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1.
Comparative analysis of the active sites of orthologous endolysins of the Escherichia lytic bacteriophages T5, RB43, and RB49.
Kutyshenko, VP, Prokhorov, DA, Mikoulinskaia, GV, Molochkov, NV, Yegorov, AY, Paskevich, SI, Uversky, VN
International journal of biological macromolecules. 2021;:1096-1105
Abstract
The methods of solution NMR, circular dichroism (CD), and differential scanning calorimetry (DSC) were used to study two zinc-containing L-alanyl-D-glutamate peptidases - endolysins of the pseudo T-even myoviruses RB43 and RB49 (EndoRB43 and EndoRB49, respectively), which are orthologous to the EndoT5, which is a zinc-containing L-alanyl-D-glutamate peptidase of the T5 siphovirus. The spatial conservation of the Zn2+-binding sites for the enzymes EndoT5, EndoRB43, and EndoRB49 was established, and the key role of Zn2+ ions in the stabilization of the spatial structures of these three peptidases was confirmed. We are showing here that the binding of the Zn2+ ion in the active center of EndoRB49 peptidase causes conformational rearrangements similar to those observed in the EndoT5 peptidase upon binding of Zn2+ and Ca2+ ions and lead to the formation of a catalytically active form of the enzyme. Therefore, the binding of the Zn2+ ion to the active site of EndoRB49 peptidase is a necessary and sufficient condition for functioning of this protein.
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2.
Comparative NanoUPLC-MSE analysis between magainin I-susceptible and -resistant Escherichia coli strains.
Cardoso, MH, de Almeida, KC, Cândido, ES, Murad, AM, Dias, SC, Franco, OL
Scientific reports. 2017;(1):4197
Abstract
In recent years the antimicrobial peptides (AMPs) have been prospected and designed as new alternatives to conventional antibiotics. Indeed, AMPs have presented great potential toward pathogenic bacterial strains by means of complex mechanisms of action. However, reports have increasingly emerged regarding the mechanisms by which bacteria resist AMP administration. In this context, we performed a comparative proteomic study by using the total bacterial lysate of magainin I-susceptible and -resistant E. coli strains. After nanoUPLC-MSE analyses we identified 742 proteins distributed among the experimental groups, and 25 proteins were differentially expressed in the resistant strains. Among them 10 proteins involved in bacterial resistance, homeostasis, nutrition and protein transport were upregulated, while 15 proteins related to bacterial surface modifications, genetic information and β-lactams binding-protein were downregulated. Moreover, 60 exclusive proteins were identified in the resistant strains, among which biofilm and cell wall formation and multidrug efflux pump proteins could be observed. Thus, differentially from previous studies that could only associate single proteins to AMP bacterial resistance, data here reported show that several metabolic pathways may be related to E. coli resistance to AMPs, revealing the crucial role of multiple "omics" studies in order to elucidate the global molecular mechanisms involved in this resistance.
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3.
Comparison of microbiological and physicochemical methods for enumeration of microorganisms.
Szermer-Olearnik, B, Sochocka, M, Zwolińska, K, Ciekot, J, Czarny, A, Szydzik, J, Kowalski, K, Boratyński, J
Postepy higieny i medycyny doswiadczalnej (Online). 2014;:1392-6
Abstract
Determination of the number of cultured bacteria is essential for scientific and industrial practice. A spread plate technique is the most common and accurate method for counting of microorganisms. However, time consuming incubation does not allow for a quick estimation of the number of bacteria in a growing culture. In the present study, the results of photometric measurements: direct optical density method (OD at 585 nm), UV absorbance at 260 and/or 280 nm of separated and lysed bacteria by sodium hydroxide and surfactant with the spread plate technique were compared. The linear regression model for bacterial strains Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli was used to compare these three methods. The UV measurement method enabled determination of the number of bacteria with similar precision. The procedure for solubilized bacteria UV measurement is robust, and is not influenced by dispersions in the original culture medium.
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4.
Container type and bactericidal activity of human milk during refrigerated storage.
Takci, S, Gulmez, D, Yigit, S, Dogan, O, Hascelik, G
Journal of human lactation : official journal of International Lactation Consultant Association. 2013;(3):406-11; quiz 424-6
Abstract
BACKGROUND Refrigeration of human milk has been recommended for its short-term storage. It has been shown that some nutritional, immunological, and bioactive properties and bactericidal activity of human milk can be altered during refrigeration. Pyrex bottles and polyethylene bags are 2 commonly used containers for human milk storage. OBJECTIVE The aim of this study was to compare the association between storage container type on the bactericidal activity of human milk for different durations of refrigeration (fresh, and at 24 and 48 hours). METHODS Forty-four samples of human milk were collected from 22 lactating mothers. Two samples of breast milk (approximately 10 mL each) were obtained by manual expression from each mother. One was collected directly into sterile Pyrex bottles and the other into polyethylene bags. One mL of human milk from each container was processed immediately after arrival to the laboratory. The remaining human milk was kept in the Pyrex and polyethylene containers at 4°C until analysis at 24 and 48 hours. The bactericidal activity of each sample was studied. A strain of Escherichia coli ATCC 25922 was used to determine the bactericidal effect of human milk. RESULTS Bactericidal activity was significantly reduced in milk samples stored in polyethylene bags compared to those stored in Pyrex bottles when milk samples were stored at 4°C for 24 and 48 hours (P < .05). CONCLUSION Short-term storage of human milk in Pyrex bottles is more appropriate than polyethylene bags for preserving its bactericidal activity against E coli.
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5.
Comparative analysis of regulatory elements between Escherichia coli and Klebsiella pneumoniae by genome-wide transcription start site profiling.
Kim, D, Hong, JS, Qiu, Y, Nagarajan, H, Seo, JH, Cho, BK, Tsai, SF, Palsson, BØ
PLoS genetics. 2012;(8):e1002867
Abstract
Genome-wide transcription start site (TSS) profiles of the enterobacteria Escherichia coli and Klebsiella pneumoniae were experimentally determined through modified 5' RACE followed by deep sequencing of intact primary mRNA. This identified 3,746 and 3,143 TSSs for E. coli and K. pneumoniae, respectively. Experimentally determined TSSs were then used to define promoter regions and 5' UTRs upstream of coding genes. Comparative analysis of these regulatory elements revealed the use of multiple TSSs, identical sequence motifs of promoter and Shine-Dalgarno sequence, reflecting conserved gene expression apparatuses between the two species. In both species, over 70% of primary transcripts were expressed from operons having orthologous genes during exponential growth. However, expressed orthologous genes in E. coli and K. pneumoniae showed a strikingly different organization of upstream regulatory regions with only 20% identical promoters with TSSs in both species. Over 40% of promoters had TSSs identified in only one species, despite conserved promoter sequences existing in the other species. 662 conserved promoters having TSSs in both species resulted in the same number of comparable 5' UTR pairs, and that regulatory element was found to be the most variant region in sequence among promoter, 5' UTR, and ORF. In K. pneumoniae, 48 sRNAs were predicted and 36 of them were expressed during exponential growth. Among them, 34 orthologous sRNAs between two species were analyzed in depth, and the analysis showed that many sRNAs of K. pneumoniae, including pleiotropic sRNAs such as rprA, arcZ, and sgrS, may work in the same way as in E. coli. These results reveal a new dimension of comparative genomics such that a comparison of two genomes needs to be comprehensive over all levels of genome organization.
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6.
Effect of cranberry drink on bacterial adhesion in vitro and vaginal microbiota in healthy females.
Jass, J, Reid, G
The Canadian journal of urology. 2009;(6):4901-7
Abstract
INTRODUCTION/OBJECTIVE Cranberries have been shown to produce urinary metabolites that influence uropathogen adhesion and prevent urinary tract infections. This study was designed to determine if consuming reconstituted, unsweetened cranberry drink from extract retained its bioactive properties by reducing uropathogen adhesion without adversely affecting urinary calcium, magnesium and the vaginal microflora. MATERIALS AND METHODS A randomized crossover study was undertaken in 12 healthy women consuming reconstituted unsweetened cranberry drink, CranActin or water. The urine was collected at 4 hours and 1 week of consumption and evaluated for antiadhesive properties and urinary pH, calcium and magnesium. Vaginal swabs were collected after 1 week of treatment to assess the vaginal microbiota by DGGE. RESULTS The resultant urine produced by subjects who consumed 500 ml reconstituted cranberry extract twice per day, significantly reduced the adherence to epithelial cells of P-fimbriated uropathogenic Escherichia coli and showed a tendency towards significance for two E. coli strains expressing fimbriae and an Enterococcus faecalis isolate. The cranberry drink treatment did not alter urinary pH, but reduced calcium and magnesium concentrations compared to water, although not to statistical significance. The reconstituted cranberry drink had no apparent detrimental effect on the vaginal microbiota. However, consuming twice daily resulted in an apparent loss of a potential pathogen from the vagina in 42% subjects. CONCLUSIONS The present findings suggest that reconstituted cranberry drink may retain the ability to reduce the risk of UTI by inhibiting pathogen adhesion while not detrimentally affecting urinary pH or vaginal microbiota, or the risk of calculi.
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7.
Comparative genomics and functional roles of the ATP-dependent proteases Lon and Clp during cytosolic protein degradation.
Chandu, D, Nandi, D
Research in microbiology. 2004;(9):710-9
Abstract
The general pathway involving adenosine triphosphate (ATP)-dependent proteases and ATP-independent peptidases during cytosolic protein degradation is conserved, with differences in the enzymes utilized, in organisms from different kingdoms. Lon and caseinolytic protease (Clp) are key enzymes responsible for the ATP-dependent degradation of cytosolic proteins in Escherichia coli. Orthologs of E. coli Lon and Clp were searched for, followed by multiple sequence alignment of active site residues, in genomes from seventeen organisms, including representatives from eubacteria, archaea, and eukaryotes. Lon orthologs, unlike ClpP and ClpQ, are present in most organisms studied. The roles of these proteases as essential enzymes and in the virulence of some organisms are discussed.
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8.
Steady-state and pre-steady-state kinetic analysis of halopropane conversion by a rhodococcus haloalkane dehalogenase.
Bosma, T, Pikkemaat, MG, Kingma, J, Dijk, J, Janssen, DB
Biochemistry. 2003;(26):8047-53
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Abstract
Haloalkane dehalogenase from Rhodococcus rhodochrous NCIMB 13064 (DhaA) catalyzes the hydrolysis of carbon-halogen bonds in a wide range of haloalkanes. We examined the steady-state and pre-steady-state kinetics of halopropane conversion by DhaA to illuminate mechanistic details of the dehalogenation pathway. Steady-state kinetic analysis of DhaA with a range of halopropanes showed that bromopropanes had higher k(cat) and lower K(M) values than the chlorinated analogues. The kinetic mechanism of dehalogenation was further studied using rapid-quench-flow analysis of 1,3-dibromopropane conversion. This provided a direct measurement of the chemical steps in the reaction mechanism, i.e., cleavage of the carbon-halogen bond and hydrolysis of the covalent alkyl-enzyme intermediate. The results lead to a minimal mechanism consisting of four main steps. The occurrence of a pre-steady-state burst, both for bromide and 3-bromo-1-propanol, suggests that product release is rate-limiting under steady-state conditions. Combining pre-steady-state burst and single-turnover experiments indicated that the rate of carbon-bromine bond cleavage was indeed more than 100-fold higher than the steady-state k(cat). Product release occurred with a rate constant of 3.9 s(-1), a value close to the experimental k(cat) of 2.7 s(-1). Comparing the kinetic mechanism of DhaA with that of the corresponding enzyme from Xanthobacter autotrophicus GJ10 (DhlA) shows that the overall mechanisms are similar. However, whereas in DhlA the rate of halide release represents the slowest step in the catalytic cycle, our results suggest that in DhaA the release of 3-bromo-1-propanol is the slowest step during 1,3-dibromopropane conversion.
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Theoretical studies on solvation contribution to the thermodynamic stability of mutants of lysozyme T4.
Deep, S, Ahluwalia, JC
Protein engineering. 2003;(6):415-22
Abstract
Atomic solvation parameters (ASPs) are widely used to estimate the solvation contribution to the thermodynamic stability of proteins as well as the free energy of association for protein-ligand complexes. In view of discrepancies in the results of free energies of solvation of folding for various proteins obtained using different atomic solvation parameter sets, systematic studies have been carried out for the calculation of accessible surface area and the changes in free energy of solvation of folding (deltaG(s,f)) for mutants of lysozyme T4 where threonine 157 is replaced by amino acids: cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, leucine, asparagine, arginine, serine and valine. The deviations of the calculated results from the experimental results are discussed to highlight the discrepancies in the atomic solvation parameter sets and possible reasons for them. The results are also discussed to throw light on the effect of chain free energy and hydrogen bonding on the stability of mutants. The octanol to water-based ASP sets 'Sch1' and 'EM' perform better than the vacuum to water-based ASP sets. The vacuum to water-based ASP sets 'Sch3' and 'WE' can be used to predict the stability of mutants if a proper method to calculate the hydrogen bond contribution to overall stability is in place.
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A spatially extended stochastic model of the bacterial chemotaxis signalling pathway.
Shimizu, TS, Aksenov, SV, Bray, D
Journal of molecular biology. 2003;(2):291-309
Abstract
We have combined two distinct but related stochastic approaches to model the Escherichia coli chemotaxis pathway. Reactions involving cytosolic components of the pathway were assumed to obey the laws of conventional stochastic chemical kinetics, while the clustered membrane receptors were represented in two-dimensional arrays similar to the Ising model. Receptors were assumed to flip between an active and an inactive state with probabilities dependent upon three energy inputs: ligand binding, methylation level due to adaptation, and the activity of neighbouring receptors. Examination of models with different lattice size and geometry showed that the sensitivity to stimuli increases with lattice size and the nearest-neighbour coupling strength up to a critical point, but this amplification was also accompanied by a proportional increase in steady-state noise. Multiple methylation of receptors resulted in diminished signal-to-noise ratio, but showed improved stability to variation in the coupling strength and increased gain. Under the best conditions the simulated output of a coupled lattice of receptors closely matched the time-course and amplitude found experimentally in living bacteria. The model also has some of the properties of a cellular automaton and shows an unexpected emergence of spatial patterns of methylation within the receptor lattice.