scholarly journals Molecular interaction of nitrate transporter proteins with recombinant glycinebetaine results in efficient nitrate uptake in the cyanobacterium Anabaena PCC 7120

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0257870
Author(s):  
Prashant Swapnil ◽  
Mukesh Meena ◽  
Ashwani K. Rai
Keyword(s):  
Amino Acids ◽  
Abc Transporter ◽  
Molecular Interaction ◽  
Transmembrane Domain ◽  
Binding Domain ◽  
Nitrate Transport ◽  
Atp Binding ◽  
Binding Motif ◽  
Transporter Proteins ◽  
Pcc 7120

Nitrate transport in cyanobacteria is mediated by ABC-transporter, which consists of a highly conserved ATP binding cassette (ABC) and a less conserved transmembrane domain (TMD). Under salt stress, recombinant glycinebetaine (GB) not only protected the rate of nitrate transport in transgenic Anabaena PCC 7120, rather stimulated the rate by interacting with the ABC-transporter proteins. In silico analyses revealed that nrtA protein consisted of 427 amino acids, the majority of which were hydrophobic and contained a Tat (twin-arginine translocation) signal profile of 34 amino acids (1–34). The nrtC subunit of 657 amino acids contained two hydrophobic distinct domains; the N-terminal (5–228 amino acids), which was 59% identical to nrtD (the ATP-binding subunit) and the C-terminal (268–591), 28.2% identical to nrtA, suggesting C-terminal as a solute binding domain and N-terminal as ATP binding domain. Subunit nrtD consisted of 277 amino acids and its N-terminal (21–254) was an ATP binding motif. Phylogenetic analysis revealed that nitrate-ABC-transporter proteins are highly conserved among the cyanobacterial species, though variation existed in sequences resulting in several subclades. Nostoc PCC 7120 was very close to Anabaena variabilis ATCC 29413, Anabaena sp. 4–3 and Anabaena sp. CA = ATCC 33047. On the other, Nostoc spp. NIES-3756 and PCC 7524 were often found in the same subclade suggesting more work before referring it to Anabaena PCC 7120 or Nostoc PCC 7120. The molecular interaction of nitrate with nrtA was hydrophilic, while hydrophobic with nrtC and nrtD. GB interaction with nrtACD was hydrophobic and showed higher affinity compared to nitrate.

scholarly journals Anabaena sp. Strain PCC 7120 hetY Gene Influences Heterocyst Development

2003 ◽  
Vol 185 (23) ◽  
pp. 6995-7000 ◽  
Author(s):  
Ho-Sung Yoon ◽  
Martin H. Lee ◽  
Jin Xiong ◽  
James W. Golden
Keyword(s):  
Atp Binding ◽  
Binding Motif ◽  
Filamentous Cyanobacterium ◽  
Hypothetical Proteins ◽  
Nitrogen Fixing ◽  
Fixed Nitrogen ◽  
Anabaena Sp ◽  
Time Required ◽  
Pcc 7120 ◽  
Heterocyst Development

ABSTRACT The filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 responds to starvation for fixed nitrogen by producing a semiregular pattern of nitrogen-fixing cells called heterocysts. Overexpression of the hetY gene partially suppressed heterocyst formation, resulting in an abnormal heterocyst pattern. Inactivation of hetY increased the time required for heterocyst maturation and caused defects in heterocyst morphology. The 489-bp hetY gene (alr2300), which is adjacent to patS (asl2301), encodes a protein that belongs to a conserved family of bacterial hypothetical proteins that contain an ATP-binding motif.

scholarly journals Contributions of Aspergillus fumigatus ATP-Binding Cassette Transporter Proteins to Drug Resistance and Virulence

2013 ◽  
Vol 12 (12) ◽  
pp. 1619-1628 ◽  
Author(s):  
Sanjoy Paul ◽  
Daniel Diekema ◽  
W. Scott Moye-Rowley
Keyword(s):  
Drug Resistance ◽  
Aspergillus Fumigatus ◽  
Abc Transporter ◽  
Abc Transporters ◽  
Sequence Similarity ◽  
Atp Binding ◽  
Content Type ◽  
Transporter Proteins ◽  
Encoding Genes ◽  
Atp Binding Cassette

ABSTRACTIn yeast cells such as those ofSaccharomyces cerevisiae, expression of ATP-binding cassette (ABC) transporter proteins has been found to be increased and correlates with a concomitant elevation in azole drug resistance. In this study, we investigated the roles of twoAspergillus fumigatusproteins that share high sequence similarity withS. cerevisiaePdr5, an ABC transporter protein that is commonly overproduced in azole-resistant isolates in this yeast. The twoA. fumigatusgenes encoding the ABC transporters sharing the highest sequence similarity toS. cerevisiaePdr5 are calledabcAandabcBhere. We constructed deletion alleles of these two different ABC transporter-encoding genes in three different strains ofA. fumigatus. Loss ofabcBinvariably elicited increased azole susceptibility, whileabcAdisruption alleles had variable phenotypes. Specific antibodies were raised to both AbcA and AbcB proteins. These antisera allowed detection of AbcB in wild-type cells, while AbcA could be visualized only when overproduced from thehspApromoter inA. fumigatus. Overproduction of AbcA also yielded increased azole resistance. Green fluorescent protein fusions were used to provide evidence that both AbcA and AbcB are localized to the plasma membrane inA. fumigatus. Promoter fusions to firefly luciferase suggested that expression of both ABC transporter-encoding genes is inducible by azole challenge. Virulence assays implicated AbcB as a possible factor required for normal pathogenesis. This work provides important new insights into the physiological roles of ABC transporters in this major fungal pathogen.

Identification of a gene cluster encoding an arginine ATP-binding-cassette transporter in the genome of the thermophilic Gram-positive bacterium Geobacillus stearothermophilus strain DSMZ 13240

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 835-840 ◽  
Author(s):  
Rebecca Fleischer ◽  
Antje Wengner ◽  
Frank Scheffel ◽  
Heidi Landmesser ◽  
Erwin Schneider
Keyword(s):  
Amino Acids ◽  
Gene Cluster ◽  
Abc Transporter ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Geobacillus Stearothermophilus ◽  
Positive Bacterium ◽  
Gram Positive ◽  
High Affinity ◽  
Atp Binding Cassette

A single gene cluster encoding components of a putative ATP-binding cassette (ABC) transporter for basic amino acids was identified in the incomplete genome sequence of the thermophilic Gram-positive bacterium Geobacillus stearothermophilus by blast searches. The cluster comprises three genes, and these were amplified from chromosomal DNA of G. stearothermophilus, ligated into plasmid vectors and expressed in Escherichia coli. The purified solute-binding protein (designated ArtJ) was demonstrated to bind l-arginine with high affinity (K d=0·39±0·06 μM). Competition experiments revealed only partial inhibition by excess l-lysine (38 %) and l-ornithine (46 %), while no inhibition was observed with l-histidine or other amino acids tested. The membrane-associated transport complex, composed of a permease (designated ArtM) and an ATPase component (designated ArtP), was solubilized from E. coli membranes by decanoylsucrose and purified by metal-affinity chromatography. The ArtMP complex, when incorporated into liposomes formed from a crude extract of G. stearothermophilus lipids, displayed ATPase activity in the presence of ArtJ only. Addition of l-arginine further stimulated the activity twofold. ATP hydrolysis was optimal at 60 °C and sensitive to the specific inhibitor vanadate. Analysis of kinetic parameters revealed a maximal velocity of ATP hydrolysis of 0·71 μmol Pi min−1 (mg protein)−1 and a K m (ATP) of 1·59 mM. Together, these results identify the ArtJMP complex as a high-affinity arginine ABC transporter.

scholarly journals Domain Interactions in the Yeast ATP Binding Cassette Transporter Ycf1p: Intragenic Suppressor Analysis of Mutations in the Nucleotide Binding Domains

2001 ◽  
Vol 183 (16) ◽  
pp. 4761-4770 ◽  
Author(s):  
Juan M. Falcón-Pérez ◽  
Mónica Martı́nez-Burgos ◽  
Jesús Molano ◽  
Marı́a J. Mazón ◽  
Pilar Eraso
Keyword(s):  
Atpase Activity ◽  
Abc Transporter ◽  
Transmembrane Domain ◽  
Substrate Binding ◽  
Nucleotide Binding ◽  
Intramolecular Interactions ◽  
Phenotypic Characterization ◽  
Atp Binding ◽  
Binding Domains ◽  
Atp Binding Cassette

ABSTRACT The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and V max of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.

scholarly journals Functional Dependence between Septal Protein SepJ from Anabaena sp. Strain PCC 7120 and an Amino Acid ABC-Type Uptake Transporter

2015 ◽  
Vol 197 (16) ◽  
pp. 2721-2730 ◽  
Author(s):  
Leticia Escudero ◽  
Vicente Mariscal ◽  
Enrique Flores
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Types ◽  
Atp Binding ◽  
Acid Uptake ◽  
Content Type ◽  
Intercellular Transfer ◽  
Functional Interactions ◽  
Pcc 7120 ◽  
Atp Binding Cassette

ABSTRACTIn the diazotrophic filaments of heterocyst-forming cyanobacteria, two different cell types, the CO2-fixing vegetative cells and the N2-fixing heterocysts, exchange nutrients, including some amino acids. In the model organismAnabaenasp. strain PCC 7120, the SepJ protein, composed of periplasmic and integral membrane (permease) sections, is located at the intercellular septa joining adjacent cells in the filament. The unicellular cyanobacteriumSynechococcus elongatusstrain PCC 7942 bears a gene,Synpcc7942_1024(here designateddmeA), encoding a permease homologous to the SepJ permease domain.Synechococcusstrains lackingdmeAor lackingdmeAand expressingAnabaenasepJwere constructed. TheSynechococcusdmeAmutant showed a significant 22 to 32% decrease in the uptake of aspartate, glutamate, and glutamine, a phenotype that could be partially complemented byAnabaenasepJ.Synechococcusmutants of an ATP-binding-cassette (ABC)-type transporter for polar amino acids showed >98% decreased uptake of glutamate irrespective of the presence ofdmeAorAnabaenasepJin the same strain. Thus,SynechococcusDmeA orAnabaenaSepJ is needed to observe full (or close to full) activity of the ABC transporter. AnAnabaenasepJdeletion mutant was significantly impaired in glutamate and aspartate uptake, which also in this cyanobacterium requires the activity of an ABC-type transporter for polar amino acids. SepJ appears therefore to generally stimulate the activity of cyanobacterial ABC-type transporters for polar amino acids. Conversely, anAnabaenamutant of three ABC-type transporters for amino acids was impaired in the intercellular transfer of 5-carboxyfluorescein, a SepJ-related property. Our results unravel possible functional interactions in transport elements important for diazotrophic growth.IMPORTANCEMembrane transporters are essential for many aspects of cellular life, from uptake and export of substances in unicellular organisms to intercellular molecular exchange in multicellular organisms. Heterocyst-forming cyanobacteria such asAnabaenarepresent a unique case of multicellularity, in which two cell types exchange nutrients and regulators. The SepJ protein located at the intercellular septa in the filaments ofAnabaenacontains a permease domain of the drug/metabolite transporter (DMT) superfamily that somehow contributes to intercellular molecular transfer. In this work, we have found that SepJ stimulates the activity of a polar amino acid uptake transporter of the ATP-binding-cassette (ABC) superfamily, which could itself affect an intercellular transfer activity related to SepJ, thus unraveling possible functional interactions between these different transporters.

scholarly journals Relative Rates of Amino Acid Import via the ABC Transporter GlnPQ Determine the Growth Performance of Lactococcus lactis

2015 ◽  
Vol 198 (3) ◽  
pp. 477-485 ◽  
Author(s):  
Faizah Fulyani ◽  
Gea K. Schuurman-Wolters ◽  
Dirk-Jan Slotboom ◽  
Bert Poolman
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Lactococcus Lactis ◽  
Abc Transporter ◽  
Essential Amino Acid ◽  
Transmembrane Domain ◽  
Affinity Constant ◽  
Remarkable Feature ◽  
Content Type

ABSTRACTThe GlnPQ transporter fromLactococcus lactishas the remarkable feature of having two substrate-binding domains (SBDs) fused to the N terminus of the transmembrane domain (TMD), and thus four SBDs are present in the homodimeric complex. Although X-ray structures and ligand binding data are available for both SBDs, little is known of how different amino acids compete with each other for transport via GlnPQ. Here we show GlnPQ has a broader substrate specificity than previously thought, with the ability to take up asparagine, glutamine, and glutamic acid, albeit via different routes and with different affinities. Asparagine and glutamine compete with each other at the level of binding to SBD1 and SBD2 (with differences in dissociation constant), but at the same time SBD1 and SBD2 compete with each other at the level of interaction with the translocator domain (with differences in affinity constant and rate of transport). Although glutamine transport via SBD1 is outcompeted by physiological concentrations of asparagine, SBD2 ensures high rates of import of the essential amino acid glutamine. Taken together, this study demonstrates that even in the presence of competing asparagine concentrations, GlnPQ has a high capacity to transport glutamine, which matches the high needs of the cell for glutamine and glutamate.IMPORTANCEGlnPQ is an ATP-binding cassette (ABC) transporter for glutamine, glutamic acid, and asparagine. The system is essential in various Gram-positive bacteria, includingL. lactisand several pathogens. Here we show how the amino acids compete with each other for binding to the multiple SBDs of GlnPQ and how these SBDs compete with each other for substrate delivery to the transporter. Overall, our results show that GlnPQ has evolved to transport diverse substrates via different paths and to optimally acquire the abundant and essential amino acid glutamine.

Crystallization and preliminary X-ray analysis of the ATP-binding domain of the ABC transporter haemolysin B fromEscherichia coli

2002 ◽  
Vol 58 (3) ◽  
pp. 539-541 ◽  
Author(s):  
László Kránitz ◽  
Houssain Benabdelhak ◽  
Carsten Horn ◽  
Mark A. Blight ◽  
I. Barry Holland ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Binding Domain ◽  
Atp Binding ◽  
X Ray ◽  
Fromescherichia Coli

scholarly journals Backbone NMR assignment of the nucleotide binding domain of the Bacillus subtilis ABC multidrug transporter BmrA in the post-hydrolysis state

2022 ◽  
Author(s):  
Victor Hugo Pérez Carrillo ◽  
Dania Rose-Sperling ◽  
Mai Anh Tran ◽  
Christoph Wiedemann ◽  
Ute A. Hellmich
Keyword(s):  
Bacillus Subtilis ◽  
Abc Transporter ◽  
Transmembrane Domain ◽  
Molecular Motor ◽  
Nucleotide Binding ◽  
Bound State ◽  
Binding Domain ◽  
Nucleotide Binding Domain ◽  
Backbone Chemical Shift ◽  
Nucleotide Interactions

AbstractATP binding cassette (ABC) proteins are present in all phyla of life and form one of the largest protein families. The Bacillus subtilis ABC transporter BmrA is a functional homodimer that can extrude many different harmful compounds out of the cell. Each BmrA monomer is composed of a transmembrane domain (TMD) and a nucleotide binding domain (NBD). While the TMDs of ABC transporters are sequentially diverse, the highly conserved NBDs harbor distinctive conserved motifs that enable nucleotide binding and hydrolysis, interdomain communication and that mark a protein as a member of the ABC superfamily. In the catalytic cycle of an ABC transporter, the NBDs function as the molecular motor that fuels substrate translocation across the membrane via the TMDs and are thus pivotal for the entire transport process. For a better understanding of the structural and dynamic consequences of nucleotide interactions within the NBD at atomic resolution, we determined the 1H, 13C and 15N backbone chemical shift assignments of the 259 amino acid wildtype BmrA-NBD in its post-hydrolytic, ADP-bound state.

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