Putative ATP-binding Cassette Transporter Research Articles

Deciphering Cellodextrin and Glucose Uptake in Clostridium thermocellum.

ABSTRACTSugar uptake is of great significance in industrially relevant microorganisms. Clostridium thermocellum has extensive potential in lignocellulose biorefineries as an environmentally prominent, thermophilic, cellulolytic bacterium. The bacterium employs five putative ATP-binding cassette transporters which purportedly take up cellulose hydrolysates. Here, we first applied combined genetic manipulations and biophysical titration experiments to decipher the key glucose and cellodextrin transporters. In vivo gene inactivation of each transporter and in vitro calorimetric and nuclear magnetic resonance (NMR) titration of each putative sugar-binding protein with various saccharides supported the conclusion that only transporters A and B play the roles of glucose and cellodextrin transport, respectively. To gain insight into the structural mechanism of the transporter specificities, 11 crystal structures, both alone and in complex with appropriate saccharides, were solved for all 5 putative sugar-binding proteins, thus providing detailed specific interactions between the proteins and the corresponding saccharides. Considering the importance of transporter B as the major cellodextrin transporter, we further identified its cryptic, hitherto unknown ATPase-encoding gene as clo1313_2554, which is located outside the transporter B gene cluster. The crystal structure of the ATPase was solved, showing that it represents a typical nucleotide-binding domain of the ATP-binding cassette (ABC) transporter. Moreover, we determined that the inducing effect of cellobiose (G2) and cellulose on cellulosome production could be eliminated by deletion of transporter B genes, suggesting the coupling of sugar transport and regulation of cellulosome components. This study provides key basic information on the sugar uptake mechanism of C. thermocellum and will promote rational engineering of the bacterium for industrial application.

Transcriptional response and functional analysis of ATP-binding cassette transporters to tannic acid in pea aphid, Acyrthosiphon pisum (Harris)

Although tannins are widely distributed in broad beans and alfalfa, the pea aphid (Acyrthosiphon pisum) can still destroy them. The ATP binding cassette (ABC) transporters participate in the metabolism of plant secondary metabolites and pesticides in insects. However, whether ABC transporter genes play a role in the metabolism of tannins in the pea aphid is unclear. Here, we found that verapamil (an ABC transporter inhibitor) significantly increased the mortality of tannic acid to pea aphid, which indicated that ABC transporter gene was related to the metabolism of tannic acid by pea aphid. Then, we identified 54 putative ABC transporter genes from the genome database of A. pisum. These genes were divided into eight subfamilies, ApABCA to ApABCH, of which subfamily G has the largest number of genes with 19, followed by the subfamily C with 14. RT-qPCR results show that the expression levels of ApABCA2, ApABCC7, ApABCG2, and ApABCG3 were highly expressed in the first instar, while those of ApABCA3, ApABCG6, ApABCG7, ApABCH3, and ApABCH4 were highly expressed in adults. Furthermore, transcription levels of many ABC transporter genes were induced by tannic acid. Especially, ApABCG17 and ApABCH2 were obviously induced after being exposed to tannic acid. Meanwhile, knockdown of ApABCG17 by RNA interference resulted in increased sensitivity of pea aphid to tannic acid. These results suggest that ApABCG17 may be involved in tannic acid metabolism in pea aphid. This study will help us to understand the mechanism of tannic acid metabolism in pea aphid, and provides a basis for further research on the physiological function of ABC transporter genes in pea aphid.

ABC transporters mined through comparative transcriptomics associate with organ-specific accumulation of picrosides in a medicinal herb, Picrorhiza kurroa.

Picrorhiza kurroa Royle ex Benth is a valuable medicinal herb of North-Western Himalayas due to presence of two major bioactive compounds, picroside-I and picroside-II used in the preparation of several hepatoprotective herbal drugs. These compounds accumulate in stolons/rhizomes; however, biosynthesized in different organs, viz., picroside-I in shoots and picroside-II in roots. As of today, no information exists on what transporters are transporting these metabolites from shoots and roots to the final storage organ, stolon, which ultimately transforms into rhizome. The ATP-binding cassette (ABC) transporters are reported to transport majority of secondary metabolites, including terpenoids in plants, therefore, we mined P. kurroa transcriptomes to identify and shortlist potential candidates. A total of 99 ABC transporter-encoding transcripts were identified in 3 differential transcriptomes, PKSS (shoots), PKSTS (stolons), and PKSR (roots) of P. kurroa, based on in silico comparative analysis and transcript abundance. 15 of these transcripts were further validated for their association using qRT-PCR in shoots, roots and stolon tissues in P. kurroa accessions varying for picroside-I and picroside-II contents. Organ-specific expression analysis revealed that PkABCA1, PkABCG1, and PkABCB5 had comparatively elevated expression in shoots; PkABCB2 and PkABCC2 in roots; PkABCB3 and PkABCC1 in stolon tissues of P. kurroa. Co-expression network analysis using ABC genes as hubs further unravelled important interactions with additional components of biosynthetic machinery. Our study has provided leads, first to our knowledge as of today, on putative ABC transporters possibly involved in long distance and local transport of picrosides in P. kurroa organs, thus opening avenues for designing a suitable genetic intervention strategy.

Fermentation Profiles, Bacterial Community Compositions, and Their Predicted Functional Characteristics of Grass Silage in Response to Epiphytic Microbiota on Legume Forages.

This study aimed to investigate the effect of epiphytic microbiota from alfalfa and red clover on the fermentative products, bacterial community compositions, and their predicted functional characteristics in Italian ryegrass silage. By microbiota transplantation and γ-ray irradiation sterilization, the irradiated Italian ryegrass was treated as follows: (1) sterile distilled water (STIR); (2) epiphytic microbiota on Italian ryegrass (IRIR); (3) epiphytic microbiota on alfalfa (IRAL); and (4) epiphytic microbiota on red clover (IRRC). The irradiated Italian ryegrass was ensiled for 1, 3, 7, 15, 30, and 60 days. STIR had similar chemical components with fresh Italian ryegrass. IRAL had higher lactic acid concentrations [64.0 g/kg dry matter (DM)] than IRIR (22.3 g/kg DM) and IRRC (49.4 g/kg DM) on day 3. IRRC had the lowest lactic acid concentrations (59.7 g/kg DM) and the highest pH (4.64), acetic acid (60.4 g/kg DM), ethanol (20.4 g/kg DM), and ammonia nitrogen (82.6 g/kg DM) concentrations and Enterobacteriaceae [9.51 log10 cfu/g fresh weight (FW)] populations among treatments on day 60. On days 3 and 60, Lactobacillus was dominant in both IRIR (42.2%; 72.7%) and IRAL (29.7%; 91.6%), while Hafnia-Obesumbacterium was predominant in IRRC (85.2%; 48.9%). IRIR and IRAL had lower abundances of “Membrane transport” than IRRC on day 3. IRIR and IRAL had lower abundances of phosphotransacetylase and putative ATP-binding cassette transporter and higher abundances of arginine deiminase on day 3. IRAL had the highest abundance of fructokinase on day 3. Overall, inoculating epiphytic microbiota from different legume forages changed the fermentative products, bacterial community compositions, and their predicted functional characteristics in Italian ryegrass silage. The microbial factors that result in the differences in fermentative profiles between legume forage and grass were revealed. Knowledge regarding the effect of epiphytic microbiota could provide more insights into the improvement of silage quality.

Identification of Novel Thermosensors in Gram-Positive Pathogens

Temperature is a crucial variable that every living organism, from bacteria to humans, need to sense and respond to in order to adapt and survive. In particular, pathogenic bacteria exploit host-temperature sensing as a cue for triggering virulence gene expression. Here, we have identified and characterized two integral membrane thermosensor histidine kinases (HKs) from Gram-positive pathogens that exhibit high similarity to DesK, the extensively characterized cold sensor histidine kinase from Bacillus subtilis. Through in vivo experiments, we demonstrate that SA1313 from Staphylococcus aureus and BA5598 from Bacillus anthracis, which likely control the expression of putative ATP binding cassette (ABC) transporters, are regulated by environmental temperature. We show here that these HKs can phosphorylate the non-cognate response regulator DesR, partner of DesK, both in vitro and in vivo, inducing in B. subtilis the expression of the des gene upon a cold shock. In addition, we report the characterization of another DesK homolog from B. subtilis, YvfT, also closely associated to an ABC transporter. Although YvfT phosphorylates DesR in vitro, this sensor kinase can only induce des expression in B. subtilis when overexpressed together with its cognate response regulator YvfU. This finding evidences a physiological mechanism to avoid cross talk with DesK after a temperature downshift. Finally, we present data suggesting that the HKs studied in this work appear to monitor different ranges of membrane lipid properties variations to mount adaptive responses upon cooling. Overall, our findings point out that bacteria have evolved sophisticated mechanisms to assure specificity in the response to environmental stimuli. These findings pave the way to understand thermosensing mediated by membrane proteins that could have important roles upon host invasion by bacterial pathogens.

Metagenomic Resolution of Functional Diversity in Copper Surface-Associated Marine Biofilms.

We used metagenomic sequencing combined with morphological and chemical analyses to investigate microbial taxa and functions related to copper-resistance and microbiologically influenced corrosion in mature copper-associated biofilms in coastal seawater for 44 months. Facultative anaerobic microbes such as Woeseia sp. were found to be the dominant groups on the copper surface. Genes related to stress response and possible heavy metal transport systems, especially RNA polymerase sigma factors (rpoE) and putative ATP-binding cassette (ABC) transport system permease protein (ABC.CD.P) were observed to be highly enriched in copper-associated biofilms, while genes encoding DNA-methyltransferase and RNA polymerase subunit were highly enriched in aluminum-associated biofilms and seawater planktonic cells, respectively. Moreover, copper-associated biofilms harbored abundant copper-resistance genes including cus, cop and pco, as well as abundant genes related to extracellular polymeric substances, indicating the presence of diverse copper-resistance patterns. The proportion of dsr in copper-associated biofilms, key genes related to sulfide production, was as low as that in aluminum biofilm and seawater, which ruled out the possibility of microbial sulfide-induced copper-corrosion under field conditions. These results may fill knowledge gaps about the in situ microbial functions of marine biofilms and their effects on toxic-metal corrosion.

Genome-wide identification of ABC transporters in monogeneans

ATP-Binding Cassette (ABC) transporters are proteins that actively mediate the transport of a wide variety of molecules, including drugs. Thus, in parasitology, ABC transporters have gained attention as potential targets for therapeutic drugs. Among the parasitic Platyhelminthes, ABC transporters have been identified and classified in a few species of Trematoda and Cestoda but not in Monogenea. Monogeneans are mainly ectoparasites of marine and freshwater fish, although they can also be found on other aquatic organisms. Severe epizootics caused by monogeneans have been reported around the world, mainly in confined and/or overcrowded fish. The purpose of this study was to identify the ABC transporters in four species of monogeneans (Gyrodactylus salaris, Protopolystoma xenopodis, Eudiplozoon nipponicum and Neobenedenia melleni) for which genomic resources are publicly available. For comparative purposes, ABC transporters were also identified in endoparasitic (Schistosoma mansoni and Echinococcus granulosus) and free-living (Macrostomun lignano and Schmidtea mediterranea) platyhelminths. Thirty-two putative ABC transporters were identified in the genome of G. salaris, 40 in the genome of P. xenopodis, 46 in the transcriptome of E. nipponicum and 9 in a rather limited ESTs set available for N. melleni. Of the eight ABC subfamilies (A–H) known in metazoans, subfamily H was the only one not found in any monogenean species. In contrast, ABCC was the best represented subfamily. Phylogenetic analyses showed a few cases of one-to-one orthologous relationships, which agree with results from other metazoan species. We found some monogenean ABC members related to subfamilies B, C and G involved in drug resistance in humans. This information may be useful for future functional studies on ABC transporters in monogeneans.

A Periplasmic Binding Protein for Pyrroloquinoline Quinone.

Pyrroloquinoline quinone (PQQ) is an essential redox cofactor in bacterial calcium- and lanthanide-dependent alcohol dehydrogenases. Although certain bacteria are known to synthesize and secrete PQQ, little is known about trafficking of this cofactor within and between cells. Here, we show that a previously uncharacterized periplasmic (solute) binding protein from Methylobacterium extorquens AM1, here renamed PqqT, binds 1 equiv of PQQ with high affinity ( Kd = 50 nM). UV-visible and spectrofluorometric titrations establish that PqqT binds an unhydrated form of PQQ with distinct spectral features from the cofactor in free solution. To our knowledge, PqqT is the first solute-binding protein identified for PQQ and the first protein implicated in cellular trafficking of the cofactor. We propose that PqqT, which is encoded adjacent to a putative ATP-binding cassette transporter in the M. extorquens genome, is involved in uptake of exogenous PQQ to supplement endogenous cofactor biosynthesis. These results support the emerging importance of PQQ transfer within microbial and microbe-host communities.

Mycobacterium tuberculosis nucleoside diphosphate kinase shows interaction with putative ATP binding cassette (ABC) transporter, Rv1273c

Protein–protein interactions are crucial for all biological processes. Compiling this network provides many new insights into protein function and gives directions for the development of new drugs targeted to the pathogen. Mycobacterium tuberculosis Nucleoside diphosphate kinase (Mtb Ndk) has been reported to promote survival of mycobacterium within the macrophage and contribute significantly to mycobacterium virulence. Hence, the present study was aimed to identify and characterize the interacting partner for Ndk. The in vitro experiments, pull down and far western blotting have demonstrated that Mtb Ndk interacts with Rv1273c, a probable drug ABC transporter ATP-binding protein annotated to export drugs across the membrane. This observation was further confirmed by molecular docking and dynamic simulations studies. The homology model of Rv1273c was constructed and docked with Mtb Ndk for protein–protein interaction analysis. The critical residues involved at interface of Rv1273c–Ndk interaction were identified. MDS and Principal Component analysis carried out for conformational feasibility and stability concluded that the complex between the two proteins is more stable as compared to apo proteins. Our findings would be expected to improve the dissection of protein–protein interaction network and significantly advance our understanding of tuberculosis infection.Communicated by Ramaswamy H. Sarma

Functional analysis of putative transporters involved in oligotrophic growth of Rhodococcus erythropolis N9T-4.

Rhodococcus erythropolis N9T-4, which is an extremely oligotrophic bacterium, can survive in a completely inorganic medium with no additional carbon source. This bacterium utilizes atmospheric CO2, but does not require any additional energy source such as light and hydrogen gas, required by autotrophic microorganisms. However, its CO2 fixation and energy-acquisition systems in the oligotrophic growth remain unrevealed. We expected N9T-4 to have the transporter(s) that imports essential compound(s) for its oligotrophic growth. Three putative ATP-binding cassette (ABC) transporters were found to be highly upregulated under oligotrophic conditions. We constructed the gene-deletion mutants of a gene encoding the substrate-binding protein for each ABC transporter (∆sbp1, ∆sbp2, and ∆sbp3). Among these mutants, ∆sbp1 showed growth defects on oligotrophic medium without carbon source. We examined the growth of the mutants on the oligotrophic medium containing 1% trehalose as a sole carbon source. The results exhibited worse growth of ∆sbp3 than that of the control strain (∆ligD), whereas intracellular trehalose content of all mutants decreased compared with that of ∆ligD. It was reported that trehalose functions as the mycolate carrier to the arabinogalactan layer in the cell wall of Mycobacterium tuberculosis. Transmission electron microscopic analysis of ∆sbp1 cells showed that an outermost envelope of the ∆sbp1 cell diminished, which was expected to be mycolate layer. From these results, we suggest that the same trehalose-recycling system as that in a Mycobacterium cell functions in the oligotrophic growth of N9T-4, and the ABC transporter comprising Sbp1 as the substrate-binding protein is strongly involved in the oligotrophic growth of N9T-4.

Antibacterial and anticancer activities of orphan biosynthetic gene clusters from Atlantis II Red Sea brine pool

BackgroundCancer and infectious diseases are problematic because of continuous emergence of drug resistance. One way to address this enormous global health threat is bioprospecting the unlikeliest environments, such as extreme marine niches, which have tremendous biodiversity that is barely explored. One such environment is the Red Sea brine pool, Atlantis II Deep (ATII). Here, we functionally screened a fosmid library of metagenomic DNA isolated from the ATII lower convective layer (LCL) for antibacterial and anticancer activities.ResultsSelected clones, 14-7E and 10-2G, displayed antibacterial effects on the marine strain Bacillus sp. Cc6. Moreover, whole cell lysates from 14-7E and 10-2G exhibited decreased cell viability against MCF-7 (39.1% ± 6.6, 42% ± 8.1 at 50% v/v) and U2OS cells (35.7% ± 1.9, 79.9% ± 5.9 at 50% v/v), respectively. By sequencing the insert DNA from 14-7E and 10-2G, we identified two putative orphan biosynthetic gene clusters. Both clusters harbored putative ATP-binding cassette (ABC) transporter permeases and S-adenosylmethionine-related genes. Interestingly, the biosynthetic gene cluster identified on 14-7E is of archaeal origin and harbors a putative transcription factor. Several identified genes may be responsible for the observed antibacterial and anticancer activities. The 14-7E biosynthetic gene cluster may be encoding enzymes producing a specialized metabolite (effect of detected genes involved in C–C bond formation and glycosylation). The bioactivity may also be due to predicted subtilases encoded by this cluster. The 10-2G cluster harbored putative glycosyltransferase and non-ribosomal peptide synthase genes; thus the observed activity of this clone could be caused by a bioactive peptide.ConclusionsThe ATII LCL prokaryotic metagenome hosts putative orphan biosynthetic gene clusters that confer antibiotic and anticancer effects. Further biochemical studies should characterize the detected bioactive components, and the potential use of 14-7E metabolite for antibiosis and 10-2G metabolite as a selective anti-breast cancer drug.

Functional characterization of BcrR: a one-component transmembrane signal transduction system for bacitracin resistance.

Bacitracin is a cell wall targeting antimicrobial with clinical and agricultural applications. With the growing mismatch between antimicrobial resistance and development, it is essential we understand the molecular mechanisms of resistance in order to prioritize and generate new effective antimicrobials. BcrR is a unique membrane-bound one-component system that regulates high-level bacitracin resistance in Enterococcus faecalis. In the presence of bacitracin, BcrR activates transcription of the bcrABD operon conferring resistance through a putative ATP-binding cassette (ABC) transporter (BcrAB). BcrR has three putative functional domains, an N-terminal helix-turn-helix DNA-binding domain, an intermediate oligomerization domain and a C-terminal transmembrane domain. However, the molecular mechanisms of signal transduction remain unknown. Random mutagenesis of bcrR was performed to generate loss- and gain-of-function mutants using transcriptional reporters fused to the target promoter PbcrA. Fifteen unique mutants were isolated across all three proposed functional domains, comprising 14 loss-of-function and one gain-of-function mutant. The gain-of-function variant (G64D) mapped to the putative dimerization domain of BcrR, and functional analyses indicated that the G64D mutant constitutively expresses the PbcrA-luxABCDE reporter. DNA-binding and membrane insertion were not affected in the five mutants chosen for further characterization. Homology modelling revealed putative roles for two key residues (R11 and S33) in BcrR activation. Here we present a new model of BcrR activation and signal transduction, providing valuable insight into the functional characterization of membrane-bound one-component systems and how they can coordinate critical bacterial responses, such as antimicrobial resistance.

Comparative transcriptome analysis of ATP-binding cassette (ABC) transporter genes in eri-silkworm, Samia cynthia ricini in response to 1-deoxynojirimycin

1-Deoxynojirimycin (DNJ) as a kind of alkaloid has been confirmed that could modulate glycometabolism and has toxicity for the eri-silkworm in our previous research. On the contrary, what is the potentially defensive mechanism when the DNJ enters the eri-silkworm. Based on comparative transcriptome sequencing, we found that ATP-binding cassette (ABC) transporter genes could be induced significantly by DNJ. In this study, a total of 16 putative ABC transporter genes were identified, which can be classified into seven subfamilies, namely one ABCA, four ABCBs, three ABCCs, two ABCDs, one ABCE, three ABCFs, and two ABCGs. Phylogenetic analysis revealed that ScABCs had strong conservation with Bombyx mori. Reverse transcription quantitative PCR (RT-qPCR) suggested that 6 ABC transporters had a strong positive correlation between RT-qPCR and transcriptome data. Additionally, S. c. ricini ABC transporter C-subfamily 4 (ScABCC4), S. c. ricini ABC transporter G-subfamily 4 (ScABCG4), S. c. ricini ABC transporter A-subfamily 3 (ScABCA3) and S. c. ricini ABC transporter C-subfamily 10 (ScABCC10) showed different expression pattern in two feed dose (1% and 2% DNJ) and three time points (6h, 12h, 48h). This study provides the first study on identification, characterization and expression patterns of ABC transporter genes in S. c. ricini response to DNJ, and lays a foundation for further understanding of their physiological roles response to the alkaloid.

Diisonitrile Natural Product SF2768 Functions As a Chalkophore That Mediates Copper Acquisition in Streptomyces thioluteus.

A nonribosomal peptide synthetase (NRPS) gene cluster (sfa) was identified in Streptomyces thioluteus to direct the biosynthesis of the diisonitrile antibiotic SF2768. Its biosynthetic pathway was reasonably proposed based on bioinformatics analysis, metabolic profiles of mutants, and the elucidation of the intermediate and shunt product structures. Bioinformatics-based alignment found a putative ATP-binding cassette (ABC) transporter related to iron import within the biosynthetic gene cluster, which implied that the product might be a siderophore. However, characterization of the metal-binding properties by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), metal-ligand titration, thin-layer chromatography (TLC), and chrome azurol S (CAS) assays revealed that the final product SF2768 and its diisonitrile derivatives specifically bind copper, rather than iron, to form stable complexes. Inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed that the intracellular cupric content of S. thioluteus significantly increased upon incubation with the copper-SF2768 complex, direct evidence for the copper acquisition function of SF2768. Further in vivo functional characterization of the transport elements for the copper-SF2768 complexes not only confirmed the chalkophore identity of the compound but also gave initial clues into the copper uptake mechanism of this nonmethanotrophic microorganism.

Mycobacterium tuberculosis universal stress protein Rv2623 interacts with the putative ATP binding cassette (ABC) transporter Rv1747 to regulate mycobacterial growth.

We have previously shown that the Mycobacterium tuberculosis universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I) of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT)-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623WT), a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623T237A) exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an M. tuberculosis Rv2623 null mutant (ΔRv2623) displays enhanced expression of phosphatidyl-myo-inositol mannosides (PIMs), while the ΔRv1747 mutant expresses decreased levels of PIMs. Animal studies have previously shown that ΔRv2623 is hypervirulent, while ΔRv1747 is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing in vivo growth phenotypes of ΔRv2623 and ΔRv1747, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747; and that Rv1747 might function as a transporter of PIMs. Because these glycolipids are major mycobacterial cell envelope components that can impact on the immune response, our findings raise the possibility that Rv2623 may regulate bacterial growth, virulence, and entry into persistence, at least in part, by modulating the levels of bacillary PIM expression, perhaps through negatively regulating the Rv1747-dependent export of the immunomodulatory PIMs to alter host-pathogen interaction, thereby influencing the fate of M. tuberculosis in vivo.

Conserved ABC Transport System Regulated by the General Stress Response Pathways of Alpha- and Gammaproteobacteria.

Brucella abortus σE1 is an EcfG family sigma factor that regulates the transcription of dozens of genes in response to diverse stress conditions and is required for maintenance of chronic infection in a mouse model. A putative ATP-binding cassette transporter operon, bab1_0223-bab1_0226, is among the most highly activated gene sets in the σE1 regulon. The proteins encoded by the operon resemble quaternary ammonium-compatible solute importers but are most similar in sequence to the broadly conserved YehZYXW system, which remains largely uncharacterized. Transcription of yehZYXW is activated by the general stress sigma factor σS in Enterobacteriaceae, which suggests a functional role for this transport system in bacterial stress response across the classes Alphaproteobacteria and Gammaproteobacteria We present evidence that B. abortus YehZYXW does not function as an importer of known compatible solutes under physiological conditions and does not contribute to the virulence defect of a σE1-null strain. The sole in vitro phenotype associated with genetic disruption of this putative transport system is reduced growth in the presence of high Li+ ion concentrations. A crystal structure of B. abortus YehZ revealed a class II periplasmic binding protein fold with significant structural homology to Archaeoglobus fulgidus ProX, which binds glycine betaine. However, the structure of the YehZ ligand-binding pocket is incompatible with high-affinity binding to glycine betaine. This is consistent with weak measured binding of YehZ to glycine betaine and related compatible solutes. We conclude that YehZYXW is a conserved, stress-regulated transport system that is phylogenetically and functionally distinct from quaternary ammonium-compatible solute importers.IMPORTANCEBrucella abortus σE1 regulates transcription in response to stressors encountered in its mammalian host and is necessary for maintenance of chronic infection in a mouse model. The functions of the majority of genes regulated by σE1 remain undefined. We present a functional/structural analysis of a conserved putative membrane transport system (YehZYXW) whose expression is strongly activated by σE1 Though annotated as a quaternary ammonium osmolyte uptake system, experimental physiological studies and measured ligand-binding properties of the periplasmic binding protein (PBP), YehZ, are inconsistent with this function. A crystal structure of B. abortus YehZ provides molecular insight into differences between bona fide quaternary ammonium osmolyte importers and YehZ-related proteins, which form a distinct phylogenetic and functional group of PBPs.

Down-regulation of aminopeptidase N and ABC transporter subfamily G transcripts in Cry1Ab and Cry1Ac resistant Asian corn borer, Ostrinia furnacalis (Lepidoptera: Crambidae).

The Asian corn borer (ACB), Ostrinia furnacalis (Lepidoptera: Crambidae), is a highly destructive pest of cultivated maize throughout East Asia. Bacillus thuringiensis (Bt) crystalline protein (Cry) toxins cause mortality by a mechanism involving pore formation or signal transduction following toxin binding to receptors along the midgut lumen of susceptible insects, but this mechanism and mutations therein that lead to resistance are not fully understood. In the current study, quantitative comparisons were made among midgut expressed transcripts from O. furnacalis susceptible (ACB-BtS) and laboratory selected strains resistant to Cry1Ab (ACB-AbR) and Cry1Ac toxins (ACB-AcR) when feeding on non-Bt diet. From a combined de novo transcriptome assembly of 83,370 transcripts, ORFs of ≥ 100 amino acids were predicted and annotated for 28,940 unique isoforms derived from 12,288 transcripts. Transcriptome-wide expression estimated from RNA-seq read depths predicted significant down-regulation of transcripts for previously known Bt resistance genes, aminopeptidase N1 (apn1) and apn3, as well as a putative ATP binding cassette transporter group G (abcg) gene in both ACB-AbR and -AcR (log2[fold-change] ≥ 1.36; P < 0.0001). The transcripts that were most highly differentially regulated in both ACB-AbR and -AcR compared to ACB-BtS (log2[fold-change] ≥ 2.0; P < 0.0001) included up- and down-regulation of serine proteases, storage proteins and cytochrome P450 monooxygenases, as well as up-regulation of genes with predicted transport function. This study predicted the significant down-regulation of transcripts for previously known Bt resistance genes, aminopeptidase N1 (apn1) and apn3, as well as abccg gene in both ACB-AbR and -AcR. These data are important for the understanding of systemic differences between Bt resistant and susceptible genotypes.

Diversity of ABC transporter genes across the plant kingdom and their potential utility in biotechnology.

BackgroundThe ATP-binding cassette (ABC) transporter gene superfamily is ubiquitous among extant organisms and prominently represented in plants. ABC transporters act to transport compounds across cellular membranes and are involved in a diverse range of biological processes. Thus, the applicability to biotechnology is vast, including cancer resistance in humans, drug resistance among vertebrates, and herbicide and other xenobiotic resistance in plants. In addition, plants appear to harbor the highest diversity of ABC transporter genes compared with any other group of organisms. This study applied transcriptome analysis to survey the kingdom-wide ABC transporter diversity in plants and suggest biotechnology applications of this diversity.ResultsWe utilized sequence similarity-based informatics techniques to infer the identity of ABC transporter gene candidates from 1295 phylogenetically-diverse plant transcriptomes. A total of 97,149 putative (approximately 25 % were full-length) ABC transporter gene members were identified; each RNA-Seq library (plant sample) had 88 ± 30 gene members. As expected, simpler organisms, such as algae, had fewer unique members than vascular land plants. Differences were also noted in the richness of certain ABC transporter subfamilies. Land plants had more unique ABCB, ABCC, and ABCG transporter gene members on average (p < 0.005), and green algae, red algae, and bryophytes had significantly more ABCF transporter gene members (p < 0.005). Ferns had significantly fewer ABCA transporter gene members than all other plant groups (p < 0.005).ConclusionsWe present a transcriptomic overview of ABC transporter gene members across all major plant groups. An increase in the number of gene family members present in the ABCB, ABCC, and ABCD transporter subfamilies may indicate an expansion of the ABC transporter superfamily among green land plants, which include all crop species. The striking difference between the number of ABCA subfamily transporter gene members between ferns and other plant taxa is surprising and merits further investigation. Discussed is the potential exploitation of ABC transporters in plant biotechnology, with an emphasis on crops.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0277-6) contains supplementary material, which is available to authorized users.

Probing allocrite preferences of 2 naturally occurring variants of the wheat LR34 ABC transporter.

For almost a century, the wheat Lr34 gene has conferred durable resistance against fungal rust diseases. While sequence homology predicts a putative ATP binding cassette transporter, the molecules that are transported (allocrites) by the encoded LR34 variants, and any associated mechanism of resistance, remain enigmatic. Here, the in vitro transport characteristics of 2 naturally occurring Lr34 variants (that differ in their ability to mediate disease resistance; Lr34sus and Lr34res) are investigated. Initially, a method to express and purify recombinant LR34Sus and LR34Res pseudo half-molecules from Saccharomyces cerevisiae, is described. Subsequently, a semi-targeted chlorophyll catabolite (CC) extraction from Lr34res-expressing wheat plants was performed based on previous reports highlighting increased accumulation of CCs in Lr34res-expressing flag leaves. Following partial biochemical characterization, this extract was applied to an LR34 in vitro proteoliposome transport assay. Mass spectroscopic analyses of transported metabolites revealed that LR34Sus imported a wheat metabolite of 618 Da and that the LR34Res transporter did not. While the identity of the LR34Sus transported metabolite remains to be confirmed and any allocrites of LR34Res remain to be detected, this work demonstrates that these variants have different allocrite preferences, a finding that may be relevant to the mechanism of disease resistance.

The putative ABC transporter encoded by the orf19.4531 plays a role in the sensitivity of Candida albicans cells to azole antifungal drugs.

ATP-binding cassette (ABC) transporters constitute a large superfamily of integral membrane proteins in prokaryotic and eukaryotic cells. In the human fungal pathogen Candida albicans, there are 28 genes encoding ABC transporters and many of them have not been characterized so far. The orf19.4531 (also known as IPF7530) encodes a putative ABC transporter. In this study, we have demonstrated that disruption of orf19.4531 causes C. albicans cells to become tolerant to azoles, but not to polyene antifungals and terbinafine. Therefore, the protein encoded by orf19.4531 is involved in azole sensitivity and we name it as ROA1, the regulator of azole sensitivity 1 gene. Consistently, we show that the expression of ROA1 is responsive to treatment of either fluconazole or ketoconazole inC. albicans In addition, through a GFP tagging approach, Roa1 is localized in a small punctuate compartment adjacent to the vacuolar membrane. However, ROA1 is not essential for the in vitro filamentation of C. albicans cells.