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PloS One 2019Streptococcus mutans primary thrives on the biofilm formation on the tooth surface in sticky biofilms and under certain conditions can lead to carious lesions on the...
Streptococcus mutans primary thrives on the biofilm formation on the tooth surface in sticky biofilms and under certain conditions can lead to carious lesions on the tooth surface. To search for a new preventive material for oral biofilm-associated diseases, including dental caries, we investigated the effects of polypyrrole, which contains an electrochemical polymer and causes protonation and incorporation of anion under low pH condition, on the biofilm formation of S. mutans and other streptococci. In this study, polypyrrole was applied in biofilm formation assays with the S. mutans strains UA159 and its gtfB and gtfC double mutant (gtfBC mutant), S. sanguinis, S. mitis and S. gordonii on human saliva and bovine serum albumin-coated 96-well microtiter plates in tryptic soy broth supplemented with 0.25% sucrose. The effects of polypyrrole on biofilm formation were quantitatively and qualitatively observed. High concentrations of polypyrrole significantly inhibited the biofilm formation of S. mutans UA159 and S. sanguinis. As an inhibition mechanism, polypyrrole attached to the surface of bacterial cells, increased chains and aggregates, and incorporated proteins involving GTF-I and GTF-SI produced by S. mutans. In contrast, the biofilm formation of gtfBC mutant, S. sanguinis, S. mitis and S. gordonii was temporarily induced by the addition of low polypyrrole concentrations on human saliva-coated plate but not on the uncoated and bovine serum albumin-coated plates. Moreover, biofilm formation depended on live cells and, likewise, specific interaction between cells and binding components in saliva. However, these biofilms were easily removed by increased frequency of water washing. In this regard, the physical and electrochemical properties in polypyrrole worked effectively in the removal of streptococci biofilms. Polypyrrole may have the potential to alter the development of biofilms associated with dental diseases.
Topics: Adult; Biofilms; Humans; Polymers; Pyrroles; Saliva; Streptococcus mutans; Young Adult
PubMed: 31774855
DOI: 10.1371/journal.pone.0225584 -
Frontiers in Microbiology 2022and species oxidize methanol pyrroloquinoline quinone-methanol dehydrogenases (MDHs). MDHs can be classified into two major groups, Ca-dependent MDH (MxaF) and...
and species oxidize methanol pyrroloquinoline quinone-methanol dehydrogenases (MDHs). MDHs can be classified into two major groups, Ca-dependent MDH (MxaF) and lanthanide (Ln)-dependent MDH (XoxF), whose expression is regulated by the availability of Ln. A set of a siderophore, TonB-dependent receptor, and an ABC transporter that resembles the machinery for iron uptake is involved in the solubilization and transport of Ln. The transport of Ln into the cytosol enhances XoxF expression. A unique protein named lanmodulin from strain AM1 was identified as a specific Ln-binding protein, and its biological function was implicated to be an Ln shuttle in the periplasm. In contrast, it remains unclear how Ln levels in the cells are maintained, because Ln is potentially deleterious to cellular systems due to its strong affinity to phosphate ions. In this study, we investigated the function of a lanmodulin homolog in strain 22A. The expression of a gene encoding lanmodulin () was induced in response to the presence of La. A recombinant LanM underwent conformational change upon La binding. Phenotypic analyses on deletion mutant and overexpressing strains showed that LanM is not necessary for the wild-type and XoxF-dependent mutant's methylotrophic growth. We found that expression was regulated by MxcQE (a two-component regulator for MxaF) and TonB_Ln (a TonB-dependent receptor for Ln). The expression level of was altered to be negatively dependent on Ln concentration in ∆ whereas it was constant in the wild type. Furthermore, when exposed to La, ∆ showed an aggregating phenotype, cell membrane impairment, La deposition in the periplasm evidenced by electron microscopy, differential expression of proteins involved in membrane integrity and phosphate starvation, and possibly lower La content in the membrane vesicle (MV) fractions. Taken together, we concluded that lanmodulin is involved in the complex regulation mechanism of MDHs and homeostasis of cellular Ln levels by facilitating transport and MV-mediated excretion.
PubMed: 35814700
DOI: 10.3389/fmicb.2022.921636 -
Frontiers in Microbiology 2021Disbalancing envelope stress responses was investigated as a strategy for sensitization of to antimicrobial agents. Seventeen isogenic strains were selected from the...
Disbalancing envelope stress responses was investigated as a strategy for sensitization of to antimicrobial agents. Seventeen isogenic strains were selected from the KEIO collection with deletions in genes corresponding to the σ, Cpx, Rcs, Bae, and Psp responses. Antimicrobial activity against 20 drugs with different targets was evaluated by disk diffusion and gradient strip tests. Growth curves and time-kill curves were also determined for selected mutant-antimicrobial combinations. An increase in susceptibility to ampicillin, ceftazidime, cefepime, aztreonam, ertapenem, and fosfomycin was detected. Growth curves for Psp response mutants showed a decrease in optical density (OD) using sub-MIC concentrations of ceftazidime and aztreonam (Δ and Δ mutants), cefepime (Δ and Δ mutants) and ertapenem (Δ mutant). Time-kill curves were also performed using 1xMIC concentrations of these antimicrobials. For ceftazidime, 2.9 log (Δ mutant) and 0.9 log (Δ mutant) decreases were observed at 24 and 8 h, respectively. For aztreonam, a decrease of 3.1 log (Δ mutant) and 4 log10 (Δ mutant) was shown after 4-6 h. For cefepime, 4.2 log (Δ mutant) and 2.6 log (Δ mutant) decreases were observed at 8 and 4 h, respectively. For ertapenem, a decrease of up to 6 log (Δ mutant) was observed at 24 h. A deficient Psp envelope stress response increased susceptibility to beta-lactam agents such as cefepime, ceftazidime, aztreonam and ertapenem. Its role in repairing extensive inner membrane disruptions makes this pathway essential to bacterial survival, so that disbalancing the Psp response could be an appropriate target for sensitization strategies.
PubMed: 33897667
DOI: 10.3389/fmicb.2021.653479 -
Infection and Immunity Jan 2022A variety of eubacteria, plants, and protozoa can modify membrane lipids by cyclopropanation, which is reported to modulate membrane permeability and fluidity. The...
A variety of eubacteria, plants, and protozoa can modify membrane lipids by cyclopropanation, which is reported to modulate membrane permeability and fluidity. The ability to cyclopropanate membrane lipids has been associated with resistance to oxidative stress in Mycobacterium tuberculosis, organic solvent stress in Escherichia coli, and acid stress in E. coli and Salmonella. In bacteria, the gene encoding cyclopropane fatty acid (CFA) synthase is induced during the stationary phase of growth. In the present study, we constructed a mutant of Salmonella enterica serovar Typhimurium 14028s ( Typhimurium) and determined the contribution of CFA-modified lipids to stress resistance and virulence in mice. Cyclopropane fatty acid content was quantified in wild-type and mutant Typhimurium. CFA levels in the mutant were greatly reduced compared to CFA levels in the wild type, indicating that CFA synthase is the major enzyme responsible for cyclopropane modification of lipids in Salmonella. Typhimurium mutants were more sensitive to extreme acid pH, the protonophore CCCP, and hydrogen peroxide compared to the wild type. In addition, mutants exhibited reduced viability in murine macrophages and could be rescued by the addition of the NADPH phagocyte oxidase inhibitor diphenyleneiodonium (DPI) chloride. Typhimurium lacking was also attenuated for virulence in mice. These observations indicate that CFA modification of lipids makes an important contribution to Salmonella virulence.
Topics: Animals; Bacterial Physiological Phenomena; Bacterial Proteins; Biosynthetic Pathways; Cyclopropanes; Disease Models, Animal; Fatty Acids; Hydrogen-Ion Concentration; Macrophages; Mice; Microbial Viability; Mutation; Oxidative Stress; Salmonella Infections; Salmonella typhimurium; Virulence
PubMed: 34662213
DOI: 10.1128/IAI.00479-21 -
Biomolecules Nov 2020The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research,... (Review)
Review
The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, β', and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, mutants lacking (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ββ' subassembly. We observed that the interaction between α2ββ' and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in . We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code's position in the reading frame of plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ββ'. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the β' subunit, and it is the same for both types of mutants.
Topics: Bacterial Proteins; DNA-Directed RNA Polymerases; Mutant Proteins; Protein Subunits; Structure-Activity Relationship; Transcription Factors
PubMed: 33238579
DOI: 10.3390/biom10111588 -
Probiotics and Antimicrobial Proteins Feb 2023The COVID-19 pandemic caused by a novel coronavirus (SARS-CoV-2) is a serious health concern in the twenty-first century for scientists, health workers, and all humans....
The COVID-19 pandemic caused by a novel coronavirus (SARS-CoV-2) is a serious health concern in the twenty-first century for scientists, health workers, and all humans. The absence of specific biotherapeutics requires new strategies to prevent the spread and prophylaxis of the novel virus and its variants. The SARS-CoV-2 virus shows pathogenesis by entering the host cells via spike protein and Angiotensin-Converting Enzyme 2 receptor protein. Thus, the present study aims to compute the binding energies between a wide range of bacteriocins with receptor-binding domain (RBD) on spike proteins of wild type (WT) and beta variant (lineage B.1.351). Molecular docking analyses were performed to evaluate binding energies. Upon achieving the best bio-peptides with the highest docking scores, further molecular dynamics (MD) simulations were performed to validate the structure and interaction stability. Protein-protein docking of the chosen 22 biopeptides with WT-RBD showed docking scores lower than -7.9 kcal/mol. Pediocin PA-1 and salivaricin P showed the lowest (best) docking scores of - 12 kcal/mol. Pediocin PA-1, salivaricin B, and salivaricin P showed a remarkable increase in the double mutant's predicted binding affinity with -13.8 kcal/mol, -13.0 kcal/mol, and -12.5 kcal/mol, respectively. Also, a better predicted binding affinity of pediocin PA-1 and salivaricin B against triple mutant was observed compared to the WT. Thus, pediocin PA-1 binds stronger to mutants of the RBD, particularly to double and triple mutants. Salivaricin B showed a better predicted binding affinity towards triple mutant compared to WT, showing that it might be another bacteriocin with potential activity against the SARS-CoV-2 beta variant. Overall, pediocin PA-1, salivaricin P, and salivaricin B are the most promising candidates for inhibiting SARS-CoV-2 (including lineage B.1.351) entrance into the human cells. These bacteriocins derived from lactic acid bacteria hold promising potential for paving an alternative way for treatment and prophylaxis of WT and beta variants.
Topics: Humans; Bacteriocins; SARS-CoV-2; Lactobacillales; COVID-19; Molecular Docking Simulation; Pandemics
PubMed: 34837166
DOI: 10.1007/s12602-021-09879-0 -
BMC Biology Dec 2020It is widely assumed that all mutant microorganisms present in a culture are able to grow and form colonies, provided that they express the features required for...
BACKGROUND
It is widely assumed that all mutant microorganisms present in a culture are able to grow and form colonies, provided that they express the features required for selection. Unlike wild-type Escherichia coli, PHO-constitutive mutants overexpress alkaline phosphatase and hence can hydrolyze glycerol-2-phosphate (G2P) to glycerol and form colonies on plates having G2P as the sole carbon source. These mutations mostly occur in the pst operon. However, the frequency of PHO-constitutive colonies on the G2P selective plate is exceptionally low.
RESULTS
We show that the rate in which spontaneous PHO-constitutive mutations emerge is about 8.0 × 10/generation, a relatively high rate, but the growth of most existing mutants is inhibited by their neighboring wild-type cells. This inhibition is elicited only by non-mutant viable bacteria that can take up and metabolize glycerol formed by the mutants. Evidence indicates that the few mutants that do form colonies derive from microclusters of mutants on the selective plate. A mathematical model that describes the fate of the wild-type and mutant populations under these circumstances supports these results.
CONCLUSION
This scenario in which neither the wild-type nor the majority of the mutants are able to grow resembles an unavoidable "tragedy of the commons" case which results in the collapse of the majority of the population. Cooperation between rare adjacent mutants enables them to overcome the competition and eventually form mutant colonies. The inhibition of PHO-constitutive mutants provides an example of mutant frequency masked by orders of magnitude due to a competition between mutants and their ancestral wild-type cells. Similar "tragedy of the commons-like" cases may occur in other settings and should be taken into consideration while estimating true mutant frequencies and mutation rates.
Topics: Escherichia coli; Microbial Interactions; Mutation; Nutrients
PubMed: 33317515
DOI: 10.1186/s12915-020-00913-1 -
Journal of Molecular Graphics &... Sep 2021SARS-CoV-2 is the causative agent of the ongoing viral pandemic of COVID-19. After the emergence of this virus, it became a global public health concern and quickly...
SARS-CoV-2 is the causative agent of the ongoing viral pandemic of COVID-19. After the emergence of this virus, it became a global public health concern and quickly evolved into a pandemic. Mexico is currently in the third position in the number of deaths due to SARS-CoV-2. To date, there have been several lineages of SARS-CoV-2 worldwide; in the Mexican population, two variants of the spike protein (S-protein) are found, localized at H49Y and D614G, which have been related to increased infectivity with respect to the wild-type S-protein. To understand how these differences impact the structural behavior of the S-protein of SARS-CoV-2, as well as binding with ACE2, we performed MD simulations combined with the molecular mechanics generalized Born/Poisson-Boltzmann surface area (MMGB(PB)SA) approach starting from X-ray crystallography data. Energetic and structural analysis showed that the differences in infectivity can be explained by differences in affinity of the protein-protein interface between the wild-type and mutant S-protein with ACE2. Conformational analysis showed that molecular recognition between the S-protein and ACE2 is linked to a decrease in the conformational flexibility of wild-type and mutant S-protein; however, an increase in the conformational mobility of ACE2 could also contribute to the binding affinity observed using the MMGB(PB)SA method.
Topics: COVID-19; Humans; Mexico; Mutant Proteins; Protein Binding; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 34242876
DOI: 10.1016/j.jmgm.2021.107970 -
International Journal of Molecular... Nov 2022Water shortages caused by climate change seriously threaten the survival and production of plants and are also one of the major environmental pressures faced by plants....
Water shortages caused by climate change seriously threaten the survival and production of plants and are also one of the major environmental pressures faced by plants. DORN1 was the first identified purinoceptor for the plant response to extracellular ATP. It has been established that DORN1 could play key roles in a series of biological activities in plants. However, the biological roles of DORN1 and the mechanism remain unclear under drought stress conditions in plants. Here, DORN1 was targeted for knockout by using the CRISPR/Cas 9 system. It was found that the loss function of DORN1 resulted in a significant decrease in the effective quantum yield of PSII [Y(II)], the photochemical quenching coefficient (qP), and the rate of photosynthetic electron transport through PSII (ETR), which reflected plants' photochemical efficiency. Whereas Y values showed obvious enhancement under drought stress conditions. Further experimental results showed that the Y, q, and ETR, which reflect plants' photochemical efficiency, increased significantly with CaCl treatment. These results indicated that the drought tolerance of the mutant was decreased, and the exogenous application of calcium ions could effectively promote the drought tolerance of the mutant. Transpiration loss controlled by stomata is closely related to drought tolerance, further, we examined the transpirational water loss in and found that it was greater than wild-type (WT). Besides, the mutant's stomatal aperture significantly increased compared with the WT and the stomata of mutant plants tend to close after CaCl treatment. Taken together, our results show that DORN1 plays a key role in drought stress tolerance in plants, which may depend on calcium and calcium-related signaling pathways.
Topics: Droughts; Calcium; Calcium Chloride; Photosynthesis; Water
PubMed: 36430696
DOI: 10.3390/ijms232214213 -
Genetics Feb 2020Plants integrate internal and external signals to finely coordinate growth and defense for maximal fitness within a complex environment. A common model suggests that...
Plants integrate internal and external signals to finely coordinate growth and defense for maximal fitness within a complex environment. A common model suggests that growth and defense show a trade-offs relationship driven by energy costs. However, recent studies suggest that the coordination of growth and defense likely involves more conditional and intricate connections than implied by the trade-off model. To explore how a transcription factor (TF) network may coordinate growth and defense, we used a high-throughput phenotyping approach to measure growth and flowering in a set of single and pairwise mutants previously linked to the aliphatic glucosinolate (GLS) defense pathway. Supporting a link between growth and defense, 17 of the 20 tested defense-associated TFs significantly influenced plant growth and/or flowering time. The TFs' effects were conditional upon the environment and age of the plant, and more critically varied across the growth and defense phenotypes for a given genotype. In support of the coordination model of growth and defense, the TF mutant's effects on short-chain aliphatic GLS and growth did not display a simple correlation. We propose that large TF networks integrate internal and external signals and separately modulate growth and the accumulation of the defensive aliphatic GLS.
Topics: Arabidopsis; Arabidopsis Proteins; Epistasis, Genetic; Fatty Acids; Gene Expression Regulation, Plant; Genotype; Glucosinolates; Mutation; Phenotype; Transcription Factors
PubMed: 31852726
DOI: 10.1534/genetics.119.302996