Using the Mesolens to observe structural changes in E. coli mature colony biofilms under different nutrient availability

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

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KUALITAS AIR SUNGAI DI KAWASAN WISATA PANTAI PETITENGET, KEROBOKAN KABUPATEN BADUNG BALI

ECOTROPHIC Jurnal Ilmu Lingkungan (Journal of Environmental Science) ◽

10.24843/ejes.2018.v12.i02.p11 ◽

2018 ◽

Vol 12 (2) ◽

pp. 226

Author(s):

Wayan Budiarsa Suyasa ◽

Sri Kunti Pancadewi G. A ◽

Iryanti E. Suprihatin ◽

Dwi Adi Suastuti G. A.

Keyword(s):

Water Quality ◽

Structural Changes ◽

Pollution Index ◽

Basic Structure ◽

Physical Damage ◽

E Coli ◽

Pollutant Source ◽

Quality Category ◽

Index Value ◽

The Impact

In order to maintain the environmental carrying capacity of coastal tourism, this research was conducted to determine the condition of river water environmental pollution in the Petitenget beach area and pollutant source activities. Determination of water quality is carried out by analyzing the water quality taken at several sampling points in the four rivers that lead to the Petitenget beach. Determined the pollution index value (IP) of the physical chemical and biological pollution parameters. The results showed that the four rivers that flow into the Petitenget Beach area had been contaminated with indications of pH, BOD, COD, ammonia, Coliform and E. coli which exceeded water quality category III class quality (PerGub Bali No 16 Year 2016). The four rivers are included in the criteria of severe contamination. The four rivers have experienced physical damage or structural changes that have very high discharge fluctuations both in quantity and quality. Slimy basic structure, smelly and slum aesthetic waters. While the indication of the impact of pollution is waste water which is directly discharged into the river from hotels, restaurants, homestays, commercial centers and settlements.

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Influence of nutrient availability on in vitro growth of major bovine mastitis pathogens

Journal of Dairy Research ◽

10.1017/s0022029921000133 ◽

2021 ◽

Vol 88 (1) ◽

pp. 80-88

Author(s):

Remo Stürmlin ◽

Josef J. Gross ◽

Olga Wellnitz ◽

Lea A. Wagner ◽

Camille Monney ◽

...

Keyword(s):

Bacterial Growth ◽

Nutrient Availability ◽

Bovine Mastitis ◽

Milk Composition ◽

B Vitamins ◽

E Coli ◽

Streptococcus Uberis ◽

Whole Milk ◽

Mastitis Pathogens

AbstractThe aim of the present study was to investigate the effects of milk composition changes on the in vitro growth of bovine mastitis pathogens. Nutritional requirements of three major bovine mastitis pathogens Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Streptococcus uberis (S. uberis) were investigated in vitro. We used ultra-high temperature (UHT) treated milk with different contents of fat, protein, and carbohydrates to test the influence of the availability of various milk constituents on pathogen growth characteristics. Additionally, the bacterial growth was investigated under experimentally modified nutrient availability by dilution and subsequent supplementation with individual nutrients (carbohydrates, different nitrogen sources, minerals, and different types of B vitamins) either to milk or to a conventional medium (thioglycolate broth, TB). Varying contents of fat, protein or lactose did not affect bacterial growth with the exception of growth of S. uberis being promoted in protein-enriched milk. The addition of nutrients to diluted whole milk and TB partly revealed different effects, indicating that there are media-specific growth limiting factors after dilution. Supplementation of minerals to diluted milk did not affect growth rates of all studied bacteria. Bacterial growth in diluted whole milk was decreased by the addition of high concentrations of amino acids in S. aureus, and by urea and additional B vitamins in E. coli and S. aureus. The growth rate of S. uberis was increased by the addition of B vitamins to diluted whole milk. The present results demonstrate that growth-limiting nutrients differ among pathogen types. Because reduced bacterial growth was only shown in diluted milk or TB, it is unlikely that alterations in nutrient availability occurring as a consequence of physiological changes of milk composition in the cow's udder would directly affect the susceptibility or course of bovine mastitis.

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NMR structure of the C-terminal domain of TonB protein from Pseudomonas aeruginosa

PeerJ ◽

10.7717/peerj.5412 ◽

2018 ◽

Vol 6 ◽

pp. e5412 ◽

Cited By ~ 4

Author(s):

Jesper S. Oeemig ◽

O.H. Samuli Ollila ◽

Hideo Iwaï

Keyword(s):

Pseudomonas Aeruginosa ◽

Structural Changes ◽

Nmr Structure ◽

Dynamics Simulation ◽

Solution Nmr ◽

Gram Negative Bacteria ◽

E Coli ◽

Terminal Domain ◽

Tonb Protein ◽

Monomeric Structure

The TonB protein plays an essential role in the energy transduction system to drive active transport across the outer membrane (OM) using the proton-motive force of the cytoplasmic membrane of Gram-negative bacteria. The C-terminal domain (CTD) of TonB protein is known to interact with the conserved TonB box motif of TonB-dependent OM transporters, which likely induces structural changes in the OM transporters. Several distinct conformations of differently dissected CTDs of Escherichia coli TonB have been previously reported. Here we determined the solution NMR structure of a 96-residue fragment of Pseudomonas aeruginosa TonB (PaTonB-96). The structure shows a monomeric structure with the flexible C-terminal region (residues 338–342), different from the NMR structure of E. coli TonB (EcTonB-137). The extended and flexible C-terminal residues are confirmed by 15N relaxation analysis and molecular dynamics simulation. We created models for the PaTonB-96/TonB box interaction and propose that the internal fluctuations of PaTonB-96 makes it more accessible for the interactions with the TonB box and possibly plays a role in disrupting the plug domain of the TonB-dependent OM transporters.

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Structural Modification of Lipopolysaccharide Conferred by mcr-1 in Gram-Negative ESKAPE Pathogens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00580-17 ◽

2017 ◽

Vol 61 (6) ◽

Cited By ~ 49

Author(s):

Yi-Yun Liu ◽

Courtney E. Chandler ◽

Lisa M. Leung ◽

Christi L. McElheny ◽

Roberta T. Mettus ◽

...

Keyword(s):

Structural Changes ◽

Lipid A ◽

Colistin Resistance ◽

Gram Negative ◽

Content Type ◽

Clinical Strains ◽

E Coli ◽

The Family ◽

Phosphoethanolamine Transferases ◽

Eskape Pathogens

ABSTRACT mcr-1 was initially reported as the first plasmid-mediated colistin resistance gene in clinical isolates of Escherichia coli and Klebsiella pneumoniae in China and has subsequently been identified worldwide in various species of the family Enterobacteriaceae. mcr-1 encodes a phosphoethanolamine transferase, and its expression has been shown to generate phosphoethanolamine-modified bis-phosphorylated hexa-acylated lipid A in E. coli. Here, we investigated the effects of mcr-1 on colistin susceptibility and on lipopolysaccharide structures in laboratory and clinical strains of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, which are often treated clinically by colistin. The effects of mcr-1 on colistin resistance were determined using MIC assays of laboratory and clinical strains of E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa. Lipid A structural changes resulting from MCR-1 were analyzed by mass spectrometry. The introduction of mcr-1 led to colistin resistance in E. coli, K. pneumoniae, and A. baumannii but only moderately reduced susceptibility in P. aeruginosa. Phosphoethanolamine modification of lipid A was observed consistently for all four species. These findings highlight the risk of colistin resistance as a consequence of mcr-1 expression among ESKAPE pathogens, especially in K. pneumoniae and A. baumannii. Furthermore, the observation that lipid A structures were modified despite only modest increases in colistin MICs in some instances suggests more sophisticated surveillance methods may need to be developed to track the dissemination of mcr-1 or plasmid-mediated phosphoethanolamine transferases in general.

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E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation and dynamics

10.1101/766725 ◽

2019 ◽

Author(s):

James Chen ◽

Saumya Gopalkrishnan ◽

Courtney Chiu ◽

Albert Y. Chen ◽

Elizabeth A. Campbell ◽

...

Keyword(s):

Rna Polymerase ◽

Conformational Changes ◽

Transcription Initiation ◽

Structural Changes ◽

E Coli ◽

Bacterial Proteins ◽

Cryo Electron Microscopy ◽

Promoter Complex ◽

Promoter Dna ◽

Induced Changes

AbstractTraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp. How TraR and its homologs regulate transcription is unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis of RNAP conformational heterogeneity revealed TraR-induced changes in RNAP dynamics. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.

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Conformational Changes of Anoplin, W-MreB1–9, and (KFF)3K Peptides near the Membranes

International Journal of Molecular Sciences ◽

10.3390/ijms21249672 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9672

Author(s):

Monika Wojciechowska ◽

Joanna Miszkiewicz ◽

Joanna Trylska

Keyword(s):

Conformational Changes ◽

Structural Changes ◽

Biological Activities ◽

Membrane Disruption ◽

Cell Penetrating Peptide ◽

Active Peptides ◽

E Coli ◽

Membrane Active Peptides ◽

Calcein Leakage ◽

Bacterial Growth Inhibition

Many peptides interact with biological membranes, but elucidating these interactions is challenging because cellular membranes are complex and peptides are structurally flexible. To contribute to understanding how the membrane-active peptides behave near the membranes, we investigated peptide structural changes in different lipid surroundings. We focused on two antimicrobial peptides, anoplin and W-MreB1–9, and one cell-penetrating peptide, (KFF)3K. Firstly, by using circular dichroism spectroscopy, we determined the secondary structures of these peptides when interacting with micelles, liposomes, E. coli lipopolysaccharides, and live E. coli bacteria. The peptides were disordered in the buffer, but anoplin and W-MreB1–9 displayed lipid-induced helicity. Yet, structural changes of the peptide depended on the composition and concentration of the membranes. Secondly, we quantified the destructive activity of peptides against liposomes by monitoring the release of a fluorescent dye (calcein) from the liposomes treated with peptides. We observed that only for anoplin and W-MreB1–9 calcein leakage from liposomes depended on the peptide concentration. Thirdly, bacterial growth inhibition assays showed that peptide conformational changes, evoked by the lipid environments, do not directly correlate with the antimicrobial activity of the peptides. However, understanding the relation between peptide structural properties, mechanisms of membrane disruption, and their biological activities can guide the design of membrane-active peptides.

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Effect of Non-Thermal Atmospheric Plasma on Food-Borne Bacterial Pathogens on Ready-to Eat Foods: Morphological and Physico-Chemical Changes Occurring on the Cellular Envelopes

Foods ◽

10.3390/foods9121865 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1865

Author(s):

Tamara Calvo ◽

Miguel Prieto ◽

Avelino Alvarez-Ordóñez ◽

Mercedes López

Keyword(s):

Salmonella Enteritidis ◽

Structural Changes ◽

Membrane Integrity ◽

Atmospheric Plasma ◽

Bacterial Cells ◽

Bacterial Inactivation ◽

Cellular Targets ◽

E Coli ◽

Physico Chemical ◽

Potential Applications

Currently, there is a need for new technological interventions to guarantee the microbiological safety of ready-to-eat (RTE) foods. Non-thermal atmospheric plasma (NTAP) has emerged as a promising strategy for inactivating microorganisms on thermo-sensitive foods, and the elucidation of its mechanisms of action will aid the rational optimization and industrial implementation of this technology for potential applications in the food industry. In this study, the effectiveness of NTAP for inactivating strains of Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes contaminating the surface of different sliced RTE foods (“chorizo”, salami, bacon, smoked salmon, tofu and apple) was investigated. In addition, to further assess the bacterial inactivation mechanisms of NTAP, the morphological and physico-chemical damages in bacterial cells were analyzed. NTAP was effective for the surface decontamination of all products tested and, especially, of cut apple, where the microbial populations were reduced between 1.3 and 1.8 log units for the two Salmonella strains and E. coli O157: H7, respectively, after 15 min of exposure. In the rest of foods, no significant differences in the lethality obtained for the E. coli O157:H7 strain were observed, with inactivation rates of between 0.6 and 0.9 log cycles after a 15-min treatment. On the other hand, the strains from the rest of pathogenic microorganisms studied were extremely resistant on tofu, where barely 0.2–0.5 log units of inactivation were achieved after 15 min of plasma exposure. S. Enteritidis cells treated for 10 min exhibited noticeable morphological and structural changes, as observed by transmission electron microscopy, which were accompanied by a loss in membrane integrity, with an increased leakage of intracellular components and uptake of propidium iodide and marked changes in regions of their FTIR spectra indicating major alterations of the cell wall components. Overall, this indicates that loss of viability was likely caused for this microorganism by a significant damage in the cellular envelopes. However, the plasma-treated cells of L. monocytogenes did not show such obvious changes in morphology, and exhibited less marked effects on the integrity of their cytoplasmic membrane, what suggests that the death of this pathogenic microorganism upon NTAP exposure is more likely to occur as a consequence of damages in other cellular targets.

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Structural analysis of an oxygen-regulated diguanylate cyclase

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s139900471501545x ◽

2015 ◽

Vol 71 (11) ◽

pp. 2158-2177 ◽

Cited By ~ 27

Author(s):

Miroslaw Tarnawski ◽

Thomas R. M. Barends ◽

Ilme Schlichting

Keyword(s):

Structural Changes ◽

Structural Information ◽

Oxygen Sensing ◽

Diguanylate Cyclase ◽

E Coli ◽

Cyclase Domain ◽

Middle Domain ◽

Ggdef Domain ◽

The Individual ◽

Related Proteins

Cyclic di-GMP is a bacterial second messenger that is involved in switching between motile and sessile lifestyles. Given the medical importance of biofilm formation, there has been increasing interest in understanding the synthesis and degradation of cyclic di-GMPs and their regulation in various bacterial pathogens. Environmental cues are detected by sensing domains coupled to GGDEF and EAL or HD-GYP domains that have diguanylate cyclase and phosphodiesterase activities, respectively, producing and degrading cyclic di-GMP. The Escherichia coli protein DosC (also known as YddV) consists of an oxygen-sensing domain belonging to the class of globin sensors that is coupled to a C-terminal GGDEF domain via a previously uncharacterized middle domain. DosC is one of the most strongly expressed GGDEF proteins in E. coli, but to date structural information on this and related proteins is scarce. Here, the high-resolution structural characterization of the oxygen-sensing globin domain, the middle domain and the catalytic GGDEF domain in apo and substrate-bound forms is described. The structural changes between the iron(III) and iron(II) forms of the sensor globin domain suggest a mechanism for oxygen-dependent regulation. The structural information on the individual domains is combined into a model of the dimeric DosC holoprotein. These findings have direct implications for the oxygen-dependent regulation of the activity of the cyclase domain.

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relA Gene control of bacterial glycogen synthesis

Canadian Journal of Biochemistry ◽

10.1139/o78-063 ◽

1978 ◽

Vol 56 (6) ◽

pp. 403-406 ◽

Cited By ~ 20

Author(s):

William A. Bridger ◽

William Paranchych

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Stationary Phase ◽

Nutrient Availability ◽

Glycogen Content ◽

Glycogen Synthesis ◽

Gene Control ◽

E Coli ◽

Stimulation Of ◽

In Cells

Starvation of Escherichia coli K12 for an amino acid results in the stimulation of bacterial glycogen synthesis in cells containing the relA+ gene, but not in cells carrying the relA− allele. Similarly, a large difference in glycogen content is demonstrable between relA+ and relA− cells in stationary phase. It is concluded that guanosine 5′,3′-bis(diphosphate) (ppGpp) or some related relA -dependent metabolite is involved in the regulation of bacterial glycogen synthesis. Detection of significant basal levels of glycogen in a relA− strain of E. coli and in unstarved relA+E. coli indicates that relA control is not absolutely required for glycogen synthesis but serves as a signal for modulation in response to nutrient availability.

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