed program for detecting the genetic determinants of non-plasmid antibiotic resistance made it possible to identify mutations of various types and significance in the porin genes of clinical isolates. These mutations led to modifications of the peptide structure of porin proteins. Single amino acid substitutions prevailed in the OpdD and OpdP porins of carbapenem-susceptible and carbapenem-resistant isolates. In the carbapenem-resistant strains, the gene encoding for OprD porin was found heavily modified, including insertions and/or deletions, which led to premature termination of porin synthesis. In several isolates resistant to meropenem, no mutations were detected in the gene encoding for OprD, which might be associated with alternative mechanisms of resistance to carbapenems.
CONCLUSION
The proposed software product can become an effective tool for deciphering the molecular genetic mechanisms of bacterial chromosomal resistance to antibiotics. Testing the program revealed differences between the occurrences of mutations significant for carbapenem resistance in the , , and genes.
Topics: Drug Resistance, Bacterial; Microbial Sensitivity Tests; Porins; Pseudomonas aeruginosa; Software
PubMed: 35265355
DOI: 10.17691/stm2021.13.6.02 -
Science (New York, N.Y.) Nov 2021Metal-binding natural products contribute to metal acquisition and bacterial virulence, but their roles in metal stress response are underexplored. We show that a...
Metal-binding natural products contribute to metal acquisition and bacterial virulence, but their roles in metal stress response are underexplored. We show that a five-enzyme pathway in synthesizes a small-molecule copper complex, fluopsin C, in response to elevated copper concentrations. Fluopsin C is a broad-spectrum antibiotic that contains a copper ion chelated by two minimal thiohydroxamates. Biosynthesis of the thiohydroxamate begins with cysteine and requires two lyases, two iron-dependent enzymes, and a methyltransferase. The iron-dependent enzymes remove the carboxyl group and the α carbon from cysteine through decarboxylation, N-hydroxylation, and methylene excision. Conservation of the pathway in and other bacteria suggests a common role for fluopsin C in the copper stress response, which involves fusing copper into an antibiotic against other microbes.
Topics: Anti-Bacterial Agents; Bacteria; Biosynthetic Pathways; Copper; Drug Resistance, Bacterial; Electron Spin Resonance Spectroscopy; Genes, Bacterial; Microbial Sensitivity Tests; Molecular Structure; Operon; Pseudomonas aeruginosa
PubMed: 34793213
DOI: 10.1126/science.abj6749 -
Infection, Genetics and Evolution :... Sep 2023Plant pathogenic Pseudomonas species use multiple classes of toxins and virulence factors during host infection. The genes encoding these pathogenicity factors are often...
Plant pathogenic Pseudomonas species use multiple classes of toxins and virulence factors during host infection. The genes encoding these pathogenicity factors are often located on plasmids and other mobile genetic elements, suggesting that they are acquired through horizontal gene transfer to confer an evolutionary advantage for successful adaptation to host infection. However, the genetic rearrangements that have led to mobilization of the pathogenicity genes are not fully understood. In this study, we have sequenced and analyzed the complete genome sequences of four Pseudomonas amygdali pv. aesculi (Pae), which infect European horse chestnut trees (Aesculus hippocastanum) and belong to phylogroup 3 of the P. syringae species complex. The four investigated genomes contain six groups of plasmids that all encode pathogenicity factors. Effector genes were found to be mostly associated with insertion sequence elements, suggesting that virulence genes are generally mobilized and potentially undergo horizontal gene transfer after transfer to a conjugative plasmid. We show that the biosynthetic gene cluster encoding the phytotoxin coronatine was recently transferred from a chromosomal location to a mobilizable plasmid that subsequently formed a co-integrate with a conjugative plasmid.
Topics: Pseudomonas; Plasmids; Virulence Factors
PubMed: 37541538
DOI: 10.1016/j.meegid.2023.105486