Membrane ABC Transporter Research Articles

Structure of the MlaC-MlaD complex reveals molecular basis of periplasmic phospholipid transport

The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD β6-β7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD.

Exploring genetic landscape of low-density polyethylene degradation for sustainable troubleshooting of plastic pollution at landfills

Plastic pollution increases globally due to the high volume of its production and inadequate mismanagement, leading to dumps in landfills affecting terrestrial and aquatic ecosystems. Landfills, as sink for plastics, leach various toxic chemicals and microplastics into the environment. We scrutinized the genetic expression for low-density polyethylene (LDPE) degradation via microorganisms to investigate cell viability and metabolic activities for biodegradation and genetic profiling. Samples were collected from the Pirana waste landfill at Ahmedabad, Gujarat, which is one of the largest and oldest municipal solid waste (MSW) dump sites in Asia. Results analyzed that isolated bacterial culture PN(A)1 (Bacillus cereus) is metabolically active on LDPE as carbon source during starvation conditions when incubated for up to 60 days, which was confirmed via 2,3,5-triphenyl-tetrazolium chloride (TTC) reduction test, reported cell viability and LDPE degradation. Abrasions, surface erosions, and cavity formations were analyzed via scanning electron microscopy (SEM), whereas the breakdown of high molecular polymers converted to low molecules, i.e., depolymerization, was also observed via Fourier-transform infrared (FTIR) spectroscopy over 90 days, along with changes in functional groups of carboxylic acids and aldehyde as well as the formation of polysulfide, aliphatic compounds, aromatic ethers, alcohols, and ether linkages. Further, transcriptomic analysis was performed via DESeq2 analysis to understand key gene expression patterns and pathways involved in LDPE degradation. During the initial phase of LDPE degradation, genes related to biological processes, like membrane transportation, ABC transporters, carbon and lipid metabolism, fatty acid degradation/oxidation, and TCA cycle, are likely to indicate pathways for stress response and molecular functions, like oxidoreductase, catalytic, lyase, transferase, and hydrolase activities were expressed. Interlinking between metabolic pathways indicates biodegradation process that mineralizes LDPE during subsequent incubation days. These pathways can be targeted for increasing the efficiency of LDPE degradation using microbes in future studies. Thus, considering microbial-mediated biodegradation as practical, eco-friendly, and low-cost alternatives, healthy biomes can degrade polymers in natural environments explored by understanding the genetic and enzymatic expression, connecting their role in the process to the likely metabolic pathways involved, thereby increasing the rate of their biodegradation.

The hemolysin A secretion system is a multi-engine pump containing three ABC transporters

Type 1 secretion systems (T1SSs) are widespread in pathogenic Gram-negative bacteria, extruding protein substrates following synthesis of the entire polypeptide. The Escherichia coli hemolysin A secretion system has long been considered a prototype in structural and mechanistic studies of T1SSs. Three membrane proteins-an inner membrane ABC transporter HlyB, an adaptor protein HlyD, and an outer membrane porin TolC-are required for secretion. However, the stoichiometry and structure of the complex are unknown. Here, cryo-electron microscopy (cryo-EM) structures determined in two conformations reveal that the inner membrane complex is a hetero-dodecameric assembly comprising three HlyB homodimers and six HlyD subunits. Functional studies indicate that oligomerization of HlyB and HlyD is essential for protein secretion and that polypeptides translocate through a canonical ABC transporter pathway in HlyB. Our data suggest that T1SSs entail three ABC transporters, one that functions as a protein channel and two that allosterically power the translocation process.

The Regulatory Protein ChuP Connects Heme and Siderophore-Mediated Iron Acquisition Systems Required for Chromobacterium violaceum Virulence.

Chromobacterium violaceum is an environmental Gram-negative beta-proteobacterium that causes systemic infections in humans. C. violaceum uses siderophore-based iron acquisition systems to overcome the host-imposed iron limitation, but its capacity to use other iron sources is unknown. In this work, we characterized ChuPRSTUV as a heme utilization system employed by C. violaceum to explore an important iron reservoir in mammalian hosts, free heme and hemoproteins. We demonstrate that the chuPRSTUV genes comprise a Fur-repressed operon that is expressed under iron limitation. The chu operon potentially encodes a small regulatory protein (ChuP), an outer membrane TonB-dependent receptor (ChuR), a heme degradation enzyme (ChuS), and an inner membrane ABC transporter (ChuTUV). Our nutrition growth experiments using C. violaceum chu deletion mutants revealed that, with the exception of chuS, all genes of the chu operon are required for heme and hemoglobin utilization in C. violaceum. The mutant strains without chuP displayed increased siderophore halos on CAS plate assays. Significantly, we demonstrate that ChuP connects heme and siderophore utilization by acting as a positive regulator of chuR and vbuA, which encode the TonB-dependent receptors for the uptake of heme (ChuR) and the siderophore viobactin (VbuA). Our data favor a model of ChuP as a heme-binding post-transcriptional regulator. Moreover, our virulence data in a mice model of acute infection demonstrate that C. violaceum uses both heme and siderophore for iron acquisition during infection, with a preference for siderophores over the Chu heme utilization system.

IL-10 contributes to gemcitabine resistance in extranodal NK/T-cell lymphoma cells via ABCC4.

BackgroundChemotherapy resistance is a main reason for treatment failure in extranodal NK/T-cell lymphoma (ENKTL). Interleukin-10 (IL-10) is closely related to the occurrence and prognosis of ENKTL. We intended to study the role and molecular mechanism of IL-10 in ENKTL resistance. MethodsFifty serum samples were collected from patients with a histologically proven diagnosis of ENKTL. Fifty healthy volunteers were enrolled as a control group. The level of serum IL-10 was detected by ELISA. The NK/T-cell lymphoma cell lines YT and NK-92 were divided into the control group (untreated), IL-10 group (treated with IL-10), IL-10 + GEM group (treated with IL-10 and gemcitabine simultaneously) and GEM group (treated with gemcitabine). A CCK8 assay and flow cytometry were employed to detect the effects of IL-10 on each group. Western blotting was applied to detect the expression of ABC membrane transporter family proteins and signaling pathway proteins in each group. ResultsSerum IL-10 levels were higher in ENKTL patients as well asin patients with ineffective treatment. The IC50 value for gemcitabine in YT and NK-92 cells increased significantly in the presence of IL-10. The effects of gemcitabine resulting in cell killing, cell cycle arrest, and apoptosis promotion were also weakened by IL-10. The expression of ABCC4, STAT1, p-STAT1, Tyk2 and p-Tyk2 was significantly increased by IL-10. ConclusionOur results indicate that IL-10 contributes to the resistance of ENKTL cells via ABCC4 and that IL-10 regulates the JAK/STAT signaling pathway in YT and NK-92 cells.

Crosstalk between Yeast Cell Plasma Membrane Ergosterol Content and Cell Wall Stiffness under Acetic Acid Stress Involving Pdr18.

Acetic acid is a major inhibitory compound in several industrial bioprocesses, in particular in lignocellulosic yeast biorefineries. Cell envelope remodeling, involving cell wall and plasma membrane composition, structure and function, is among the mechanisms behind yeast adaptation and tolerance to stress. Pdr18 is a plasma membrane ABC transporter of the pleiotropic drug resistance family and a reported determinant of acetic acid tolerance mediating ergosterol transport. This study provides evidence for the impact of Pdr18 expression in yeast cell wall during adaptation to acetic acid stress. The time-course of acetic-acid-induced transcriptional activation of cell wall biosynthetic genes (FKS1, BGL2, CHS3, GAS1) and of increased cell wall stiffness and cell wall polysaccharide content in cells with the PDR18 deleted, compared to parental cells, is reported. Despite the robust and more intense adaptive response of the pdr18Δ population, the stress-induced increase of cell wall resistance to lyticase activity was below parental strain levels, and the duration of the period required for intracellular pH recovery from acidification and growth resumption was higher in the less tolerant pdr18Δ population. The ergosterol content, critical for plasma membrane stabilization, suffered a drastic reduction in the first hour of cultivation under acetic acid stress, especially in pdr18Δ cells. Results revealed a crosstalk between plasma membrane ergosterol content and cell wall biophysical properties, suggesting a coordinated response to counteract the deleterious effects of acetic acid.

Metabolomic insights into the browning of the peel of bagging \u2018Rui Xue\u2019 apple fruit

BackgroundBagging is one of the most important techniques for producting high-quality fruits. In the actual of cultivating, we found a new kind of browning in peel of apple fruit that occurs before harvest and worsen during storage period. There are many studies on metabonomic analysis of browning about storage fruits, but few studies on the mechanism of browning before harvest.ResultsIn this study, five-year-old trees of ‘Rui Xue’ (CNA20151469.1) were used as materials. Bagging fruits without browning (BFW) and bagging fruits with browning (BFB) were set as the experimental groups, non-bagging fruits (NBF) were set as control. After partial least squares discriminant analysis (PLS-DA), 50 kinds of metabolites were important with predictive VIP > 1 and p-value < 0.05. The most important differential metabolites include flavonoids and lipids molecules, 11 flavonoids and 6 lipids molecules were significantly decreased in the BFW compared with NBF. After browning, 11 flavonoids and 7 lipids were further decreased in BFB compared with BFW. Meanwhile, the significantly enriched metabolic pathways include galactose metabolism, ABC membrane transporter protein, flavonoid biosynthesis and linoleic acid metabolism pathways et al. Physiological indicators show that, compared with NBF, the content of malondialdehyde (MDA), hydrogen peroxide (H2O2), superoxide anion (O2−) in peel of BFW and BFB were significantly increased, and the difference of BFB was more significant. Meanwhile, the antioxidant enzyme activities of BFW and BFB were inhibited, which accelerated the destruction of cell structure. In addition, the metabolome and physiological data showed that the significantly decrease of flavonoid was positively correlated with peel browning. So, we analyzed the expression of flavonoid related genes and found that, compared with NBF, the flavonoid synthesis genes MdLAR and MdANR were significantly up-regulated in BFW and BFB, but, the downstream flavonoids-related polymeric genes MdLAC7 and MdLAC14 were also significantly expressed.ConclusionsOur findings demonstrated that the microenvironment of fruit was changed by bagging, the destruction of cell structure, the decrease of flavonoids and the increase of triterpenoids were the main reasons for the browning of peel.

Engineering endogenous ABC transporter with improving ATP supply and membrane flexibility enhances the secretion of \u03b2-carotene in Saccharomyces cerevisiae

BackgroundProduct toxicity is one of the bottlenecks for microbial production of biofuels, and transporter-mediated biofuel secretion offers a promising strategy to solve this problem. As a robust microbial host for industrial-scale production of biofuels, Saccharomyces cerevisiae contains a powerful transport system to export a wide range of toxic compounds to sustain survival. The aim of this study is to improve the secretion and production of the hydrophobic product (β-carotene) by harnessing endogenous ABC transporters combined with physiological engineering in S. cerevisiae.ResultsSubstrate inducibility is a prominent characteristic of most endogenous transporters. Through comparative proteomic analysis and transcriptional confirmation, we identified five potential ABC transporters (Pdr5p, Pdr10p, Snq2p, Yor1p, and Yol075cp) for β-carotene efflux. The accumulation of β-carotene also affects cell physiology in various aspects, including energy metabolism, mitochondrial translation, lipid metabolism, ergosterol biosynthetic process, and cell wall synthesis. Here, we adopted an inducible GAL promoter to overexpress candidate transporters and enhanced the secretion and intracellular production of β-carotene, in which Snq2p showed the best performance (a 4.04-fold and a 1.33-fold increase compared with its parental strain YBX-01, respectively). To further promote efflux capacity, two strategies of increasing ATP supply and improving membrane fluidity were following adopted. A 5.80-fold increase of β-carotene secretion and a 1.71-fold increase of the intracellular β-carotene production were consequently achieved in the engineered strain YBX-20 compared with the parental strain YBX-01.ConclusionsOverall, our results showcase that engineering endogenous plasma membrane ABC transporters is a promising approach for hydrophobic product efflux in S. cerevisiae. We also highlight the importance of improving cell physiology to enhance the efficiency of ABC transporters, especially energy status and cell membrane properties.

Redox-related Molecular Mechanism of Sensitizing Colon Cancer Cells to Camptothecin Analog SN38.

The aim of this study was to elucidate the possibility of sensitizing colon cancer cells to the chemotherapeutic drug SN38 and investigate its mechanism of action after combined treatment with electroporation (EP). Cells were treated with SN38, EP and their combination for 24/48 h. The cell viability, actin cytoskeleton integrity, mitochondrial superoxide, hydroperoxides, total glutathione, phosphatidyl serine expression, DNA damages and expression of membrane ABC transporters were analyzed using conventional analytical tests. The combination of EP and SN38 affected cell viability and cytoskeleton integrity. This effect was accompanied by: (i) high production of intracellular superoxide and hydroperoxides and depletion of glutathione; (ii) increased DNA damage and apoptotic/ferroptotic cell death; (iii) changes in the expression of membrane ABC transporters - up-regulation of SLCO1B1 and retention of SN38 in the cells. The anticancer effect of the combined treatment of SN38 and EP is related to changes in the redox-homeostasis of cancer cells, leading to cell death via apoptosis and/or ferroptosis. Thus, electroporation has a potential to increase the sensitivity of cancer cells to conventional anticancer therapy with SN38.

Developmental patterns in human blood\u2013brain barrier and blood\u2013cerebrospinal fluid barrier ABC\xa0drug transporter expression

When drugs exert their effects in the brain, linear extrapolation of doses from adults could be harmful for children as the blood–brain barrier (BBB) and blood–CSF barrier (BCSFB) function is still immature. More specifically, age-related variation in membrane transporters may impact brain disposition. As human data on brain transporter expression is scarce, age dependent [gestational age (GA), postnatal age (PNA), and postmenstrual age (PMA)] variation in immunohistochemical localization and staining intensity of the ABC transporters P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins 1, 2, 4, and 5 (MRP1/2/4/5) was investigated. Post mortem brain cortical and ventricular tissue was derived from 23 fetuses (GA range 12.9–39 weeks), 17 neonates (GA range 24.6–41.3 weeks, PNA range 0.004–3.5 weeks), 8 children (PNA range 0.1–3 years), and 4 adults who died from a wide variety of underlying conditions. In brain cortical BBB, immunostaining increased with age for Pgp and BCRP, while in contrast, MRP1 and MRP2 staining intensity appeared higher in fetuses, neonates, and children, as compared to adults. BCSFB was positively stained for Pgp, MRP1, and MRP2 and appeared stable across age, while BCRP was not detected. MRP4 and MRP5 were not detected in BBB or BCSFB. In conclusion, human BBB and BCSFB ABC membrane transporters show brain location and transporter-specific maturation.

Characterization of the Efflux Capability and Substrate Specificity of Aspergillus fumigatus PDR5-like ABC Transporters Expressed in Saccharomyces cerevisiae.

This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. TheA. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine β-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable.IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance.Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.

Molecular response mechanism in Escherichia coli under hexabromocyclododecane stress

The effects of hexabromocyclododecane (HBCD) on the relationship between physiological responses and metabolic networks remains unclear. To this end, cellular growth, apoptosis, reactive oxygen species, exometabolites and the proteome of Escherichia coli were investigated following exposure to 0.1 and 1 μM HBCD. The results showed that although there were no significant changes in the pH value, apoptosis and reactive oxygen species under HBCD stress, cell growth was inhibited. The metabolic network formed by glycolysis, oxidative phosphorylation, amino acids biosynthesis, membrane proteins biosynthesis, ABC transporters, glycogen storage, cell recognition, compound transport and nucleotide excision repair was disrupted. Cell chemotaxis and DNA damage repair were the effective approaches to alleviate HBCD stress. This work improves our understanding of HBCD toxicity and provides insight into the toxicological mechanism of HBCD at the molecular and network levels.

Comparison of cytotoxicity and membrane efflux pump inhibition in HepG2 cells induced by single-walled carbon nanotubes with different length and functional groups

Environmental risk of single-walled carbon nanotubes (SWCNTs) is receiving increasing attentions owing to their wide study and application. However, little is known on the influence of length and functional groups on SWCNT cytotoxicity. In this study, six types of SWCNTs with different functional groups (pristine, carboxyl group and hydroxyl group) and lengths (1–3 μm and 5–30 μm) were chosen. Cytotoxicities in human hepatoma HepG2 cells induced by these SWCNTs were compared based on cell viability, oxidative stress, plasma membrane fluidity and ABC transporter activity assays. Results showed that all the SWCNTs decreased cell viability of HepG2, increased intracellular reactive oxygen species (ROS) level, and damaged plasma membrane in a concentration-dependent manner. Long SWCNTs had stronger cytotoxic effects than short SWCNTs, which might be due to weaker aggregation for the long SWCNTs. Functionalization changed the toxic effects of the SWCNTs, and different influence was found between long SWCNTs and short SWCNTs. Moreover, the six types of SWCNTs at low concentrations changed plasma membrane fluidity, inhibited transmembrane ABC transporter (efflux pump) activity, and acted as chemosensitizer to improve the sensitivity of cells to arsenic, indicating the chemosensitive effect should be considered as toxic endpoint of SWCNTs. Comparison of different toxic endpoints among the six types of SWCNTs showed that short hydroxyl-SWCNT might be safer than other SWCNTs. This study provides insights into toxicities of SWCNTs, which is of great value for the risk assessment and application of SWCNTs.

Transcriptional profiling of contrasting genotypes revealed key candidates and nucleotide variations for drought dissection in Camellia sinensis (L.) O. Kuntze

Tea is popular health beverage consumed by millions of people worldwide. Drought is among the acute abiotic stress severely affecting tea cultivation, globally. In current study, transcriptome sequencing of four diverse tea genotypes with inherent contrasting genetic response to drought (tolerant & sensitive) generated more than 140 million reads. De novo and reference-based assembly and functional annotation of 67,093 transcripts with multifarious public protein databases yielded 54,484 (78.2%) transcripts with significant enrichment of GO and KEGG drought responsive pathways in tolerant genotypes. Comparative DGE and qRT analysis revealed key role of ABA dependent & independent pathways, potassium & ABC membrane transporters (AtABCG22, AtABCG11, AtABCC5 & AtABCC4) and antioxidant defence system against oxidative stress in tolerant genotypes, while seems to be failed in sensitive genotypes. Additionally, highly expressed UPL3HECT E3 ligases and RING E3 ligases possibly enhance drought tolerance by actively regulating functional modification of stress related genes. Further, ascertainment of, 80803 high quality putative SNPs with functional validation of key non-synonymous SNPs suggested their implications for developing high-throughput genotyping platform in tea. Futuristically, functionally relevant genomic resources can be potentially utilized for gene discovery, genetic engineering and marker-assisted genetic improvement for better yield and quality in tea under drought conditions.

Oestrogen-related receptor alpha mediates chemotherapy resistance of osteosarcoma cells via regulation of ABCB1.

Chemotherapy resistance is one of the major challenges for the treatment of osteosarcoma (OS). The potential roles of oestrogenic signals in the chemoresistance of OS cells were investigated. As compare to the parental cells, the doxorubicin and cisplatin (CDDP) resistant OS cells had greater levels of oestrogen‐related receptors alpha (ERRα). Targeted inhibition of ERRα by its specific siRNAs or inverse agonist XCT‐790 can restore the sensitivity of OS resistant cells to chemotherapy. This might be due to that si‐ERRα can decrease the expression of P‐glycoprotein (P‐gp, encoded by ABCB1), one important ABC membrane transporter for drug efflux. XCT‐790 can decrease the transcription and mRNA stability of ABCB1, while had no effect on protein stability of P‐gp. ERRα can bind to the transcription factor of SP3 to increase the transcription of ABCB1. Furthermore, XCT‐790 treatment decreased the expression of miR‐9, which can bind to the 3′UTR of ABCB1 and trigger its decay. Collectively, we found that ERRα can regulate the chemoresistance of OS cells via regulating the transcription and mRNA stability of ABCB1. Targeted inhibition of ERRα might be a potential approach for OS therapy.

ABC membrane transporters and their multifunctional nature

ABC transporters are the largest family of transport proteins, which mediate the active translocation of a wide spectrum of molecules through the cell membrane, using energy from hydrolysis of ATP. They can act as importers (in Procaryota exclusively) or exporters (in both Procaryota and Eucaryota) of specified substrates. Despite a quite conservative structure model, ABC transporters are diverse in terms of functions performed in the body. These proteins are important elements of the blood-organ barriers, they maintain lipid homeostasis, participate in cellular immune response but also are involved in the development of multidrug resistance as well as affect the biology of tumour cells. Additionally, they play the role of ion channels or regulators of their activity. The activity of ABC transporters is also correlated with the occurrence of certain disease entities, such as cystic fibrosis, Dubin-Johnson syndrome, Tangier disease, persistent hyperinsulinemic hypoglycemia of infancy (PHHI) and neoplasia. Due to their therapeutic potential, ABC transporters are becoming increasingly popular. Knowledge of the mechanisms of action of ABC transporters and their regulatory systems will provide the basis for the development of personalized medicine, which may be significant especially in the context of cancer treatment.

The S-layer biogenesis system of Synechocystis 6803: Role of Sll1180 and Sll1181 (E. coli HlyB and HlyD analogs) as type-I secretion components for Sll1951 export

Multiple secretion pathways are known for export of protein(s) forming the S-layer in bacteria. The unicellular model cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter S. 6803) also possesses a well-defined S-layer composed of Sll1951 protein. However, the mechanism of its secretion is not completely understood. In the present study, the putative T1SS (Type I secretion system) components, Sll1180 and Sll1181 [inner membrane ABC transporter and membrane fusion protein (MFP), respectively] were characterized for their role in Sll1951 secretion. The corresponding ORFs i.e. sll1180 and sll1181 were inactivated by insertion of a spectinomycin resistance gene. The viability of the homozygous mutants of both the genes indicated dispensability of the corresponding proteins under the experimental conditions. Interestingly, the culture supernatants of the mutants i.e. Δsll1180 and Δsll1181, lacked Sll1951 as observed on SDS-PAGE and confirmed by mass spectrometry. Immunofluorescence delineated a distinct outer ring of Sll1951 in S. 6803 cells only that was further iterated by transmission and scanning electron microscopy. The loss of S-layer imparted an aggregative phenotype to both the mutants. Surprisingly, Δsll1181 cells showed increased sensitivity to different antibiotics indicating a role in multidrug efflux. This is the first report establishing Sl1180 and Sll1181 proteins as partners of the previously characterized Slr1270, for Sll1951 secretion and thus S-layer biogenesis in S. 6803. Sll1181 (in conjunction with Slr1270) also acts as MFP in multidrug efflux along with a yet uncharacterized inner membrane protein.

Control of hmu Heme Uptake Genes in Yersinia pseudotuberculosis in Response to Iron Sources.

Despite the mammalian host actively sequestering iron to limit pathogenicity, heme (or hemin when oxidized) and hemoproteins serve as important sources of iron for many bloodborne pathogens. The HmuRSTUV hemin uptake system allows Yersinia species to uptake and utilize hemin and hemoproteins as iron sources. HmuR is a TonB-dependent outer membrane receptor for hemin and hemoproteins. HmuTUV comprise a inner membrane ABC transporter that transports hemin and hemoproteins from the periplasmic space into the bacterial cytoplasm, where it is degraded by HmuS. Here we show that hmuSTUV but not hmuR are expressed under iron replete conditions, whereas hmuR as well as hmuSTUV are expressed under iron limiting conditions, suggesting complex transcriptional control. Indeed, expression of hmuSTUV in the presence of inorganic iron, but not in the presence of hemin, requires the global regulator IscR acting from a promoter in the intergenic region between hmuR and hmuS. This effect of IscR appears to be direct by binding a site mapped by DNaseI footprinting. In contrast, expression of hmuR under iron limiting conditions requires derepression of the ferric uptake regulator Fur acting from the hmuR promoter, as Fur binding upstream of hmuR was demonstrated biochemically. Differential expression by both Fur and IscR would facilitate maximal hemin uptake and utilization when iron and heme availability is low while maintaining the capacity for periplasmic removal and cytosolic detoxification of heme under a wider variety of conditions. We also demonstrate that a Y. pseudotuberculosis ΔiscR mutant has a survival defect when incubated in whole blood, in which iron is sequestered by heme-containing proteins. Surprisingly, this phenotype was independent of the Hmu system, the type III secretion system, complement, and the ability of Yersinia to replicate intracellularly. These results suggest that IscR regulates multiple virulence factors important for Yersinia survival and growth in mammalian tissues and reveal a surprising complexity of heme uptake expression and function under differing conditions of iron.

GeneGini: Assessment via the Gini Coefficient of Reference "Housekeeping" Genes and Diverse Human Transporter Expression Profiles.

SummaryThe expression levels of SLC or ABC membrane transporter transcripts typically differ 100- to 10,000-fold between different tissues. The Gini coefficient characterizes such inequalities and here is used to describe the distribution of the expression of each transporter among different human tissues and cell lines. Many transporters exhibit extremely high Gini coefficients even for common substrates, indicating considerable specialization consistent with divergent evolution. The expression profiles of SLC transporters in different cell lines behave similarly, although Gini coefficients for ABC transporters tend to be larger in cell lines than in tissues, implying selection. Transporter genes are significantly more heterogeneously expressed than the members of most non-transporter gene classes. Transcripts with the stablest expression have a low Gini index and often differ significantly from the “housekeeping” genes commonly used for normalization in transcriptomics/qPCR studies. PCBP1 has a low Gini coefficient, is reasonably expressed, and is an excellent novel reference gene. The approach, referred to as GeneGini, provides rapid and simple characterization of expression-profile distributions and improved normalization of genome-wide expression-profiling data.

Structural Basis for the Serratia marcescens Lipase Secretion System: Crystal Structures of the Membrane Fusion Protein and Nucleotide-Binding Domain.

Serratia marcescens secretes a lipase, LipA, through a type I secretion system (T1SS). The T1SS for LipA, the Lip system, is composed of an inner membrane ABC transporter with its nucleotide-binding domains (NBD), LipB, a membrane fusion protein, LipC, and an outer membrane channel protein, LipD. Passenger protein secreted by this system has been functionally and structurally characterized well, but relatively little information about the transporter complex is available. Here, we report the crystallographic studies of LipC without the membrane anchor region, LipC-, and the NBD of LipB (LipB-NBD). LipC- crystallographic analysis has led to the determination of the structure of the long α-helical and lipoyl domains, but not the area where it interacts with LipB, suggesting that the region is flexible without LipB. The long α-helical domain has three α-helices, which interacts with LipD in the periplasm. LipB-NBD has the common overall architecture and ATP hydrolysis activity of ABC transporter NBDs. Using the predicted models of full-length LipB and LipD, the overall structural insight into the Lip system is discussed.