scholarly journals Molecular Characterization of the Hemin Uptake Locus (hmu) from Yersinia pestis and Analysis ofhmu Mutants for Hemin and Hemoprotein Utilization

1999 ◽  
Vol 67 (8) ◽  
pp. 3879-3892 ◽  
Author(s):  
Jan M. Thompson ◽  
Heather A. Jones ◽  
Robert D. Perry
Keyword(s):  
Yersinia Pestis ◽  
Parent Strain ◽  
Gram Negative Bacteria ◽  
Uptake System ◽  
Constitutive Activity ◽  
Gene Products ◽  
Lower Affinity ◽  
High Concentrations ◽  
Lethal Doses

ABSTRACT Sequence analysis of the hemin uptake locus (hmu) ofYersinia pestis revealed five genes, hmuRSTUV, required for use of hemin and hemoproteins as iron sources. The translated gene products have homologies with proteins of the hemin transport genes of several gram-negative bacteria. Promoters were identified upstream of hmuP′R (p1) and upstream ofhmuS (p2); p1, which contains a Fur box, is regulated by iron and Fur, while p2 exhibits weak, but constitutive, activity. HmuR, which has homology with TonB-dependent outer membrane (OM) receptors, is localized to the OM of Y. pestis and is required for utilizing hemin and all hemoproteins under iron-depleted conditions. The proposed ABC transporter, HmuTUV, is necessary for use of hemin, hemin-albumin, and myoglobin, but not hemoglobin, hemoglobin-haptoglobin, or heme-hemopexin, as iron sources. In the absence of HmuTUV, HmuS, a cytoplasmic protein, is involved in use of hemoglobin and heme-hemopexin. In mice, the 50% lethal doses ofY. pestis ΔhmuP′RSTUV mutants injected subcutaneously or retro-orbitally did not differ from that of the Hmu+ parent strain. Thus, the hmu system is not essential for infection in mice via these routes. Growth studies showed that a ΔhmuP′RSTUV mutant could grow in iron-depleted medium containing high concentrations of hemoglobin, suggesting that an Hmu-independent, lower-affinity hemoglobin uptake system may exist.

scholarly journals Characterization of simultaneous uptake of xylose and glucose in Caldicellulosiruptor kronotskyensis for optimal hydrogen production

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Thitiwut Vongkampang ◽  
Krishnan Sreenivas ◽  
Jonathan Engvall ◽  
Carl Grey ◽  
Ed W. J. van Niel
Keyword(s):  
Glucose Uptake ◽  
Growth Patterns ◽  
Sugar Uptake ◽  
Uptake System ◽  
Sugar Mixtures ◽  
Xylose Uptake ◽  
Growth Profiles ◽  
High Concentrations ◽  
Xylose Transporter

Abstract Background Caldicellulosiruptor kronotskyensis has gained interest for its ability to grow on various lignocellulosic biomass. The aim of this study was to investigate the growth profiles of C. kronotskyensis in the presence of mixtures of glucose–xylose. Recently, we characterized a diauxic-like pattern for C. saccharolyticus on lignocellulosic sugar mixtures. In this study, we aimed to investigate further whether C. kronotskyensis has adapted to uptake glucose in the disaccharide form (cellobiose) rather than the monosaccharide (glucose). Results Interestingly, growth of C. kronotskyensis on glucose and xylose mixtures did not display diauxic-like growth patterns. Closer investigation revealed that, in contrast to C. saccharolyticus, C. kronotskyensis does not possess a second uptake system for glucose. Both C. saccharolyticus and C. kronotskyensis share the characteristics of preferring xylose over glucose. Growth on xylose was twice as fast (μmax = 0.57 h−1) as on glucose (μmax = 0.28 h−1). A study of the sugar uptake was made with different glucose–xylose ratios to find a kinetic relationship between the two sugars for transport into the cell. High concentrations of glucose inhibited xylose uptake and vice versa. The inhibition constants were estimated to be KI,glu = 0.01 cmol L−1 and KI,xyl = 0.001 cmol L−1, hence glucose uptake was more severely inhibited by xylose uptake. Bioinformatics analysis could not exclude that C. kronotskyensis possesses more than one transporter for glucose. As a next step it was investigated whether glucose uptake by C. kronotskyensis improved in the form of cellobiose. Indeed, cellobiose is taken up faster than glucose; nevertheless, the growth rate on each sugar remained similar. Conclusions C. kronotskyensis possesses a xylose transporter that might take up glucose at an inferior rate even in the absence of xylose. Alternatively, glucose can be taken up in the form of cellobiose, but growth performance is still inferior to growth on xylose. Therefore, we propose that the catabolism of C. kronotskyensis has adapted more strongly to pentose rather than hexose, thereby having obtained a specific survival edge in thermophilic lignocellulosic degradation communities.

scholarly journals Characterization of phylogenetically distant members of the adenylate cyclase family from mycobacteria: Rv1647 from Mycobacterium tuberculosis and its orthologue ML1399 from M. leprae

2005 ◽  
Vol 387 (2) ◽  
pp. 541-551 ◽  
Author(s):  
Avinash R. SHENOY ◽  
Nandini P. SREENATH ◽  
Mohana MAHALINGAM ◽  
Sandhya S. VISWESWARIAH
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Adenylate Cyclase ◽  
Biochemical Properties ◽  
The Other ◽  
Gene Products ◽  
Class Iii ◽  
Expression And Purification ◽  
Mycobacterium Tuberculosis H37rv ◽  
High Concentrations

Analysis of the genome sequence of Mycobacterium tuberculosis H37Rv has identified 16 genes that are similar to the mammalian adenylate and guanylate cyclases. Rv1647 was predicted to be an active adenylate cyclase but its position in a phylogenetically distant branch from the other enzymes characterized so far from M. tuberculosis makes it an interestingly divergent nucleotide cyclase to study. In agreement with its divergence at the sequence level from other nucleotide cyclases, the cloning, expression and purification of Rv1647 revealed differences in its biochemical properties from the previously characterized Rv1625c adenylate cyclase. Adenylate cyclase activity of Rv1647 was activated by detergents but was resistant to high concentrations of salt. Mutations of substrate-specifying residues to those present in guanylate cyclases failed to convert the enzyme into a guanylate cyclase, and did not alter its oligomeric status. Orthologues of Rv1647 could be found in M. leprae, M. avium and M. smegmatis. The orthologue from M. leprae (ML1399) was cloned, and the protein was expressed, purified and shown biochemically to be an adenylate cyclase, thus representing the first adenylate cyclase to be described from M. leprae. Importantly, Western-blot analysis of subcellular fractions from M. tuberculosis and M. leprae revealed that the Rv1647 and ML1399 gene products respectively were expressed in these bacteria. Additionally, M. tuberculosis was also found to express the Rv1625c adenylate cyclase, suggesting that multiple adenylate cyclase proteins may be expressed simultaneously in this organism. These results suggest that class III cyclase-like gene products probably have an important role to play in the physiology and perhaps the pathology of these medically important bacteria.

scholarly journals Enolase isoenzymes in adult and developing Xenopus laevis and characterization of a cloned enolase sequence

1988 ◽  
Vol 251 (1) ◽  
pp. 31-39 ◽  
Author(s):  
N Segil ◽  
A Shrutkowski ◽  
M B Dworkin ◽  
E Dworkin-Rastl
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Xenopus Laevis ◽  
Adult Brain ◽  
Gene Products ◽  
Adult Liver ◽  
Acidic Form ◽  
High Concentrations ◽  
Enolase Gene Eno1

As part of a study of glycolysis during early development we have examined the pattern of expression of enolase isoenzymes in Xenopus laevis. In addition, the nucleotide sequence of a cDNA clone coding for the complete amino acid sequence of one enolase gene (ENO1) in X. laevis was determined. X. laevis ENO1 shows highest homology to mammalian non-neuronal enolase. Analysis of enolase isoenzymes in X. laevis by non-denaturing electrophoresis on cellulose acetate strips revealed five isoenzymes. One form was present in all tissues tested, two additional forms were expressed in oocytes, embryos, adult liver and adult brain, and two further forms were restricted to larval and adult muscle. Since enolase is a dimer, three different monomers (gene products) could account for the observed number of isoenzymes. This pattern of enolase isoenzyme expression in X. laevis differs from that of birds and mammals. In birds and mammals the most acidic form is neuron-specific and there is only one major isoenzyme expressed in the liver. RNAase protection experiments showed the presence of ENO1 mRNA in oocytes, liver and muscle, suggesting that it codes for a non-tissue-restricted isoenzyme. ENO1 mRNA concentrations are high in early oocytes, decrease during oogenesis and decrease further after fertilization. Enolase protein, however, is maintained at high concentrations throughout this period.

scholarly journals Characterization of Simultaneous Uptake of Xylose and Glucose in Caldicellulosiruptor Kronotskyensis for Optimal Hydrogen Production

2021 ◽  
Author(s):  
Thitiwut Vongkampang ◽  
Krishnan Sreeni ◽  
Jonathan Engvall ◽  
Carl Grey ◽  
Ed van Niel
Keyword(s):  
Glucose Uptake ◽  
Bioinformatic Analysis ◽  
Growth Patterns ◽  
Sugar Uptake ◽  
Uptake System ◽  
Sugar Mixtures ◽  
Xylose Uptake ◽  
Growth Profiles ◽  
High Concentrations

Abstract BackgroundCaldicellulosiruptor kronotskyensis has gained interest for its ability to grow on various lignocellulosic biomass. The aim of this study was to investigate the growth profiles of C . kronotskyensis in the presence of mixtures of glucose-xylose. Recently, we characterized a diauxic-like pattern for C. saccharolyticus on lignocellulosic sugar mixtures. In this study we aimed to investigated further whether C . kronotskyensis has adapted to uptake glucose in the disaccharide form (cellobiose) rather than the monosaccharide (glucose). ResultsInterestingly, growth of C . kronotskyensis on glucose and xylose mixtures did not display diauxic-like growth patterns. Closer investigation revealed that, in contrast to C. saccharolyticus , C . kronotskyensis does not possess a second uptake system for glucose. Both C. saccharolyticus and C . kronotskyensis share the characteristics of preferring xylose over glucose. Growth on xylose was twice as fast (μ max = 0.57 h -1 ) as on glucose (μ max = 0.28 h -1 ). It was found that C . kronotskyensis takes up glucose and xylose simultaneously with the same transporter. A study of the sugar uptake was made with different glucose-xylose ratios to find a kinetic relationship between the two sugars for transport into the cell. High concentrations of glucose inhibited xylose uptake and vice versa. The inhibition constants were estimated to be K I,glu = 0.01 cmol·L -1 and K I,xyl = 0.001 cmol·L -1 , hence glucose uptake was more severely inhibited by xylose uptake. Bioinformatic analysis indicated the lack of another sugar uptake system in C . kronotskyensis as compared to C. saccharolyticus . Therefore, it was investigated whether glucose uptake by C . kronotskyensis was in the form of cellobiose. Indeed, cellobiose is taken up faster than glucose, nevertheless, the growth rate on each sugar remained similar. ConclusionsC . kronotskyensis possesses a xylose transporter that might take up glucose at an inferior rate even in the absence of xylose. Alternatively, glucose can be taken up in the form of cellobiose, but growth performance is still inferior to growth on xylose. Therefore, we propose that the catabolism of C . kronotskyensis has adapted more strongly to pentose rather than hexose thereby having obtained a specific survival edge in thermophilic lignocellulosic degradation communities.

scholarly journals Characterization of the Binding Protein-Dependent Cellobiose and Cellotriose Transport System of the Cellulose Degrader Streptomyces reticuli

1999 ◽  
Vol 65 (6) ◽  
pp. 2636-2643 ◽  
Author(s):  
Andreas Schlösser ◽  
Jens Jantos ◽  
Karl Hackmann ◽  
Hildgund Schrempf
Keyword(s):  
Abc Transporter ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Transport Systems ◽  
Uptake System ◽  
Gene Products ◽  
Abc Transport ◽  
Insertional Inactivation ◽  
Atp Binding Protein

ABSTRACT Streptomyces reticuli has an inducible ATP-dependent uptake system specific for cellobiose and cellotriose. By reversed genetics a gene cluster encoding components of a binding protein-dependent cellobiose and cellotriose ABC transporter was cloned and sequenced. The deduced gene products comprise a regulatory protein (CebR), a cellobiose binding lipoprotein (CebE), two integral membrane proteins (CebF and CebG), and the NH2-terminal part of an intracellular β-glucosidase (BglC). The gene for the ATP binding protein MsiK is not linked to the ceb operon. We have shown earlier that MsiK is part of two different ABC transport systems, one for maltose and one for cellobiose and cellotriose, in S. reticuli and Streptomyces lividans. Transcription of polycistronic cebEFG and bglC mRNAs is induced by cellobiose, whereas the cebR gene is transcribed independently. Immunological experiments showed that CebE is synthesized during growth with cellobiose and that MsiK is produced in the presence of several sugars at high or moderate levels. The described ABC transporter is the first one of its kind and is the only specific cellobiose/cellotriose uptake system of S. reticuli, since insertional inactivation of the cebEgene prevents high-affinity uptake of cellobiose.

Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Rémi Terrasse ◽  
Jayesh Arun Bafna ◽  
Lorraine Benier ◽  
Mathias Winterhalter
Keyword(s):  
Outer Membrane ◽  
Lipid Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Multi Drug Resistance ◽  
Gram Negative Bacteria ◽  
Membrane Channels ◽  
Gram Negative ◽  
Electrophysiological Characterization

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we extract, purify and reconstitute them into artificial planar membranes. To avoid this time-consuming procedure, here we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer which moreover allows also to some extend the characterization of membrane protein channels in their native environment. Two major membrane channels from <i>Escherichia coli</i>, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution methods, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin on the LPS side yields somewhat higher association (<i>k<sub>on</sub></i>) and lower dissociation (<i>k<sub>off</sub></i>) rates compared to LPS-free reconstitution. We conclude that using outer membrane vesicles is a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

scholarly journals Adsorption Studies on Magnetic Nanoparticles Functionalized with Silver to Remove Nitrates from Waters

Water ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 1757
Author(s):  
Yesica Vicente-Martínez ◽  
Manuel Caravaca ◽  
Antonio Soto-Meca ◽  
Miguel Ángel Martín-Pereira ◽  
María del Carmen García-Onsurbe
Keyword(s):  
Magnetic Nanoparticles ◽  
Nitrate Content ◽  
Adsorption Efficiency ◽  
Experimental Conditions ◽  
Dependent Behavior ◽  
Before And After ◽  
Naoh Solution ◽  
High Concentrations ◽  
Interference Studies

This paper presents a novel procedure for the treatment of contaminated water with high concentrations of nitrates, which are considered as one of the main causes of the eutrophication phenomena. For this purpose, magnetic nanoparticles functionalized with silver (Fe3O4@AgNPs) were synthesized and used as an adsorbent of nitrates. Experimental conditions, including the pH, adsorbent and adsorbate dose, temperature and contact time, were analyzed to obtain the highest adsorption efficiency for different concentration of nitrates in water. A maximum removal efficiency of 100% was reached for 2, 5, 10 and 50 mg/L of nitrate at pH = 5, room temperature, and 50, 100, 250 and 500 µL of Fe3O4@AgNPs, respectively. The characterization of the adsorbent, before and after adsorption, was performed by energy dispersive X-ray spectroscopy, scanning electron microscopy, Brunauer-Emmett-Teller analysis and Fourier-transform infrared spectroscopy. Nitrates can be desorbed, and the adsorbent can be reused using 500 µL of NaOH solution 0.01 M, remaining unchanged for the first three cycles, and exhibiting 90% adsorption efficiency after three regenerations. A deep study on equilibrium isotherms reveals a pH-dependent behavior, characterized by Langmuir and Freundlich models at pH = 5 and pH = 1, respectively. Thermodynamic studies were consistent with physicochemical adsorption for all experiments but showed a change from endothermic to exothermic behavior as the temperature increases. Interference studies of other ions commonly present in water were carried out, enabling this procedure as very selective for nitrate ions. In addition, the method was applied to real samples of seawater, showing its ability to eliminate the total nitrate content in eutrophized waters.

Sign in / Sign up

Export Citation Format

Share Document