Two Unconventional Metallothioneins in the Apple Snail Pomacea bridgesii Have Lost Their Metal Specificity during Adaptation to Freshwater Habitats

Metallothioneins (MTs) are a diverse group of proteins responsible for the control of metal homeostasis and detoxification. To investigate the impact that environmental conditions might have had on the metal-binding abilities of these proteins, we have characterized the MTs from the apple snail Pomacea bridgesii, a gastropod species belonging to the class of Caenogastropoda with an amphibious lifestyle facing diverse situations of metal bioavailability. P. bridgesii has two structurally divergent MTs, named PbrMT1 and PbrMT2, that are longer than other gastropod MTs due to the presence of extra sequence motifs and metal-binding domains. We have characterized the Zn(II), Cd(II), and Cu(I) binding abilities of these two MTs after their heterologous expression in E. coli. Our results have revealed that despite their structural differences, both MTs share an unspecific metal-binding character, and a great ability to cope with elevated amounts of different metal ions. Our analyses have also revealed slight divergences in their metal-binding features: PbrMT1 shows a more pronounced Zn(II)-thionein character than PbrMT2, while the latter has a stronger Cu(I)-thionein character. The characterization of these two unconventional PbrMTs supports the loss of the metal-binding specificity during the evolution of the MTs of the Ampullariid family, and further suggests an evolutionary link of this loss with the adaptation of these gastropod lineages to metal-poor freshwater habitats.

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Iron is a ligand of SecA-like metal-binding domains in vivo

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012611 ◽

2020 ◽

Vol 295 (21) ◽

pp. 7516-7528

Author(s):

Tamar Cranford-Smith ◽

Mohammed Jamshad ◽

Mark Jeeves ◽

Rachael A. Chandler ◽

Jack Yule ◽

...

Keyword(s):

Sodium Azide ◽

Metal Binding ◽

Cytoplasmic Membrane ◽

E Coli ◽

Binding Domains ◽

Equilibrium Binding ◽

Plausible Model ◽

Metal Binding Domain ◽

Linker Domain

The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modeling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo.

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Characterization of the DNA- and Metal-Binding Properties of Vibrio anguillarum Fur Reveals Conservation of a Structural Zn2+ Ion

Journal of Bacteriology ◽

10.1128/jb.182.21.6264-6267.2000 ◽

2000 ◽

Vol 182 (21) ◽

pp. 6264-6267 ◽

Cited By ~ 27

Author(s):

Ekaterina E. Zheleznova ◽

Jorge H. Crosa ◽

Richard G. Brennan

Keyword(s):

Metal Binding ◽

Iron Uptake ◽

Vibrio Anguillarum ◽

Zinc Ion ◽

Ferric Uptake Regulator ◽

Binding Properties ◽

E Coli ◽

Solution Studies

ABSTRACT The ferric uptake regulator, Fur, represses iron uptake and siderophore biosynthetic genes under iron-replete conditions. Here we report in vitro solution studies on Vibrio anguillarum Fur binding to the consensus 19-bp Escherichia coli iron box in the presence of several divalent metals. We found that V. anguillarum Fur binds the iron box in the presence of Mn2+, Co2+, Cd2+, and to a lesser extent Ni2+ but, unlike E. coli Fur, not in the presence of Zn2+. We also found that V. anguillarum Fur contains a structural zinc ion that is necessary yet alone is insufficient for DNA binding.

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Biochemical characterization of fruit juice for impact of storage on commercial beverages

Asian Journal of Dairy and Food Research ◽

10.18805/ajdfr.dr-1182 ◽

2017 ◽

Vol 36 (04) ◽

Author(s):

Sujata Sethy

Keyword(s):

Biochemical Characterization ◽

Cadmium Content ◽

Sunset Yellow ◽

E Coli ◽

Duration Of Storage ◽

Food Colour ◽

Microbial Analysis ◽

The Impact

The study was performed to evaluate the impact of storage time on safety and hygienic quality of commercial fruit beverage on the basis of market survey. Three most popular brands and three least popular brands of fruit beverages like squash and syrup were selected and stored for nine months at room temperature. The samples were subjected to chemical analysis for food colour, preservative (SO2), heavy metals (Pb and Cd), pectin and microbial analysis for bacteria, fungus, yeast and E. coli at bimonthly interval. Lead content in all the beverages and synthetic permitted food colours like sunset yellow 136.5 ppm in squash A and tartazine 133.2 ppm in squash B and 310.9 ppm in Syrup F was found which exceeded the permissible limit. Preservative (SO2) and cadmium content remained within the acceptable limit. Total bacterial and fungal count increased sharply with the advancement of storage particularly from 7th month onwards. Duration of storage had no effect on the colour and heavy metal content of the beverages.

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Solution and solid-state characterization of a dicopper receptor for large substrates

Canadian Journal of Chemistry ◽

10.1139/v02-065 ◽

2002 ◽

Vol 80 (5) ◽

pp. 496-498 ◽

Cited By ~ 9

Author(s):

Greg JE Davidson ◽

Andrew J Baer ◽

Adrien P Côté ◽

Nicholas J Taylor ◽

Garry S Hanan ◽

...

Keyword(s):

Metal Binding ◽

Central Cavity ◽

X Ray ◽

X Ray Crystallography ◽

Binding Domains ◽

Rigid Spacer ◽

Complex Coordination ◽

Solid State Characterization ◽

Large Central Cavity

Ligand 2 contains two metal-binding domains separated by a rigid spacer and assembles into a dicopper receptor with a large central cavity with no evidence of catenation.Key words: copper complex, coordination, X-ray crystallography, molecular recognition.

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Non-O157 Verotoxin-Producing Escherichia coli: A Problem, Paradox, and Paradigm

Experimental Biology and Medicine ◽

10.1177/153537020322800402 ◽

2003 ◽

Vol 228 (4) ◽

pp. 333-344 ◽

Cited By ~ 46

Author(s):

Karl A. Bettelheim

Keyword(s):

Escherichia Coli ◽

Animal Health ◽

Domestic Animals ◽

Emerging Pathogens ◽

E Coli ◽

The Impact ◽

Identification And Characterization

The problems associated with identification and characterization of non-O157 verotoxin-producing Escherichia coli (VTEC) are discussed. The paradox of VTEC is that most reports of human illnesses are associated with serotypes such as O157:H7, O111:H– (nonmotile), O26:H11, and O113:H21, which are rarely found in domestic animals. However, those VTEC serotypes commonly found in domestic animals, especially ruminants, rarely cause human illnesses. When they cause human illnesses, the symptoms are similar to those caused by the serotypes E. coli O157:H7, O111:H–, O26:H11, and O113:H21. The impact of VTEC on human and animal health is also addressed. The VTEC and their toxicity are considered as a paradigm for emerging pathogens. The question on how such pathogens could arise from a basic commensal population is also addressed.

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Evolution, purification, and characterization of RC0497: a peptidoglycan amidase from the prototypical spotted fever species Rickettsia conorii

Biological Chemistry ◽

10.1515/hsz-2018-0389 ◽

2020 ◽

Vol 401 (2) ◽

pp. 249-262 ◽

Cited By ~ 3

Author(s):

Jignesh G. Patel ◽

Hema P. Narra ◽

Krishna Mohan Sepuru ◽

Abha Sahni ◽

Sandhya R. Golla ◽

...

Keyword(s):

Cell Wall ◽

Spotted Fever ◽

Protein A ◽

Rickettsia Conorii ◽

Spotted Fever Group ◽

Purification And Characterization ◽

E Coli ◽

Binding Domains ◽

Α Helix

AbstractRickettsial species have independently lost several genes owing to reductive evolution while retaining those predominantly implicated in virulence, survival, and biosynthetic pathways. In this study, we have identified a previously uncharacterized Rickettsia conorii gene RC0497 as an N-acetylmuramoyl-L-alanine amidase constitutively expressed during infection of cultured human microvascular endothelial cells at the levels of both mRNA transcript and encoded protein. A homology-based search of rickettsial genomes reveals that RC0497 homologs, containing amidase_2 family and peptidoglycan binding domains, are highly conserved among the spotted fever group (SFG) rickettsiae. The recombinant RC0497 protein exhibits α-helix secondary structure, undergoes a conformational change in the presence of zinc, and exists as a dimer at higher concentrations. We have further ascertained the enzymatic activity of RC0497 via demonstration of its ability to hydrolyze Escherichia coli peptidoglycan. Confocal microscopy on E. coli expressing RC0497 and transmission immunoelectron microscopy of R. conorii revealed its localization predominantly to the cell wall, septal regions of replicating bacteria, and the membrane of vesicles pinching off the cell wall. In summary, we have identified and functionally characterized RC0497 as a peptidoglycan hydrolase unique to spotted fever rickettsiae, which may potentially serve as a novel moonlighting protein capable of performing multiple functions during host-pathogen interactions.

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Preparation and characterization of a new mutant homolog of chemotaxis protein CheY from anaerobic hyperthermophilic microorganism Thermotoga naphthophila

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20196501041 ◽

2019 ◽

Vol 65 (1) ◽

pp. 41-50

Author(s):

D.V. Grishin ◽

Ju.A. Gladilina ◽

D.D. Zhdanov ◽

M.V. Pokrovskaya ◽

I.Yu. Toropygin ◽

...

Keyword(s):

Recombinant Proteins ◽

Bacterial Chemotaxis ◽

Laboratory Strain ◽

Expression Vectors ◽

Cultivation Conditions ◽

Kinetics Parameters ◽

E Coli ◽

Hyperthermophilic Microorganism ◽

The Impact

Using genetic engineering methods the expression vectors structures have been designed to produce recombinant proteins TnaCheY and Tna CheY-mut, the homologues of the chemotaxis protein CheY from the hyperthermophilic organism Thermotoga naphthophila in Escherichia coli BL21(DE3) cells. The cultivation conditions of transformed strains were optimized. The influence of episomal expression of the heterologous chemotaxis protein CheY on growth kinetics parameters of the culture of mesophilic bacteria E. coli was studied. The optimal purification flowchart of the obtained proteins using thermolysis is proposed. Using the E. coli BL21(DE3) laboratory strain as an example, the possibility of employment the episomal expression of such proteins to control the cultivation and production time of pharmaceutically and industrially valuable metabolites due to the impact on some stages of the bacterial chemotaxis is experimentally proved.

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Conserved Dynamic Mechanism of Allosteric Response to L-arg in Divergent Bacterial Arginine Repressors

Molecules ◽

10.3390/molecules25092247 ◽

2020 ◽

Vol 25 (9) ◽

pp. 2247

Author(s):

Saurabh Kumar Pandey ◽

Milan Melichercik ◽

David Řeha ◽

Rüdiger H. Ettrich ◽

Jannette Carey

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Large Scale ◽

Rotational Transition ◽

Sequence Motifs ◽

E Coli ◽

Dna Binding Domains ◽

Binding Domains ◽

Distinct Sequence ◽

Dynamics Simulations

Hexameric arginine repressor, ArgR, is the feedback regulator of bacterial L-arginine regulons, and sensor of L-arg that controls transcription of genes for its synthesis and catabolism. Although ArgR function, as well as its secondary, tertiary, and quaternary structures, is essentially the same in E. coli and B. subtilis, the two proteins differ significantly in sequence, including residues implicated in the response to L-arg. Molecular dynamics simulations are used here to evaluate the behavior of intact B. subtilis ArgR with and without L-arg, and are compared with prior MD results for a domain fragment of E. coli ArgR. Relative to its crystal structure, B. subtilis ArgR in absence of L-arg undergoes a large-scale rotational shift of its trimeric subassemblies that is very similar to that observed in the E. coli protein, but the residues driving rotation have distinct secondary and tertiary structural locations, and a key residue that drives rotation in E. coli is missing in B. subtilis. The similarity of trimer rotation despite different driving residues suggests that a rotational shift between trimers is integral to ArgR function. This conclusion is supported by phylogenetic analysis of distant ArgR homologs reported here that indicates at least three major groups characterized by distinct sequence motifs but predicted to undergo a common rotational transition. The dynamic consequences of L-arg binding for transcriptional activation of intact ArgR are evaluated here for the first time in two-microsecond simulations of B. subtilis ArgR. L-arg binding to intact B. subtilis ArgR causes a significant further shift in the angle of rotation between trimers that causes the N-terminal DNA-binding domains lose their interactions with the C-terminal domains, and is likely the first step toward adopting DNA-binding-competent conformations. The results aid interpretation of crystal structures of ArgR and ArgR-DNA complexes.

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Iron is a ligand of SecA-like metal-binding domains in vivo

10.1101/613315 ◽

2019 ◽

Cited By ~ 1

Author(s):

Tamar Cranford-Smith ◽

Mohammed Jamshad ◽

Mark Jeeves ◽

Rachael A. Chandler ◽

Jack Yule ◽

...

Keyword(s):

Sodium Azide ◽

Metal Binding ◽

Cytoplasmic Membrane ◽

E Coli ◽

Binding Domains ◽

Equilibrium Binding ◽

Plausible Model ◽

Metal Binding Domain ◽

Linker Domain

ABSTRACTThe ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modelling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo.

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Cadmium Uptake, MT Gene Activation and Structure of Large-Sized Multi-Domain Metallothioneins in the Terrestrial Door Snail Alinda biplicata (Gastropoda, Clausiliidae)

International Journal of Molecular Sciences ◽

10.3390/ijms21051631 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1631 ◽

Cited By ~ 2

Author(s):

Veronika Pedrini-Martha ◽

Simon Köll ◽

Martin Dvorak ◽

Reinhard Dallinger

Keyword(s):

Metal Binding ◽

Potential Role ◽

Gene Activation ◽

Cd Stress ◽

Tissue Concentrations ◽

Binding Domains ◽

Terrestrial Snails ◽

The Impact ◽

Cd Exposure

Terrestrial snails (Gastropoda) possess Cd-selective metallothioneins (CdMTs) that inactivate Cd2+ with high affinity. Most of these MTs are small Cysteine-rich proteins that bind 6 Cd2+ equivalents within two distinct metal-binding domains, with a binding stoichiometry of 3 Cd2+ ions per domain. Recently, unusually large, so-called multi-domain MTs (md-MTs) were discovered in the terrestrial door snail Alinda biplicata (A.b.). The aim of this study is to evaluate the ability of A.b. to cope with Cd stress and the potential involvement of md-MTs in its detoxification. Snails were exposed to increasing Cd concentrations, and Cd-tissue concentrations were quantified. The gene structure of two md-MTs (9md-MT and 10md-MT) was characterized, and the impact of Cd exposure on MT gene transcription was quantified via qRT PCR. A.b. efficiently accumulates Cd at moderately elevated concentrations in the feed, but avoids food uptake at excessively high Cd levels. The structure and expression of the long md-MT genes of A.b. were characterized. Although both genes are intronless, they are still transcribed, being significantly upregulated upon Cd exposure. Overall, our results contribute new knowledge regarding the metal handling of Alinda biplicata in particular, and the potential role of md-MTs in Cd detoxification of terrestrial snails, in general.

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